Desarrollo y evaluación toxicológica de compuestos aliáceos para su aplicación en el ámbito agroalimentario

UNIVERSIDAD DE SEVILLA FACULTAD DE FARMACIA DEPARTAMENTO DE NUTRICIÓN Y BROMATOLOGÍA, TOXICOLOGÍA Y MEDICINA LEGAL “Desarrollo y evaluación toxicológica de compuestos aliáceos para su aplicación en el ámbito agroalimentario” “Development and toxicological evaluation of alliaceous compounds for application in the agri-food sector” Memoria que presenta el Graduado Antonio Cascajosa Lira para optar al título de Doctor por la Universidad de Sevilla con la Mención Internacional Sevilla, 2024 UNIVERSIDAD DE SEVILLA Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal Facultad de Farmacia C/ Profesor García González, nº 2 41012 Sevilla (España) Teléfono: 954 55 67 62 Fax: 954 55 64 22 Dª SILVIA PICHARDO SÁNCHEZ, Catedrática de Universidad y Directora del Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal de la Universidad de Sevilla, INFORMA: Que la tesis Doctoral titulada “Desarrollo y evaluación toxicológica de compuestos aliáceos para su aplicación en el ámbito agroalimentario” presentada por el graduado Antonio Cascajosa Lira para optar al grado de Doctor por la Universidad de Sevilla con la Mención Internacional, ha sido realizada en el área de Toxicología de este Departamento bajo la dirección de la Dra. Ana María Cameán Fernández y la Dra. Ángeles Jos Gallego. Así mismo, informa de la estancia internacional predoctoral llevaba a cabo en el Centro Interdisciplinar de Investigación Marina e Ambiental (CIIMAR) bajo la dirección del Dr. Alexandre Campos. Y para que conste, firmo el presente en Sevilla a fecha de firma. UNIVERSIDAD DE SEVILLA Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal Facultad de Farmacia C/ Profesor García González, nº 2 41012 Sevilla (España) Teléfono: 954 55 67 62 Fax: 954 55 64 22 Dª Ana María Cameán Fernández y Dª Ángeles Jos Gallego, Catedráticas de Universidad del Departamento de Nutrición y Bromatología, Toxicología y Medicinal Legal de la Universidad de Sevilla, INFORMAN: Que siendo directoras de la Tesis Doctoral del graduado Antonio Cascajosa Lira, con título: “Desarrollo y evaluación toxicológica de compuestos aliáceos para su aplicación en el ámbito agroalimentario”, avalan la idoneidad de la presentación de la misma en la modalidad de compendio por publicaciones, e indican que las aportaciones publicadas aceptadas para su publicación cumplen con los criterios de la evaluación publicada por la CNEI, así: El trabajo “Safety assessment of propyl-propane-thiosulfonate (PTSO): 90-days oral subchronic toxicity study in rats” fue publicado en. Food and Chemical Toxicology, 144, 111612. Q1 (14/144) en FOOD SCIENCE AND TECHNOLOGY. Índice de impacto: 6,0. El trabajo “Mutagenicity and genotoxicity assessment of a new biopreservative product rich in Enterocin AS-48” fue publicado en Food and Chemical Toxicology, 146, 111846. Q1 (14/144) en FOOD SCIENCE AND TECHNOLOGY. Índice de impacto: 6,0. El trabajo “Genotoxicity evaluation of propyl-propane-thiosulfinate (PTS) from Allium genus essential oils by a combination of micronucleus and comet assays in rats” fue publicado en Foods, 10(5), 989. Q1 (35/144) en FOOD SCIENCE AND TECHNOLOGY. Índice de impacto: 5,6. El trabajo “Simultaneous determination of Allium compounds (Propyl propane thiosulfonate and thiosulfinate) in animal feed using UPLC-MS/MS” fue publicado en Food and Chemical Toxicology, 157, 112619. Q1 (34/144) en FOOD SCIENCE AND TECHNOLOGY. Índice de impacto: 5,6. El trabajo “Acute and subchronic 90-days toxicity assessment of propyl-propane- thiosulfinate (PTS) in rats” fue publicado en Food and Chemical Toxicology, 161, 112827. Q1 (20/94) en TOXICOLOGY. Índice de impacto: 4,3. UNIVERSIDAD DE SEVILLA Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal Facultad de Farmacia C/ Profesor García González, nº 2 41012 Sevilla (España) Teléfono: 954 55 67 62 Fax: 954 55 64 22 El trabajo “In vitro toxicity studies of bioactive organosulfur compounds from Allium spp. with potential application in the agri-food industry: A review” fue publicado en Foods, 11(17), 2620. Q1 (34/142) en FOOD SCIENCE AND TECHNOLOGY. Índice de impacto: 5,2 El trabajo “Genotoxicity evaluation of two derived products from Allium extracts: spropylmercaptocysteine and s-propyl mercaptoglutathione.” Fue publicado en Food Bioscience, 53, 102671. Q1(36/173) en FOOD SCIENCE & TECHNOLOGY. Índice de impacto: 4,8 El trabajo “Identification of in vitro metabolites of an Allium organosulfur compound and environmental toxicity prediction as part of its risk assessment” fue publicado en Environmental Research, 229, 116001. Q1 (15/403) en PUBLIC, ENVIRONMENTAL & OCCUPATIONAL HEALTH. Índice de impacto: 7,7 El trabajo “Risk assessment and environmental consequences of the use of the Alliumderived compound propyl-propane thiosulfonate (PTSO) in agrifood applications” fue publicado en Environmental Research, 236, 116682. Q1 (15/403) en PUBLIC, ENVIRONMENTAL & OCCUPATIONAL HEALTH. Índice de impacto: 7,7 El trabajo “Two-Generation Toxicity Study of the Antioxidant Compound Propyl- Propane Thiosulfonate (PTSO)” fue publicado en Antioxidants, 13(3), 350. Q1 (8/72) en CHEMISTRY, MEDICINAL. Índice de impacto: 6,0. El trabajo “Protective effects of sulforaphane against toxic substances and contaminants: A systematic review” fue publicado en Phytomedicine, 130, 155731. Q1. (19/354) en PHARMACOLOGY & PHARMACY. Índice de impacto: 6,5. UNIVERSIDAD DE SEVILLA Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal Facultad de Farmacia C/ Profesor García González, nº 2 41012 Sevilla (España) Teléfono: 954 55 67 62 Fax: 954 55 64 22 Este trabajo ha sido realizado en el Área de Toxicología del Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal de la Facultad de Farmacia de la Universidad de Sevilla, y se ha financiado con los siguientes Proyectos de Investigación: • Plan Estatal 2013-2016 Retos - Colaboración Empresa. ECOFEED. RTC-2017- 6199-2. Alimentación sostenible de bovino de cebo con menor generación de gas metano y obtención de carne fresca y derivados cárnicos sostenibles medioambientalmente. (Investigadora principal: Ana Mª Cameán Fernández) • PAIDI: Actividades de Transferencia 2017. AT17_5323_USE. Desarrollo y evaluación toxicológica de compuestos aliáceos para su aplicación como aditivos en nutrición animal (Investigadora principal: Ana Mª Cameán Fernández) • PAIDI 2018: Proyectos I+D+i. P18-TP-2147. Estudios toxicológicos para evaluar la seguridad de compuestos azufrados como potenciales aditivos sensoriales en nutrición animal (Investigadoras principales: Ana Mª Cameán Fernández y Silvia Pichardo Sánchez). • Horizonte 2020. TOXICROP. H2020-823860. Cyanotoxins in irrigation waters: surveillance, risk assesssment, and innovative remediation proposals TOXICROP. (Investigadora principal (US): Ana Mª Cameán Fernández). El doctorando Antonio Cascajosa Lira ha disfrutado de una ayuda para contratos predoctorales para la formación de profesores universitarios, bajo la modalidad de contrato predoctoral, de Personal Investigador en Formación (PIF), con referencia FPU 2019-01247. AGRADECIMIENTOS Después de cinco años de trabajo y crecimiento personal, este sueño se ha convertido en realidad. Este recorrido ha sido, sin duda, uno de los mayores desafíos de mi vida, lleno de aprendizajes y momentos de incertidumbre, pero también de una profunda satisfacción al ver cómo cada paso me acercaba un poco más a este momento. No puedo continuar sin antes expresar mi más sincero agradecimiento a todas las personas que, de una forma u otra, han estado presentes a lo largo de este proceso. Este logro no es solo mío, es el reflejo de todo el apoyo, la paciencia y el amor que he recibido de aquellos que han creído en mí, incluso cuando yo mismo lo dudaba. Primero, quiero expresar mi más profundo agradecimiento a mis directoras de tesis, quienes no solo me guiaron, sino que me acompañaron en cada paso de este camino con una paciencia infinita. A la Dra. Ana Cameán, te agradezco de corazón por confiar en mí desde el inicio y brindarme la oportunidad de emprender este viaje, incluso en los momentos más difíciles. Gracias por tu sabiduría, por ser una guía en los momentos de duda y por enseñarme a nunca conformarme, siempre buscando ser mejor. A la Dra. Ángeles Jos, gracias por estar a mi lado, compartiendo horas interminables para sacar esta Tesis adelante. Tu pasión por la investigación es contagiosa, y no solo me has enseñado, sino que también me has inspirado a seguir adelante cuando las cosas se complicaban. Tu apoyo constante y tu cercanía han sido un pilar fundamental para mí. De verdad, a las dos, gracias por creer en mí y por ser no sólo mentoras, sino también compañeras en esta aventura. Sin vosotras, nada de esto hubiera sido posible. Quiero expresar mi más sincero agradecimiento a quienes forman parte de este maravilloso grupo de investigación. A la Dra. Silvia Pichardo, mis palabras de gratitud no son suficientes para expresar cuánto valoro todo lo que has hecho por mí durante esta tesis. Trabajar contigo en equipo ha sido maravilloso. Siempre has estado dispuesta a brindar un consejo acertado, una solución creativa y un apoyo constante en cada obstáculo que se presentaba en el camino. A la Dra. Anabel Prieto, te agradezco por tu apoyo incondicional, tanto en lo laboral como en lo personal. Tu capacidad para trabajar con calma y claridad ha sido un ejemplo para mí. A la Dra. María Puerto, gracias por transmitir tu pasión por la investigación y por los consejos a largo de esta etapa. A la Dra. Remedios Guzmán, quien ha estado a mi lado desde mis inicios como alumno interno. Gracias por acompañarme siempre y ayudarme a crecer tanto en lo académico como en lo personal. Para mi eres un ejemplo a seguir, tu orientación ha sido clave para que pudiera avanzar con seguridad. A la Dra. Leticia Diez-Quijada, con quien compartí mis primeros momentos en el laboratorio de toxicología, gracias por ser una fuente de inspiración. Gracias, sinceramente, a todas ustedes. Cada una, con su forma de ser, ha dejado una huella imborrable en mí y en este trabajo. Quiero agradecer de corazón a Félix por su constante ayuda y disposición a lo largo de este proyecto. Siempre has estado disponible para echar una mano cuando más lo necesitaba, y eso ha sido clave para que todo saliera adelante sin contratiempos. Por supuesto, quiero expresar mi más profundo agradecimiento a la Dra. Conchi y al Dr. Óscar, quienes, con una sonrisa han estado a mi lado en cada paso de este camino. Su apoyo y energía han hecho que incluso los momentos más difíciles se sintieran más livianos. A mis compañeros Cris y Toño, no tengo palabras suficientes para agradecer los momentos inolvidables que hemos compartido. Han sido más que colegas, han sido amigos que siempre llevaré en mi corazón. Cada risa, cada desafío superado juntos, es un recuerdo que atesoro profundamente. También quiero agradecer de corazón a las chicas de Nutri: Marta, Marina, Ángeles, Pilar y Rocío, que siempre han estado al otro lado del laboratorio. Gracias por su cercanía, sus sonrisas y por esos momentos de apoyo y complicidad que han sido tan importantes en esta etapa. También quiero expresar mi agradecimiento a la empresa DOMCA y a todo su equipo por el apoyo brindado durante este proceso. A Enrique Guillamón, Alberto Baños, José Manuel de la Torre y Arancha Aguinaga, gracias por los recursos, la confianza y el respaldo que me han ofrecido en cada etapa. Su colaboración ha sido fundamental para que esta Tesis pudiera salir adelante. Quiero extender mi más sincero agradecimiento a los miembros de la Universidad de Córdoba (UCO) que han colaborado en esta tesis. A todo el equipo de investigación dirigido por la Dra. Rosario Moyano, gracias por su apoyo constante. A los Dres. Nahúm Ayala y Ana Molina, y a Maribel Barasona, les agradezco su dedicación, su tiempo y por cada aportación que han hecho para que este proyecto avanzara. Quiero extender mi agradecimiento a los servicios generales de la universidad (CITIUS), y en especial a Maru, Rocío, Modesto y Laura. Su apoyo ha sido invaluable, y siempre han estado dispuestos a ayudar, resolviendo nuestros problemas en tiempo récord. Su dedicación y eficiencia han hecho una gran diferencia en nuestro trabajo, y no puedo dejar de agradecerles su compromiso y profesionalidad. Quiero expresar mi más sincero agradecimiento al equipo del Instituto de Recursos Naturales y Agrobiología de Sevilla, en especial a los Dres. José Antonio y Nicasio, por su colaboración y apoyo a lo largo de este proceso. También quiero agradecer a los Dres. Alexandre Campos y Vitor Vasconcelos, quienes han colaborado conmigo desde el Centro Interdisciplinar de Investigação Marinha e Ambienta durante mi estancia predoctoral, que han enriquecido enormemente mi conocimiento y experiencia. A la Dra. Giorgiana, gracias por toda la ayuda y el apoyo que me has brindado. Tu disposición para ayudar ha sido un verdadero alivio y un pilar en este camino. Trabajar con todos estos investigadores ha sido un verdadero privilegio, y su colaboración ha sido fundamental para el desarrollo de esta tesis. Sin su esfuerzo y compromiso, este logro no habría sido posible. Quiero agradecer de corazón a mis amigos, quienes se han convertido en una parte fundamental de mi vida durante estos años académicos. Su amistad, que se ha fortalecido en este tiempo, siempre ha sido un refugio en los momentos más difíciles, logrando sacarme una sonrisa cuando más lo necesitaba. Gracias, Camila, Joselu, Raúl, Pablo, Juanmi y Fran, por estar siempre ahí y hacer este camino mucho más llevadero con su apoyo y compañía. Todo esto no hubiera sido posible sin mi familia. Quiero dedicar unas palabras muy especiales a mis padres. A mi padre, quien ya no está físicamente conmigo, pero cuya memoria recuerdo en cada paso que doy. Gracias, papá, por todo lo que me enseñaste, por ser mi inspiración y por darme las bases sobre las que hoy me sostengo. Aunque no estés aquí para compartir este logro, sé que tu amor y sabiduría me han acompañado siempre. A mi madre y a mi hermana, gracias por vuestra fortaleza inquebrantable durante estos años. Habéis sido un pilar fundamental en mi vida, enfrentando cada desafío con valentía y amor. Mi familia siempre es la fuerza para seguir adelante y alcanzar mis metas. También tengo que agradecer a la persona que me ha acompañado todo este tiempo, gracias, José Andrés por ser tan atento conmigo, cuidarme y estar siempre a mi lado apoyándome en todo momento. Te quiero. VI VII ÍNDICE DE FIGURAS Y TABLAS/ FIGURES AND TABLES INDEX Figura 1. A) Emisiones de gases de efecto invernadero por año y B) por ramas de actividad. ......................................................................................................................... 24 Figura 2. Proceso de biosíntesis de los diferentes compuestos organosulfurados. ......... 28 Figura 3. Estructura química del propil propano tiosulfonato (PTSO) .......................... 38 Figura 4. Estructura química del propil propano tiosulfinato (PTS). ............................. 38 Figura 5. Formación del sulforafano in vivo a través de su precursor ............................ 41 Figura 6. Estructura molecular de la enterocina AS-48 .................................................. 42 Figura 7. Esquema de la evaluación toxicológica a seguir para aditivos en piensos (según EFSA, 2017) .................................................................................................................... 51 Figura 8. Esquema de la evaluación toxicológica a seguir para productos fitosanitarios (según Reglamento 1107/2009) ....................................................................................... 53 Tabla 1. Artículos de revisión en los que se tratan los diferentes beneficios para la salud de los compuestos OSCs. ................................................................................................. 29 Tabla 2. Resumen de los estudios toxicológicos llevados a cabo hasta ahora de las sustancias que se abordan en la presente tesis doctoral. .................................................. 55 VIII IX LISTA DE ABREVIATURAS • ADG: Ganancia diaria de peso • ADME: Absorción, Distribución, Metabolismo y Excreción • ALT: Alanina Aminotransferasa (Alanine Aminotransferase) • ALB: Albúmina • ALKP: Fosfatasa Alcalina • AMS: sulfuro de alilo y metilo • AMSO: Alil metil sulfoxido • AMSO2: Alil metil sufonato • AOAC: Association of Official Analytical Chemists • AS-48: Enterocina AS-48 • AST: Aspartato Aminotransferasa • BCM: Bromoclorometano • BNMN: Células binucleadas con micronúcleos • BUN: Nitrógeno Ureico en Sangre • CAR: Carvacrol • CAR-R: Bacterias resistentes a carbapenem • CD-PTS: Complejo cyclodextrin-PTS • CE: Comisión europea • CE50: Concentración efectiva media • CIN: Cinamaldehido • CL50: Concentración letal media • CPT: Contenido proteico total • CYN: Cilindrospermopsina • DAS: Sulfuro de dialilo • DADS: Dialil disulfuro • DMTS: Dimetil trisulfuro • DPDS: Dipropil disulfuro • DENV: Virus del Dengue • DL50: Dosis Letal Media • DMI: Ingesta de materia seca • DMT: Dosis Máxima Tolerable • DPPH: 2,2-difenil-1- picrilhidrazilo • EFSA: Autoridad Europea de Seguridad Alimentaria • Endo-III: Endonucleasa-III • ERO: Especies Reactivas de Oxígeno • EU: Unión Europea • FPG: Formamidopirimidina ADN glicosilasa • GBE: extracto de brócoli rico en glucorafanina • GLUC: Glucosa en Sangre • GST: Glutatión S-transferasa • GSSP: - S-propilglutatión • HCT: Hematocrito • HGB: Hemoglobina • HPP: Procesamiento por Altas Presiones • HPLC: Cromatografía Líquida de Alta Resolución • HRGC-MS: Cromatografía de gases de alta resolución acoplada a espectrometría de masas • IPCC: Panel Intergubernamental sobre el Cambio Climático • Keap1: Proteína asociada a ECH similar a Kelch • LC50: Concentración Letal Media (Lethal Concentration 50) • LC-MS: Cromatografía Líquida acoplada a Espectrometría de Masas • LMR: Límites Máximos de Residuos • LOEC: Concentración de efecto observado más bajo X • LPO: Peroxidación lipídica • MCH: Hemoglobina Corpuscular Media • MCHC: Concentración Media de Hemoglobina Corpuscular • MDA: Malondialdehido • MIC: Concentración Inhibitoria Mínima • MN: Micronúcleo • NCE: Eritrocito normocromático • NEMs: Nuevos enfoques metodológicos • NDI: Índice de división nuclear • NO: Óxido nítrico • NOAEL: Nivel sin efecto adverso observado • NOEC: Concentración sin efecto observado • Nrf2: Factor 2 relacionado con el factor nuclear eritroide 2 • ODS: Objetivos de Desarrollo Sostenible • OECD: Organización para la Cooperación y el Desarrollo Económico • OMS: Organización Mundial de la Salud • OMG: Organismo Modificado Genéticamente • OSCs: Compuestos organosulfurados • PRAN: Plan Nacional Resistencia Antibióticos • PCE: Eritrocitos Policromáticos • PT: Tiempo de Protrombina • PTS: Propil propano tiosulfinato • PTSO: Propil propano tiosulfonato • RBC: Glóbulos Rojos • RDW: Ancho de Distribución de los Glóbulos Rojos • ROS: Especies Reactivas de Oxígeno • RSM: Metodología de Superficie de Respuesta • SAC: S-alilcisteína • SAMC: S-Alilmetilcisteína • SARS-CoV: Coronavirus del Síndrome Respiratorio Agudo Severo • SFE: Extracción con Fluidos Supercríticos • SFN: Sulforafano • TOT PROT: Proteína Total • UE: Unión Europea • VFA: Ácidos grasos volátiles • VIH: Virus de la Inmunodeficiencia Humana • VO: Vía Oral • VSH: Virus del Sarcoma de Hardy • XO: Xantina oxidasa 1 I. RESUMEN/ SUMMARY Resumen/Summary 3 I. RESUMEN Los compuestos organosulfurados (OSCs) son metabolitos secundarios producidos por diferentes especies del género Allium que poseen importantes actividades biológicas, como antimicrobianas, antioxidantes, antiinflamatorias, antidiabéticas, anticarcinogénicas y antiespasmódicas. En los últimos años, se ha promovido su uso en la industria agroalimentaria como sustitutos de los conservantes sintéticos, lo que aumenta la exposición acumulativa potencial para los consumidores. Antes de su aplicación en esta industria, es necesario realizar una evaluación de seguridad, conforme a lo estipulado por la Autoridad Europea de Seguridad Alimentaria (EFSA). Este trabajo revisa la literatura científica sobre la evaluación de toxicidad in vitro de los OSCs, siguiendo las directrices PRISMA para revisiones sistemáticas. Actualmente, hay muy pocos estudios in vitro que investiguen la toxicidad potencial de los OSCs, estando centradas la mayoría de las investigaciones en evaluar la citotoxicidad para esclarecer sus efectos antiproliferativos, y enfocándose en aspectos terapéuticos mediante líneas celulares cancerosas como modelo experimental principal. El análisis llevado a cabo demostró que el disulfuro de dialilo (DADS) es el compuesto más estudiado, seguido por el trisulfuro de dialilo (DATS), el sulfuro de dialilo (DAS), la alicina y el ajoeno. Por otro lado, y siguiendo también las directrices PRISMA, se han revisado los efectos protectores del OSC conocido como sulforafano (SFN). Las investigaciones científicas indican que el hígado y el sistema nervioso son los órganos diana de mayor atención, posiblemente debido al papel clave del estrés oxidativo en enfermedades hepáticas y neurodegenerativas. Sin embargo, también se han demostrado actividades protectoras en los pulmones, el corazón, el sistema inmunológico, los riñones y el sistema endocrino. El SFN ejerce sus efectos protectores al activar la vía Nrf2, que potencia las defensas antioxidantes y reduce el estrés oxidativo, además de suprimir la inflamación al disminuir la producción de interleucinas. Así mismo, el SFN inhibe la apoptosis a través de la caspasa 3 y aumenta los niveles de Bcl2. En general, el SFN demuestra mecanismos multifacéticos para contrarrestar los efectos adversos de agentes tóxicos. El SFN tiene potenciales aplicaciones clínicas como agente quimioprotector, aunque se necesitan más estudios para establecer dosis seguras en humanos. El análisis bibliográfico realizado ha dado lugar a dos publicaciones científicas: Resumen/Summary 10 Resumen/Summary 11 I. SUMMARY Organosulfur compounds (OSCs) are secondary metabolites produced by different Allium species that possess important biological activities, such as antimicrobial, antioxidant, anti-inflammatory, antidiabetic, anticarcinogenic and antispasmodic. In recent years, their use has been promoted in the agri-food industry as substitutes for synthetic preservatives, increasing the potential cumulative exposure for consumers. Prior to their application in this industry, a safety assessment, as stipulated by the European Food Safety Authority (EFSA), is required. A review of the scientific literature regarding the in vitro toxicity assessment of CSOs, following the PRISMA guidelines for systematic reviews, has been performed. Currently, there are very few in vitro studies investigating the potential toxicity of OSCs, with most research focused on evaluating cytotoxicity to elucidate their antiproliferative effects, focusing on therapeutic aspects using cancer cell lines as the main experimental model. The results showed that diallyl disulfide (DADS) is the most studied compound, followed by diallyl trisulfide (DATS), diallyl sulfide (DAS), allicin and ajoene. On the other hand, and also following PRISMA guidelines, the protective effects of the OSCs known as sulforaphane (SFN) have been reviewed. Scientific research indicates that the liver and the nervous system are the target organs of greatest attention, possibly due to the key role of oxidative stress in liver and neurodegenerative diseases. However, protective activities have also been demonstrated in the lungs, heart, immune system, kidneys and endocrine system. SFN exerts its protective effects by activating the Nrf2 pathway, which enhances antioxidant defenses and reduces oxidative stress, as well as suppressing inflammation by decreasing the production of interleukins. Likewise, SFN inhibits apoptosis by preventing caspase 3 cleavage and increasing Bcl2 levels. Overall, SFN demonstrates multifaceted mechanisms to counteract the adverse effects of toxic agents. SFN has potential clinical applications as a chemoprotective agent, although further studies are needed to establish safe doses in humans. The literature review conducted has resulted in two scientific publications: In vitro toxicity studies of bioactive organosulfur compounds from Allium spp. with potential application in the agri-food industry: A review. (Cascajosa-Lira et al., 2022, Foods, 11(17), 2620). Resumen/Summary 12 Protective effects of sulforaphane against toxic substances and contaminants: A systematic review. (Cascajosa-Lira et al., 2024, Phytomedicine, 130, 155731). To ensure consumer health, a genotoxicity evaluation of the substances and their metabolites is necessary, following the recommendations of the EFSA. Tests were performed to evaluate the toxicological profile of PTSO derivatives, including cytotoxicity tests, Ames test (OECD 471), micronucleus test (OECD 478) and comet assay. The results showed that there were no cytotoxic effects in Caco-2 cells exposed to the metabolites S-propylmercaptocysteine (CSSP) and S-propylmercaptoglutation (GSSP) at concentrations from 0 to 450 μM. The mutagenicity index remained between 0.6 and 1.4 for both compounds, with no mutagenic effects at the concentrations tested. In the micronucleus and comet assays, no genotoxic or oxidative damage was observed. Therefore, it is concluded that these compounds are not genotoxic under the conditions evaluated, indicating that their presence in food and feed does not represent a risk, although further studies are required to complete their safety profile. In addition, a new biopreservative derived from the fermentation of a dairy byproduct by the Enterococcus faecalis UGRA10 strain has been evaluated. This product has a broad antibacterial spectrum due to the presence of Enterocin AS-48. To evaluate its potential as a food additive, mutagenicity and genotoxicity tests were performed using the Ames assay on 5 Salmonella strains (OECD 471) and the micronucleus assay (OECD 478) on L5178Y/Tk ± cells. The results of the Ames test showed no mutagenicity at the concentrations tested, and no genotoxicity was evident in the micronucleus test. The negative results in both tests highlight the good safety profile of the by-product and support its use as an additive, although further toxicological studies are needed before approval and commercial application. These genotoxicological studies have resulted in two scientific publications: Mutagenicity and genotoxicity assessment of a new biopreservative product rich in Enterocin AS-48. (Cascajosa-Lira et al., 2020, Food and Chemical Toxicology, 146, 111846). Genotoxicity evaluation of two derived products from Allium extracts: S- propylmercaptocysteine and S-propyl mercaptoglutathione. (Cascajosa-Lira et al., 2023, Food Bioscience, 53, 102671). Resumen/Summary 13 Since OSCs will be mainly used as additives in animal nutrition, a method for their analysis has been validated in feed. The extraction parameters for the analysis of PTSO and PTS in feed matrices by solid-liquid extraction and quantification by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLCMS/MS) have been optimized. Optimization was carried out using response surface methodology (RSM) in a Box-Behnken experimental design, improving the sample/solvent ratios and evaporation temperature at the rotary evaporator. The method was validated for three concentration levels for both compounds: PTSO (100, 500, 1000 ng/g) and PTS (500, 1150, 2300 ng/g). The best recoveries of PTSO and PTS were obtained using 12.5 mL of 100% acetonitrile, stirring for 15 min and with an evaporation temperature of 20 °C. The validated method was applied to detect and quantify these compounds in different feed matrices. This is the first study that simultaneously analyze PTSO and PTS at low concentrations, employing a validated method using the sensitive technique UPLC-MS/MS. The validation of this method resulted in the following scientific publication: Simultaneous determination of Allium compounds (Propyl propane thiosulfonate and thiosulfinate) in animal feed using UPLC-MS/MS. (Cascajosa-Lira et al., 2021, Food and Chemical Toxicology, 157, 112619). In addition to monitoring the additive, it is also important to know its metabolic pathways. Therefore, we aimed to study the biotransformation of PTSO, combining in silico and in vitro techniques. In silico ecotoxicity studies were carried out to consider the environmental impact of the compound before its commercial use. An analytical method was developed and validated to determine the parent compound and its metabolites by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Phase I and II metabolism of PTSO was predicted using the Meta- Pred web server. In phase I, rat (male and female) and human liver microsomes were incubated with PTSO and an NADPH regeneration system. In phase II, microsomes were incubated with PTSO and glutathione (GSH) or uridine diphosphoglucuronic acid. Analysis revealed the presence of PTS generated by a redox reaction in phase I, and two phase II conjugates: GSSP and CSSP. In addition, ADME parameters and potential ecotoxicity of PTSO and its metabolites were predicted by in silico software. The results of the in silico ecotoxicity study evidenced that metabolism induced the formation of detoxified metabolites of the parent compound, except dimercaprol and 3- Resumen/Summary 14 mercaptopropane-1,2-diol. This biotransformation study has resulted in the following scientific publication: Identification of in vitro metabolites of an Allium organosulfur compound and environmental toxicity prediction as part of its risk assessment. (Cascajosa-Lira et al., 2023, Environmental Research, 229, 116001). In vivo toxicokinetic studies were performed with the objective to evaluate the ADME of PTSO in rats. For this purpose, rats of the Sprague Dawley strain received an oral dose of 175 mg/kg by gastric gavage. Plasma and tissue samples (brain, stomach, liver, liver, spleen, kidney, lung and testis) were collected at specific intervals (10 min, 20 min, 30 min, 1h, 2h, 4h, 6h, 8h and 24h) and analyzed by Gas Mass Chromatography (GC-MS), Pyrolysis Gas Chromatography-Mass Chromatography (PyGC-MS) and UHPL-MS/MS to detect PTSO and its metabolites. PTSO was not detected in plasma or solid tissues during the entire sampling period; however, phase I and II metabolites were identified in both matrices. The toxicokinetic profile of CSSP, a significant metabolite, showed a well-correlated pattern. In the stomach, CSSP reached peak concentrations of 495.63±6.53 ng/mL, while lower concentrations of 123.59±8.35 ng/mL were observed in plasma. In addition, CSSP demonstrated high water solubility and rapid excretion, with a plasma half-life of 0.66±0.05 hours. The results obtained led to the next submitted publication: Acute study and the absorption, distribution, and metabolomic profile of the natural compound propyl-propane-thiosulfonate from Allium cepa in rats. (Cascajosa-Lira et al., submitted). In view of the inconclusive results in the in vitro genotoxic studies of PTS (available in the scientific literature and performed by our research group), an in vivo study was carried out. In this work, the in vivo genotoxicity of PTS was evaluated in male and female rats, following OECD guidelines. After oral administration of doses of 5.5, 17.4 and 55.0 mg/kg PTS, a combination of the bone marrow micronucleus assay (OECD 474) and the standard and enzyme-modified comet assay (OECD 489) were performed. After necropsy, histopathological studies were performed. The results showed no in vivo genotoxicity of PTS at any of the doses tested, evidenced by the absence of increased micronucleus, as well as DNA strand breaks or oxidative damage in the comet assays. Histopathological study revealed that only the highest dose tested (55.0 mg/kg) in the Resumen/Summary 15 liver and all dose groups in the stomach presented minimal pathological lesions in the organs studied. Consequently, this work confirms that PTS is not genotoxic at the doses evaluated. This work resulted in the following scientific publication: Genotoxicity evaluation of Propyl-Propane-Thiosulfinate (PTS) from Allium genus essential oils by a combination of micronucleus and comet assays in rats. (Cascajosa-Lira et al., 2021, Foods, 10(5), 989). In addition, in vivo prolonged exposure tests, specifically 90-day subchronic studies were conducted to evaluate both PTSO and PTS. In these studies, OECD guideline 408 was followed. Doses of 0, 14, 28 and 55 mg/kg/day were administered to groups of 40 male and 40 female rats. No clinical signs, mortality, changes in body weight, food consumption or feed conversion efficiency were detected. No alterations in hematological or biochemical parameters outside the normal ranges were observed. The histopathological study showed no differences in organ weights or morphological or histological changes. Based on these results, the no observed adverse effect level (NOAEL) was ≥55 mg/kg/day for both compounds. These trials resulted in the following scientific publications: Safety assessment of propyl-propane-thiosulfonate (PTSO): 90-days oral subchronic toxicity study in rats. (Cascajosa-Lira et al., 2020, Food and Chemical Toxicology, 144, 111612). Acute and subchronic 90-days toxicity assessment of propyl-propane- thiosulfinate (PTS) in rats. (Cascajosa-Lira et al., 2022, Food and Chemical Toxicology,161, 112827). In order to complete the evaluation of the in vivo toxicological profile of PTSO, a two-generation study was conducted for reproductive and developmental effects (OECD 416). The study evaluated the potential reproductive, teratogenic and heritable effects of PTSO in two generations of CD1 mice (80 per sex per generation). The mice were exposed to three dose levels of PTSO (14, 28 and 55 mg/kg/day) during the pre-mating, gestation and lactation phases. No clinical changes or mortality related to PTSO administration were observed. Although there were some variations in body weight and food consumption, they were not sex- or dose-dependent. Both parental generations (F0 and F1) showed normal reproductive performance, and the offspring (F1 and F2) were born without abnormalities. Sex hormone levels (progesterone, testosterone, estradiol, Resumen/Summary 16 FSH and LH) remained within the normal range. Although significant changes in sperm analysis were observed in the F0 group, there were no variations in the F1 group and no alterations in fertility. Organ weights, both absolute and relative, and histopathological study showed no significant alterations in males and females. In conclusion, PTSO is not considered to be reproductive or developmentally toxic under the conditions evaluated, which supports its safety profile for agri-food applications. This study resulted in the following scientific publications: Risk assessment and environmental consequences of the use of the Alliumderived compound propyl-propane thiosulfonate (PTSO) in agrifood applications. (Cascajosa-Lira et al., 2023, Environmental Research, 236, 116682) Two-generation toxicity study of the antioxidant compound propyl-propane thiosulfonate (PTSO). (Cascajosa-Lira et al., 2024, Antioxidants, 13(3), 350.) On the other hand, and since the rabbit breeding industry is growing due to the increasing demand for healthy and sustainable meat, the application of PTSO and PTS as sensory additives in nutrition for this species has been carried out. This study investigated the effects of PTSO and PTS supplementation in farm rabbits over a 76-day period, evaluating growth, health, muscle metabolism and gut microbiota parameters. The results showed significant improvements in the growth of rabbits fed Allium extract, including higher final weight, increased daily weight gain (ADG) and lower mortality. Biochemical analysis revealed normal values in the treated group, while muscle analysis confirmed the presence of some PTSO metabolites. Gut microbiota analysis showed clear differences between the control and treated groups. This study highlights the potential benefits of PTSO and PTS supplementation to improve growth, health and gut microbiota composition in farm rabbits, suggesting its efficacy as a feed additive. This study led to the next submitted publication: Effects of dietary of an Allium extract on growth, biochemistry, metabolism, and gut microbiota of rabbits (Oryctolagus cuniculus). (Cascajosa-Lira et al., submitted). Resumen/Summary 17 19 II. INTRODUCCIÓN/ INTRODUCTION Introducción/Introduction 26 Una opinión que se está consolidando entre los consumidores según los estudios de tendencias de mercado (Calsamigla, 2018). Además de la ganadería sostenible, muchos consumidores demandan productos alimentarios libres de aditivos o residuos sintéticos. En la búsqueda de nuevos aditivos, se están explorando compuestos o extractos de plantas con capacidad anti-metanogénica. Los compuestos polifenólicos y organosulfurados parecen tener un futuro prometedor, ya que muchos de estos compuestos también poseen propiedades antioxidantes y antimicrobianas. Estas propiedades pueden beneficiar a los humanos a través de la cadena alimentaria o actuar como conservantes en la carne. Sin embargo, al manejar estos extractos, es importante considerar factores como la estandarización y la estabilidad de los mismos para asegurar su eficacia y seguridad. La ganadería sostenible es fundamental para abordar los desafíos interrelacionados de la salud humana, animal y ambiental, que son el núcleo del principio "One Health" (Buttke et al., 2011). Este enfoque reconoce que la salud de las personas está intrínsecamente ligada a la salud de los animales y del medio ambiente, y que, para mejorar la salud de unos, es necesario considerar a todos. En el contexto de la ganadería, esto significa adoptar prácticas que minimicen el impacto ambiental, mejoren el bienestar animal y aseguren la seguridad alimentaria, lo que a su vez tiene beneficios directos para la salud humana. Además, la ganadería sostenible apoya directamente los Objetivos de Desarrollo Sostenible (ODS) de la Agenda 2030 de las Naciones Unidas. Por ejemplo, contribuye al ODS 2 (Hambre Cero) al mejorar la eficiencia y la productividad de la producción de alimentos, asegurando que haya suficientes productos animales para alimentar a una población mundial en crecimiento sin comprometer los recursos del planeta. También promueve el ODS 12 (Producción y Consumo Responsables) al incentivar métodos de producción que minimicen el desperdicio y utilicen los recursos de manera más eficiente. Así mismo, la reducción de las emisiones de gases de efecto invernadero y la protección de los ecosistemas son claves para el ODS 13 (Acción por el Clima), lo que subraya la importancia de la ganadería sostenible en la lucha contra el cambio climático. Introducción/Introduction 27 3. Compuestos organosulfurados. Características y propiedades Los compuestos organosulfurados (OSCs, por sus siglas en inglés) se subdividen en compuestos de azufre alílico, indoles, isotiocianatos y sulforafano, conocidos históricamente por sus características terapéuticas. El átomo de azufre es responsable de las características particulares de los OSCs, lo que los hace útiles en el tratamiento de enfermedades mediadas por estrés oxidativo. Los vegetales de los géneros Brassica (Crucíferas) y Allium, como coliflor, brócoli, repollo, cebolla y principalmente ajo son las principales fuentes de OSCs. El fuerte sabor de la cebolla se debe a la presencia de OSCs, específicamente mono-, di- y tri-sulfuros, tiosulfonatos y tiosulfinatos. Los glucosinolatos, conocidos como precursores de los isotiocianatos, se encuentran en el género Brassica (Crucíferas). Durante la masticación o el daño causado por insectos, los glucosinolatos experimentan un proceso de hidrólisis por la enzima mirosinasa, en el que se producen isotiocianatos, tiocianatos y diversos compuestos bioactivos. En los vegetales del género Allium, los OSCs se dividen en 1) compuestos volátiles de azufre o solubles en aceite, como los sulfuros dialil, responsables de su sabor y aroma picantes, 2) compuestos menos olorosos y solubles en agua, incluidos la S-alilcisteína (SAMC) o S-alilmetilcisteina (SAC), que se encuentran en extractos acuosos de ajo (Figura 2). Los OSCs en el ajo se activan cuando se machaca o se corta en trozos en un período corto de tiempo. El ajo crudo machacado contiene altas concentraciones de alicina, que es responsable de sus principales funciones potencialmente farmacológicas. Introducción/Introduction 28 Figura 2. Proceso de biosíntesis de los diferentes compuestos organosulfurados. 3.1. Efectos sobre la salud Los OSCs han demostrado efectos fisiológicos contra la inflamación crónica y aguda en estudios in vitro y también en humanos. Los principales efectos sobre la salud Introducción/Introduction 29 de los OSCs han sido recogidos en la literatura científica en diferentes artículos de revisión y se han compilado en la tabla 1. Tabla 1. Artículos de revisión en los que se tratan los diferentes beneficios para la salud de los OSCs. ENFERMEDADES PRINCIPALES EFECTOS REFERENCIAS Enfermedades cardiovasculares: hipertensión, hiperlipidemia, diabetes, síndrome metabólico, aumento de la actividad plaquetaria e hipertensión pulmonar. La evidencia existente hasta la fecha sugiere que los OSCs tienen la capacidad de prevenir la producción de especies reactivas de oxígeno (ERO), aumentar el estado antioxidante, aumentar la biodisponibilidad del óxido nítrico, prevenir la inflamación vascular, reducir el contenido de lípidos e inhibir la agregación plaquetaria. Los resultados obtenidos en humanos son inconsistentes, probablemente debido a diferencias en las preparaciones de ajo. Vazquez-Prieto et al., 2010 Mirhadi et al., 2024 Síndrome metabólico: Obesidad, dislipidemia, hipertensión y diabetes mellitus tipo 2. Los OSCs son efectivos para reducir mediadores internos de la hipertensión en el cuerpo, como el óxido nítrico (NO), la renina/angiotensina, la expresión del factor nuclearkB y el estrés oxidativo. Además, los extractos y compuestos puros poseen propiedades beneficiosas para reducir el colesterol, los triglicéridos y las lipoproteínas de baja densidad. Algunos estudios han reportado que el género Allium es efectivo en la pérdida de peso y la diabetes tipo 2 a través de propiedades antioxidantes, reducción de la glucosa en sangre y resistencia a la insulina, y aumento de la sensibilidad a la insulina. Sin embargo, se necesitan más ensayos clínicos para determinar la efectividad terapéutica del género Allium y sus componentes activos. Borah et al., 2021 Dwivedi et al., 2023 Taleghani et al., 2024 Neurodegeneración: falta de coordinación, memoria y cognición. Los OSCs mejoraron significativamente los parámetros de comportamiento y estrés oxidativo, así como los cambios histopatológicos en el nervio ciático. Reduce los niveles de malondialdehído Dwivedi et al., 2023 Shukla et al., 2024 Introducción/Introduction 30 (MDA), mejora la disfunción cognitiva y previene la lesión neuronal. El efecto neuroprotector de los OSCs en las habilidades de aprendizaje y memoria se ha evaluado y se demostró que podría mejorar los déficits de aprendizaje y memoria . Infecciones víricas: VIH, VSH, SARS-CoV, CMV, DENV y PIV- 3. Actividad antiviral potencial al bloquear la entrada viral en las células huésped, inhibir enzimas virales clave como la ARN polimerasa y la transcriptasa inversa, y regular la transcripción de genes virales. Además, se ha observado que los OSCs tienen efectos inmunomoduladores que contribuyen a mitigar las infecciones virales. Rouf et al., 2020 3.2. Actividad antioxidante El exceso de radicales libres juega un papel importante en el daño celular y desencadena el estrés oxidativo, que puede llevar a enfermedades graves como el cáncer, el infarto de miocardio y enfermedades neurodegenerativas. Estos radicales son especies moleculares que tienen electrones desapareados en orbitales atómicos, los cuales pueden fácilmente aceptar o donar electrones a otras moléculas, haciéndolos inestables y reactivos. La enzima xantina oxidasa (XO) puede desencadenar la formación de radicales anión superóxido. Por lo tanto, se necesitan inhibidores para reducir la producción de ácido úrico y EROs mediante la inhibición de la actividad de la XO (Kurnia et al., 2023). Un género ampliamente conocido por contener metabolitos secundarios con potencial antioxidante es Allium. Según los estudios científicos, estas especies contienen varios compuestos esenciales y activos como flavonoides, compuestos de azufre, ácido pirúvico, taninos, antocianinas y saponinas, que poseen actividad biológica, incluyendo antioxidante. Estos compuestos activos se pueden encontrar en todas las partes de las especies de Allium, como bulbos, hojas, pieles y raíces (Batiha et al., 2020; Țigu et al., 2021). Las investigaciones sobre A. cepa realizadas por Ramesh et al. (2018) concluyeron que el extracto de hoja de A. cepa contiene varios grupos de compuestos con potencial antioxidante, como alcaloides, flavonoides, terpenoides, aminoácidos y carotenoides. Otro estudio también reportó que se han aislado con éxito varios compuestos derivados Introducción/Introduction 31 de la quercetina de A. sativum. Otra especie, A. ascalonicum, también contiene varios grupos de compuestos como alcaloides, saponinas y trazas de flavonoides en sus hojas, aunque la investigación en hojas de A. ascalonicum sólo se ha realizado hasta el nivel de extracto. Los estudios previos se han limitado a informar sobre la actividad antioxidante in vitro usando métodos comunes como la actividad de captura de radicales (2,2-difenil- 1-picrilhidrazilo) DPPH y de radicales hidroxilo. Sin embargo, la capacidad antioxidante de los compuestos quercetina 3-O-β-d-xilopiranósido, quercetina 3-O-β-d- xilopiranósido, kaempferol 3-O-β-d-glucopiranósido y 2,4-dimetiltiophene en inhibir la actividad de la enzima XO no ha sido demostrada. Estos cuatro compuestos son conocidos por tener buena actividad antioxidante in vitro y se encuentran en el género Allium, especialmente en los bulbos y hojas (Major et al., 2018; Yuasa et al., 2021). El mecanismo de acción antioxidante de los compuestos OSCs es tanto directo como indirecto. Por un lado, tienen la capacidad de reducir las ERO y la peroxidación lipídica (LPO). Por otro lado, tienen capacidad para activar el factor de transcripción Nrf2 (factor 2 relacionado con el eritroide nuclear), que desempeña un papel crucial en la respuesta antioxidante celular. Estos compuestos interactúan con la proteína Keap1 (proteína asociada a ECH similar a Kelch), un modulador negativo de Nrf2, lo que permite la liberación y activación de Nrf2. Una vez activado, Nrf2 induce la transcripción de una amplia gama de genes involucrados en el mantenimiento del equilibrio redox celular, la reducción de la inflamación y la promoción de la respiración y biogénesis mitocondrial. Entre los compuestos OSCs naturales con actividad antioxidante destacan: el sulforafano, la alicina, S-alilcisteína, tiosulfonatos y tiosulfinatos. Introducción/Introduction 32 3.3. Actividad antimicrobiana Los compuestos antimicrobianos principales en especies del género Allium frescas se forman al degradarse los S-alquil(L)-cisteína sulfóxidos por la acción de la enzima aliinasa después de que sus tejidos se dañen. Estos compuestos sulfurados no se encuentran de forma natural en las plantas como otros metabolitos secundarios antimicrobianos (fenoles, terpenoides, alcaloides, ácidos orgánicos, y polipéptidos) (Kyung et al., 2012). La actividad antimicrobiana de Allium se debe principalmente a los tiosulfinatos y sus productos de transformación. Otros compuestos como péptidos, flavonoides, fenoles, alcaloides, y saponinas también contribuyen. Los tiosulfinatos, incluyendo la alicina, inhiben microorganismos al reaccionar con grupos SH de proteínas celulares, generando disulfuros mixtos. Diversas especies de Allium, como el ajo (A. sativum), cebolla (A. cepa), chalote (A. ascalonicum), ajo elefante (A. ampeloprasum) y ajo silvestre (A. ursinum), han mostrado actividad antimicrobiana. Estas especies contienen diferentes tipos y niveles de sulfóxidos, principalmente aliína en el ajo y ajo gigante, isoaliína en la cebolla y chalote, y metiína en el cebollino y cebollino chino. La cantidad de sulfóxidos varía según factores genéticos y condiciones de almacenamiento postcosecha. Los extractos de Allium muestran actividad antimicrobiana contra bacterias, hongos, virus y parásitos. El ajo y la cebolla son los más estudiados. Los extractos de Allium inhiben el crecimiento de bacterias Gram-positivas, Gram-negativas y bacterias ácido-resistentes, aunque generalmente se cree que las bacterias Gram-negativas son más sensibles a los principios activos del ajo (Yoshida et al., 1987; Leuter et al., 1996; Chung et al., 2008; Kang et al., 2010). Varios estudios han demostrado la actividad antibacteriana de los OSCs contra diversas bacterias Gram positivas y Gram negativas. Se ha reportado que tienen un efecto antibacteriano contra Escherichia coli, Bacillus anthracis, Photobacterium damselae subsp. piscicida, Streptococcus iniae, Candida albicans, Enterococcus faecalis y Bacillus subtilis entre muchas otras bacterias (Llana-Ruiz-Cabello et al., 2015; Putnik et al., 2019). Según Snoussi et al. (2016), todos los extractos de Allium ricos en OSCs presentaron actividad antibacteriana con una relación dosis-actividad, mostrando la mayor inhibición a 50 mg/mL. El mecanismo de acción antibiótico de los compuestos OSCs se basa en su alta permeabilidad a través de las membranas de fosfolípidos y su interacción con ciertas enzimas que contienen tiol en los microorganismos. Esta reacción provoca la inhibición Introducción/Introduction 33 de los sistemas enzimáticos dependientes de tiol que podrían explicar su actividad contra una amplia gama de bacterias Gram negativas y Gram positivas. Con respecto a la actividad antifúngica, los extractos de bulbos de chalote inhiben casi todos los hongos a nivel del 0,5%. Los aceites de Allium y sus sulfuros inhiben más a los hongos que a las bacterias. El ajo y sus componentes activos son más antifúngicos que antibacterianos, con compuestos antimicrobianos como sulfocompuestos heterocíclicos, alcohol alílico y ácido 3-(aliltrisulfanil)-2-aminopropanoico (Choi et al., 2015). Además, el género Allium también presenta compuestos con actividad antiviral. La alicina y sus productos de transformación, como los polisulfuros de alilo y el ajoeno, muestran actividad antiviral. El ajoeno tiene más actividad antiviral que la alicina. Extractos de ajo inhiben la penetración y proliferación del virus de la gripe en células renales caninas y el virus del mosaico de la papa in vivo e in vitro (Mehrbod et al., 2009; Mohamed, 2010). 3.4. Actividad antiparasitaria Los extractos de ajo (Allium sativum) muestran una notable actividad inhibidora contra varios parásitos, incluyendo Leishmania donovani, Spironucleus vortens, y Eimeria papillata. Esta capacidad antiparasitaria se atribuye principalmente a compuestos como la alicina, los ditienos y el ajoeno (Sharma et al., 2010). El crecimiento de S. vortens es inhibido por alicina, ditienos y ajoeno, aunque con valores de Concentración Inhibitoria Mínima (MIC) mucho más altos que los reportados para la mayoría de bacterias, hongos y protozoos. Esto indica un nivel inusualmente alto de tolerancia de S. vortens a los productos del género Allium. Se cree que el átomo de carbono en la vecindad del átomo de azufre del tiosulfínato es atacado por grupos SH, lo que puede ser el mecanismo subyacente de esta inhibición (Millet et al., 2011). El extracto de ajo ha demostrado eficacia en la inhibición de L. donovani, el agente causante de la leishmaniasis. Aunque los detalles específicos del mecanismo no se detallan en el texto original, se puede inferir que los compuestos sulfurados presentes en el ajo, como la alicina, juegan un papel crucial en esta actividad antiparasitaria (Sharma et al., 2010). Introducción/Introduction 34 Eimeria papillata, un parásito que causa coccidiosis en aves, también es susceptible a los extractos de ajo. La actividad antiparasitaria contra E. papillata sugiere que los compuestos activos del ajo pueden interferir en los procesos vitales del parásito, posiblemente afectando la integridad de la membrana celular y otras funciones críticas (Dkhil et al., 2011). El ajoeno, un compuesto derivado del ajo, inhibe el crecimiento de Trypanosoma cruzi, el agente causante de la enfermedad de Chagas. Esta inhibición va acompañada de una reducción significativa en el contenido de fosfatidilcolina en las células del parásito y una alteración en las estructuras de las membranas intracelulares. Esto sugiere que el ajoeno puede desorganizar las membranas celulares del parásito, afectando su viabilidad y capacidad de proliferación (Urbina et al., 1993). Se ha demostrado que los OSCs como PTSO y PTS promueven el aumento en la probabilidad de supervivencia contra P. damselae subsp. piscicida y la reducción de los parásitos de branquias S. chrysophrii en doradas (Cabello-Gómez et al., 2022). Tanto PTSO como PTS tienen una fuerte capacidad para ejercer un efecto preventivo sobre la invasión celular a concentraciones bajas, pero concentraciones más altas de cada compuesto no impidieron la penetración de parásitos en las células. Se sugiere que, a concentraciones bajas, el PTSO ejerce un efecto inmunomodulador que inhibe la expresión de genes relacionados con la inflamación y el daño oxidativo, mientras que las concentraciones más altas provocan cambios en el metabolismo para proteger la supervivencia celular, reduciendo la capacidad preventiva frente a agentes parásitos (Aguinaga-Casañas et al., 2022). 3.5. Actividad anti-metanogénica Las plantas del género Allium han demostrado ser efectivas en la reducción de emisiones de metano en rumiantes, lo que es de gran relevancia para la mitigación del cambio climático. Un estudio realizado con Allium mongolicum Regel mostró que suplementar la dieta de terneros con 800 mg/kg de peso corporal de extractos frescos de esta planta, que contiene alicina natural y es rica en flavonoides, alcaloides, ácidos orgánicos y aceites esenciales, redujo las emisiones de metano por ingesta de materia seca en más del 20%. Este efecto se debe a la presencia de compuestos bioactivos que alteran la fermentación en el rumen, disminuyendo la producción de gases sin afectar negativamente el patrón de fermentación ruminal. Estos resultados destacan el potencial Introducción/Introduction 35 de los extractos de Allium como una estrategia sostenible para reducir las emisiones de metano en la ganadería (Lambo et al., 2024). Los OSCs del género Allium han demostrado tener actividad anti-metanogénica in vitro e in vivo. En un conjunto de estudios que investigaron los efectos de compuestos como PTS y PTSO en la producción de metano en el rumen de cabras, se realizaron experimentos tanto in vitro como in vivo. En el primer experimento in vitro, se incubaron cuatro concentraciones (40, 80, 160 y 320 µl/L) de varios compuestos en líquido ruminal diluido durante 24 horas, utilizando dietas con diferentes fuentes de almidón y proteína. Se observó que la producción total de gas disminuyó linealmente con todos los compuestos, excepto con eugenol (EUG) y la combinación de PTS+PTSO. La concentración total de ácidos grasos volátiles (VFA) y la relación acetato disminuyeron significativamente con PTS, PTSO y bromoclorometano (BCM) (Martínez-Fernández et al., 2013). Adicionalmente, un segundo experimento in vitro con cultivos puros de metanógenos probó PTS y BCM contra tres cepas arqueales, observándose una reducción sustancial en la producción de metano con ambos compuestos (Martínez-Fernández et al., 2015). Basándose en estos resultados, se seleccionaron dos concentraciones de PTS, carvacrol (CAR), cinamaldehído (CIN), BCM (160 y 320 µl/L), PTSO (40 y 160 µl/L) y dialil disulfuro (DADS) (80 y 320 µl/L) para un segundo experimento in vitro de 72 horas, que mostró una reducción lineal en la producción de metano (P⩽0.009) con los compuestos, siendo las mayores reducciones observadas con PTS (hasta un 96%), DADS (62%) y BCM (95%) (Martínez-Fernández et al., 2013). En el experimento in vivo, dos grupos de 16 cabras adultas no preñadas fueron estudiadas durante 9 días para evaluar el efecto de añadir PTS (50, 100 y 200 mg/l de contenido ruminal por día) y BCM (50, 100 y 160 mg/L de contenido ruminal por día). Se observó una reducción lineal (33% y 64%, respectivamente) en la producción de metano por unidad de consumo de materia seca, menor que la inhibición máxima observada in vitro (Martínez-Fernández et al., 2014). Un segundo ensayo in vivo de 36 días evaluó el efecto del complejo cyclodextrin- PTS complex (CD-PTS) (200 mg de componente activo/l de contenido ruminal por día) en la producción de metano, patrones de fermentación, abundancia microbiana y utilización de nutrientes en cabras alimentadas con heno de alfalfa y concentrado. Doce Introducción/Introduction 42 prometedora en la búsqueda de prácticas agrícolas y ganaderas más sostenibles y saludables. Uno de ellos es la enterocina AS-48 (Figura 6), que es una bacteriocina circular producida por la cepa Enterococcus faecalis UGRA10, aislada de un queso de leche de oveja cruda (Cebrian et al., 2012). Este péptido antimicrobiano exhibe una amplia actividad contra bacterias Gram positivas, destacando especies como Listeria, Bacillus, Enterococcus, Planococcus, ycobacterium, Corynebacterium y Nocardia. Aunque también afecta a diversas especies de bacterias Gram negativas, siendo éstas mucho menos sensibles debido al efecto protector de la membrana externa. Recientemente, se ha descrito la actividad de la enterocina AS-48 contra protozoos flagelados como Leishmania y Trypanosoma, lo que amplía su potencial aplicación en el control de patógenos. Su actividad antimicrobiana la convierte en un bioconservante alimentario prometedor, siendo investigada su aplicación en una amplia gama de productos, incluyendo carnes, productos lácteos, mariscos y alimentos de origen vegetal. Sin embargo, a pesar de su potencial, hay pocos estudios sobre su perfil toxicológico hasta la fecha. Estudios in vitro e in vivo han demostrado que la enterocina AS-48 exhibe baja actividad hemolítica en sangre, no induce acumulación de nitritos en macrófagos RAW no estimulados y posee baja citotoxicidad en varias líneas celulares humanas. Además, estudios en embriones de pez cebra expuestos a dosis bajas no mostraron anomalías visibles y se estableció una concentración letal 50 (CL50) entre 3,0 y 6,4 μM (Cebrian et al., 2019). Estudios subcrónicos en ratones alimentados con enterocina AS-48 durante 90 días no revelaron efectos adversos en la salud animal (Baños et al., 2019). Sin embargo, se requieren estudios de genotoxicidad y mutagenicidad para respaldar su seguridad antes de su aprobación y comercialización como aditivo alimentario. Figura 6. Estructura molecular de la enterocina AS-48. Tomada de Cao et al., (2022) Introducción/Introduction 43 6. Proceso de autorización de nuevos aditivos en piensos y productos fitosanitarios 6.1. Reglamento (CE) No 1831/2003. Autorización de aditivos en piensos La producción ganadera desempeña un papel fundamental en la agricultura de la Comunidad Europea, dependiendo en gran medida de la disponibilidad de piensos seguros y de alta calidad. La libre circulación de alimentos y piensos seguros es esencial para el mercado interior, contribuyendo significativamente a la salud y bienestar de los ciudadanos, y favoreciendo sus intereses sociales y económicos. Las políticas comunitarias deben asegurar un alto nivel de protección para la vida y la salud humana. Para salvaguardar la salud humana, la sanidad animal y el medio ambiente, los aditivos para alimentación animal deben ser evaluados mediante un procedimiento comunitario antes de su comercialización, uso o transformación. Aunque los alimentos para animales domésticos no afectan a la cadena alimentaria ni el medio ambiente de las tierras agrícolas, es necesario introducir disposiciones específicas para sus aditivos. El Reglamento (CE) no 178/2002 establece que los alimentos y piensos importados deben cumplir con los requisitos de la legislación alimentaria comunitaria o condiciones equivalentes. Por lo tanto, las importaciones de aditivos para alimentación animal deben cumplir condiciones similares a las de los aditivos producidos en la Comunidad Europea. La acción comunitaria en salud humana, sanidad animal y medio ambiente debe basarse en el principio de precaución. Conforme al artículo 153 del reglamento, la Comunidad promoverá el derecho de los consumidores a la información. La aplicación de la Directiva 70/524/CEE ha evidenciado la necesidad de revisar las normas sobre aditivos para proteger mejor la sanidad animal, la salud humana y el medio ambiente, teniendo en cuenta los avances tecnológicos y científicos que han introducido nuevos tipos de aditivos. Este Reglamento también debe aplicarse a las mezclas de aditivos vendidas al consumidor final, y su comercialización y uso deben ajustarse a las condiciones de autorización de cada aditivo. Las premezclas no deben considerarse preparaciones bajo la definición de aditivo. Sólo los aditivos autorizados según el procedimiento de este Reglamento podrán comercializarse, utilizarse o transformarse para la alimentación animal. Se deben definir categorías de aditivos para facilitar su evaluación y autorización. Aminoácidos, sales de aminoácidos, sustancias análogas, urea y derivados, actualmente cubiertos por la Directiva 82/471/CEE, deben incluirse como una categoría de aditivos para piensos y transferirse al ámbito del presente Reglamento. Introducción/Introduction 44 Las normas de ejecución relativas a la solicitud de autorización de aditivos para alimentación animal deben considerar los diferentes requisitos de documentación aplicables a los animales productores de alimentos y a otros animales. La evaluación científica de los aditivos para piensos debe ser realizada por la Autoridad Europea de Seguridad Alimentaria (EFSA), y las solicitudes deben incluir estudios sobre los residuos para evaluar los límites máximos de residuos (LMR). La Comisión debe elaborar directrices para la autorización de aditivos en colaboración con la EFSA, prestando atención a la extrapolación de resultados a especies menores. Se prevé un procedimiento de autorización simplificado para aditivos que ya han superado el procedimiento para uso alimentario según la Directiva 89/107/CEE. La evaluación científica del riesgo debe complementarse con factores sociológicos, económicos y medioambientales, y la autorización debe ser otorgada por la Comisión. Para proteger el bienestar animal y la seguridad del consumidor, se incentivará a los solicitantes a solicitar prórrogas de autorización para especies menores, ofreciendo un año adicional de protección de datos. La Comisión debe tener la competencia para autorizar aditivos, establecer condiciones de uso y mantener un registro de aditivos autorizados, en colaboración con los Estados miembros. Los titulares de autorizaciones deben aplicar planes de vigilancia postcomercialización para identificar efectos imprevistos en la salud humana, animal o medio ambiente, utilizando sistemas de rastreo de productos. Las autorizaciones deben revisarse periódicamente para adaptarse a los avances tecnológicos y científicos. Debe establecerse un registro de aditivos autorizados con información específica disponible públicamente. Normas transitorias deben permitir que aditivos ya presentes en el mercado continúen mientras se evalúan conforme al nuevo Reglamento. Se deben prohibir los antibióticos promotores del crecimiento, permitiendo tiempo para desarrollar alternativas. Algunas sustancias con efectos coccidiostáticos e histomonostáticos deben ser tratadas como aditivos para piensos. El etiquetado detallado de productos debe facilitar la elección informada del usuario final y minimizar obstáculos al comercio. Los aditivos para piensos modificados genéticamente deben seguir un procedimiento de autorización adicional al Reglamento (CE) no 1829/2003. Los artículos 53 y 54 del Reglamento (CE) no 178/2002 establecen procedimientos para medidas de emergencia cuando un pienso constituya un riesgo grave. Las medidas necesarias para la ejecución del presente Reglamento deben seguir la Decisión 1999/468/CE. Los Estados Introducción/Introduction 45 miembros deben establecer sanciones eficaces y disuasorias para las infracciones de este Reglamento. Según el reglamento 1999/468/CE no se comercializará, transformará o utilizará un aditivo para alimentación animal a menos que: a) se disponga de una autorización concedida de acuerdo con el presente Reglamento; b) se cumplan las condiciones de utilización establecidas en el presente Reglamento; y c) se cumplan los requisitos de etiquetado establecidos en el presente Reglamento. Para experimentos con fines científicos, los Estados miembros podrán autorizar el uso de sustancias como aditivos que no estén autorizadas a nivel comunitario, con excepción de los antibióticos, siempre que dichos experimentos se realicen conforme a los principios y condiciones previstos en la Directiva 87/153/CEE, la Directiva 83/228/CEE, o las normas mencionadas en el apartado 4 del artículo 7 del presente Reglamento, y siempre que exista una supervisión oficial adecuada. Los animales en cuestión sólo podrán utilizarse para la producción de alimentos si las autoridades determinan que no habrá efectos adversos en la sanidad de los animales, en la salud humana ni en el medio ambiente. Cualquier persona que desee obtener una autorización de un aditivo para alimentación animal o de un nuevo uso de un aditivo para alimentación animal presentará una solicitud conforme al artículo 7 del presente Reglamento. Sólo se concederá, rechazará, prorrogará, modificará, suspenderá o revocará una autorización por los motivos y conforme a los procedimientos establecidos en el presente Reglamento o de conformidad con los artículos 53 y 54 del Reglamento (CE) no 178/2002. El solicitante de una autorización o su representante deberá estar establecido en la Comunidad. No se autorizará un aditivo para piensos si el solicitante de la autorización no demuestra adecuada y suficientemente, conforme a las medidas de ejecución establecidas en el artículo 7, que, utilizado según las condiciones establecidas en el reglamento de autorización, el aditivo cumple los requisitos de que no tendrá un efecto adverso para la sanidad animal, la salud humana o el medio ambiente, no será presentado de manera que induzca a error al consumidor, no perjudicará al consumidor influyendo negativamente en las características distintivas de los productos animales ni inducirá a error respecto a dichas características. Además, deben poseer al menos una de las siguientes características: influir positivamente en las características del pienso, en las características de los productos animales, en el color de los pájaros y peces ornamentales, satisfacer las necesidades alimenticias de los animales, influir positivamente en las Introducción/Introduction 46 repercusiones medioambientales de la producción animal, influir positivamente en la producción, la actividad o el bienestar de los animales, especialmente actuando en la flora gastrointestinal o la digestibilidad de los piensos, o tener un efecto coccidiostático o histomonostático. Los antibióticos distintos de los coccidiostáticos o de los histomonóstatos no se autorizarán como aditivos para alimentación animal. 6.2. Solicitud de autorización de aditivos en piensos La solicitud de autorización prevista en el artículo 4 debe presentarse a la Comisión, que la remitirá a la EFSA ("la Autoridad"). La Autoridad acusará recibo de la solicitud en quince días y compartirá la información con los Estados miembros y el público, respetando la confidencialidad. El solicitante debe enviar directamente a la Autoridad información detallada sobre el aditivo para alimentación animal, incluidos su nombre, dirección, identificación del aditivo, método de producción, estudios realizados y condiciones de comercialización. Además, debe declarar que ha enviado muestras al laboratorio comunitario de referencia. Para aditivos relacionados con organismos modificados genéticamente (OMG), se requiere una propuesta de seguimiento y un resumen del expediente. La Comisión, tras consultar a la Autoridad, establecerá normas de ejecución y orientaciones específicas para la autorización de aditivos, diferenciando los requisitos según el tipo de animales y utilizando procedimientos simplificados para aditivos autorizados en alimentación humana. La EFSA ha publicado una guía para la evaluación toxicológica de aditivos en piensos: ” Guidance on the assessment of the safety of feed additives for the consumer” (2012). 6.3. Reglamento (CE) No 1107/2009. Autorización de productos fitosanitarios. La producción vegetal es esencial en la Comunidad, y el uso de productos fitosanitarios es fundamental para proteger los vegetales contra organismos nocivos y mejorar la producción agrícola. No obstante, estos productos pueden tener efectos adversos en la producción vegetal y conllevar riesgos para humanos, animales y el medio ambiente, especialmente si se comercializan sin pruebas y autorizaciones oficiales o se usan incorrectamente. El presente Reglamento busca garantizar una alta protección de la salud humana y animal y del medio ambiente, mientras se salvaguarda la competitividad de la agricultura comunitaria. Aplicando el principio de cautela, este Reglamento exige que la industria demuestre que las sustancias o productos no son nocivos para la salud ni tienen Introducción/Introduction 47 efectos inaceptables en el medio ambiente. Para eliminar obstáculos en el comercio de productos fitosanitarios por las diferentes protecciones en los Estados miembros, el Reglamento establece normas armonizadas para la aprobación de sustancias activas y la comercialización de productos, fomentando la libre circulación y disponibilidad en los Estados miembros. Una sustancia sólo se incluirá en un producto fitosanitario si se demuestra un claro beneficio para la producción vegetal y no se esperan efectos adversos. La decisión sobre la aceptabilidad de estas sustancias debe ser a nivel comunitario con criterios armonizados, aplicables desde la primera aprobación y en renovaciones. Se promueve el desarrollo de métodos de ensayo sin animales para sustituir estudios actuales. Por previsibilidad, eficiencia y coherencia, se establece un procedimiento detallado para aprobar sustancias activas, con información clara de los interesados y evaluación por un Estado miembro y la EFSA. La EFSA evalúa riesgos, mientras que la Comisión gestiona riesgos y decide sobre las sustancias. Se debe asegurar la transparencia del proceso. El presente Reglamento se aplicará a los productos, en la forma en que se suministren al usuario, que contengan o estén compuestos de sustancias activas, protectores o sinergistas, y que estén destinados a: proteger los vegetales o productos vegetales de organismos nocivos o evitar su acción, salvo cuando se utilicen principalmente por motivos de higiene; influir en los procesos vitales de los vegetales, como el crecimiento, de forma distinta a los nutrientes; mejorar la conservación de productos vegetales, siempre que no estén sujetos a disposiciones comunitarias especiales sobre conservantes; destruir vegetales o partes de vegetales no deseados, excepto algas, a menos que los productos se apliquen en el suelo o el agua para proteger los vegetales; y controlar o evitar el crecimiento no deseado de vegetales, excepto algas, a menos que los productos se apliquen en el suelo o el agua para proteger los vegetales. Estos productos se denominarán "productos fitosanitarios". Además, este Reglamento será aplicable a las sustancias, incluidos los microorganismos, que ejerzan una acción general o específica contra los organismos nocivos en los vegetales, partes de vegetales o productos vegetales, denominadas en adelante "sustancias activas". El presente Reglamento se aplicará a: Introducción/Introduction 48 a) sustancias o preparados, denominados "protectores", que se añaden a un producto fitosanitario para eliminar o reducir sus efectos fitotóxicos en determinadas plantas; b) sustancias o preparados, denominados "sinergistas", que, aunque presenten poca o ninguna actividad en los efectos indicados en el apartado 1 del reglamento, puedan aumentar la actividad de las sustancias activas de un producto fitosanitario; c) sustancias o preparados, denominados "coformulantes", que se usen o estén destinados a usarse en un producto fitosanitario o en un adyuvante, pero que no sean sustancias activas, protectores o sinergistas; y d) sustancias o preparados, denominados "adyuvantes", que consistan en coformulantes, o preparados que contengan uno o varios coformulantes, en la forma en que se suministren al usuario y se comercialicen para que éste los mezcle con un producto fitosanitario, mejorando su eficacia u otras propiedades plaguicidas. Los residuos de productos fitosanitarios derivados de una aplicación conforme a las buenas prácticas fitosanitarias y bajo condiciones de uso realistas deberán cumplir los siguientes requisitos: a) no deben tener efectos nocivos en la salud humana, incluidos los grupos vulnerables, ni en la salud animal, considerando los efectos acumulativos y sinérgicos conocidos y evaluados con métodos científicos aceptados por la Autoridad, ni afectar las aguas subterráneas; b) no deben tener efectos inaceptables en el medio ambiente. Para residuos significativos desde el punto de vista toxicológico, ecotoxicológico, medioambiental o del agua potable, deben existir métodos estándar de medición y normas analíticas disponibles. Los productos fitosanitarios aplicados bajo buenas prácticas fitosanitarias y condiciones de uso realistas deberán cumplir los siguientes requisitos: a) ser suficientemente eficaces; b) no tener efectos nocivos inmediatos o retardados en la salud humana, incluidos los grupos vulnerables, o en la salud animal, directamente o a través del agua potable (considerando las sustancias derivadas del tratamiento del agua), alimentos, piensos o aire, ni consecuencias en el lugar de trabajo u otros efectos indirectos, considerando los efectos acumulativos y sinérgicos conocidos y evaluados con métodos científicos aceptados por la Autoridad, ni afectar las aguas subterráneas; c) no tener efectos inaceptables en los vegetales o productos vegetales; d) no causar Introducción/Introduction 49 sufrimientos ni dolores innecesarios a los vertebrados que se pretenda combatir con ellos; e) no tener efectos inaceptables en el medio ambiente, considerando especialmente su destino y distribución en el entorno, el impacto en especies no objetivo y su repercusión en la biodiversidad y ecosistemas. Cuando una sustancia activa sea necesaria para controlar un riesgo fitosanitario grave que no pueda ser gestionado por otros medios disponibles, incluidos métodos no químicos, podrá aprobarse dicha sustancia durante un período limitado de hasta cinco años, siempre y cuando su uso esté sujeto a medidas de mitigación de riesgos para minimizar la exposición de humanos y del medio ambiente. Los niveles máximos de residuos para estas sustancias se establecerán según el Reglamento (CE) no 396/2005. Esta excepción no se aplicará a sustancias clasificadas como carcinógenas de categoría 1A, carcinógenas de categoría 1B sin valor umbral, o tóxicas para la reproducción de categoría 1A según el Reglamento (CE) no 1272/2008. Los Estados miembros podrán autorizar productos fitosanitarios con estas sustancias activas únicamente cuando sea necesario para controlar el riesgo fitosanitario grave en su territorio, y deberán elaborar un plan de eliminación progresiva para controlar el riesgo por otros medios, incluidos métodos no químicos, que será transmitido inmediatamente a la Comisión. 6.4. Solicitud de autorización de productos fitosanitarios El productor de la sustancia activa debe presentar una solicitud de aprobación o modificación de las condiciones de aprobación ante un Estado miembro ("el Estado miembro ponente"), junto con un expediente resumido y un expediente completo, conforme al artículo 8 del reglamento No 1107/2009. Si falta alguna parte de los expedientes, se debe justificar científicamente que la sustancia activa cumple los criterios del artículo 4. Una asociación de productores puede presentar una solicitud conjunta. El Estado miembro ponente evaluará la solicitud, a menos que otro Estado miembro se encargue de ello, y la evaluación puede realizarse conjuntamente por varios Estados miembros. El solicitante puede solicitar confidencialidad para cierta información, y los Estados miembros decidirán sobre ello. Además, el solicitante debe proporcionar una lista completa de los ensayos y estudios presentados. Por último, cabe mencionar que la EFSA no ha publicado ninguna guía para la evaluación toxicológica de productos fitosanitarios. Introducción/Introduction 50 7. Estudios toxicológicos necesarios para la autorización de aditivos en piensos y fitosanitarios La seguridad de los aditivos de piensos se evalúa en base a los estudios toxicológicos realizados in vitro e in vivo en animales de laboratorio. El conjunto básico de estudios toxicológicos que se incluyen en la guía de la EFSA (2017) comprende estudios sobre genotoxicidad y toxicidad oral subcrónica. Esto puede ser completado según sea necesario con estudios sobre toxicidad oral crónica, toxicidad reproductiva incluyendo la toxicidad del desarrollo prenatal, y carcinogenicidad. Se deben llevar a cabo estudios adicionales que proporcionen información necesaria para la evaluación de la seguridad de la sustancia activa y sus residuos si existe algún motivo de preocupación. Esto podría incluir estudios in vitro e in silico (por ejemplo, la comparación entre sustancias con estructuras químicas similares) para mejorar la comprensión de los mecanismos de toxicidad y comparar la toxicidad de la sustancia madre con la de sus metabolitos. Los estudios toxicológicos sobre metabolitos particulares pueden ser necesarios si no se puede demostrar la proximidad metabólica entre la especie objetivo y la especie de prueba de laboratorio. Además, si hay datos toxicológicos disponibles de estudios en humanos, estos deben ser tenidos en cuenta (Figura 7). Introducción/Introduction 51 Figura 7. Esquema de la evaluación toxicológica a seguir para aditivos en piensos (Elaboración propia en base a EFSA, 2017). Creado con BioRender.com. El estudio de la seguridad de sustancias que podrían emplearse como fitosanitarios se lleva a cabo de acuerdo con la normativa europea actual, en particular, el Reglamento (CE) Nº 1107/2009, de fecha 21 de octubre de 2009 y el reglamento (CE) Nº 283/2013 del 1 de marzo de 2013. La lista de los ensayos que deben realizarse se encuentran en el punto 6, parte A del reglamento Nº 283/2013. Según estos reglamentos para la Introducción/Introduction 58 genotoxicidad, inducción de mutaciones o estudios sobre mecanismos de muerte celular (Bácskay et al., 2018). Entre los diversos métodos disponibles para evaluar la viabilidad y la muerte celular, destacan la reducción del Bromuro de 3-(4,5-dimetiltiazol-2-ilo)-2,5- difeniltetrazol (MTT) y la sal de tetrazolio 3-[4,5-dimetiltiazol-2-il]-5-[3-carboximetoxi- fenil]-2-[4-sulfofenil]-2H-tetrazolio (MTS) para evaluar la funcionalidad de las mitocondrias (Baltrop et al., 1991) y la cuantificación del contenido proteico total (CPT) para medir la proliferación celular (Bradford, 1976). Al seleccionar las líneas celulares para llevar a cabo la evaluación, Gosslau (2016) sugiere realizar un pequeño cribado inicial en líneas celulares de interés y luego llevar a cabo ensayos más específicos en líneas que representen tejidos u órganos diana del efecto. 8.3. Genotoxicidad y mutagenicidad Para evaluar la genotoxicidad y mutagenicidad, se debe realizar una combinación seleccionada de diferentes pruebas de genotoxicidad. Las pruebas in vitro deben realizarse con y sin sistema de activación metabólica S9, y se debe tener en cuenta la compatibilidad del material de prueba, como por ejemplo la volatibilidad y solubilidad, con el modelo in vitro. En este sentido, la EFSA recomienda las siguientes dos pruebas in vitro como primer paso (Comité Científico de la EFSA, 2011): • La prueba de reversión de mutación bacteriana: Ames (directriz de la OCDE 471). • Ensayo de micronúcleos en células de mamífero in vitro (directriz de la OCDE 487). Esta combinación de pruebas cumple con los requisitos básicos para cubrir los tres puntos finales genéticos con el número mínimo de pruebas; la prueba de reversión de mutación bacteriana cubre mutaciones genéticas y la prueba de micronúcleos in vitro cubre tanto aberraciones cromosómicas estructurales como numéricas. El test de Ames (guía OCDE 471 (2020)) es un método in vitro diseñado para evaluar el potencial mutagénico de una sustancia, ya sea como compuesto puro o en mezclas complejas. Este ensayo detecta la capacidad de la sustancia para revertir mutaciones en los genes responsables de la biosíntesis de histidina o triptófano en Salmonella typhimurium o Escherichia coli, respectivamente, restaurando así la capacidad normal de estas bacterias para sintetizar estos aminoácidos. Por lo tanto, el ensayo de Ames permite Introducción/Introduction 59 identificar mutaciones puntuales causadas por sustituciones, adiciones o eliminaciones de uno o varios pares de bases en el ADN, lo que revela transiciones o transversiones y cambios en el marco de lectura como mecanismos de producción de la mutación. Además, este método es rápido, sencillo de realizar y fácil de reproducir, lo que lo convierte en una herramienta adecuada para evaluar la mutagenicidad de las sustancias. El ensayo de micronúcleos descrito en la guía OCDE 487 (2023) permite detectar sustancias que inducen la formación de MN en células que han completado una división nuclear y se encuentran en la fase binucleada del ciclo celular, lo que indica la presencia de fragmentos cromosómicos o cromosomas completos adicionales. Esta técnica está ganando popularidad debido a su rapidez, facilidad de análisis y capacidad para detectar compuestos aneugénicos y clastogénicos. Esto la hace más atractiva que el tradicional ensayo de aberraciones cromosómicas. Además, existen otros ensayos de genotoxicidad complementarios a esta batería principal, como es el caso del ensayo de cometa estándar y modificado con enzimas. Esta técnica permite detectar sustancias que provocan roturas en el ADN de células eucariotas (Collins, 2004). En el caso de la modificación con enzimas, el ensayo se lleva a cabo utilizando enzimas específicas de reparación con actividad glicosilasa o endonucleasa, como la formamidopirimidina ADN glicosilasa (FPG) o la endonucleasa-III (Endo-III), para detectar daño oxidativo en el material genético, lo que permite evaluar el efecto genotóxico indirecto de sustancias con potencial prooxidante (Collins, 2004). En comparación con otros métodos, el ensayo cometa presenta ciertas ventajas, como una alta sensibilidad, rapidez, requisitos de muestra pequeños, la obtención de datos a nivel de células individuales y la posibilidad de llevarse a cabo en cualquier célula eucariota, tanto in vitro como in vivo (Collins, 2004; Azqueta et al., 2013). En caso de resultados positivos en la batería in vitro, se deben revisar todos los datos relevantes disponibles sobre la sustancia de prueba, y, se deben realizar uno o más estudios in vivo adecuados para evaluar si el potencial genotóxico observado in vitro se manifiesta in vivo, demostrando de esa manera una correlación correcta de los resultados. Los estudios de mutagenicidad llevados a cabo en los OSCs de interés son el test de Ames y el ensayo de linfoma de ratón (MLA) en presencia y ausencia de la fracción microsomal S9. En el caso del PTSO y PTS ya se han llevado a cabo ambos estudios. El PTSO no mostró un aumento en el número de revertientes en la prueba de Ames con cepas Introducción/Introduction 60 bacterianas, incluso en la concentración más alta probada (20 μM), tanto en presencia como en ausencia de S9. Sin embargo, en el ensayo de linfoma de ratón (MLA) con células L5178Y Tk⁺/⁻, aunque no se encontraron diferencias significativas en las frecuencias de mutaciones inducida tras 4 horas de tratamiento, se observaron aumentos significativos en las frecuencias de mutaciones inducidas en ensayos de 24 horas, en un rango de concentraciones de 2,5 a 20 μM. Estos resultados sugieren que la mutagenicidad del PTSO puede depender de la duración de la exposición y de la concentración utilizada (Mellado-García et al., 2016a). Los estudios sobre la mutagenicidad del PTS indicaron que, en las cepas bacterianas de S. typhimurium TA97, TA98 y TA100, no hubo un aumento en el número de colonias revertientes a ninguna de las concentraciones de PTS probadas, con o sin el sistema de activación metabólica S9. En la cepa TA102, se observó una disminución significativa en las colonias en ausencia de S9 (8,75-140 mM) y en presencia de S9 (8,75-35 mM). La cepa TA104 mostró un aumento en las colonias revertientes a las dos concentraciones más bajas en presencia de S9. El índice mutagénico fue inferior a 2 en todas las cepas, indicando que el PTS no es mutagénico en estas condiciones. En el ensayo de linfoma de ratón con células L5178Y Tk+/- tratadas con PTS durante 4 y 24 horas, no se encontraron diferencias significativas en las frecuencias de mutación inducidas. Los controles positivos confirmaron la validez de los ensayos (Mellado-García et al., 2017). Adicionalmente, se han realizado estudios de mutagenicidad/genotoxicidad para evaluar el extracto de brócoli rico en glucorafanina (GBE). En la prueba de Ames, se expusieron cuatro cepas de prueba de Salmonella typhimurium (TA97, TA98, TA100 y TA102) hasta 5000 µg de SFN/placa en presencia y ausencia del sistema de activación metabólica S9. Los resultados no mostraron actividad mutagénica (Zhou et al., 1015). Por otro lado, los estudios de genotoxicidad realizados incluyen el ensayo de micronúcleos en la línea celular L5178Y Tk⁺/⁻ y el ensayo cometa, tanto en su versión estándar como modificada con enzimas, en la línea celular Caco-2. En el caso del PTSO, en el ensayo de MN, las concentraciones seleccionadas se basaron en la concentración efectiva media (EC50) de 0-40 μM. En ausencia de la mezcla de activación metabólica S9, no se observó un aumento significativo en la frecuencia de células binucleadas con micronúcleo (BNMN%) ni en la frecuencia total de micronúcleos, y tampoco hubo variaciones significativas en el índice de división nuclear (NDI). Sin embargo, en presencia de la mezcla S9, se detectó un aumento muy significativo (P ≤ 0.001) en la Introducción/Introduction 61 frecuencia de células binucleadas con micronúcleos (BNMN %) a partir de 15 μM, sugiriendo que los metabolitos de PTSO pueden inducir la formación de micronúcleos y, por ende, causar daño genético. En el ensayo cometa, el PTSO no indujo rupturas de cadenas de ADN en células Caco- 2 a ninguna de las concentraciones probadas (0-50 μM) después de 24 o 48 horas de exposición. Además, la digestión enzimática de los núcleos con Endo III y FPG no reveló un aumento en el daño del ADN en células tratadas con PTSO, demostrando que no provoca daño genotóxico indirecto por oxidación del ADN (Mellado-García et al., 2016a). Los estudios sobre la genotoxicidad del PTS muestran que este compuesto induce daños genéticos en células L5178Y Tk+/-. Se observó un aumento significativo en el BNMN % a la concentración más alta probada (17,25 mM) en ausencia del sistema de activación metabólica S9. Además, se registraron incrementos significativos a concentraciones de 20 y 25 mM en presencia de S9. No se encontraron variaciones significativas en el índice de división nuclear (NDI) en comparación con el grupo control en ninguna de las concentraciones probadas, tanto en presencia como en ausencia de S9 (Mellado-García et al., 2017). En el ensayo estándar del cometa, el PTS no indujo rupturas de ADN en células Caco- 2 a concentraciones de 70 y 140 mM tras 24 y 48 horas de exposición. Sin embargo, se observaron aumentos significativos en las rupturas de ADN a 280 mM PTS después de ambos tiempos de exposición en comparación con el grupo control. La cantidad de ADN en la cola del cometa fue aproximadamente el doble a 280 mM PTS tras 48 horas en comparación con la misma concentración después de 24 horas. No se observó un aumento en el daño al ADN tras la digestión enzimática de los núcleos con Endo III o FPG en células expuestas a PTS. Los controles positivos en todos los experimentos mostraron aumentos significativos en el porcentaje de ADN en la cola del cometa, confirmando la eficacia del ensayo (Mellado-García et al., 2017). Por tanto, es necesario una evaluación in vivo de los OSCs de interés para garantizar la seguridad de su uso tras los resultados contradictorios encontrados in vitro. 8.4. Ecotoxicidad La evaluación de la ecotoxicidad in vitro propuesta en esta Tesis Doctoral se realiza en dos niveles: microalgas y dafnias. Introducción/Introduction 62 Los primeros estudios se realizan en microalgas de agua dulce. Durante un período de 72 horas normalmente, los organismos de prueba en crecimiento exponencial son expuestos a la sustancia en cultivos por lotes. La respuesta del sistema se manifiesta como una reducción del crecimiento en una serie de cultivos de algas expuestos a diversas concentraciones del producto a ensayar. Esta respuesta se evalúa como una función de la concentración de exposición en comparación con el crecimiento promedio de cultivos de control no expuestos. El indicador del ensayo es la inhibición del crecimiento, expresada como el aumento logarítmico de la biomasa (tasa de crecimiento específica promedio) durante el período de exposición. Además, se puede calcular la concentración que produce un x% de inhibición del crecimiento (por ejemplo, 50%), expresada como la ErCx (por ejemplo, ErC50). Otro parámetro de respuesta utilizado es el rendimiento, que se define como la biomasa al final del período de exposición menos la biomasa al inicio del mismo. A partir del rendimiento registrado en una serie de soluciones de prueba, se puede calcular la concentración que produce un x% de inhibición del rendimiento (por ejemplo, 50%), expresada como la EyCx (por ejemplo, EyC50). Además, se pueden determinar estadísticamente la concentración de efecto observado más bajo (LOEC) y la concentración sin efecto observado (NOEC). Estos datos proporcionan información valiosa para la evaluación del riesgo ambiental y la regulación de sustancias potencialmente peligrosas (OECD 201, 2011). En el siguiente paso de la evaluación de la ecotoxicidad, se procede al estudio de la sustancia utilizando dafnias jóvenes, las cuales tienen menos de 24 horas de edad al inicio del ensayo. Estos organismos se someten a la exposición a la sustancia a diferentes concentraciones a lo largo de un período de 48 horas. Posteriormente, los resultados obtenidos son analizados meticulosamente para calcular la Concentración Efectiva Media (CE50) tanto a las 24 como a las 48 horas. La determinación de la CE50 en el período inicial de 24 horas reviste una importancia fundamental para comprender los posibles efectos agudos de la sustancia en este nivel trófico, junto con los estudios en microalgas, proporcionando información de relevancia para la evaluación del riesgo ambiental y la regulación de sustancias de potencial riesgo para el ecosistema acuático (OECD 202, 2004). Introducción/Introduction 63 9. Evaluación toxicológica in vivo de compuestos organosulfurados para las aplicaciones propuestas 9.1. Genotoxicidad Para evaluar la genotoxicidad in vivo la EFSA recomiendan las siguientes pruebas como estudios de seguimiento a los ensayos in vitro en caso de obtener resultados no concluyentes: • El ensayo de micronúcleos en eritrocitos de mamífero (directriz de la OECD 474, 2016), • El ensayo de mutación genética en células somáticas y germinales de roedores transgénicos (directriz de la OECD 488, 2022), • El ensayo Cometa in vivo (directriz de la OECD 489, 2016). La prueba de micronúcleos in vivo cubre los marcadores de aberraciones cromosómicas estructurales y numéricas y es un seguimiento adecuado para clastógenos y aneugénicos in vitro. Puede haber circunstancias en las que un ensayo de aberración cromosómica en médula ósea de mamífero in vivo (OECD 475, 2014) pueda ser una prueba de seguimiento alternativa. En los estudios de genotoxicidad in vivo, en caso de resultados negativos, es importante obtener evidencia de la exposición de células diana y puede ser necesario considerar otros tejidos relevantes (por ejemplo, tejidos de contacto para sustancias altamente reactivas que no están disponibles sistémicamente). Las pruebas en roedores transgénicos pueden detectar mutaciones puntuales y pequeñas deleciones y no tienen restricciones de tejido. El ensayo cometa in vivo se considera una prueba indicadora útil en términos de sensibilidad a las sustancias que causan mutaciones genéticas y/o aberraciones cromosómicas estructurales y puede aplicarse a muchos tejidos diana. Con el fin de reducir el número de animales utilizados, se puede considerar un estudio de roedores único que combine el análisis de diferentes parámetros (por ejemplo, MN y ensayo cometa). En situaciones específicas, se puede considerar el uso de otros estudios (por ejemplo, estudios mecanicistas) para definir el potencial genotóxico in vivo de una sustancia. La genotoxicidad de los OSCs in vivo está poco estudiada. El PTSO ha sido evaluado por su potencial genotóxico en estudios recientes utilizando varios ensayos (Mellado- García et al., 2015). En el ensayo de genotoxicidad y citotoxicidad en médula ósea de ratas, se analizó la proporción de eritrocitos policromáticos (PCE) entre el total de Introducción/Introduction 64 eritrocitos y el porcentaje de micronúcleos (% MN). Los resultados indicaron que no hubo diferencias significativas en la proporción de PCE/total de eritrocitos entre los grupos experimentales y los controles negativos o positivos. Además, el PTSO no indujo la frecuencia de MN en eritrocitos inmaduros a ninguna de las dosis probadas (Mellado- García et al., 2016b). En el ensayo estándar del cometa y su versión modificada por enzimas, el PTSO no provocó rupturas de cadenas de ADN en la sangre, el estómago y el hígado de ratas a ninguna de las dosis administradas. La evaluación del daño oxidativo se realizó mediante la versión modificada del ensayo del cometa, en la que las muestras fueron tratadas con las enzimas reparadoras Endo III y FPG, que introducen rupturas en sitios de ADN con lesiones oxidativas. No se observaron bases oxidadas en estos órganos cuando se utilizaron Endo III y FPG a ninguna de las dosis probadas en ambos sexos. Estos resultados sugieren que el PTSO no provoca daño significativo en el ADN ni induce lesiones oxidativas en los tejidos evaluados (Mellado-García et al., 2016b). 9.2. Toxicidad aguda y subcrónica Los ensayos de toxicidad aguda en roedores son fundamentales en la evaluación inicial de la seguridad de una sustancia química o un producto. Estos estudios proporcionan información sobre los posibles efectos adversos que una sustancia puede tener cuando se administra en una sola dosis aguda. La importancia de estos ensayos radica en varios aspectos. Primero, permiten una evaluación rápida de la seguridad de la sustancia al brindar información sobre los posibles efectos adversos en un corto período de tiempo, generalmente en un plazo de 24 a 72 horas después de la administración. Esto ayuda a tomar decisiones sobre la necesidad de realizar estudios adicionales de toxicidad a largo plazo. Segundo, estos ensayos permiten determinar la dosis letal aguda (DL50), que es un parámetro importante para clasificar el grado de toxicidad de una sustancia y para establecer niveles de exposición seguros para los seres humanos y el medio ambiente. Tercero, los ensayos de toxicidad aguda permiten evaluar una amplia gama de efectos agudos, como cambios en el comportamiento, signos clínicos de toxicidad, efectos en órganos y sistemas específicos, y mortalidad, lo que proporciona información sobre la toxicidad inmediata de una sustancia. Para evaluar la toxicidad de manera aguda, se debe seguir la guía de la OECD 425 (2022). Introducción/Introduction 65 Para investigar el potencial tóxico subcrónico de la sustancia activa, se debe realizar al menos un estudio en una especie de roedor con una duración de al menos 90 días. Si la información de un estudio en roedores no es una base adecuada para la evaluación del riesgo para el consumidor, puede ser necesario realizar un estudio adicional en una especie no roedora. El producto de prueba debe administrarse por vía oral (preferiblemente incorporado a la dieta) con al menos tres niveles de dosis además de un grupo control para evaluar una respuesta dosis-dependiente. La dosis más alta utilizada normalmente debería estar asociada con la evidencia de efectos adversos. El nivel de dosis más bajo no debería producir ninguna evidencia de toxicidad. Los protocolos para estos estudios deben cumplir con las directrices de la OCDE 408 (2018) (roedores) o 409 (no roedores) (1998). Anteriormente, se ha realizado un ensayo subcrónico de un extracto de Allium rico en PTSO llamado Proallium. En un ensayo subcrónico de 13 semanas, se administraron diferentes dosis de Proallium AP® (25, 100 y 400 mg/kg/día) a ratas para evaluar sus efectos sobre diversos parámetros biológicos. Los resultados indican que no hubo diferencias significativas en el peso corporal final, la ganancia de peso y el consumo total de alimentos entre los grupos tratados y los grupos control, tanto en machos como en hembras. Los cambios semanales en el peso corporal y el consumo de alimentos y agua tampoco mostraron diferencias significativas entre los grupos (Mellado-García et al., 2016b). En cuanto a los parámetros hematológicos y bioquímicos, la administración del extracto no produjo cambios en los parámetros hematológicos como los conteos de glóbulos rojos (RBC), la cantidad de hemoglobina (HGB), el hematocrito (HCT), el volumen corpuscular medio (MCV), la hemoglobina corpuscular media (MCH), la concentración media de hemoglobina corpuscular (MCHC), el número de plaquetas (PLT), el ancho de distribución de los glóbulos rojos (RDW) y el tiempo de protrombina (PT). Tampoco se detectaron diferencias significativas en los conteos de leucocitos totales y diferenciales. Se observó una leve disminución en los niveles de glucosa en sangre (GLUC) en los machos tratados con la dosis media (100 mg/kg/día), pero este cambio no se consideró biológicamente significativo. No se encontraron cambios significativos en los marcadores de función hepática como la aspartato aminotransferasa (AST), la alanina aminotransferasa (ALT), la fosfatasa alcalina (ALKP), la proteína total (TOT PROT) y la albúmina (ALB) ni en los parámetros de función renal (BUN, creatinina), aunque hubo Introducción/Introduction 66 un aumento significativo en los valores de BUN en los machos tratados con la dosis más alta (400 mg/kg/día) en comparación con la dosis más baja (25 mg/kg/día), pero este cambio tampoco se consideró biológicamente significativo. La necropsia macroscópica y el peso de los órganos tampoco revelaron diferencias significativas en los pesos absolutos y relativos de los órganos, ni en las relaciones peso de órgano/peso corporal y peso de órgano/peso cerebral entre los grupos tratados y los controles. El examen histopatológico de los órganos internos (cerebro, hígado, corazón, bazo, riñón, timo, adrenales, testículos, epidídimo, útero y ovarios) no mostró diferencias entre los grupos tratados y no tratados en ambos sexos. En conclusión, la administración subcrónica del extracto de Allium a ratas no produjo efectos adversos significativos en términos de peso corporal, consumo de alimentos y agua, parámetros hematológicos y bioquímicos, peso y estructura de los órganos, lo que sugiere que el extracto es seguro a las dosis ensayadas. Sin embargo, es fundamental ensayar en futuros estudios los compuestos purificados del extracto. En lo que respecta al SFN, hay pocos estudios de su toxicidad aguda y subcrónica. Scola et al. (2017) observaron toxicidad severa, como sedación profunda, ataxia, ptosis y temblores en ratones expuestos de forma aguda a altas dosis de SFN administradas por vía intraperitoneal (i.p.). En este estudio, la dosis de 300 mg de SFN/kg resultaron en la muerte de todos los animales dentro de los primeros 180 minutos. Síntomas similares se reportaron con dosis de 250 mg/kg, con siete de doce ratones muriendo dentro de los 240 minutos. A una dosis de 200 mg/kg, se observaron sedación y ptosis en todos los animales, con seis de doce ratones muriendo durante la primera noche. A dosis más bajas, como 150 mg/kg, la sedación persistió, pero no se observó mortalidad. Además, los autores determinaron la dosis letal media (DL50) en 212,67 mg/kg. Por otro lado, Zhou et al. (2015) estudiaron los efectos de un extracto de brócoli rico en glucorafanina en ratas expuestas oralmente durante 30 días a dosis de 3 g/kg/día (equivalente a 390 mg/kg/día de glucorafanina, precursor de SFN). Durante el estudio de alimentación de 30 días, no se observó mortalidad ni hallazgos clínicos adversos relacionados con el tratamiento. Los animales en todos los grupos mostraron actividades y crecimiento normales. Los pesos corporales no difirieron significativamente entre los grupos tratados con SFN y los grupos control, aunque hubo una ligera disminución en el consumo de alimentos en los machos de dosis alta. Sin embargo, esta disminución se asoció con un aumento en la tasa de utilización de alimentos, y la ganancia de peso Introducción/Introduction 67 corporal se mantuvo similar a la de los grupos control. Se observaron fluctuaciones menores en los parámetros de hematología y química clínica, pero éstas estuvieron dentro del rango de control histórico y no mostraron relaciones claras dosis-respuesta. Los parámetros de química clínica mostraron cambios mínimos dentro del rango de control histórico del laboratorio de pruebas, sin diferencias significativas en los indicadores de toxicidad hepática o renal. Los pesos de los órganos aumentaron en los machos de dosis alta para el bazo y los riñones, mientras que el peso absoluto de los testículos se redujo ligeramente. Sin embargo, todos los valores de peso de los órganos se mantuvieron dentro del rango de control histórico. Las evaluaciones macroscópicas en la necropsia no revelaron anormalidades, y las evaluaciones histológicas de varios tejidos mostraron estructuras normales sin anormalidades observables relacionadas con el tratamiento con SFN. Cabe destacar que estos estudios no han ensayado sustancias puras sino extractos de Allium o brócoli. El estudio de sus componentes activos aislados es importante para obtener resultados precisos y consistentes en estudios científicos. Los extractos vegetales contienen una mezcla compleja de compuestos, lo que puede dificultar la identificación de los efectos específicos de cada componente. En cambio, el uso de sustancias puras permite aislar y estudiar directamente los efectos individuales de los compuestos activos, como la alicina en el ajo o el SFN en el brócoli. Esto no sólo facilita la comprensión de sus mecanismos de acción, sino que también mejora la reproducibilidad de los experimentos y la interpretación de los resultados. Además, el ensayo de sustancias puras elimina la variabilidad inherente a los extractos vegetales, que pueden presentar diferencias en la concentración de compuestos activos debido a factores como la procedencia, el método de extracción y las condiciones de almacenamiento. Por tanto, la investigación con sustancias puras es esencial para el desarrollo de aplicaciones terapéuticas seguras y efectivas. 9.3. Toxicocinética Para la evaluación de la seguridad de los aditivos, es imprescindible abordar sus parámetros toxicocinéticos in vivo. Estos parámetros delinean el trayecto que los agentes químicos siguen a través del organismo, incluyendo su absorción, distribución, metabolismo y excreción. Los datos derivados de estos estudios son de particular relevancia en investigaciones sobre toxicidad aguda y crónica de los aditivos, especialmente cuando se administran en dosis elevadas. Dado que la dosis de un Justificación y objetivos/ Significance and purposes 75 III. JUSTIFICACIÓN Y OBJETIVOS/ SIGNIFICANCE AND PURPOSES Como se ha mencionado anteriormente, los OSCs tienen potencial para ser utilizados como aditivos en piensos para animales con el objetivo de mejorar su conservación y propiedades organolépticas Debido a estas aplicaciones, tanto los animales como los consumidores estarán expuestos a estos compuestos. La exposición oral, tanto de los animales al pienso como de los consumidores a los animales alimentados con dicho pienso, así como su aplicación en el medio ambiente, hace imprescindible evaluar la toxicidad de estos compuestos antes de su autorización a nivel europeo. Después de realizar una primera revisión bibliográfica sobre los OSCs de interés para esta tesis doctoral (PTSO, PTS, CSSP, GSSP y SFN), se encontraron resultados contradictorios respecto a la toxicidad in vitro en presencia de la activación metabólica S9. Esta discrepancia pone de manifiesto la necesidad de llevar a cabo una evaluación más exhaustiva de los mismos. Por lo tanto, se desarrolló un primer enfoque experimental utilizando líneas celulares intestinales humanas (Caco-2) y de linfoma de ratón (L5178Y tk +/-), para investigar los metabolitos de algunos OSCs, como GSSP y CSSP. Adicionalmente, se ha llevado a cabo la evaluación de la mutagenicidad, citotoxicidad y genotoxicidad de un nuevo aditivo no organosulfurado, la enterocina AS-48, mediante métodos reconocidos como el ensayo de Ames, el ensayo de MN y el ensayo cometa estándar y modificado con enzimas. Estos ensayos proporcionan una visión detallada de los posibles efectos adversos a nivel genético y celular, permitiendo identificar riesgos potenciales antes de su implementación en la industria. Además, es relevante desarrollar de nuevas técnicas analíticas para detectar y cuantificar los OSCs en matrices alimenticias (piensos) y muestras biológicas (plasma, estómago, etc.), ya que ello es crucial para garantizar la precisión y fiabilidad de las evaluaciones toxicológicas. Estas técnicas avanzadas permiten un monitoreo más eficiente y exacto de los compuestos en diversas muestras, lo que es fundamental para estudios de seguridad alimentaria y salud pública. Justificación y objetivos/ Significance and purposes 76 Así mismo, los estudios toxicocinéticos, que investigan la absorción, distribución, metabolismo y excreción (ADME) de los OSCs objeto de estudio y sus metabolitos principales, son necesarios para comprender el comportamiento biológico de estos compuestos. Esta información es indispensable para evaluar el riesgo potencial de los aditivos, asegurando que su uso sea seguro tanto para los animales como para los humanos. En una segunda instancia, se llevó a cabo una primera evaluación in vivo en ratas con exposiciones agudas (≤ 3 días) para obtener una valoración preliminar de la toxicidad y genotoxicidad de estos compuestos, como PTS. Además, se evaluó a largo plazo la toxicidad de los OSCs por vía oral mediante ensayos de toxicidad subcrónica durante 90 días en ratas (OECD, 408) y de toxicidad reproductiva (OECD, 416) durante dos generaciones en ratón. Los estudios subcrónicos permiten identificar efectos tóxicos que pueden no manifestarse en estudios a corto plazo, proporcionando una visión más completa de la seguridad de los compuestos con una exposición prolongada. Así mismo, los estudios de toxicidad reproductiva a lo largo de dos generaciones son importantes para evaluar los posibles efectos a largo plazo en la fertilidad, el desarrollo embrionario y el crecimiento de la descendencia, aspectos críticos que no pueden ser evaluados adecuadamente en estudios de corta duración. Estos enfoques permiten establecer un perfil toxicológico más robusto y fiable, asegurando que los OSCs se puedan utilizar de manera segura en aplicaciones animales o humanas. Todos estos ensayos se realizaron siguiendo las recomendaciones de la EFSA y utilizando las diferentes guías de la OECD correspondientes. Por otro lado, también se ha llevado a cabo la evaluación de la potencial ecotoxicidad de los aditivos utilizando modelos in vitro e in vivo, como algas, dafnias y peces (en curso), siendo esencial para asegurar que estos compuestos no presenten riesgos significativos para el medio ambiente. Estos ensayos permiten identificar posibles efectos adversos en diferentes niveles de la cadena trófica acuática, proporcionando una comprensión integral de su seguridad. Por último, el uso del PTSO y PTS en nuevas especies de animales de granja, como el conejo, es esencial para ampliar el conocimiento sobre la aplicación de estos aditivos en nutrición animal. Es por ello, que se realizó un estudio que abarcó una evaluación exhaustiva de sus efectos en la bioquímica, la microbiota, la eficiencia alimentaria y la presencia de metabolitos en las partes comestibles del animal, Justificación y objetivos/ Significance and purposes 77 específicamente el músculo. Estos datos son fundamentales para determinar la seguridad y eficacia de los aditivos en especies animales menos estudiadas. En conjunto, la investigación propuesta es fundamental para desarrollar compuestos seguros y efectivos para la nutrición animal, contribuyendo a la mejora de la salud animal, la sanidad vegetal y la seguridad alimentaria. La integración de estos enfoques proporciona una base sólida para la validación científica de nuevas sustancias con aplicaciones agroalimentarias, asegurando su sostenibilidad y aceptación en el mercado. Por todo ello, en la presente tesis doctoral el objetivo general es contribuir al desarrollo y evaluación toxicológica de distintos compuestos aliáceos con potencial aplicación en el ámbito agroalimentario. Para conseguir este objetivo general se abordarán los siguientes objetivos específicos: En relación a su aplicación en nutrición animal: 1. Realizar una revisión bibliográfica sobre el perfil toxicológico y efectos beneficiosos de compuestos aliáceos con potencial uso en la industria agroalimentaria. 2. Evaluación de la citotoxicidad, genotoxicidad y mutagenicidad in vitro de los metabolitos del PTSO además de otro nuevo compuesto natural no organosulfurado (Enterocina AS-48). 3. Desarrollar y validar un método para la determinación de PTSO y PTS en matrices de alimentación animal mediante UHPLC-MS/MS (Orbitrap). 4. Investigar in vitro los potenciales procesos de biotransformación hepáticos que puede experimentar el PTSO empleando modelos subcelulares de rata y humanos. 5. Investigar la absorción, distribución, metabolismo y excreción (ADME) in vivo en rata (protocolo OECD 417) de PTSO. 6. Evaluar in vivo la genotoxicidad del PTS mediante el uso del ensayo cometa (OECD 489) y micronúcleos (OECD 474). 7. Evaluar la toxicidad general subcrónica in vivo de PTSO y PTS, en rata, mediante un estudio a dosis repetidas de 90 días (protocolo OECD 408). 8. Investigar la potencial toxicidad reproductiva in vivo del PTSO sobre dos generaciones mediante un estudio multigeneracional (protocolo OECD 416). Justificación y objetivos/ Significance and purposes 78 9. Investigar los efectos de la aplicación dietética de extractos de Allium sobre el crecimiento, la bioquímica, el metabolismo y la microbiota intestinal de conejos. El trabajo experimental ha sido realizado en el Área de Toxicología del Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal de la Facultad de Farmacia de la Universidad de Sevilla. Además, se ha contado con el uso de los servicios de Biología, Espectrometría de Masas, Invernadero y Microscopía del Centro de Investigación, Tecnología e Innovación de la Universidad de Sevilla (CITIUS). La síntesis química de los OSCs estudiados y extractos naturales se ha desarrollado en estrecha colaboración con el Ldo. Enrique Guillamón, y los Dres. Alberto Baños, José Manuel de la Torre y María Arántzazu Aguinaga de DMC Research Center S.L.U. La elaboración y purificación de la Enterocina AS-48 se llevó a cabo gracias a la colaboración de la Dra. Eva Valdivia, del Departamento de Microscopía de la Universidad de Granada. Así mismo, los estudios en animales de laboratorio y los estudios histopatológicos se realizaron en colaboración con el Área de Toxicología y el Departamento de Anatomía y Anatomía Patológica Comparadas de la Universidad de Córdoba, con la valiosa participación de la Dra. Rosario Moyano, la Dra. Ana Molina y el Dr. Nahúm Ayala. Del mismo modo, los estudios en animales de granja se llevaron a cabo gracias a la colaboración con el grupo Martini, en Italia, formado por Andrea Ricci, Andrea Frabetti y Andrea Barausse. Finalmente, el Dr. José A. González-Pérez y el Dr. Nicasio T. Jiménez-Morillo han colaborado activamente en el desarrollo de la técnica de pirólisis analítica utilizada para el análisis de OSCs en muestras biológicas. Los estudios para evaluar el potencial quimioprotector de los OSCs frente a CYN han sido posibles Justificación y objetivos/ Significance and purposes 79 gracias a una estancia internacional en el CIIMAR (Oporto, Portugal), bajo la supervisión de su director Dr. Vitor Vasconcelos y del Dr. Alexandre Campos. Siguiendo la normativa de la Universidad de Sevilla, el resumen, la justificación y objetivos, y las conclusiones se redactan tanto en español como en inglés para optar a la “Mención Internacional en el Título de Doctor”. Justificación y objetivos/ Significance and purposes 80 As mentioned above, OSCs have the potential to be used as additives in animal feed to improve its preservation and organoleptic properties Due to these applications, both animals and consumers will be exposed to these compounds. The oral exposure of both animals to the feed and consumers to the animals fed with this feed, as well as their application in the environment, makes it essential to evaluate the toxicity of these compounds before their authorization at European level. After a first literature review on the OSCs of interest for this PhD thesis (PTSO, PTS, CSSP, GSSP and SFN), contradictory results were found regarding in vitro toxicity in the presence of S9 metabolic activation. This discrepancy highlights the need for further evaluation. Therefore, a first experimental approach was developed using human intestinal (Caco-2) and mouse lymphoma (L5178Y tk +/-) cell lines to investigate the metabolites of some OSCs, such as GSSP and CSSP. Additionally, evaluation of the mutagenicity, cytotoxicity and genotoxicity of a new non-organosulfurized additive, enterocin AS-48, by recognized methods such as the Ames assay, micronucleus assay and the standard and enzyme-modified comet assay, has been carried out is crucial to ensure its safety. These assays provide detailed insight into potential adverse effects at the genetic and cellular level, allowing potential risks to be identified prior to implementation in the industry. In addition, new analytical techniques have been developed to detect and quantify OSCs in food matrices (feed) and biological samples (plasma, stomach, etc.), as this is crucial to ensure the accuracy and reliability of toxicological evaluations. These advanced techniques allow more efficient and accurate monitoring of compounds in various samples, which is essential for food safety and public health studies. Likewise, toxicokinetic studies, which investigate the absorption, distribution, metabolism and excretion of the target OSCs and their major metabolites, are necessary to understand the biological behavior of these compounds in organisms. This information is indispensable to evaluate the potential risk of the additives, ensuring that their use is safe for both animals and humans. In a second instance, a first in vivo evaluation was carried out in rats with acute exposures (≤ 3 days) to obtain a preliminary assessment of the toxicity and genotoxicity of these compounds, as PTS. In addition, the long-term toxicity of OSCs by the oral route Justificación y objetivos/ Significance and purposes 81 was evaluated by subchronic toxicity tests for 90 days in rats (OECD, 408) and reproductive toxicity (OECD, 416) for two generations in mice. Subchronic studies allow the identification of toxic effects that may not manifest themselves in short-term studies, providing a more complete picture of the safety of compounds with prolonged exposure. Likewise, reproductive toxicity studies over two generations are important to assess potential long-term effects on fertility, embryonic development and offspring growth, critical aspects that cannot be adequately evaluated in short-term studies. These approaches allow a more robust and reliable toxicological profile to be established, ensuring that OSCs can be used safely in animal or human applications. All these tests were performed following EFSA recommendations and using the different OECD guidelines. On the other hand, the evaluation of the potential ecotoxicity of additives has also been carried out using in vitro and in vivo models, such as algae, daphnia and fish, being essential to ensure that these compounds do not present significant risks to the environment. These tests allow the identification of possible adverse effects at different levels of the aquatic trophic chain, providing a comprehensive understanding of their safety. Finally, the application of the use of PTSO and PTS in new species of farm animals, such as rabbits, is essential to expand the knowledge on the application of these additives in animal nutrition. For this reason, a study was carried out that included an exhaustive evaluation of their effects on biochemistry, microbiota, feed efficiency and the presence of metabolites in the edible parts of the animal, specifically the muscle. These data are critical to determine the safety and efficacy of additives in less studied animal species and to ensure that the final products are safe for human consumption. Taken together, these studies are critical to develop safe and effective compounds for animal nutrition, contributing to the improvement of animal health, plant health and food safety. The integration of these approaches provides a solid basis for the scientific validation of new substances with agri-food applications, ensuring their sustainability and market acceptance. Therefore, the general objective of this doctoral thesis is to contribute to the development and toxicological evaluation of different alliaceous compounds with Justificación y objetivos/ Significance and purposes 82 potential application in the agri-food sector. In order to achieve this general objective, the following specific objectives will be addressed: In relation to their application in animal nutrition: 1. To carry out a literature review on the toxicological profile and beneficial effects of alliaceous compounds with potential use in the agri-food industry. 2. Evaluation of the in vitro cytotoxicity, genotoxicity and mutagenicity of PTSO metabolites in addition to another new natural non-organosulfur compound (Enterocin AS-48). 3. To develop and validate a method for the determination of PTSO and PTS in animal feed matrices by UHPLC-MS/MS (Orbitrap). 4. To investigate in vitro the potential hepatic biotransformation processes that PTSO may undergo using rat and human subcellular models. 5. To investigate the in vivo rat absorption, distribution, metabolism and excretion (ADME) (OECD 417 protocol) of PTSO. 6. To evaluate in vivo the genotoxicity of PTS using the comet assay (OECD 489) and micronucleus (OECD 474). 7. To evaluate the in vivo subchronic general toxicity of PTSO and PTS, in rat, by means of a 90-day repeated dose study (OECD 408 protocol). 8. To investigate the potential in vivo reproductive toxicity of PTSO on two generations by means of a multigenerational study (protocol OECD 416). 9. To investigate the effects of the dietary application of Allium extracts on growth, biochemistry, metabolism and intestinal microbiota of rabbits. The experimental work has been carried out in the Toxicology Area of the Department of Nutrition and Bromatology, Toxicology and Legal Medicine of the Faculty Justificación y objetivos/ Significance and purposes 83 of Pharmacy of the University of Seville. In addition, the Biology, Mass Spectrometry, Greenhouse and Microscopy services of the Center for Research, Technology and Innovation of the University of Seville (CITIUS) have been used. The chemical synthesis of the studied OSCs and natural extracts has been developed in close collaboration with Enrique Guillamón, Dr. Alberto Baños, Dr. José Manuel de la Torre, Dr. María Arántzazu Aguinaga from DMC Research Center S.L.U. The elaboration and purification of Enterocin AS-48 was carried out thanks to the collaboration of Dr. Eva Valdivia, from the Microscopy Department of the University of Granada. Likewise, studies in laboratory animals and histopathological studies were carried out in collaboration with the Toxicology Area and the Department of Comparative Anatomy and Pathological Anatomy of the University of Cordoba, with the valuable participation of Dr. Rosario Moyano, Dr. Ana Molina and Dr. Nahúm Ayala. Similarly, studies in farm animals were carried out thanks to the collaboration with the Martini group, in Italy, formed by Andrea Ricci, Andrea Frabetti and Andrea Barausse. Finally, Dr. José A. González-Pérez and Dr. Nicasio T. Jiménez-Morillo have actively collaborated in the development of the analytical pyrolysis technique used for the analysis of OSCs in biological samples. The studies to evaluate the chemoprotective potential of OSCs against CYN have been possible thanks to an international research stay at CIIMAR (Porto, Portugal), under the supervision of Dr. Vitor Vasconcelos and Dr. Alexandre Campos. Following the regulations of the University of Seville, the abstract, the justification and objectives, and the conclusions are written in both Spanish and English to qualify for the “International Mention in the PhD Degree”. Foods 2022, 11, 2620 2 of 25 such as antiviral, antimicrobial, antioxidant, antiprotozoal, antidiabetic, anticarcinogenic, antispasmodic, antimutagenic, anti-amnesic, antiasthmatic, anti-inflammatory, neuroprotective, hepatoprotective, hypotensive, immunomodulatory, hypoglycemic, as well as their prebiotic properties [2,4–12]. In relation to antibacterial activity, gram-positive bacteria seem to be more sensitive than gram-negative bacteria to these compounds. This could be due to lipopolysaccharides present in the outer membrane of Gram-negative bacteria. However, the mechanism of action is not yet sufficiently clarified, and it seems that it is associated with the inactivation of thiolic bacterial enzymes. Moreover, the mechanism of the anti-inflammatory activity of some OSCss can be associated with the inhibition of TNF-α-initiated secretion of pro-inflammatory cytokines from epithelial digestive cells. In addition, compounds such DADS, DATS and SAC inhibit the formation of inflammatory lipopolysaccharide by repressing NF-кB and MAPK signaling pathways [2]. Recently, Rochetti et al. [13] carried out an extensive phytochemical investigation of nine Allium species, highlighting a promising nutraceutical potential of these species. These properties give the Allium genus diverse uses in several areas of knowledge, and although traditionally this genus has already been used medicinally, in recent years its use has incremented in the agri-food industry [14]. Thus, Allium extracts and OSCss can be potentially used in food and feed sectors as a substitute for synthetic preservatives due to their significant antimicrobial properties [11,15]; nevertheless, their use in food is limited due to their organoleptic properties [1]. The use of OSCs and Allium extracts to control food spoilage have also been studied [16,17]. Several OSCs, such as propylpropane-thiosulfinate (PTS) and propyl-propane thiosulfonate (PTSO) have a potential application in maize storage, mainly due to their antifungal and antimycotoxigenic activity. They could be used in concentrations in the order of parts per million to reduce up to 90% the concentrations of mycotoxins produced by Fusarium [18]. Moreover, due to their antioxidant and antibacterial activities, PTSO and PTS inserted in a synthetic matrix have been also proposed for usage in active food-packaging to better preserve salads [19,20]. As a consequence of their new applications, the potential accumulative exposure to consumers has increased, and a safe range of concentrations for their use in the industry should be established to reach the market and avoid risks for consumers (Table 1). Table 1. Organosulfur compounds present in Allium species, chemical structure and main properties. Name Chemical Structure Mode of Action Reference Antibacterial: in vitro activity against Gram +: E-Ajoene Z-Ajoene MIC = 5–250 µg/mL and Gram –: MIC = 100–500 µg/mL [21] Antifungal: MIC = 15–50 µg/mL Antioxidant enzyme induction: NAD(P)H: quinone oxidoreductase-1 (NQO1) [22] Alliin Antioxidant: superoxide scavenging activity [23] Allicin Antibacterial: in vitro activity against Staphylococcus strains MIC = 600 µg/mL and Gram-bacteria: MIC = 4–300 µg/mL [24–26] Antifungal: MIC = 1.52–6.25 µg/mL [27] Antioxidant activity in vivo: SOD and GSH-Px activities increased. Radical scavenging ability of hydroxyl radical increase with Allicin Antibacterial activity against S. aureus: [24,28,29] Di-allyl-disulphide (DADS) MIC = 4 µg/mL and Helicobacter pylori MIC = 200 µg/mL [25,30] Antifungal activity against Aspergillus spp: MIC = 8–12 µg/mL and Candida spp: MIC = 4–12 µg/mL [30] Foods 2022, 11, 2620 3 of 25 Table 1. Cont. Name Chemical Structure Mode of Action Reference Antioxidant in vivo activity [31,32] Antibacterial activity against S. aureus: Di-allyl-sulphide (DAS) MIC = 20 µg/mL and H. pylory MIC = 4 µg/mL [25,30] Antifungal activity against Aspergillus spp: MIC= 40–64 µg/mL and Candida spp: MIC= 32–72 µg/mL [30] Antioxidant in vivo activity [33] Antibacterial activity against S. aureus: Di-allyl-trisulphide (DATS) MIC = 2 µg/mL and H. pylory MIC = 25 µg/mL [25,30] Antifungal activity against Aspergillus spp: MIC = 2–8 µg/mL and Candida spp: MIC = 1–8 µg/mL [30] Dipropyl disulphide (DPDS) Dipropyl sulphide (DPS) Antioxidant in vitro activity [34] Antioxidant in vitro activity [16] Antioxidant in vitro activity [16] Propyl-propane- tiosulphonate (PTSO) Antibacterial activity against Gram +: MCB = 0.5–10 µg/mL and Gram-: MCB = 1.25–10 µg/mL Antifungal: MFC against Verticillium dahliae = 19.53–39.06 µg/mL MFC90 against Candida spp. = 64–128 µg/mL Antiprotozoal and Antiparasitic: reduce the number of apicomplexa in monogastric animals. Reducing a plurality of aquatic parasites in aquatic animals. [20,35,36] [11,37] [38,39] Antioxidant [20] Propyl-propane- tiosulphinate (PTS) Antibacterial activity against Gram -: MCB = 128–1024 µg/mL and Gram +: MCB = 128 µg/mL Antifungal activity MFC against Verticillium dahliae = 78.13 µg/mL MFC90 against Candida spp = 128 µg/mL Antiprotozoal and antiparasitic: reduce the number of Apicomplexa in monogastric animals. Reducing a plurality of aquatic parasites in aquatic animals. [11] [34,40] [38,39] S-allylcysteine (SAC) Antioxidant activity by scavenging ROS [41] Vinyldithiin Antioxidant activity [42] Furthermore, synthetic chemical additives, such as butylhydroxytoluene (BHT) and Butylhydroxyanisole (BHA), have shown problems regarding their safety, including carcinogenicity and sensitization in consumers [43]. For this reason, there is a call for replacement with natural and safe alternatives of natural origin, such as the bacteriocins [44] like Nisin (E234), a peptide produced from Lactococcus lactis, vegetable extracts such as citrus extracts rich in flavonoids [44] or essential oils (EO) with preservative properties, which have been reported to have antimicrobial and antioxidant properties [45]. In the case of garlic EOs, some authors demonstrated their preservation and antimicrobial capacities (including PTSO and PTS in its composition) [46,47]. However, important adverse effects produced by these compounds have also been described such as allergic reactions, gastrointestinal tract injury, weight loss, anemia, and toxicity to the liver, heart, and kidney [16]. Thus, garlic has been classified as a type I allergen. Because of this all, to carry out all possible applications in the agri-food industry, it is necessary that OSCs pass a safety assessment as specified by the European Food Safety Authority (EFSA) [48–51]. This safety evaluation includes a wide array of tests both in vitro and in vivo. The “Guidance for submission for food additive evaluations” by EFSA [48] Foods 2022, 11, 2620 4 of 25 described the need of a toxicological evaluation in the following core areas: genotoxicity, toxicokinetics, toxicity comprising chronic, subchronic and carcinogenicity, developmental and reproductive toxicity. For the toxicological research, a tiered approach is followed, initially using fewer complex tests to obtain hazard data. These are then evaluated to determine if they are sufficient for risk assessment or, if not, for designing studies at higher tiers [48]. Therefore, in vitro studies are usually the initial step in the toxicological evaluation of any compound. Thus, the objective of this work is to provide a systematic overview of the scientific literature of OSCs with antibacterial, antifungal, and other properties present in the Allium genus (Table 1) in regard to their in vitro toxicity evaluation. To achieve this purpose, the authors have followed the PRISMA 2020 guidelines for systematic reviews. This information could contribute to the safe use of these compounds in the agri-food sector. 2. Materials and Methods This research was performed according to the Preferred Reporting Items for a Systematic Review and Meta-analysis (PRISMA) statement [52]. The question to be answered was: Do Allium compounds have toxic activity in vitro? 2.1. Protocol and Registration The protocol for the present systematic review was not registered in any Systematic Review and Meta-analysis database. 2.2. Eligibility and Exclusion Criteria International studies were considered. The eligibility criteria of the present systematic review were as follows: Inclusion criteria: (1) articles on Allium toxicity in vitro; (2) articles published prior to 21 September 2021; and (3) articles reporting comprehensive results and/or information on the field. Exclusion criteria: (1) unsystematic and narrative reviews; (2) articles published in a language other than English; (3) proceedings of conferences and dissertations; (4) books or book chapters; (5) editorial material; (6) articles dealing with Allium in vitro in which the test item is not a naturally occurring alliaceous compound (e.g., synthetically modified allicin) in the genus Allium or when the test has no toxicological relevance (e.g., protective effects of organosulfur compounds towards N-nitrosamine- induced DNA damage) as represented in the flowchart (Figure 1). 2.3. Information Sources and Search Strategy The electronic research databases Web of Science, Scopus, Science Database and PubMed were searched on 21 September 2021. The search identified articles published from inception to 21 September 2021 inclusive. The Boolean strings chosen were: (“Propyl thiosulfinate oxide” OR “propyl-propane-thiosulfonate” OR “propyl propane thiosulfinate” OR “propyl-propane-thiosulfinate” OR “organosulfur compound*” OR diallyl* OR allicin OR alliin OR ajoene OR “dipropyl disulphide” OR “dipropyl sulphide” OR propiin) AND (toxicity OR cytotoxicity OR genotoxicity) AND (“in vitro” OR “cell line”). The searches included works published in all languages. The Web of Science database option search was “theme” in all databases. The Scopus database options search were: “title, abstract and keywords”. The Science Database option search was “all fields except full text (NOFT)” and the PubMed option search was “all fields”. 2.4. Study Selection Once the selection criteria have been established, a three-step process was performed to review all records according to the eligibility criteria: first was reading the title, second, the abstract, and third, the entire text of the publication. The works obtained by the four databases were crossed with the EndNote X9 (Bld 12062) software to identify possible duplicates and to classify the works according to the exclusion and inclusion criteria. Two authors (PA-M and AC-L) formed the review team to implement measures to reduce Foods 2022, 11, 2620 5 of 25 random mistakes and bias at all review phases and independently examined titles, abstracts and full texts of the articles for possible addition. Conflicts on whether a given reference should be incorporated or not were determined through discussion. Figure 1. PRISMA flowchart of article selection. 2.5. Data Extraction and Data Items The data items included for data extraction were: Assays performed, experimental model, concentration ranges and time exposure, and main results. This data extraction form is presented in Table 2. 2.6. Risk of Bias in Individual Studies Bias can be judged to be a systematic mistake that can lead to an underestimation or overestimation of the true result [53]. The risk of bias for each incorporated work was evaluated using The Methods Guide for Comparative Effectiveness Reviews [54]. The characteristics of bias considered are shown in Table 3. Each risk of bias question scored 2 (fully reported), 1 (partially reported) or 0 (unclear/not reported). Scores for each risk of bias question were added together to give a total score between 0 and 10. A score of 0–4 was considered a high risk of bias; 5–7 was considered a moderate risk of bias; 8–10 was considered a low risk of bias. The risk of bias assessment was performed by the reviewer team. Differences were resolved by discussion between the three reviewers (AC-L, AC and AIP). 2.7. Results Construction and Statistical Analysis Compiled information and findings in the studies were examined independently by the reviewer team in order to find conflicts in the extracted data, should they be present. Foods 2022, 11, 2620 6 of 25 3. Results 3.1. Search Results The procedure of article selection is shown in the flow chart of Figure 1. The research database Web of Science returned 461 works; Scopus returned 190 works; Science Database returned 9 works and PubMed returned 287 works. The 947 works found by the four databases combined were crossed with the EndNote X9 (Bld 12062) software to identify possible duplicates. A total of 307 works were eliminated in this stage. After evaluating the abstract of each of the remaining articles, those that were associated with the subject of the study (43) were selected. According with the flow diagram (Figure 1), some articles were excluded since they had no toxicological importance, e.g., “Effect of allicin on promastigotes and intracellular amastigotes of Leishmania donovani and L. infantum”. It should be noted that some of the studies could be introduced in more than one exclusion group but the final criterion was agreed by discussion of the review team. Finally, a total of 43 works were found to be eligible for the present systematic review following the full-text eligibility assessment. 3.2. Study Characteristics and Results of Individual Studies The characteristics and main findings of the 43 selected articles in the present systematic review, such as Assays performed, experimental model, concentration ranges and time exposure and main results, are summarized in Table 2. Thus, the main tests carried out have focused on investigating cytotoxicity, cell apoptosis and ROS production against antioxidant assays. The MTT assay highlights how to determine the EC50 of these compounds in different cellular models. The flow cytometry is used for different determinations such as apoptosis, cell cycle, mitochondrial membrane potential and ROS. On the other hand, assays related to the mutagenicity and genotoxicity showed less interest. In this sense, Ames test, MN, comet assay and MLA have been performed. Regarding the experimental models, different cell lines have been used. Melanoma, lymphoma and gastrointestinal tract cells were the ones most often selected (Figure 2). The 43 selected articles were published between 1990 and 2021. According to the corresponding authors addresses, the articles were published in 19 different countries: 8 in China, 6 in Korea, 4 in USA, 3 in Spain, 3 in Poland, 2 in India, 2 in Taiwan, 2 in Germany, 2 in UK, 2 in Venezuela, 1 in Brazil, 1 in Israel, 1 in Egypt, 1 in Japan, 1 in Iran, 1 in France, 1 in Croatia, 1 in Turkey and 1 in Italy. The 43 included articles were published in 33 different journals, Food and Chemical Toxicology being the journals with most publications. 3.3. In Vitro Studies of OSCs from Allium spp. Focused on Safety Evaluation for Agri-Food Application Of the 43 studies selected, only 4 have been performed specifically to explore the safety of OSCs from Allium spp. for agri-food applications [16,17,55,56] (see Table 2). Mellado- García et al. [17–55] studied the genotoxicity potential of PTSO and PTS using in vitro test battery including Salmonella typhimurium reverse mutation assay (Ames Test), The cytokinesis-block micronucleus cytome assay (MN) and single cell gel electrophoresis assay (comet assay). Both compounds reported negative results by Ames Test and genotoxic effects were described in similar concentration for PTSO and PTS by MN assay. However, MLA and comet assays showed contradictory results between these compounds. No cytotoxicity or mutagenicity of DPS, DPDS, and mixtures were reported by Llana-Ruiz- Cabello et al. [16]. In relation to polymer films of Allium cepa L., no induction of MN was observed, although the HTP films showed signs of mutagenicity by the Ames Test [56]. Foods 2022, 11, 2620 7 of 25 Figure 2. Graphical model: (A) reported beneficial health effects, (B) in vitro toxicological evaluation and (C) reported results. Foods 2022 , 11, 2620 8 of 25 Time Exposure ↓ ± strains TA98 or TA100 Allium Products (Pure Compound or Extract) Table 2. Overview of the studies reporting the in vitro toxicological evaluation of OSCs from Allium spp. Assays Performed Experimental Model Concentration Ranges and Main Results Reference Ajoene Allicin Ajoene DDS DAS Survival by MTT assay Total cell protein Metabolic activity by MTT assay Cell death by trypan blue assay Levels of GSH, GSSG and acidic aminoacids Glu and Asp Clonal survival Mass growth rate. Anchorage-Independent Growth. FS4 BHK21 BJA-B BJA-B cells Control and differentiated HT29 cells 5–20 µg/mL for 48 h. 173 and 82 nmol/mL for 0–6 h. 385 and 150 fmol/cell for 48 h. 1–3 µg/mL DDS 24 h 100– 300 µg/mL DAS 24 h after which the cells were incubated in fresh medium for 10–12 days. Cells were exposed to 24-h intervals for seven days. EC50(ajoene): FS4 (36 µM) > BHK21 (30 µM) > BJA-B (12 µM). ED50 (ajoene): FS4 (450 fmol/cell) > BHK21 (190 fmol/cell) > BJA-B (85 fmol/cell). ED50(allicin): FS4 (720 fmol/cell) > BHK21 (430 fmol/cell) > BJA-B (185 fmol/cell). In both experiments, cell viability in a dose and time dependent manner. In the first minutes after exposure, GSH decreased and GSSG increased. The further course strongly depended on the dose. The Glu pool showed an immediate increase and in a later stage decreased. The Asp pool showed the contrary pattern. Dq (concentration at which no cell killing occurs) 0.10 0.03 µg/mL for DAS and was not found for DDS. D37 (dose required to reduce survival from 100% to 37%) are 2.93 ± 0.14 µ g/mL for DAS and 164 ± 12 µ g/mL for DDS. Cytotoxicity DADS > DAS. [57] [58] [59] Clonal survival DAS, DADS Induction of chromosome aberrations Induction of SCEs CHO 100–600 µg/mL DAS 2–10 µg/mL DDS Ames test was performed with S9 Both compounds induced chromosome aberrations and SCEs (DADS > DAS) +S9: reduction of the induction of SCEs by both compounds, and enhanced the generation of aberrations by DADS. DAS, DPS, DPDS: increased activation of BaP, CP, N-PiP and [60] DAS, DADS, DPS, DPDS Ames test Ames test: Salmonella typhimurium Cytotoxicity by MTT assay and microsomes from DAS-, DADS-, DPS-, DPDS-treated rats (1 mmol/Kg) PhIP, while DADS only increased mutagenicity of PhIP. In contrast, some OCS inhibited the mutagenicity of different mutagens, while other enhanced it. The cell growth was significantly inhibited by DAS and DADS [61] DAS, DADS, garlic extract DADS Western blot analysis of Bcl-2, Bax and p53 expression Northern blot analysis Apoptosis by acridine orange staining Cell viability and apoptosis by flow cytometry Oxidative stress (carbonylated proteins, MDA) Western blot analysis p-53-wild type H460 and p-53-null type H1299 non-small cell lung cancer cells (NSCLC) SH-SY5Y DAS and DADS (0–25 µM) and garlic extract (0–200 µg/mL) for 1 h 50 µM for 12 and 24 h 50 µM up to 2 h and slightly inhibited by garlic extract. The OSCs compounds and garlic extract have apoptotic potential on lung cancer cells, and the mechanism was regulated through p53-dependent or p-53 independent related Bax/Bcl-2 dual pathway. Blockage in G2/M phase DADS induced a ROS-mediated activation of JNK/c-Jun pathway in neuroblastoma cells, and this activation led to apoptosis. [62] [63] Foods 2022 , 11, 2620 9 of 25 Exposure ↑ Allium Products (Pure Compound or Extract) Table 2. Cont. Assays Performed Experimental Model Concentration Ranges and Time Survival by MTT assay Apoptosis by flow cytometry 10–200 µM for 2 h. 10, 25 µM for 2 h pretreatment and treated with 100 µM H2O2 for 30 min. Main Results Reference Cellular viability was not affected up to 25 µM DAS. apoptotic cells at 100 µM of treatment and at 25 µM of pretreatment versus H2O2 treatment in these cells. ↑ the expressions of p85a PI3K, phosphorylated Akt and DADS Cell signals by western blot analysis of phosphorylated forms of GSK-3β and Akt, and p85a PI3K Free radical levels and membrane lipid peroxidation Cell proliferation by MTT assay Morphological apoptosis DNA fragmentation assay N18D3 25, 100 µM for 2 h treatment with and without post-treatment of 100 µM H2O2 for 30 min. phosphorylated GSK-3 in N18D3 cells pretreated with 25 µM (2 h) and subsequently exposed to 100 µM H2O2 (30 min). Treatment with 100 µM reduced these biomarkers in N18D3 cells. the levels of free radicals and membrane lipid peroxidation a concentration-dependent manner. Allicin caused inhibition of cellular growth in a concentration- and time-dependent manner. DNA fragmentation and morphological changes (degeneration of neuritis, shrinkage of cell bodies and condensation of nuclei) in cells exposed to 5–20 µg/mL for 24 h. [64] Allicin Cell cycle by flow cytometry Caspase-3 determination Expression of Cyt c, Bax and AIF by Western blot analysis AGS 5–100 µg/mL for 6, 12, 24 and 48 h. 45.2% apoptotic cells. in the sub-G1 DNA content. in the proportion in cells of S phase after exposure to 20 µg/mL of allicin for 24 h. Allicin results in the release of Cyt c and in increase of mitochondrial Bax protein level. Caspase-3 activation and cleavage of PARP were not detected. [65] Ajoene MTS/PMS chromogenic assay Cell adhesion assay B16/BL6 HT-29 A549 MDA-MB-231 PANC-1 SKBR-3 NIH 3T3 3T3/HER2 Splenocytes B16/BL6-LEC1 10–100 µM for 24 h 1–100 µM for 24 h IC50(B16/BL6) = 18 µM IC50(HT-29) = 19 µM IC50(A549) = 41 µM IC50(MDA-MB-231) = 7 µM IC50(PANC-1) = 38 µM IC50(SKBR-3) = 19 µM IC50(NIH 3T) = 17 µM IC50(3T3/HER2) = 9 µM IC50(Splenocytes) ≥100 µM B16/BL6 29% inhibition at 10 µM [66] ↑ ↑ ↓ Foods 2022 , 11, 2620 10 of 25 Time Exposure ↓ ↓ ↓ ↓ ↑ ↓ Allium Products (Pure Compound or Extract) Table 2. Cont. Assays Performed Experimental Model Concentration Ranges and Cell viability by trypan blue exclusion assay Wright staining and ApopTag assay for apoptosis ROS production Main Results Reference DAS, DADS, DATS Intracellular free [Ca2+] by Fura-2 assay mRNA expression of β-actin, bax, bcl-2, calpastatin phosphorylation of stress kinases analysis Western Blot analysis analysis Mitocondrial membrane potential Caspase 3 and 9 activity GST activity T98G U87MG 100 µM DAS 100 µM DADS 25 µM DATS for 24 h The three garlic compounds induced cytotoxic effects via ROS production, increase in endoplasmic reticulum (ER) stress, decrease mitochondrial membrane potential, activation of stress kinases and cysteine proteases. Depolymerizing effect of allicin in a concentration-dependent [67] In vitro tubulin polymerization assay and image analysis Cell viability by MTS assay NIH-3T3 0.2–25 µM manner until 25 µM. Disruptive effect of allicin increases with the duration of incubation. 5 and 10 µmol/L cell viability and this cytotoxic effect was not prevented by the addition of mevalonate or GGPP. Ajoene (5 µmol/l) in combination with atorvastatin [68] Ajoene (≥98%) DADS Apoptosis by flow cytometry Microscopic evaluation Cell viability by MTT assay Apoptosis by fluorescence microscopy and flow cytometry p-ERK and ERK protein levels by Western blot analysis Cell viability by trypan blue exclusion assay ALP and LDH assays ROS generation B16F10 1, 5, 10 µmol/L for 24 h 50–400 µmol/l for 24 h. MCF-7 200 µmol/l for 6, 12, 24 and 48 h. 5–75 µM for 0–24 h (0.1 mmol/L) or pravastatin (0.1 mmol/L) cell viability in a synergistic way. Apoptosis observed by diminution in cell volume, condensation of cytoplasm. cellular viability and apoptosis in concentration-dependent manner. These effects were observed mainly from 200 µmol/l of treatment. Inhibition of ERK and activation of SAPK/JNK and p38. 50 µM cell viabilityALP and LDH decreased with time. ROS increased.~55% apoptosis. Cell cycle arrest at G2/M [69] [70] DAS DADS Apoptosis by flow cytometry analysis Cell cycle analysis DNA fragmentation analysis Immunoblot analysis of caspase-3, NF-κB, ERK-2 MTT assayApoptosis by flow cytometry, DNA fragmentation assay and morphology analysisWestern blot analysis of the expression of phosphor-MAPKs (ERK, p38) Colo 320 DM HL-60 50 µM for 6, 12 h 50 µM for 12 h 50 µM for 12 h 1.25–20 mg/L for 96 h 5–20 mg/L for 24 h 10 mg/L for 24 h Oligonucleosomal-laddering, characteristic of apoptosis.Higher protein expression of caspase-3 and NF-κB and lower expression of ERK-2. 10 mg/L 59.6% cell viability Apoptosis was evidenced in a concentration-dependent manner by different assays. Inhibition of ERK and activation of p38 [71] [72] Allicin Foods 2022 , 11, 2620 11 of 25 Time Exposure ↑ ↑ Allium Products (Pure Compound or Extract) Table 2. Cont. Assays Performed Experimental Model Concentration Ranges and Main Results Reference DATS n-DADS s-DADS DATS DAS DATS MTT assay Apoptosis by flow cytometry Expression of Bcl-2, Bax, Bcl-xL/Bcl-xS, Cyt c, caspase 9 and poly(ADP-ribose) polymerase by western blotting. Activity of caspase 3 MTT assay Cell cycle analysis Apoptosis by flow cytometry and by fluorescence microscopy after staining with Hoechst 33,258 Cell viability by trypan blue assay ROS by fluorescence microscopy and flow cytometry Mitochondrial ROS levels assay Immunoblot analysis Cell viability, cell cycle and apoptosis by PI staining by flow cytometric assay DNA damage by Comet assay and DAPI staining Flow cytometric assay for the production of Ca2+ and the level of mitochondrial membrane potential Western blot of apoptotic associated proteins Microarray assay Comet assay Apoptosis and ROS by flow cytometry Immunoblotting for phosphoolorect-ERK1/2 (P-ERK1/2 A375 M14 MCF-7 MDA-MB-231 HeLa PC-3 cells transfected with the plasmid encoding p66ShcS36A or an empty pcDNA3.1 vector 5–60 µM for 24, 48, 72 h Exposure to IC50 for 72 h: A375 11.7 µM and M14 14.1 µM IC50 for 24, 48, 72 h IC50 for 16, 24, 36, 48 h 0.01–2.00 mmol/L 48 for 72 h. 0.01, 0.05, 0.25 mmol/L for 48 h. 0.05 mmol/L for 48 h, microscopy 10–100 µM for 16 h 10–80 µM for 1 h 10, 50 µM for 1 h 10–100 µM for 16 h 25–100 µM for 24 h 75 µM for 0–2 h 75 µM for 0–72 h 5 µM DAS for 24 h After 24 of transfection, cells were treated with DATS (0–40 µM) IC50(A375) = 11.7 µM IC50(M14) = 14.1 µM percentage of apoptosis Reduced Bcl-2 and Bcl-xL expression Increase in caspase-3 activity with time s- and n-DADS present concentration- and time-dependent inhibitory effects and similar cytotoxicity in MCF-7 cells. Apoptosis from 0.01 mmol/L for 48 h. The percentages of cells in G0/G1-, S- and G2/Mphase did not differ from each other. Apoptotic cell death in concentration- and time-dependent manner was observed with cell shrinkage and cytoplasmic membrane blebbing. ROS with activation of ASK1 and a downstream signal transduction JNK (C-Jun N-terminal kinase)-Bim pathway at 50–80 µM. DNA damage and fragmentation. Induced apoptosis and decreased the viability in concentration- and time-dependent manner. Induced cell cycle arrest increasing G0/G1 cell population and decreasing G2/M and S cell population. Decreased levels of mitochondrial membrane potential and promoted the levels of Ca2+ DAS promoted the levels of Fas, FasL and caspase-8, Bax, cytochrome c, Apaf-1, Bid, caspase-9 and -3. 28 genes were expressed at least by 2-fold compared with the untreated control cells. DATS increased p66Shc phosphorylation at serine 36, which was abolished by JNK inhibitor, and DATS-induced ROS formation was abolished in cells expressing p66ShcS36A variant. In cells expressing this variant, DATS-induced Akt dephosphorilation was reduced. The signaling pathway with P66Shc could be indispensable for DATS-induced prostate cancer cell death by modulating the Akt activity and ROS generation. [73] [74] [75] [76] [77] Foods 2022, 11, 2620 18 of 25 43 studies have been performed specifically to explore the safety of OSCs for further agrifood applications [16,17,55,56] Regarding results obtained for specific OSCs, DADS in relation to genotoxicity aspects has been reported to induce chromosome aberrations and sister chromatid exchanges in a Chinese hamster’s ovary cell line (CHO) [60]. Additionally, it increased the mutagenicity of 2-amino-1-methyl-6-phenylimidazol [4,5-b] pyridine when the Ames test was performed with S9 fraction from rats exposed to DADS. Filomeni et al., [64] and Kim et al. [65] explored the effects of DADS on two different cellular models of the nervous system and obtained different results. Whereas the first one suggested a pivotal role for oxidative stress in DADS-induced apoptosis on SH-SY5Y cells and pointed out a potential use as antiproliferative agent in cancer therapy, the second one observed opposite results on N18D3 cells, depending on the concentration used, with a protective effect at low concentrations. Recently, important toxic effects have been reported of this compound associated to high doses [105]. For these reasons, more DADS toxicity studies are necessary to guarantee its safe use as an anticancer agent. Two other studies [70,75] investigated its effects on a breast cancer cell line (MCF-7) and obtained similar results, inhibition of cell proliferation and apoptosis induction, with Lei et al. [70] providing mechanistic clues (inhibition of ERK and activation of SAPK/JNK and p38 pathways). Apoptosis was also observed in other different cell types where the antiproliferative effects of DADS were investigated such as p53-wild type H460 and p53- null type H1299 non-small-cell lung cancer cells [63], in human glioblastoma cells [68], human leukemia cells [72], human esophageal carcinoma cells [84,85] or primary effusion lymphoma cells [87]. All these reports support the potential use of DADS as chemotherapeutic agent. Results reported for DATS are similar to those discussed for DADS, as DATS have also shown to induce cytotoxicity, ROS production or apoptosis (also evidenced by changes in the expression of related genes and proteins) in different cell types such as NIH-3T3, MCF-7, PC-3, AGS, U87M6, PNT-1A, MDA-MB468, MDA-MD231, A172, U343 and T98G. Only Das et al. [68] and Shigemi et al. [87] evaluated both DADS and DATS, and compared the results obtained. Both of them observed similar results: DATS was more potent than DADS and DAS for induction of cell death with involvement of mitochondria and ROS production. With respect to DAS, it induced genotoxic effects similarly to DADS [60,62], but it was less cytotoxic to CHO cells and it increased the activation of a higher number of mutagens. Again, several studies in a variety of cell lines showed its antiproliferative effect [59,63,68,71,77,87]). It has been reported that garlic compounds (DAS, DADS, DATS) do not require a p53-dependent pathway for mediation of apoptosis [68]. Moreover, its potential to reduce the tissue injury caused by ethanol was also demonstrated [92]. In order to compare the effects produced by each of these compounds, differences have been found, mainly due to the diverse experimental models and conditions used (concentration, time of exposure, biomarkers, etc.). Studies evaluating allicin mainly reported cytotoxicity [57], and apoptosis [66,83,86,90] as adverse outcomes, mediated by different key events such as altered genes and protein expression or cell cycle changes. Scharfenberg et al. [57] were the only ones that studied not only allicin but also its decomposition product ajoene and observed that allicin was less toxic than ajoene in three different cell lines. This compound, ajoene, was also investigated by different authors [58,67,73] that observed cytotoxicity (with different cell lines showing different sensitivity) and apoptosis. Regarding cytotoxicity assays, there are only a few cell lines listed in these studies that are included in the guidelines by OECD guidance for toxicological evaluation of chemicals. Most of them are cancer cell lines, and the effects reported in these findings correspond to a therapeutic anticancer effect and not to a cytotoxic evaluation. In this sense, more studies focused on the toxic effect of OSCs isolated are necessary. Moreover, those OSCs with the fewer number of studies available in the scientific literature were also those mainly focused on safety issues in relation to their use in the Foods 2022, 11, 2620 19 of 25 agri-food sector. Thus, neither mutagenicity by the Ames test nor cytotoxicity in the human intestinal carcinoma Caco-2 cell line was observed for DPS and DPDS [16]. Additionally, a complete battery of genotoxicity tests were performed for PTSO [17] and PTS [55]. Genotoxicity assessment plays a key role in the safety evaluation required by EFSA guidelines for the submission of dossiers of different substances, such as food and feed additives, etc. [48,50], with the basic battery performed with in vitro tests. However, results of this review showed that only few assays have been carried out and some studies do not include the basic battery of tests required by the EFSA. In this regard and taking into account that specific OSCs that have been mainly investigated for their chemotherapeutic potential such as DADS, DATS, DAS, etc., show also interesting activities for their use in the food industry (antimicrobial, antioxidant or antifungal activities, among others, see Table 1), the thorough study of their genotoxicity would be worthy of research. Moreover, advanced in vitro models (i.e., 3D) could provide new data to support in vitro-in vivo data extrapolation for OSCs in general, and the testing of relevant concentrations used in the agri-food sector would allow to consider both efficacy and safety aspects. Thus, in vitro assays on their own can still provide valuable information to contribute to the commercial use of OSCs. 5. Conclusions In general, there are very few in vitro studies focused on investigating the potential toxicity of OSCs. Most research studies aimed at evaluating only the cytotoxicity of OSCs on different cellular models to elucidate antiproliferative effects of these compounds and justify their potential as chemoprotective agent against carcinogenesis. This makes it difficult to assess the safety of the use of these compounds for a correct risk assessment. In addition, it limits the preliminary information needed to proceed with an in vivo toxicity assessment. Therefore, other cellular models such non-cancer cell lines should be included to ensure a correct in vitro toxicity evaluation of these compounds. Specifically, considering that genotoxicity assessment plays a key role in the safety evaluation required by EFSA; more genotoxicity studies of OSCs are necessary to guarantee consumer safety before their use as a potential natural additive in the food industry. Author Contributions: Conceptualization, A.C.-L., A.B., E.G., A.J. and A.M.C.; methodology, A.C.-L. and P.A.-M.; formal analysis, A.C.-L., P.A.-M., A.I.P., A.J. and A.M.C., investigation, A.C.-L., P.A.-M., A.I.P., A.J. and A.M.C.; data curation, A.C.-L., P.A.-M., A.I.P., A.J. and A.M.C.; writing—original draft preparation, A.C.-L., P.A.-M., A.I.P., A.B., E.G., A.J. and A.M.C.; writing—review and editing, A.C.-L., A.I.P., A.B., E.G., A.J. and A.M.C.; supervision, A.J.; project administration, A.M.C.; funding acquisition, A.M.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Consejería de Economía y Conocimiento of Junta de Andalucía (AT17_5323_USE and P18-TP-2147) and Antonio Cascajosa Lira thanks the Spanish Ministerio de Universidades for the funding FPU grant (FPU2019/01247). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study is contained within the article. Acknowledgments: The authors wish to thank the Consejería de Economía y Conocimiento of Junta de Andalucía (AT17_5323_USE and P18-TP-2147) for the financial support. Antonio Cascajosa Lira thanks the Spanish Ministerio de Universidades for the funding FPU grant (FPU2019/01247). Conflicts of Interest: The authors declare no conflict of interest. The co-authors Alberto Baños and Enrique Guillamón affiliated to DMC Research Center declare that they have no conflict of interest. Abbreviations 3T3-HER2: Mice fibroblast cell line; 3T3-L1: Mice fibroblast cell line; 5-Fu: 5-fluorouracil; A172: Human brain cell line; A375: Human melanoma cells; A549: Adenocarcinomic human alveolar basal Foods 2022, 11, 2620 20 of 25 epithelial cells; AGS: Human gastric carcinoma cell line; AIF: Apoptosis inducing factor; AMPK: AMP-activated protein kinase; ALP: Alkaline phosphatase activity; Asp: Aspartate; ATRA: All-trans retinoic acid; B16/BL6: Melanoma mouse cell line; B16F10: Murine melanoma cell; BC2: Human hepatoma cell line; BC3: Primary effusion lymphoma cell line; BCBL1: Human lymphoma cell line; BGC-823: Human gastric cancer cell line; BHK21: Baby hamster kidney-derived cell line; BJA-B: Burkitt lymphoma-derived cell line; Caco-2: Human colorectal adenocarcinoma cell line; CHO: Chinese hamster ovary cells; CRC: Human colon cancer cell line; CTX: Cyclophosphamide; Cyt c: Cytochrome c; DADS: Diallyl disulfide; DAS: Diallyl sulfide; DATS: Diallyl trisulfide; DLD-1: Colorectal adenocarcinoma cell line; DG75: Human lymphoma cell line; DPS: Dipropyl sulphide; DPDS: Dipropyl disulfide; ECA109: Human esophageal carcinoma cell line; ED50: amount of test compound required to reduce cell viability to 50%; ERK: Stress-activated protein kinase extracellular signalregulated kinase; ERK-2: Extracellular regulatory kinase-2; EROD: Ethoxyresorufin O-deethylase; FHC: Normal human colon cell line; FS4: Human foreskin-derived cell line; GGPP: Geranylgeranylpyrophosphate; Glu: L-glutamate; GM-07492: Primary human skin fibroblasts; GSH: Reduced Glutathione; GSH-Px: Glutathione peroxidase; GSK-3: Glycogen syntethase kinase-3; GSSG: Oxidized glutathione; HBL6: Human lymphoma cell line; HEL: Human erythroleukemia cell line; HeLa: Cervical cancer cell line; HepG2: Human liver cancer cell line; HL-60: Human leukemia cell line; HT29: Human colorectal cell line; HTC116: Human colon cancer cell line; Http: Unwashed hydrothermally treated pulp; JNK/c-Jun: c-jun terminal kinase; M14: Human melanoma cell line; MAPKs; Mitogenactivated protein kinases; MCB: Minimal Bactericidal Concentration; MCF-7: Human breast cancer cell line; MDA: Malondialdehyde; MDA-MB-231: Human breast cell line; MDA-MB-468: Human breast cell line; MFC: Minimum fungicidal concentration; MIC: Minimal Inhibitory Concentration; MGC-803: Human gastric cancer cell line; MLA: Mouse lymphoma thymidine-kinase assay; MMP: Mitocondrial membrane potential; MN: Micronucleus; MROD: Methoxyresorufin O-demethylase; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NF-κB: Nuclear factor enhancing the kappa light chains of activated B cells; NIH-3T3: Mice fibroblasts cell line; NR: neutral red; L02: Human normal liver cell line; LDH: Lactate dehydrogenase activity; LEC1: Leuco-phytohemagglutinin resistant cell line; OCIM-1: Human leukemia cell line; p38: Mitogen-activated protein kinase; PAGE: Protein extracts and polyacrylamide gel electrophoresis; PANC-1: Human pancreatic carcinoma; PC: Protein content; PC-3: Human prostate adenocarcinoma cell line; PI3K/Akt: Phosphatidylinositol 3-kinase; PNPH: p-Nitrophenol hydroxylase; PROD: pentoxyresorufin O-dealkylase; PTS: Propyl-propane thiosulphinate; PTSO: Propyl-propane thiosulphonate; ROS: Reactive oxygen species; Ramos: Epstein-Barr virus-negative Burkitt lymphoma cell line; RAW 264.7: Mouse macrophage cell line; ROS: reactive oxygen species; RT-PCR: Quantitative realtime polymerase chain reaction; SAPK: stress-activated protein kinase; SCEs: sister chromatid exchanges; SGG-7901: Human gastric cancer cell line; SiRNA: small interfering RNA; SKBR-3: Human breast cancer cell line; SOD: Superoxide dismutase; SW620: Human colorectal cancer cell line; T98G: Glioblastoma cell line; TRAIL: Tumor necrosis factor-related apoptosis-induced ligand; U251: Human glioma cell line; U2OS: Human osteosarcoma cell line; U343: Human glioblastoma cell line; U87MG: Human glioblastoma cell line http:HTP: Washed hydrothermally treated pulp. 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Dutta, A.; Dahiya, A.; Prakash, A.; Agrawala, P.K. Acute toxicity of diallyl sulfide derived from Allium sativum (garlic) in mice and its possible mechanisms. Phytomed. Plus 2021, 1, 100084. [CrossRef] CAPÍTULO 2 / CHAPTER 2 Antonio Cascajosa Lira, Ana Isabel Prieto, Silvia Pichardo, Ángeles Jos, Ana M. Cameán. Protective effects of sulforaphane against toxic substances and contaminants: A systematic review This article was published in Phytomedicine, 130, Antonio Cascajosa Lira, Ana Isabel Prieto, Silvia Pichardo, Ángeles Jos, Ana M. Cameán, Protective effects of sulforaphane against toxic substances and contaminants: A systematic review, 155731, Copyright Elsevier (2024) 119 A. Cascajosa-Lira et al. Table 1 (continued ) Phytomedicine 130 (2024) 155731 (continued on next page) 6 Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference with the modulation of the intrinsic Nrf2/ARE and NF- κ B pathway. Cisplatin 500 µ g/kg/d for 3 days Cuprizone (CPZ) 2 mg/kg/day for two weeks Rats were pretreated i.p. with SFN (500 µ g for 3 days) and then exposed to cisplatin (single i.p. injection, 10 mg/kg) and sacrificed 3 days later. Rats were fed a CPZ-contained diet (0.2 %) for four weeks after which they were SFN i.p. administrated (2 mg/kg/day) for two weeks. Wistar rats SFN prevented cisplatin-induced liver damage and attenuates histopathological alterations of the ALT and AST enzymes observed in exposed rats. SFN protected against cisplatininduced oxidative stress in the rat liver by reversing the increase in LPO and protein carbonyl content and the decrease in GSH and antioxidant enzymes (CAT, SOD, GPx, GR). SFN has a high scavenging capacity for free radicals such as peroxynitrite anion, superoxide anion, singlet oxygen, and peroxyl radicals, and prevented mitochondrial alteration of oxygen uptake and decreased complex I activity. Wistar rats SFN treatment reversed all the biochemical alterations produced by CPZ such as ALT and AST. In addition, SFN produced in decrease a LPO and enhanced T-AOC levels and CAT activities in rats pretreated with CPZ, reducing CPZ-induced oxidative stress in hepatic tissue. Gaona-gaona et al., 2011 Fouad et al., 2023 D-galactosamine (GalN) and lipopolysaccharide (LPS) 3 mg/kg The rats were divided into 4 groups: control, SFN treated and 2 groups i. p. injected with GalN (300 mg/kg) and LPS (30 μ g/kg), one of them also exposed i.p. to SFN. Male Wistar rats SFN provided protection against GalN/LPS-induced fulminant liver failure. This was demonstrated with reduced mortality rates, reduced serum AST and ALT activities, restored SOD, CAT, GPx and Oh-1 activities, and improved pathological liver changes. Sayed et al., 2014 Ethanol In vitro: 6 μ M In vivo: 0.05 g/kg In vitro: cells were treated with or without 100 mM ethanol in the absence or presence of SFN. In vivo: mice were gavaged twice a day with 30 % ethanol at a dose of 3 g/kg b.w. for 5 days. Some mice were injected i.p. with SFN, at the dose of 0.05 g/kg once a day for 5 days. In vitro: HepG2, E47 cells In vivo: SV129 humanised male CYP2E1 knockinknocking mice In vitro SFN elevated Nrf2 levels and decreased lipid accumulation in cells cultured with ethanol. In vivo, SFN activated Nrf2, increased levels of Nrf2 target heme oxygenase- 1 and decreased oxidant stress as shown by the decrease in LPO and 3- nitrotyrosine protein adducts and increased GSH levels. It decreased the levels of triglycerides and cholesterol, and Oil Red O staining. It proved to be an effective in vivo inhibitor of acute ethanol-induced fatty liver in mice. Similarly, in vitro, SFN elevated Nrf2 levels and decreased lipid accumulation in cells cultured with ethanol. Zhou et al., 2014 Ethanol 0.1 ml/g b.w. SFN Six groups of mice were established: normal control group (A), model control (B), bifendate pill (BDP) intervention group (C,) aerobic exercise intervention group (D), SFN intervention group (E) and group treated with SFN integrated with aerobic exercise (F). Mice in groups A and B were intragastrically administrated with 50 % ethanol for 8 ml/kg b.w. for 12 weeks. From the fifth week, the intervention group C was given 0.1 ml/g b.w. of BDP before ethanol, while the same amount of SFN solution was given to intervention groups E and F; and group B received an equal amount of distilled water. The training time, in groups D and F, was 60 min a day, 5 days a week and 8 weeks in total. Mice SPF SFN administration has a stronger treatment effect than aerobic exercise alone, and the combination of SFN and aerobic exercise has the strongest protective effect on acute alcoholic hepatic injury in mice. The SOD activity in liver experienced significant increases and the MDA and triglyceride (TG) experienced significant decreases in the SFN group and the group treated with SFN administration integrated with aerobic exercise. VLDL in the serum of mice also experienced significant decreases in this latter group. Thus, both SFN and aerobic exercise can impose a certain protective effect on acute alcoholic liver injury in mice and help reducing the damage caused by alcohol in liver cells. Wang and Zhou, 2020 A. Cascajosa-Lira et al. Table 1 (continued ) Phytomedicine 130 (2024) 155731 7 Concentration or (H 2 O 2 ) Hydrogen peroxide (H 2 O 2 ) Lipopolysaccharide (LPS) concentrations of SFN and / or Selenium (Se) for different times. 5 µM Cells were exposed to several concentrations of SFN for 24 h and were treated with 700 µM H 2 O 2 for another 24 h. 0.13- 0.39 mg/kg Mice were treated with LPS (15 mg / kg i.p.) and with SFN at different concentrations (0.13 or 0.26 or 0.39 mg / kg i.v.) 12 h after LPS injection. TrxR-1 expression in HHL-5 cells. SFN can protect against H 2 O 2 -induced cell death, and this protection was also synergically enhanced with Se. By using siRNA to kill TrxR-1 or Nrf2, the protection of SFN (5 µ M) was reduced. SFN-induced TrxR-1expression was associated with significant levels of Nrf2 translocation into the nucleus. HHL5 and HepG2 cells Pretreatment with low concentration of SFN ( ≤ 5 µ M) reduced the cytotoxicity induced by H 2 O 2; moreover SFN reduced DNA damage caused by H 2 O 2 in HepG2 but not in HHL5 cells. The Nrf2 / GSH signaling pathways play a crucial role in the protective effects of SFN against H 2 O 2. Male c57BL/6 mice SFN decreased the mortality and the lethal liver injury previously increased by LPS at the highest concentration tested. SFN decreased serum levels of ALT and AST. Furthermore, SFN decreased the expression of inflammatory cytokines and the expression of the toll-like receptor 4 (TLR4) protein. Liu et al., 2019 Lee et al., 2020 Lipopolysaccharide (LPS) 2 µ M Cells were treated with SFN for 24 h and subsequently they were exposed to 1 μ g/ml LPS for 24 h. Hep G2 cells SFN suppressed hepcidin secretion LPS-induced transcription and interleukin-6 (IL-6) reducing the inflammatory responses in these cells without decreasing the cells viability. Al-Bakheit and Abu-Qatouseh, 2020 Microcystin-LR (MC-LR) 10 μ M HepG2, BRL-3A and NIH 3 T3 cells were treated with 10, 5 or 40 μ M MC-LR for 24 h with and without pretreatment with SFN (1 – 20 μ M) for 12 h. HepG2, BRL-3A and NIH 3 T3 cell lines SFN protects in a time- and concentrationdependent way against the cytotoxicity caused by MC-LR. SFN produced a significant increase in intracellular GSH levels in HepG2 cells, increased the number of cell colonies, and reduced cell death by apoptosis in all cell lines tested. Gan et al., 2010 Microcystin-LR (MC-LR) Olanzapine (OLZ) 5 μ mol/animal Mice were injected daily i.p. for 10 days with 40 and 50 MC-LR; with and without SFN (12 h prior to exposure to MC-LR). 90 mg/kg/d Mice were treated with 8 mg/kg/ d of OLZ by osmotic minipump for 4 weeks and SFN (90 mg/kg/d) by gavage starting 1 week prior to administration of OLZ. Male BALB/c mice SFN prevents MC-LR-induced hepatotoxicity and animal death in mice. In fact, it can block MC-LR- induced apoptosis. Furthermore, SFN stabilised Nrf2 in vivo and inhibited LPO and GSH reduction. C57BL/6 mice SFN can partially prevent the dysregulated glucose and lipid metabolism produced by OLZ together with a high-fat diet, representing protection against liver injury. SFN increased Nrf2 target gene (NQO1) and decreased OLZ-produced 4-HNE adducts produced by OLZ. Sun et al., 2011 Isaacson et al., 2020 Vanadium (VOSO4) 5 µ M Cells were exposed to VOSO4, SFN, and a mixture of both. HepG2, Caco-2, and Vero cells Intracellular ROS, DNA, and lysosomal oxidative damage underlined the indirect antioxidant activity of SFN, confirmed by the increase in GSH. SFN neutralised the metal-induced imbalance of redox homoeostasis Visalli et al., 2017 4-HNE: 4-Hydroxynonenal; ADH: aldehyde dehydrogenase; Akt: serine/threonine protein kinase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; APAP: Acetaminophen; Bax: bcl-2-like protein 4; BRL-3A: fibroblast-like cell isolated from the liver of a rat; Caco-2: human colon adenocarcinoma; CAT: catalase; galN: D -galactosamine; GPx: glutathione peroxidase; GR: glutathione reductase; GSH: glutathione; GST: glutathione-S-transferase; GSTM3: Glutathione S-transferases mu3; HepaRG: human hepatoma-derived cell line; HepG2: human liver cancer cell line; HMOX1: heme oxygenase-1; IL: interleukin; LPS: bacterial lipopolysaccharide; LPO: lipid peroxidation; LO2 cells: Human hepatocyte cells; LX-2: Hepatic stellate cells; MC-LR: Microcystin-LR; MDA: malondialdehyde; mTOR: mammalian target of rapamycin; NF- κ B: Nuclear factor kappa light chain enhancer of activated B cells; NIH 3 T3: embryonic mouse fibroblast cell line; Nrf2: nuclear factor erythroid 2- related factor 2; NOX1: NADPH oxidase 1; NOX4: NADPH oxidase 4; NQO1: NAD(P)H dehydrogenase [quinone] 1; OLZ: olanzapine; ROS: reactive oxygen species; SOD: superoxide dismutase; T-AOC: total antioxidant capacity; Tbil: Total bilirrubine; TLR4: toll-like receptor 4; TNF α : tumour necrosis factor alpha; Vero cells: monkey kidney epithelial cell line. Toxic substance Sulforaphane dose Exposure conditions Experimental model Main results Reference Hydrogen peroxide 2,5 – 80 µ M Cells were exposed to several HHL-5 cells SFN and Se synergistically induced Li et al., 2012 A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 8 Fig. 3. SFN acts as an indirect antioxidant through the Nrf2/ Keap1/ARE signaling pathway. Created with BioRender.com. CAT – Catalase; GSH – Glutathione; GPx - Glutathione Peroxidase; GR - Glutathione Reductase; GST - Glutathione S-Transferase; HMOX-1 - Heme Oxygenase-1; NQO1 - NAD(P)H Quinone Dehydrogenase 1; SOD - Superoxide Dismutase. Created with BioRender.com. Fig. 4. Mechanism of action of SFN in liver. Created with BioRender.com. hepcidin and IL-6; consequently, the inflammatory response and cancer progression could be inhibited (Al-Bakheit et al., 2020). Additionally, SFN has been able to repair DNA and lysosomal oxidative damage (Visalli et al., 2017). SFN has been shown to inhibit the catalytic activity in isolated microsomes (oxidation of para nitrophenol) and to block the genotoxicity of nitrosodimethylamine, a substrate for oxidation by CYP2E1. Therefore, Nrf2 activation coupled with possible inhibition of CYP2E1 was believed to make SFN an attractive chemical to blunt the toxic actions associated with CYP2E1 (Zhou et al., 2014) (Fig. 4). Regarding in vivo studies, SFN has been assayed in the following models: Wistar rats (mainly males), SFP mice, C57BL/6 J mice, Kunming mice, BALB/C mice. The doses tested varied widely from 500 µ g/kg to 100 mg/kg. These studies have investigated the effects of SFN on countering the toxic actions of d-galactosamine and lipopolysaccharide (LPS) (Sayed et al., 2014; Lee et al., 2019), drugs such as acetaminophen (Noh et al., 2015), cuprizone (Fouad et al., 2023), olanzapine (Isaacson et al., 2020) and cisplatinum (Gaona-Gaona et al., 2011); metalloids such as arsenic (Thanga-pandiyan et al., 2019), plasticisers such as bisphenol-A (Hong et al., 2023), metals such as cadmiun (he et al., 2021), ethanol (Zhou et al., 2014; Wang and Zhou, 2020), cyanotoxins such as Microcystin-LR (Sun et al., 2011). Similar to the in vitro findings, these toxics induced an inflammatory responses accompanied by prooxidant conditions in vivo models. Furthermore, MC-LR primarily elicits toxic effects in the liver due to the use of specific transporters. The primary route of administration for SFN was oral, with intraperitoneal (i.p.) injection being the secondary option. In vivo research validates the mechanism of action, with its primary attributes being the antioxidant and anti-inflammatory activities, which are responsible for the protective effects. When exposed to certain toxins, cells can become more vulnerable to increased permeability, leading to elevated levels of plasma enzymes from the liver such as alanine aminotransferase (ALT), aspartate aminotransferase (ASP), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total bilirubin, and gamma-glutamyl transferase (GGT). Furthermore, the intestinal membrane becomes more susceptible A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 9 to potential transfer of LPS from the intestinal microbiota to the liver, thereby triggering inflammatory processes. SFN can effectively decrease permeability, restore cell membrane integrity, and decrease the infiltration of enzymes and inflammatory substances. On the other hand, SFN could restore antioxidant enzyme levels when they are affected by toxics, primarily mediated by the increase in Nrf2 factor. Regarding histopathological studies, SFN-pretreated animals exposed to Cd show markedly attenuated abnormalities such as vacuolization, inflammatory cell infiltration, sinusoidal dilatation, and distinct damage of cytoplasmic organelles (He et al., 2021). Recently, Hong et al. (2023) reported that SFN demonstrated a mitigating effect on lipid metabolism disorder and stress of the ER induced by BPA in both in vitro and in vivo models. In vitro findings suggested that SFN reduced lipid accumulation in hepatocytes and lowered the levels of crucial lipogenic enzymes by inhibiting the ER pathways. However, in vivo, SFN did not cause a reduction in the liver/body weight ratio in animals treated with BPA. New research in order to elucidate the role of ER stress in vivo is needed. Neuroprotective effects of sulforaphane The main neuroprotective effects of SFN reported in the selected literature are reviewed in Table 2. Its efficacy has been demonstrated using both in vitro and in vivo models, in vitro models being the most common. The effects of SFN are mainly related with the antiinflammatory and antioxidant activity that prevents neuronal degeneration. Studies carried out in vitro have been performed by SFN exposure to several cell lines and primary cultures from different parts of the nervous system: SH-SY5Y (Lee et al., 2014; Zhou et al., 2016; Brasil et al., 2023), neural crest cells (Li et al., 2019), BV-2 cells (Konwinski et al., 2014; Wang et al., 2020; Yang et al., 2023), N2A cells (Kwak et al., 2007), primary microglial and cortical neurons (Yang et al., 2023), or primary astrocytes (Bergstrom et al., 2011). The in vitro protective effects of SFN are investigated against proteins such as amyloid beta (Yang et al., 2023) or prion (Lee et al., 2014), insecticides such as chlorpyrifos (Brasil et al., 2023) or rotenone (Zhou et al., 2016), herbicides such as paraquat (Mizumo et al., 2011), chemicals such as H 2 O 2 (Konwinski et al., 2004; Kwak et al., 2007; Bergmenton et al., 2011; Mizuno et al., 2011) and Lipopolysaccharide (Holloway et al., 2016; Wang et al., 2020). The majority of neurotoxic agents under investigation act as acetylcholinesterase (AchE) inhibitors, encompassing insecticides and herbicides. Additionally, research has also focused on toxics implicated in cognitive dysfunction, such as amyloid beta and prions. There is only one study using an ex vivo model, with organotypic cultured rat hippocampal tissue from rats exposed to scopolamine (SCOP) and SFN (Park et al., 2021). In this study, the authors showed that SFN exhibited a concentration-dependent increase in overall fEPSP (field excitatory postsynaptic potential) after high-frequency stimulation and mitigated the interference of SCOP-induced fEPSP in the CA1 area of the hippocampus. Furthermore, SFN prevented the long-term potentiation (LTP) and cognitive abilities induced by cholinergic and muscarinic receptor blockade. These findings indicate that SFN mitigates the decline induced by SCOP in short-term working memory, long-term spatial memory, and avoidance memory in rats. These effects are correlated with the induction of brain-derived neurotrophic factor (BDNF) and cAMP response element binding protein (CREB) expression in the hippocampus, along with the enhancement of synaptic activity. Therefore, SFN merits further investigation as a potential agent for preventing and treating Alzheimer ’ s disease (AD) or disorders related to learning and memory deficits in individuals affected by neurodegenerative disorders (Park et al., 2021) (Fig. 5). In terms of in vivo studies, only three different models have been used: C57BL/6 mice, Wistar and Sprague-Daley rats. The in vivo protective effects of SFN were investigated against 6-hydroxydopamine (Morroni et al., 2013), cis-platinum (Fouad et al., 2022), ethanol (Li et al., 2019; Xu et al., 2020), drugs such as pilcarpine (Folbergrov a ´ et al., 2023), and lipopolysaccharide (LPO) (Holloway et al., 2016; Wang et al., 2020). Similar to what has been studied in vitro, toxic agents studied in vivo also caused mainly cognitive and coordination dysfunctions. The protective effect of SFN in vivo has been shown to have 3 main pathways: 1) by antioxidant effects, 2) by anti-inflammatory effects, and 3) by improving motor coordination and reflexes. SFN exhibited a multifaceted protective role in various contexts. In the context of neurodegenerative oxidative damage to the basal ganglia, SFN restored nigral GSH levels and enhanced GST and GR, thus increasing the antioxidant potential (Morroni et al., 2023). Furthermore, SFN inhibited ROS production and MDA accumulation in mice with rotenone-induced dopaminergic neural loss (Zhou et al., 2016). Consequently, SFN could serve as a prevention of neurodegenerative processes such as Parkinson ’ s disease and improve locomotor activity. Furthermore, SFN exerted anti-inflammatory effects on LPS by suppressing NF κ B signaling, as reported by Wang et al. (2020). Additionally, SFN demonstrated its efficacy in mitigating alcohol-induced loss of righting reflex (LORR) duration without affecting latency, as observed in the study by Xu et al. (2020). Taking into account the in vitro and in vivo information reported, the entire mechanism of action is proposed in Fig. 5. Lastly, SFN is involved in the prevention of adverse effects caused by cis-platinum. SFN increased in AchE activity and restored redox status by regulating LPO, NO, and GSH levels (Fouad et al., 2022) after exposure to pilocarpine and LiCl. The protective effects of SFN extended to the hippocampal and the dentate gyrus, where it prevented damage induced by oxidative stress (Folbergrov a ´ et al., 2023). Nephroprotective effects of sulforaphane Table 3 includes the nephroprotective effects of SFN found in the scientific literature, in both in vitro and in vivo models. The antiinflammatory and antioxidant activities of SFN may be responsible of its efficient prevention of renal injury. In relation to this protective effect, more in vivo studies have been found than in vitro ones, in contrast to what has been found in the previous tissues. Only one in vitro study has been found that attributes protective effects of SFN using LLC-PK1 cells against the toxic effects of cis-platinum by restoring mitochondrial membrane potential ( Guerrero-Beltr a ´ n et al., 2010). The rest of the studies have been carried out in Wistar rats, mainly testing protective effects against metals and their compounds: cadmium (Li et al., 2015), mercury ( Guo, 2016), cis-platinum ( Guerrero-Beltr a ´ n et al., 2010 ; 2012) and calcium oxalate (Liu et al., 2012). The toxic effects induced by these metals at the renal level caused mainly renal failure, glomerulopathies, and tubulopathies. All the studies highlight a common mechanism of action: SFN restores the levels of urinary blood urea nitrogen (BUN) and restores the enzymatic activities of NAG, LDH, ALP, SOD, and GSH-Px. Additionally, because of its antioxidant effects, it is able to decrease the high levels of MDA produced by toxic substances. Cardioprotective effects of sulforaphane Table 4 presents a synthesis of the cardioprotective effects of SFN, including evidence derived from both in vitro and in vivo models. Table 4 consolidates the key findings from the selected studies to provide a comprehensive overview of SFN ’ s impact on cardiovascular health. These effects are mainly ascribed to the antioxidant activity of SFN, demonstrating its efficacy in preventing damage to the heart and aorta. There is only one in vitro study using primary cultured cells from rat. A mechanism of action against H 2 O 2 -produced oxidative stress has been demonstrated in which the presence of antioxidant enzymes is increased when cells are treated with SFN and estradiol (E2) (Angeloni et al., 2017). All the in vivo studies have been conducted in C57BL/6 J mice. The toxic substances assayed were as follows: cuprizone (CPZ), methamphetamine (MA) and angiotensin II (Ang II) to induce hypertension. A. Cascajosa-Lira et al. Phytomedicine 130 (2024) 155731 (continued on next page) 10 Table 2 Overview of studies reporting the protective effects of SFN in neuronal. Concentration or Amyloid-beta (A β ) and A β 25 – 35 fibrils 1 or 10 μ M Primary cells: treatment with different concentrations of either A β , or 50 μ M A β together with 1 μ M SFN for 3, 6, 12, or 24 h. BV-2 cells: 50 μ M A β with & without 10 μ M SFN for 30 h. Primary microglia and primary cortical neurones from 1-day-old C57BL/6 J mice. Mouse microglial cell line (BV-2) by 6-OHDA. SFN indirectly attenuated microgliamediated neurotoxicity. SFN inhibited the activation of cytostatic autophagy and the NLRP3 inflammasome, as well as the production of pro-inflammatory cytokines and the polarisation of M1. This is mainly related to the decrease in intracellular ROS in A β -activated microglia. Yang et al., 2023 Chlorpyrifos (CPF) 0.5 – 5 μ M SFN 6 h before exposure to 100 µ M chlorpyrifos for 3 or 24 h Cis-platinum 2 mg/kg The SFN was intranasally administered for 5 days followed by a single i.p. dose of 12 mg/kg/week of Cis-platinum on the sixth day, followed by 3 successive days of intranasally administration of SFN. Ethanol 1 μ M NCCs were pretreated with or without 1 μ M SFN for 24 h, followed by concurrent exposure to 1 μ M SFN and 50 or 100 mM ethanol. Ethanol 5 mg/kg SFN was i.p. administered (5 mg / kg / day, 5 consecutive days), and then 20 % EtOH v / v (1.75 g/kg for ataxia, 3 g/kg for hypothermia and 4.0 g/kg for LORR) was injected i.p. SH-SY5Y cells SFN prevented CPF-induced mitochondrial dysfunction and redox impairment. Anti-inflammatory effects stabilising IL-1 β levels. HMOX-1 mediates mitochondrial protection. Wistar rats The increase in AchE activity caused by Cisplatinum was inhibited by SFN which decreased LPO, NO, and GSH in the brain. Regressing lesions were found in the cerebral cortex of rats in which only sporadic necrosis of neurons was found. Neural crest cells (NCCs) SFN significantly reduced ethanol-induced apoptosis in NCC cells. SFN also decreased ethanol-induced changes in the expression of E-cadherin and vimentin, and restored EMT in NCCs exposed NCCs. SFN decreased ethanol-induced reduction of H3K4me3 in the promoter regions of the Snail1 gene, restored the expression of Snail1 and down-regulated Snail1 gene E- cadherin. Male C57BL/6 J mice SFN significantly shortened the duration of alcohol-induced LORR in mice but did not affect the latency. It reduced the alcoholinduced temperature change, while it did not affect the basal core temperature of the mice. In the ataxia test, SFN increased the latency of falling from the rotarod dowel after alcohol injection. However, it did not affect the basal coordination of the mice. SFN reversed CUS-potentiated alcoholinduced hypothermia and rescued the reduced alcohol induced ataxia. It increased CAT activity in the hippocampus of CUS mice. Brasil et al., 2023 Fouad et al., 2022 Li et al., 2019 Xu et al., 2020 Hydrogen peroxide (H 2 O 2 ) Hydrogen peroxide (H 2 O 2 ) 1- 10 µ M SFN: briefly (1 – 4 h), constantly (24 h) or repeatedly (4 h daily, up to 4 days) and 50 – 75 μ M H 2 O 2 : (30 min-5 h) 5 µ M Cells were treated with SFN and other compounds (OPZ, D3T) in Primary astrocyte cultures from Sprague- Dawley rats Murine BV-2 microgial cells Elevate levels of GSH. Prolonged induction of NQO1. Attenuation of Nrf2-dependent HMOX1 induction. NQO1 accumulation and continuous induction of GSH after daily SFN stimulations, resulting in prolonged protection against superoxides. Induction of the expression of antioxidant detoxification proteins by SFN (and other Bergstrom et al., 2011 Konwinski et al., 2004 Toxic substance Sulforaphane dose Exposure conditions Experimental model Main results Reference 6-hydroxydopamine (6- 5 mg/kg 4 groups of mice: 2 received a 6- Male C57BL/6 J mice 6-OHDA-induced rotations and deficits in Morroni et al., OHDA) OHDA injection in the left striatum; motor coordination that were ameliorated 2013 the other 2 received saline solution. by SFN. After brain lesions, SFN was In addition, SFN protected 6-OHDA- administered (5 mg/kg) or vehicle in induced apoptosis by blocking DNA both lesioned and sham mice. fragmentation and caspase-3 activation. Injections were made twice a week These results were further supported by for 4 weeks. The groups are: 6- immunohistochemical findings in the OHDA/VH; 6-OHDA/SFN; Sham/ substantia nigra that showed that SFN VH; Sham/SFN. protected neurons from neurotoxic effects of 6-OHDA. SFN increased the GSH content in mice with 6-OHDA lesion compared to 6-OHDA/ VH mice, and GST (41 %) and GR (69 %) activities. SFN treatment could dramatically downregulate ERK1 / 2 phosphorylation induced A. Cascajosa-Lira et al. Table 2 (continued ) Phytomedicine 130 (2024) 155731 (continued on next page) 11 Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference Hydrogen peroxide (H 2 O 2 ) Hydrogen peroxide (H 2 O 2 ) and paraquat DMSO for 24 h. Cells were treated with H 2 O 2 (400 µ M, 4 h. Furthermore, cells were treated with SF, OPZ or D3T 5 µ M and LPS (80 ng/ ml, 24 h) Up to 2.50 µ M Cells were treated with SFN for 18 h- 20 h followed by graded concentrations of H 2 O 2 for additional 18 h. 0 – 10 µ M Cultures were incubated in the presence or absence of SFN, 6-HITC, or BSO for 24 h. Cultures were exposed to H 2 O 2 or paraquat for 24 h, and then cell viability was determined by LDH release assay. Neuro2A murine neuroblastoma cells Primary neuronal cultures of rat striatum compounds, OPZ, D3T) was correlated with increased resistance of cells to H 2 O 2 - induced toxicity, as well as elimination of intracellular ROS in response to LPS treatment, during microgial cell activation. SFN enhanced the 26 proteasome expression of the catalytic subunits, as well as proteasomal peptidase activities in the cells. SFN protected cells against H 2 O 2 - mediated cytotoxicity in a manner dependent on proteosomal function. Inhibition of proteasome activities decreased the protective effects of SFN, as well as protein oxidation. Pretreatment with SFN and 6-HITC protected against H 2 O 2 - and paraquatinduced cytotoxicity in a concentrationdependent manner. SFN and 6-HITC induced Nrf2 translocation into the nucleus and increased the expression of γ -GCS, a ratelimiting enzyme in GSH synthesis and content. Exposure to BSO, an irreversible inhibitor of γ -GCS, suppressed the protective effects of both compounds. On the contrary, SFN and 6-HITC increased the expression of heme oxygenase-1 (HO-1) in neurons. Kwak et al., 2007 Mizuno et al., 2011 LiCl + pilocarpine 5 mg/kg SFN was administered in two doses, 48 and 24 h before the administration of 127 mg/kg of LiCl i.p. 35 mg/kg of Pilocarpine was administered i.p. 24 h later. Wistar rats Pretreatment with SFN did not change latency, character, duration, or severity of seizures and mortality. Complete prevention of the increase in hippocampal fields and the dentate gyrus was found. Folbergrov a ´ et al., 2023 Lipopolysaccharide (Escherichia coli serotype 0111:B4) Lipopolysaccharide (LPS) 5 and 50 mg / kg i.p. In vitro 0 – 40 µ M SFN In vivo 1 mg/kg/day SFN Mice were exposed to 5 mg/kg and 50 mg/kg of SFN dissolved in corn oil by injection 24 h prior to exposure to 0.5 mg/kg of LPS. In vitro Cells were pre-incubated with SFN or 1 % DMSO for 1 h followed by postincubation with LPS for 24 h. In vivo Three groups: control group, LPS (1 mg/kg/day LPS i.p.) group; and LPS + SFN group (1 mg/kg/day SFN 30 min before injection of LPS). All treatments were held for 5 days. Wild-type (WT) C57BL/6 J mice and Nfe2l2tm1Ywk knockout (Nrf2-KO) mice. In vitro BV2 cells In vivo SPF mice SFN may reduce cerebral inflammation in sepsis produced by LPS by mediating its anti-inflammatory and antioxidant effects through Keap1/Nrf2 transcriptional activation of the antioxidant system and via the NF κ B pathway. In vitro The modulation of TRAF6 and RIPK1 ubiquitination by Cezanne played a key role in the mechanism of SFN inhibiting NF- κ B pathway. In vivo SFN improved LPS-induced neurocognitive dysfunction in rats. It inhibited neuroinflammation by suppressing NF- κ B signalling activation induced by LPS. Holloway et al., 2016 Wang et al., 2020 Prion protein (PrP) 1 – 10 µ M Cells were treated with several concentrations of SFN and PrP (106 – 126 sequence) for up to 36 h. SH-SY5Y cells SFN prevented prion-mediated cell apoptosis by decreasing the previous increase in PrP-positive annexin V cells by PrP, and by decreasing LDH release levels. The TUNEL assay also revealed that SFN inhibited PrP -induced apoptosis. SFN induced autophagy, decreasing PeP- induced neurotoxicity. ATG% eliminates the blocked neuroprotective effects of SFN. Activation of the AMPK pathway regulated the neuroprotective effects of SFN-induced autophagy. Lee et al., 2014 Rotenone In vitro: 10 µ M In vivo: 50 mg/kg In vitro: cells were treated with SFN for 2 h and then exposed to rotenone (0.5 and 1 µ M) for 24 h. In vivo: control group; SFN group injected i.p. every other day; Rotenone group 30 mg/kg orally & daily for 60 days and Rotenone + SFN group in a similar way In vitro: SH-SY5Y In vivo: Male C57BL/6 J mice SFN exerted a neuroprotective effect that involved Nrf2-dependent reductions in oxidative stress, mTOR-dependent inhibition of neuronal apoptosis, and restoration of normal autophagy. Thus, SFN inhibited locomotor activity deficiency and dopaminergic neuronal loss, inhibited ROS production, MDA accumulation, and increased GSH levels. It also increased the expression of Nrf2, HO-1 and NQO1. It inhibited the mTOR- mediated signalling pathway of p70S6K Zhou et al., 2016 A. Cascajosa-Lira et al. Table 2 (continued ) Phytomedicine 130 (2024) 155731 12 Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference and 4E-BP1. SFN might have a role in preventing Parkinson ’ s disease. Scopolamine (SCOP) Ex vivo: 1, 10, and 100 μ M, In vivo: 15 mg/ kg. - The hippocampal slices were treated with SFN (1, 10, and 100 μ M), SCOP (300 μ M) or SFN + SCOP [SFN (10 μ M) + SCOP (300 μ M)], dissolved in artificial cerebrospinal fluid (aCSF), 10 min after recording. - After 5 days of adaptation, rats were treated with SFN (15 mg / kg, ip for 14 days. Treatment with SCOP (1.5 mg / kg, i.p.m) was started on the 11th day and continued for 9 days, and behavioural tests were performed from the 11th day to the 18th day. On day 19, rats were sacrificed, and hippocampi were removed for molecular analysis. Organotypic Cultured rat hippocampal tissues (OHSCs) from rats (ex vivo) and in vivo rats SFN increased total fEPSP in a dosedependent manner after high-frequency stimulation and attenuated SCOP-induced interference of the fEPSP in the CA1 area. SFN also restored cognitive function and inhibited memory impairment as indicated by the alleviation of the negative neurological effects of SCOP, that is, a lower ratio of spontaneous alternation in the Y-maze, a reduced latency in the passive avoidance test, and an increased navigation time in the Morris water maze. Park et al., 2021 4E-BP1: Eukaryotic translation initiation factor 4E binding protein 1; 6-OHDA: 6hydroxydopamine; 6-HITC: 6-(methylsulifnyl) hexyl isotiocyanate; Ache: acetylcholinesterase; AMPK: protein kinase activated with adenosine 5 ′ -monophosphate (AMP)-activated protein kinase; BSO: l-buthionine-sulfoximine; CPF: Chlorpyrifos; D3T: 3-H-1,2.dithiole-3-thione; EMT: epithelial-mesenchymal transition; ERK1/2: extracellular signal-regulated kinases 1 and 2; IL: interleukin;; fEPSP: total field excitatory postsynaptic potential; GR: glutathione reductase; GSH: glutathione; GSK-3b: glycogen synthase kinase 3 beta; GST: glutathione-S-transferase; H3k4me3: Trimethylation of histone H3 lysine 4; HMOX-1: heme oxygenase 1 gene; LDH: Lactate dehydrogenase; LPS: bacterial lipopolysaccharide; LPO: lipid peroxidation; LORR: Loss of the righting reflex; NCCs: Neural Crest Cells; NLRP3: pyrin domain-containing 3; NF-kB: nuclear factor kappa-light-chain enhancer of activated B cells; NQO1: NAD(P)H dehydrogenase [quinone] 1; NO: nitric oxide; MDA: malondialdehyde; mTOR: mammalian target of rapamycin; OPZ: Oltipraz; ROS: reactive oxygen species; SCOP: scopolamine; SH-SY5Y: neuroblastoma cell line; SNAI1: snail family transcriptional repressor 1; TRAF6: Tumour necrosis factor receptor associated factor 6; RIPK1: Receptor-interacting serine / threonine protein kinase 1; P70S6K: Ribosomal protein S6 kinase beta-1 protein; VCAM-1: Vascular cell adhesion protein 1; γ -GCL: γ Glutamate – cysteine ligase. Fig. 5. Mechanism of action of the protective effects of SFN in the nervous system. Created with BioRender.com. A. Cascajosa-Lira et al. Table 3 Phytomedicine 130 (2024) 155731 13 Overview of studies reporting the protective effects of SFN on kidney. dose model chloride (CdCl 2 ) Calcium oxalate times a week. Subsequently, two hours later, three groups received i.p. injections of CdCl 2 at doses of 3 μ mol/kg, 6 μ mol/kg, and 9 μ mol/kg, respectively, five times a week. 0.2 mg/kg Rats with kidney stone model were injected with 0.2 mg/kg of SFN. increases in urinary protein and BUN levels, along with elevated NAG, LDH, and ALP in urine, as well as higher levels of MDA in the renal cortex. At the same time, there was a notable decrease in the content of GSH and the activities of SOD and GSH-Px in the renal cortex. However, in rats pretreated with SFN, these changes showed significant improvement. Wistar rats Urine levels of oxalic acid, Ca2 + , and MDA were significantly lower compared to the model group; consequently, the formation of calcium oxalate kidney stones was inhibited. Liu et al., 2020 Cisplatin 0.5 – 5 µ M for 24 h 500 µg/kg Cells were treated with SFN (0 – 5 µ M) for 24 h and then 40 µ M of cisplatin was added for another 24 h. Rats were exposed to a single dose of cisplatin by injection (7.5 mg/kg). SFN (500 µg/kg i.v.) was administered twice: 24 h before exposure and also 24 h after exposure to toxicants and the animals were killed three days after injection of cisplatin. LLC-PK1 cells Wistar rats Protection against mitochondrial alterations produced by cisplatin in both LLC-PK1 cells (loss of membrane potential) and isolated mitochondria (inhibition of mitochondrial calcium uptake, cytochrome c release of cytochrome c, and decrease in GSH content, aconitase activity, adenosine triphosphate (ATP) content, and oxygen consumption). Prevention of cisplatin-induced increase in ROS production and decrease in NQO1 and γ -GCL activities. Guerrero-Beltr a ´ n et al., 2010 Cisplatin 500 µ g/kg Rats were exposed to a single dose of cisplatin by injection (7.5 mg/kg). SFN (500 µg/kg i.v.) was administered twice: 24 h before exposure and also 24 h after exposure to toxicants and the animals were killed three days after injection of cisplatin. Wistar rats SFN prevented cisplatin-induced renal injury by modulating the activation of various cell death and pro-inflammatory pathways (p53, JNK, p38- α , TNF- α and NF- κ B) and impairments of key pro-survival signaling mechanisms (ERK and p38- β ). Guerrero-Beltr a ´ n et al., 2012 Mercury chloride (HgCl 2 ) 2 mg/kg Rats received 2.2, 4.4 and 8.8 mg/kg of HgCl 2 and 2 mg/kg of SFN in the intervention groups. Wistar rats Urinary protein and BUN levels, along with kidney cortical GSH and MDA levels, were notably higher compared to the SFN intervention group. On the contrary, the SFN group exhibited significant increases in urinary LDH, ALP, NAG, as well as kidney cortical SOD and GSH-Px activities. Guo, 2016 ALT: alanine aminotransferase; GPx: glutathione peroxidase; GSH: glutathione; JNK: Mitogen-Activated Pro-tein Kinases; BUN: Blood Ureic Nitrogen; ERK1/2: extracellular signal-regulated kinases 1 and 2; LDH: lactate dehydro-genase; LLC-PK1: Lilly Laboratories Culture-Porcine Kidney 1; NAG: N-acetylglutamate synthase; NF-kB: nuclear factor of activated B cells; NQO1: NAD(P)H dehydrogenase [quinone] 1; p38: tumour protein; p53: tumour protein; SOD: Superoxide dismutase; TNF α : tumour necrosis factor alpha; γ -GCL: γ Glutamate – cysteine ligase. In these studies, as in the in vitro studies, there is an increase in the activity of antioxidant defenses mediated mainly by the activation of Nrf2 factor (Xin et al., 2018; Wang et al., 2022b; Fouad, 2023; Yu et al., 2023). Pulmonary protection of sulforaphane Table 5 provides a summary of the main findings regarding the pulmonary protective effects of SFN, encompassing the most significant results from the selected studies. Again, the effects are predominantly attributed to the antioxidant and anti-inflammatory activities of SFN, showing its effectiveness in preventing pulmonary system injuries. In vitro studies have employed only two types of experimental models: bronchial epithelial cell lines (Wang et al., 2018; Gasparello et al., 2021; Quin et al., 2021) and alveolar epithelial cells (Lv et al., 2020). However, there is a variety of substances tested in vitro: proteins such as the SARS-COV-2 protein (Gasparello et al., 2021), metals such as cadmium (Wang et al., 2018), potassium dichromate (Lv et al., 2020) and particulate matter (PM.5) (Quin et al., 2021). On the other hand, in vivo studies in rat and female mice (CD1 strain) have been applied against the detrimental effects of potassium dichromate and benzo(a) pyrene, respectively. Most of these toxins induce an inflammatory state and cause oxidative stress. The antioxidant efficacy of SFN has been demonstrated by its ability to increase Nrf2 and HO-1 (Wang et al., 2018), to decrease MDA and ROS (Quin et al., 2021) and therefore increase the capacity of antioxidant enzymes (Kalpana Deepa Priya et al., 2011). Furthermore, the anti-inflammatory effect has been demonstrated by decreasing the expression of proinflammatory interleukins (IL-6 and IL-8) and other markers (Lv et al., 2020; Gasparello et al., 2021). Gastrointestinal protection induced by sulforaphane Table 6 provides a detailed summary of the findings, highlighting the significant protective effects of SFN on the gastrointestinal system as evidenced by the selected in vivo studies. In these studies, Sprague Dawley and Wistar rats have been chosen as experimental models, together with mice of the C57BL/6 J strain and Syrian hamsters. In this type of studies, the following substances have been used to induce models of gastrointestinal disease in animals: acetic acid (Alattar et al., 2022) or sodium dextran sodium sulfate (DSS) (Wagner et al., 2013; Wu et al., 2023; Holman et al., 2023; Zhang et al., 2023) to produce intestinal colitis, acetylsalicylic acid (ASA) (Zeren et al., 2016) to produce gastric ulcers and N-nitroso-bis(2-oxopropyl) amine (BOP) (Kuroiwa et al., 2006) to produce pancreatic cancer. SFN has exerted a protective effect against all these compounds and in all disease models assayed. The effects of SFN in colitis are mainly due to its anti-inflammatory and protective action on the intestinal Toxic substance Sulforaphane Concentration or Exposure conditions Experimental Main results Reference Cadmium 1 mg/kg Rats were treated with 1 mg/kg SFN i.p. three Wistar rats The high dose of cadmium exhibited significant Li, 2015 A. Cascajosa-Lira et al. Table 4 Phytomedicine 130 (2024) 155731 14 = = Overview of studies reporting the protective effects of SFN on heart. Concentration or Angiotensin II (Ang II) subcutaneously with Ang II (0.5 mg/kg b. w.) or an equivalent volume of 0.9% physiological stroke saline solution every other day for 2 months, with or without SFN treatment 5 days a week for 3 months. 5 mg/kg Exp1. Mice were injected with 0.5 mg / kg of Ang II every other day for 2 months, with / without injection of SFN 5 days a week for 3 months. In the end some mice were euthanized and others kept for additional 3 months without SFN. Exp2. Nrf2-KO and WT in a similar way but suppressing the last 3 months of treatment. Exp3. Ang II or vehicle for 2 months and then 1 month without treatment. (Nrf2-KO) mice Male C57BL/6 J mice: Nrf2-TG, Nrf2-KO and wild type However, the protective effect of SFN on Ang II-induced aortic damage and upregulation of genes downstream of Nrf2 was not observed in Nrf2-KO mice. SFN induced up-regulation of aortic Nrf2 and inhibited the accumulation of ERK, GSK-3 β , and Fyn in the nuclei. SFN can prevent Ang II-induced cardiomyopathy by activating Nrf2- mediated exogenous antioxidant defences, and that up-regulation and activation of Nrf2 by SFN are achieved partially through the Akt/GSK-3 β /Fyn pathway. Xin et al., 2018 Cuprizone (CPZ) 2 mg/kg/day for two weeks Rats were fed a CPZ-contained diet (0.2 %) for four weeks after which they were SFN i. p. administrated (2 mg/kg/day) for two weeks. Wistar rats SFN resulted in a reduction in LPO and an enhancement of total antioxidant capacity levels and CAT activities in rats pretreated with CPZ, thus alleviating CPZ-induced oxidative stress in cardiac tissues. Fouad, 2023 Hydrogen peroxide H 2 O 2 Methamphetamine (MA) 0.1 – 0.5 μ M SFN for 24 in the absence or presence of physiological concentrations of E2 (10 – 50 nM) and 100 μ M H 2 O 2 for 30 min. 10 mg/kg Mice received SFN i.p. once a day or SFN + 2 mg/kg MA i.p. twice a day for 5 days. Primary cardiomyocytes cultures from Sprague – Dawley rats Adult male C57BL/6 J mice The protective effect of cotreatment is not mediated by ER. Cotreatment of SFN and E2 synergistically up-regulated phase II enzymes and activated pro-survival signaling pathways. The simultaneous presence of ERK1/2 and Akt inhibitors leads to a significant reduction in cardiomyocyte viability with respect to SFN + E2-treated cells before H2O2-induced damage. SFN reduced LDH release. Nrf2 attenuated MA-induced myocardial injury by regulating oxidative stress and apoptosis, thus playing a protective role. In particular, MA effects (MDA, SOD, protein expression levels of Caspase-3 and Bax, protein expression levels of Bcl-2) were reversed by Nrf2 activation. Angeloni et al., 2017 Yu et al., 2023 Akt: serine/threonine protein kinase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; b.w.: body weight; CPZ: cuprizone; E2: 17- β -estradiol; ERK1/2: extracellular signal-regulated kinases 1 and 2; Fyn: tyrosine specific phospho-transferase; GSK-3b: glycogen synthase; Nrf2: nuclear factor; LDH: Lactate dehydrogenase; T-AOC: total antioxidant capacity; P13K: fosfatidilinositol 3 kinasa. microbiota, decreasing the expression and production of interleukins and other inflammatory markers (Wagner et al., 2013; Wu et al., 2023). Furthermore, this anti-inflammatory effect is also responsible for the reduction of gastric ulcers together with its antioxidant effect, increasing the activity of enzymes such as SOD and GPX and decreasing the expression of inflammatory markers such as NO and NF- κ B gene (Zeren et al., 2016). Finally, a time-dependent anticancer effect ameliorating pancreatic hyperplasia has been demonstrated (Kuroiwa et al., 2006). Inmunoprotective effects of sulforaphane Table 7 provides a summary of the main findings regarding the immunoprotective effects of SFN, including the most significant results from the selected in vitro and in vivo studies. In general, the immunoprotective mechanism of action consists mainly of a synergy between antioxidant effects and inflammatory marker-reducing effects (Xu et al., 2024). On the one hand, the activation of the Nrf2 factor, which through keap1 reduces ROS and cell apoptosis. On the other hand, it inhibits the gene expression of genes related to inflammatory factors such as VCAM-1, E-selectin. This in turn contributes to the reduction of proinflammatory cytokines and NO release. SFN produces a down-regulation of genes related to carcinogenic processes such as miR-19a, miR-19b, PTEN and p21. At the genetic level, it has also been shown to reduce the % of micronuclei. Thus, the mechanism of action of SFN on the immune system consists mainly in the reduction of inflammatory cytokines and NO (Holloway et al., 2016; Ruhee et al., 2019). Moreover, antioxidant effects have been also described, although to a lesser degree. In the case of immune system models used, there is an imbalance between in vitro (n 6) and in vivo (n 1) assays. In vitro assays have mainly used human lymphocytes and monocytes from volunteers, but they have been also performed on cell lines such as MCF-7, RAW 264.7, GLC and HBMEC-3. The protective effects of SFN against metals such as cadmium, inflammatory chemicals such as LPS or hydrogen peroxide, plasticizers such as butylbenzyl phthalate, and physical agents such as gamma radiation have been studied. On the other hand, there is only one in vivo study performed in C57BL/6 J mice exposed to LPS (Holloway et al., 2016). Other protective effects of sulforaphane Table 8 presents a thorough overview of other protective effects of SFN different from those mentioned above. SFN has protective activity against numerous chemical and physical agents and on many different tissues such as the skin, endocrine system, reproductive system, eyes, bones, and muscles. As shown above, the models used are very varied, Toxic substance Sulforaphane dose Exposure conditions Experimental model Main results Reference Angiotensin II (Ang 0.5 mg / kg b.w. Four groups: control, SFN, Ang II, and Ang Wild-type (WT) C57BL/ SFN treatment prevented aortic damage Wang et al., II) SFN II + SFN (Ang II/SFN). Mice were treated 6 J and Nrf2-knockout through Nrf2 activation in WT mice. 2022b A. Cascajosa-Lira et al. Table 5 Phytomedicine 130 (2024) 155731 15 Overview of the studies reporting the protective effects of SFN on toxicants with pulmonary effects. Toxic substance Sulforaphane Concentration or dose Exposure conditions Experimental model Main results Reference Arsenic 10 mg/kg The animals were exposed to 4.8 mg/m 3 of synthetic dust (with 10 % arsenic trioxide (2 – 3 µ m) for 30 min/day. SFN was injected every other day until the end of the experiment (14 days). Nrf2-WT and Nrf2-KO mice Exposure to inhaled arsenic resulted in pathological alterations, oxidative DNA damage, and mild apoptotic cell death in the lung; all of which were blocked by SFN in a Nrf2-dependent manner. Zheng et al., 2012 Benzo(a) pyrene (BaP) 9 µmol/mice/day Animals were exposed to the toxic (100 mg/ kg of BaP i.p.) thrice a week for 20 weeks. Moreover, two animal groups were asigned for SFN treatment and BaP: Pre-treatment group was administrated with SFN alternate days for two weeks before BaP and Posttreatment group was exposed to SFN alternate days from the 12th week to the 20th week of BaP treatment. Swiss albino CD1 female mice SFN attenuated the oxidative stress caused by BaP modulating SOD, CAT, GR, GPx, and G6PDH activity, as well as attenuating the GSH and LPO content in the lung. SFN restored the enzymatic activities in charge of the electronic transport of mitochondria in lung. Post-treated SFN animals shows highly reduced immunoreactivity for the Bcl2 protein. Kalpana Deepa Priya et al., 2011 Cadmium (Cd) 0, 2.5, 5 and 10 µ M SFN Cells were transformed by exposure to Cd for 5 months. Furthermore, both transformed and untransformed cells were exposed to SFN for 24 h. BEAS-2BR cells In cadmium-transformed BEAS-2BR cells, SFN restored autophagy, decreased Nrf2, and decreased apoptosis resistance. In untransformed cells, SFN induced Nrf2 to decrease ROS and possibly malignant cell transformation. They concluded that SFN protected against Cd-induced lung carcinogenesis. Wang et al., 2018 K 2 Cr 2 O 7 4 mg/kg In vivo rats were divided in 4 groups: control; SFN, K2Cr2O7, and K 2 Cr 2 O 7 + SF. Rats in SFN group were subcutaneously injected with SFN solution and i.p. injected with saline solution every day for 35 d Rats in the chromium group were i.p. injected with K 2 Cr 2 O 7 solution (4 mg/kg) and subcutaneously with sterile saline solution. Rats in the K 2 Cr 2 O 7 * SFN group were i.p. injected with the chromium salt and sub. injected with SF. In vitro MLE-12 cells were treated with K 2 Cr 2 O 7 (1 µ g/ml) for 24 h after pretreatment with 0.1 µ M for 30 min. In vivo Wistar rats In vitro mouse alveolar type II epithelial cell line (MLE-12). SFN prevented oxidative stress, histopathological lesions, inflammation, apoptosis, and changes in Akt and GSK-3b levels in vivo and in vitro. However, SFN cannot play the protective effect against K 2 Cr 2 O 7 - induced cell injury after treatment with an Aktspecific inhibitor (MK-2206 2HCl) in MLE-12 cells. Lv et al., 2020 Particulate matter (PM.5) (0.25 – 16 µ M) After incubation for 24 h, cells were washed and incubated with PM.5 (100 – 400 µ g/ml) and/or SFN (0.25 – 16 µ M) for 24, 48 and 72 h. Human bronchial epithelial (HBE) cells PM2.5 generated ROS and inflammatory responses. Pretreatment with SFN alleviated these negative effects: levels of MDA and ROS decreased, LDH release decreased, and cell apoptosis reducing the apoptosis ratio in the cells. SFN decreased inflammation by lowering the secretion of some inflammatory cytokines (IL- 6, IL-8). Pre-incubation with SFN increased Nrf2 and HO-1. Qin et al., 2021 S-protein of SARS-CoV- 2 5 and 10 μ M for 24 h. IB3 – 1 cells were treated with 5, 15 o 50 nM S-protein in the presence of 5 and 10 μ M SFN for 24 h. Bronchial epithelial IB3 – 1 cells Treatment with SFN reversed the up-regulation of IL-6 and IL-8 induced by SARS-CoV-2 Spike protein in IB3 – 1 cells. Furthermore, SFN- mediated inhibitory effects were also observed for PDGF, IL-9, G-CSF, GM-CSF, IFN- γ , MCP-1 and MIP-1 β . Gasparello et al., 2021 CAT: catalase; G6PDH: Glucose-6-phosphate de-hydrogenase; GCLM: glutamate cysteine ligase modifier subunit; G-CSF: granulocyte colony stimulating factor; GMCSF: granulocyte-macrophage colony-stimulating factor; GPx: glutathione peroxidase; GR: glutathione reductase; GSH: glutathione; GSK-3b: glycogen synthase; HMOX1: heme oxygen-ase-1; IL: interleukin; MLE-12: mouse alveolar type II epithelial cell line; LDH: Lactate dehydrogenase; LPO: lipid peroxidation; MDA: malondialdehyde; SARS-CoV-2: coronavirus 2 of severe acute respiratory syndrome coronavirus 2; PM.5: particulate matter. both in vitro and in vivo. In vitro, cell lines, primary cultures, and zebrafish larvae have been used. On the other hand, the experimental in vivo models are Wistar rats and mice of several strains (CD1, C5BL/6 J, B6129SF1 and ICR). In addition, non-rodent species such as Oreochromis niloticus, Drosophila melanogaster and Marsuperanus japonicus have been also employed. In relation to protective effects on the skin, SFN shows antioxidant, anti-inflammatory and anticarcinogenic effects against radiation and irritants (Abel et al., 2011; Klesczynski et al., 2013; Mathew et al., 2014) in vitro. Similarly, in assays using eye-related models, SFN is also capable of protecting against irradiating and oxidizing agents. Its mechanism of action is based on enhancing Nrf2-mediated antioxidant and anti-inflammatory activity, demonstrated only in vitro models (Chang et al., 2020; Yang et al., 2021). Regarding muscle and bone tissues, antioxidant activity has been demonstrated using both in vitro and in vivo assays. In addition, in muscle, SFN has the ability to restore protein synthesis and increase regenerative capacity (Hoon Son et al., 2017). In the endocrine system, SFN has masculinization ability to restore the development in mice exposed to vinclozolin (Amato et al., 2022). However, the mechanism of action by which it protects against endocrine disruption is not clear or established. SFN also exhibits the ability to restore metabolic problems in the pancreas (Song et al., 2009) and lipid metabolism through inhibition of LKB1/AMPK by increasing lipophagy and mitochondrial fatty acid metabolism (Zhang et al., 2014). Foods 2021, 10, 989 12 of 15 factor, when test substances were less toxic in vivo, detoxification processes are usually implicated [31,32]. In general, the detoxification system can play a fundamental role in the impact of genotoxic or mutagenic compounds in different ways: (a) by avoiding the formation of the final mutagen by metabolic sequestration of the promutagen or (b) by inactivation of the active metabolite after its creation [31]. In this sense, several authors have reported the metabolization of OSC present in the oil section of Allium sp. such as DADS (diallyl disulfide) by oxidation (cytochrome P450) to produce diallyl sulfoxide (DASO) and allyl sulfone (DASO2). Moreover, various in vivo studies have found that DAS (diallyl sulfide) and DADS can modulate the phase I and phase II metabolizing enzymes [40,41]. According to our hypothesis of the metabolization of OSC compounds as a possible detoxification route, Mellado-García et al. [31] reported the presence of degradation products of PTSO in the stomach of rats exposed, after the application of pyrolysis techniques, whereas in the liver, only one was identified in comparison to control rats, indicating the influence of the distribution and metabolism processes. Further research focused on metabolism processes and toxicokinetic aspects of PTS are necessary to define metabolic derivates of this organosulfur compound and to assess its safety to be applied as a feed additive. In addition, following the guidance of EFSA for feed additives, the subchronic toxicity test should be performed through a 90-day assay. 5. Conclusions The combined in vivo MN-comet assay protocol showed that PTS did not produce genotoxic effects in the stomach, liver, blood cells, and bone marrow of male and female rats orally (gavage) exposed to 5.5, 17.4, and 55.0 mg/kg b.w. In addition, no oxidative DNA damage was induced by PTS when the enzymes FPG and Endo III were applied in the nucleus isolated from the cells of the liver, stomach, and blood. The histopathological study revealed only minimal hypertrophy of the mucus cells and flaking epithelium in the stomach and slight lesions in the liver, and occasionally, minimal inflammatory infiltrates in rats exposed to the highest dose (55.0 mg of PTS/kg). Consequently, the absence of in vivo genotoxic effects of PTS supports its approval as a feed additive or active agent in food contact materials, although more studies are needed to obtain a global picture of its safety profile. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/foods10050989/s1, Table S1: Measures of the% DNA in tail for the liver of the standard comet assay and the modified comet assay with enzymes in their different treatments: buffer F, FPG, Endo III, Table S2: Measures of the% DNA in tail for the stomach of the standard comet assay and the modified comet assay with enzymes in their different treatments: buffer F, FPG, Endo III, Table S3: Measures of the% DNA in tail for the blood of the standard comet assay and the modified comet assay with enzymes in their different treatments: buffer F, FPG, Endo III. Author Contributions: Conceptualization: Á.J. and A.M.C.; methodology, A.C.-L., M.P., A.I.P., L.D.- Q.J., R.M., and V.M.-H.; software, A.C.-L., M.P., L.D.-Q.J., and V.M.-H.; formal analysis, A.C.-L., M.P., and V.M.-H.; investigation, A.C.-L., M.P., A.I.P., L.D.-Q.J., S.P., R.M., and V.M.-H.; resources, A.B., E.G., Á.J., and A.M.C.; writing—original draft preparation, A.C.-L.; V.M.-H., and A.M.C.; writing—review and editing, A.M.C., Á.J., S.P., R.M., A.B., E.G., and V.M.-H.; supervision, S.P., Á.J., and A.M.C.; project administration, A.M.C., Á.J., and S.P.; funding acquisition, A.B., E.G., A.M.C., and S.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministerio de Ciencia e Innovación (Project RTC-2017-6199-2), and by the Junta de Andalucía (Projects AT 2017-5323 and P18-TP-2147) for its financial support. Antonio Cascajosa Lira thanks the Spanish Ministerio de Universidades for the funding FPU grant (FPU2019-01247). Leticia Diez-Quijada Jiménez thanks the Spanish Ministerio de Economía, Industría y Competitividad for the grant FPI (BES-2016-078773). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the The Ethics Committee on Animal Experimentation of the University of Sevilla approved this in vivo experiment (09/03/2016/026). Foods 2021, 10, 989 13 of 15 Informed Consent Statement: Not applicable. Acknowledgments: Thanks go to the Spanish Ministerio de Ciencia e Innovación (Project RTC-2017-6199- 2), and to the Junta de Andalucía (Projects AT 2017-5323 and P18-TP-2147) for its financial support. Antonio Cascajosa Lira thanks the Spanish Ministerio de Universidades for the funding FPU grant (FPU2019-01247). Leticia Diez-Quijada Jiménez thanks the Spanish Ministerio de Economía, Industría y Competitividad for the grant FPI (BES-2016-078773). The authors wish to thank the Microscopy service of CITIUS and the animal housing from the Facultad de Farmacia in the Universidad de Sevilla. Conflicts of Interest: The authors declare no conflict of interest. References 1. Farhat, Z.; Hershberger, P.A.; Freudenheim, J.L.; Mammen, M.J.; Hageman Blair, R.; Aga, D.S.; Mu, L. Types of garlic and their anticancer and antioxidant activity: A review of the epidemiologic and experimental evidence. Eur. J. Nutr. 2021. [CrossRef] 2. Bosem, S.; Laha, B.; Banerjee, S. Anti-inflammatory activity of isolated allicin from garlic with post-acoustic waves and microwave radiation. J. Adv. Pharm. Res. 2013, 13, 512–514. 3. Vezza, T.; Algieri, F.; Garrido-Mesa, J.; Utrilla, M.P.; Rodríguez-Cabezas, M.E.; Baños, A.; Guillamón, E.; García, F.; Rodríguez- Nogales, A.; Gálvez, J. 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Safety assessment of propyl-propane-thiosulfonate (PTSO): 90-days oral subchronic toxicity study in rats This article was published in Food and Chemical Toxicology, 144, Antonio Cascajosa Lira, Ana Isabel Prieto, Alberto Baños, Enrique Guillamón, Rosario Moyano, Angeles Jos, Ana M. Cameán, Safety assessment of propyl-propane-thiosulfonate (PTSO): 90-days oral subchronic toxicity study in rats, 11612, Copyright Elsevier (2020) 255 + - Food and Chemical Toxicology 144 (2020) 111612 Safety assessment of propyl-propane-thiosulfonate (PTSO): 90-days oral subchronic toxicity study in rats Antonio Cascajosa Lira a , Ana Isabel Prieto a , Alberto Ba n ˜ os b , Enrique Guillam o ´ n b , Rosario Moyano c , Angeles Jos a , * , Ana M. Came ´ an a a Area of Toxicology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain b DMC Research Center SLU, Camino de Jayena s/n, Alhedin, 18620, Granada, Spain c Department of Pharmacology, Toxicology and Legal and Forensic Medicine, University of Cordoba, Campus de Rabanales Carretera Madrid-Cadiz s/n, Spain A R T I C L E I N F O Keywords: Subchronic toxicity 90-day Organosulfur compounds Allium Propyl-propanethiosulfonate A B S T R A C T Propyl-propane-thiosulfonate (PTSO) is one of the main organosulfur compounds present in Allium essentials oil. Different applications in the food sector have been proposed for PTSO, such as food and feed additive and as active packaging. However, the authorization of its use depends on its toxicity profile. Thus, as a part of its safety assessment, in this work a repeated dose 90-day oral toxicity study has been conducted for the first time in rats following the OECD guideline 408. PTSO was administered to groups of 10 male and 10 female rats at dose levels of 0, 14, 28, and 55 mg/kg/day. No clinical signs or mortality and no changes in body weight, food consumption and feed conversion efficiency were detected through the study. Moreover, no treatment-related changes in hematological and biochemical parameters were observed, for either sex or dose groups. The histopathology study performed revealed no differences in organ weights, and no morphological and histopathological changes were observed. Based on these results, the no-observed-adverse-effect level (NOAEL) of PTSO was judged to be ≥ 55 mg/kg/day for both sexes. 1. Introduction Essentials Oils (EO) extracts from plants have been the focus of numerous studies due to their potential in the pharmaceutical and food industries. Several works and patents have been developed specifically for the application of EO and their components into the food sector (Ribeiro-Santos et al., 2017; Llana-Ruiz-Cabello et al., 2015a; Maisanaba et al., 2017). Globally, their relatively safe status, properties and acceptance by consumers which demand natural compounds to replace synthetic ones have piqued the interest of industries and consumers (Sacchetti et al., 2005; Benkeblia and Lanzotti, 2007; Debiagi et al., 2014). Among those beneficial plants, Allium sp. is a genus well-known for its antimicrobial, antiviral, antiprotozoal, antifungal or antioxidant properties (among others). These properties are mainly due to their content of organosulfur compounds (OSC), which are secondary phytochemical metabolites, biosynthesized for defensive purposes against biotic and abiotic stressors. They are mainly formed for the action of the enzyme alliin (stored in vacuoles) on cytoplasmic compounds like alk(en)yl cysteine sulfoxides (ACOs) once the vegetable tissue is hurt (Putnik et al., 2019) (Fig. 1). Otherwise, these compounds are not available in intact cells as they are toxic for the plant (Ramirez et al., 2017). One of the main components of Allium essential oil is propylpropane-thiosulfonate (PTSO), that corresponds to the molecular formula C 6 H 14 O 2 S 2 (Fig. 1). This compound has been stabilized and characterized by DMC Research Center SLU (Granada, Spain) to be used for different applications, taking advantage of its beneficial properties. Thus, this product has been reported to show mainly antioxidant and antimicrobial activities, being able to inhibit the growth of Gram ( ) and Gram ( ) bacteria as well as molds and yeast (Peinado et al., 2012, 2013; Llana-Ruiz-Cabello et al., 2015b). Its antibacterial activity in vitro in humans has also been demonstrated (Sorlozano-Puerto et al., 2018). Proallium AP®, a commercial Allium sp. extract with a 14.5% PTSO content, has been proposed as a biopreservative in active food packaging for human food commodities mainly due to its antioxidant and antimicrobial activity (Llana-Ruiz-Cabello et al., 2018). The packaging material used in these systems can incorporate components intended to be released into the food from the package, allowing foods to arrive at the * Corresponding author. Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, C/ Profesor García Gonz a ´ lez, n º 2. C.P. 41012, Seville. Spain. E-mail address: [email protected] (A. Jos). https://doi.org/10.1016/j.fct.2020.111612 Received 24 June 2020; Received in revised form 9 July 2020; Accepted 11 July 2020 Available online 29 July 2020 0278-6915/© 2020 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox A.C. Lira et al. Food and Chemical Toxicology 144 (2020) 111612 2 Fig. 1. Biochemical pathways and chemical structures of organosulfur compounds. (Alk(en)yl cysteine sulfoxides (ACOs); Propyl-propane-thiosulfonate (PTSO); Propyl-propane-thiosulfinate (PTS)). Fig. 2. Mean body weights (g) of A) male and B) female rats orally exposed to 0, 14, 28 and 55 mg/kg b.w./day PTSO and control rats for 90 days. consumers with their original or enhanced organoleptic properties, with longer shelf-life and safety (Ribeiro-Santos et al., 2017). Previously, another study carried out by Seydim & Sarikus (2006) tested the antimicrobial activity of garlic EO in combination with oregano EO in films made with whey protein isolate, and showed antimicrobial activity in a Fig. 3. Mean body weight gain (%) of A) male and B) female rats orally exposed to 0, 14, 28 and 55 mg/kg b.w./day PTSO and control rats for 90 days. concentration of 4% (w/v). But the incorporation of garlic EO and their components in active food packaging can result in a higher human exposure and consequently, more research is needed to stablish the safety concentration. An additional proposed application for PTSO is as sensory additive in animal nutrition, improving the palatability of feed, and also as a zootechnical additive, being an alternative to the use of A.C. Lira et al. Table 1 Food and Chemical Toxicology 144 (2020) 111612 3 ± ± Effect of 90 days oral exposure to PTSO on body weight and food consumption in rats. Values represent the mean ± SD of 10 rats/sex/group. Differences between control and treated groups for male and female rats were evaluated by Kruskal-Wallis test (K.W.) or by ANOVA test (F values). MALE FEMALE PARAMETERS Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 (0 mg/Kg/day) (14 mg/Kg/ day) (28 mg/Kg/ day) (55 mg/Kg/ day) (0 mg/Kg/day) (14 mg/Kg/ day) (28 mg/Kg/ day) (55 mg/Kg/ day) Inicial body weight (g) N = 10 N = 10 N = 10 N = 10 N = 10 N = 10 N = 10 N = 10 320.1 ± 10.8 317.4 ± 8.7 321.4 ± 14.0 320.3 ± 14.7 226.5 ± 9.2 228.6 ± 14.6 227.3 ± 10.1 225.3 ± 12.3 F(36.3) = 0.19 p = 0.90; N.S. F(36.3) = 0.14 p = 0.94; N.S. Final body weight (g) 633.8 ± 38.3 623.1 ± 28.7 611.5 ± 26.5 625.9 ± 48.1 326.8 ± 11.5 338.0 ± 29.1 321.1 ± 23.8 321.3 ± 18.3 F(36.3) = 0.64 p = 0.59; N.S. F(36.3) = 1.33 p = 0.28; N.S. Body weight gain 313.7 ± 35.4 305.7 ± 29.5 290.1 ± 29.2 305.6 ± 38.2 100.3 ± 13.5 109.4 ± 19.9 93.8 ± 23.3 96.0 ± 15.5 F(36.3) = 0.88 p = 0.46; N.S F(36.3) = 1.40 p = 0.26; N.S. Total feed intake (g) 2977.2 ± 197.2 2998.0 ± 207.0 2822.1 ± 249.2 2810.5 ± 155.5 1948.4 ± 174.2 1994.9 ± 258.8 1928.3 ± 210.7 1888.3 ± 142.6 KW = 7.17 p < 0.066; N.S KW = 1.94 p = 0.58; N.S. Feed conversion ratio 9.6 ± 0.8 9.9 ± 1.4 9.8 ± 1.0 9.3 ± 0.9 19.8 ± 3.2 18.5 ± 2.5 21.3 ± 3.7 20.3 ± 4.6 F(36.3) = 0.65 p = 0.59; N.S. KW = 2.51 p = 0.47; N.S. Values are mean ± SD for 10 rats/sex/group. F: Statistics ANOVA test; K.W: Kruskal-Wallis Statistic; N.S.: Not Significant. antibiotics (Peinado et al., 2012), contributing to reduce resistance generated by their excessive use in livestock. Since the ban in the European Union (EU) of the use of antibiotics as growth promoters, the search for new alternative products that ensure similar production levels and food security without generating unwanted effects, including human resistances, has been fostered, being additives of natural origin a good alternative. The regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 sets the parameters of use of the additives used in animal nutrition. Recent studies have shown that some feed additives can help animals to maintain good physiological conditions and improve animal welfare. This has led to an amendment in the abovementioned regulation on 12th June 2019, establishing new functional groups of feed additives to improve physiological condition of animals. In this regard, other properties demonstrating the potential use of PTSO in animal nutrition sector are the anti-methanogenic effect described during the fermentation process in rumen ( Martínez-Fern a ´ ndez et al., 2013) and the immunomodulatory and anti-inflammatory properties (Vezza et al., 2019). However, the successful development of any application of this compound must be accompanied by an extensive toxicological evaluation, that guarantees its safety for the final consumers, both humans and livestock. In this regard, the toxicological profile of PTSO has been investigated by cytotoxicity assays in human cell lines (Llana-Ruiz-Cabello et al., 2015b) as well as its genotoxicity and mutagenicity in vitro (Mellado-- García et al., 2015) and genotoxicity in vivo (Mellado-Garcia et al., 2016b). Furthermore, its acute toxicity in vivo has been also evaluated (Llana-Ruiz-Cabello et al., 2015b). Notwithstanding, safe doses of use have been not yet established for PTSO. Thus, a repeated dose 90-days oral toxicity study in rodents of PTSO would be necessary to clearly characterize its toxicity, being also a requirement in the authorization application processes of the European Food Safety Authority (EFSA, 2012, 2016a; 2016b, 2017). The aim of the present study is, therefore, to further explore the toxicity potential of PTSO and, for the first time, to conduct a subchronic dietary toxicity assay of PTSO in rats following internationally recognized test guidelines (Organisation for Economic Co-operation and Development, OECD 408, 2018). Considering these facts, in the present study several parameters have been evaluated, including body weight changes, food and water consumption, feed conversion efficiency, organ weight ratios and biochemistry and hematology parameters. In addition the histopathology of various tissues has been also studied. The results from the complete assessment of this subchronic study would allow to get an estimation of a non-observed-adverse-effect-level (NOAEL) of exposure to establish safety conditions for human exposure to PTSO. 2. Materials and methods 2.1. Test item and doses preparation PTSO was supplied by DMC RC SLU (Granada, Spain) with a 96% of purity. Commercial powder neutral gelatin from pork protein (Jesus Navarro S.A., Alicante, Spain) was employed as the vehicle for the test substance in all groups including the controls. For the 90-days study, the doses were prepared daily for each animal during the 13 weeks, and on Fridays they were also prepared for the weekend. The dose was mixed in 3 mL of liquid gelatin. The volumes of PTSO ( μ L) to add to the gelatin depended on the dose selected for each group, and the gelatin could solidify at 4 ◦ C overnight. Homogeneity of the dietary dose formulations and their stability were confirmed to be at least 5 days. 2.2. Animals conditions and husbandry services The rats were supplied by Charles River laboratories S.L. (Kings, NY, USA), 40 males and 40 females of Sprague-Dawley strain. They were approximately 7 weeks old and were stabilized for an acclimatization period of 7 days during which they were examined by a veterinary surgeon. When the first week of dosage began the rats body weight mean was 320 g 11.3 for males and 227 11.9 for females. Animals were individually housed in cages type 3H with Souralit 29/ 12 plus (souralit S.L., Gerona, Spain) aspen wood bedding and food completely available without restriction using standard dry pellet diet for rodents Scientific (Panlab, S.L.U., Cornella de Llobregat, Barcelona, Spain). They were kept in a room with controlled conditions of hygiene behind a barrier system, a range of temperature of 21 ± 2 ◦ C, with a 10 – 15 air changes per hour, and a relative humidity between 30 and 70% under 12 h light/dark cycle. Each cage contained an information card which contained study code (19-CAM-11-animal number), sex, dose, group, and individual animal identification. Community tap water (EMACSA, Cordoba Water company, C o ´ rdoba, Spain), filtered and autoclaved was available ad libitum. 2.3. Study design The maximum tolerable dose (MTD) for PTSO in rats orally exposed A.C. Lira et al. Food and Chemical Toxicology 144 (2020) 111612 10 Fig. 4. Histopathological study of liver, kidney, spleen, heart, brain, pituitary, stomach, intestines, lung, testes, and and skeletal muscle of untreated Sprague Dawley rats, control (A), and treated (B) with PTSO (55 mg/kg/day) for 90 days (bars = 50 μ m). In the liver, normal hepatic cords and normal polyhedral hepatocytes with central nucleus and clear cytoplasm are seen in control and exposed animals. In the kidney, the renal parenchyma with normal glomeruli and renal tubules is shown in control rats (A), as well as in rats treated with the highest dose of PTSO (B). Detail of the apparently normal spleen parenchyma in control and treated rats (A, B). In the heart, normal cardiac fibers were observed in all groups (A, B). In the brain, the motions of the cerebral cortex are normal (A, B). Details of the apparently normal pituitary are observed in control and treated rats (A, B). Detail of the stomach with apparently normal mucous and glandular cells in control and treated rats (A, B). Intestinal villi with abundant apparently normal enterocytes are shown in all groups. Detail of the control bronchial epithelium, without alterations in the bronchi and alveoli in the untreated rats (A) as well as in the rats treated with the highest dose (B). Male rat testes showed normal seminiferous tubules and interstitial space (A) that is maintained in treated male rats (B). The ovaries of the treated and control female rats (A, B) presented normal follicles in all groups. Detail of the normal striated skeletal muscle of the treated and control rats (A, B). A.C. Lira et al. Food and Chemical Toxicology 144 (2020) 111612 11 (Llana-Ruiz-Cabello et al., 2015b). Mellado-García et al. (2016a) observed that in rats treated with 55 mg/kg (3 doses at 0, 24 and 45h and euthanized at 48h) an increase in the glycogen storage was noticeable in the liver and also a slight degenerative process in the chief cells of the stomach. In this regard, at the dose levels assayed, histopathological lesions were absent. This could be explained by the exposure way employed in this study, using gelatin as vehicle and with the feed. In the previous trials, oral gavage with a stomach tube after a fasting period was used following the recommendations of the corresponding OCDE guidelines. The bolus could have a more deleterious effect on the gastrointestinal system as there is a direct contact. Differences in toxicity between PTSO and Proallium AP® could be explained as the toxicity shown by components of an essential oil can be modulated by the other constituents by synergistic/antagonistic phenomena (Escobar et al., 2015; Pavlidou et al., 2004). The interest of PTSO in comparison to Proallium® is based on its different chemical properties (higher hydrophilia) and in its higher efficiency on its antioxidant and antimicrobial properties. Other authors have shown the potential toxicity of aqueous extracts of different medicinal plants, including Allium sativum, in Wistar rats. Thus, Sulaiman et al. (2014) administered orally to the animals 10 mg/kg of A. sativum extract for 30 days and observed alteration in the activities of marker enzymes: AST increased in liver, kidney and heart, ALT in serum and liver, and ALP activity was reduced in serum, heart, kidney and liver. They concluded that caution was required in using unrefined extracts of these herbs in traditional settings. There are scarce in vivo toxicity data regarding other OSC. Thus, Guyonnet et al. (2000) demonstrated the effects of some of them (DAS, DADS, dipropylsulfiide (DPS) and dipropyl disulfide (DPDS)) on the activation of several mutagens in male Wistar rats exposed to 1 mmol/kg by gavage for 4 days. They explained the results based on the induction of cytochrome (CYP) and phase II enzymes activities. This effect, the alteration of CYP activity, was pointed out by other authors as well (Davenport and Wargovich, 2005). Moreover, they observed hepatotoxicity induced by DAS (bile duct obstruction, hyperproliferation and focal points of necrosis) in rats gavaged daily with 200 mg/kg for 1, 4, or 8 weeks. On the contrary, 8 weeks of exposure to lower doses (50 and 100 mg/kg) did not induced liver histophatological damage. This suggest that liver could be the target organ of OSC as both, DAS (Davenport and Wargovich, 2005) and PTSO (Llana-Ruiz-Cabello et al., 2015b), have shown liver toxicity when a threshold dose is exceeded. Also, Wu et al. (2001) exposed rats orally to garlic oil (GO, 200 mg/kg) and 3 allyl compounds, DAS (20 and 80 mg/kg), DADS (80 mg/kg), and diallyl trisulfide (DATS, 70 mg/kg) 3 times a week for 6 weeks and examined the antioxidation system in rat livers and red blood cells. They found that GO, DADS and DATS significantly induced the glutathione content (GSH) in blood cells but neither GO nor any of its OSC affected the GSH-related antioxidant enzymes. Hepatic GSH was not influenced by garlic components. But DADS and DATS significantly increased the activity of GSH-reductase and GSH-transferase and decreased GSH peroxidase. In the present study the hematological parameters were not influenced by the PTSO exposure and scarce scientific data dealing with PSTO are available to compare. 5. Conclusions In conclusion, the results obtained confirm the already reported safety profile of PTSO for some food applications at the conditions considered. Thus, PTSO did not promote toxic effects as seen from body weight changes, food and water consumption, feed conversion efficiency, biochemical and blood parameters as well as organ toxicity and histological examinations of main organs that could eventually be affected by its subchronic administration (90 days). NOAEL was estimated to be ≥ 55 mg/kg/day. CRediT authorship contribution statement Antonio Cascajosa Lira: Formal analysis, Investigation, Writing - original draft. Ana Isabel Prieto: Formal analysis, Writing - review & editing. Alberto Ba n ˜ os: Resources, Writing - review & editing. Enrique Guillam o ´ n: Resources, Writing - review & editing. Rosario Moyano: Investigation, Resources. Angeles Jos: Writing - original draft, Writing - review & editing, Supervision. Ana M. Came a ´ n: Writing - review & editing, Supervision, Project administration, Funding acquisition. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to acknowledge the Spanish Ministerio de Ciencia e Innovacio ´ n (Project RTC-2017-6199-2), and to the Junta de Andalucía (Project AT 2017-5323 ) for its financial support. References Benkeblia, N., Lanzotti, V., 2007. Allium thiosulfinates: chemistry, biological properties and their potential utilization in food preservation. Food 1, 193 – 201. Davenport, D.M., Wargovich, M.J., 2005. Modulation of cytochrome P450 enzymes by organosulfur compounds from garlic. Food Chem. Toxicol. 43, 1753 – 1762. https:// doi.org/10.1016/j.fct.2005.05.018. Debiagi, F., Kobayashi, R.K.T., Nakazato, G., Panagio, L.A., Mali, S., 2014. Biodegradable active packaging based on cassava bagasse, polyvinylalcohol and essential oils. Ind. Crop. 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The immunomodulatory properties of propyl-propane thiosulfonate contribute to its intestinal anti-inflammatory effect in experimental colitis. Mol. Nutr. Food Res. 63 (5), e1800653 https://doi.org/10.1002/mnfr.201800653. Wu, C.C., Sheen, L.Y., Chen, H.W., Tsai, S.J., Lii, C.K., 2001. Effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells. Food Chem. Toxicol. 39 https://doi.org/10.1016/S0278-6915(00)00171-X, 633-569. 271 CAPÍTULO 10 / CHAPTER 10 Antonio Cascajosa Lira, Silvia Pichardo, Alberto Baños, Enrique Guillamón, Verónica Molina-Hernández, Rosario Moyano, Angeles Jos, Ana M. Cameán. Acute and subchronic 90-days toxicity assessment of propyl-propane- thiosulfinate (PTS) in rats This article was published in Food and Chemical Toxicology, 161, Antonio Cascajosa Lira, Silvia Pichardo, Alberto Baños, Enrique Guillamón, Verónica Molina-Hernández, Rosario Moyano, Angeles Jos, Ana M. Cameán, Acute and subchronic 90-days toxicity assessment of propyl-propane-thiosulfinate (PTS) in rats, 112827, Copyright Elsevier (2022) Food and Chemical Toxicology 161 (2022) 112827 Acute and subchronic 90-days toxicity assessment of propyl-propane-thiosulfinate (PTS) in rats Antonio Cascajosa-Lira a , Silvia Pichardo a , * , Alberto Ba n ˜ os b , Enrique Guillam o ´ n b , Ver o ´ nica Molina-Hern a ´ ndez c , Rosario Moyano c , A ´ ngeles Jos a , Ana M Came a ´ n a a A ´ rea de Toxicología, Facultad de Farmacia, Universidad de Sevilla, Profesor García Gonz a ´ lez n 2, 41012, Seville, Spain b DMC Research Center, Camino de Jayena, 82, 1862, Alhendín, Spain c Departamento de Anatomía y Anatomía Patolo ´ gica Comparadas y Toxicología, Facultad de Veterinaria, Universidad de C ´ ordoba, Campus de Rabanales, 14014, C o ´ rdoba, Spain A R T I C L E I N F O Handling Editor: Dr. Jose Luis Domingo Keywords: Subchronic toxicity 90-Days Organosulfur compounds Allium Propyl-propane-thiosulfinate A B S T R A C T The organosulfur compounds (OSC) extracted from Allium spp. exhibit antibacterial, antifungal, and antioxidant properties. The agri-food industry is taking advantage of these properties by using them as natural feed and food additives. In the present work, an acute and a subchronic 90-days toxicity studies have been conducted for the first time to assess the safety of the OSC propyl-propane-thiosulfinate (PTS). Both studies were carried out following the Organization for Economic Co-operation and Development test guidelines (425 and 408, respectively). The acute study provided a maximum tolerated dose (MTD) of 175 mg/kg and the subchronic study established the Non Observed Adverse Effect Level (NOAEL) ≥ 55 mg/kg body weight (b.w.)/day in both sexes. In addition, the subchronic study performed on rats exposed to 14, 28 and 55 mg/kg b.w./day PTS, revealed no changes in any of the hematological parameters measured as well as no differences in body weight and water/ food consumption. However, biochemical parameters were altered in some groups, although they were not biologically significant (Ca 2 + in female rats, and the thyroids hormones T3 and T4 in rat males). Furthermore, the histopathological assessment evidenced no abnormality on the gastrointestinal, respiratory, lymphoid, urinary, circulatory, nervous, musculoskeletal, and reproductive systems. 1. Introduction Organic sulfur compounds (OSCs) are phytochemical molecules present in many species of the Allium genus. This genus includes more than 600 species, among them, the edibles highlight: garlic (A. sativum) onion (A. cepa), leek (A. porrum), chive (A. schoenoprasum), shallot (A. ascalonicum), and giant flowering onion (A. giganteum). The OSCs are well-known for their biological properties, among which antibacterial, antifungal, antioxidants, antiviral, antiprotozoal, anti-inflammatory properties stand out (Farhat et al., 2021; Putnik et al., 2019; Vezza et al., 2019). In onion, the most common OSCs are isoalliin (S-prope- nyl-L-cysteine sulfoxide) that changes into methiin (S-methyl-L-cysteine sulfoxide), also present in garlic; and propiin (S-propyl-L-cysteine sulfoxide) that, due to the action of alliinase, leads to propyl-propane- thiosulfinate (PTS) (C 6 H 14 O 1 S 2 ) ( Guillam o ´ n et al., 2021). Their properties are of great interest to the agri-food industry, in which there has recently been a high boom in the demand for additives of natural origin (Cascajosa-Lira et al., 2020a). Additionally, in the last years the number of research and patents of OSCs have increased: the use of dialkyl thiosulfonate or thiosulfinate to reduce the number of apicomplexa in animals (Bravo and Lillehoj, 2013) or the use of PTS and propyl propane thiosulfonate (PTSO) for the prevention and reduction of parasites in aquatic animals ( Ba n ˜ os Arjona et al., 2016). PTS has demonstrated antifungal activity against Verticillium dahliae in olive trees suggested that this compound could be used as natural tool for Crop Pest Management ( Falc o ´ n-Pi n ˜ eiro et al., 2021). Moreover, PTS has shown beneficial effects on goats as an inhibitor of methanogenesis ( Martínez-Fern a ´ ndez et al., 2013 , 2015). Another use for PTS is as sensory additive in animal nutrition, improving the palatability of feed and being an alternative to the use of antibiotics in farm animals. It has been suggested to be used as an alternative of antibiotics (Peinado et al., 2012, 2013) supporting the decrease in bacteria resistance generated by their excessive application in farm animals. The regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 * Corresponding author. A ´ rea de Toxicología, Facultad de Farmacia, Universidad de Sevilla, Profesor García Gonz a ´ lez n 2, 41012, Seville, Spain. E-mail address: spi[email protected] (S. Pichardo). https://doi.org/10.1016/j.fct.2022.112827 Received 22 October 2021; Received in revised form 7 December 2021; Accepted 17 January 2022 Available online 22 January 2022 0278-6915/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox A. Cascajosa-Lira et al. Food and Chemical Toxicology 161 (2022) 112827 2 ± Fig. 1. Flowchart of the subchronic toxicity study. September 2003 sets the parameters of handling of the additives used in animal nutrition. With respect to this legislation, the use of antibiotics as growth promoters is forbidden; the development of alternative products to replace those antibiotics is expected. In addition, OSCs or essential oils are being employed as additives in food and feed (Cascajosa-Lira et al., 2021a), either because of their flavoring character or their antioxidant or antimicrobial properties (Llana-Ruiz-Cabello et al., 2015; Maisanaba et al., 2017). In this sense, they can be a good alternative for additives of chemical origin, such as sulfites, used in meats, wines or juices that cause allergic reactions in some consumers (EFSA, 2016a) and also could degrade molecules with nutritional value such as thiamine or vitamin B1 (Singh et al., 2005). For this reason, there is an increased demand of new products that guarantee safety and similar production levels, without generating unwanted effects, such as human resistances or side effects, being additives of natural origin a good alternative. In this regard, it is necessary to have a comprehensive knowledge of its toxicological profile to guarantee its safety. Regarding the toxicological profile of PTS, at the present time there are available cytotoxicity assays in mammalian cells as well as mutagenicity and genotoxicity assessment in vitro (Mellado-García et al., 2017) and an in vivo genotoxicity study in rats (Cascajosa-Lira et al., 2021). The European Food Safety Authority (EFSA) required a 90-days oral toxicity study among the toxicological assays for the approval of food and feed additives (EFSA 2012; 2016b, 2017) following the guideline 408 of the Organization for Economic Cooperation and Development (OECD). Recently, several subchronic studies in rats treated with OSC such as PTSO (Cascajosa-Lira et al., 2020b) and Allium extract (Mellado-García et al., 2016) have been published. Moreover, different essential oils have shown antioxidant and antimicrobial characteristics that are as well of interest in the agri-food industry (Lira et al., 2020; Llana-Ruiz-Cabello et al., 2017; Mellado-García et al., 2016; Mishra et al., 2018; Preece et al., 2021). However, the acute and subchronic studies of PTS itself has not been conducted so far. Therefore, the objectives of this work were to carry out, for the first time, an acute toxicity study (OECD 425, 2008) and a 90-day subchronic toxicity study by oral route in rats (OECD 408, 2018) in order to study the possible adverse effects in long terms of PTS by establishing the NOAEL (no observed adverse effect level). 2. Materials and methods 2.1. Dose preparation and acute toxicity study design PTS was supplied by DMC Research Center (Granada, Spain) with a 76% of purity (cod. 19.10.2020). The results of the PTS analysis by UPLC-MS-MS were reported in Cascajosa-Lira et al. (2021a). In order to determine the maximum tolerable dose (MTD), the acute oral toxicity study (Up and Down procedure, OECD 425, 2008) was carried out. Initially, one rat was administered by gastric tube with a single dose of 175 mg/kg body weight (b.w.) PTS. Then, the single doses to the consecutive rats were administered in increasing/decreasing order: 550 and 2000 mg/kg b.w. PTS depending on the morbidity or mortality of the animals. Mortality and clinical sign were observed for two weeks before sacrifice. If animals showed moribund signs or obvious severe pain, then they were humanely sacrificed. Three animals were dosed with 175 mg/kg b.w., 3 were exposed to 550 mg/kg b.w., and only 1 animal was treated with 2000 mg/kg b.w. 2.2. Subchronic study design A total of 80 rats (40 males and 40 females with similar body weight mean in a range of 20% as mentioned in the guideline) were randomly distributed into eight groups (10 rats/sex/group of doses). The distribution of rats in this study is described in Fig. 1. The highest dose was set at 55 mg/kg b.w. accordingly with the MTD and the palatability of the product, and descending doses were selected using a factor of root of ten according to the guideline (OECD 408, 2018) recommendations: 14, 28, 55 mg/kg b.w. Therefore, 3 groups of rats from each sex were exposed to 14, 28, 55 mg/kg b.w./day PTS. The commercial powder neutral gelatin from pork protein was employed as the vehicle for the test substance in all groups including the control. The dose was mixed in 3 mL of liquid gelatin. PTS was included in the gelatin that solidified at 4 ◦ C overnight following Cascajosa-Lira et al. (2020b). In addition, the vehicle-control group were fed with gelatin without PTS. A. Cascajosa-Lira et al. Food and Chemical Toxicology 161 (2022) 112827 3 ± 2.3. Animal conditions and husbandry services All animals received human care in accordance with the guidelines for the protection of animals used for the science purposes (Directive, 2010/63/EU, Decision, 2020/569/UE, and RD 1386/2018). All procedures have been approved by the Ethical Animal Experimentation Committee of the University of C o ´ rdoba and by the Junta de Andalucía (project nº 22/12/2020/154). The rats, Sprague-Dawley strain, were 7 weeks old. Initially, all of them become stable for an acclimatization phase of 7 days. Animals were individually housed in type 3H cages with aspen wood bedding and food completely available using standard dry pellet diet for rodents. They were kept in a room with controlled conditions of hygiene behind a barrier system, a range of temperature of 21 2 ◦ C with a 10 – 15 air changes per hour and a relative humidity between 30 and 70% under 12 h light/dark cycle. 2.4. Clinical observation and ophthalmic examination Examination included observations of skin and fur, eye and mucous membranes, respiratory and circulatory effects, automatic effects as salivation, central nervous system, motor activity, including tremors and convulsions, changes in motor activity, gait and posture, reactivity to handling or sensory stimuli, and normally or strange behavior. For the ophthalmic examination, before and after treatment, the following organs were also observed: cornea, crystalline lens, conjunctivae, sclera, iris, and fundus of all animals. 2.5. Body weight, food, and water consumption These three parameters were weekly recorded in order to avoid the stress of the animals and the means per groups and sex were calculated. The total food consumed per cage was recorded and weekly mean intake per rat was calculated, required to determinate feed ratio conversion efficiency (FCE) according to Cascajosa-Lira et al. (2020b) by the ratio of food intake (g)/weight gained (g). 2.6. Haematology, biochemistry and, endocrinology As recommended OECD 408 (2018), once the animals were sacrificed under anesthesia effects, blood samples were extracted from the heart by an intracardiac injection. Then, the hematological parameters were determined on an automatic hematological analyzer Cell-Dyn 3700 (Abbot, GMI, MI, USA): red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hematoglobin (MCH), mean corpuscular hematoglobin concentration (MCHC), blood platelet count (PLT), red cell volume distribution (RDW), prothrombin time (PT), cefaline time (CT), white blood cell count (WBC), neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO) and basophils (BA). The standard serum biochemistry parameters were analyzed with an automatic chemistry analyzer Cobas 6000 (Roche Diagnostics, IN, USA), to determinate these biochemistry parameters: glucose (GLUC), blood urea nitrogen (UREA), creatine (CREAT), bile acids (BILI-T), total cholesterol (CHOL), high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides (TRIGL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), albumin (ALB), total protein (TOT PROT), sodium (Na + ), potassium (K + ) and calcium (Ca 2 + ) ions. The hormones: triiodothyronine (T3) and thyroxine (T4) were also measured using an analyzer Cobas 6000 (Roche Diagnostics, IN, USA). Thyroid stimulating hormone (TSH) was measured by a commercial ELISA kit from Demeditec (DEV9977 TSH rat ELISA, Demeditec Diagnostics GmbH, Germany). 2.7. Necropsy, organ weight and histopathology All animals survived the complete treatment. Then, rats were fasted overnight (for 18 h before the sacrifice) and deeply anaesthetized with isofluorane. Later, they were euthanized with CO 2 and exsanguinated by intracardiac injection according to directive 2010/63 UE. All rats received a complete pathology examination through the necropsy. The following tissues and organs were collected from animals and weighed wet in situ after dissection: brain, liver, heart, spleen, kidneys, thymus, adrenal glands, uterus with cervix and ovaries (females) and testes, epididymis and seminal gland (males). For the histopathological assay, tissues from the gastrointestinal system (liver, pancreas, glandular and nonglandular stomach, and small and large intestines), from the respiratory, lymphoid, urinary, circulatory, musculoskeletal, and nervous systems (lung, spleen, kidney, heart, skeletal muscle, cerebellum, and cerebrum), and the reproductive system (ovary and testes) were taken from the control and the PTS administered rats (14, 28 and 55 mg/kg b.w./day). All the tissue samples were fixed in 10% buffered formalin for 24h and routinely processed and embedded in paraffin wax. Tissue sections (4 μ m thick) were stained with haematoxylin and eosin (H&E). 2.8. Statistical analysis Statistical analysis was carried out using Graph-Pad Prism 9 software (GraphPadSoftware Inc., La Jolla, USA). Continuous variables were summarized using mean and standard deviation (SD) and were reported by sex and dosage. One-way analysis of variance (ANOVA) was carried out to test differences in continuous variables. Normality assumption was tested using Kolmogorov-Sminorv ’ s test. If non-normality was observed, comparison was performed with Kruskal-Wallis test. If those tests were statistically significant, multiple comparisons were performed using Tukey-Kramer/Dunn ’ s Multiple Comparisons Tests. 3. Results 3.1. Acute study. LD50 and MTD Following the guidelines of the OECD 425, the first dose assayed was 175 mg/kg b.w. given by gavage. Considering the results obtained in each step the dose increased to 550 and 2000 mg/kg b.w. The results obtained in each dose were the following. None of the rats orally exposed to 175 mg/kg b.w. died or experienced any clinical injury. Different responses were recorded in rats exposed to 550 mg/kg b.w.; one rat was sacrificed for human reasons; another rat survived with severe irreversible signs of toxicity; and the other rat survived showing light signs of toxicity. In addition, two out of the three rats exposed to 550 mg/kg b.w. underwent a decrease in weight gain above 20%. The rat dosed with 2000 mg/kg b.w. of PTS died immediately after exposure. Consequently, the LD50 of PTS was stablished at 550 mg/kg b.w., and MTD at 175 mg/kg b.w. However, due to palatability acceptance of the product, the dose for the subchronic study was lowered at 55 mg PTS/kg b.w. as previously explained in section 2.2. 3.2. Clinical and ophthalmological observation No changes in gait, posture, or handling response, abnormal, clonic, or tonic movements, and stereotypes or bizarre behavior was recorded in any group. The ophthalmological examination performed at the end of the study showed no abnormalities in all doses and sex groups during the study period. Some contact injuries were observed in seven rats in the 28 and 55 mg/kg b.w./day dose groups on back, face and hands. No other clinical observations were noted. A. Cascajosa-Lira et al. Food and Chemical Toxicology 161 (2022) 112827 4 Table 1 Effect of 90 days oral exposure to PTS on body weight and food consumption in Sprague Dawley. Values are mean ± SD for 10 rats/sex/group. F: Statistics ANOVA test; K.W: Kruskal-Wallis Statistic; N.S.: Not Significant. MALE FEMALE PARAMETERS Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 (0 mg/kg/day) (14 mg/kg/ day) (28 mg/kg/ day) (55 mg/kg/ day) (0 mg/kg/day) (14 mg/kg/ day) (28 mg/kg/ day) (55 mg/kg/ day) Inicial body weight (g) Final body weight (g) N ¼ 10 N ¼ 10 N ¼ 10 N ¼ 10 N ¼ 10 N ¼ 10 N ¼ 10 N ¼ 10 360.5 ± 12.18 313.00 ± 31.34 309.80 ± 13.35 317.10 ± 14.12 223.40 ± 12.22 222.00 ± 13.03 229.20 ± 12.15 220.40 ± 13.83 F(36.3) ¼ 0.5309 p ¼ 0.6640; N.S. F(36.3) ¼ 0.8938 p ¼ 0.4537; N.S. 647.40 ± 48.14 623.00 ± 63.24 645.00 ± 37.96 658.50 ± 35.15 334.70 ± 23.35 345.00 ± 19.29 351.50 ± 27.50 334.80 ± 17.59 F(36.3) ¼ 0.9836 p ¼ 0.4118; N.S. KW ¼ 2.697 p ¼ 0.4408; N.S. Body weight gain (g) 340.90 ± 43.83 310.00 ± 64.22 335.20 ± 29.85 341.40 ± 30.93 121.30 ± 17.60 123.00 ± 9.78 122.30 ± 18.00 114.40 ± 12.08 Total feed intake (g) F(36.3) ¼ 1.118 p ¼ 0.3546; N.S. F(36.3) ¼ 0.7174 p ¼ 0.5481; N.S. 3241.10 ± 314.04 3165.60 ± 293.63 3304.60 ± 249.09 3485.40 ± 263.36 2127.80 ± 215.17 2171.80 ± 219.48 2210.20 ± 291.03 2049.40 ± 208.61 Feed conversion F(36.3) ¼ 2.358 p ¼ 0.0879; N.S. F(36.3) ¼ 0.8560 p ¼ 0.4727; N.S. 9.56 ± 0.71 10.55 ± 1.93 9.90 ± 0.77 10.25 ± 0.86 17.79 ± 2.54 17.72 ± 1.99 18.32 ± 3.20 18.00 ± 1.73 F(36.3) ¼ 1.315 p ¼ 0.2845; N.S. F(36.3) ¼ 0.1258 p ¼ 0.9442; N.S. Values are mean ± SD for 10 rats/sex/group. The differences between control and treated groups for male and female rats were evaluated by Kruskal-Wallis test (K.W.) or by ANOVA-Tukey test (F values). N.S. not significant. Fig. 2. Mean body weights (g) of A) male and B) female rats orally exposed to 14, 28 and 55 mg/kg b.w./day PTS and control rats for 90 days. Values are mean ± standard deviation (SD) for 10 rats/sex/group. The differences between control and treated groups for male and female rats were evaluated by ANOVA- Tukey tests. 3.3. Body weight, body weight gain, food consumption, feed conversion efficiency, and water consumption There are no significant differences between groups of doses in the final body weight, body weight gain, total food intake or feed conversion efficiency (Table 1). The mean body weight per week of male and female rats exposed to PTS (14, 28 and 55 mg/kg b.w./day) was not significantly changed through the complete experiment in males (Fig. 2A) and Fig. 3. Mean body weight gain (%) of A) male and B) female rats orally exposed to 14, 28 and 55 mg/kg b.w./day PTS and control rats for 90-day. Values are mean ± SD for 10 rats/sex/group. The differences between control and treated groups for male and female rats were evaluated by Kruskal-Wallis test or ANOVA-Tukey tests. females (Fig. 2B). Similarly, no significant changes were observed in % body weight gain in males (Fig. 3A) and females (Fig. 3B). There were significant differences in food consumption in control and 14 mg/kg b. w./day groups in comparison to 55 mg/kg b.w./day in males for three weeks as were shown in Fig. 4A. In contrast, there were no significant ratio A. Cascajosa-Lira et al. Food and Chemical Toxicology 161 (2022) 112827 5 Fig. 4. Mean Food consumption (g) of A) male and B) female rats orally exposed to 14, 28 and 55 mg/kg b.w./day of PTS and control rats for 90-day. Values are mean ± SD for 10 rats/sex/group. The differences between control and treated groups for male and female rats were evaluated by Kruskal-Wallis test or ANOVA-Tukey tests. The significance levels observed are α p < 0,05 when 14 mg/kg b.w./day, and 55 mg/kg b.w/day. groups were compared. & p < 0,05 when control group and 55 mg/kg were compared. changes in female groups (Fig. 4B). The water consumption in male rats did not show any significant differences (Fig. 5A). But in the other hand, female rats exposed to 28 and 55 mg/kg b.w./day showed significant differences in comparison with control group in the second week and in the eleventh week respectively (Fig. 5B). 3.4. Biochemistry and haematology Haematology parameters are shown in Table 2. There were no significant differences in any parameter measured for male and female rats. In addition, there were no significant differences in total and differential leukocyte counts for rats fed with PTS for both sexes (Table 3). Clinical biochemistry parameters of rats exposed to all levels of PTS doses (14, 28, 55 mg/kg b.w.) are shown in Table 4. These results revealed significant differences in Ca 2 + in female rats exposed to 14 mg/ kg b.w./day in comparison with control group. In addition, thyroids hormones showed significant differences in male rats. T3 levels revealed significant increase in rats exposed to 14 mg/kg b.w./day Besides, T4 levels are also increased in the same group of dosage and additionally in 55 mg/kg b.w./day in comparison with control group in male rats. 3.5. Necropsy and organ weights The macroscopy examination of the organs and tissue did not show any remarkable damage in any dosage level assayed of PTS. The organ weight (brain, liver, heart, spleen, kidney, thymus, adrenals, testes, Fig. 5. Mean water consumption (mL) of A) male and B) female rats orally exposed to 14, 28 and 55 mg/kg b.w./day of PTS and control rats for 90-day. Values are mean ± SD for 10 rats/sex/group. The differences between control and treated groups for male and female rats were evaluated by Kruskal-Wallis test or ANOVA-Tukey tests. The significance levels observed are #p < 0,05 when control group and 28 mg/kg b.w./day were compared & p < 0,05 when control group and 55 mg/kg b.w./day were compared. epididymis, seminal gland, uterus, and ovaries) of male and female rats of control and treated groups are shown in Table 5 as well as the ratio of the weight of the organ with respect to the total weight in Table 6 and with respect to the brain weight in Table 7. Subchronic administration of PTS did not revealed any significant difference in the organ weight or ratios. 3.6. Histopathology The histopathology of the organs from the gastrointestinal, respiratory, lymphoid, urinary, circulatory, nervous, musculoskeletal, and reproductive systems were firstly assessed in the control and 55 mg/kg b.w./day PTS groups including male and female rats. The results obtained from the histopathological study revealed no observable pathological features in the rats administered with 55 mg/kg b.w./day PTS dose compared with the controls in any studied organs. Moreover, the comparative study between male and female rats no displayed histopathological differences neither. The representative histological images of the gastrointestinal system are included in Fig. 6. Thus, control and 55 mg/kg b.w./day PTS male and female rats presented a histologically normal liver with hepatocytes extending from the portal spaces to central veins without pathological features. In addition, pancreases showed both endocrine and exocrine components with normal appearance no displaying any pathological change in the samples from the control and 55 mg/kg b.w./day PTS male and female rats. Both glandular and non-glandular (squamous) stomachs did not display pathological changes in any group studied showing an A. Cascajosa-Lira et al. Food and Chemical Toxicology 161 (2022) 112827 12 Fig. 6. Representative histological images of the gastrointestinal organs comparing both control and 55 mg/kg b.w./day PTS rats for 90 days. H&E stain. 5. Conclusions Administration of PTS to male and female rats in the diet for 90-days, following the OECD 408 guideline, caused no significant changes in any hematological and biochemical parameters measured. Moreover, no differences in body weight and water/food consumption, as well as none histopathological abnormalities were recorded. Therefore, the NOAEL Fig. 7. Representative histological images of organs from respiratory, lymphoid, renal, circulatory, musculoskeletal, and nervous systems comparing both control and 55 mg/kg b.w./day PTS rats. H&E stain. [Document text truncated for crawler view.]