2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-7-oxo-8-oxa-3,5-dithia-4-phosphatetradecanoate 4-oxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD Guideline 421 Reproduction / Developmental Toxicity Screening Test (gavage, rats) - read across

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

4. DATA MATRIX

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system 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

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

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

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. 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

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS AND SURVIVAL: Yes
All rats were observed twice daily, once in the morning and once in the afternoon‘ for mori bundity and mortalitv. Individual detailed clinical observations were recorded weekly (prior to test article administration during the treatment period). Each male and female was also observed for signs of toxicity at the time of dosing and approximately 1-2 hours following dose administration. AlI significant findings were recorded. Observations included, but were not limited to, evaluations for changes in appearance of the skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system functions, somatomotor activity and behavior patterns. 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.

BODY WEIGHT: Yes
Individual male body weights were recorded weekly, beginning prior to treatment on the first day of dose administration, throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded weekly, beginning on the first day of dose administration, until evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.

FOOD CONSUMPTION: Yes
Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17and 20 and on lactation days 1 and 4. Following mating, food consumption for males was measured on a weekly basis until the scheduled euthanasia.

Oestrous cyclicity (parental animals):

Not done

Sperm parameters (parental animals):

Not done

Litter observations:

LITTER VIABILITY AND DEATHS
Each litter was examined dailv for survival, and all deaths were recorded. A daily record of litter size was maintained. Intact offspring dying were necropsied using a fresh dissection technique including the heart and major vessels Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by Ithe gross findings. The carcass of each pup was then discarded.

CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS
Pups were individually weighed on PND 1 and 4.

SEX DETERMINATION
Pups were individually sexed on PND 1 and 4.

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. Females that delivered were euthanized on lactation day 4, the number of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on postmating day 25 (females with evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski. 1964).

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Brain
Epididymides
Kidnevs
Liver
Ovaries with oviducts
Pituitary gland
Testes

HISTOPATHOLOGY
Ovaries with oviducts, pituitary gland, testes, epididymides were prepared for microscopic examination. 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. The vagina and cervix were also examined from each female in the 10 and 50 mg/kg/day 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

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. In addition one female in the 150 mg/kg/day group was euthanized on lactation day 1 due to total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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. 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 14-17 and 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) during gestation days 17-20.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males:
An increase in food consumption was observed during the post-mating period. It was attributed to the weight loss.

Females:
No statistical difference in food consumption was attributed to treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

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 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.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
No test article-related effects on F0 reproductive performance were observed at any dosage level.
Male and female mating indices were 100.0% in all groups.
Male fertility indices were 91.7, 100.0, 83.3 and 100.0% and female fertility indices were 91.7, 100.0, 83.3 and 83.3% in the control, 10, 50 and 150 mg/kg/day groups, respectively.
Male copulation indices were 91.7, 100.0, 83.3 and 100.0% and female conception indices were 91.7, 100.0, 83.3 and 83.3% in the same respective groups.
No statistically significant differences were noted between the control and test article-treated groups. Males that did not sire a litter numbered 1, 0, 2 and 0 in the control, 10, 50 and 150 mg/kg/day groups, respectively.
Females that had evidence of mating but were nongravid numbered 1, 0, 2 and 2 in the same respective groups.
The mean numbers of days between pairing and coitus in the test article-treated groups were similar to the control group value. None of these differences were statistically significant.
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).
Mean live litter size on 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.

GESTATION LENGTH AND PARTURITION
Mean gestation lengths in the 10, 50 and 150 mg/kg/day groups were similar to those in the control group.
Signsof dystocia were observed in female no. 74656 (euthanized in extremis on gestation day 22 in the 150 mg/kg/day group). On the day of expected parturition, this female was hypoactive, had an unkempt appearance, piloerection, soft stool, drooping eyelids, hypothermia, was recumbent and unresponsive to handling. In addition, 2 other females in this group were found dead 1 day prior to the expected day of parturition (gestation day 21).

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

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

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The percentage of males at birth in the treated groups was unaffected.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Pups (Iitters) that were found dead numbered 2(2), 2(2), 2(2) and 23(4) in the control‘10, 50 and 150 mg/kg/day 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.

A reduction (not statistically significant) in postnatal survival in the 150 mg/kg/day group (93.8% per litter) was observed on PND 0 (relative to the number born) compared to the control group value (98.9% per litter). Mean postnatal survival in this group (82.1 % per litter) was reduced during PND 0-1 (not statistically significant) compared to the control group value (100.0% per litter). This decrease was largely due to female no. 74640 that had total litter loss on PND 1 (17 pups): however, dead pup was found in each of 3 other litters at that time. Postnatal survival in the 150 mg/kg/day group was similar to that in the control group during PND-4. However. when the entire postnatal period (birh to PND 4) was evaluated, postnatal survival in this 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights in the 150 mg/kg/day group pups were decreased (10.0%-17.2%, males and 13.6%-18.3%, females) compared to the control group values 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.

Mean male and female pup body weights and body weight changes in the 10 and 50 mg/kg/day groups were unaffected during PND 1-4. No statistically significant differences from the control group were noted.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Of the pups (litters) found dead during PND 0-4 numbered 0(0), 1(1) and 18(2) were too autolyzed to examine and 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 internal findings were noted.

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

In this study 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, developmental, systemic and neonatal toxicity resulting from exposure to 2-ethylhexyl mercaptoactetate when administered orally by gavage to rats.

Executive summary:

In an OECD guideline 421 study, groups of 12 male and female rats were exposed to doses of EHTG of 0, 10, 50 and 150 mg/kg bw/day by gavage (WIL, 2005). 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. All animals were observed twice daily for mortality and morbidity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. All F0 females were allowed to deliver and rear their pups until lactation day 4. The F0 females were euthanized on lactation day 4 and the F0 males were euthanized following completion of the necropsies of the F0 females. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. 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. F1 clinical observations and body weights were recorded on postnatal days (PND) 1 and 4. Pups were euthanized on PND 4 following an external examination and discarded without further evaluation.

Three males and 3 females in the 150 mg/kg-bw/day group were found dead or euthanized in extremis. Prior to death/euthanasia, 2 of these males and 1 female had marked body weight losses; these males also had decreased defecation, unkempt appearance and/or red or yellow material on various body surfaces. The female that was euthanized in extremis had signs of dystocia. These premature deaths were attributed to the test article. In addition, 1 female in the 150 mg/kg-bw/day group was euthanized due to total litter loss on lactation day 1. All other animals survived to the scheduled necropsies. There were no test article-related clinical findings in the 10 mg/kg-bw/day group males and females or in the 50 mg/kg-bw/day group females. There were no test article-related effects on male and female mating and fertility indices, male copulation index or female conception index. The mean number of days between pairing and coitus in the test article-treated groups were similar to the control group value.

Mean body weights and/or body weight gains in the 150 mg/kg-bw/day group males were generally reduced throughout the study. Mean food consumption in these males was similar to that in the control group during the pre- and post-mating periods. During the pre-mating period, no test article-related effects on body weight gain and food consumption were observed in the 150 mg/kg-bw/day group females. However, mean maternal body weight gain in these females was lower during gestation days 17-20; the reduction was attributed to a female with a small litter size and 1 of the females that was found dead on gestation day 21. Gestation food consumption was unaffected by test article administration in this group. During lactation, mean body weight gain and food consumption in the 150 mg/kg/day group were similar to that in the control group. There were no test article-related effects on mean body weights or food consumption in the 10 and 50 mg/kg-bw/day group males and females throughout the study.

Mean gestation lengths in the test article-treated groups were similar to that in the control group. Two females in the 150 mg/kg-bw/day group were found dead on gestation day 21, near the time of expected parturition, 1 female was euthanized in extremis on gestation day 22 with signs of dystocia, and 1 female was euthanized due to total litter loss on lactation day 1.

Mucification of the cervical and vaginal epithelium was noted microscopically in the 150 mg/kgbw/ 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. Although this finding itself was not considered abnormal morphologically, its presence on lactation day 4 was clearly increased compared to the control group. 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-bw/day group was considered test article-related. No test article-related effects on the mean numbers of corpora lutea, implantation sites or unaccounted-for sites were observed at any dosage level. Slight reductions in the mean numbers of corpora lutea and implantation sites were observed in the 150 mg/kg-bw/day group. The decreases resulted in a reduced mean number of pups born and corresponded to lower mean maternal body weight gain late in gestation. These slight reductions in mean numbers of corpora lutea and implantation sites and the number of pups born was not considered test article-related as these decreased mean values were attributed primarily to a single female with only 8 corpora lutea.Macroscopic changes in the liver that correlated to hepatocellular vacuolization were observed in 2 males and 1 female in the 150 mg/kg/day that were found dead or euthanized in extremis. At the scheduled necropsies, no test article-related macroscopic lesions were observed, including in the liver. However, mean relative (to final body and brain weight) liver weights in the 150 mg/kg/day group males and females were increased, and the increase was considered related to the lesions observed in the unscheduled death animals. Mean relative (to final body weight) kidney weight was increased in the 150 mg/kg/day group males. EHTG reduced post-natal survival at 150 mg/kg-bw/day but was not teratogenic. There were no test article-related effects on the general physical condition of the pups or pup body weights in the 10 and 50 mg/kg/day groups. At the necropsy of pups that were found dead, there were no internal findings that were related to parental treatment with the test article.At doses producing maternal mortality, EHTG is considered to have a reproductive effect (dystocia in the 150 mg/kg-bw/day group was considered test article-related). Within the limits of the experimental design, a dosage level of 50 mg/kg-bw/day was considered to be the NOAEL for reproductive effects and systemic (maternal) toxicity resulting from exposure to EHTG when administered orally by gavage to rats.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]
The read across substance, 2-ethylhexyl mercaptoacetate, is a presursor and impurity in the commercial form of the registered substance (product name D 16-051). More importantly, 2-ethyl hexyl mercaptoacetate is also an expected hydrolysis degradation product of the registered substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]
The commercial form of the registered substance (product name D 16-051), contains approximately 4% 2-ethylhexyl mercaptoacetate.

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]
In addition to being a precursor and impurity in the commercial form of the registered substance, 2-ethylhexyl mercaptoacetate is an expected hydrolysis degradation product of the registered substance. It is therefore considered to be a relevant surrogate substance.

4. DATA MATRIX
Genotoxicity:
The registered substance was negative in the Ames test at concentrations up to 5000 µg/plate with and without metabolic activation, and did not produce cytogenetic changes in the bone marrow of Chinese hamster treated by oral gavage up to a limit dose of 3,000 mg/kg.

The read across substance, 2-ethylhexyl mercaptoacetate was negative in the in vitro gene mutations assays on bacteria (Ames test) and mammalian cells (mouse lymphoma assay), both in the presence and absence of metabolic activation, and was negative in the in vivo mouse micronucleus test.

Repeated Dose Toxicity:
Registered substance - The ‘No Observed Effect Level’ (NOEL) for systemic  toxicity in a 28-day oral gavage study in rats was 100 mg/kg bw/day.

Read across substance - Administration of 2-ethylhexyl mercaptoacetate in the diet of rats for 28 days did not lead to a proliferation of hepatic peroxisomes, and did not produce any treatment-related effects. The 28 day NOAEL was 0.2% EHTG in the diet (the highest dose tested; 168 mg/kg bw for males; 173 mg/kg bw for females).

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

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

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The surrogate substance 2-ethylhexyl mercaptoacetate (EC No. 231 -626 -4), which is an impurity in D16-051 and also an expected hydrolysis degradation product of D 16-051, was tested in an OECD 421 guideline study. In this study 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, developmental, systemic and neonatal toxicity resulting from exposure to 2-ethylhexyl mercaptoactetate when administered orally by gavage to rats.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

In an OECD guideline 421 study of the surrogate substance 2 –ethylhexyl mercaptoacetate (an impurity and expected degradation product of D 16-051), 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, developmental, systemic and neonatal toxicity resulting from exposure to 2-ethylhexyl mercaptoactetate when administered orally by gavage to rats.

Justification for classification or non-classification

Based on data available for the surrogate substance 2 –ethylhexyl mercaptoacetate (an impurity and expected degradation product of TGEPO), TGEPO is not classified for reproductive toxicity according to Regulation (EC) No 1272/2008.