2-ethylhexyl mercaptoacetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 421 study on 2-ethylhexyl mercaptoacetate (EHTG)

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 (Bowman, 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/kg-bw/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.

 

OECD 421 study on sodium mercaptoacetate (sodium thioglycolate)

In a reproduction/developmental screening test performed according to the OECD Guideline # 421, four groups of 12 male and 12 female Sprague-Dawley rats received sodium thioglycolate (purity 98.9% pure), daily, by oral (gavage) administration, 10 weeks before mating and through mating and, for the females, through gestation until day 5 post-partum, at dose-levels of 0, 20, 40 or 80 mg/kg bw/d (Davies, 2010).

Clinical signs and mortality were checked daily. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. The animals were paired for mating and the dams were allowed to litter and rear their progeny until day 5post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth, pup clinical signs were recorded daily and pup body weights were recorded on days 1 and 5post-partum. The males were sacrificed after completion of the mating period and the females on day 5 post-partum (or on day 25post-coitumfor females which did not deliver). The body weight and selected organs (brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles and testes and uterus) were weighed and a macroscopic post-mortem examination of the principal thoracic and abdominal organs and a microscopic examination of selected organs (macroscopic lesions, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, testes, and uterus) were performed. In the females, which were apparently non-pregnant, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique. Epididymal sperm was sampled for motility, morphology and count and testicular sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted. The pups were sacrificed on day 5post-partumand were carefully examined for gross external abnormalities and a macroscopic post-mortem examination was performed.

Two males (weeks 11 and 13) and one female (week 4) given 80 mg/kg/day were found dead during the pre-mating or mating periods with no clinical signs observed before death and no relevant post-mortem findings. These deaths are considered to be treatment-related. Three out of 11 surviving females given 80 mg/kg/day were found dead on day 23 post-coitum, all having delivered pups, although one female had one fetus in the vagina and still had 11 dead fetuses in the uterine horns at necropsy. Another pregnant female with dead and live fetuses in the uterine horns was sacrificed on day 23 post-coitum because of poor clinical condition. At 80 mg/kg/day, one additional female was prematurely sacrificed on day 1 post-partum because all the pups were dead and another female was found dead on day 2 post-partum.

One female given 40 mg/kg/day was found dead on day 22 post-coitum, pregnant with dead fetuses in the uterine horns.

Ptyalism was observed at 40 and 80 mg/kg/day with a dose-related incidence and may be related to the taste of the test item.

There were no significant effects of treatment on mean body weight gains; all sodium thioglycolate-treated male and female groups had body weight gains similar to those of the control group throughout the study. There were no effects of treatment on mean food consumption, except a slight lowering of food consumption during the lactation period for the two females given 80 mg/kg/day (-10%).

There were no effects of treatment with sodium thioglycolate on vaginal cyclicity, with mean cycle lengths of 4 to 5 days in all groups including control. All pairs mated and the majority of the females were pregnant. There were no effects of treatment on the mean number of days taken to mate.

Females given 80 mg/kg/day had a significantly longer gestation period (22.8, p<0.01, vs. 21.6 days), a non-significantly lower number of corpora lutea (mean of 6.7, ns, vs.8.5) and a significantly lower number of implantations (10.3, p<0.001,vs.16.5) and pups (9.0, p<0.01,vs.14.7). One female had total resorptions and one litter died on day 1 post-partum.

There were no treatment-related pup clinical signs or necropsy findings. Pups treated at 40 or 80 mg/kg/day had higher mean body weight gains than the controls between day 1 and day 5 post-partum.

There were no effects of treatment on sperm morphology, motility or counts. The mean liver and kidneys weights were slightly but statistically significantly higher for males given 80 mg/kg/day (+15% for liver and +13% for kidneys in absolute weights). Higher liver weights correlated with a trend towards increased glycogen content at this dose-level and was considered to be related to the test item administration. For the higher kidney weights, a relationship to treatment was considered to be equivocal as there were no histopathological correlates. The mean absolute seminal vesicle weights were statistically significantly lower for all male groups in a dose-related manner (absolute weights were -17%, -19% and -35% at 20, 40 and 80 mg/kg/day, respectively). This correlated with a slight decrease in secretory content in the seminal vesicles observed at microscopic examination of the males given 80 mg/kg/day. In the absence of atrophy of seminal epithelium at microscopic examination, these minor findings were not considered to be adverse.

The dose-level of 80 mg/kg/day was considered to be higher than the Maximum Tolerated Dose for a dosing period of 13 weeks as there were two males and one female found dead during the premating or mating periods. In addition, treatment at this lethal dose was associated with delayed delivery as four females were found dead or prematurely sacrificed after the normal period for delivery and had not delivered all the pups. Administration of sodium thioglycolate at this lethal dose of 80 mg/kg/day was also associated with the death or premature sacrifice of two additional females during the peri-natal period (from day 1 to day 2post-partum). The female sacrificed was killed on day 1post-partumbecause all its litter died. There were no effects on male or female mating behavior or fertility and the test item was considered not to hinder embryo-fetal development. The test item at the mid and high dose-levels caused increased pup body weight gain after birth but there were no relevant pup clinical signs orpost-mortemfindings. In males, when compared to controls, liver and kidneys weights were slightly but significantly higher. There were no relevant macroscopic or microscopic findings nor any effects on sperm morphology, motility or counts.

At 40 mg/kg/day, one pregnant female with dead fetuses in the uterine horns was found dead on day 22post-coitum. There were no effects of treatment on body weight, food consumption, male or female mating behavior and fertility and pregnancy parameters. There were no adverse effects of treatment on the pup body weight gain after birth.

There were no effects of treatment at 20 mg/kg/day.

Under the experimental conditions of this study:

.  the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day (based on deaths at 40 and 80 mg/kg/day),

.  male reproductive performance was not affected by treatment with sodium thioglycolate. Dosing at 40 and 80 mg/kg/day resulted in deaths in late gestation associated with delayed delivery and a No Observed Effect Level (NOEL) for female reproductive performance was therefore set at 20 mg/kg/day,

.  the NOEL for toxic effects on progeny was set at 40 mg/kg/day, based on the dead litter at 80 mg/kg/day.

A proper evaluation of the reproductive performance of females given 80 mg/kg/day was not possible because of the numerous deaths observed in this group in the peri-natal period.

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:

From 9 february to 6 July 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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 (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

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 post-mating 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).

Key result

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 p ups): 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

Key result

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

Key result

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

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.

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 he 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.
49 of

Executive summary:

In an OECD guideline 421 study, groups of 12 male and female rats were exposed to doses of EHTG of0, 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/kg-bw/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:

reproductive toxicity, other

Type of information:

other: Corap assessment

Adequacy of study:

other information

Reliability:

other: Substance evaluation under CoRAP

Executive summary:

Excerpt from the CoRAP evaluation report:

One of the reasons for the selection of 2-EH was health hazard concern. It was noted that the developmental effects were observed in the pre-natal developmental toxicity studies.

The effects on the reproduction of 2-EH were evaluated in CoRAP in 2015:

•       The available data does not raise concern that 2-EH would affect fertility and sexual function.

•       The results of animal studies provide evidence of an adverse effect of 2-EH on development at very high doses [> 1000 mg/kg bw/d] causing strong toxic effects in dams, therefore they can be considered as a secondary non-specific consequence of other toxic effects. Evaluation of the available information shows that no maternal toxicity or slight maternal toxicity was observed with in animal studies and no developmental toxicity warranting classification is observed.

It was concluded that this concern could be removed because outcome of a full evaluation of the available information shows that doses of 2-EH are not toxic or are only slightly toxic to maternal animals and no developmental toxicity warranting classification is observed.

7.7.1.   Toxicity to reproduction (effects on fertility and developmental toxicity)

 

One of the reasons for the selection of 2-EH was health hazard concern. It was noted in the screening that developmental effects were observed in the pre-natal developmental toxicity studies. Therefore, a full evaluation of the available information was required in order to assess whether the observed developmental effects are the result of maternal toxicity. This part of the document reviews the available study reports in order to assess reproductive toxicity of 2-EH, and assess whether a proposal of harmonised C&L is needed for this endpoint.

Fertility

 

There is no study aimed at assessment of effect of 2-EH on fertility and sexual function on animal. Also observations on humans are not available.

 

However, taking into account that di (2 -ethylhexyl) terephthalate (DEHT) is metabolised to 2 -ethylhexan-1-ol (2-EH) and terephthalic acid, the 2- generation reproduction toxicity study of Faber et al. (2007) with DEHT provide some information on effect of 2- EH on fertility and sexual function. The 2 -ethylhexan-1 -ol and terephthalic acid are thus available in the body after di (2 -ethylhexyl) terephthalate (DEHT) application. Terephthalic acid has been shown to not affect fertility and sexual function (study report, 2003). Lack of effects of DEHT on fertility and sexual function found in the study of Faber et al. (2007) confirms findings of study report study (2003) and gives no indication that 2-EH would affect fertility and sexual function.

 

This conclusion is further supported by the results of repeated dose toxicity studies. The results of 90-day oral gavage study (Astil et al. , 1996) indicate that 2-EH at dose 250 and 500 mg/kg/day, thus comparable to the doses of 2-EH estimated in Faber et al. study (2007), does not affect morphology of testes or ovaries in rats and in mice. The relative weight of testes at a dose of 500 mg/kg/day was increased and that of ovaries decreased in rats only at dose 250 mg/kg /day, but not at a dose of 500 mg/kg. The relative weight of the testes (related to body weight) of 500 mg/kg/day group was slightly increased (5.5 % compared to control group). However, neither the absolute testes weight nor the relative weight of the testes related to brain weight did show any significant changes. Thus, the weight changes in testes can be attributed to the decreased body weight in males of high dose group (93 % of control value) and were not considered as adverse. There was decreased body weight gain in male and female rats at 500 mg/kg amounting to weight losses of 7% in males and 6% in females by Week 13. Due to low intensity, lack of dose response relationship and due to lack of histopathological changes in testes and ovaries, the changes in the weight of testes and ovaries alone are not regarded as adverse and do not warrant classification for fertility and sexual function. The weight of these reproductive organs were not affected in mice administered 2-EH by gavage at doses 250 and 500mg mg/kg, day for 13 weeks (Astil, 1996).

 

The effects of 2-EH on the testis are of interest because of the testicular atrophy and Sertoli cell damage produced in rats by high doses of DEHP, of which 2-EH is a metabolite (Gray and Gangolli, 1986). Sjoberg et al. (1986) showed that 350 mg of 2EH/kg/day administered to Sprague-Dawley (SD) rats by oral gavage for 5 days had no effect on testis weight and produced no effects on the seminiferous tubule. Similarly, concentrations of 0.2 mM of 2-EH were without effect on Sertoli cells from SD rats in primary culture studies (Gray and Beamond 1984). In the 13-week study in rats (Astil et al., 1996) there was a slight increase in relative testis weight at 500 mg/kg, not correlated with any morphological changes, and there were no gross or microscopic changes in testes of mice at 500 mg/kg. Therefore it is thus unlikely that the effects of DEHP on the testis are attributable to 2-EH.

 

The available data does not raise concern that 2-EH would affect fertility and sexual function.

 

Developmental toxicity

 

There are several studies to evaluate developmental toxicity of 2-EH: two studies in mice (study report 1991; Hardin et al. 1987) and four studies in rats (Hellwig and Jäckh, 1997; Ritter et al. 1987, Tyl et al. 1992, Nelson et al. 1989).

 

Mice

 

The first one, GLP and OECD TG 414 compliant study (study report 1991 reported also as NTP, 1991) provided evidence that 2-EH in doses 17, 59, and 191 mg/kg bw/day does not induce embryo or fetal toxicity in mice.

 

In the second mice study (Hardin et al. 1987) in which mice were given 1525 mg/kg on day 6 till day 13 of gestation the following developmental effects were observed: decreased number of viable litters and pups per liter, decreased birth weight and weight gain for pups. However these effects should be considered as a secondary non-specific consequence of other toxic effects in dams, because 2-EH at a dose applied in this study caused 34% mortality in the exposed mice. In addition, short term toxicity study in mice using similar dose of 1500 mg/kg/d for (study report, 1992b) provided evidence that 2- EH at this repeated dose level causes damage of several organs (stomach, liver, kidney) which may affect intrauterine and early postnatal development of pups.

 

Rats

 

There were two oral studies (Hellwig and Jäckh, 1997; Ritter et al. 1987), one dermal study ( Tyl et al. 1992) and one inhalation study (Neslon et al. 1989) of developmental toxicity of 2-EH in rats.

 

In the Hellwig and Jäckh oral study (1997) performed in accordance with GLP and OECD TG 414 (except that 10 animals instead of 20 was used per group) 2-EH at dose of 1300 mg/kg/d caused intrauterine deaths of embryos and pups, reduced foetal weight, increased incidence of internal and skeletal malformations as well as of skeletal variations and retardation. However, 2-EH at the dose of 1300 mg/kg/d was also very toxic to dams; 6 out of 10 treated dams were found dead before the end of the study. Therefore high maternal toxicity was most probably responsible for some effects such as increased intrauterine deaths or reduced foetal weight, as well as for some of the internal and skeletal malformations observed in that group, although their incidence was very low. 2-EH did not induced developmental toxicity at a dose of 130 mg/kg/d, while at a dose of 650 mg/kg/d the foetal weight slightly reduced, but still within historical control level. Therefore it may be concluded that in this study 2-EH did not exert developmental toxicity in doses not lethal to dams.

 

The oral study of Ritter et al. (1987) has limited reliability because the study design was not similar to that required by OECD TG 414 or method B.31 (Council Regulation (EC) No 440/2008). In addition, the observed results were not consistent with those observed in other studies. In spite of high doses used (800 and 1600 mg/kg/d) the study did not provide any information on maternal toxicity and no embryo or pup mortality was observed. Malformations in fetuses following single treatment with 2-EH at a dose approximately 1600 mg/kg included hydronephrosis (7.8% of live fetuses), tail defects (4.9% of live fetuses), limb defects (9.7% of live fetuses). Such defects were not observed after 2-EH treatment in studies of Hellwig and Jäckh (1997) and study report (1991) or in other studies. Taking into account a purpose of the study focused on clarification of developmental toxicity of di(2-ethylhexyl) phthalate (DEHP, use of only 7 females in a group, application of 2-EH only as a single dose at 12 day of gestation, lack of compliance of study design with OECD TG 414, lack of consistency of effects observed with other studies, it is doubtful whether study of Ritter et al. (1987) should be taken as a reliable source of information on developmental toxicity of 2-ethylhexanol.

 

The embryonic or fetal development of rats was not affected in the dermal developmental toxicity study (Tyl et al., 1992) conducted under GLP and with a design compliant with OECD TG 414 in which 2-EH was administered at doses 252, 840, and 2520 mg/kg bw/day, although the systemic maternal toxicity was observed at the highest dose.

 

No developmental toxicity was noted in an inhalation study (Nelson et al., 1989), in which female rats were exposed 2-EH at 850 mg/m³ during days 0-19 of gestation. At this exposure level 2-EH was moderately toxic to dams as can be judged based on reduction of feed consumption and body weight gain during gestation.

 

Based on the existing body of evidence it is concluded that at doses not lethal to mothers, in studies performed in compliance with methodological requirements, 2-EH does not induce developmental toxicity in mice and rats. Only at high doses, which were lethal to dams 2-EH increases intrauterine lethality of embryos and pups and leads to retardation of development. Even at these lethal doses the increase of fetal malformations is very low and no dose-response relationship is seen.

 

It is concluded that these developmental effects at doses highly toxic to dams are secondary non-specific consequence of other toxic effects in dams, and they do not justify classification of 2-EH for developmental toxicity. Several studies (study report 1991 reported also as NTP, 1991; Hellwig and Jäckh, 1997; Tyl et al., 1992 and Nelson et al., 1989) provided evidence that 2-EH is not a developmental toxicant in rats and mice.

 

2-ethylhexanoic acid (2-EHA)

 

The substance evaluation for 2-EHA is currently ongoing and the conclusions of this evaluation will be published on ECHA website once the evaluation is concluded.

 

2-ethylhexanoic acid (2-EHA) (EC No205-743-6, CAS No 149-57-5) ), which is major urinary metabolite of 2-ethyl-1-hexanol in rats (Albro, 1975; Deisinger et al. 1993;1994) has harmonised classification as Repr. Cat. 3; R63 Possible risk of harm to the unborn child, which has been transposed to Repr. 2 H361d *** in Table 3.1 List of harmonised classification and labelling of hazardous substances of Annex VI of the Regulation 1272/2008. In the study by Deisinger et al. (1994), the main metabolites in urine of orally treated rats were 2-ethylhexanoic acid, 5-hydroxy-2-ethylhexanoic acid, 6- hydroxy-2-ethylhexanoic acid and 2-ethyl-1,6-hexane diacid. Together, they represented 37 - 45% of the administered dose. Minor metabolites were 5-hydroxy-2-ethylhexanoic acid as well as lactones of 5-hydroxy-2-ethylhexanoic acid and 2-ethyl-5-hexanoneacid. They represented 3 - 5% of the administered dose. About 1% of the administered dose was recovered as 2-ethylhexanol. All these compounds were predominantly excreted as glucuronides (Deisinger et al.,1994). Albro (1975) reported the formation of about 50% 2-ethylhexanoic acid following a single oral exposure of rats to 275 mg/kg.

 

1.  The range-finding study in Fischer 344 rats showed significant maternal toxicity (death in seven of eight dams) and statistically significant reduction of maternal weight gain at 1000 mg/kg for GD 6-9. Indications of maternal toxicity were also observed at 500 mg/kg, including not statistically significant weight gain reduction and clinical sings of toxicity.

 

In the main developmental toxicity study, groups of 25 pregnant Fischer 344 rats per dose level received daily doses of 0, 100, 250 and 500 mg/kg 2-EHA (nominal in corn oil) by oral gavage from gestational day 6 to 15 (study report, 1988c; 1988d; study report, 1993 ) the clinical signs of maternal toxicity were only observed at the high-dose level (500 mg/kg) and included hypoactivity, ataxia, audible respiration, ocular discharge and periocular encrustations. No mortality and no effects on body weight were observed. Liver weight (absolute and relative) was significantly increased in the high-dose group.

 

Foetal effects: There were no changes in the incidence of resorptions and dead foetuses or in the percentage of viable foetuses. Foetal body weights (males and females) per litter were significantly reduced at 500 mg/kg, but these findings may be confounded by the slightly larger mean litter size. No significant differences in the incidence of external, skeletal or visceral malformations were observed among all groups.

 

There was a reduction in ossification of the axial and appendicular skeletons at 500 mg/kg. An increase in the number of foetuses with unossified anterior arch of the atlas and proximal phalanges of the forelimb and hindlimb was also observed at 250 mg/kg.

NOAEL of 250 mg/kg for maternal toxicity of 2-EHA was obtained, based on clinical signs of toxicity and increased liver weights. For developmental toxicity, a NOAEL of 100 mg/kg was established, based on reduced skeletal ossification at a dose of 250mg/kg/d. (study report, 1988c; 1988d; study report, 1993). However, minor developmental changes, when there is only a small reduction in foetal/pup body weight or retardation of ossification when seen in association with maternal toxicity, do not necessarily warrant classification as developmental toxicant (point 3.7.2.4.3. of the CLP Regulation).

2. The range-finding study in pregnant rabbits treated with 500 and 1000 mg/kg 2-EHA showed high toxicity. Mortality was observed at the high and mid doses. No changes in resorptions, deaths or malformed foetuses occurred. No external malformations were observed in foetuses at any of the treated groups (study report, 1988c; 1988d; study report, 1993).

In the definitive study, groups of 15 pregnant rabbits per dose level were administered, by gavage, daily doses of 0, 25, 125 and 250 mg/kg 2-EHA in corn oil on gestational days 6 to 18. In this study, mortality was recorded at 125 and 250 mg/kg (one female each) on days 15 and 16 of gestation, respectively. One abortion was observed on gestational day 27 at 125 mg/kg. A significant reduction in body weight gain and food consumption was observed in the high-dose group during the post-treatment period (gestational days 18 to 29). At necropsy, no gross pathology, no changes in corrected body or gestational weights or in absolute and relative liver weights were observed.

 

Foetal effects: There was no increase of resorptions and dead foetuses or changes in the percentage of viable foetuses. No effects on foetal body weights and sex ratios were observed and no differences in malformations or variations were seen either.

3.  In addition, a non-GLP developmental toxicity study, equivalent or similar to OECD 414, has been reported in the IUCLID dataset and considered as the supporting study (Pennanenet al., 1992). Groups of 20 or 21 female Wistar rats per dose level received daily doses of 100, 300 and 600 mg/kg 2-EHA as sodium salt via drinking water, during gestational days 6 to 19. Control animals received deionized water.

 

Body weight of dams suffered a slight decrease at the high-dose level from day 13 onward. At termination, statistically significant reductions in mean body weight and corrected maternal body weight gain were observed. In the same dose group, a decrease of 20% in the consumption of drinking water containing 2-EHA was seen from day 6, compared to the control group. No differences in food consumption were observed at any dose level. No maternal toxicity was noted at the low- and mid-dose groups.

In the mid- and high-dose groups the placental weight was also statistically significant reduced. No changes in gravid uterus weight were observed. At necropsy, no gross pathological changes in the organs of the dams occurred.

 

Foetal effects: The number of implantations, living foetuses or resorptions were not affected by treatment with 2-EHA . No dead foetuses were seen either in treated or control groups. Significant decreases in mean foetal body weight per litter were observed at 600 mg/kg. At 300 mg/kg, the mean body weight of female foetuses was also decreased.

 

Dose-dependent increases in the number of foetuses with skeletal or visceral anomalies were observed at all dose levels, compared to controls. It has to be pointed out that the number of litters affected by these alterations has not been indicated. Clubfoot (congenital deformity of the foot, which is twisted out of shape or position) was the most severe skeletal malformation, occurred in all treatment groups, being only statistically significant at the two highest doses.

Those results justified the EU harmonised classification of 2-ethylhexanoic acid as Repr. 2 H361d: Suspected of damaging the unborn child based on the observed developmental effects in animals, such as fetotoxicity and skeletal malformations (clubfoot) in rat following oral doses given on days 6-19 of gestation.

 

The analysis of data on of 2-EHA indicate that potency of its developmental toxicity in rats is rather low, while it does not exert developmental toxicity in rabbits, since no embryonal or foetal effects were observed in rabbits given 2-EHA et doses of 25, 125, 250, 500 and 1000 mg/kg (study report, 1993). In rats the developmental toxicity is shown as retardation in ossification of skeletal system in fetuses of rats exposed at doses of 250 and 500 mg/kg/d. (study report (1993)) or as a decreased foetal body weight, and increases incidence skeletal or visceral anomalies and of clubfoot in foetuses of females rats exposed to EHA at 300 and 600 mg/kg.

 

Assuming that in the worst case 50 % of orally given 2-EH is metabolised to 2-EHA in order to reach developmental toxicity the doses of 2-EH would have to be at the level of 600–1200 mg/kg, which are known to be rather highly toxic to adult rats (Hellwig and Jäckh, 1997; study report, 1991). Therefore the data showing developmental toxicity of 2-EHA do not provide sufficient evidence of developmental toxicity of the parent substance.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
- Breeder: Charles River Laboratories France, L'Arbresle, France
- Age at the beginning of the treatment period: 6 weeks old 
- Weight at the beginning of the treatment period: ca. 205 g for the  males and ca. 160 g for the females
- Acclimation: 7-days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature : 22 ± 2°C
- Relative humidity : 50 ± 50%
- Light/dark cycle : 12h/12h (7:00 - 19:00)
- Ventilation : about 12 cycles/hour of filtered, non-recycled air.

HOUSING
The animals were housed individually in polycarbonate cages or in  wire-wesh cages. Autoclaved wood shavings were provided as nesting  material, a few days before delivery and during the lactation period

FOOD and WATER
- Food: SSNIFF R/M-H pelleted maintenance diet ad libitum
- Water: filtered (0.22 µm filter) tap water ad libitum

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

TREATMENT
- Vehicle: degassed purified water, obtained by reverse osmosis 
- Dosage form preparation: solution in the vehicle at 4, 8 and 16 mg/mL,  expressed as active substance. 
- Volume: 5 ml/kg

Details on mating procedure:

MATING
- Mating procedure: one female was placed with one male from the same  dose-level group. If necessary, the estrous cycle stage was determined  from a fresh vaginal lavage (stained with methylene blue), each morning  during the mating period, until the females mated

PARTURITION
Females were allowed to drop their litters normally and rear their  progeny until sacrifice

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On weeks 1, 4, 8 and 12. There was a satisfactory agreement (± 10%) between the nominal and  actual concentrations.

Duration of treatment / exposure:

Exposure period: * Males: during 10w before mating, the mating period (2w) and until sacrifice.
* Females: during 10 w before mating, the mating period, pregnancy and lactation until day 4 post partum.
Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Duration of test: 16 weeks

Frequency of treatment:

7 days per week

Dose / conc.:

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

Remarks:

16 mg/kg bw/d as mercaptoacetic acid

Dose / conc.:

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

Remarks:

32 mg/kg bw/d as mercaptoacetic acid

Dose / conc.:

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

Remarks:

64 mg/kg bw/d as mercaptoacetic acid

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected by the Study Monitor based on the results of a preliminary 2-week toxicity study (CIT/Study No. 30720 TSR). In this study sodium thioglycolate in purified water was planned to be administered at dose-levels of 15, 30, 60 and 75 mg/kg/dayfor 14 days. After no effects were observed at all dose-levels, the group previously given 15 mg/kg/day was administered with 100 mg/kg/day on days 8, 9 and 10 and then 150 mg/kg/day on days 11, 12 and 13, the other groups were treated as scheduled until day 14. When treated at 150 mg/kg/day, one male was sacrificed on day 13 following marked clinical signs and three females were found dead on day 14. The male and two of the females had spleens reduced in size. Male terminal body weight gains were slightly lower than controls at 15/100/150 mg/kg/day while females showed a body weight gain comparable to controls until day 11 and a mean terminal body weight loss (-10%) when the dose level was increased to 150 mg/kg/day. Food consumption was lower for this group for both sexes over the last 3-day period when the dose level was increased to 150 mg/kg/d. At necropsy, some animals in the 60, 75 and 15/100/150 mg/kg/day dose-groups had irregular colored kidneys, while at 75 mg/kg/day marked lobular pattern of the liver was noted with paleness (also seen at 15/100/150 mg/kg/day). Mean ovary weights were lower at 15/100/150 mg/kg/day and mean uterus weights were lower at 75 and 15/100/150 mg/kg/day. 150 mg/kg/day was considered to exceed the maximum tolerated dose because mortality occurred after 3 days of treatment. 80 mg/kg/day was selected as a top dose level for the OECD 421 study considering the limited effects observed at 75 mg/kg/day.

Positive control:

No apprpriate

Parental animals: Observations and examinations:

- Morbidity and mortality: at least twice a day
- Clinical signs: at least once a day
- Body weight:          
. males: on day 1, then once a week until sacrifice         
. females: on day 1, then once a week until mated, then on days 0, 7, 14  and 20 pc and on days 1 and 5 pp (postpartum).
- Food consumption         
. males: once a week (except during the mating period) until sacrifice         
. females: once a week during the premating period and then on the  following intervals: days 0-7, 7-10, 10-14, 14-17 and 17-20 pc, and days  1-5 pp.

Oestrous cyclicity (parental animals):

Each morning for three weeks before the start of the pairing period. 

Sperm parameters (parental animals):

The left epididymis was removed, weighed (total and cauda separately) and  sperm from the cauda was sampled for motility, morphology investigations  and epididymal sperm count. The left testis was weighed and ground. The resulting preparation was  diluted and sperm heads resistant to homogenization (i.e. elongated  spermatids and mature spermatozoa) was counted in a Neubauer cell.

Litter observations:

- Litter size: total litter size and numbers of pups of each sex were  recorded as soon as possible after birth. The litters were observed daily.
- Clinical signs: daily
- Body weight: days 1 and 5 pp

Postmortem examinations (parental animals):

PATHOLOGY
- Sacrifice
. males: after the end of the mating period,
. females: on day 5 pp,
. females which had not delivered by day 25 pc: on day 25 pc
. females which did not mate: 24 days after the end of the mating period,
- Organ weights: 
Brain, epididymides, heart, kidneys, liver, ovaries,  prostate, seminal vesicles and testes, uterus
- Macroscopic post-mortem examination: on all parent animals. In all  females, the number of implantation sites and corpora lutea were  recorded. 
- Preservation of tissues: macroscopic lesions, ovaries, prostate,  seminal vesicles, uterus (horns and cervix), vagina, brain, heart, liver  and kidneys, in 10% buffered formalin. Testes and epididymides, in  Bouin's fluid        
- Microscopic examination: macroscopic lesions,  epididymides, heart,  kidneys, liver, ovaries, prostate*, seminal vesicles*, testes, and  uterus (* in the control and high-dose groups only).

Postmortem examinations (offspring):

PATHOLOGY
- Sacrifice
. pups: on day 5 pp.
-  A macroscopic examination was performed for all pups, including those found dead or prematurely sacrificed. Macroscopic lesions were preserved in 10% buffered formalin (or another appropriate fixative).

Statistics:

- Data other than organ weights:
Mean values were compared by one-way variance analysis and Dunnett test, (mean values being considered as normally distributed, variances being considered as homogeneous). Values compared by Fisher exact probability test are presented as percentages.
- Organ weights:
Dunn test or Student test (2 groups) or Dunnett test (3 or more groups) or Mann-Whitney/Wilcoxon test

Reproductive indices:

Pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

Post-implantation loss:
Number of implantation sites - Number of live concepti
_____________________________________________ x 100
Number of implantations

Mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

Fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

Gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females

Offspring viability indices:

Live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

Viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All males and females given 80 mg/kg/day were observed to have ptyalism from week 2 of dosing generally until the end of the study. The number of females affected during gestation (8/11) and lactation (2/4) was less than the number affected during pre-pairing (11/12).
7/12 males and 5/12 females given 40 mg/kg/day experienced ptyalism towards the end of the pre pairing dosing period (week 9 onwards) and, for males, until the end of the study, but only 3/12 females were affected during gestation and 1/11 had ptyalism during lactation.
No clinical signs were observed at 20 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

. 80 mg/kg/day:
One female (K20428) was found dead on day 23 of dosing. No clinical signs other than ptyalism had been observed prior to death. No abnormalities were observed at post-mortem examination.
One male (K20347) was found dead on day 74 of dosing. No clinical signs other than ptyalism were observed prior to death. The male had not mated prior to death. At necropsy, the stomach mucosa had brown/black areas.
One male (K20349) was found dead on day 90 of dosing. No clinical signs other than ptyalism were observed prior to death. At necropsy, the right kidney had a dilated pelvis.
One female (K20421) was found dead on day 23 post-coitum with 17 live pups in the bedding. At necropsy, thick, blackish contents were observed in the vagina.
One female (K20422) was found dead on day 23 post-coitum with 4 dead pups and one live pup in the bedding and one pup in the vagina. At necropsy, there were 11 dead fetuses in the uterine horns.
One female (K20426) was found dead on day 23 post-coitum with 11 live pups and five dead pups in the bedding. Difficulty to deliver was noted for the female prior to death. At necropsy, the lungs were reddish in color and there were serous contents in the thoracic cavity.
One female (K20427) was prematurely sacrificed on day 23 post-coitum with signs of piloerection, cold to the touch, pallor of eye recorded only on the same day. No pups were delivered prior to death. At necropsy, there were 15 dead fetuses and one live fetus and one late resorption in the uterine horns and the liver was pale.
One female (K20429) was prematurely sacrificed on day 1 post-partum because all the pups were dead. Prior to sacrifice, emaciated appearance, piloerection and round back were observed. No abnormalities were observed at post-mortem examination.
One female (K20423) was found dead on day 2 post-partum with no clinical signs other than ptyalism observed prior to death. At necropsy, the urinary bladder was dilated.
. 40 mg/kg/day:
One female (K20415) was found dead on day 22 post-coitum. At necropsy, the female was pregnant with 16 dead fetuses in the uterine horns.
The high rate of mortality in the treated females, with presence of implantation scars, late resorptions and/or dead fetuses in the uterine horns, was considered to be treatment-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See Table 1
The mean body weight gain over the 10-week pre-mating period was similar to that of the controls for both males and females at all dose-levels. Females given 80 mg/kg/day had a slight lowering of body weight gain between day 50 and day 64 but this did not affect the overall mean gain.
There were no effects of treatment on body weight gains during gestation. Females treated at all dose-levels showed an apparently increased body weight gain during the lactation period, however there were two control females which lost weight during this period and when they are excluded there are no significant differences between the groups (there were only two females in the group mean at 80 mg/kg/day so the mean value is not as reliable as the other groups).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Male food consumption at all dose-levels was similar to that of the controls during the first 5 weeks of dosing (ranging from -3% to +4% difference) and was then statistically significantly higher at 80 mg/kg/day for the last 5 weeks of the pre mating period (+10% to +14%).
Female food consumption at all dose-levels was similar to that of the controls throughout the pre mating and gestation periods (ranging from -5% to +16% difference). Females given 80 mg/kg/day had slightly lower mean food consumption during lactation (-10%), but given the low number of females considered in this dose group, these changes were considered to bear no biological significance.

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):

. Males
Systemic (scheduled sacrifices and unscheduled sacrifices)
* Heart
Minimal or slight myocardial degeneration/necrosis was seen in the two males given 80 mg/kg/day and found dead at day 74 or 90.
As these findings were recorded focally and moreover also found in the control animals which survived (2/12 males), they were considered without relationship to treatment. This finding is commonly seen in untreated Sprague-Dawley rats of this age (Greaves, 2007).
* Liver
A trend in marginally higher concentration of glycogen content was observed in the liver of the surviving males given 80 mg/kg/day.
Incidence and mean severity [affected and (affected + non affected)] of glycogen content in the liver of decedents and surviving males.

Dose-level (mg/kg/day) 0 20 40 80
Number of males with glycogen content
Status: found dead - - - 0/2
Number of males with glycogen content
Status: scheduled sacrifice 10/12 10/12 8/12 9/10
Mean severity (affected animals) (3) (2.5) (2.1) (3.9)
Mean severity (affected + non affected) 2.5 2.1 1.4 3.5
-: not applicable.

This finding was considered to be related to the test item administration in view of the effect of SODIUM THIOGLYCOLATE on the carbohydrate metabolism. However these marginal differences at microscopic examination of the liver given the high dose-level were considered not to be adverse.
In addition, a relationship to the slightly higher liver weights at 80 mg/kg/day in the males which survived and which were not fasted before sacrifice was considered to be uncertain as the marginally lower glycogen content at 40 mg/kg/day in the males was not correlated with lower liver weights.
* Kidneys
Acidophilic globules were noted in the cortical tubular epithelium of the kidneys of the males given 80 mg/kg/day and of their respective controls. Incidence and severity were similar in both groups. It was therefore considered not to be toxicologically meaningful and related to the intra tubular alpha micro-globulin deposits in renal cortex which can be found at such incidence and severity in treated as well as untreated control male rats.

Incidence and mean severity (affected and affected + non affected) of acidophilic globules in the kidneys of decedents and surviving males

Dose-level (mg/kg/day) 0 80
Number of males with acidophilic globules
(mean severity)
Status: found dead - 1/2 (2.0)
Number of males with acidophilic globules
(mean severity)
Status: scheduled sacrifice 9/12 (1.7) 8/10 (1.9)
Total number of males and mean severity 9/12 (1.7) 9/12 (1.9)
Mean severity (affected + non affected) 1.3 1.4
-: not applicable.

* Genital Organs
Testes/Epididymides
The microscopic examination of the PAS/Hematoxylin stained testes and epididymides with knowledge of the different stages of maturation of the seminiferous tubules did not reveal any disturbance in the treated males. There was no evidence of degenerative changes or delay in sexual maturity in the treated males from any group compared with the respective controls. The higher number of males given 40 mg/kg/day with focal tubular atrophy was considered to be fortuitous and correspond to the junction between seminiferous tubules and tubulu recti, as it was almost always found near albuginea (subcapsular).
Seminal vesicles
Slight reduced secretory content was noted in 5/10 surviving males given 80 mg/kg/day. It correlated with lower weights at necropsy and was considered to be treatment-related. Reduced secretory content, most often unilateral and of minimal or slight severity, was also observed in 5/12 males given 20 mg/kg/day. Unilateral reduced secretory content was noted at minimal severity in 3/12 males given 40 mg/kg/day, while unilateral slight or moderate reduced secretory content was observed in two other males given 40 mg/kg/day. Except for male K20328 given the low dose-level, the individual seminal vesicle weights of the males given 20 or 40 mg/kg/day were of same magnitude as those of the controls.
It was considered that the reduced secretory content at 20 and 40 mg/kg/day was not treatment related.
In addition, as no atrophy of seminal epithelium could be observed in any treated group, the reduced secretory content together with lower seminal vesicle weights at 80 mg/kg/day was considered not to be adverse. This was confirmed by the absence of any abnormality in the sperm parameters.

- Females
Systemic (scheduled sacrifices and unscheduled sacrifices)

* Heart
Minimal myocardial degeneration/fibrosis was seen in 1/7 females prematurely killed. As this finding was recorded focally, and moreover found in one control female that survived, it was not considered to be related to the test item administration.

* Liver
Slight or marked peri/medio-lobular vacuolated hepatocytes were seen in 3/7 females found dead or prematurely sacrificed given 80 mg/kg/day. In 1/7 females marked vacuolated hepatocytes were associated with minimal hepatocellular degeneration/necrosis and minimal granulocyte infiltration. A relationship to treatment was considered to be equivocal as such observations were not found in any surviving female given 80 mg/kg/day. In control and treated (80 mg/kg/day) groups, half of the females (almost all surviving and one found dead) showed similar severity of glycogen content (see table below).

Incidence and mean severity (affected and (affected + non affected)) of glycogen content in the liver of decedents and surviving females

Dose-level (mg/kg/day) Number of females with glycogen content
(mean severity)
0 80
Status: found dead - 1/7 (3.0)
Status: scheduled sacrifice 6/12 (2.3) 4/5 (2.8)
Total number of females (affected animals) 6/12 (2.3) 5/12 (2.8)
Mean severity (affected + non affected) 1.2 1.2
-: not applicable.

* Kidneys
Slight bilateral vacuolated cortical tubules, tubular dilatation and unilateral, minimal, focal cortical necrosis were observed in the kidneys of female K20427 given 80 mg/kg/day and sacrificed moribund. Slight proteinaceous casts were noted in the kidneys of female K20421 from the same group. None of these were noted in any of the surviving females. All were thus considered to be of no toxicological importance.

* Uterus/vagina
Microscopic examination of uterus and/or vagina was suggestive of on-going pregnancy in the females found dead or prematurely sacrificed or evidence that females had not delivered. It was recorded in the table 4.
No relevant microscopic observations were recorded in the ovaries.

All the other microscopic findings noted in the prematurely killed, found dead or surviving males and females given 80 mg/kg/day or from the control group, including those observed in the prostate of the males, were those which can be found spontaneously in the untreated laboratory rat of this strain and were considered to bear no relationship to treatment with the test item.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no effects of treatment with sodium thioglycolate on vaginal cyclicity, with mean cycle lengths of 4 to 5 days in all groups including control.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

More than 96% of the sperm were morphologically normal in the groups given SODIUM THIOGLYCOLATE and more than 95% of the sperm were motile. There were therefore no effects of treatment at any dose-level.
All values for epididymidal and testicular sperm counts are similar to, or greater than, those observed in controls, there were therefore no effects of treatment.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- Mating data:
With the exception of male K20347 (given 80 mg/kg/day) which was found dead after 3 days of pairing and had not mated, all pairs mated. The mean number of days taken to mate was slightly higher for the group given 40 mg/kg/day but as this was related to the contribution of two females and as it was not also observed at 80 mg/kg/day, it was considered not to be related to treatment.
- Fertility data:
There was one non-pregnant female in each of the groups given vehicle or 20 mg/kg/day and two non-pregnant females in the group given 80 mg/kg/day. This was considered not to be related to treatment with the test item.
- Delivery data: See Table 2
The duration of gestation was statistically significantly longer for the females given 80 mg/kg/day than for the other groups and the number of females surviving delivery was markedly lower.
The mean number of corpora lutea was lower in females given 80 mg/kg/day resulting in a lower number of implantations and fetuses, although implantation losses were comparable with the controls and observed only in one female with total resorptions.
Pup survival was slightly worse at 80 mg/kg/day, mainly due to one female found dead so her litter was sacrificed and one female with a dead litter. The remaining two females lost only three pups between them.
The percentage of male pups was slightly low at 80 mg/kg/day (ns), when compared with the controls, however there were less pups in this group.

Dose descriptor:

NOEL

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect of any king

Remarks on result:

other: 16 mg/kg bw/d as mercaptoacetic acid

Dose descriptor:

LOAEC

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on deaths at 40 and 80 mg/kg/day during the premating period and/or parturition

Remarks on result:

other: 32 mg/kg bw/d as mercaptoacetic acid

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

One pup in the control group had a haematoma on the head and one pup in the 40 mg/kg/day dose-group had a necrosed tail. As these were isolated findings, they were considered to be spontaneous in origin.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Pup survival was slightly worse at 80 mg/kg/day, mainly due to one female was found dead so her litter was sacrificed and one female had a dead litter. The remaining two females lost only two pups between them.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pups of dams treated at 40 or 80 mg/kg/day showed an increased mean body weight gain from day 1 to day 5 post-partum (ranging from +14% to +51% difference). The pups of dams treated at 80 mg/kg/day also had a higher mean birth weight than the controls (+12% for males and +14% for females) but there were fewer pups per litter in this group which is likely to have had an impact. Since this increased body weight gain is dose-related it is considered to be related to treatment with the test item.

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):

One pup in the 40 mg/kg/day dose-group had a small liver with whitish areas. One pup in the 20 mg/kg/day group had whitish areas on the liver.
These findings were not observed in the 80 mg/kg/day dose-group. The examination of the historical control data shown that whitish area have already been observed in control animals in 2 OECD 421 studies with frequencies of 1/125 (0.8%) and 1/113 (0.9%), in consequence this effect was not considered to be treatment-related.

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: 32 mg/kg bw/d as mercaptoacetic acid

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other: 64 mg/kg bw/d as mercaptoacetic acid

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

40 other: mg/kg bw/day (actual dose received) (32 mg/kg bw/d as mercaptoacetic acid)

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:

yes

Table 1: main differences in mean body weight and body weight gain (in grams)

Sex	Male				Female
Dose-level (mg/kg/day)	0	20	40	80	0	20	40	80
Pre-mating
. days 1-71	+315	+305 (-3%)	+304 (-3%)	+315 (0%)	+133	+130 (-2%)	+125 (-6%)	+129 (-3%)
Gestation
. days 0-20p.c.	/	/	/	/	+120	+128 (+7%)	+127 (+6%)	+112 (-7%)
Lactation
. days 1-5 p.p.	/	/	/	/	+11	+20 (+82%)	+18 (+64%)	+13 (+18%)

/: not recorded

Table 2: delivery data

Dose-level (mg/kg/day)	0	20	40	80
Duration of gestation (days)	21.6	21.4	21.5	22.8**
. Number of females delivering on day 21p.c.	4	7	6
. Number of females delivering on day 22p.c.	7	4	5	2
. Number of females delivering on day 23p.c.				1
. Number of females delivering on day 24p.c.				1
Number of females surviving delivery/number of pregnant females	11/11	11/11	11/12	4/8^a
Mean number ofcorpora lutea	17.0	15.7	14.9	13.2
Mean number of implantations	16.5	15.4	14.4	10.3#
Mean number of live pups delivered	14.7	14.7	13.4	9.0**

**: p<0.01, #: p<0.001.

a: excluding female K20431 which had total resorptions.

Table 3: differences (expressed in %) noted between treated and control animals in the absolute and relative organ weights

 

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	20	40	80	20	40	80
Body weight	-1	-2	0	-1	0	-4
- Kidneys
. absolute	-3	-1	+13**	0	+1	-5
. relative	-2	0	+13**	+1	+1	-1
- Liver
. absolute	-1	+1	+15*	+1	+9	+1
. relative	0	+3	+15**	+2	+9	+5
- Prostate
. absolute	+4	+1	-10
. relative	+4	+3	-11
- Seminal vesicles
. absolute	-17*	-19*	-35**
. relative	-16	-18	-35**
- Uterus
. absolute				-3	+1	+161
. relative				-1	+2	+170

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Table 4

Females prematurely killed or found dead

 

Dose-level (mg/kg/day)	Day*	Status**	40		80
Female Number/status at necropsy			Macroscopic finding	Microscopic findings	Macroscopic finding	Microscopic findings
K20415	97	Dead during pregnancy (22)	Dead fetuses (16)	Gestational uterus Marked vaginal mucification	-	-
K20421	102	Dead during pregnancy (23)	-	-	Implantation scars (18) Black thick vaginal content	Moderate vaginal mucification
K20422	96	Dead during pregnancy (23)	-	-	Dead fetuses (11) Implantation scars (6)	Gestational uterus Remnants of membranes and ombilical cord
K20423	98	Dead during lactation (2)	-	-	No macroscopic observation	Trophoblast remnants
K20426	97	Dead during pregnancy (23)	-	-	Implantation scars (16)	Gestational uterus
K20427	98	Prematurely sacrificed during pregnancy (23)	-	-	Alive fetus (1) Dead fetuses (15) Late resorption (1)	Gestational uterus
K20429	101	Prematurely sacrificed during lactation (1)			No macroscopic observation	Slight neutrophil infiltration with necrosis and giant cells with hypertrophic nuclei

*: day of autopsy, **: gestational day or dead during lactation.

(n): number of alive or dead fetuses, late resorptions or implantation scars in uterine horns.

 

Females killed after delivery

 

Dose-level (mg/kg/day)	Day*	Status**	40		80
Female Number/status at necropsy			Macroscopic finding	Microscopic findings	Macroscopic finding	Microscopic findings
K20431	99	Final sacrifice on lactation day 5	-	-	Late resorption (1) Thick black horn and vagina content	Minimal endometrium atrophy (epithelium and stroma) moderate mucification of vagina

*: day of autopsy,  **: gestational day or dead during lactation.

(n): number of resorptions.

Conclusions:

Under the experimental conditions of this study:
. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day (based on deaths at 40 and 80 mg/kg/day),
.   male reproductive performance was not affected by treatment with sodium thioglycolate. Dosing at 40 and 80 mg/kg/day resulted in deaths in late gestation associated with delayed delivery and a No Observed Effect Level (NOEL) for female reproductive performance was therefore set at 20 mg/kg/day,
.   the NOEL for toxic effects on progeny was set at 40 mg/kg/day, based on the dead litter at 80 mg/kg/day.
A proper evaluation of the reproductive performance of females given 80 mg/kg/day was not possible because of the numerous deaths observed in this group in the peri-natal period.

Executive summary:

In a reproduction/developmental screening test performed according to the OECD Guideline # 421, four groups of 12 male and 12 female Sprague-Dawley rats received sodium thioglycolate (purity 98.9% pure), daily, by oral (gavage) administration, 10 weeks before mating and through mating and, for the females, through gestation until day 5 post-partum,at dose-levels of 0, 20, 40 or 80 mg/kg bw/d (0, 16, 32 and 64 mg/kg bw/d as mercaptoacetic acid).

Clinical signs and mortality were checked daily. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. The animals were paired for mating and the dams were allowed to litter and rear their progeny until day 5 post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth, pup clinical signs were recorded daily and pup body weights were recorded on days 1 and 5 post-partum. The males were sacrificed after completion of the mating period and the females on day 5 post-partum (or on day 25 post-coitum for females which did not deliver). The body weight and selected organs (brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles and testes and uterus) were weighed and a macroscopic post-mortem examination of the principal thoracic and abdominal organs and a microscopic examination of selected organs (macroscopic lesions, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, testes, and uterus) were performed. In the females, which were apparently non-pregnant, the presence of implantation scars on the uterus was checked using ammonium sulphide staining technique. Epididymal sperm was sampled for motility, morphology and count and testicular sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted. The pups were sacrificed on day 5 post-partum and were carefully examined for gross external abnormalities and a macroscopic post-mortem examination was performed.

Two males (weeks 11 and 13) and one female (week 4) given 80 mg/kg/day were found dead during the pre-mating or mating periods with no clinical signs observed before death and no relevant post-mortem findings. These deaths are considered to be treatment-related. Three out of 11 surviving females given 80 mg/kg/day were found dead on day 23 post-coitum, all having delivered pups, although one female had one fetus in the vagina and still had 11 dead fetuses in the uterine horns at necropsy. Another pregnant female with dead and live fetuses in the uterine horns was sacrificed on day 23 post-coitum because of poor clinical condition. At 80 mg/kg/day, one additional female was prematurely sacrificed on day 1post-partumbecause all the pups were dead and another female was found dead on day 2 post-partum.

One female given 40 mg/kg/day was found dead on day 22 post-coitum, pregnant with dead fetuses in the uterine horns.

Ptyalism was observed at 40 and 80 mg/kg/day with a dose-related incidence and may be related to the taste of the test item.

There were no significant effects of treatment on mean body weight gains; all sodium thioglycolate-treated male and female groups had body weight gains similar to those of the control group throughout the study. There were no effects of treatment on mean food consumption, except a slight lowering of food consumption during the lactation period for the two females given 80 mg/kg/day (-10%).

There were no effects of treatment with sodium thioglycolate on vaginal cyclicity, with mean cycle lengths of 4 to 5 days in all groups including control. All pairs mated and the majority of the females were pregnant. There were no effects of treatment on the mean number of days taken to mate.

Females given 80 mg/kg/day had a significantly longer gestation period (22.8, p<0.01,vs. 21.6 days), a non-significantly lower number of corpora lutea (mean of 6.7, ns, vs. 8.5) and a significantly lower number of implantations (10.3, p<0.001,vs.16.5) and pups (9.0, p<0.01, vs. 14.7). One female had total resorptions and one litter died on day 1 post-partum.

There were no treatment-related pup clinical signs or necropsy findings. Pups treated at 40 or 80 mg/kg/day had higher mean body weight gains than the controls between day 1 and day 5 post-partum.

There were no effects of treatment on sperm morphology, motility or counts. The mean liver and kidneys weights were slightly but statistically significantly higher for males given 80 mg/kg/day (+15% for liver and +13% for kidneys in absolute weights). Higher liver weights correlated with a trend towards increased glycogen content at this dose-level and was considered to be related to the test item administration. For the higher kidney weights, a relationship to treatment was considered to be equivocal as there were no histopathological correlates. The mean absolute seminal vesicle weights were statistically significantly lower for all male groups in a dose-related manner (absolute weights were -17%, -19% and -35% at 20, 40 and 80 mg/kg/day, respectively). This correlated with a slight decrease in secretory content in the seminal vesicles observed at microscopic examination of the males given 80 mg/kg/day. In the absence of atrophy of seminal epithelium at microscopic examination, these minor findings were not considered to be adverse.

The dose-level of 80 mg/kg/day was considered to be higher than the Maximum Tolerated Dose for a dosing period of 13 weeks as there were two males and one female found dead during the premating or mating periods. In addition, treatment at this lethal dose was associated with delayed delivery as four females were found dead or prematurely sacrificed after the normal period for delivery and had not delivered all the pups. Administration of sodium thioglycolate at this lethal dose of 80 mg/kg/day was also associated with the death or premature sacrifice of two additional females during the peri-natal period (from day 1 to day 2post-partum). The female sacrificed was killed on day 1post-partumbecause all its litter died. There were no effects on male or female mating behavior or fertility and the test item was considered not to hinder embryo-fetal development. The test item at the mid and high dose-levels caused increased pup body weight gain after birth but there were no relevant pup clinical signs or post-mortem findings. In males, when compared to controls, liver and kidneys weights were slightly but significantly higher. There were no relevant macroscopic or microscopic findings nor any effects on sperm morphology, motility or counts.

At 40 mg/kg/day, one pregnant female with dead fetuses in the uterine horns was found dead on day 22post-coitum. There were no effects of treatment on body weight, food consumption, male or female mating behavior and fertility and pregnancy parameters. There were no adverse effects of treatment on the pup body weight gain after birth.

There were no effects of treatment at 20 mg/kg/day.

Under the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day (16 mg/kg bw/d as mercaptoacetic acid) (based on deaths at 40 and 80 mg/kg/day),

. male reproductive performance was not affected by treatment with sodium thioglycolate. Dosing at 40 and 80 mg/kg/day resulted in deaths in late gestation associated with delayed delivery and a No Observed Effect Level (NOEL) for female reproductive performance was therefore set at 20 mg/kg/day (16 mg/kg bw/d as mercaptoacetic acid),

. the NOEL for toxic effects on progeny was set at 40 mg/kg/day (32 mg/kg bw/d as mercaptoacetic acid), based on the dead litter at 80 mg/kg/day.

A proper evaluation of the reproductive performance of females given 80 mg/kg/day was not possible because of the numerous deaths observed in this group in the peri-natal period.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

A simulated gastric hydrolysis study of EHTG found that under low pH conditions (1.2 @ 37°C similar to mammalian gastric systems) EHTG is hydrolytically unstable with a half live of 6.8 hours (Groult, 2013). It means that EHTG is partially hydrolyzed in the stomach before to be absorbed. As any esters, EHTG is further hydrolysed in several tissues by carboxylesterasesto the corresponding acid and alcohol, namely mercaptoacetic acid (thioglycolic acid, TGA; CAS No. 68-11-1) and 2-ethylhexanol (2-EH, CAS No. 104-76-7). After oral administration of ³⁵S-EHTG, the radioactivity was quickly and extensively excreted and the intact substance was neither found in excrements (urine and faeces) nor in organs indicating a total hydrolysis in vivo (Seigler et al., 1971). Therefore, the systemic toxicity of EHTG is resulting from the toxicity of 2-EH and TGA.

 

Toxicological data are available for mercaptoacetic acid and 2-ethylhexanol from the REACH registration dossier no.01-2119494933-24-0000 and 01-2119487289-20-0000, respectively. The comparison of the acute oral toxicity data shows that the LD50s are in the same range of dose levels for EHTG (1.5 mmol/kg bw) and TGA and its salts (0.5-2.2 mmol/kg bw) and well lower than the LD50 of 2-EH (15.7 mmol/kg bw). Therefore, it appears that the acute systemic toxicity of EHTG is essentially driven by the toxicity of the mercaptoacetic moiety.

 

The effects of EHTG on the reproductive parameters are thought to result from the parental toxicity induced by 2-EH and/or TGA.

In the OECD guideline 421 study on EHTG (Bowman, 2005), parental systemic toxicity was observed at 150 mg/kg/day. Three males and 3 females 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.

 

The effects on the reproduction of 2-EH were evaluated in CoRAP in 2015 and the conclusion was:

-         The available data does not raise concern that 2-EH would affect fertility and sexual function.

-         The results of animal studies provide evidence of an adverse effect of 2-EH on development at very high doses [> 1000 mg/kg bw/d] causing strong toxic effects in dams, therefore they can be considered as a secondary non-specific consequence of other toxic effects. Evaluation of the available information shows that no maternal toxicity or slight maternal toxicity was observed with in animal studies and no developmental toxicity warranting classification is observed.

Therefore, 2-EH do not have a significant effect in the parental and reproductive toxicities at these dose levels of EHTG.

 

At the opposite, the mercaptoacetic moiety have similar parental toxicity and reproductive effects than EHTG, in the same dose range (on a molar basis).

 

In an OECD guideline 421 study on sodium mercaptoacetate (Davies, 2006), parental toxicity was evident at the high dose levels. Two males and one female given 80 mg/kg bw/d were found dead during the pre-mating or mating periods with no clinical signs observed before death and no relevant post-mortem findings. Four females at 80 mg/kg/d and one at 40 mg/kg bw/d were found dead or were prematurely sacrificed because of difficulties to deliver.

 

In both OECD 421 studies on EHTG and TGA, the NOAELs for parental toxicity and reproductive performance were similar, around 0.2 mmol/kg bw/day.

As reported in the registration dossier of mercaptoacetic acid, mercaptoacetate and 2-mercaptoethanol are known to induce fatty liver in rodents after an acute administration (Nordmann and Nordmann, 1971; Sabourault et al., 1976, 1979). Several experiments have shown that the toxicity of 2-mercaptoethanol was in fact due to its oxidation into mercaptoacetate through the combined effects of alcohol and aldehyde dehydrogenases. It has been demonstrated that mercaptoacetate and/or 2-mercaptoethanol induced an inhibition of the ß-oxidation of fatty acids (Bauché et al., 1977, 1981, 1982 and 1983). This inhibition induced secondary effects like a decrease of blood glucose and liver glycogen, blood and hepatic ketone bodies and liver acetyl-CoA and an increase of plasma free fatty acids and liver triglycerides and acyl-CoA and an enhancement of hepatic pyruvate content (Freeman et al, 1956; Nordmann and Nordmann, 1971; Sabourault et al., 1976, 1979). The fatty liver induced by mercaptoacetate was mainly due to an inhibition of acyl-CoA dehydrogenase activity (Bauché et al., 1981) and consequently to a marked depression of the ß-oxidation pathway.

In a study on the acute effects of sodium mercaptoacetate (Grosdidier, 2011) on blood glucose level, it appears that fasted animals are more sensitive to the toxic and hypoglycaemic effects of mercaptoacetate than non-fasted animals. In fasted animals, an initial rise of the glycaemia is followed by a severe hypoglycaemia until the re-feeding of the animals. In the non-fasted animals, no initial rise was observed and the hypoglycaemia was moderate when compared to fasted animals. The initial glucose rise in the fasted animals suggests that mercaptoacetate stimulates the glycogenolysis, then when the glycogene is depleted, the blood sugar level decreased. The administration of glucose to fasted animals prevents the blood glucose decrease. The effect of fasting was also observed in repeated dose toxicity studies. Mortality and toxicity was observed at the dose level of 150 mg/kg bw/d administered by gavage in the 14-day range-finding and the OECD 421 studies (Bowman, 2005a and 2005b) whereas, no mortality and toxic effect was observed in the 28-day repeated dose dietary study (Mallet, 1988) at a mean daily intake of 170 mg/kg bw/d. In a gavage study, at the time of treatment (morning) the animals could at a stage of fasting more or less pronounced (rats eat during the night) whereas in the dietary study food is absorbed with EHTG, preventing a potential hypoglycemia.

 

From the mechanism of action of mercaptoacetate, it appeared that the parental toxicity observed in the OECD 421 studies with EHTG and TGA resulted from the repeated acute toxic effect of dose levels close to the acute lethal dose levels. In addition, pregnant females at the time of delivery are more sensitive to the systemic toxic effects of mercaptoacetate. Indeed, when starting the labour for parturition, pregnant females are prone not to eat and accordingly when the labour starts, some females could be in an advanced stage of fasting at the time of treatment. In addition, the labour is a high energy consuming effort, which could increase the glycogen depletion. All together, the prolonged fasting period, the glycogen depletion and the inhibition of the fatty acids oxidation, could induce a lethal hypoglycemia. Secondary effects this toxicity on the surviving dams could be a lack of nesting/nursing behavior and this causes death of some pups which have been delivered.

Justification for classification or non-classification

According to REGULATION (EC) No 1272/2008 "an increased incidence of mortality among the treated dams over the controls shall be considered evidence of maternal toxicity if the increase occurs in a dose-related manner and can be attributed to the systemic toxicity of the test material. Maternal mortality greater than 10 % is considered excessive and the data for that dose level shall not normally be considered for further evaluation". Maternal mortality greater than 10% and toxicity was observed at the dose level of 150 mg/kg bw/day in the screening reproductive toxicity studies.

Therefore, considering the lack of both maternal and reproductive toxicity at the dose level of 50 mg/kg bw/day, no classification is warranted for reproductive and developmental toxicity under CLP and DSD.