Benzenethiol

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: the National Toxcology Program's Reproductive Assessment by Continuous Breeding protocol

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzenethiol

EC Number:

203-635-3

EC Name:

Benzenethiol

Cas Number:

108-98-5

Molecular formula:

C6H6S

IUPAC Name:

benzenethiol

Details on test material:

Thiophenol (CAS No. 108-98-5, R.O.W. Sciences ID No. 1(03) was purchased from Aldrich Chemical Company (St Louis, Missouri) and provided by the NTP through Research Triangle Institute (RTI), (Research Triangle Park, North Carolina). All bulk chemical analyses and test formulations analyses were conducted by RTI. Upon receipt, two 5 g samples of the bulk test article were collected into glass bottles and sealed with Teflon coated lids then stored under nitrogen in the freezer protected from light for possible analysis. A bulk test article sample of 5 g was collected and sent for purity and stability testing within 30 days of receipt and thereafter at 24 ±2 week intervals. The identity of the compound was confirmed by infrared and nuclear magnetic resonance spectrometry. The purity of the test article was estimated by high performance liquid chromatography. Prior to Stability studies conducted on com oil solutions containing 0.2 mg/ml of thiophenol indicated no significant loss of chemical after 30 days of storage under nitrogen at I-3°C. Under conditions which simulate animal dosing (room temperature and exposed to air in a hood), the dosage formulation (0.2 mg/ml), lost 11.7% of thiophenol content over 3 hours. Upon receipt, the test article was stored at room temperature. Prior to the initiation of this . study, the test article was refrigerated at 1-3OC in a sealed container under nitrogen and protected from light. The test article was analyzed every 24± 2 weeks to verify stability. Bulk chemical reanalysis upon completion of the study indicated that the purity of the test article did not appreciably alter during the course of the study (Table A55).
The analytical chemistry reports are in Volume IT, Appendix 4. Upon completion of the study, the remaining bulk test article was returned to RTI.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were housed two/three per cage by sex from receipt to randomization and during the Task 4 holding phase; two per cage (one male and one female) during Task 2, Task 3 crossover, and Task 4 cohabitation; and housed individually at all other times. All animals were housed in polycarhonate cages 9"L x 81h"W x 8"H (single-housed) or 19"L x 101h"W x 8"H (group-housed) suspended on stainless-steel racks with an Edstrom automatic watering system providing filtered tap water. Racks were equipped with filter paper liners.
Polycarbonate caging contained Beta ChipQD heat treated hardwood laboratory bedding. Pelleted Zeigler™ NIH 07 Small Animal Feed was available ad libitum in stainless-steel hanging feeders, and used within five months of the milling date. The feed was analyzed for nutrients, aflatoxins, nitrosamines, heavy metals, chlorinated hydrocarbons, organophosphates, PCBs, nitrites, nitrates, estrogenic activity, BHA, BHT, total bacterial plates, coliforms, E. coli, and salmonella by the vendor and the results approved by NTP prior to release. A water quality sample was analyzed for total dissolved solids, heavy metals, chlorinated hydrocarbons, organophosphates, nitrates, nitrites, microbiological content, and total tribalomethanes at least semi-annually to conform with the Safe Drinking Water Act. All of the feed and water contaminants were at levels not expected to interfere with the study.
A 12-hour light/12-hourdark cycle was maintained throughout the study. During the study, the temperature range in the animal room was 68-74°F and the relative percent humidity was 30-70%.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The animals received
daily doses of thiophenol via oral gavage at 0, 9, 18, or 35 mglkg/day until the day before euthanasia/necropsy.
Control animals received the corn oil vehicle only.

Details on mating procedure:

After the weaning of the final litter, crossover mating trial was conducted to
determine the affected sex. For the female crossover mating, naive males were cohabitated with the 0 or 35 mglkg
females for one week (1 male to 1 female). The females were tested for vaginal sperm and/or copulatory plug
beginning the day following the start of cohabitation. The animals were separated when either sperm-positive, a
copulatory plug was observed, or after 7 days, whichever came first.
For the male crossover mating, naive females were cohabitated with the 0 or 35 mglkg males for one week
(1 male to 1 female). The naive females were tested for vaginal sperm and/or copulatory plug beginning the day
following the start of cohabitation. The animals were separated when either sperm-positive, a copulatory plug was
observed, or after 7 days, whichever came first.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability studies conducted on com oil solutions containing 0.2 mg/ml of thiophenol indicated no significant
loss of chemical after 30 days of storage under nitrogen at I-3°C. Under conditions which simulate animal dosing
(room temperature and exposed to air in a hood), the dosage formulation (0.2 mg/ml), lost 11.7% of thiophenol
content over 3 hours. Upon receipt, the test article was stored at room temperature. Prior to the initiation of this
. study, the test article was refrigerated at 1-3OC in a sealed container under nitrogen and protected from light. The
test article was analyzed every 24± 2 weeks to verify stability. Bulk chemical reanalysis upon completion of the
study indicated that the purity of the test article did not appreciably alter during the course of the study (Table A55).
The analytical chemistry reports are in Volume IT, Appendix 4. Upon completion of the study, the remaining
bulk test article was returned to RTI.

Duration of treatment / exposure:

F0 were dosed seven days of premating exposure to thiophenol by oral gavage and until euthanasia/necropsy.
F1 were dosed with the same level of thiophenol as their parents received from after the weaning fo the day before euthanasia/necropsy.

Frequency of treatment:

one adminisitration per day

Details on study schedule:

Task2 - Task 2 consisted of a control group and three treated groups (20 animal/sex/group). The animals received
daily doses of thiophenol via oral gavage at 0, 9, 18, or 35 mglkg/day until the day before euthanasia/necropsy.
Control animals received the corn oil vehicle only. At the initiation of treatment (Study Day 1) the animals were
singly housed. On Study Day 8, male and female rats from the same group were cohabitated (1 male to 1 female)
for 16 weeks. All litters produced during the continuous breeding phase were evaluated on PND 1 and then
euthanized by decapitation and discarded without necropsy. The total number of pups born, number of live and
dead pups, number of male and female pups, and total pup weight of each sex were obtained. Dam and sire weights
were obtained following delivery (pND 1).
At the end of the 16 week continuous breeding phase, ~e pairs were separated and dosing continued.
Litters born (Fl ) after the continuous breeding phase (final litter) were reared by the dam until weaning on PND
21. The total number of pups, number of live and dead pups, number of male and female pups, and total pup
weight of each sex were obtained on PND 1, 4, 7, 14, and 21. The dam and sire were weighed following delivery.
The dam was also weighed on PND 4, 7, 14, and 21. Feed consumption measurements for lactating dams were
obtained on PND 1, 4, 7, 11, 14, 18, and 21.
All animals were observed twice daily for mortality and signs of toxicity. Body weights were obtained at
randomization,.at initiation of treatment, and weekly thereafter. Physical examinations were performed weekly and
feed consumption was measured during Study Weeks 1, 6, 12 and 18 (males only).
Task3 - After the weaning of the final litter from Task 2, the Task 3 crossover mating trial was conducted to
determine the affected sex. For the female crossover mating, naive males were cohabitated with the 0 or 35 mglkg
females for one week (1 male to 1 female). The females were tested for vaginal sperm and/or copulatory plug
beginning the day following the start of cohabitation. The animals were separated when either sperm-positive, a
copulatory plug was observed, or after 7 days, whichever came first. The females were allowed to litter and the
total number of pups born, number of live and dead pups, number of male and female pups, and total pup weight
of males and females were obtained. The dam and sire weights were obtained following delivery. All pups were
euthanized by decapitation on PND 1 and discarded without necropsy. Vaginal smears were collected from all
control and high-dose females for 12 days beginning Study Day 202 and ending Study Day 213.
For the male crossover mating, naive females were cohabitated with the 0 or 35 mglkg males for one week
(1 male to 1 female). The naive females were tested for vaginal sperm and/or copulatory plug beginning the day
following the start of cohabitation. The animals were separated when either sperm-positive, a copulatory plug was
observed, or after 7 days, whichever came first. The females were allowed to litter and the total number of pup's
born, number of live and dead pups, number of male and female pups, and total pup weight of males and females
were obtained. The dam and sire weights were obtained following delivery. All pups were euthanized by
decapitation on PND 1 and discarded without necropsy. All females were euthanized by carbon dioxide
asphyxiation and a uterine exam was performed to determine number of uterine implantation sites and corpora lutea.
The corpora lutea were regressed by the time of necropsy, making them very difficult to count. Since the corpora
lutea counts and pre-implantation loss data were not suitable for reporting in this study, only the number of
resorptions and post-implantation loss were reported. The difference between number of implantation sites and total
number of pups born was used to calculate the number of resorptions.
All animals were observed twice daily for mortality and signs of toxicity. Naive males and females were
not treated and were weighed at randomization, cohabitation (not required by protocol), littering, and termination.
The naive animals received physical exams weekly. Naive males were euthanized by carbon dioxide asphyxiation
after the crossover mating trial and discarded withoutnecropsy.
Task4 - On PND 21, the F1 pups were separated from the Fo dam. Two male and two female pups in each litter
were randomly selected, weighed, and assigned a permanent animal number and tattooed. Oral gavage dosing of
thiophenol in the com oil was initiated on PND 22. Selected weanlings were reared until 81 ±10 days of age.
Approximately one week before PND 81 ± 10, at least one'male and one female from each litter were
selected to obtain 20 breeding pairs. Sibling matings were avoided. Breeding pairs were cohabitated for seven days
then separated and singly housed. All litters produced were evaluated on PND 1 and then etithanized by
decapitation. The total number of pups, number of live and dead pups, number of male and female pups, and total
pup weight of each sex were obtained. The dam and sire were weighed following delivery. Vaginal smears were
obtained from all females once daily for 12 days beginning 34 days after cohabitation.
All animals were observed twice daily for mortality and signs of toxicity. Animals were weighed weekly
during the rearing period to calculate dose volumes. Body weights were obtained weekly and feed consumption was
measured during Weeks 2 and 4 of the F1 fertility assessment. Physical examinations were performed weekly during
Task 4.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0m g/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
9 mg/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
18 mg/kg
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
35 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

20 animals

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted using the National Toxicology Program's Reproductive Assessment by Continuous Breeding (RACB) protocol. The specific study design for the protocol has been published previously (Lamb, 1985; Reel et aI., 1985; Heindel et al., 1989). The RACB protocol is divided into four Tasks (Figure 1).
In the present study, Tasks 2-4 were conducted. Task 1 was not conducted since previous studies conducted at R. O.W. Sciences, Inc. provided sufficient
data to select dose levels. Based on the acute oral LD50 of 46.2 mglkg (Fairchild and Stokinger, 1958), and the increased mortality in the 40 mglkg/day females and the increased liver weights at 30 mglkg/day noted in a previously conducted RACB study, the high-dose was selected to be 35 mglkg/day, with the remaining dose levels at 18 and 9 mglkg/day. Task 2 dose levels were set so that the highest dose was expected to depress weight gain by approximately 10% and permit greater than 90% survival. The middle dose was selected to produce little or no systemic toxicity, whereas the low dose was designed to be a no~effect level.
Task 2, the continuous breeding phase, consisted of a control group and three treated groups (20 pairs/group). Following seven days of premating exposure to thiophenol by oral gavage, the animals were housed as breeding pairs for 112 days (16 weeks). Litters produced during the cohabitation period were counted and weighed by sex on PND 1 and then euthanized. At the end of the 112 days, the pairs were separated with continued dosing. Any litters born (F1) after the continuous breeding phase were reared by the dam until weaning on PND 21. Selected weanlings were reared in the same sex groups until 81 ±10 days of age. F[ animals were dosed, after weaning. with the same level of thiophenol as their parents received during Task 2. These F[ animals were used for assessment of second-generation reproductive toxicity (see Task 4 below).
Because decreased pup weights were observed during Task 2, a one-week crossover mating trial (Task 3) was performed on the parental animals from the control and high-dose groups to determine the affected sex. Lowand mid-dose animals were maintained at the same dosing regimen until the control and high-dose animals were euthanized/necropsied. After Task 3 crossover mating, Fa (Task 2/3) animals were necropsied and terminal body weights and organ weights were obtained, sperm were analyzed, and reproductive tissues were saved.
Task 4, the assessment of the Fj generation, was conducted using offspring from all four dose groups. At sexual maturity (81 ±10 days of age), twenty control animals of each sex and 20 treated animals of each sex in each dose group were randomly assigned to breeding pairs, avoiding sibling matings, and cohabited for seven days then separated. Offspring were counted and weighed by sex on PND 1. At necropsy, terminal body weights and organ weights were obtained, speml were analyzed. and reproductive tissues were saved in fixative.

Examinations

Parental animals: Observations and examinations:

Task2 - Fo. CONTINUOUS BREEDING PHASE-• Litter data •. Feed consumption • Body weight
The total number of pups, number of live and dead pups, number of male and female pups, and total pup
weight of each sex were obtained on PND 1, 4, 7, 14, and 21. The dam and sire were weighed following delivery.
The dam was also weighed on PND 4, 7, 14, and 21. Feed consumption measurements for lactating dams were
obtained on PND 1, 4, 7, 11, 14, 18, and 21.
All animals were observed twice daily for mortality and signs of toxicity. Body weights were obtained at
randomization,.at initiation of treatment, and weekly thereafter. Physical examinations were performed weekly and
feed consumption was measured during Study Weeks 1, 6, 12 and 18 (males only).

Task3- Fo FEMALE CROSSOVER MATING-• Litter data • 12 days vaginal smears • Necropsy treated felnales • Organ weights . • Histopathology
Task3- FoMALE CROSSOVER MATING - • Litter data • Necropsy treated males • Organ weights . • Sperm evalWitions • Histopathology

Task4- F1 FERTILITY ASSESSMENT - • Organ weights • .SPerm evaluations - • Histopathology

Sperm parameters (parental animals):

Sperm motion parameters were measured using the right cauda epididymis (in phosphate buffered saline
and bovine serum albumin) with the CellSoft™ computer-assisted sperm motion analysis system (Cryo Resources,
Ltd., New York). Epididymal sperm density and morphology were determined. The sperm morphology samples
were obtained from the left cauda epididymis and the sperm density samples were obtained from the right cauda
epididymis. The left cauda epididymis was used to prepare the sperm morphology slides because the right cauda
epididymis was placed in PBS/BSA for computer assisted sperm. analysis and this buffer interferes with the staining
of the sperm. The right testis was frozen for evaluation of spermatid head counts. Three hundred ILl of sperm
suspension and 300 ILl of TNE buffer were placed in an individually labelled tube, frozen and shipped on dry ice
to Dr. Schrader at NIOSH (Cincinnati, Ohio) for analysis of chromatin structure.
Animals found dead or euthanized in extremis during the study (all tasks) were subject to a gross necropsy
which is described in the protocol (Appendix 2). No histopathology was performed on these tissues

Litter observations:

Task2 - Fo. CONTINUOUS BREEDING PHASE-
The total number of pups, number of live and dead pups, number of male and female pups, and total pup
weight of each sex were obtained on PND 1, 4, 7, 14, and 21. The dam and sire were weighed following delivery.
The dam was also weighed on PND 4, 7, 14, and 21. Feed consumption measurements for lactating dams were
obtained on PND 1, 4, 7, 11, 14, 18, and 21.
All animals were observed twice daily for mortality and signs of toxicity. Body weights were obtained at
randomization,.at initiation of treatment, and weekly thereafter. Physical examinations were performed weekly and
feed consumption was measured during Study Weeks 1, 6, 12 and 18 (males only).

Postmortem examinations (parental animals):

Following the completion of the Task 3 crossover mating, terminal body weights were obtained from all
surviving Task 2/3 animals. All animals were euthanized via carbon dioxide asphyxiation and animals not scheduled
to be necropsied were discarded. A gross necropsy was performed on all of the control anilnaIs and the first
surviving 10 per sex from each of the treated groups. Necropsies were performed by trained personnel from
Pathology Associates International under the direct supervision of a Board-certified pathologist. Necropsies included
examination of the external surface of the body. all orifices, and the cranial, thoracic, and abdominal cavities and
their contents.
The following organs were weighed: liver, kidneys, right cauda epididymis, right epididymis, prostate,
seminal vesicles with coagulating glands, right .testis, and ovaries. The liver, kidneys, prostate, seminal vesicles
with coagulating glands, uterus, cervix, and vagina were preserved in 10% neutral-buffered formalin for possible
histopathological examination. The ovaries were preserved in Bouin's for 24-48 hours and then transferred to 70%
ethanol. The left testis and epididymis from all necropsied males were fixed in 4% paraformaldehyde for 48-72
bours and then transferred to phosphate buffered saline. The kidneys and livers of the first 10 control animals/sex
with liver andJor kidney lesions and all treated animals were selected to be microscopically examined based on
increased liver and kidney weights. These tissues were processed through paraffin, sectioned at 5-6 microns, sta.i.iJ.ed
with hematoxylin and eosin and evaluated. The left testis and epididymis from the first ten males per group were
processed through glycol methacrylate (GMA), sectioned at 3 microns, and evaluated.
Sperm motion parameters were measured using the right cauda epididymis (in phosphate buffered saline
and bovine serum albumin) using the CellSoftTM computer-assisted sperm motion analysis- system (Cryo Resources,
Ltd., New York). Sperm density and morphology were determined using the right cauda epididymis: The right
testis was frozen for evaluation of spermatid head counts.

Postmortem examinations (offspring):

Animals were weighed and then euthanized via CO2 asphyxiation. Animals not scheduled to be necropsied
were discarded. A gross necropsy was performed on all control animals and the first 10 surviving animals from
the treated groups. Necropsies were performed by trained personnel from Pathology Associates International under
the direct supervision of a Board-certified pathologist. Necropsies included examination of the external surface of
the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.
The following organs were weighed: liver, kidneys, right cauda epididymis, right epididymis, prostate,
seminal vesicles with coagulating glands, right testis, and ovaries. The liver, kidneys, prostate, seminal vesicles
with coagulating glands, uterus, cervix, and vagina were preserved in 10% neutral-buffered formalin for possible
histopathological examination. The ovaries were preserved in Bouin's for 24-48 hours and then transferred to 70%
ethanol. The left testis and epididymis from all necropsied males were fixed in 4% paraformaldehyde for 48-72
hours and then transferred to phosphate buffered saline. The kidneys, liver, and one ovary from the first 10
surviving control animals and all necropsied treated animals were processed through paraffin, sectioned at 5-6
microns, stained with hematoxylin and eosin and evaluated. The left testis and epididymis from the first 10
surviving control animals and all necropsied treated animals were processed through glycol methacrylate (GMA),
sectioned at 3 microns, and evaluated. These tissues were selected for microscopic examination based on increased
liver, kidney, and testis weights.
Sperm motion parameters were measured using the right cauda epididymis (in phosphate buffered saline
and bovine serum albumin) with the CellSoft™ computer-assisted sperm motion analysis system (Cryo Resources,
Ltd., New York). Epididymal sperm density and morphology were determined. The sperm morphology samples
were obtained from the left cauda epididymis and the sperm density samples were obtained from the right cauda
epididymis. The left cauda epididymis was used to prepare the sperm morphology slides because the right cauda
epididymis was placed in PBS/BSA for computer assisted sperm. analysis and this buffer interferes with the staining
of the sperm. The right testis was frozen for evaluation of spermatid head counts. Three hundred ILl of sperm
suspension and 300 ILl of TNE buffer were placed in an individually labelled tube, frozen and shipped on dry ice
to Dr. Schrader at NIOSH (Cincinnati, Ohio) for analysis of chromatin structure.
Animals found dead or euthanized in extremis during the study (all tasks) were subject to a gross necropsy
which is described in the protocol (Appendix 2). No histopathology was performed on these tissues.

Statistics:

Data from Tasks 2, 3, and 4 were statistically analyzed by Analytical Sciences Inc. (Durham, North
Carolina). Most hypotheses are tested using the nonparametric multiple comparisons procedure of Dunn (1964) or
S~irley (1977), as modified by Williams (1986). Shirley's test was designed to detect treatment related differences
when the response to treatment consistently increased (or decreased) with increasmg dose. Although the test
employs a smoothing algorithm to adjust for dose-response inversions, Dunn's test was more appropriate if the
departure from monotonicity was severe. Jonckheere's test (1954) was used to ascertain whether there was
sufficient evidence of a dose-related response to apply Shirley's test. If the p-value from Jonckheere's test was less
than 0.01, Shirley's test was used; otherwise, Dunn's test was applied.
In the crossover mating trial (Task 3), the Kruskal-Wallis test (Kruskal-Wallis, 1952) was used to test
equality of response among dose groups, while multiple comparison tests use the method of Dunn. Task 2 data
were analyzed first with Jonckheere's test and then with either Shirley's or Dunn's test. Task 3 dam weights were
analyzed with Dunn's test.
For data expressed as a proportion, such as number fertile/number cohabitated, the Cochran-Armitage test
(Armitage, 1971) was used to test for a dose-related trend, and pairwise comparisons were performed using a chi-
square test (Conover, 1971). In Task 3, where dose groups do not represent increasing dose levels, a chi-square
test was used to test simultaneous equality of response across groups and for pairwise comparisons.
Since the number of pups in a litter may influence the average pup weight, a parametric analysis of
covariance (Neter and Wasserman, 1974) was used to test overall equality in average pup weight, after adjustment
for average litter size. Pairwise comparisons were performed using Dunnett's test (1955).

Reproductive indices:

(Task 2)
Average Litters per Pair,Average Live Pups per Litter,Proportion of Pups Born Alive,Adjusted Live Pup Weighrt
,Cumulative Days to Litter
(Task 4) Pregnancy Index,Live Pups per Litter,Proportion of Pups Born Alive
,Adjusted Live Pup Weighta,Days to Litter,Estrous Cycle Length

Offspring viability indices:

Fertility Index of male. Crossover Mating - Naive Females x Treated Males (Task 3)
Live Pups per Litter
Proportion of Pups Born Alive
Fertility Index of female Crossover Mating - Naive Males x Treated Females (Task 3)
Live Pups per Litter
Proportion of Pups Born Alive

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

rough haircoat, hunched posture, red discharge from the eyes, dyspnea, thinness, diarrhea, lacrimation, rales, ulcer, tissue masses, discoloration, abscess, vaginl discharge, prolapsed vagina, etc.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fo 35 mg/kg males were 7-15% less than controls. Fo female body weights were not adversely effected by thiophenol.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Fo 35 mg/kg males were 7-15% less than controls. Fo female body weights were not adversely effected by thiophenol.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The livers and kidneys revealed treatment-related lesions in the Fo males and females. These lesions primarily involved centrilobular hepatocellular hypertrophy and renal tubule degeneration and associate changes (renal tubule dilatation).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of the vaginal smears revealed no differences between the control and high-dose groups in the amount of time spent in the different estrous stages or cycle length

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

treatment-related decreases (5-6%) in percent motile sperm in the 18 and 35 mg/kg groups compared to controls.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

increase in the incidence of enlarged and pitted kidneys in the 35 mg/kg males at necropsy

Details on results (P0)

At necropsy, liver and kidney weights increased with increasing dose: liver weights (relative to body weight) of the 9, 18, and 35 mglkg animals were increased by 20,35, and 50% (males) and 11, 18,36% (females), respectively. Relative kidney weights of the 9, 18, and 35 mg/kg animals were increased by 30, 53, and 104% (males) and 8, 5, 20% (females), respectively. There was a treatment-related increase in the incidence of enlarged and pitted kidneys in the Fo males at necropsy. Increased incidences of renal tubule degeneration (30%,35 %, and 40%, respectively) were observed in 9, 18, and 35 mg/kg Fo males and females. Centrilobular hepatocellular hypertrophy was observed in the 18 and 35 mg/kg Fo males and 9, 18, and 35 mg/kg Fo females.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 9 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical observations noted during Task 4 included alopecia, thinness, hunched posture, abrasions, discharge from the eyes, rough haircoat, swelling, and tissue masses.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No differences were observed in the mean average litters per pair, proportion of pups born alive, or sex ratio (number of males/total number of pups) (Table 2-2). The number of live pups per litter was comparable between the 0, 9, and 18 mg/kg dose groups

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of the 35 mglkg F, males were 11-17% less than controls on Task Weeks 2 and 4, and at delivery and necropsy.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

revealed no differences between dose groups in pregnancy index, number of live pups per litter, proportion of pups bom alive, sex ratio of pups, dain weight at delivery, and average days to litter.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

relative liver weights of the 9, 18, and 35 mg/kg animals were increased males and females. Relative kidney weights of the 9, 18, and 35 mg/kg animals were increased males and females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, the liver and kidneys were also enlarged in a treatment-related fashion for both sexes in the FI animals.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

tubule degeneration was observed in 9, 18, and 35 mg/kg FI males and 18 and 35 mg/kg FI females.·Centrilobular hepatocellular hypertrophy was observed in the 9, 18, and 35 mg/kg Fl males and females.

Details on results (F1)

In the F1 mating trial, live F2 pup weight was decreased by 9 and 12%in the 18 and 35 mglkg dose groups,
respectively, when compared to controls. Other endpoints were unchanged.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 9 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: As observed in the Fo animals, renal tubule degeneration was observed in 9, 18, and 35 mg/kg FI males and 18 and 35 mg/kg FI females. Centrilobular hepatocellular hypertrophy was observed in the 9, 18, and 35 mg/kg Fl males and females.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

The potential reproductive toxicity of thiophenol in Sprague-Dawley rats was evaluated using the Reproductive Assessment by Continuous Breeding (RACB) protocol. Based on acute oral LD50 of 46.2 mg/kg and the increased mortality in the 40 mg/kg/day females and the increased liver weights at 30 mg/kg/day noted during a previous RACB study with thiophenol, dose levels for the continuous breeding phase for this study were set at 9, 18, 35mg/kg. Male and female Sprague-Dawley rats were exposed to thiophenol in com oil by oral gavage. During 16 weeks of cohabitation, live pup weight adjusted for litter size was decreased by 4 and 6% in the 9 and 35 mg/kg dose groups, respectively. The number of live pups was slightly but not significantly decreased (7 %) at 35 mg/kg. No differences were observed in the pregnancy index, cumulative days to litter, mean average litters per pair, proportion of pups born alive, or sex ratio of pups. A crossover mating trial (Task 3) revealed the females as the affected sex. When naive males were mated with control or 35 mg/kg females, the mean live pup weight and adjusted live pup weight were reduced in the 35 mg/kg group by 8-9%. No other treatment-related effects were seen. When naive females were mated with control or 35 mg/kg males, reproductive parameters were comparable between dose groups.

Throughout the study, the body weights of the Fo 35 mg/kg males were 7-15% less than controls. Fo female body weights were not adversely effected by thiophenol. At necropsy, liver and kidney weights increased with increasing dose: liver weights (relative to body weight) of the 9, 18, and 35 mg/kg animals were increased by 20,35, and 50% (males) and 11, 18, 36% (females), respectively. Relative kidney weights of the 9, 18, and 35 mg/kg animals were increased by 30, 53, and 104% (males) and 8, 5, 20% (females), respectively. There was a treatment-related increase in the incidence of enlarged and pitted kidneys in the Fo males at necropsy. Increased incidences of renal tubule degeneration (30%, 35 %, and 40%, respectively) were observed in 9, 18, and 35 mg/kg Fo males and females. Centrilobular hepatocellular hypertrophy was observed in the 18 and 35 mg/kg Fo males and 9, 18, and 35 mg/kg Fo females.

Evaluation of epididymal computer-assisted sperm analysis (CASA) data revealed treatment-related decreases (5-6%) in percent motile sperm in the 18 and 35 mg/kg groups compared to controls. Other reproductive endpoints at necropsy were comparable among dose groups.

In Task 4 (second generation evaluation), the pup weights of the 35 mg/kg FI males and females were decreased by 13-16% during Postnatal Day (PND) 4 and 7 of lactation, however, no differences were observed on PND 1, 14 or 21. There was no treatment-related increase in pre-weaning mortality of the F1 animals.

In the F1 mating trial, live F2 pup weight was decreased by 9 and 12%in the 18 and 35 mglkg dose groups, respectively, when compared to controls. Other endpoints were unchanged.

Body weights of the 35 mglkg F1, males were 11-17% less than controls on Task Weeks 2 and 4, and at delivery and necropsy. At necropsy, the liver and kidneys were also enlarged in a treatment-related fashion for both sexes in the FI animals: relative liver weights of the 9, 18, and 35 mg/kg animals were increased by 18,37, and 62% (males) and 14,17,43% (females), respectively. Relative kidney weights of the 9, 18, and 35 mg/kg animals were increased by 52, 67, and 163%(males) and 12, 6, 26% (females), respectively. There was a treatment-related increase in the incidence of enlarged, pale, and soft kidneys in the FI males at necropsy. As observed in the Fo animals, renal tubule degeneration was observed in 9, 18, and 35 mg/kg FI males and 18 and 35 mg/kg F1 females.· Centrilobular hepatocellular hypertrophy was observed in the 9, 18, and 35 mg/kg Fl males and females. Microscopic evaluation of the left testis from F1 males revealed a treatment-related increase in inhibited spermiation of the stageVIII-X tubules. No F1 sperm endpoints were significantly changed. Results of this study show that thiophenol is not a selective reproductive toxicant, because the minor effects on development (decreased live pup weight during the crossover mating) occurred concomitant with, or at doses greater than, those doses that produce hepatic or renal effects. The no-observable-adverse-effect level (NOAEL) was not established in this study as the 9 mg/kg animals displayed increased liver and kidney weights (absolute and relative) and treatment-related microscopic lesions. A maximum tolerated dose (MID) was reached for the 35 mg/kg Po and F1 generation animals, based on decreased body weights (males only), increased liver and kidney weights. and treatment-related renal and hepatic lesions.