Sa e y assessmen o p opyl-p opane- hiosul ona e (PTSO): 90-days o al subch onic
oxici y s udy in a s
Au o es: An onio Cascajosa Li a1, Ana Isabel P ie o1, Albe o Baños2, En ique
Guillamón2, Rosa io Moyano3 , Angeles Jos1*, Ana M. Cameán1.
1A ea o Toxicology, Facul y o Pha macy, Uni e si y o Se illa, Se illa, Spain.
2 DMC Resea ch Cen e SLU, Camino de Jayena s/n, Alhedin, 18620 G anada, Spain.
3 Depa men o Pha macology, Toxicology and Legal and Fo ensic Medicine,
Uni e si y o Co doba, Campus de Rabanales Ca e e a Mad id-Cadiz s/n
*Co esponding au ho : Ángeles Jos Gallego
A ea o Toxicology, Facul y o Pha macy, Uni e sidad de Se illa. C/ P o eso Ga cía
González, nº 2. C.P. 41012, Se ille. Spain.
E-mail add ess: [email p o ec ed]
Manusc ip Ti le Page
- A 90 days o al oxici y s udy was conduc ed wi h p opyl-p opane- hiosul ona e
- PTSO did no induce oxic e ec s a he highes dose assayed (55 mg/Kg/day)
- NOAEL o PTSO was es ima ed o be ≥ 55 mg/Kg/day
Highligh s ( o e iew)
Abs ac
P opyl-p opane- hiosul ona e (PTSO) is one o he main o ganosul u compounds
p esen in Allium essen ials oil. Di e en applica ions in he ood sec o ha e been
p oposed o PTSO, such as ood and eed addi i e and as ac i e packaging. Howe e ,
he au ho iza ion o i s use depends on i s oxici y p o ile. Thus, as a pa o i s sa e y
assessmen , in his wo k a epea ed dose 90-day o al oxici y s udy has been conduc ed
o he i s ime in a s ollowing he OECD guideline 408. PTSO was adminis e ed o
g oups o 10 male and 10 emale a s a dose le els o 0, 14, 28, and 55 mg/Kg/day. No
clinical signs o mo ali y and no changes in body weigh , ood consump ion and eed
con e sion e iciency we e de ec ed h ough he s udy. Mo eo e , no ea men - ela ed
changes in hema ological and biochemical pa ame e s we e obse ed, o ei he sex o
dose g oups. The his opa hology s udy pe o med e ealed no di e ences in o gan
weigh s, and no mo phological and his opa hological changes we e obse ed. Based on
hese esul s, he no-obse ed-ad e se-e ec le el (NOAEL) o PTSO was judged o be
≥ 55 mg/Kg/day o bo h sexes.
Keywo ds: Subch onic oxici y; 90-day; O ganosul u compounds; Allium; P opyl-
p opane hiosul ona e
Abs ac
1
1. In oduc ion
Essen ials Oils (EO) ex ac s om plan s ha e been he ocus o nume ous s udies
due o hei po en ial in he pha maceu ical and ood indus ies. Se e al wo ks and
pa en s ha e been de eloped speci ically o he applica ion o EO and hei componen s
in o he ood sec o (Ribei o-San os e al., 2017; Llana-Ruiz- Cabello e al., 2015;
Maisanaba e al., 2017). Globally, hei ela i ely sa e s a us, p ope ies and accep ance
by consume s which demand na u al compounds o eplace syn he ic ones ha e piqued
he in e es o indus ies and consume s (Sacche i e al., 2005; Benkeblia and Lanzo i,
2007; Debiagi e al., 2014). Among hose bene icial plan s, Allium sp. is a genus well-
known o i s an imic obial, an i i al, an ip o ozoal, an i ungal o an ioxidan p ope ies
(among o he s). These p ope ies a e mainly due o hei con en o o ganosul u
compounds (OSC), which a e seconda y phy ochemical me aboli es, biosyn hesized o
de ensi e pu poses agains bio ic and abio ic s esso s. They a e mainly o med o he
ac ion o he enzyme alliin (s o ed in acuoles) on cy oplasmic compounds like
alk(en)yl cys eine sul oxides (ACOs) once he ege able issue is hu (Pu nik e al.,
2019) (Fig. 1). O he wise, hese compounds a e no a ailable in in ac cells as hey a e
oxic o he plan (Rami ez e al., 2017).
One o he main componen s o Allium essen ial oil is p opyl-p opane- hiosul ona e
(PTSO), ha co esponds o he molecula o mula C6H14O2S2 (Fig. 1). This compound
has been s abilized and cha ac e ized by DMC Resea ch Cen e SLU ( G anada, Spain)
o be used o di e en applica ions, aking ad an age o i s bene icial p ope ies. Thus,
his p oduc has been epo ed o show mainly an ioxidan and an imic obial ac i i ies,
being able o inhibi he g ow h o G am (–) and G am (+) bac e ia as well as molds and
yeas (Peinado e al., 2012, 2013; Llana-Ruiz-Cabello e al., 2015). I s an ibac e ial
ac i i y in i o in humans has also been demons a ed (So lozano-Pue o e al., 2018).
Manusc ip o e ision (CLEAN VERSION) Click he e o iew linked Re e ences
2
P oallium AP®, a comme cial Allium sp. ex ac wi h a 14.5% PTSO con en , has
been p oposed as a biop ese a i e in ac i e ood packaging o human ood
commodi ies mainly due o i s an ioxidan and an imic obial ac i i y (Llana-Ruiz-
Cabello e al., 2018). The packaging ma e ial used in hese sys ems can inco po a e
componen s in ended o be eleased in o he ood om he package, allowing oods o
a i e a he consume s wi h hei o iginal o enhanced o ganolep ic p ope ies, wi h
longe shel -li e and sa e y (Ribei o-San os e al., 2017). P e iously, ano he s udy
ca ied ou by Seydim & Sa ikus, (2006) es ed he an imic obial ac i i y o ga lic EO in
combina ion wi h o egano EO in ilms made wi h whey p o ein isola e, and showed
an imic obial ac i i y in a concen a ion o 4% (w/ ). Bu he inco po a ion o ga lic EO
and hei componen s in ac i e ood packaging can esul in a highe human exposu e
and consequen ly, mo e esea ch is needed o s ablish he sa e y concen a ion. An
addi ional p oposed applica ion o PTSO is as senso y addi i e in animal nu i ion,
imp o ing he pala abili y o eed, and also as a zoo echnical addi i e, being an
al e na i e o he use o an ibio ics (Peinado e al., 2012), con ibu ing o educe
esis ance gene a ed by hei excessi e use in li es ock. Since he ban in he Eu opean
Union (EU) o he use o an ibio ics as g ow h p omo e s, he sea ch o new
al e na i e p oduc s ha ensu e simila p oduc ion le els and ood secu i y wi hou
gene a ing unwan ed e ec s, including human esis ances, has been os e ed, being
addi i es o na u al o igin a good al e na i e. The egula ion (EC) No 1831/2003 o he
Eu opean Pa liamen and o he Council o 22 Sep embe 2003 se s he pa ame e s o
use o he addi i es used in animal nu i ion. Recen s udies ha e shown ha some eed
addi i es can help animals o main ain good physiological condi ions and imp o e
animal wel a e. This has led o an amendmen in he abo emen ioned egula ion on 12 h
June 2019, es ablishing new unc ional g oups o eed addi i es o imp o e

3
physiological condi ion o animals. In his ega d, o he p ope ies demons a ing he
po en ial use o PTSO in animal nu i ion sec o a e he an i-me hanogenic e ec
desc ibed du ing he e men a ion p ocess in umen (Ma ínez-Fe nández e al. 2013 )
and he immunomodula o y and an i-in lamma o y p ope ies (Vezza e al., 2019).
Howe e , he success ul de elopmen o any applica ion o his compound mus be
accompanied by an ex ensi e oxicological e alua ion, ha gua an ees i s sa e y o he
inal consume s, bo h humans and li es ock.
In his ega d, he oxicological p o ile o PTSO has been in es iga ed by
cy o oxici y assays in human cell lines (Llana-Ruiz-Cabello e al., 2015) as well as i s
geno oxici y and mu agenici y in i o (Mellado-Ga cía e al., 2015) and geno oxici y in
i o (Mellado-Ga cia e al., 2016b). Fu he mo e, i s acu e oxici y in i o has been also
e alua ed (Llana-Ruiz-Cabello e al., 2015). No wi hs anding, sa e doses o use ha e
been no ye es ablished o PTSO. Thus, a epea ed dose 90-days o al oxici y s udy in
oden s o PTSO would be necessa y o clea ly cha ac e ize i s oxici y, being also a
equi emen in he au ho iza ion applica ion p ocesses o he Eu opean Food Sa e y
Au ho i y (EFSA, 2012; 2016a; 2016b; 2017).
The aim o he p esen s udy is, he e o e, o u he explo e he oxici y po en ial o
PTSO and, o he i s ime, o conduc a subch onic die a y oxici y assay o PTSO in
a s ollowing in e na ionally ecognized es guidelines (O ganisa ion o Economic
Co-ope a ion and De elopmen , OECD 408, 2018). Conside ing hese ac s, in he
p esen s udy se e al pa ame e s ha e been e alua ed, including body weigh changes,
ood and wa e consump ion, eed con e sion e iciency, o gan weigh a ios and
biochemis y and hema ology pa ame e s. In addi ion he his opa hology o a ious
issues has been also s udied. The esul s om he comple e assessmen o his
4
subch onic s udy would allow o ge an es ima ion o a non-obse ed-ad e se-e ec -
le el (NOAEL) o exposu e o es ablish sa e y condi ions o human exposu e o PTSO.
2. Ma e ials and Me hods
2.1. Tes i em and doses p epa a ion
PTSO was supplied by DMC RC SLU (G anada, Spain) wi h a 96% o pu i y.
Comme cial powde neu al gela in om po k p o ein (Jesus Na a o S.A., Alican e,
Spain) was employed as he ehicle o he es subs ance in all g oups including he
con ols.
Fo he 90-days s udy, he doses we e p epa ed daily o each animal du ing he
13 weeks, and on F idays hey we e also p epa ed o he weekend. The dose was mixed
in 3 mL o liquid gela in. The olumes o PTSO (µL) o add o he gela in depended on
he dose selec ed o each g oup, and he gela in could solidi y a 4 ºC o e nigh .
Homogenei y o he die a y dose o mula ions and hei s abili y we e con i med o be
a leas 5 days.
2.2. Animals condi ions and husband y se ices
The a s we e supplied by Cha les Ri e labo a o ies S.L. (Kings, NY, USA), 40
males and 40 emales o Sp ague-Dawley s ain. They we e app oxima ely 7 weeks old
and we e s abilized o an acclima iza ion pe iod o 7 days du ing which hey we e
examined by a e e ina y su geon. When he i s week o dosage began he a s body
weigh mean was 320 g ± 11.3 o males and 227 ± 11.9 o emales.
5
Animals we e indi idually housed in cages ype 3H wi h Sou ali 29/12 plus
(sou ali S.L., Ge ona, Spain) aspen wood bedding and ood comple ely a ailable
wi hou es ic ion using s anda d d y pelle die o oden s Scien i ic (Panlab, S.L.U.,
Co nella de Llob ega , Ba celona, Spain). They we e kep in a oom wi h con olled
condi ions o hygiene behind a ba ie sys em, a ange o empe a u e o 21±2ºC, wi h a
10-15 ai changes pe hou , and a ela i e humidi y be ween 30-70% unde 12 h
ligh /da k cycle. Each cage con ained an in o ma ion ca d which con ained s udy code
(19-CAM-11-animal numbe ), sex, dose, g oup, and indi idual animal iden i ica ion.
Communi y ap wa e (EMACSA, Co doba Wa e company, Có doba, Spain), il e ed
and au ocla ed was a ailable ad libi um.
2.3. S udy design
The maximum ole able dose (MTD) o PTSO in a s o ally exposed o PTSO
(ga age) was p e iously se a 55 mg/kg by Llana-Ruiz-Cabello e al., (2015) ollowing
he OECD 425 (2008) es guideline (o al oxici y s udy: Up and Down p ocedu e), and
i was used as a e e ence o es ablish he es doses. Acco dingly, his dose was selec ed
as he highes one o be es ed, and also descending doses using a 2- old in e al ac o
acco ding o he guideline OECD 408 (2018) ecommenda ions: 14, 28, 55 mg/kg/day.
Ra s (10/sex/g oup) we e o ally adminis e ed he selec ed doses and he con ol g oup
ecei ed only he ehicle (po k gela ine).
This s udy was pe o med a he Cen al Se ice o Expe imen al Animals om
he Uni e si y o Co doba (SAE, Co doba, Spain) in which all animals ecei ed human
ca e in acco dance wi h he guidelines o he p o ec ion o animals used o he science
6
pu poses (Di ec i e, 2010/63 EU, Decision, 2012/707/UE, and RD 53/2013). All
p ocedu es ha e been app o ed by he E hical Animal Expe imen a ion Commi ee o
he Uni e si y o Có doba and by he Jun a de Andalucía (p ojec nº 20/10/2015-348).
2.4. Clinical obse a ions
Each animal was obse ed wice daily o mo bidi y and mo ali y and once
daily o clinical signs, such as changes in skin, u , eyes o mucous memb anes;
sec e ions; changes in gai , pos u e, o handling esponse; abno mal, clonic, o onic
mo emen s, and s e eo ypes o biza e beha io . Oph halmic examina ions we e
pe o med on all animals be o e ini ia ing he s udy and in he con ol and in he highes
dose g oup a he end o ea men .
2.5. Body weigh , ood and wa e consump ion
These h ee pa ame e s we e checked weekly in o de o a oid s ess. The mean
body weigh pe g oup and sex we e calcula ed weekly and p io o nec opsy om
indi idual animals’ da a, as well as he ood and wa e consump ion. The o al ood
consumed pe cage was eco ded and he weekly mean in ake pe a was calcula ed.
The eed con e sion e iciency (FCE) a io was de e mined acco ding o Escoba e al.,
(2015) by he a io o ood in ake (g)/ weigh gained (g).
2.6. Hema ology and Biochemis y
Blood samples we e ex ac ed om he hea by an in aca diac injec ion unde
ligh iso luo ane anes hesia a week 13. Then, he hema ological pa ame e s we e
13
The esul s ob ained ag ee wi h hose o Mellado e al. (2016b) who e alua ed he
sa e y o P oallium AP®, an Allium-based comme cial p oduc in a 90-days eeding
s udy wi h a s. PTSO is ac ually he majo o ganosul u compound p esen in P oallium
AP® (14.5%). Simila ly, nei he clinical signs no any o he changes on gene al,
biochemical, hema ological o his opa hological pa ame e s we e de ec ed, and he
au ho s de i ed a NOAEL highe han 400 mg/Kg/day a he condi ions assayed. Bo h
s udies show a good co ela ion as in he p esen s udy he es i em had a 7- old highe
con en o PTSO han P oallium® AP (100 e sus 14.5%) and he NOAEL de i ed o
P oallium was 7- old highe . This sugges s ha PTSO has an impo an ole on he
oxici y o Allium ex ac s. Ac ually, a MTD o 55 mg/kg in a s o PTSO has been
es ablished as p e iously indica ed. And highe doses es ed acco ding o he OECD 425
guideline (2000 mg/kg and 175 mg/kg) esul ed in he dea h o he animal and e iden
hepa o oxici y. On he con a y, a single dose o 55 mg/kg did no induce ema kable
damage (Llana-Ruiz-Cabello e al, 2015). Mellado-Ga cía e al. (2016a) obse ed ha in
a s ea ed wi h 55 mg/kg (3 doses a 0, 24 and 45h and eu hanized a 48h) an inc ease
in he glycogen s o age was no iceable in he li e and also a sligh degene a i e p ocess
in he chie cells o he s omach.
In his ega d, a he dose le els assayed, his opa hological lesions we e absen . This
could be explained by he exposu e way employed in his s udy, using gela in as ehicle
and wi h he eed. In he p e ious ials, o al ga age wi h a s omach ube a e a as ing
pe iod was used ollowing he ecommenda ions o he co esponding OCDE
guidelines. The bolus could ha e a mo e dele e ious e ec on he gas oin es inal
sys em as he e is a di ec con ac .
Di e ences in oxici y be ween PTSO and P oallium AP® could be explained as he
oxici y shown by componen s o an essen ial oil can be modula ed by he o he

14
cons i uen s by syne gis ic/an agonis ic phenomena (Escoba e al., 2015; Pa lidou e
al., 2004). The in e es o PTSO in compa ison o P oallium® is based on i s di e en
chemical p ope ies (highe hyd ophilia) and in i s highe e iciency on i s an ioxidan
and an imic obial p ope ies.
O he au ho s ha e shown he po en ial oxici y o aqueous ex ac s o di e en
medicinal plan s, including Allium sa i um, in Wis a a s. Thus, Sulaiman e al. (2014)
adminis e ed o ally o he animals 10 mg/kg o A. sa i um ex ac o 30 days and
obse ed al e a ion in he ac i i ies o ma ke enzymes: AST inc eased in li e , kidney
and hea , ALT in se um and li e , and ALP ac i i y was educed in se um, hea ,
kidney and li e . They concluded ha cau ion was equi ed in using un e ined ex ac s
o hese he bs in adi ional se ings.
The e a e sca ce in i o oxici y da a ega ding o he OSC. Thus, Guyonne e al.
(2000) demons a ed he e ec s o some o hem (DAS, DADS, dip opylsul iide (DPS)
and dip opyl disul ide (DPDS)) on he ac i a ion o se e al mu agens in male Wis a
a s exposed o 1 mmol/kg by ga age o 4 days. They explained he esul s based on
he induc ion o cy och ome (CYP) and phase II enzymes ac i i ies. This e ec , he
al e a ion o CYP ac i i y, was poin ed ou by o he au ho s as well (Da enpo and
Wa go ich, 2005). Mo eo e , hey obse ed hepa o oxici y induced by DAS (bile duc
obs uc ion, hype p oli e a ion and ocal poin s o nec osis) in a s ga aged daily wi h
200 mg/kg o 1, 4, o 8 weeks. On he con a y, 8 weeks o exposu e o lowe doses (50
and 100 mg/kg) did no induced li e his opha ological damage. This sugges ha li e
could be he a ge o gan o OSC as bo h, DAS (Da enpo and Wa go ich, 2005) and
PTSO (Llana-Ruiz-Cabello e al, 2015), ha e shown li e oxici y when a h eshold
dose is exceeded. Also, Wu e al. (2001) exposed a s o ally o ga lic oil (GO, 200
mg/Kg) and 3 allyl compounds, DAS (20 and 80 mg/kg), DADS (80 mg/kg), and diallyl
15
isul ide (DATS, 70 mg/kg) 3 imes a week o 6 weeks and examined he an ioxida ion
sys em in a li e s and ed blood cells. They ound ha GO, DADS and DATS
signi ican ly induced he glu a hione con en (GSH) in blood cells bu nei he GO no
any o i s OSC a ec ed he GSH- ela ed an ioxidan enzymes. Hepa ic GSH was no
in luenced by ga lic componen s. Bu DADS and DATS signi ican ly inc eased he
ac i i y o GSH- educ ase and GSH- ans e ase and dec eased GSH pe oxidase. In he
p esen s udy he hema ological pa ame e s we e no in luenced by he PTSO exposu e
and sca ce scien i ic da a dealing wi h PSTO a e a ailable o compa e.
5. Conclusions
In conclusion, he esul s ob ained con i m he al eady epo ed sa e y p o ile o
PTSO o some ood applica ions a he condi ions conside ed. Thus, PTSO did no
p omo e oxic e ec s as seen om body weigh changes, ood and wa e consump ion,
eed con e sion e iciency, biochemical and blood pa ame e s as well as o gan oxici y
and his ological examina ions o main o gans ha could e en ually be a ec ed by i s
subch onic adminis a ion (90 days). NOAEL was es ima ed o be ≥ 55 mg/Kg/day.
Con lic o In e es
The au ho s decla e ha he e a e no con lic s o in e es .
Acknowledgemen s
The au ho s would like o acknowledge he Spanish Minis e io de Ciencia e Inno ación
(P ojec RTC-2017-6199-2), and o he Jun a de Andalucía (P ojec AT 2017-5323) o
i s inancial suppo .
16
17
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RBC: E y h ocy e coun ; HGB: hemoglobin; HCT: hema oc i ; MCV: mean co puscula olume; MCH: mean co puscula hemoglobin; MCHC: mean co puscula hemoglobin concen a ion; PLT:
pla ele ( h ombocy e) coun ; RDW: ed blood cell dis ibu ion wid h; T PRO: p o h ombin ime; T CEF: cephalin ime. F: S a is ics ANOVA es ; K.W: K uskal-WallisS a is ic; N.S.: No Signi ican ;
&Signi ican ly di e en in compa ison o g oup 4 when p<0.05; # signi ican ly di e en in compa ison o g oup 3 when p<0.05.

Table 3
WBC: o al leukocy e coun ; NE: neu ophil; LY: lymphocy e; MO: monocy e; EO: eosinophil; BA: basophil; F: S a is ics ANOVA es ; K.W: K uskal-Wallis S a is ic; N.S.: No Signi ican .
DIFFERENTIAL WHITE BLOOD CELLS COUNT DATA SUMMARY
MALE
FEMALE
G oup 1
G oup 2
G oup 3
G oup 4
G oup 1
G oup 2
G oup 3
G oup 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)
N=10
N=10
N=10
N=10
N=10
N=10
N=10
N=10
WBC
MEAN
11.43
11.84
12.03
11.70
7.19
6.02
6.83
4.54
103/µL
ST. DEV.
3.38
1.70
1.88
3.80
3.67
1.93
2.16
2.70
KW= 0.60 p=0.90; N.S.
F(34.3)=1.86 p=0.16; N.S.
NE
MEAN
16.43
14.59
16.07
16.95
30.24
31.03
24.66
24.99
%
ST. DEV.
5.07
2.80
3.53
5.88
27.30
15.46
15.28
11.31
KW=1.47 p=0.69; N.S.
F(34.3)=0.35 p=0.79; N.S.
LY
MEAN
71.87
80.02
78.09
74.52
64.73
62.00
68.32
67.00
%
ST. DEV.
14.48
4.25
5.78
8.01
26.04
17.24
14.42
12.00
KW= 5.07 p=0.17; N.S.
F(34.3)=0.25 p=0.86; N.S.
MO
MEAN
3.01
1.52
2.87
2.94
1.44
1.07
0.75
1.57
%
ST. DEV.
2.24
1.81
2.33
2.16
1.66
0.74
0.77
1.78
KW=2.71 p=0.44; N.S.
KW=2.22 p=0.53; N.S.
EO
MEAN
3.38
3.22
2.03
4.58
3.17
6.33
4.52
6.94
%
ST. DEV.
2.33
1.69
0.58
2.88
2.52
4.53
2.90
5.23
KW=6.70 p=0.08; N.S.
KW=3.14 p=0.37; N.S.
BA
MEAN
0.57
0.70
0.95
1.07
0.41
0.54
0.80
0.51
%
ST. DEV.
0.50
0.44
1.39
1.09
0.41
0.41
0.65
0.39
KW=1.42p=0.70; N.S.
F(34.3)= 1.01 p=0.40; N.S.
Table 3
Table 4
CLINICAL BIOCHEMISTRY DATA SUMMARY
MALE
FEMALE
G oup 1
G oup 2
G oup 3
G oup 4
G oup 1
G oup 2
G oup 3
G oup 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)
N=10
N=10
N=10
N=10
N=10
N=10
N=10
N=10
GLUC
MEAN
143.50
146.50
128.40
123.60
125.60
121.60
105.20
104.00
mg/dL
ST. DEV.
22.73
27.44
26.31
20.30
16.64
17.20
27.59
44.93
F(36.3)=2.12 p=0.12; N.S.
KW=4.90 p=0.18; N.S.
UREA
MEAN
35.22
31.67
33.99
35.37
30.26
31.87
34.90
34.22
mg/dl
ST. DEV.
4.83
2.95
4.37
3.16
4.63
3.36
7.02
4.14
F(36.3)=1.92 p=0.14; N.S.
F(36.3)=1.85 p=0.16; N.S.
CREAT
MEAN
0.30
0.28
0.29
0.29
0.34
0.35
0.35
0.34
mg/dL
ST. DEV.
0.04
0.02
0.03
0.03
0.06
0.03
0.07
0.02
F(36.3)=0.48 p=0.70; N.S.
F(36.3)=0.26 p=0.86; N.S.
BILI-T
MEAN
0.21
0.31
0.21
0.22
0.26
0.28
0.25
0.26
mg/dL
ST. DEV.
0.02
0.32
0.02
0.03
0.03
0.02
0.03
0.01
KW=1.41 p=0.70; N.S.
F(36.3)=2.15 p=0.11; N.S.
CHOL
MEAN
107.50
95.40
93.50
88.50**
104.20
101.80
112.40
99.20
mg/dL
ST. DEV.
13.83
15.36
10.60
8.50
11.12
20.75
20.58
15.36
F(36.3)=4.25 p<0.01**
F(36.3)=1.08 p=0.36; N.S.
TRIGL
MEAN
156.40
163.40*
131.80
123.00
78.60
104.80
84.40
77.80
mg/dL
ST. DEV.
33.81
36.16
27.59
20.96
18.87
28.06
21.53
16.94
KW=10.48 p<0.05*
F(36.3)=1.85 p=0.16; N.S.
AST
MEAN
147.52
145.76
135.39
183.73
264.52
246.91
251.94
264.62
U/L
ST. DEV.
17.74
26.66
17.66
101.03
94.90
92.11
73.01
122.10
KW=3.44 p=0.33; N.S.
KW= 0.39 p=0.95; N.S.
ALT
MEAN
30.46
38.36
33.42
31.44
38.31
35.40
37.03
35.22
U/L
ST. DEV.
6.26
16.86
6.09
2.42
10.34
17.81
5.26
10.55
KW=3.12 p=0.38; N.S.
KW=2.41 p=0.49; N.S.
ALKP
MEAN
91.20
98.20
88.30
89.60
62.00
62.90
66.40
60.60
U/L
ST. DEV.
9.62
18.50
26.97
15.21
12.14
9.99
17.22
9.13
Table 4
GLUC: glucose; CREAT: c ea inine; Bili-T: Bili ubin, o al; CHOL:choles e ol, o al; TRIGL: iglyce ides; AST:aspa a e amino ans e ase; ALT: alanine amino ans e ase; ALKP: alkaline phospha ase; ALB:albumin; TOT PROT: p o ein, o al;
Na+:sodium; K+:po assium; Ca++: calcium.
F: S a is ics ANOVA es ; K.W:K uskal-WallisS a is ic; N.S.: No Signi ican
*Signi ican ly di e en om con ol. *when p<0.01
**Signi ican ly di e en om con ol. **when p<0.01
KW=0.56 p=0.65; N.S.
F(36.3)=0.39 p=0.76; N.S.
ALB
MEAN
3.93
3.88
3.98
4.00
4.80
4.59
4.66
4.72
g/dl
ST. DEV.
0.47
0.25
0.32
0.29
0.35
0.34
0.59
0.34
KW=1.54 p=0.67; N.S.
F(36.3)=0.47 p=0.70; N.S.
TOT PROT
MEAN
6.24
6.26
6.26
6.20
6.46
6.78
6.78
6.54
g/dl
ST. DEV.
0.34
0.48
0.37
0.29
0.44
0.35
0.68
0.51
F(36.3)=0.06 p=0.98; N.S.
F(36.3)=1.05 p=0.38; N.S.
Na+
MEAN
134.80
133.70
133.60
132.80
142.60
139.60
140.20
139.40
mmol/L
ST. DEV.
10.40
3.59
3.24
4.08
4.62
7.12
11.64
7.78
KW=2.00 p=0.57; N.S.
KW=1.65 p=0.65; N.S.
K+
MEAN
7.87
8.89
7.90
8.38
9.68
12.81
14.93
10.34
mmol/L
ST. DEV.
1.58
1.62
0.77
1.29
6.97
6.18
7.61
5.67
F(36.3)=1.26 p=0.30; N.S.
KW=6.35 p=0.10; N.S.
Ca++
MEAN
10.87
10.28
10.82
10.51
11.30
11.42
10.76
11.25
mg/dL
ST. DEV.
1.04
0.66
0.64
0.47
1.06
1.26
0.87
1.36
KW=4.43 p=0.22; N.S.
KW=2.34 p=0.51; N.S.
Table 5
F: S a is ics ANOVA es ; K.W: K uskal-Wallis S a is ic; N.S.: No Signi ican . * Signi ican ly di e en om g oup 3 in compa ison o g oup 1 when p<0.05.
ORGAN WEIGHT DATA SUMMARY
MALE
FEMALE
G oup 1
G oup 2
G oup 3
G oup 4
G oup 1
G oup 2
G oup 3
G oup 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)
N=10
N=10
N=10
N=10
N=10
N=10
N=10
N=10
BODY W.
MEAN
633.80
623.10
611.5
625.90
BODY W.
MEAN
326.80
338.00
321.10
321.30
(g)
ST. DEV.
38.25
28.71
26.50
48.14
(g)
ST. DEV.
11.50
29.08
23.81
18.31
F(36.3)=0.19 p=0.90; N.S.
F(36.3)=1.34 p=0.29; N.S.
BRAIN
MEAN
2.09
2.13
2.12
2.07
BRAIN
MEAN
1.96
2.03
2.01
2.06
(g)
ST. DEV.
0.21
0.14
0.17
0.23
(g)
ST. DEV.
0.21
0.10
0.12
0.15
KW=0.09 p=0.99; N.S.
KW=2.35 p=0.50; N.S.
LIVER
MEAN
21.06
18.49
18.40
18.41
LIVER
MEAN
8.41
9.54
8.90
8.48
(g)
ST. DEV.
4.86
1.45
1.77
2.40
(g)
ST. DEV.
1.36
1.09
1.08
0.83
F(36.3)=1.99 p=0.13; N.S.
F(36.3)=2.21 p=0.10; N.S.
HEART
MEAN
2.14
1.98
1.82*
1.99
HEART
MEAN
1.21
1.28
1.20
1.22
(g)
ST. DEV.
0.18
0.19
0.19
0.32
(g)
ST. DEV.
0.13
0.13
0.14
0.16
F(36.3)=3.11 *p<0.05
F(36.3)=0.67 p=0.59; N.S.
SPLEEN
MEAN
1.17
1.16
1.16
1.45
SPLEEN
MEAN
0.80
0.86
0.74
0.75
(g)
ST. DEV.
0.10
0.15
0.14
0.93
(g)
ST. DEV.
0.11
0.19
0.07
0.09
KW=0.2426 p=0.9704; N.S.
F(36.3)=1.71 p=0.18; N.S.
KIDNEYS
MEAN
4.05
4.10
3.99
4.06
KIDNEYS
MEAN
2.13
2.30
2.15
2.06
(g)
ST. DEV.
0.30
0.41
0.37
0.28
(g)
ST. DEV.
0.23
0.23
0.20
0.14
F(36.3)=0.17 p=0.92; N.S.
F(36.3)=2.48 p=0.08; N.S.
THYMUS
MEAN
0.88
0.80
0.83
0.71
THYMUS
MEAN
0.67
0.70
0.66
0.60
(g)
ST. DEV.
0.25
0.12
0.15
0.27
(g)
ST. DEV.
0.08
0.13
0.20
0.10
F(36.3)=1.20 p=0.32; N.S.
F(36.3)=1.02 p=0.40; N.S.
TESTES
MEAN
3.92
3.95
3.77
3.83
UTE./CERV.
MEAN
0.87
0.76
1.03
0.94
(g)
ST. DEV.
0.44
0.29
0.32
0.28
(g)
ST. DEV.
0.24
0.17
0.81
0.32
F(36.3)=0.62 p=0.61; N.S.
KW=1.32 p=0.73; N.S.
EPIDIDIMS
MEAN
1.88
2.05
2.36
2.07
OVARIES
MEAN
0.26
0.31
0.23
0.25
(g)
ST. DEV.
0.24
0.52
0.98
0.28
(g)
ST. DEV.
0.0.8
0.10
0.06
0.07
KW=4.00 p=0.26; N.S.
F(36.3)= 1.90 p=0.15; N.S.
ADRENALS
MEAN
0.11
0.13
0.12
0.12
ADRENALS
MEAN
0.15
0.12
0.10
0.12
(g)
ST. DEV.
0.04
0.05
0.07
0.05
(g)
ST. DEV.
0.08
0.04
0.02
0.08
KW=0.72 p=0.87; N.S.
KW=4.95 p=0.18; N.S.
Table 5
Table 6
F: S a is ics ANOVA es ; K.W: K uskal-Wallis S a is ic; N.S.: No Signi ican .
ORGAN WEIGHT/BODY WEIGHT RATIO DATA SUMMARY
MALE
FEMALE
G oup 1
G oup 2
G oup 3
G oup 4
G oup 1
G oup 2
G oup 3
G oup 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)
N=10
N=10
N=10
N=10
N=10
N=10
N=10
N=10
BRAIN
MEAN
0.33
0.34
0.36
0.33
BRAIN
MEAN
0.60
0.60
0.63
0.64
(%)
ST. DEV.
0.03
0.03
0.06
0.04
(%)
ST. DEV.
0.07
0.05
0.6
0.05
KW=4.99 p=0.17; N.S.
F(36.3)=1.17 p=0.34; N.S.
LIVER
MEAN
3.31
2.97
3.22
2.93
LIVER
MEAN
2.58
2.82
0.77
2.64
(%)
ST. DEV.
0.71
0.15
0.71
0.21
(%)
ST. DEV.
0.42
0.23
0.27
0.24
KW=2.03 p=0.57; N.S.
F(36.3)=1.47 p=0.24; N.S.
HEART
MEAN
0.33
0.31
0.32
0.32
HEART
MEAN
0.37
0.38
0.38
0.38
(%)
ST. DEV.
0.04
0.02
0.05
0.05
(%)
ST. DEV.
0.04
0.05
0.06
0.05
F(36.3)=0.64 p=0.59; N.S.
KW=1.12 p=0.77; N.S.
SPLEEN
MEAN
0.18
0.19
0.20
0.23
SPLEEN
MEAN
0.25
0.25
0.23
0.23
(%)
ST. DEV.
0.02
0.02
0.03
0.14
(%)
ST. DEV.
0.03
0.05
0.02
0.03
KW=2.15 p=0.54; N.S.
F(36.3)=0.76 p=0.52; N.S.
KIDNEYS
MEAN
0.63
0.65
0.70
0.65
KIDNEYS
MEAN
0.65
0.68
0.67
0.64
(%)
ST. DEV.
0.05
0.05
0.15
0.06
(%)
ST. DEV.
0.07
0.06
0.06
0.06
KW=1.42 p=0.70; N.S.
KW=2.53 p=0.47; N.S.
THYMUS
MEAN
0.14
0.13
0.15
0.11
THYMUS
MEAN
0.21
0.21
0.21
0.19
(%)
ST. DEV.
0.02
0.02
0.05
0.04
(%)
ST. DEV.
0.03
0.04
0.06
0.03
KW=4.80 p=0.19; N.S.
F(36.3)=0.68 p=0.57; N.S.
TESTES
MEAN
0.62
0.63
0.66
0.61
UTE./CERV.
MEAN
0.27
0.23
0.32
0.29
(%)
ST. DEV.
0.07
0.05
0.14
0.07
(%)
ST. DEV.
0.07
0.05
0.25
0.10
KW=1.16 p=0.76; N.S.
KW=2.47 p=0.48; N.S.
EPIDIDIMS
MEAN
0.30
0.32
0.41
0.33
OVARIES
MEAN
0.08
0.09
0.07
0.08
(%)
ST. DEV.
0.04
0.08
0.18
0.05
(%)
ST. DEV.
0.02
0.03
0.02
0.02
KW=5.544 p=0.1361; N.S.
KW=3.53 p=0.32; N.S.
ADRENALS
MEAN
0.02
0.02
0.02
0.02
ADRENALS
MEAN
0.05
0.04
0.03
0.04
(%)
ST. DEV.
0.01
0.01
0.01
0.01
(%)
ST. DEV.
0.02
0.01
0.01
0.02
KW=0.45 p=0.93; N.S.
KW=4.49 p=0.21; N.S.
Table 6

Table 7
F: S a is ics ANOVA es ; K.W: K uskal-Wallis S a is ic; N.S.: No Signi ican .
ORGAN WEIGHT/BRAIN WEIGHT RATIO DATA SUMMARY
MALE
FEMALE
G oup 1
G oup 2
G oup 3
G oup 4
G oup 1
G oup 2
G oup 3
G oup 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)
N=10
N=10
N=10
N=10
N=10
N=10
N=10
N=10
LIVER
MEAN
1011.66
874.10
876.04
892.82
LIVER
MEAN
437.12
470.10
444.59
411.74
(%)
ST. DEV.
229.58
103.29
131.13
92.16
(%)
ST. DEV.
104.65
48.18
56.83
41.47
KW=4.08 p=0.25; N.S.
F(36.3)=1.27 p=0.30; N.S.
HEART
MEAN
103.79
93.72
86.79
97.94
HEART
MEAN
62.93
63.41
60.00
59.58
(%)
ST. DEV.
16.99
12.97
10.89
23.79
(%)
ST. DEV.
13.12
8.56
8.83
9.81
KW=6.19 p=0.10; N.S.
F(36.3)=0.37 p=0.78; N.S.
SPLEEN
MEAN
56.36
54.78
55.10
55.42
SPLEEN
MEAN
41.83
42.21
37.10
36.61
(%)
ST. DEV.
7.85
6.83
7.14
7.75
(%)
ST. DEV.
9.96
9.33
3.40
6.17
F(36.3)=0.084 p=0.97; N.S.
F(36.3)=1.52 p=0.23; N.S.
KIDNEYS
MEAN
195.30
193.90
190.21
198.22
KIDNEYS
MEAN
110.18
113.37
107.39
100.25
(%)
ST. DEV.
22.29
27.64
30.06
26.91
(%)
ST. DEV.
18.71
12.13
13.40
12.68
F(36.3)=0.15 p=0.93; N.S.
KW=5.94 p=0.11; N.S.
THYMUS
MEAN
42.63
37.87
39.25
34.33
THYMUS
MEAN
34.57
34.51
33.29
28.27
(%)
ST. DEV.
13.08
6.61
7.85
12.04
(%)
ST. DEV.
5.25
5.96
12.14
4.26
F(36.3)=1.13 p=0.35; N.S.
KW=5.75 p=0.12; N.S.
TESTES
MEAN
189.99
187.00
179.45
187.33
UTE./CERV.
MEAN
44.81
37.40
51.51
46.40
(%)
ST. DEV.
33.59
23.50
24.34
28.69
(%)
ST. DEV.
12.42
8.40
40.13
18.00
F(36.3)=0.26 p=0.85; N.S.
KW=2.62 p=0.45; N.S.
EPIDIDIMS
MEAN
89.92
96.89
112.09
100.46
OVARIES
MEAN
13.04
15.32
11.31
12.10
(%)
ST. DEV.
7.49
24.61
46.97
12.46
(%)
ST. DEV.
3.734
5.41
3.20
3.43
KW=3.96 p=0.27; N.S.
F(36.3)=1.85 p=0.16; N.S.
ADRENALS
MEAN
5.42
5.40
5.91
5.75
ADRENALS
MEAN
8.00
6.10
4.82
5.98
(%)
ST. DEV.
2.59
2.83
3.99
2.77
(%)
ST. DEV.
4.87
2.02
1.33
3.97
KW=0.50 p=0.92; N.S.
KW=4.27 p=0.23; N.S.
Table 7
Figu e 1 Click he e o access/download;Figu e; igu a 1. ajo. i
Figu e 2 Click he e o access/download;Figu e;Figu e 2. Body weigh . i
Figu e 3 Click he e o access/download;Figu e;Figu e 3. Mean body
weigh gain. i