Identification of in vitro metabolites of an Allium organosulfur compound and environmental toxicity prediction as part of its risk assessment

1 This is an Accepted Manuscript of an article published by Elsevier in Environmental Research 229, 116001 on 15 July 2023, available at: https://doi.org/10.1016/j.envres.2023.116001 . It is deposited under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. 1 2 Identification of in vitro metabolites of an Allium organosulfur compound and 3 environmental toxicity prediction as part of its risk assessment 4 Antonio Cascajosa-Lira1, Concepción Medrano-Padial1, Silvia Pichardo1*, José Manuel de la Torre2, 5 Alberto Baños2, Ángeles Jos1, Ana M. Cameán1. 6 1 Área de Toxicología, Facultad de Farmacia, Universidad de Sevilla, Profesor García González n 2, 41012 7 Seville, España. 8 2 DMC Research Center, Camino de Jayena, 82, 18620 Granada, España. 8 9 10 *Corresponding author: Silvia Pichardo 11 Área de Toxicología, Facultad de Farmacia, Universidad de Sevilla, Profesor García González n 2, 41012 12 Seville, España. 13 E-mail address: [email protected] 14 Telephone number: (0034) 639513371 15 Fax number: (0034) 954556422 16 2 17 Abstract 18 Propyl-propane-thiosulfonate (PTSO) is an organosulfur compound found in Allium spp. 19 Due to its antioxidant and antimicrobial activities, PTSO has been proposed for 20 applications in the agri-food sector, such as feed additive. However, its use with 21 commercial purposes depends on its toxicity evaluation. The present work aimed to 22 perform a pilot-study of toxicokinetic profile of PTSO combining in silico and in 23 vitro techniques, important steps in the risk assessment process. In silico ecotoxicity 24 studies were also performed considering the importance of the environmental impact of 25 the compound before its commercial use. First, an analytical method has been developed 26 and validated to determine the original compound and its metabolites by ultra- 27 performance liquid chromatography-tandem mass spectrometry. The phase I and II 28 metabolism of PTSO was predicted using Meta-Pred Web Server. For the phase I 29 metabolism, rat (male and female) and human liver microsomes were incubated with 30 PTSO and NADPH regeneration system. Furthermore, in the phase II, microsomes were 31 incubated with PTSO and glutathione or uridine 5’- diphosphoglucuronic acid. The 32 analysis revealed the presence of propylpropane thiosulfinate (PTS) originated by redox 33 reaction in phase I, and two conjugates from the phase II: S-propylmercaptoglutathione 34 (GSSP) and S-propylmercaptocysteine (CSSP). Additionally, considering the 35 environmental fate of PTSO and its metabolites, the ADME parameters and the potential 36 ecotoxicity were also predicted using in silico softwares. The results of the ecotoxicity in 37 silico study evidenced that the metabolism induced the formation of detoxified 38 metabolites from the parent compound, except for dimercaprol and 3- 39 mercaptopropane1,2-diol. Further in vivo assays are needed to confirm this prediction. 40 Keywords: Toxicokinetics, Allium, additive, bioactive compounds, metabolites, 41 UHPLC-MS/MS. 3 42 Funding sources 43 - Junta de Andalucía: Project P18-TP-2147. 44 - Spanish Ministerio de Universidades: FPU2019-01247 granted to Antonio 45 Cascajosa-Lira. 4 46 1. Introduction 47 Organosulfur compounds (OSC) are phytochemical molecules present in many 48 species of the Allium genus as secondary metabolites (González-Ramírez et al., 2022). 49 This genus includes more than 600 species, among them, the most common types of 50 edibles: garlic (A. sativum), onion (A. cepa), leek (A. porrum), chive (A. schoenoprasum), 51 shallot (A. ascalonicum), and giant flowering onion (A. giganteum) (Demir et al., 2022). 52 The OSCs are generated by an enzymatic cascade as a result of physical aggression to the 53 plant like cut and crush. The alliinase degrades initial compounds as alk(en)yl cysteine 54 sulfoxides (ACSOs) giving rise to new molecules: sulfonic acids, thiosulfonates, 55 thiosulfinates, diallyl, monodi-, and tri-sulphides, vinyldithiins, etc. (Poojary et al., 56 2017). These molecules are well-known for their biological properties, such as 57 antibacterial, antifungal, antioxidant, antiviral, antiprotozoal, etc. (Putnik et al., 2019; 58 Farhat et al., 2021) awaking the interest of the agri-food industry due to their diverse 59 spectrum of biological applications (Baños et al., 2019; Cascajosa-Lira et al., 2021a, 60 2021b, 2020a, 2020b). In this sense, during the last decade the number of patents and 61 research of OSCs related to sustainable livestock have increased: the use of dialkyl 62 thiosulfonate or thiosulfinate to reduce the number of apicomplexa and other parasites in 63 animals (Bravo and Lillehoj, 2013; Aguinaga-Casañas et al., 2022; Cabello-Gómez et al., 64 2022) or the use of propyl propanethiosulfinate (PTS) and propyl propanethiosulfonate 65 (PTSO) for the prevention and reduction of parasites in aquatic animals contributing to 66 the elimination of residues generated by antibiotics and antiparasitics (Baños-Arjona et 67 al., 2016). 68 Propyl-propane-thiosulfonate (PTSO, C6H14O2S2) is one of these interesting 69 compounds which has been characterized by DMC Research Center (Granada, Spain). 70 Because of its properties, it has been proposed in different applications in the agri-food 5 71 sector (Peinado et al., 2012, 2013; Llana-Ruiz-Cabello et al., 2015). Since the regulation 72 (EC) No 1831/2003 of the European Parliament of the Council of 22 September 2003 73 forbids the use of antibiotics as growth promoters in farm animals, PTSO is considered 74 an interesting alternative (Peinado et al., 2012, 2013). It has been proved that this 75 compound when used as a sensory additive in animal nutrition, improves the palatability 76 of feed and decreases bacteria resistance caused in animals by the excessive use of 77 antibiotics (Peinado et al., 2012,2013). Moreover, there is an increase in the demand of 78 new natural additives to avoid synthetic preservatives, consequently, the OSCs isolated 79 and essential oils are being used for this purpose in meats or sauces because of their 80 antimicrobial properties, their flavoring character or antioxidant ability (Llana-Ruiz- 81 Cabello et al., 2015; Maisanaba et al., 2017). Considering all this background, PTSO 82 could be a good substitute for additives of artificial origin. 83 Before its industrial use, the European Food Safety Authority (EFSA) requests 84 toxicological studies to establish the main toxicological profile of PTSO (EFSA 2012). 85 There are already some experimental studies performed with PTSO to assess its toxicity: 86 cytotoxicity assays in mammalian cells, mutagenicity, and genotoxicity assessment in 87 vitro (Mellado-García et al., 2015), in vivo genotoxicity study in Sprague-Dawley rats 88 (Mellado-García et al., 2016), and an in vivo subchronic 90-days study (Cascajosa-Lira 89 et al., 2020a). The results suggest a safe use of PTSO so far. Moreover, among other 90 studies, toxicokinetic assays are also required to support the understanding of the 91 mechanisms of toxicity of the tested compound (OECD TG 417, 2010). Thus, it is 92 necessary to have a comprehensive knowledge of the toxicokinetic profile of PTSO as 93 well as a validated method to analyze the parent compound and its metabolites to ensure 94 its safe use. Regarding the analytical profile of PTSO, in the last years, new methods have 95 emerged for the determination of this compound in different food and feed matrices (Abad 6 96 et al., 2016; Cascajosa-Lira et al., 2021a). Some metabolites have been characterized and 97 monitored in food and feed supplemented with PTSO (Abad et al., 2016, 2017). However, 98 these studies are still scarce, and more information is needed. In this regard, it is 99 appropriated to perform a pilot in vitro study with microsomes from several species and 100 both sexes, to address the potential gender and species differences, needed in the 101 assessment of dose dependency of metabolism in relation to exposure levels. In addition, 102 microsomes may be useful to identify the specific microsomal enzymes involved in the 103 metabolism of the substance which can be relevant in species extrapolation (Chavan et 104 al., 2018). 105 Metabolism plays a central role in regulating the toxicity of a variety of chemicals. 106 Relatively innocuous substances can be converted to highly toxic metabolites. The 107 present work aimed to perform an in silico study of PTSO and its metabolites. Moreover, 108 the ADME parameters were also predicted. In addition, the environmental fate of PTSO 109 is expected since one of its more promising uses is as feed additive. In order to complete 110 the in silico toxicity assessment, the ecotoxicity of products derived from PTSO was 111 assayed to predict their impact in the environment. As far as we know, there are not data 112 about this subject in the scientific literature. 113 Taking into account all this background, the objectives of this work were to carry 114 out, for the first time, an in vitro metabolism study for PTSO, using female and male rats 115 and human microsomes, in order to identify and characterize the metabolites of PTSO 116 and to know potential differences in the metabolism linked to the species or sex. In 117 addition, an appropriate analytical method to determine the metabolites as part of the risk 118 and safety assessment by UHPLC-MS/MS has been validated. Finally, the in silico 119 120 ADME and toxicity data of the main metabolites identified were predicted. 7 121 2. Materials and Methods 122 2.1. Materials and reagents 123 PTSO (99% Ref. 311220), propyl-propane thiosulfinate (PTS) (99% Ref. 124 150622), S-propylglutathione conjugate (GSSP) (99% Ref.160720), and S- 125 propylcysteine (CSSP) (99% Ref.170720) were supplied by DMC Research center. 126 Testosterone and 7-hidroxicumarine and reduced glutathione were obtained from Sigma- 127 Aldrich (St. Louis, MO, USA). HPLC grade methanol (MeOH), acetonitrile (ACN), and 128 other solvents were purchased from Merck (Darmstadt, Germany). Pooled Rat Liver 129 Microsomes (RLM) of both sexes and Human Liver microsomes (HLM), Uridine 130 diphosphate (UDP) reaction mix solution, and NADPH Regenerating System were 131 purchased from Corning Gentest (Woburn, Massachusetts, USA). Ultrapure water (18.2 132 MΩ cm resistivity) purified by the NANOpure DiamondTM (Barnstead, USA) system 133 was used. 134 2.2. Validation of the analytical method 135 The analytical method used to determine PTSO and its metabolites was validated 136 attending to the following parameters: limits of detection and quantification (LOD, LOQ), 137 working range, trueness, and precision, according to different analytical guidelines 138 (AOAC, 2016; González et al., 2010; González and Herrador, 2007). 139 About this, known metabolites such PTS, GSSP, and CSSP were validated in a 140 concentration range of 0.01-0.1-1 mg L-1. Furthermore, PTSO was validated in a 141 concentration range of 1-2.5-5-10 mg L-1. Lower concentrations were employed in the 142 case of metabolites since they will be always found in a lower concentration than the 143 parent compound. Stock solutions of the different analytes were prepared in ACN 50%. 8 144 The LOD and LOQ values were obtained using the standard deviation (SD), 145 calculated from the calibration curve using the residual standard deviation of the 146 regression line. Likewise, the response and the slope of the calibration curves, following 147 the equation 3xSD/slope and 10xSD/slope, for LOD and LOQ, respectively (ICH 148 Harmonised Tripartite Guideline, 2005). The recoveries were calculated for each 149 concentration in triplicate per day, on 3 different days, including the working range, 150 according to the protocol described by Cascajosa-Lira et al., (2021a). After that, the 151 results were evaluated considering the respective tabulated reference values at each 152 concentration level, according to AOAC (2016). 153 2.3. In silico analysis of human metabolism 154 Meta-Pred Web Server was used as an open access service to predict the site of 155 metabolism of PTSO based on their structure–activity relationship algorithms. The 156 chemical structure was submitted and the probability of being metabolized by several 157 isoforms of cytochrome (CYP) (CYP1A2, CYP2C19, CYP2D6, CYP2D9, and CYP3A4) 158 and by uridine 5'-diphospho-glucuronosyltransferase (UGT) was calculated. 159 2.4. In vitro metabolism 160 The assays were performed following the methodology of Lee et al., (2015) with 161 some modifications (Feng et al., 2021; Rani et al., 2018). Three different groups were set: 162 one for phase I (oxidation) and two for phase II (conjugation with glutathione or 163 glucuronic acid). In all the experiments rats microsomes for both sexes, male (MRLM) 164 and female (FRLM), and human microsomes (HLM) were employed. 165 The metabolism reaction mixture (total volume 1 mL) consisted of MRLM, 166 FRLM or HLM in 100 mM phosphate buffer saline (PBS) (pH 7.4). In the case of phase 167 I metabolism study, this mixture was preincubated at 37°C for 5 min; then, PTSO was 9 168 added, and the reaction was initiated by the addition of nicotinamide adenine dinucleotide 169 phosphate oxidase (NADPH)-regenerating solution containing 1.3 mM NADP+, 3.3 mM 170 glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, and 3.3 mM 171 MgCl2. 172 In order to study the possible conjugation with glutathione in phase II, after the 173 oxidation process, 5 mM of glutathione was added. Glucuronidation was determined in 174 the presence of UDP–glucuronic acid under similar reaction conditions, except that UDP– 175 glucuronosyltransferase was added to the reaction mixture which included 50 mM Tris– 176 HCl (pH 7.5), 8 mM MgCl2, 25 g/mL alamethicin, and 2 mM uridine 5’- 177 diphosphoglucuronic acid (UDPGA). 178 Positive controls have been used to test the activity of the enzymes in charge of 179 metabolism (CYP450 and glucuronosyltransferase). The positive control for oxidative 180 process (phase I) was testosterone in a final concentration of 20 M, and 7- 181 hydroxycoumarine for glucuronidation (phase II) in a final concentration of 1 M. A 182 positive control is unnecessary in the conjugation with GSH, as the reaction can take place 183 in the presence or absence of glutathione S-transferase. 184 185 2.5. Sample preparation 186 Samples of each experiment were collected (50 µL) at each time (0, 0.5, 1.5, 10, 187 15, 30, 60, 120 min) of the reaction, and immediately the same volume of ice-cold ACN 188 was added in order to stop the metabolic reaction. Then, samples were vortex for 2-3 min 189 and stored in ice until their analysis. 190 2.6. UHPLC MS/MS equipment and conditions 16 337 PTS (EFSA, 2012). Other frequent metabolic pathways found in our study were 338 dehydrogenation, reduction, and nitro-reduction. Among all the metabolites found in 339 phase I, 5 of them are present when using rats and human microsomes (M3, M5, M8, M9 340 and, M12). Moreover, 2 of them are present only in rats microsomes (M6 and M7), 341 probably due to the different metabolic pathways that occur between species. Regarding 342 sex differences in metabolism, there are 5 metabolites (M1, M2, M4, M10, M11) 343 appearing exclusively on one of the two microsomes. It has been observed, for example, 344 that female rats have higher CYP3A4 and CYP2D6 enzyme activity in the liver, so it is 345 likely that the differences observed in this study are due to different CYP expression 346 (Soldin and Mattison, 2009). In this regard, although sex differences in the metabolism 347 of many xenobiotics have been described, sex differences in the metabolism of 348 organosulfur compounds from Allium have not yet been described in the literature. 349 The comparison of the present results is complicated due to the scarcity of 350 metabolic studies of Allium compounds, specifically thiosulfonates, reported in the 351 scientific literature. Previous metabolic studies carried out to date are focused on volatile 352 organic compounds (Scheffler et al., 2016a, b; Lawson et al., 2018), leaving aside water- 353 soluble and non-volatile organic compounds which are the majority in metabolic 354 processes. Metabolites from garlic similar to PTSO were found in human urine by other 355 authors (Scheffler et al., 2016b) such as allyl methyl sulphide (AMS), allyl methyl 356 sulfoxide (AMSO), allyl methyl sulfone (AMSO2) and dimethyl trisulphide (DMTS) 357 analyzed by means of high-resolution gas chromatography coupled to mass spectrometer 358 (HRGC-MS). However, these compounds were not found in the present work, probably 359 because volatile compounds are difficult to determine by UPLC-MS/MS. In addition, 360 there are many compounds not identified in the present work which can be found in the 361 table 5. 17 362 In the in silico analysis of OSCs present in garlic, epoxidation was the most 363 probable reaction that yields AMS and DAS (Diaz et al., 2022). In addition, 364 hydrogenation and sulfoxidation present a low probability of occurring in AMS, and only 365 hydrogenation reaction was predicted to happen in DAS. In this sense, hydrogenation 366 367 reaction is also observed in our study, reported as reduction. 368 3.4. Identification of phase II metabolites. 369 As shown in figure 3B, the 7-hydroxycoumarine (positive control in phase II) 370 concentration decreased in a time-dependent manner, demonstrating that there is UGT- 371 conjugation activity in the microsomes at the times tested. In the conjugation with GSH 372 no positive control is needed since the reaction can occur with and without the presence 373 of glutathione s-transferase (GST) (Klaasen, 2019). 374 In the phase II biotransformation study of PTSO, a total of 53 metabolites were 375 identified after the analysis of samples from RLMM, RLMF and HLM by UHPLC- 376 MS/MS. All the metabolites included in table 4 underwent phase I reactions (mainly nitro- 377 reduction and desaturation) before the phase II metabolism. The metabolites were 378 conjugated with many substances, the frequency of appearance was the following: 379 arginine, glutamine, stearic acid, sulphate, ornitine, glucoside and glycine/cysteine. 380 Among all conjugated metabolites detected in phase II, 6 of them were identified in the 381 three microsomes used (M14, M24, M39, M47, M50, and M56) being all of them 382 consequence of different conjugate reactions: with glutamine, ornithine, glutathione, 383 stearic acid, cysteine, and methylation. Regarding differences between male and female 384 microsomes rat, there are 37 metabolites identified (table 4) present only in one sex. 385 Moreover, there are 6 metabolites only detected in human microsomes (M31, M34, M37, 18 386 M43, M44, and M57), being all of them consequence of dehydration or hydration 387 reactions followed by conjugation with aminoacids or conjugation with stearic acid. 388 Previous in vivo studies of DAS and its putative metabolites, diallyl sulfoxide 389 (DASO) and diallyl sulfone (DASO2), have reported glutathione and N-acetyl conjugates 390 in rats (Jin and Baillie, 1997). In view of this, it is important to note that other researchers 391 have identified N-acetyl-S-allyl-L-cysteine in human urine after garlic consumption 392 (Jandke and Spiteller, 1987, De Rooij et al. 1996). These metabolites are similar to the 393 ones found in the present work, since DASO and DASO2 are analogues to PTS and PTSO, 394 respectively (with allyl-methyl groups instead of propyl groups), so the presence of 395 glutathione and cysteine conjugates may be synthesized from the water-soluble S-allyl 396 cysteine catalyzed by N-acetyl transferase and seems to be a characteristic metabolic 397 pathway in Allium organosulfur compounds (Amano et al., 2015). In this sense, S- 398 propylcysteine (identified in our study as M50) has been also detected in fresh shallot and 399 black onion (Moreno-Rojas et al., 2018). Other studies in which garlic OSCs such as 400 DADS (Germain et al., 2002) was administered to rats, have shown their 401 biotransformation to volatile thiosulfonates and thiosulfinates such as AMSO2 and 402 AMSO. However, no studies of phase II biotransformation of these substances have been 403 performed. 404 Taking into account all the metabolites detected after PTSO biotranformation in 405 vitro assays performed with MRLM, FRLM and HLM microsomes and their 406 characterization by UHPLC-MS/MS in the present work, a proposed metabolic route has 407 been designed including only the metabolites with a well-known chemical structure 408 (Figure 4). This transformation pathway of PTSO includes hydroxylation, reduction, and 409 desaturation in phase I, and conjugation with glutathione and cysteine in phase II. As far 410 as we know, this is the first approach of the metabolic pathway of PTSO. The only 19 411 metabolite included in the figure that was not detected in our study was dipropyl 412 disulphide (DPDS). The transformation of PTS into DPDS has been described by 413 Teyssier and Siess, (2000). In another recent study, DPDS was also detected in liver 414 samples (Garcia-Nicolas et al., 2023). In our study, DPDS was not detected probably due 415 to its volatile nature, being needed a GC-MS analysis as performed by these authors. 416 Recently, in the case of another OSC such as allicin, thioacrolein (M110, in the 417 present work) has been recently proposed as its metabolite (Mengers et al., 2022). Little 418 is known about the metabolic pathways related to thiosulfonates, and even though the 419 EFSA has already published a document related to the biotransformation of organosulfur 420 compounds (EFSA, 2012), only one thiosulfonate is included (methyl 421 methanethiosulfonate). Therefore, the metabolic route of thiosulfonates needs to be 422 clarified. In the present study, the study of microsomal metabolism associated with the 423 thiosulfonate present in Allium genus, PTSO, led to the successful identification of 184 424 organosulfur-derived metabolites. Moreover, most of the previous studies carried out in 425 vivo to identify the Allium compounds metabolites employed plant extracts or garlic 426 supplements (Ruiz et al., 1997; Suarez et al., 1999; Rosen et al., 2000; Scheffler et al., 427 2016a, 2016b) instead of pure compounds, such as the one used in the present work. 428 Consequently, these studies have difficulties to confirm if the metabolites found are 429 generated by individual metabolism or originated due to the action of plant enzymes in 430 431 the oral cavity (Taucher et al., 1996; Buhr et al., 2009). 432 3.5. In silico ADME and toxicity prediction studies based on QSAR 433 The results of ADME and general toxicity prediction probabilities of PTSO and 434 metabolites are present in table 6. Regarding absorption process, PTSO presents an in 435 silico prediction of 0.005 % of probability of being absorbed, which is very low 20 436 considering that this compound accomplishes with Lipinski´s rules: the molecular weight 437 is less than 500 Da, it has less than five hydrogen bond donors, less than ten hydrogen 438 acceptor bonds and finally, calculated ClogP value is less than five (1.83). Therefore, it 439 can be expected that both PTSO and its metabolites will have poor glycoprotein binding 440 and remain in their free form available for crossing biological barriers. In fact, as table 6 441 shows, only one metabolite shows a BBB penetration probability less than 50% (S- 442 propylglutathione). In the next step, elimination of PTSO as well as its metabolites, shows 443 similar clearance: 9.40±2.52 mL min-1 kg-1 and similar half-time life: 0.75±0.13h. 444 Despite the in silico prediction of toxicity reveals certain probability of developing 445 mutagenic activity in the AMES test for PTSO (table 6), this finding has been refuted by 446 Mellado-García et al. (2015) demonstrating that PTSO has no mutagenic effects in the 447 AMES test carried out in the laboratory. In addition, the phase II metabolites GSSP and 448 CSSP showed very low probability of mutagenicity, which has been confirmed recently 449 in the Ames test (Cascajosa-Lira, 2021c). Regarding oral toxicity, the probability of 450 highly toxic effect by oral route is less than 50% in 6 of 9 compounds, being the predicted 451 value for the PTSO the lowest (0.094). Thus, PTSO as well as the potential metabolites 452 derived from it seem to describe a safe toxicological profile by predicting toxicity using 453 in silico tools. Previous in vivo studies following the OECD 425 guideline in rats orally 454 fed with PTSO resulted in a maximum tolerated dose (MTD) of 55 mg/kg bw (Llana- 455 Ruiz-Cabello et al., 2015). Although oral LD50 for PTSO has not been reported so far, it 456 seems that this could be set between 200-2000 mg kg-1 bw, which is in agreement with 457 the low probability of highly toxic effect found in the in silico study. 458 The predicted ecotoxicity of the PTSO and its detected metabolites is shown in figure 459 5, after the application the software EPI Suite™ (version 4.1). In general, most of the 460 metabolites have been found to be less toxic than the parent compound, except for 21 461 dimercaprol and 3-mercaptopropane1,2-diol. The latter metabolites are, in general, more 462 toxic than PTSO. By contrast, the higher detoxification rate was observed in the 463 conjugated metabolites, S-propylglutathione and S-propylcysteine, followed by the 464 metabolite 2-(methylthio)ethanol. In the case of S-allyl 1-propanesulfinothioate and 465 propylpropanethiosulfinate, both metabolites exerted similar toxicity than the parent 466 compound in all the assays. No previous in vivo studies have been reported focused on 467 the ecotoxicity of these metabolites. This prediction needs to be confirmed with further 468 469 in vivo assays. 470 4. Conclusions 471 The present work proposes for the first time a metabolic pathway for the thiosulfonate 472 PTSO present in many species of the genus Allium. The in vitro metabolism studies 473 carried out with rats and human microsomes have identified many metabolites in phase I, 474 being the dehydration the most frequently reaction found. In the phase II studies, 475 conjugation also takes places with many amino acids, highlighting the conjugation with 476 glutamine. In these in vitro metabolism assays, differences with species and gender have 477 been observed, being more evident in the case of phase II reactions. Moreover, it is also 478 important to highlight that differences due to the gender were more remarked. The in 479 silico toxicity studies revealed low rat oral acute toxicity for PTSO and its metabolites. 480 In general, the in silico ecotoxicity study revealed that the metabolism of PTSO leads to 481 detoxified metabolites, except for dimercaprol and 3-mercaptopropane1,2-diol, although 482 further in vivo assays are needed to confirm this prediction. 483 484 Conflict of Interest 22 485 The authors declare that there are no conflicts of interest. 486 Acknowledgements 487 The authors would like to thank the Junta de Andalucía (Project P18-TP-2147) for its 488 financial support. Antonio Cascajosa Lira thanks the Spanish Ministerio de Universidades 489 for the funding FPU grant (FPU2019-01247). The authors wish to thank the Mass 490 Spectrometry service of CITIUS (Centro de Investigación, Tecnología e Innovación 491 Universidad de Sevilla). 492 23 493 References 494 Abad, P., Arroyo-Manzanares, N., García-Campaña, A.M. (2016). A rapid and simple 495 UHPLC-ESI-MS/MS method for the screening of propyl propane thiosulfonate, a 496 new additive for animal feed. Analytical Methods, 8, 3730–3739. 497 https://doi.org/10.1039/c6ay00219f 498 Abad, P., Arroyo-Manzanares, N., Gil, L., García-Campaña, A.M. (2017). Use of onion 499 extract as a dairy cattle feed supplement: Monitoring propyl propane thiosulfonate 500 as a marker of its effect on milk attributes. 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