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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 9 february to 6 July 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-ethylhexyl mercaptoacetate
- Physical state: colourless liquid
- Stability under test conditions: considered stable
- Storage condition of test material: at room temperature under nitrogen

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, North Carolina, USA
- Age at initiation of dose administration: approximately 10 weeks old
- Weight at initiation of dose administration: Males: 322.9-387.3 g; Females: 210.3-261.5 g;
- Housing: individually
- Diet: Certified Rodent Labdiet 5002 ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-21.6
- Humidity (%): 35.7-40.7
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 february 2005 To: 6 July 2005

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test article formulations were weight/volume (test article/vehicle) formulations. They were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored at room temperature. They were stirred continuously throughout the
preparation, sampling and dose administration procedures. They were visually found to be homogeneous and anatically confirmed to be homogeneous.


VEHICLE
- Justification for use and choice of vehicle (if other than water): no
- Concentration in vehicle: 0 (vehicle group), 2 (low), 10 (middle) and 30 mg/mL (high concentration group)
- Amount of vehicle (if gavage): Dosage volume was 5 mL /kg for all groups
- Purity: 100%
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): in plastic maternity cages with nesting material, ground corncob bedding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Resuspension homogeneity and 11-day stability were established in a previous study. Samples for concentration analysis were collected from the
middle stratum of each formulation (including control) every 2 weeks.
Duration of treatment / exposure:
Exposure period: Males received 15 daily doses prior to mating. Males were dosed throughout the mating period for a total of 54 doses.
Females received a minimum of 15 daily doses prior to pairing and were dosed through lactation.
Duration of test substance (TS) exposure: males 54 days; females 41-44 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 50 and 150 mg/kg bw /day
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on the results of a previous study (See Bowman (2005)/Oral 14 day repeated 02). In that study, 1 female in the 150 mg/kg/d group was found dead on study day 2. Effects on the mean bodyweight, on food consumption and liver and kidney weights were also observed at this level in both sexes. Increased liver and kidney weights were also observed in the males of the 50 mg/kg/d group.
- Rationale for animal assignment: no (randomization).
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data. Cage-board was changed 3 times per week.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly and twice daily for mortality or signs of moribundity


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly until pairing; not recorded during mating period; then weekly for males and
on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.

WATER CONSUMPTION : No
Oestrous cyclicity (parental animals):
During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. Individual gestation length was calculated using the date delivery started.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight
Litter observations:
Pups were examined for gross malformations, and the number of stillborns and alive pups were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at day 54 after the first day of treatment
- Maternal animals: All surviving animals at post-mating day 25 or at lactation day 4


GROSS NECROPSY
- Gross necropsy consisted of examination of external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic,
abdominal and pelvic cavities, including viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
Ovaries with oviducts, pituitary gland, testes, epididymides were prepared for microscopic examination and weighed. Brain, kidneys and liver were weighed. Coagulating glands, mammary gland, prostate gland, seminal vesicle, uterus with vagina and cervix and all gross lesions were prepared for microscopic examination.
Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups and females in the low- and mid-dose
groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age following an external examination and discarded without further evaluation.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test
group to the control group by sex. Statistical analyses were not conducted if the number of animals was two or less. Data obtained from nongravid
females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
Parental mating, fertility, conception and copulation indices were analyzed using the Chi-square test with Yates' correction factor
(Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption,
offspring body weights and body weight changes, gestation length, number of implantation sites, number of corpora lutea, number of pups born,
live litter size on Post Natal Day (PND) 0, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals values were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran. 1980) to determine intergroup differences. If the ANOVA revealed
statisticallv significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA test
(Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance,
Dunn's test (Dunn, 1964) was used to compare the test groups to the control group.
Reproductive indices:
Mating, fertility and copulation/conception indices were calculated.
Offspring viability indices:
Mean live litter size and postnatal survival were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Three males in the 150 mg/kg/day group were found dead or euthanized in extremis due to excessive body weight loss on study days 4 or 14. Two of them had decreased defecation and an unkempt appearance the day of or prior to euthanasia on study day 14.
Three females in the 150 mg/kg/day group were found dead or euthanized in extremis on gestation day 21 or 22. On gestation day 22, one female
exhibited signs of dystocia: hypoactivity, an unkempt appearance, piloerection, soft stool, drooping eyelids, hypothermia (cool to the touch) and was recumbent and unresponsive to handling; consequently, this female was euthanized later that day.

Salivation and/or clear material around the nose and/or mouth was observed in the 50 mg/kg/day group males and the 150 mg/kg/day group
males and females at the time of dose administration and/or approximately 1-2 hours following dose administration.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
Mean body weight gain was reduced (statistically significant, p<0.01) in the 150 mg/kg/day group during the pre-mating period (study days 0-14) compared to that in the control group, resulting in reduced (not statistically significant) mean body weight gains when the entire treatment period
(study days 0-54) was evaluated. An increase in food consumption was observed during the post-mating period. It was attributed to the weight loss.
Mean body weights and body weight gains in the 10 and 50 mg/kg/day group males were similar to those in the control group throughout the study. None of the differences were statistically significant.

Females:
No statistical change was elicited during pre-mating and lactation periods.
During gestation days 17-20, mean body weight gains in the 150 mg/kg/day group females were reduced compared to those in the control group.
The difference was statistically significant (p<0.05).
No statitical difference in food consumption was attributed to treatment.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Mean gestation lengths in the 10, 50 and 150 mg/kg/day groups were similar to those in the control group. No statistically significant differences
were noted.
In one female at 150 mg/kg bw/d, signs of dystocia were observed. In addition, 2 other females in this group were found dead l day prior to the expected day of parturition (gestation day 21).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No significant effect of treatment on testes and epididymides weight was observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test article-related effects on F0 reproductive performance were observed at any dosage level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Test article-related increases in mean relative (to final body and brain weight) liver weights were observed in the 150 mg/kg/day group males and
females. The difference from the control group relative (to final body weight) value was statistically significant (p<0.01) in the males and females. The increase in mean relative liver weight corresponded to the microscopic findings observed in the 150 mg/kg/day group animals that were found
dead or euthanized in extremis. An increase in mean kidney weight (absolute and relative to final body and brain weights) was observed in the 150 mg/kg/day group males; the difference from the control group relative (to final body weight) value was statistically significant (p<0.01).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Three males and 3 females in the 150 mg/kg/day group were found dead or euthanized in extremis during the study.
One male was found dead on study day 4 and had no gross pathology findings. The two other males were euthanized in extremis on study day 14; both males had pale livers. One also had white areas in the liver.
One dead female had a white area in the liver and dark red discoloration of the lungs; there were no other test article-related macroscopic findings.
The findings in the liver corresponded to increases in the mean relative liver weights in males and females in the 150 mg/kg/day group at the
scheduled necropsy, and were therefore considered test article-related.
No other significant findings were observed by macroscopic examination.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The 2 males that were euthanized in extremis and 1 of the females that was found dead had test article-related findings (periportal or generalized
hepatocellular vacuolation) in the liver. This finding was characterized by the presence of numerous small, round, clear vacuoles within the
hepatocellular cytoplasm, which distended the affected cells and gave the cytoplasm a foamy appearance. Affected cells were limited to the areas
immediately surrounding portal triads (periportal hepatocellular vacuolation) or affected hepatocytes in a diffuse manner without apparent zonal
distribution (generalized hepatocellular vacuolation). This finding correlated with the macroscopic findings (white areas or pale liver) observed
in these animals that were found dead/euthanized in extremis and with increased relative liver weights observed in animals at the scheduled
necropsy.
Livers were not scheduled to be retained/examined at the scheduled necropsy. Therefore, further investigation of this finding at the
scheduled necropsy was not possible.

Mucification of the cervical and vaginal epithelium was noted microscopically in the 150 mg/kg/day group gravid females that died or were euthanized in extremis prior to the scheduled necropsy on lactation day 4. This finding may be a morphologically normal peri-parturitional phenomenon, but since there were no control group animals examined at that age, this cannot be unequivocally established. However. there was a dose-related increase in incidence and severity of mucification compared to the control group at the scheduled necropsy on lactation day 4 (1/11 (9.1%),
3/12 (25%), 3/10 (30%) and 4/6 (67%) of the gravid females in the control, 10, 50 and 150 mg/kg/day groups, respectively). Taken in context of the maternal mortality, dystocia and adverse effects on pup growth and survival, mucification of the vaginal epithelium in the 150 mg/kg/day group was considered test article-related.

All other microscopic changes were consistent with normal background lesions in clinically normal rats of the strain and age used in this study, and were considered to be spontaneous and/or incidental in nature and unrelated to test article administration.


Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Mean live litter size on Post Natal Day (PND) 0 in the 150 mg/kg/day group (12.7 pups per dam) was decreased compared to the control group value (15.3 pups per dam). The difference was not statistically significant.

When the entire postnatal period (birth to PND 4) was evaluated, postnatal survival in the 150 mg/kg bw/d group (73.2% per litter) was reduced compared to that in the control group (98.9% per litter). The difference was statistically significant (p<0.05).
The effects on postnatal survival in this 150 mg/kg/day group were considered test article-related.

The mean number of pups born in the 150 mg/kg/day group (13.3 per dam) was slightly (not statistically significant) lower than the control group
value (15.5 per dam). This reduction was due to one female that delivered only 8 pups: this female also had only 8 corpora lutea. Therefore, the
reduction in the mean number of pups born (not survival) in the 150 mg/kg/day group was not considered to be treatment-related, although there was clear maternal toxicity (mortality) in this dose group.

The percentage of males at birth in the 150 mg/kg/d group was unaffected by administration of the test article to the parental animals.

The mean number of pups born, live litter size and the percentage of males at birth in the 10 and 50 mg/kg/day groups were similar to the control
group values. Postnatal survival in these groups were unaffected by test article administration.


CLINICAL SIGNS (OFFSPRING)
2(2), 2(2), 2(2) and 23(4) pups (litters) were found dead in the control, 10, 50 and 150 mg/kg/d groups, respectively. In the 150 mg/kg/day group, 4 pups were missing and presumed to have been cannibalized and 7 pups were small; these findings were considered to be test article-related. The general physical condition of pups in the 10 and 50 mg/kg/day groups was unaffected by test article administration.

BODY WEIGHT (OFFSPRING)
Mean body weights in the 150 mg/kg/day group pups were decreased compared to the control group values (10.0% - 17.2% for males and 13.6% - 18.3% for females on PND 1 and 4); the majority of the differences were statistically significant (p<0.05). Mean male and female pup body weight gains
during PND 1-4 in this group were reduced compared to those in the control group: the difference was not statistically significant. The reductions in pup body weights and body weight gains were attributed to the test article.

GROSS PATHOLOGY (OFFSPRING)
Of the pups (litters) found dead during PND 0-4, 1(1), 0(0), 1(1) and 18(2) were too autolyzed to examine in the control, 10, 50 and 150 mg/kg/day groups, respectively.
Of the remaining pups (litters), 1(1), 2(2), 1(1) and 5(2) had no presence of milk in the stomach in the control, 10, 50 and 150 mg/kg/day groups, respectively.
No other gross pathology findings were noted.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: viability index

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 Mean Body Weights (g) – Parental Males

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Males

Initial

357.1 +/- 16.6

356.0 +/- 13.8

355.5 +/- 14.6

353.7 +/- 13.9

Week 1

389.9 +/- 23.7

384.3 +/- 20.4

383.1 +/- 16.6

369.4 +/- 25.3

Week 2

419.6 +/- 29.6

411.3 +/- 24.6

411.0 +/- 16.3

367.1 +/- 54.9**

Week 3

436.4 +/- 16.6

427.8 +/- 28.7

427.0 +/- 17.8

405.8 +/- 21.9

Week 4

463.6 +/- 33.4

453.7 +/- 30.9

451.3 +/- 18.5

427.0 +/- 21.7*

Week 5

489.1 +/- 39.0

477.4 +/- 32.9

470.2 +/- 26.8

449.1 +/- 27.9

*Statistically significant difference from the control group (p<0.05) using Dunnett’s test.

**Statistically significant difference from the control group (p<0.01) using Dunnett’s test.

Table 2 Mean Body Weights changes (g) – Statistical changes in Parental animals

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Males

Day 0-7

32.9 +/- 10.2

28.3 +/- 8.4

27.6 +/- 7.9

17.0 +/- 17.7**

Day 0-14

62.3 +/- 15.8

55.3 +/- 13.6

55.6 +/- 10.5

14.8 +/- 51.8**

Females

Day 0-7

9.1 +/- 5.2

8.9 +/- 7.6

9.6 +/- 7.1

0.4 +/- 11.7*

Day 7-14

8.4 +/- 8.2

9.2 +/- 5.7

9.2 +/- 5.9

26.8 +/- 19.8**

Gestation Day 17-20

44 +/- 6.6

43 +/- 8.4

47 +/- 7.3

30 +/- 21.8*

*Statistically significant difference from the control group (p<0.05) using Dunnett’s test.

**Statistically significant difference from the control group (p<0.01) using Dunnett’s test.

Table 3 Relative Organ Weights – Statistical changes in Parental animals scheduled for necropsy

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Organ Weight (g) to bodyweight

Males

Kidney

0.714 +/- 0.048

0.694 +/- 0.043

0.739 +/- 0.070

0.795 +/- 0.067**

Liver

3.77 +/- 0.32

3.72 +/- 0.31

3.98 +/- 0.38

4.33 +/- 0.34**

Females

Kidney

0.695 +/- 0.046

0.700 +/- 0.058

0.698 +/- 0.043

0.763 +/- 0.046

Liver

4.48 +/- 0.42

4.41 +/- 0.35

4.45 +/- 0.34

5.20 +/- 0.42**

**Statistically significant difference from control group (p<0.01) using Dunnett’s test.

Table 4 Postnatal survival rates (Birth to Postnatal Day 4)

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

% per litter

Birth to Postnatal Day 4

98.9 +/- 2.5

97.9 +/- 3.9

98.7 +/- 2.7

73.2 +/- 33.9

**Statistically significant difference from control group (p<0.01) using Dunnett’s test.

Table 5 Mean Body Weights (g) – Offspring

Exposure Group

Control

10 mg/kg/d

50 mg/kg/d

150 mg/kg/d

Males

Postnatal Day 1

7.0 +/- 0.6

6.8 +/- 0.4

7.1 +/- 0.8

6.3 +/- 0.7

Postnatal Day 4

9.9 +/- 1.1

9.2 +/- 0.7

9.6 +/- 1.4

8.2 +/- 0.7*

Females

Postnatal Day 1

6.6 +/- 0.8

6.4 +/- 0.3

6.7 +/- 0.7

5.7 +/- 0.7*

Postnatal Day 4

9.3 +/- 1.2

8.7 +/- 0.7

9.2 +/- 1.1

7.6 +/- 1.3**

*Statistically significant difference from control group (p<0.05) using Dunnett’s test.

Applicant's summary and conclusion

Conclusions:
A dosage level of 50 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity resulting from exposure to 2-ethylhexyl mercaptoactetate when administered orally by gavage to rats.
Executive summary:

This study was designed to provide information on the potential adverse effects of oral exposure 2-ethylhexyl mercaptoacetate on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition and lactation of the Fo generation and development of offspring from conception through day 4 of postnatal life.

2-ethylhexyl mercaptoacetate, in the vehicle, corn oil, was administered orally by gavage once daily to 3 groups of Crl:CD (SD) rats, each group consisting of 12 males and 12 females. Dosage levels were 10, 50 and 150 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 12 rats/sex received the vehicle on a comparable regimen. Males received 15 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 54 doses. Females received a minimum of 15 daily doses prior to pairing and were dosed through lactation day 3 for a total of 41-44 doses.

In this study, severe parental systemic toxicity was observed in the 150 mg/kg/day group. It was characterized by: mortality, moribundity, decreased mean body weight gain, decreased consumption of feed, increased liver and kidney weight, or hepatocellular vacuolization in at least one sex of the F0 animals; and increased mucification of the cervical and vaginal epithelium in post-partum F0 dams. Decreased viability and growth of the F1 animals through post-partum day 4 also occurred at the 150 mg/kg/day dose. Within the limits of the experimental design, a dosage level of 50 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity resulting from exposure to 2-ethylhexyl mercaptoactetate when administered orally by gavage to rats.