Mercaptoacetic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Mercaptoacetic acid and its salts are not considered to be reproductive toxicants.

 

In a 2-generation reprotoxicity study following the OECD guideline # 416 with sodium mercaptoacetate, in view of the maternal mortalities and liver effects in males and females observed at 40 mg a. i. /kg/day, the dose-level of 20 mg a. i. /kg/day (16 mg/kg bw/d as mercaptoacetic acid) was considered to be the NOEL for parental toxicity, female fertility and gestation of each generation and for development, growth and survival of the progeny. It is probable that the small effects observed on pup survival at 40 mg a. i. /kg/day were secondary to the severe and lethal effects observed in the pregnant dams at that dose level. The NOEL for male fertility and female mating behaviour was higher or equal than 40 mg a. i. /kg/day (32 mg/kg bw/d as mercaptoacetic acid).

In a reproduction/developmental toxicity screening test performed with sodium mercaptoacetate following the OECD guideline # 421, with and extended pre-mating period of treatment. High mortality was observed in both males and females at 80 mg/kg bw. The NOAEL for parental toxicity was considered to be 20 mg/kg bw/d (based on deaths at 40 and 80 mg/kg bw/d) (16 mg/kg bw/d as mercaptoacetic acid), the NOAEL for male reproductive performance was higher than 80 mg/kg/day (64 mg/kg bw/d as mercaptoacetic acid), the NOAEL for female reproductive performance (mating, fertility and delivery) was considered to be 20 mg/kg bw/d (based on deaths at 40 and 80 mg/kg bw/d) (16 mg/kg bw/d as mercaptoacetic acid) and the NOAEL for toxic effects on progeny was 40 mg/kg bw/d (based on the dead litter at 80 mg/kg bw/d) (32 mg/kg bw/d as mercaptoacetic acid).

 

2-generation study (OECD 416)

The effects of Sodium mercaptoacetate, on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrous cycle, mating behavior, conception, gestation, parturition, lactation and weaning, and on the growth and development of the offspring was evaluated over 2 generations (Davies, 2010a). This study was performed under GLP compliance and following the OECD Guideline for Testing of Chemicals No. 416 (Two-Generation Reproduction Toxicity Study), 22ndJanuary 2001.

Three groups of 25 male and 25 female Sprague-Dawley rats received sodium mercaptoacetate, daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. Sodium mercaptoacetate was administered as a solution in degassed purified water, by oral gavage, at dose-levels of 10, 20 or 40 mg a. i. /kg/day (a. i. = active ingredient, equivalent to 8, 16 and 32 mg/kg bw/d as mercaptoacetic acid). Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded weekly. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening, surface righting, cliff avoidance and air righting). At the end of the treatment period or prior to premature sacrifice, the F0 animals were blood sampled for analysis of hematology and blood biochemistry parameters, including ß-hydroxybutyrate and acetoacetate determination. The animals were not fasted before blood collection. After weaning of the pups, the males and females of the F0 generation were sacrificed. Sperm analysis was performed on the first 10 males of the control and high-dose groups (groups 1 and 4) and since no treatment-related effects were observed this was not performed on males of the other groups. A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs and macroscopic lesions of all groups and for the control and high-dose groups the heart, kidneys and liver were also examined. The liver of all intermediate-dose group animals was also examined. Of the progeny of the F0 generation, one or two males and one or two females per litter were selected to constitute the F1 generation of groups of 25 male and 25 female rats in the control, low and intermediate groups and 27 male and 27 female rats in the high-dose group (this to compensate in advance for mortality around the time of delivery, a known effect of the test item). Three groups received the test item, Sodium mercaptoacetate, daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. Due to the test item inducing known difficulties around the time of delivery, all mated females, including controls, were not treated from day 19 of gestation until day 1 of lactation in order to see if this would reduce the mortality. The test item was administered as a solution in degassed purified water, by oral gavage, at dose-levels of 10, 20 or 40 mg a. i. /kg/day. Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once at weaning before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded weekly. Each animal was assessed for sexual maturity (balanopreputial separation or vaginal opening) and the day of age and body weight were recorded on the day each animal was positive. At 4 weeks of age, the animals were assessed for auditory function (acoustic startle response) and pupil constriction, and weeks of age the spontaneous locomotor activity was measured using an automated infra-red sensor equipment. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 19 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening, surface righting, cliff avoidance and air righting). In addition, the anogenital distance of all pups was measured on the day after birth and the presence of nipples was checked in all males on post-natal day 12 or 13. At weaning of the pups, the males and females of the F1 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs and any macroscopic lesions of all groups. In addition, pups of both the F0 and F1 animals were submitted for a macroscopic examination. One randomly selected pup/sex/litter for F1 and F2 litters, all pups found dead or prematurely sacrificed and any pups showing external abnormalities or clinical signs were weighed and then submitted for a macroscopic examination of the principal thoracic and abdominal organs with special attention paid to the reproductive organs.

There were no effects of treatment at 10 or 20 mg a. i. /kg/day.

At 40 mg a. i. /kg/day, the males and females showed no effects of treatment during the pre-mating, mating or gestation phases. At delivery, four females were found dead; one on gestation day 21 and three on gestation day 22. One of these females found dead on gestation day 22 had delivered 12 live and one dead pup before dying, the other three females had not started delivery and had dead fetuses in the uterine horns at necropsy. On lactation day 1, one female, showing signs of poor condition (piloerection, blood and placentae in the bedding), cannibalized her 10 pups (it is likely that more pups were born and cannibalized before being noticed because there were five more implantation sites in the uterine horns than pups). It is not known whether the pups were alive or dead prior to cannibalism, however the adverse outcome is considered to be a maternal effect since no fetuses remained in the uterine horns at necropsy; the female did deliver all pups starting delivery on gestation day 21. No effects of treatment were observed in the F0 generation animals during the remainder of the lactation period other than a slightly lower mean body weight gain of the females during the first 4 days of lactation. There were no effects on sperm parameters in the control or high-dose group males. There were no treatment-related effects on organ weights at any dose-level. There were no treatment-related microscopic changes in testis, epididymis, prostate, coagulating glands or seminal vesicles. There were possible treatment-related changes in the uterus of one 40 mg a. i. /kg/day female (one female which had only implantation scars thought to be left from early resorptions); thrombosis of the mesometrial remnant, organized by granulation tissue composed of fibroplasia, fibrin plugs, thrombi and neovascularization. Golden pigment-laden cells and pyknotic trophoblastic cells were observed. female had hemorrhage of a mesometrial triangle. Minimal to moderate periportal hepatocellular microvacuolation was observed at 40 mg a. i. /kg/day in 2/25 males and 6/25 females, and in 4/6 prematurely sacrificed/found dead females suggesting mild liver toxicity at this dose-level. No control animals had the same finding.

Female plasma fatty acid concentration was statistically significantly decreased however there were no effects of treatment with the test item on plasma acetoacetate or ß-hydroxybutyrate concentrations indicating that the animals were not in ketosis.

There were no effects of treatment on F1 pups in the 10 or 20 mg a. i. /kg/day groups.

At 40 mg a. i. /kg/day, the pups had a 9.6% mortality rate during the first 4 days of lactation (cannibalism and being found dead); neither the pups nor the F0 dams showed particular clinical signs (except one female which had piloerection and cannibalized all pups in the litter). There was a possible, very slight, delay in physical development; the majority of the pups achieved tooth eruption, eye opening and auditory canal opening on the same day as the majority of the pups from the other groups but there was a higher percentage of pups achieving these landmarks on later days than in the other groups. The females of the F1 generation started treatment (on day 22 of age) with a statistically significantly lower mean body weight than the controls. Mean body weight gain and mean food consumption were both also statistically significantly lower for the first week of treatment. During gestation, the females had a slightly lower body weight gain. Treatment was stopped on gestation day 19 and delivery passed without problem in all females. However, two females were prematurely sacrificed on day 2 or day 5 of lactation due to death of the litter. One of the females had thrombosis of mesometrial triangles. Treatment was re-started on lactation day 1 and the number of pup deaths was higher when compared with the controls. This was mainly due to one female which had three found dead pups and cannibalized 13 pups over several days until lactation day 5 when all pups were dead. Another female also had total litter death but delivered only one pup. The total number of dead pups in this group was similar to that of the controls when the female with the 16 dead pups is excluded (15 dead pupsvs.the control group), but whereas the control pup deaths were concentrated in four litters, at 40 mg a. i. /kg/day the pup deaths were spread over seven litters. The females had not had any noticeable problems during delivery and were not showing any clinical signs during the first days of lactation except for a possible effect on maternal nesting and nursing behavior since the pups were often observed to be cold, even those that survived. There were no treatment-related effects on organ weights at any dose-level. There were no effects on sperm parameters in the control or high-dose group males. There were no treatment-related microscopic changes in testis, epididymis, prostate, coagulating glands or seminal vesicles of the males or in the ovaries, oviducts, uterus and vagina of the 40 mg a. i. /kg/day females.

The female F2 pups had a slightly lower mean body weight at the end of lactation (-5%) but there were no effects on male or female F2 pup physical development in terms of eye opening, tooth eruption or auditory canal opening or on reflex development.

At 40 mg a. i. /kg/day, it is concluded that the test item has no effect on non-pregnant, naïve, adult rats but that it causes maternal toxicity and death of susceptible pregnant females around the time of delivery. Effects on the dam include lack of nesting/nursing behavior and this causes death of the pups which have been delivered. If treatment is stopped just prior to delivery, the females may survive delivery but pup death may still occur and pup clinical signs of coldness suggest that maternal nesting/nursing behavior is still impaired by treatment with the test item and affect pup survival. There is evidence that female rats are more affected by test item treatment than males as shown by lower F1 female body weight and body weight gain.

Minimal to moderate periportal heptocellular microvacuolation was observed in females and some male F0 animals treated at 40 mg a. i. /kg/day suggestive of mild hepatotoxicity and especially in dams (4/6) found dead or prematurely sacrificed at time of parturition. Sodium mercaptoacetate is known to induce fatty liver via aninhibition of the ß-oxidation of fatty acids.

There were no effects on sperm parameters in the control or high-dose group males.

There were no effects of treatment on any parameters measured in either males or females with the test item at 10 or 20 mg a. i. /kg/day.

Under the experimental conditions of this study, and in view of the maternal mortalities and liver effects in males and females observed at 40 mg a. i. /kg/day, the dose-level of 20 mg a. i. /kg/day (16 mg/kg bw/d as mercaptoacetic acid) was considered to be the No Observed Effect Level (NOEL) for parental toxicity, female fertility and gestation of each generation and for development, growth and survival of the progeny. It is probable that the small effects observed on pup survival at 40 mg a. i. /kg/day were secondary to the severe and lethal effects observed in the pregnant dams at that dose level. The No Observed Effect Level (NOEL) for male fertility and female mating behaviour was higher or equal than 40 mg a. i. /kg/day (32 mg/kg bw/d as mercaptoacetic acid).

 

Reproduction/developmental screening test (OECD 421)

In a extended reproduction/developmental screening test performed according to the OECD Guideline # 421, four groups of 12 male and 12 female Sprague-Dawley rats received sodium mercaptoacetate (purity 98.9% pure), daily, by oral (gavage) administration, 10 weeks before mating and through mating and, for the females, through gestation until day 5post-partum,at dose-levels of 0, 20, 40 or 80 mg/kg bw/d (equivalent to 0, 16, 32 and 64 mg/kg bw/d as mercaptoacetic acid) (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 5post-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 23post-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 23post-coitumbecause 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 2post-partum.

One female given 40 mg/kg/day was found dead on day 22post-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 mercaptoacetate-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 mercaptoacetate 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 1post-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 5post-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 mercaptoacetate 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 mercaptoacetate. 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 February 2009 - 21 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guideline (deviations were not considered to have compromised the validity of the study and results); adequate coherence between data, comments and conclusions

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

the temperature and the relative humidity were sometimes outside the target range + deviations to study plan

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

yes

Remarks:

idem above

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 B.35

Deviations:

yes

Remarks:

idem above

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: the males were approximately 6 weeks old and the females were approximately 5 weeks old
- Weight: males: mean body weight of 175 g (range: 156 g to 202 g) /females: mean body weight of 109 g (range: 92 g to 125 g)
- Fasting period before study: no
- Housing: the F0 males and females and the F1 generation after weaning were individually housed, except during pairing, in wire-mesh cages.
A metal tray containing autoclaved sawdust was placed under each cage.
Towards the end of the gestation period, and with their litter during lactation, the F0 and F1 females were housed in polycarbonate cages containing
autoclaved sawdust. Autoclaved wood shavings were provided as nesting material, a few days before delivery and during the lactation period.
- Diet (e.g. ad libitum): all animals had free access to SSNIFF R/M-H pelleted maintenance diet distributed weekly
- Water (e.g. ad libitum): the animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): 12 h cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: 10 February 2009 To: 21 October 2009.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

VEHICLE
- Nature: degassed purified water obtained by reverse osmosis and subsequently degassed by sonication for at least 15 minutes and finally saturated
with nitrogen gas for at least 15 minutes. This was stored under nitrogen atmosphere.
- Concentration in vehicle: 2, 4 and 8 mg a.i./mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution. The required quantity of test item was mixed with the required quantity of vehicle in order to prepare a
solution at the highest required concentration (8 mg a.i./mL). The low and intermediate concentrations (2 and 4 mg a.i./mL) were prepared by dilution of the high concentration with the vehicle.
The test item dosage forms were prepared weekly by the CIT Pharmacy under nitrogen atmosphere and were stored, in brown glass bottles, at +4°C
and under nitrogen atmosphere until treatment.
All concentrations and dose-levels in this study are expressed as active ingredient.

Details on mating procedure:

- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal lavage (day of confirmed mating was designated day 0 post-coitum)
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): housed individually in polycarbonate cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method: HPLC/UV
The test item concentration in samples of each control and test item dosage form prepared for use in weeks 1, 4, 8, 12, 16, 20, 24, 28, 32 and 36 was determined.

Duration of treatment / exposure:

Each animal was given the appropriate dosage form once a day, at approximately the same time each day, 7 days a week, according to the following
schedule:
- in the males:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- until sacrifice (after weaning of the pups).

- in the females:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- during pregnancy,
- during lactation until day 21 post-partum inclusive,
- females with no delivery were treated until the day prior to sacrifice.

Day 1 corresponds to the first day of treatment period.

Frequency of treatment:

Once daily.

Details on study schedule:

- F1 parental animals not mated until 9 to 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 22 days of age.
- Age at mating of the mated animals in the study: between 12 and 14 weeks

Dose / conc.:

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

Remarks:

8 mg/kg bw/d as mercaptoacetic acid

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

No. of animals per sex per dose:

For dose-levels of 0, 10 and 20 mg a.i./kg/day: 25 animals per sex and per dose
For dose-level of 40 mg a.i./kg/day: 25 (P0) or 27 (F1) animals per sex.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected on the basis of the results of previous studies:
- an OECD 421 study using dose-levels of 20, 40 and 80 mg/kg/day (CIT/Study No. 30721 RSR) in which 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 13-week toxicity study in rats using dose-levels of 7, 20 and 60 mg/kg/day (CIT/Study No. 38414 TCR) in which mortality occurred at
60 mg/kg/day and a few hematology, blood biochemistry and microscopic effects were observed at 20 mg/kg/day.

- Rationale for animal assignment: random.

Positive control:

None.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and
on days 0, 7, 14 and 20 post-coitum and days 1, 4, 7, 14 and 21 post-partum.
The female prematurely sacrificed was weighed prior to sacrifice.

FOOD CONSUMPTION: once a week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No.

LABORATORY INVESTIGATIONS (F0 generation)
- Blood sampling
Blood samples were taken from the orbital sinus of non-fasted animals (5 to 6 hours after treatment) under light isoflurane anesthesia, and collected into tubes containing the appropriate anticoagulant. All samples for hematology, blood biochemistry and ß-hydroxybutyrate and acetoacetate analysis were taken at the same moment for the males. The females were sampled either on day 21 post partum or on day 24 post-coitum if no delivery had occurred
- Hematology: peripheral blood
The following parameters were determined for all surviving animals towards the end of the treatment period, 5 to 6 hours after treatment, on day 24 post-coitum for all females not having delivered and for moribund animal T20401 (4F) prior to sacrifice on day 1 post-partum.
Erythrocytes (RBC)
Hemoglobin (HB)
Mean cell volume (MCV)
Packed cell volume (PCV)
Mean cell hemoglobinconcentration (MCHC)
Mean cell hemoglobin (MCH)
Thrombocytes (PLT)
Leucocytes (WBC)
Differential white cellcount with cell morphology
. neutrophils (N)
. eosinophils (E)
. basophils (B)
. lymphocytes and large unstained cells (L+LUC)
. monocytes (M)
Prothrombin time (PT)
- Blood biochemistry
The following parameters were determined for all surviving animals towards the end of the treatment period, 5 to 6 hours after treatment, on day 24 post-coitum for all females not having delivered and for moribund animal T20401 (4F) prior to sacrifice on day 1 post-partum.
Chloride (Cl-)
Glucose (GLUC)
Urea (UREA)
Creatinine (CREAT)
Triglycerides (TRIG)
Aspartateaminotransferase (ASAT)
Alanineaminotransferase (ALAT)
Free fatty acids (ACGR)
Lactate (LACT)
ß hydroxybutyrate
acetoacetate

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning as follows:
- during the last 3 weeks of the pre-mating period,
- during the mating period, until the females were mated.

Sperm parameters (parental animals):

Parameters examined in F0 and F1 male parental generations (on the first ten surviving F0 males and the first ten surviving F1 males of the control
and high-dose groups):
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal
sperm reserve, sperm motility, sperm morphology.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 post-partum: yes
- If yes, maximum of 8 male and 8 female pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring of F0 and F1 generations: number and sex of pups, stillbirths, live births, postnatal mortality,
presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: reflex development, physical development, presence of
nipples in males (progeny of F1).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities in:
- pups found dead,
- pups prematurely sacrificed,
- pups culled on post natal day 4 (PND 4),
- pups sacrificed on PND 22.

yes, for internal abnormalities in:
- pups showing external abnormalities or clinical signs,
- pups found dead or prematurely sacrificed,
- one randomly selected F1 and F2 pup/sex/litter sacrificed on PND 22.

Possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: in all surviving animals, after weaning of the litters
- Maternal animals: in all surviving animals, at the weaning of the litters. Females which did not deliver were sacrificed on day 25 post-coitum after body weight recording. Females with litter dying entirely were sacrificed as appropriate

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 (table procedure) were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed on PND 4 and PND 22, all F2 offspring were sacrificed at the weaning of the litters.
- The following animals were subjected to postmortem examinations (gross external abnormalities):
. pups found dead,
. pups prematurely sacrificed,
. pups culled on PND 4,
. pups sacrificed on PND 22.

GROSS NECROPSY (progeny of the F0 and F1 generations)
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Gross necrospy was performed in:
. pups showing external abnormalities or clinical signs,
. pups found dead or prematurely sacrificed,
. one randomly selected F1 and F2 pup/sex/litter sacrificed on PND 22.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.

Reproductive indices:

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

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

Fertility index:
Number of pregnant females
_______________________________ x 100
Number of mated females

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

Lactation index on day 21 post-partum:
Number of surviving pups on day 21 post-partum
________________________________________ x 100
Number of surviving pups on day 4 post-partum

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no premature deaths in the groups treated at 0, 10 or 20 mg a.i./kg/day.
At 40 mg a.i./kg/day, four females were found dead; one on gestation day 21 (T20408) and three on gestation day 22 (T20410, T20418 and T20420). None of the females had clinical signs prior to death except that female T20420 had just completed delivery and had given birth to 12 live pups and one dead pup. At necropsy there were 13 implantation scars on the uterine horns which matches the number of pups delivered. The other females had not started delivery and had dead fetuses in the uterine horns at necropsy.
Female T20420, who had just delivered, had a hemorrhage of one mesometrial triangle in the uterus which could have contributed to the death.
In females T20410 and T20418 some mesometrial triangles were present in the histological sections of the uterus but there were no microscopic findings including hemorrhage which could explain the deaths.
Another female treated at 40 mg a.i./kg/day (female T20401) was prematurely sacrificed on lactation day 2 because all pups were cannibalized on lactation day 1. The female still had piloerection, blood, placentae and fetuses in the bedding (but not in the uterine horns) on lactation day 2 indicating poor clinical condition of the dam after the pups had been born. At microscopic examination, septicemia, peritonitis and abscesses in the mesometrial triangles, thought to be of uterine origin, were observed.
It is concluded that the test item causes mortality of susceptible dams around the time of delivery. In a few females which deliver, nesting/nursing behavior is impaired which causes the pups to die or be cannibalized.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, it was considered that there were no effects of treatment with the test item.
All female test item-treated groups had similar mean body weight gains to the controls during the pre-pairing, gestation and lactation phases, except during the first 4 days of lactation where the group treated at 40 mg a.i./kg/day had a statistically significantly lower mean body weight gain (5 g vs. 13 g, p<0.01). There were three females in group 4 which lost weight during this period but the majority of the females in this group gained little weight. This may be related to difficulties at time of delivery and a longer recovery time at this dose-level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of males and females treated with test item was comparable with that of the controls throughout the premating, gestation and lactation periods at all dose-levels.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated at 40 mg a.i./kg/day had a statistically significantly increased hemoglobin level in the blood when compared with the controls (15.5 g/dL vs. 15.0 g/dL, p<0.05). This treatment related increase was not observed in the females treated at the same dose-level. None of the other red blood cell parameters showed differences to the controls so a relationship to treatment of this isolated parameter is doubtful and of no biological significance. In addition, the hemoglobin concentration was within Historical data range for male rats of this approximate age (13.3 to 17.2 g/dL).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Sex Male Female
Dose-level (mg a.i/kg/day) 0 10 20 40 0 10 20 40
Urea(mmol/L) 5.2 4.9 4.8 4.7* 8.4 8.4 7.9 8.2
Fatty acids(mmol/L) 0.08 0.10 0.10 0.08 0.07 0.06 0.05 0.04**
Acetoacetate(µmol/L) 57.3 55.4 57.1 63.7 58.2 55.7 61.0 65.4
ß-hydroxybutyrate(µmol/L)64.3 64.8 59.8 56.8 62.7 61.1 62.0 63.5
Statistically significant *: p<0.05, **: p<0.01.

Males treated at 40 mg a.i./kg/day had a statistically significantly decreased urea concentration when compared with the controls, but of no biological significance. Females treated at the same dose-level had a statistically significantly decreased fatty acid concentration when compared with the controls. Neither of these differences was observed in the other sex or at the other dose-levels.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled death
Treatment-related changes were seen in the uterus from some prematurely sacrificed/found dead females treated at 40 mg a.i./kg/day and could be related to mortality around the time of delivery.
Females T20408, T20410 and T20418, given the test item at 40 mg a.i./kg/day were found dead on day 21, 22 and 22 of gestation, respectively. No microscopic findings could explain the death of these animals. Female T20410 and T20418 presented in the uterus some mesometrial triangles. This normal structure of both fetal/maternal origin is part of the placenta that infiltrates the uterine wall (rat hemochorial placentation) and is involved in the normal gestation. It is characterized by invasion of trophoblastic cells in the uterine wall (mesometrium, myometrium and endometrium), forming large "lakes" of blood, consisting in vascular structures lined by inconstant bumpy cells and glycogen type cells. The interstitium in these locations presented glycogen type, trophoblastic and granulated cells.
Female T20420 given 40 mg a.i./kg/day was found dead on day 22 of gestation. At microscopic examination, mesometrial triangles were seen in the uterus along with minimal hyperplasia of the endometrial epithelium. In the center of one of these mesometrial triangles, slight necrosis of the endometrium (stroma, epithelium and glands) was observed with subsequent hemorrhage that collected in the lumen of the uterus and that was also found in the lumen of the vagina. This bleeding could have contributed to the death of the rat. A relationship to treatment could not be ruled out.
Female T20401, treated at 40 mg a.i./kg/day and prematurely sacrificed on day 2 of lactation presented microscopic findings suggestive of septicemia and peritonitis. The surface of the liver and, at a lesser severity of the kidneys, presented a diffuse acute suppurative and necrotic inflammation with presence of bacterial colonies. These findings correlated with the irregular discoloration and green discoloration of these two organs seen at necropsy along with the green pouch seen in the liver. In the liver, the inflammation extended into the adjacent parenchyma, resulting in hepatocellular necrosis and infiltration of the parenchyma by mixed inflammatory cells. Minimal multifocal cortical necrosis of the left adrenal correlated with the white discoloration seen macroscopically. The origin of the inflammatory process was though to be the uterus. In the uterus, severe abscessation of the mesometrial triangle was seen in continuation with the outer layer of the uterus. It consisted of very large abscesses characterized by central coagulation necrosis of the vascular and connective tissues, degenerated neutrophils and fibrin exudation. Bacterial colonies were numerous within the abscesses and in vascular structure (bacteremia / septicemia). Mesothelial cells are bumpy and prominent (hyperplasia and hypertrophy). No luminal inflammation (pyometra) was evident but necrotic debris of the endometrial epithelium along with degenerated neutrophils and fibrin can be seen. This inflammatory change was also associated with mesometrial thrombosis in the abscesses and in the adjacent tissue and with slight necrosis of the superficial and glandular endometrial epithelium. A hemorrhagic collection from these lesions was seen in the lumen of uterus and drained through the cervix into the vagina. Adrenal cortical hyperplasia and severe lymphoid atrophy in the thymus were attributed to the stress associated with the poor clinical condition. These findings had contributed to the poor clinical condition of the female. In the absence of control animals at a similar stage of gestation, a relationship between the uterine lesions and the treatment with Sodium thioglycolate could not be ruled out, although the infection seen in the uterus could be a contributing factor.

Terminal sacrifice
Males F0
No treatment-related microscopic changes were noted in the testis, epididymis, prostate, coagulating glands, or seminal vesicles in males given 40 mg a.i./kg/day.
Females F0
Changes that could be related to treatment were seen in the uterus of one female given 40 mg a.i./kg/day.
Qualitative evaluation of the ovaries, uterus and vagina The histomorphological characteristics of the estrous cycle were present in almost the equal of treated and control females (11/20 versus 15/25, respectively). Microscopic findings suggestive of delayed / disturbance of estrous cycle (mucification of the vaginal epithelium), suggesting that they had not started cycling again, were present in 10/25 control females and 9/20 treated females.
Minimal to slight greenish pigment laden macrophages located in the endometrium, myometrium and mesometrium were seen in 18/25 control females and in 10/20 treated females. These macrophages were frequently associated with minimal to moderate remnant of the mesometrial triangle. These mesometrial remnants were seen in 21/25 control females and 14/20 treated females.
In one female (T20412) given 40 mg a.i./kg/day, thrombosis of the mesometrial remnant organized by a well developed granulation tissue composed of fibroplasia, fibrin plugs, thrombi and neovascularization (a large part of these vessels most likely belonged to the placentation). Golden pigment laden macrophages were numerous. Pyknotic trophoblastic cells were seen. This granulation tissue probably corresponded to a scar of the placentation that correlated with implantation scars seen at necropsy. These changes were suggestive of early resorption. As no control female was sacrificed at the same period of the study, a definitive relationship could not be stated.
From the quantitative analysis (ovaries), minimal changes of the number of primordial follicles and corpora lutea were observed in the treated females. The minor differences were due, at least in part, to the inter-individual variability.
Treatment-related microscopic change was seen in the liver of both males and females given 40 mg a.i./kg/day and consisted of periportal hepatocellular microvacuolation.
Minimal to moderate periportal hepatocellular microvacuolation was noted in the liver of 2/25 males and 6/25 females treated at 40 mg a.i./kg/day.
Among affected females, 4/6 were found dead or prematurely sacrificed and 2/6 were sacrificed at the end of the treatment period. This finding was characterized by densely packed microvacuoles within the cytoplasm and correlated with the accentuated lobular pattern noted in one female at necropsy. This change most likely corresponded to steatosis (neutral lipids). Considering the very low incidence in males given 40 mg a.i./kg/day, a relationship to treatment was uncertain. In females, the incidence and severity were higher than in males and although the affected females were mainly the prematurely sacrificed or found dead females, a relationship to treatment could not be excluded.
Minimal increased incidence of hepatocellular degeneration/necrosis was seen in treated females. Due to the multifocal isolated distribution of limited areas, a relationship to treatment was considered unlikely.

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

The mean number of cycles per female was minimally lower at all dose-levels when compared with the controls. This is partly due to the females with abnormal cycling, although there were not sufficient numbers of females per group nor a dose-relationship to conclude that there was a treatment-related effect.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects of treatment with the test item on sperm parameters.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

No problems were encountered during the mating period; all groups mated within a mean of 3 days and the number of pregnant females in each group was considered to be within normal limits.
At 40 mg a.i./kg/day, four females were found dead on gestation day 21 or 22 and another female (T20412) did not deliver and was found to have only implantation scars at necropsy. The body weight of this female 20 days after mating was similar to that of non-pregnant females so it was considered that the implantation scars remained after early resorptions had occurred.
The mean number of implantation sites was similar among all groups. The post-implantation loss and the mean number of pups delivered were minimally higher or lower, respectively, in all test item-treated groups when compared with the controls (the high-dose group was outside historical data range for both the post-implantation loss and the number of pups born but in the absence of a significant difference when compared with the controls of this study, this was considered not to be relevant). Female T20401 (40 mg a.i./kg/day) had five more implantation sites than pups born but was prematurely sacrificed on lactation day 2 because of cannibalism of the litter on lactation day 1. The implantation sites were counted at necropsy but there were no resorptions. It is likely that this female delivered five pups which were never seen because they were cannibalized after birth. In this case, the post-implantation loss is skewed by differences in the pup numbers that were not due to mortality in utero. At the dose-levels of 10 and 20 mg a.i./kg/day respectively, female T20366 had 12 fewer pups than implantation sites and female T20395 had six fewer pups than implantation sites. Overall, it was considered that there were no effects of treatment on in utero implantation loss.
A high number of pups died in the group treated at 40 mg a.i./kg/day. Of the 21 pups which died during lactation, 16 were from two litters. Female T20401, who had piloerection and hypoactivity before delivery and piloerection and blood, fetuses and placentas in the bedding for several days after delivery, cannibalized all the pups on lactation day 1. It is not possible to know whether the pups were born healthy and correctly formed or whether they died before being cannibalized since a macroscopic examination was not possible. Another six of the dead pups were from female T20404 who showed no clinical signs but cannibalized two pups (the other four were found dead, one having shown pallor on the day prior to death). The remaining five pups were scattered among five different litters.
The five dead pups at 20 mg a.i./kg/day were from five different litters as were the 13 dead pups at 10 mg a.i./kg/day.
Overall, the pup deaths in the control group and the group treated at 20 mg a.i./kg/day were scattered one pup per affected litter, but at 10 and 40 mg a.i./kg/day the deaths tended to be concentrated in two litters. With the exception of female T20401 discussed above, there was nothing remarkable about these females in terms of clinical signs, gestational body weight or number of pups. Some cannibalized pups were cold to the touch and pale the day before death but the found dead pups, with one exception, did not show any clinical signs prior to death.

Key result

Dose descriptor:

NOEL

Remarks:

parental toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Remarks on result:

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

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male (T20115) was found dead on day 46 of treatment without having shown any clinical signs prior to death. This male had no macroscopic or microscopic findings that could have contributed to the death but did have marked infiltration of the prostatic interstitium by mononuclear inflammatory cells.
Two females treated at 40 mg a.i./kg/day (T20518 and T20520) were sacrificed on days 2 or 5 of lactation, respectively, due to dead litter. Neither dam showed clinical signs although the pups of female T20520 were cold to the touch indicating possibly poor maternal nesting behavior.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no effects of treatment with the test item on mean male body weight or body weight gain the during the 10-week premating treatment period.
Females treated at 40 mg a.i./kg/day started the F1 generation with a statistically significantly lower mean body weight than the controls. The female pups had finished lactation with a mean body weight which was 4% lower than that of the controls. After selection the mean body weight was 7% lower than that of the controls. Mean body weight gain during the first week of treatment was statistically significantly lower than that of the controls and the lower mean body weight continued until the fourth week of treatment, by which time it was only slightly lower than that of the controls and it remained minimally lower until the end of the premating period (-3% on day 71). During the gestation period, females treated at 40 mg a.i./kg/day gained less weight than the controls and had a mean body weight difference of -5% on gestation day 20, however greater body weight gains during lactation reduced the deficit.
It is considered that there was a minimal effect of treatment with the test item on body weight of the F1 generation females at 40 mg a.i./kg/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on mean male food consumption.
Females treated at 40 mg a.i./kg/day had a statistically significantly lower mean food consumption during the first week of treatment (13 g/animal/day, p<0.05, vs. 15 g/animal/day), but thereafter mean food intake was comparable with the controls.

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:

no effects observed

Description (incidence and severity):

No changes in organ weights attributable to the treatment with Sodium thioglycolate.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Unscheduled death
One control male was found dead on day 46. No macroscopic findings that could have contributed to the death of the rat were observed at necropsy.
Females T20518 and T20520 were prematurely sacrificed on days 2 and 5 of lactation, respectively, due to dead litter. No abnormalities were observed at necropsy.
Terminal sacrifice
No treatment-related macroscopic findings were observed in F1 parents given Sodium thioglycolate at 10, 20 or 40 mg a.i./kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled death
Control male T20115, found dead on day 46, presented marked mononuclear inflammatory cells infiltration of the prostatic interstitium. No microscopic findings that could have contributed to the death of the animal were observed.
Female T20518, prematurely sacrificed on day 2 of lactation, presented moderate multifocal remnants of mesometrial triangles, indicative of gestation. The uterus of female T20520, given 40 mg a.i./kg/day and prematurely sacrificed on day 5 of lactation, showed large mesometrial triangles. The mesometrial triangles presented thrombosis and were largely replaced by granulation tissue. These changes could be related to difficulties seen during delivery that could be treatment-related.
Terminal sacrifice
Males F1
No treatment-related microscopic changes were noted in the testis, epididymis, prostate, coagulating glands, or seminal vesicles in males given 40 mg a.i./kg/day.
Females F1
No treatment-related microscopic changes were noted in ovaries, oviducts, uterus or vagina in females given 40 mg a.i./kg/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no effects of treatment with the test item on estrous cycles.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects of treatment with the test item on sperm parameters.

Description (incidence and severity):

One male treated at 10 mg a.i./kg/day and three males treated at 40 mg a.i./kg/day did not mate during the 14 days of pairing with the female. All females were re-paired with males that had previously mated and mating occurred within 5 days. The number of males not mating at 40 mg a.i./kg/day was rather high (male mating index = 89%, compared to the background data mean of 99%). Males T30139 (10 mg a.i./kg/day), T20194 and T20197 (40 mg a.i./kg/day) had no abnormal macroscopic findings at necropsy but male T20183 (40 mg a.i./kg/day) had small right testis and epididymis at necropsy and little seminal liquid with virtually no spermatozoa at sperm analysis.
Five females treated at 40 mg a.i./kg/day mated but were not pregnant. Two of the females were not cycling (in diestrus for 9 or 12 days) and one female was paired with a male which was later found to have aspermia/oligospermia. None of the other males had microscopic findings which were considered to have impaired fertility.
Overall, the time taken to mate was prolonged in these two groups because of the males which did not mate but it was considered that treatment with the test item did not specifically delay mating.
The mean duration of gestation was comparable to that of the controls for all groups.
The mean numbers of implantation sites and pups born were comparable with the controls at all dose-levels. The mean post-implantation loss at 10 and 20 mg a.i./kg/day was slightly higher than the controls, and outside Historical data range, but the group treated at 40 mg a.i./kg/day had a lower mean post-implantation loss than the controls, and was within Historical data range, so it was considered that this increase at the low and intermediate dose-levels was incidental.
At 40 mg a.i./kg/day, two entire litters died. Female T20518 had only one pup which died on lactation day 2 without any observed clinical signs. There were no recorded implants in the uterine horns at sacrifice although there were greenish pigmen-laden macrophages and necrosis of the endometrium in the uterus.
Female T20520 had 16 pups; one was found dead on lactation day 2, two were cannibalized on lactation day 3, four were cannibalized and two were found dead on lactation day 4 and the remaining seven were cannibalized on lactation day 5. The majority of the pups were observed to be cold on lactation day 3 which indicated lack of nesting/nursing by the dam or inability or lack of desire to nurse in the pups. The dam showed no signs of poor clinical condition.
The remaining 14 dead pups from the 40 mg a.i./kg/day group were spread between seven litters (between one and five dead pups per litter).
Four litters (between one and eight dead pups per litter). The groups treated at 10 or 20 mg a.i./kg/day had fewer dead pups than the control group.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

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

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Some cannibalized pups were cold to the touch and pale the day before death but the found dead pups, with one exception, did not show any clinical signs prior to death.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The pup deaths in the control group and the group treated at 20 mg a.i./kg/day were scattered one pup per affected litter, but at 10 and 40 mg a.i./kg/day the deaths tended to be concentrated in two litters.

Dose-level (mg a.i./kg/day) 0 10 20 40
Number of cannibalized pups 1 6 2 13
Number of cannibalized pups with clinical signs 1 4 0 0
Number of pups found dead 4 6 3 8
Number of found dead pups with clinical signs 0 0 0 1
Number of prematurely sacrificed pups 0 1a 0 0
a: pup 14 of female T20360 with necrosed right hindlimb, pallor, thin appearance.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of treatment with the test item on mean body weight or body weight gain

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:

no effects observed

Description (incidence and severity):

It was considered that the difference in age at balanopreputial separation was not related to treatment with the test item.
It was considered that there were no effects on the age of vaginal opening at any dose-level.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No changes in organ weights attributable to the treatment with Sodium thioglycolate were noted

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no relevant findings at necropsy in F1 pups found dead or sacrificed at weaning at any dose-level.

Histopathological findings:

no effects observed

Description (incidence and severity):

No treatment-related observations were noted.

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

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

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The majority of the cannibalized pups were observed to be cold on lactation day 3 which indicated lack of nesting/nursing by the dam or inability or lack of desire to nurse in the pups. In the litters where cannibalized and found dead pups occurred, the other pups often showed the same clinical signs, generally coldness.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 40 mg a.i./kg/day, two entire litters died. Female T20518 had only one pup which died on lactation day 2 without any observed clinical signs.
Female T20520 had 16 pups; one was found dead on lactation day 2, two were cannibalized on lactation day 3, four were cannibalized and two were found dead on lactation day 4 and the remaining seven were cannibalized on lactation day 5.
The remaining 14 dead pups from the 40 mg a.i./kg/day group were spread between seven litters (between one and five dead pups per litter). In the control group, the 16 dead pups were spread between four litters (between one and eight dead pups per litter). The groups treated at 10 or 20 mg a.i./kg/day had fewer dead pups than the control group.

Dose-level (mg a.i./kg/day) 0 10 20 40
Number of cannibalized pups 10 4 4 16
Number of cannibalized pups with clinical signs 4 1 0 14
Number of pups found dead 6 5 7 15
Number of found dead pups with clinical signs 4 3 2 4
Number of prematurely sacrificed pups 0 0 2a 0
a: female T20481: pups 3 and 10 because of tremors, locomotory difficulties, piloerection and emaciation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of treatment with the test item on mean body weight or body weight gain

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:

no effects observed

Description (incidence and severity):

No changes in organ weights attributable to the treatment with Sodium thioglycolate were noted

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic findings were noted in F2 pups.

Histopathological findings:

no effects observed

Description (incidence and severity):

No treatment-related observations were noted

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other: 16 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:

no

Conclusions:

Sodium thioglycolate, was administered once daily by oral gavage to males and females for 10 weeks prior to pairing, during mating, gestation and lactation until weaning of the pups. Selected pups formed the F1 generation and were treated with the test item once daily by oral gavage for 10 weeks, from day 22 of age until the start of the pairing period, during mating, gestation and lactation until weaning of the F2 pups. The dose-levels for both generations were 10, 20 and 40 mg a.i./kg/day (a.i. = active ingredient). Due to mortalities around the time of delivery in the F0 females, the F1 generation females were not treated from gestation day 19 until lactation day 1.
At 40 mg a.i./kg/day, it is concluded that sodium thioglycolate has no effect on non-pregnant, naïve, adult rats but that it causes maternal toxicity and death of susceptible pregnant females around the time of delivery. Effects on the dam include lack of nesting/nursing behavior and this causes death of the pups which have been delivered. If treatment is stopped just prior to delivery, the females may survive delivery but pup death may still occur and pup clinical signs of coldness suggest that maternal nesting/nursing behavior is still impaired by treatment with the test item and affect pup survival.
There is evidence that female rats are more affected by sodium thioglycolate treatment than males as shown by lower F1 female body weight and body weight gain.
Minimal to moderate periportal heptocellular microvacuolation was observed in F0 animals treated at 40 mg a.i./kg/day suggestive of mild
hepatotoxicity and especially in dams found dead or prematurely sacrificed at the time of parturition (out of the eight animals presenting this finding, four were found dead or prematurely sacrificed dams). Sodium thioglycolate is known to induce fatty liver via an inhibition of the ß-oxidation of fatty acids.

There were no effects on sperm parameters in the control or high-dose group males of either generation.
There were no effects of treatment on any parameters measured in either males or females with the test item at 10 or 20 mg a.i./kg/day.

Under the experimental conditions of this study, and in view of the maternal mortalities and liver effects in males and females observed at 40 mg a.i./kg/day, the dose-level of 20 mg a.i./kg/day (16 mg/kg bw/d as mercaptoacetic acid) was considered to be the No Observed Effect Level (NOEL) for parental toxicity, female fertility and gestation of each generation and for development, growth and survival of the progeny. It is probable that the small effects observed on pup survival at 40 mg a.i./kg/day were secondary to the severe and lethal effects observed in the pregnant dams at that dose level.

The No Observed Effect Level (NOEL) for males fertility and female mating behaviour was higher or equal than 40 mg a.i./kg/day (32 mg/kg bw/d as mercaptoacetic acid).

Executive summary:

The objective of this study was to provide general information concerning the effects of the test item,Sodium thioglycolate,on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrous cycle, mating behavior, conception, gestation, parturition, lactation and weaning, and on the growth and development of the offspring over 2 generations. This study was performed under GLP compliance and following the OECD Guideline for Testing of Chemicals No. 416 (Two-Generation Reproduction Toxicity Study), 22^ndJanuary 2001.

Three groups of 25 male and 25 female Sprague-Dawley rats received the test item, Sodium thioglycolate (batch No.25462), daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. The test item was administered as a solution in degassed purified water, by oral gavage, at dose-levels of 10, 20 or 40 mg a.i./kg/day (a.i. = active ingredient) (16, 32 and 64 mg/kg bw/d as mercaptoacetic acid). Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded weekly. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening, surface righting, cliff avoidance and air righting). At the end of the treatment period or prior to premature sacrifice, the F0 animals were blood sampled for analysis of hematology and blood biochemistry parameters, including ß-hydroxybutyrate and acetoacetate determination. The animals were not fasted before blood collection. After weaning of the pups, the males and females of the F0 generation were sacrificed. Sperm analysis was performed on the first 10 males of the control and high-dose groups (groups 1 and 4) and since no treatment-related effects were observed this was not performed on males of the other groups. A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs and macroscopic lesions of all groups and for the control and high-dose groups the heart, kidneys and liver were also examined. The liver of all intermediate-dose group animals was also examined.

Of the progeny of the F0 generation, one or two males and one or two females per litter were selected to constitute the F1 generation of groups of 25 male and 25 female rats in the control, low and intermediate groups and 27 male and 27 female rats in the high-dose group (this to compensate in advance for mortality around the time of delivery, a known effect of the test item). Three groups received the test item, Sodium thioglycolate(batch No.26461), daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. Due to the test item inducing known difficulties around the time of delivery, all mated females, including controls, were not treated from day 19 of gestation until day 1 of lactation in order to see if this would reduce the mortality. The test item was administered as a solution in degassed purified water, by oral gavage, at dose-levels of 10, 20 or 40 mg a.i./kg/day. Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once at weaning before the beginning of the treatment period and then once a week. Body weight and food consumption were recorded weekly. Each animal was assessed for sexual maturity (balanopreputial separation or vaginal opening) and the day of age and body weight were recorded on the day each animal was positive. At 4 weeks of age, the animals were assessed for auditory function (acoustic startle response) and pupil constriction, andweeks of age the spontaneous locomotor activity was measured using an automated infra-red sensor equipment. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 19 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening, surface righting, cliff avoidance and air righting). In addition, the anogenital distance of all pups was measured on the day after birth and the presence of nipples was checked in all males on post-natal day 12 or 13. At weaning of the pups, the males and females of the F1 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs and any macroscopic lesions of all groups. In addition, pups of both the F0 and F1 animals were submitted for a macroscopic examination. One randomly selected pup/sex/litter for F1 and F2 litters, all pups found dead or prematurely sacrificed and any pups showing external abnormalities or clinical signs were weighed and then submitted for a macroscopic examination of the principal thoracic and abdominal organs with special attention paid to the reproductive organs.

There were no effects of treatment at 10 or 20 mg a.i./kg/day.

At 40 mg a.i./kg/day, the males and females showed no effects of treatment during the pre-mating, mating or gestation phases. At delivery, four females were found dead; one on gestation day 21 and three on gestation day 22. One of these females found dead on gestation day 22 had delivered 12 live and one dead pup before dying, the other three females had not started delivery and had dead fetuses in the uterine horns at necropsy. On lactation day 1, one female, showing signs of poor condition (piloerection, blood and placentae in the bedding), cannibalized her 10 pups (it is likely that more pups were born and cannibalized before being noticed because there were five more implantation sites in the uterine horns than pups). It is not known whether the pups were alive or dead prior to cannibalism, however the adverse outcome is considered to be a maternal effect since no fetuses remained in the uterine horns at necropsy; the female did deliver all pups starting delivery on gestation day 21. No effects of treatment were observed in the F0 generation animals during the remainder of the lactation period other than a slightly lower mean body weight gain of the females during the first 4 days of lactation. There were no effects on sperm parameters in the control or high-dose group males.

There were no treatment-related effects on organ weights at any dose-level. There were no treatment-related microscopic changes in testis, epididymis, prostate, coagulating glands or seminal vesicles. There were possible treatment-related changes in the uterus of one 40 mg a.i./kg/day female (female T20412 which had only implantation scars thought to be left from early resorptions); thrombosis of the mesometrial remnant, organized by granulation tissue composed of fibroplasia, fibrin plugs, thrombi and neovascularization. Golden pigment-laden cells and pyknotic trophoblastic cells were observed. The found dead females had no microscopic findings which could explain the deaths but one female had hemorrhage of a mesometrial triangle. Minimal to moderate periportal hepatocellular microvacuolation was observed at 40 mg a.i./kg/day in 2/25 males and 6/25 females, and in 4/6 prematurely sacrificed/found dead females suggesting mild liver toxicity at this dose-level. No control animals had the same finding.

Female plasma fatty acid concentration was statistically significantly decreased however there were no effects of treatment with the test item on plasma acetoacetate or ß-hydroxybutyrate concentrations indicating that the animals were not in ketosis.

 

There were no effects of treatment on F1 pups in the 10 or 20 mg a.i./kg/day groups.

At 40 mg a.i./kg/day, the pups had a 9.6% mortality rate during the first 4 days of lactation (cannibalism and being found dead); neither the pups nor the F0 dams showed particular clinical signs (except female T20401 which had piloerection and cannibalized all pups in the litter). There was a possible, very slight, delay in physical development; the majority of the pups achieved tooth eruption, eye opening and auditory canal opening on the same day as the majority of the pups from the other groups but there was a higher percentage of pups achieving these landmarks on later days than in the other groups. The females of the F1 generation started treatment (on day 22 of age) with a statistically significantly lower mean body weight than the controls. Mean body weight gain and mean food consumption were both also statistically significantly lower for the first week of treatment. During gestation, the females had a slightly lower body weight gain. Treatment was stopped on gestation day 19 and delivery passed without problem in all females. However, two females were prematurely sacrificed on day 2 or day 5 of lactation due to death of the litter. One of the females had thrombosis of mesometrial triangles. Treatment was re-started on lactation day 1 and the number of pup deaths was higher when compared with the controls. This was mainly due to one female which had three found dead pups and cannibalized 13 pups over several days until lactation day 5 when all pups were dead. Another female also had total litter death but delivered only one pup. The total number of dead pups in this group was similar to that of the controls when the female with the 16 dead pups is excluded (15 dead pupsvs.the control group), but whereas the control pup deaths were concentrated in four litters, at 40 mg a.i./kg/day the pup deaths were spread over seven litters. The females had not had any noticeable problems during delivery and were not showing any clinical signs during the first days of lactation except for a possible effect on maternal nesting and nursing behavior since the pups were often observed to be cold, even those that survived. There were no treatment-related effects on organ weights at any dose-level. There were no effects on sperm parameters in the control or high-dose group males. There were no treatment-related microscopic changes in testis, epididymis, prostate, coagulating glands or seminal vesicles of the males or in the ovaries, oviducts, uterus and vagina of the 40 mg a.i./kg/day females.

The female F2 pups had a slightly lower mean body weight at the end of lactation (-5%) but there were no effects on male or female F2 pup physical development in terms of eye opening, tooth eruption or auditory canal opening or on reflex development.

At 40 mg a.i./kg/day, it is concluded that the test item has no effect on non-pregnant, naïve, adult rats but that it causes maternal toxicity and death of susceptible pregnant females around the time of delivery. Effects on the dam include lack of nesting/nursing behavior and this causes death of the pups which have been delivered. If treatment is stopped just prior to delivery, the females may survive delivery but pup death may still occur and pup clinical signs of coldness suggest that maternal nesting/nursing behavior is still impaired by treatment with the test item and affect pup survival. There is evidence that female rats are more affected by test item treatment than males as shown by lower F1 female body weight and body weight gain. Minimal to moderate periportal heptocellular microvacuolation was observed in females and some male F0 animals treated at 40 mg a.i./kg/day suggestive of mild hepatotoxicity and especially in dams (4/6)found dead or prematurely sacrificed at time of parturition. Sodium thioglycolate is known to induce fatty liver via aninhibition of the ß-oxidation of fatty acids.

There were no effects on sperm parameters in the control or high-dose group males.

There were no effects of treatment on any parameters measured in either males or females with the test item at 10 or 20 mg a.i./kg/day.

 

Under the experimental conditions of this study, and in view of the maternal mortalities and liver effects in males and females observed at 40 mg a.i./kg/day, the dose-level of 20 mg a.i./kg/day (16 mg/kg bw/d as mercaptoacetic acid) was considered to be the No Observed Effect Level (NOEL) for parental toxicity, female fertility and gestation of each generation and for development, growth and survival of the progeny. It is probable that the small effects observed on pup survival at 40 mg a.i./kg/day were secondary to the severe and lethal effects observed in the pregnant dams at that dose level.

 

The No Observed Effect Level (NOEL) for males fertility and female mating behaviour was higher or equal than 40 mg a.i./kg/day (32 mg/kg bw/d as mercaptoacetic acid).

 

Reference 2

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

16 mg/kg bw/day

Study duration:

subchronic

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

Description of key information

Mercaptoacetic acid and its salts are not considered to be developmental toxicants.

The developmental toxicity of sodium and ammonium mercaptoacetate has been investigated in standard oral and dermal studies in rats and/or rabbits compliant or comparable to OECD guideline # 414. Ammonium mercaptoacetate was administered by gavage to pregnant Wistar rats from gestational days 6-19. At 75 mg/kg bw/d, two animals died. The body weight, food and water consumption of the dams were not affected by the treatment. No embryo/fetal toxicity, or treatment-related teratogenicity was observed in any group. The NOAELs for maternal and embryo-foetal toxicity were 15 (12.6 mg/kg bw/day as mercaptoacetic acid) and 75 mg/kg bw/d (63.3 mg/kg bw/day as mercaptoacetic acid), respectively. In another study, sodium mercaptoacetate was topically applied to pregnant Sprague-Dawley rats from gestational days 6-19 and to pregnant New Zealand White rabbits from gestational days 6-29. In rats, there was one reported maternal death at 200 mg/kg bw/d. Feed consumption, water consumption, and body weights of the dams were significantly increased. The body weights of the foetuses were significantly lower than the controls at 200 mg/kg bw/d, however there was no other evidence of embryo/foetal toxicity. In rabbits, moderate to severe erythema occurred at the dosing site in all groups, however no maternal systemic toxicity, embryo/foetal toxicity, or treatment-related teratogenicity were observed in any group. The NOAEL for maternal and developmental toxicity was 100 mg/kg bw/d (80.7 mg/kg bw/day as mercaptoacetic acid) in rats and >= 65 mg/kg bw/d (52.5 mg/kg bw/day as mercaptoacetic acid) in rabbits. No teratogenic effects were observed.

 

Oral route

Main study

The developmental toxicity potential of ammonium mercaptoacetate was evaluated in rats in a study performed according to the OECD Guideline #414 (Gleich, 1998). Ammonium mercaptoacetate was administered by gavage to 3 groups of 25 pregnant Wistar rats from gestation days (GD) 6 to 19 at daily doses of 0, 3, 15, and 75 mg/kg bw/d (0, 2.5, 12.6 and 63.3 mg/kg bw/day as mercaptoacetic acid). Day 0 of pregnancy was designated as the day of confirmed mating. Clinical signs including mortality and evidence of abortion were checked daily. Body weight was determined on GD0, 6 and the daily until GD20. Food and water consumption was recorded at designated intervals during pregnancy. On day 20 of pregnancy, females were killed. The terminal body weights of the dams were recorded. The gravid uterus was weighed and foetuses removed by hysterectomy. Females were examined macroscopically. Litter parameters were recorded: number of corpora lutea, implantation sites, early and late resorptions, and dead and live foetuses. Foetuses were weighed, sexed, and submitted to external examination. About half of them were examined for skeletal malformations and half for organ malformations. Rooting in the bedding material was seen in all rats in the high dose group. Two animals in the high dose group died on GD 20, one day after the last administration of the test material. These deaths were regarded to be treatment related. Eighteen to 24 rats out of 25 per group proved to be pregnant. The body weight development, food and water consumption were not affected by treatment. The terminal body weights were similar in all groups. The uterus weights in the high dose group were slightly, but not significantly lower than in the control or the other groups. This finding was regarded to be accidental. The numbers of corpora lutea, implantations and live foetuses were not affected. The numbers of dead foetuses, and complete, early and late resorptions were not increased. The sex distribution was not affected in any of the groups. The weights of the foetuses in the low, mid, and high dose groups were similar to the control and not affected by treatment. The frequency of all malformations was in a normal range and not increased. The NOAELs for maternal and embryo-foetal toxicity were 15 (12.6 mg/kg bw/day as mercaptoacetic acid) and higher than 75 mg/kg bw/d (63.3 mg/kg bw/day as mercaptoacetic acid), respectively. No teratogenic effects were observed.

 

Range-finding study

A preliminary study has been conducted for evaluating the potential reprotoxic effects of ammonium thioglycolate in rats and for evaluating the correct dose to administrate to rats in the core study (Gleich, 1998). Ammonium thioglycolate was administered by gavage to groups of 5 pregnant Wistar rats from gestation days (GD) 6 to 19 at daily doses of 1, 10, 50, 100, and 150 mg/kg. The control rats group were treated with the vehicle, demineralized water. The rats were killed on GD 20. The foetuses were removed, weighed, and examined for macroscopic malformations.

The numbers of corpora lutea, implantations and live foetuses per dam were not affected at the doses up to 50 mg/kg. At 50 mg/kg, the animals showed no signs of intoxication and only very slightly reduced body weight gain during pregnancy.

At 100 mg/kg, the animals showed signs of intoxication and 3/5 animals died. In the 2 surviving rats at 100 mg /kg, the number of corpora lutea and implantations was unaffected, but the foetal loss was increased. The foetuses in the 100 mg/kg dose group were lighter, but not malformed. Then the high dose for the main study should be lower than 100 mg/kg. At 150 mg/kg, all animals died.

In conclusion, the high dose for the main study should be higher than 50 mg/kg and lower thant 100 mg/kg.

 

Rabbit studies

 

Main study (DRAFT RESULTS)

The objective of this study was to evaluate the developmental and maternal toxicity in pregnant female rabbits and developing embryos/fetuses after oral gavage treatment with test item Sodium mercaptoacetate (Thiocare®H102 46% ) in pregnant rabbits from gestation day (GD) 6 to 28, acoording to OECD guideline 414. 
The dose levels for this study were selected based on the results of the Dose Range Finding study (DRF) study N6282. In the DRF study, dose levels of 0, 25, 50, 75 and 100 mg/kg/day were evaluated in pregnant rabbits (6 rabbits/group) treated with the test item or vehicle from GD 6 to GD 28 at the dose volume of 2 mL/kg body weight. At 25 mg/kg/day, there was reduction in body weight and food consumption. Doses of 50, 75 and 100 mg/kg/day resulted in unscheduled mortalities. Based on the results of the DRF study in pregnant rabbits, considering treatment related deaths at ≥50 mg/kg/day, a high dose of 30 mg/kg/day was selected for the definitive study. A mid dose of 15 mg/kg/day and a low dose of 5 mg/kg/day were selected to provide a graded response to the test item.
In the definitive study, 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred. Test item in vehicle (Milli-Q water) was administered at 5, 15 and 30 mg/kg/day at the dose volume of 2 mL/kg body weight for the low, mid, and high dose groups, respectively. The vehicle control group received the vehicle only.
The following parameters and end points were evaluated in this study: Maternal parameters - clinical signs, body weight, body weight gain, food consumption, gross pathology, gravid uterine weight, number of corpora lutea, implantation sites, early and late resorptions, pre and post implantation loss and fetal parameters (total number of live fetuses, fetal weights, sex, and sex ratio). All the fetuses were examined for external malformations. All the live fetuses were examined for visceral and skeletal variations and malformations.
During the conduct of the experiment, homogeneity and active ingredient analyses were carried out from the dose formulation samples collected prior to initiation of treatment and termination of treatment. The results of formulation analyses were within the acceptable limits.

Main findings from the study are summarized below:
Mortality, clinical signs, and gross necropsy changes: There were no death in any group.The treatment resulted in clinical signs of hypoactivity in 2/23 rabbits and one rabbit showed posture- sitting with head hung down at 30 mg/kg/day. There were incidences of abortions at all the tested doses (two rabbits in vehicle control group, one each in 5 and 15 mg/kg/day and three rabbits at 30 mg/kg/day). There were no gross pathological findings that could be attributed to the test item.
Body weight and food consumption: No effect was observed at 5 mg/kg/day.The significantly lower maternal body weight and body weight gains observed during later periods of gestation at 15 and 30 mg/kg/day were associated with lower food consumption and are considered treatment-related changes.
Maternal developmental parameters: No significant changes in any maternal developmental toxicity endpoint were observed at 5 and 15 mg/kg/day. At 30 mg/kg/day, the percentage of late resorptions were higher (16.65 vs. 3.08% in vehicle control), which in turn resulted in higher post-implantation loss (15.59% vs. 6.39% in vehicle control). These findings were considered treatment-related. No gross pathological changes of the placenta were observed in any of the animals.
Litter Parameters: No effect was observed at 5 and 15 mg/kg/day. At 30 mg/kg/day, the mean fetal weight of males (-17%), females (-17%) and combined sex (-18%) were significantly lower, compared to the vehicle control group and attributed to secondary to lower maternal body weights and food consumption. At 15 mg/kg/day, a significant reduction in fetal weight was observed in females (-9%) compared to the respective control value.
Fetal examination: The results from external, visceral, and skeletal examinations did not reveal any significant effects that could be attributed to the test item.
In conclusion, oral administration of Sodium mercaptoacetate (Thiocare®H102 46%) at 0, 5, 15 and 30 mg/kg/day to pregnant rabbits during GD 6 to 28 resulted in treatment-related clinical signs of hypoactivity and posture- sitting with head hung down at 30 mg/kg/day. The significantly lower maternal body weights towards the later part of gestation at 15 and 30 mg/kg/day were associated with lower food consumption are considered treatment-related changes.
At 30 mg/kg/day, the percentage of resorptions were higher, which in turn resulted in higher post-implantation loss. The secondary to lower maternal body weights and food consumption. On gross pathology evaluation of rabbits, no abnormality was detected up to the high dose of 30 mg/kg/day. Fetal external, visceral, and skeletal examination revealed no signs of teratogenicity up to 30 mg/kg/day.

 

Dermal route

Rat studies

Main study

The developmental toxicity of sodium thioglycolate was evaluated in pregnant rats in a study performed according to a method comparable to the OECD guideline # 414 (NTP, 2001; Tyl, 2001; Tyl et al., 2003). Pregnant Sprague-Dawley rats were exposed topically, 6 hr/day, to sodium thioglycolate (99% pure) in vehicle (95% ethanol:distilled water, 1:1) from gestational day (gd) 6 through 19 at dose levels of 0, 50, 100, and 200 mg/kg bw/d. Twenty-five timed-mated female rats were assigned to each group and monitored at regular intervals throughout gestation for clinical signs (including dosing site condition), feed and water intake, and body weight. At necropsy on gestational day 20; the following were recorded: maternal clinical condition; body, liver and gravid uterine weights; and pregnancy status. The number of ovarian corpora lutea and uterine implantations (resorbed, dead, or live foetuses) was recorded. All live foetuses were counted, weighed, and examined for external alterations, including cleft palate. Approximately 50% of the live rat foetuses per litter were sexed internally and examined for visceral alterations. These foetuses were decapitated and the heads fixed, decalcified, and examined for soft tissue craniofacial alterations. All foetal carcasses were eviscerated (and rat foetuses not scheduled for a visceral examination were sexed internally), macerated, and stained with alizarin red S and alcian blue. Intact rat foetuses (those not decapitated) were examined for ossified and cartilaginous skeletal alterations.

Sodium thioglycolate treatment was associated with one maternal death at 200 mg/kg/d (only clinical observations at the dosing site preceded death). Other effects, dependent on dose and exposure duration, included body weight and weight gain reductions, changes in relative feed and water intake, and discoloration and slight erythema at the application site. Feed consumption was significantly increased above the control at 50 (5.4%) and 100 (6.1%) mg/kg bw/d, but not at 200 mg/kg bw/d (2.2%). Maternal water consumption was significantly increased at 200 mg/kg bw/d (12.7%) and slightly (but not statistically significantly) increased at 50 (3.2%) and 100 (5.1%) mg/kg bw/d. Maternal body weights and body weight changes were decreased only at 200 mg/kg bw/d. At scheduled necropsy, there were no macroscopic indications of organ toxicity. In addition, organ weights and gravid uterine weights were equivalent across groups; maternal body weight was significantly reduced at 200 mg/kg/d. Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. A significant, dose-related, upward trend was present for % foetuses with skeletal variations per litter, but there were no significant pairwise comparisons to the vehicle control group value and the % were in the range of the historical control data . Body weights of male and female foetuses per litter at 200 mg/kg bw/d were significantly lower than control values. Based on the study findings, a no observed adverse effect level (NOAEL) could be identified at 100 mg/kg/day (80.7 mg/kg bw/d as mercaptoacetic acid) for maternal and developmental toxicity.

 

Range finding study

In a range-finding study (NTP, 2000), pregnant Sprague-Dawley-derived (CD®) rats were topically exposed to sodium thioglycolate (50, 250, 350, and 525 mg/kg/day) in vehicle (95% ethanol:distilled water mixed 1: 1) or vehicle alone for six hours per day, from gestational day (gd) 6 through 19 (all doses) or gd 1 through 19 (0 and 525 mg/kg/day). Thirteen timed-mated females were assigned to each group and monitored at regular intervals throughout gestation for clinical signs, food and water intake, and body weight. At necropsy of surviving dams on gd 20, maternal clinical condition (body, liver, and gravid uterine weights) and pregnancy status were recorded. The number of resorbed, dead, or live fetuses was also recorded. Live fetuses were weighed, sexed, and examined for external morphological abnormalities, including cleft palate.

Sodium thioglycolate was either lethal or produced severe maternal morbidity in a dose­related manner. Dams manifested lethality and morbidity soon after the onset of maternal exposure to 350 and 525 mg/kg/day in the gd 6-19 exposure group and at 525 mg/kg/day in the gd 1-19 exposure group. All surviving dams in these groups (and in the gd 1-19 control group) were terminated by gd 12 for humane reasons. At the lower doses, one dam died on gd 10 after four days exposure to 250 mg/kg/day. No deaths or severe toxicity occurred in the 50 mg/kg/day group.

The severity and incidence of nonlethal effects were also dose and exposure dependent. Effects included body weight reduction, intermittent changes in food and water intake, skin irritation, reduced activity, and piloerection. Maternal body weight and body weight change were decreased at >= 250 mg/kg/day, whereas at 50 mg/kg/day, they were similar to the control values.

At scheduled necropsy, relative liver weight (% sacrifice weight) was significantly increased at 250 mg/kg/day. The gravid uterine weight at 250 mg/kg/day was approximately 42% lower than the control value.

Prenatal viability and embryo/fetal growth were reduced at 250 mg/kg/day, whereas there were no treatment-related effects on external fetal morphology. Three litters at 250 mg/kg/day were fully resorbed (versus none at 0 or 50 mg/kg/day). Fetal body weights from dams exposed to 250 mg/kg/day were lower (p< 0.05) relative to the control group values.

In summary, maternal toxicity was evident at 250 mg/kg/day (expressed as reduced body weights, weight gain, and feed consumption, and one death). Above 250 mg/kg/day, the dams manifested severe morbidity and death. Developmental toxicity at 250 mg/kg/day was expressed as significantly reduced fetal body weights ( for all fetuses and male fetuses but not for female fetuses), and slightly reduced live litter size, increased resorptions, and three fully resorbed litters, but no evidence of fetal treat­ment-related external malformations or variations.

 

Rabbit studies

Main study

The developmental toxicity of sodium thioglycolate was evaluated in pregnant rabbits in a study performed according to a method comparable to the OECD guideline # 414 (NTP, 2001; Tyl, 2001; Tyl et al., 2003). Pregnant New Zealand White Rabbits were exposed topically, 6 hr/day, to sodium thioglycolate (99% pure) in vehicle (95% ethanol:distilled water, 1:1) from gestational day (GD) 6 through 29 at dose levels of 0, 10, 15, 25 or 65 mg/kg bw/d (equivalent to 0, 8.1, 12.1, 20.2 and 52.5 mg/kg bw/d as mercaptoacetic acid). The dose levels were selected on the basis of a range finding study showing maternal mortality at dose levels >= 100 mg/kg/day. Twenty-four naturally mated female rabbits (in two replicates of 12) were assigned to each group andmonitored at regular intervals throughout gestation for clinical signs (including dosing site condition), feed and water intake, and body weight. At necropsy on gestational day 30; the following were recorded: maternal clinical condition; body, liver, kidney and gravid uterine weights; and pregnancy status. The number of ovarian corpora lutea and uterine implantations (resorbed, dead, or live foetuses) was recorded. All live foetuses were counted, weighed, and examined for external alterations, including cleft palate. Heads fixed, decalcified, and examined for soft tissue craniofacial alterations. All foetal carcasses were eviscerated (and rat foetuses not scheduled for a visceral examination were sexed internally), macerated, and stained with alizarin red S and alcian blue. Intact rat foetuses (those not decapitated) andall rabbit foetuseswere examined for ossified and cartilaginous skeletal alterations.

No maternal deaths were associated with sodium thioglycolate treatment. One female in the 25 mg/kg bw/d group delivered early and one doe in the 65 mg/kg bw/d group was removed due to a preexisting condition (trichobezoar or hairball in the stomach). Clinical observations were almost exclusively limited to effects of treatmentat the dosing site (skin erythema) in all groups. Maternal body weight and body weight gain were equivalent across dose groups for all intervals measured except for body weight gain for gestational days 12-15. There was a trend for decreased body weight gain for this interval, which was significant in the high dose group when compared to controls. There were no consistent treatment related reductions in feed consumption. At scheduled necropsy, there was no effect of treatment on terminal maternal body weight. In addition, organ weights and gravid uterine weights were equivalent across groups. Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. Body weights of male and female foetuses per litter and percent males and females per litter were equivalent across dose groups. The maternal NOAEL for systemic toxicity was at or above 65 mg/kg bw/d (52.5 mg/kg bw/d as mercaptoacetic acid); for local toxicity at the dosing site, it was below 10 mg/kg bw/. The NOAEL for developmental toxicity was at or above 65 mg/kg bw/d (52.5 mg/kg bw/d as mercaptoacetic acid).

 

Range finding study

A developmental toxicity screen was conducted in New Zealand White rabbits (NTP, 2000). Naturally mated female New Zealand White rabbits (8/group) were dosed topically with sodium thioglycolate at 25, 50, 100, 150, and 175 mg/kg/day or vehicle control (ethanol 95%: distilled water, 1: 1) for a minimum of 6 hours per day on gestational days (gd) 6 through 29. Dose volume was 0.5 ml/kg. Eight time-mated rabbits were assigned to each group and monitored throughout gestation for clinical signs, food intake, and body weight. At necropsy (gd 30), the following were recorded: maternal clinical condition; body, liver, paired kidney and gravid uterine weights; and pregnancy status. The number of resorbed, dead, or live fetuses was also recorded. Live fetuses were weighed and examined for external morphological abnormalities, including cleft palate.

At 100, 150 and 175 mg/kg/day, excessive maternal toxicity (morbidity and mortality) resulted in termination of surviving does in these groups by gd 15. No maternal deaths or severe toxicity occurred at 25 or 50 mg/kg/day. There was a significant reduction in body weight gain for gd 6-9 for the 150 and 175 mg/kg/day groups, but no reduced body weight or weight gain during gestation (gd 0 to 30), treatment or for any other time interval for does in the 25 and 50 mg/kg/day groups. Feed consumption was not affected by treatment. Edema and erythema at the dosing site were seen in all treatment groups until sacrifice with dose-related increases in incidence and severity. Moderate to severe erythema was seen at >= 50 mg/kg/day. Other signs of toxicity included clinical weight loss (defined as >=150 g in any weigh period) and lethargy at 100-175 mg/kg/day. There were no effects of treatment at scheduled necropsy for body weight, gravid uterine weight, and liver or kidney weights at 25 or 50 mg/kg/day. Prenatal viability and embryo/fetal growth were not statistically affected. However, the number of live fetuses per litter showed a significant downward trend across dose groups. Percent nonlive implants per litter increased from 1.1 % in the controls to 25.3% at 50 mg/kg/day. However, this parameter was not statistically significant (due to small sample sizes and large variance). There was no effect of treatment on fetal external morphology.

Based on the maternal mortality and morbidity, the maximal tolerated dose for the main developmental toxicity study was between 50 and 100 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Only draft final study report available at submission.

Reliability:

1 (reliable without restriction)

Justification for type of information:

Following ECHA 's communication CCH-D-2114541355-52-01/F:
A PNDT study according to the test method OECD TG 414 must be performed in rabbit as preferred non-rodent species. The study shall be performed with oral administration of the analogue substance ammonium sulfanylacetate (EC 226-540-9).
The study has been conducted on sodium sulfanylacetate (EC 206-696-4), by the registrants of the substance (Bruno Bock Chemische Fabrik GmbH & Co. KG).
At the moment only the draft study report is available.


HYPOTHESIS FOR THE ANALOGUE APPROACH

- The target and source substances are grouped based on similar molecular structure and functionality.

- The target and source substance have similar physico-chemical properties.

- The target and source substances have similar reactivity :
o At acidic pH (for example the pH stomach of 1.2), more than 99% of the product will be present as an acidic form.
o At neutral pH, diluted solutions of thioglycolic acid and its salts undergo full dissociation into the thioglycolate anion (HS-CH2-COO-) and the respective cations (H+, NH4+ or Na+).

- The target and source substances have similar toxicity :
o The toxicity of each compound is driven by the thioglycolate anion.
o The effect of the counter-ion (sodium or ammonium) on the systemic toxicity of the thioglycolic salts is not expected to be significant.
o The acute and repeated dose toxicity of sodium sulphate and ammonium chloride, sulphate and phosphate is moderate, with effective dose levels far higher than those of the thioglycolates.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KCC BIO LABS, Sy No.8/1, Tondagere Village, Tavarakere Post, Hebburu Hobli Tumakuru- 572120 Karnataka, India

- Age at study initiation: 8 - 9 months

- Weight at study initiation:
Mean body weight Body weight range
G1 – 3.7606 ± 0.171 3.4146 to 4.0913
G2 – 3.7541 ± 0.166 3.4738 to 4.0832
G3 – 3.7533 ± 0.160 3.4347 to 4.9955
G4 – 3.7577 ± 0.178 3.3405 to 4.0942

- Fasting period before study: none

- Housing:
Standard laboratory conditions, air conditioned with adequate fresh air supply (12 - 15 air changes/hour). Environment: with temperature 19 to 21°C, relative humidity 64 to 65 %, with 12 hours light and 12 hours dark cycle.
The maximum and minimum temperature and relative humidity in the experimental rooms were (females and males were housed in different rooms) recorded once daily. The relative humidity in the experimental rooms was calculated daily from dry and wet bulb temperature recordings.

- Diet (e.g. ad libitum):
Rabbit feed manufactured by Special Diets Services, P.O Box 705, Witham, Essex, CM8 3AD, England (Batch No.5286) was provided ad libitum in stainless steel feed hoppers to rabbits.

- Water (e.g. ad libitum):
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter-cum-purifier (Eureka Forbes Ltd, Mumbai, India) was provided ad libitum in polycarbonate bottles with stainless steel sipper tubes to rabbits.

- Acclimation period:
After clinical examination for good health and suitability for the study, the rabbits were acclimatized for five days before initiation of mating. During the acclimatization period, all rabbits were observed at least once daily. Females used in this study were nulliparous and non-pregnant.


IN-LIFE DATES:
Acclimatization : Start: 05 May 2022 / End: 09 May 2022
Co-habitation : Start: 10 May 2022 / End: 19 May 2022
Treatment : Start: 16 May 2022 / End: 26 June 2022
Sacrifice : Start: 08 June 2022 / End: 17 June 2022

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q water. The concentration and stability of the test item in the vehicle (Milli-Q water) was performed at dose concentrations of 1.0 0 mg/mL and 150 mg/mL. The test item was found to be stable in the vehicle up to 24 h.

Details on exposure:

VEHICLE
- Concentration in vehicle: 2.5 / 7.5 / 15 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item dose formulations were prepared daily prior to treatment and used within the stability period.
For homogeneity and concentration analysis of the test item, the prepared formulations were sampled at the initiation (16 May 2022) of treatment (GD 6) and at termination (14 June 2022) of treatment period (GD 28). Prepared formulations were samples in duplicate sets (one set for analysis and one set as back-up). Dose formulation samples were analysed for Thiocare®H102 46% formulation content using analytical validated method.
Dose formulations were considered acceptable as the mean percent recovery results were within 70 to 120% at each dose level and relative standard deviation (% RSD), of assay was less than or equal to 20%.

Details on mating procedure:

During the mating period, females were cohabited randomly with males in a 1:1 ratio. After confirmation of mating visually, the Gestation Day 0 (GD 0) was recorded for each individual rabbit.

Duration of treatment / exposure:

From GD 6 to GD 28 of presumed gestation at approximately the same time each day (varying by ± 3 hours).

Frequency of treatment:

Daily

Duration of test:

GD 0 to GD 29

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control group

Dose / conc.:

5 mg/kg bw/day

Remarks:

4 mg/kg/day as mercaptoacetic acid

Dose / conc.:

15 mg/kg bw/day

Remarks:

12 mg/kg/day as mercaptoacetic acid

Dose / conc.:

30 mg/kg bw/day

Remarks:

24 mg/kg/day as mercaptoacetic acid

No. of animals per sex per dose:

23 rabbits/group

Control animals:

yes, concurrent vehicle

Details on study design:

A preliminary dose-range-finding (DRF) study in pregnant rabbits was carried out using 6 rabbits per group . The rabbits were treated at doses of 25 (G2), 50 (G3), 75(G4) and 100 (G5) mg/kg/day along with vehicle control (Milli-Q water) group at a dose volume of 2 mL/kg body weight.

The results were as follows:

Oral administration of Thiocare® H102 46% at =50 mg/kg/day to pregnant rabbits resulted in unscheduled mortalities which required humane removal of rabbits at =75 mg/kg/day. At 25 mg/kg/day, there were no clinical signs or mortalities. The mean body weight and body weight gains were lower with significant reduction in the food consumption, indicative of maternal toxicity. Maternal parameters (uterine weight, number of corpora lutea, implantations, early and late resorptions, and pre- and post-implantation loss) and litter parameters (mean fetal weights) were comparable to control. There was one doe at 25 mg/kg/day which had complete resorption. There were no external fetal malformations observed at 25 and 50 mg/kg/day doses. The gross pathology findings in stomach (red foci in glandular mucosa) and kidneys (red foci) at =50 mg/kg/day were related to test item administration.

Based on these results of dose range finding study in pregnant rabbits and considering treatment related deaths at =50 mg/kg/day, following doses are proposed for the definitive study in pregnant rabbits in consultation with sponsor:

G1 - Vehicle control - 0 mg/kg/day
G2 - Low dose - 5 mg/kg/day
G3 - Mid dose - 15 mg/kg/day
G4 - High dose - 30 mg/kg/day

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rabbits were observed for morbidity and mortality twice daily, i.e., once in the morning and once in the afternoon.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during the treatment period of the study: pre-dose and post dose (within 1-2 hours of administration).

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes for the following intervals: GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29


WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Gross pathological changes of all external and visceral organs of rabbits, including uterine contents were recorded

OTHER:
Aborted rabbit was necropsied, and gross observations observed were recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placenta, pregnancy status

Uteri that appeared non-gravid were subjected to 10% ammonium sulfide staining to observe implantation sites (identified as pregnant animals) or to confirm the non-pregnant status

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all live fetuses]
- Head examinations: Yes: [half per litter ]
- Anogenital distance of all live rodent pups: No

Statistics:

The numerical results were evaluated by an appropriate statistical method using the litter as the unit for data analysis.

The following statistical tests were used:
Data was captured using the ProvantisTM laboratory information management system (LIMS), parameters such as maternal body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss, number of implantations, sex ratio, number of corpora lutea, early and late resorptions was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. Data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), if data found to be nonhomogeneous or of nonnormal data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group.

The incidence of does with resorptions was tested for using Chi-square test followed by Fisher’s exact test for group association.

The incidence of fetus and litter (incidence and percent) observations for external, visceral, and skeletal was tested for Cochran Armitage trend test and pair wise comparison was be done by Fisher’s exact test.

Descriptive statistics Mean, SD, Percentages & Numbers was be presented by Treatment group and Day.

All hypothesis testing wase carried out at the 5% (two-sided) significance level unless otherwise specified. Significant differences are designated throughout the report as below:

*: Statistically significant difference from the control group at p < 0.05

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs or mortalities at 5 and 15 mg/kg/day. At 30 mg/kg/day, hypoactivity was observed on two rabbits one rabbit RBa8653 was found hypoactive on GD 25 and aborted on GD 28, and another rabbit RBa8654 was hypoactive from GD 15-20 and 25 to 27 and posture sitting with head hung down from GD 15 to 18.

Mortality:

no mortality observed

Description (incidence):

The number of rabbits sacrificed at term were 21, 22, 22 and 20 (G1 to G4), of which 1, 3, 2 and 1 rabbits were non-pregnant and the number of pregnant rabbits were 20, 19, 20 and 19, at 0, 5, 15 and 30 mg/kg/day, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean maternal body weights and body weight gains during the different days of gestation at 5 mg/kg/day were statistically comparable to vehicle control group.
At 15 mg/kg/day, the mean body weights towards the later part of gestation GD 27 and 29 were statistically significantly lower by -5% when compared to the vehicle control group.

At 30 mg/kg/day, the mean body weights and gains towards the later part of gestation GD 27 and 29 were statistically significantly lower by -6 and -7%, and -282%, respectively when compared to the vehicle control group.
Maternal body weight gain corrected for uterine weight was also statistically significantly lower by -5 and -7% at 15 and 30 mg/kg/day, respectively, compared to the vehicle control group. Corrected body weight gain was statistically significantly lower at 15 and 30 mg/kg/day by -206 and -399%, respectively.

The significant decreases seen in the body weights towards the later part of gestation at 15 and 30 mg/kg/day associated with lower food consumption are considered treatment-related changes.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption during the different days of gestation period at 5 mg/kg/day were statistically comparable to vehicle control group except during GD 9-12 and the reduction was statistically significantly lower by -6%.
Food consumption was significantly lower during GDs 9-12, 12-15, 15-18, 21 24 and 27-29 with a reduction of -5 to -17% at 15 mg/kg/day, and GDs 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 with a reduction of -4 to -61% at 30 mg/kg/day compared to vehicle control group.
The overall food consumption was significantly lower during GDs 6-29 and 0 29 at 15 and 30 mg/kg/day, with a reduction of -9 and -7% and -26 and 22%, respectively compared to the vehicle control group.
The significant decreases seen in the body weights towards the later part of gestation at 15 and 30 mg/kg/day associated with lower food consumption are considered treatment-related changes.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological findings observed at necropsy in the vehicle control and test item treated rabbits including aborted rabbit.

Neuropathological findings:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There were incidences of abortion of 2, 1, 1, and 3 at 0, 5, 15 and 30 mg/kg/day.
3 females were prematurely sacrificed because of abortion.

Pre- and post-implantation loss:

effects observed, treatment-related

Total litter losses by resorption:

effects observed, treatment-related

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg/day, significant increase in late resorptions (16.65 vs. 3.08% in vehicle control) resulting in higher post-implantation loss (15.59% vs. 6.39% in vehicle control). One rabbit (RBa8646) had complete resorptions (with all late resorptions). These findings were considered treatment-related.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The maternal parameters comprising mean number of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss, and rabbits with resorptions were statistically comparable between the vehicle control and rabbits treated up to 15 mg/kg/day.
Gross evaluation of the placentas did not reveal any findings in any rabbits at any tested dose levels.

Dose descriptor:

NOAEL

Remarks on result:

other: not determined as only the draft study report was available at submission

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 15 mg/kg/day, a significant reduction in fetal weight was observed in females (-9%) compared to the respective control value.

At 30 mg/kg, the mean fetal weight of males (-17%), females (-17%) and combined sex (-18%) were significantly lower than the vehicle control group. This is considered secondary to lower maternal body weights and food consumption.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test-item-related abnormalities were observed during external observation of the fetuses at any of the doses. There was an incidence of a small fetus (anomaly) in one litter (1/141 at 15 mg/kg/day dose group and fore limb moderate flexion at wrist in one fetus (1/124 in one litter). This was considered an incidental finding and was consistent with our historical control.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test-item-related skeletal malformations observed in fetuses of does treated up to 30 mg/kg/day. All skeletal findings were comparable to the concurrent or historical controls.

Following significant skeletal observations were made:

Significantly higher incidences of extra lumbar centra and arch #8 (44.7%, 63/141 fetuses), 13th rib extra bilateral (22.7%, 32/141) and hypoplastic sternum #5 (11.4%, 16/141 fetuses) were found at 15 mg/kg/day. These observations in rabbit fetuses are considered transient and resolve as the organism matures.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no test-item-related visceral malformations observed in fetuses of doe’s treated up to 30 mg/kg/day. Incidences of liver median lobe extra lobation (variation) at 15 mg/kg/day and hypoplastic gallbladder (anomaly) were observed in test-item-treated groups. These findings were in line with the in-house historical control data and considered spontaneous and not related to the test item treatment.
The number of fetal heads subjected to sectioning was 64, 66, 65, and 59 from 0, 5, 15 and 30 mg/kg/day dose groups, respectively. Fetal heads in all dose groups were normal when subjected to Wilson’s razor-blade sectioning

Details on embryotoxic / teratogenic effects:

The litter parameters comprising total number of fetuses, mean litter size, mean fetal weight, and sex ratio were comparable between the vehicle control group and rabbits treated at 5 mg/kg/day.

At 15 mg/kg/day, a significant reduction in fetal weight was observed in females (-9%) compared to the respective control value.

At 30 mg/kg, the mean fetal weight of males (-17%), females (-17%) and combined sex (-18%) were significantly lower than the vehicle control group. This is considered secondary to lower maternal body weights and food consumption.

Dose descriptor:

NOAEL

Remarks on result:

other: not determined as only the draft study report was available at submission

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, oral administration of Sodium mercaptoacetate at 0, 5, 15 and 30 mg/kg/day to pregnant rabbits during GD 6 to 28 resulted in treatment-related hypoactivity at 30 mg/kg/day. The significantly lower maternal body weights towards the later part of gestation at 15 and 30 mg/kg/day were associated with lower food consumption are considered treatment-related non-adverse changes. At 30 mg/kg/day, the percentage of resorptions were higher, which in turn resulted in higher post-implantation loss. The mean fetal weights of males, females, and combined sex were significantly lower, compared to the vehicle control group and considered secondary to lower maternal body weights and food consumption. Gross pathology evaluation of rabbits revealed no abnormality up to the high dose of 30 mg/kg/day. Fetal external, visceral, and skeletal examination revealed no signs of teratogenicity up to 30 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the developmental and maternal toxicity in pregnant female rabbits and developing embryos/fetuses after oral gavage treatment with test item Thiocare®H102 46% in pregnant rabbits from gestation day (GD) 6 to 28. This study provided a rational basis for risk assessment in humans and helped to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rabbits.

 

The dose levels for this study were selected based on the results of the Dose Range Finding study (DRF) study N6282. In the DRF study, dose levels of 0, 25, 50, 75 and 100 mg/kg/day were evaluated in pregnant rabbits (6 rabbits/group) treated with the test item or vehicle from GD 6 to GD 28 at the dose volume of 2 mL/kg body weight. At 25 mg/kg/day, there was reduction in body weight and food consumption. Doses of 50, 75 and 100 mg/kg/day resulted in unscheduled mortalities. Based on the results of the DRF study in pregnant rabbits, considering treatment related deaths at ≥50 mg/kg/day, a high dose of 30 mg/kg/day was selected for the definitive study. A mid dose of 15 mg/kg/day and a low dose of 5 mg/kg/day were selected to provide a graded response to the test item.

 

In the definitive study, 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred. Test item in vehicle (Milli-Q water) was administered at 5, 15 and 30 mg/kg/day at the dose volume of 2 mL/kg body weight for the low, mid, and high dose groups, respectively. The vehicle control group received the vehicle only.

The following parameters and end points were evaluated in this study: Maternal parameters - clinical signs, body weight, body weight gain, food consumption, gross pathology, gravid uterine weight, number of corpora lutea, implantation sites, early and late resorptions, pre and post implantation loss and fetal parameters (total number of live fetuses, fetal weights, sex, and sex ratio). All the fetuses were examined for external malformations. All the live fetuses were examined for visceral and skeletal variations and malformations.

During the conduct of the experiment, homogeneity and active ingredient analyses were carried out from the dose formulation samples collected prior to initiation of treatment and termination of treatment. The results of formulation analyses were within the acceptable limits.

 

Main findings from the study are summarized below:

 

Mortality, clinical signs, and gross necropsy changes:

There were no death in any group.

The treatment resulted in  clinical signs of hypoactivity in 2/23 rabbits and one rabbit showed posture- sitting with head hung down at 30 mg/kg/day. There were incidences of abortions at all the tested doses (two rabbits in vehicle control group, one each in 5 and 15 mg/kg/day and three rabbits at 30 mg/kg/day). There were no gross pathological findings that could be attributed to the test item.

 

Body weight and food consumption:

No effect was observed at 5 mg/kg/day.

The significantly lower maternal body weight and body weight gains observed during later periods of gestation at 15 and 30 mg/kg/day were associated with lower food consumption and are considered treatment-related changes.

 

Maternal developmental parameters:

No significant changes in any maternal developmental toxicity endpoint were observed at 5 and 15 mg/kg/day. At 30 mg/kg/day, the percentage of late resorptions were higher (16.65 vs. 3.08% in vehicle control), which in turn resulted in higher post-implantation loss (15.59% vs. 6.39% in vehicle control). These findings were considered treatment-related.

No gross pathological changes of the placenta were observed in any of the animals.

 

Litter Parameters:

No effect was observed at 5 and 15 mg/kg/day.

At 30 mg/kg/day, the mean fetal weight of males (-17%), females (-17%) and combined sex (-18%) were significantly lower, compared to the vehicle control group and attributed to secondary to lower maternal body weights and food consumption.

At 15 mg/kg/day, a significant reduction in fetal weight was observed in females (-9%) compared to the respective control value.

Fetal examination: The results from external, visceral, and skeletal examinations did not reveal any significant effects that could be attributed to the test item.

 

Conclusion

In conclusion, oral administration of Thiocare®H102 46% at 0, 5, 15 and 30 mg/kg/day to pregnant rabbits during GD 6 to 28 resulted in treatment-related clinical signs of hypoactivity and posture- sitting with head hung down at 30 mg/kg/day. The significantly lower maternal body weights towards the later part of gestation at 15 and 30 mg/kg/day were associated with lower food consumption are considered treatment-related changes.

Reference 2

Endpoint:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Strain of rats: Hsd/Cpb: WU
Source: Fa. Harlan Winkelmann GmbH, 33178 Borchert
Age: 9-10 weeks old
Acclimatization period: 6 days
Weight range at study initiation: 177-213 g Diet: Altromin Standard Diet TPF N 1234, ad libitum
Water: Tap water, ad libitum

Husbandry:
Housing: Makrolon type III cages
Illumination: artificial lighting from 6.00 a.m. - 6.00 p.m.
Temperature: 21-23.5°C
Relative humidity: 51-66%Vehicle: demineralized water

Route of administration:

oral: gavage

Vehicle:

other: demineralized water

Details on exposure:

- Rate of solution preparation: daily
- Amount of vehicle: 10 ml/kg (base on weight on GD6)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and homogeneity of ammonium thioglycolate in preparations administred were determined once during the study, on the second day of treatment. The results showed values ranging from 96.5 % to 100 % of the nominal concentrations

Details on mating procedure:

- Groups of four sexually matures, virgin females were left over night with one stock stud.
- Proof of pregnancy: sperm in vaginal smear (GD 0)

Duration of treatment / exposure:

gestation days 6 - 19

Frequency of treatment:

daily

Duration of test:

Duration of test: 15 days - dosing GD 6 -19, animals were killed on day 20

Dose / conc.:

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

Remarks:

2.5 mg/kg bw/day as mercaptoacetic acid

Dose / conc.:

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

Remarks:

12.6 mg/kg bw/day as mercaptoacetic acid

Dose / conc.:

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

Remarks:

63.3 mg/kg bw/day as mercaptoacetic acid

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

Clinical signs including mortality and evidence of abortion were checked daily.
Body weight was determined on GD0, 6 and daily until GD20.
Food and water consumption were recorded at designated intervals during pregnancy.
On day 20 of pregnancy, females were killed. The terminal body weights of the dams were recorded. Females were examined macroscopically.

Litter parameters were recorded: number of corpora lutea, implantation sites, early and late resorptions, and dead and live foetuses.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes:

Fetal examinations:

Foetuses were weighed, sexed, and submitted to external examination. About 1/2 of them were examined for skeletal malformations and 1/2 for organ malformations.The foetus sex was determined on the base of measurment of anogenital indice and by inspection of gonads.

Statistics:

yes: Dunnett test or Fischer-Pitman permutation test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

As a reaction to treatment, all rats of the high dose group showed rooting in the bedding material for about 15 minutes after dosing on different days between GD 8 and 19.
Furthermore, Hair loss was observed in 1/25 rat of the low, 6//25rats of the mid and 2/25 rats of the high dose groups. In the mid and high dose groups one rat each had a scabby skin wound. These findings are regarded as being spontaneous and not as treatment related.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two animals in the high dose group died. Theywere found dead on GD 20

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development in the low, mid and high dose groups corresponded to that in the control

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption in the low, mid and high dose groups did not differ significantlyfrom the control

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

The water consumption in the low and mid dose groups did not differ significantly from the control. In the high dose group the water consumption dropped significantly when the females became pregnant. This lasted until GD 9.Since the animals were treated from GD 6 to 19, thisdrop in water consumption is assumed to be coincidental and not treatment related.

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:

no effects observed

Description (incidence and severity):

The uterus weights in the low and mid dose groups were similar to the control, but were slightly and not significantly lower in the high dose group. This finding is assumed to be accicental

Gross pathological findings:

not examined

Description (incidence and severity):

Dams 88 and 89 were found dead on GD 20. The dead bodies were pale and the livers showed lobulation. All other organs were without any pathological findings. There was no indication of maltreatment. Both animals were pregnant. The fetuses weighed 2. 0 to 2.8 gram. One litter was stained and the other cut into transversal sections. The fetuses proved to be normally developed and ossified in accordance with their age and weight. A cause of death could not be established. It must be assumed that the two deaths were treatment related.
There were no abnormal findings in the other dams

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

The uterus weights in the high dose group were slightly, but not significantly lower than in the control or the other groups. This finding is regarded to be accidental. The numbers of corpora lutea, implantations and live foetuses were not affected.
The numbers of dead foetuses, complete, early and late resorptions were not increased. The sex distribution was not affected in any of the groups.

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Remarks on result:

other: 12.6 mg/kg bw/day as mercaptoacetic acid

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The weights of the foetuses in the low, mid, and high dose groups were similar to the control and not affected by treatment. The frequency of all malformations was in a normal range and not increased.

Type of investigation and fndings, number of fetuses affected
Group 1
Asymmetric sternebrae 2-5 in 12111 (dam number/fetus number)
Asymmetric sternebrae 2-5 in 22/13
Group 2
Aplasia of the left testis in 47/5
Ribs bent in the paravertebral line and thickened in 48/8
Group 3
Aplasia of the righ testis in 51/12
Split sternebrae 2-4 in 62/8
Asymmetric sternebrae 2-5 in 66/3
Ribs bent in the paravertebral line and thickened in 68/8
Fetus 73/2 with an additional pair of hind extremities, grown together, originating from the abdominal wall, about 1 mm to the left of the navel. Apart from this, the appearance of the fetus is normal. Evaluation of the additional pair of hind extremities after skeletal staining: 2 rudimentary pelvic bones, growntogether, 2 femurs, tibia and fibula twice, 3 metatarsalia twice.
Group 4
Fetus 97/5 with decreased size of caudal pole of left kidney.
The type and frequency of the malformations listed above were in a normal range and not dose dependently increased.

Skeletal variations
The ossification status of the fetuses in the low, mid, and high dose groups was comparable. There was no indication of treatment related effects.

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: 63.3 mg/kg bw/day as mercaptoacetic acid

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

The doses of 3 and 15 mg ammonium thioglycolate/kg were systemically tolerated by the rats.
At dose level of 75 mg/kg, two dams died on GD 20. Rooting in the bedding material had been seen, but no maternotoxic effects.
The no observed effect level (NOEL) for embryo-fetal toxicity was 75 mg/kg.
No teratogenic effects were observed.

Executive summary:

The potential of ammonium mercaptoacetate to induce developmental toxicity after maternal exposure during the critical period of organogenesis was evaluated in rat according to OECD Guideline N° 414 (22nd January 2001) and in compliance with Good Laboratory Practices.

Ammonium mercaptoacetate was administered orally by gavage to four groups of 25 bred female Sprague-Dawley rats once daily from gestation days 6 through 19. Dosage levels were 0, 3, 15, and 75 mg/kg bw/d ( 2.5, 12.6 and 63.3 mg/kg bw/day as mercaptoacetic acid).

The fetuses were delivered by caesarean section on GD 20 and examined for macroscopic malformations. About 1/2 of them were examined for skeletal malformations and 1/2 for organ malformations. Clinical signs including mortality and evidence of abortion were checked daily. Body weight was determined on GD0, 6 and the daily until GD20. Food and water consumption was recorded at designated intervals during pregnancy. On day 20 of pregnancy, females were killed. The terminal body weights of the dams were recorded. The gravid uterus was weighed and foetuses removed by hysterectormy. Females were examined macroscopically.

The doses of 3 and 15 mg ammonium thioglycolate/kg were systemically tolerated by the rats. All animals in the low and medium level dose group survived to the scheduled necropsy. At the dose level of 75 mg/kg, two dams died on GD20, one day after the last administration of test material. These death were considered treatment related. At this dose level, rooting in the bedding material had been seen in all rats, but no maternotoxic effects. No treatment related internal findings were observed at any dose level. No clinical signs that could be attributed to ammonium mercaptoacetate were observed in the treated groups. Body weight and food consumption were not affected by treatment. Intrauterine growth and survival were unaffected by ammonium mercaptoacetate administration at all dose levels. Any significant or relevant foetal external, soft tissue and skeletal malformations were observed at any dose level. The developmental variations expressed in the treated groups were generally similar to those present in the control group or occurred in a manner that was not dose related. No teratogenic effects were observed

Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 15 mg/kg/d (12.6 mg/kg bw/day as mercaptoacetic acid) for maternal toxicity. A NOAEL has been determined at 75 mg/kg/d (63.3 mg/kg bw/day as mercaptoacetic acid) for embryo-foetal toxicity.

Reference 3

Endpoint:

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Test animals:         
- Source: Charles River Laboratories (Raleigh, NC).        
- Age: no data         
- Weight at dosing: ca. 250 g        
-  Acclimation period: no data        
- Diet: Purina Certified Rodent Chow (No. 5002), ad libitum        
-  Water: Tap water, ad libitum         
- Housing: singly housed in solid-bottom  polycarbonate cages with stainless steel wire lids with Sani-Chips certified hardwood bedding

Environmental conditions:         
- Temperature: 70.0-72.81 F         
- Humidity: 28.0-58 %         
- Air changes: no data         
- Photoperiod: Alternating 12-hour light and dark cycles

Route of administration:

dermal

Vehicle:

other: 95% ethanol:distilled water, 1:1

Details on exposure:

Treatment: 1.5 ml/kg of test material in vehicle or vehicle alone was applied  directly to a 3x3 inch shaved area on the dorsum of the rat with a glass  syringe fitted with a 16-gauge, stainless-steel, ball-tipped dosing  needle. The formulation was allowed to remain on the animal for a minimum  of 6 hr/treatment day. At the end of each 6-hr period, the application  site was wiped with warmwater-soaked gauze to remove residual vehicle or  sodium thioglycolate.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prepared weekly based on stability information and were verified to be  within 92.7-103.5 % of target.

Duration of treatment / exposure:

gestation days 6-19

Frequency of treatment:

daily

Duration of test:

necropsy on GD 20

Dose / conc.:

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

Remarks:

40.3 mg/kg bw/d as mercaptoacetic acid

Dose / conc.:

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

Remarks:

80.7 mg/kg bw/d as mercaptoacetic acid

Dose / conc.:

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

Remarks:

161.4 mg/kg bw/d as mercaptoacetic acid

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Range-finding study:
Dose selection in rats was based on a 14-day pilot dermal toxicity study (NTP, 1999) on nonpregnant CD rats, five per group and four groups (0, 6, 60, 600 mg/kg/day), for a preliminary estimate of appropriate dose range and duration. In that study, there was one treatment-related death at 600 mg/kg/day on the last day of administration (Day 14) and no other effects at that dose or at either of the other doses. Because deaths occurred late in the oral dosing period of ammonium thioglycolate in Wistar rats ( Gleich, 1998) and in this pilot dermal study, the possibility existed that prolonged exposure was necessary to assess toxicity. Therefore, a second range-finding study (NTP, 2000) was carried out in pregnant rats (13 per group) with two exposure durations: GD 1-19 (for high dose and vehicle control) and GD 6-19 (for all four doses and vehicle control). The doses selected were 0, 50, 250, 350, and 525 mg/kg/day. Pregnant CD (Sprague-Dawley) rats were topically exposed to sodium thioglycolate in vehicle (95% ethanol:distilled water mixed 1:1), unoccluded, for 6 hr/day and monitored at regular intervals throughout gestation for clinical signs, food and water intake, and body weight. At necropsy of surviving dams on gd 20, maternal clinical condition (body, liver, and gravid uterine weights) and pregnancy status were recorded. The number of resorbed, dead, or live fetuses was also recorded. Live fetuses were weighed, sexed, and examined for external morphological abnormalities, including cleft palate.
Sodium thioglycolate was either lethal or produced severe maternal morbidity in a dose­related manner. Dams manifested lethality and morbidity soon after the onset of maternal exposure to 350 and 525 mg/kg/day in the gd 6-19 exposure group and at 525 mg/kg/day in the gd 1-19 exposure group. All surviving dams in these groups (and in the gd 1-19 control group) were terminated by gd 12 for humane reasons. At the lower doses, one dam died on gd 10 after four days exposure to 250 mg/kg/day. No deaths or severe toxicity occurred in the 50 mg/kg/day group.
The severity and incidence of nonlethal effects were also dose and exposure dependent. Effects included body weight reduction, intermittent changes in food and water intake, skin irritation, reduced activity, and piloerection. Maternal body weight and body weight change were decreased at >= 250 mg/kg/day, whereas at 50 mg/kg/day, they were similar to the control values.
At scheduled necropsy, relative liver weight (% sacrifice weight) was significantly increased at 250 mg/kg/day. The gravid uterine weight at 250 mg/kg/day was approximately 42% lower than the control value.
Prenatal viability and embryo/fetal growth were reduced at 250 mg/kg/day, whereas there were no treatment-related effects on external fetal morphology. Three litters at 250 mg/kg/day were fully resorbed (versus none at 0 or 50 mg/kg/day). Fetal body weights from dams exposed to 250 mg/kg/day were lower (p< 0.05) relative to the control group values.
In summary, maternal toxicity was evident at 250 mg/kg/day (expressed as reduced body weights, weight gain, and feed consumption, and one death). Above 250 mg/kg/day, the dams manifested severe morbidity and death. Developmental toxicity at 250 mg/kg/day was expressed as significantly reduced fetal body weights ( for all fetuses and male fetuses but not for female fetuses), and slightly reduced live litter size, increased resorptions, and three fully resorbed litters, but no evidence of fetal treat­ment-related external malformations or variations.

Maternal examinations:

Females were monitored at regular intervals throughout gestation for clinical  signs (including dosing site condition), feed and water intake, and body  weight. At necropsy on gd 20, the following were recorded: maternal  clinical condition; body, liver and gravid uterine weights; and pregnancy  status. 

Ovaries and uterine content:

The number of ovarian corpora lutea and uterine implantations (resorbed,  dead, or live fetuses) was recorded.

Fetal examinations:

All live foetuses were counted,  weighed, and examined for external alterations, including cleft palate. Approximately 50% of the live foetuses per litter were sexed internally  and examined for visceral alterations. These foetuses were decapitated  and the heads fixed, decalcified, and examined for soft tissue  craniofacial alterations. All foetal carcasses were eviscerated (and  foetuses not scheduled for a visceral examination were sexed internally),  macerated, and stained with alizarin red S and alcian blue. Intact  foetuses (those not decapitated) were examined for ossified and  cartilaginous skeletal alterations.

Statistics:

The unit for statistical measurement was the pregnant female or the  litter. Quantitative continuous data (e.g., maternal body weights, foetal  body weights, feed consumption, etc.) were compared among treatment  groups, per species, by parametric statistical tests whenever Bartlett  test for homogeneity of variance was not significant.  General linear models (GLM) procedures were applied to the ANOVA and the  tests for linear trend. Before GLM analysis, an arc sine-square root  transformation was carried out on all litter-derived percentage data  (e.g., % resorptions/litter, % malformations/litter, % variations/litter)  (Snedecor and Cochran, 1967). For litter-derived percentage data, the  ANOVA was weighted according to litter size. When a significant (p <  0.05) main effect for dose occurred, Dunnett's multiple comparison test  (Dunnett, 1955, 1964) was used to compare each treatment group to the  control group for that measure. A one-tailed (i.e., Dunnett's test) was used for all pairwise comparisons  to the vehicle control group, except that a two-tailed test was used for maternal body and organ weight  parameters, maternal feed consumption, fetal body weight, and percent  males per litter.

Clinical signs:

not specified

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Clinical signs appeared by 24 hr after the first treatment, predominantly as alterations at the dosing site. For instance, patches of discoloration were observed at the dosing site, which were often scaly in appearance.
Also, increased incidences of very slight edema and erythema were observed in the high dose group. Effects did not worsen over time after multiple daily applications.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female in the high dose group was found dead on the morning of GD 18. Necropsy findings included blood at vaginal opening, resorbed conceptuses, pale liver, and an ulceration of the stomach.
At termination (GD 20), there were 21–25 litters per group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weights were significantly reduced in the 200 mg/kg/day group from GD 9 through termination, as were weight changes during treatment and gestation, and gestational weight change corrected for gravid uterine weight.
Body weights and weight changes showed dose-related decreasing trends from GD 6 through sacrifice (also during pretreatment, GD 0–6), as well as for the treatment period, gestation, and for corrected weight gains. Maternal body weight change was significantly reduced at the high dose for GD
6–9 (data not shown), with a significant decreasing trend across dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For the entire dosing period (GD 6–20), maternal relative feed consumption (g of feed/kg of body weight/day) was significantly increased at 50 (5.4%) and 100 (6.1%) mg/kg/day and slightly (but not statistically significantly) increased at 200 (2.2%) mg/kg/day.
Maternal relative feed consumption was also significantly increased at 50 and 100 mg/kg/day for GD 9–12, 12–15, and 15–18; at100 mg/kg/day for GD 18–19 and 19–20; and also at 200 mg/kg/day for GD 15 through 18.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption in gm/kg/day showed increasing trends from GD 12–20, with significant increases occurring only in high dose group. For the entire dosing period (GD 6–20), relative maternal water consumption (gm/kg/day) was increased (but not statistically significantly) at 50 (3.2%) and 100 (5.1%) mg/kg/day and was significantly increased (12.7%) at 200 mg/kg/day.

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:

no effects observed

Description (incidence and severity):

Maternal organ weights indicated no treatment-related differences

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

There was an apparent increase in the incidence of resorptions at the high dose. Relative to the control value, the value of the high dose group was higher by nearly two-fold, but not statistically significantly different, with a large variance due to one dam (No. 31) with 100% prenatal loss (her uterus contained one implant site and no live fetuses).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

body weight and weight gain

mortality

Remarks on result:

other: 80.7 mg/kg bw/d as mercaptoacetic acid

Abnormalities:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The fetal body weights per litter were significantly reduced at the high dose for sexes separately (8.3% reduction for males, 7.1% for females) and for genders combined (data not shown). The fetal body weights per litter of the intermediate dose groups were unaffected.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Prenatal viability was unaffected by maternal exposure to sodium  thioglycolate. The incidences of foetal external, visceral, and skeletal  alterations were also unaffected. A significant, dose-related, upward  trend was present for % foetuses with skeletal variations per litter, but  there were no significant pairwise comparisons to the vehicle control  group value. The historical data presented in the study report shown a  range of 0.58 to 25.65 % for this parameter, with 9 out of 21 studies  (43%) with a % higher than 15.1% (the highest value observed at 200 mg/kg  bw/d). Accordingly, this effect was not considered as treatment-related.  Body weights of male and female foetuses per litter at 200 mg/kg/day were  significantly lower than control values.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

80.7 mg/kg bw/d as mercaptoacetic acid

Abnormalities:

no effects observed

Developmental effects observed:

no

Maternal Toxicity in CDs Rats Exposed to Sodium Thioglycolate by Topical Application on Gestational Days 6 Through 19

                                           

	Sodium thioglycolate (mg/kg/day, dermal)
	0	50	100	200
Maternal pregnancy status
Treated, n	25	25	25	25
Removed, n	0	0	0	0
Dead or euthanized, n	0	0	0	1a
Pregnant at sacrifice, n(%)	25(100)	23(92)	23(92)	21(88)b
Maternal body weight changes (g)c,d
Pretreatment wt. gain (GD 0–6)	32.2±1.3h	29.21±1	28.4±2.1	26.5±1.6
Gestation weight gain (GD 0–20)	135.6±3.3i	129.7±4.4	127.0±5.0	112.5±5.9k
Treatment weight gain (GD 6–20)	110.1±3.1i	106.5±4.0	104.8±3.8	92.7±5.7j
Corrected weight gaine	49.6±1.8i	45.6±2.4	43.9±3.3	34.8±2.8k
Gravid uterine wt.	86.0±3.2	84.1±3.1	83.0±3.1	77.6±4.9
Maternal organ weightsc,f
Liver Absolute (g)	17.78±0.23	17.64±0.40	17.69v0.29	17.02±0.27
Relative (% sacrifice wt.)f	4.53±0.04	4.56±0.07	4.62±0.05	4.65±0.08
Kidney Absolute (g)	2.22±0.04	2.19±0.05	2.26±0.05	2.20±0.04
Relative (% sacrifice wt.)f	0.57±0.01h	0.57±0.01	0.59±0.01	0.60±70.01
Maternal feed consumptionc,g
Pretreatment period (gd 0–6)
Absolute (g/day)	23.4±0.5	23.2±0.6	22.6±0.6	22.5±0.4
Relative (g/kg/day)	85.0±1.4	84.6±1.7	83.3±2.0	83.6±1.3
Treatment period (GD 6–20)
Absolute (g/day)	26.3±0.3	27.5±0.4	27.3±0.5	25.5±0.4
Relative (g/kg/day)	77.1±0.5	81.3±0.9k	81.8±0.8k	78.8±0.8
Maternal water consumptionc,g
Pretreatment period (gd 0–6)
Absolute (g/day)	35.7±1.2	34.2±0.9	32.4±1.1	34.1±0.9
Relative (g/kg/day)	129.8v4.3	125.2v2.9	119.0±3.5	126.9±3.1
Treatment period (gd 6–20)
Absolute (g/day)	43.6±1.3	44.1±1.3	44.4±1.1	46.2±1.2
Relative (g/kg/day)	127.1±3.2i	131.2±3.3	133.6±2.6	143.3v3.9k

aDam 4 was found dead on the morning of gd 18.

bDam 31 had only one implantation site, and no live fetuses on gd 20.

cIncludes all dams pregnant at sacrifice; mean 7 SEM; gd, gestational day.

dBody weights were recorded in the morning of each designated gestational day.

eWeight change during gestation minus gravid uterine weight.

fCalculated using body weight at the time of sacrifice on gd 20.

gRelative feed and water consumption (g/kg/day) were calculated using appropriate body weights.

hp<0.05; Test for linear trend.

ip<0.01; Test for linear trend.

jp<0.05; Dunnett’s test

k p<0.01; Dunnett’s test.

Developmental Toxicity in CDsRat Fetuses Following Maternal Exposure to Sodium Thioglycolate by Topical. Application on Gestational Days 6 Through 19

	Sodium Thioglycolate (mg/kg/day, dermal)
	0	50	100	200
All littersa,b	25	23	23	21
No. corpora lutea/dam	17.5±0.4	16.7±0.5	16.4±0.5	16.8±0.7
No. implantation sites/litter	15.6±0.5	15.1±0.6	14.7±0.7	15.4±v0.8
% preimplantation loss/litter	11.2±2.7	10.3±2.4	11.6±3.6	10.5±4.1
% resorptions/litter	3.9±1.3	4.6±1.4	3.0±0.9	11.2±5.1
% litters with resorptions	40	48	35	52
% late fetal deaths/litter	0.0±0.0	0.3±0.3	0.0±0.0	0.0±0.0
% litters with late fetal deaths	0	4	0	0
Live littersb,c	25	23	23	20d
No. live fetuses/litter	15.0±0.6	14.4±0.6	14.2±0.6	15.1±0.6
Avg. male fetal body wt./litter (g)	3.73±0.06f	3.70±0.05	3.75±0.08	3.42±0.08g
Avg. female fetal body wt./litter (g)	3.50±0.05e	3.56±0.05	3.61±0.10	3.25± 0.08g
% male fetuses/litter	45.173.6	52.373.5	50.673.2	52.5±2.3
% externally malformed fetuses/litter	0.0±0.0	0.0±0.0	0.9±0.7	0.3±0.3
% viscerally malformed fetuses/litter	0.0±0.0	0.5±0.5	0.0±0.0	2.2±1.7
% skeletally malformed fetuses/litter	2.3±1.8	2.2±1.0	1.2±0.8	4.5±2.6
% malformed fetuses/litter	1.1±0.9	1.4±0.6	1.5±0.7	3.4±2.2
% fetuses with external variations/litter	0.5±0.5	0.5±0.4	0.0±0.0	0.3±0.3
% fetuses with visceral variations/litter	3.3±1.3	5.0±1.5	3.7±1.7	3.7±2.2
% fetuses with skeletal variations/litter	3.9±1.3f	6.4±2.3	11.2±3.0	15.1±4.2
% fetuses with variations/litter	4.1±1.0	6.3±1.4	7.3±1.7	9.9±2.6

aIncludes all dams pregnant at sacrifice; litter size¼no. implantation sites per dam.

bReported as the mean 7 SEM.

cIncludes only dams with live fetuses; litter size¼no. live fetuses per dam.

dOne female at 200 mg/kg/day had a fully resorbed litter at scheduled necropsy.

ep<0.05; Test for linear trend.

fp<0.01; Test for linear trend.

gp<0.05; Dunnett’s test.

Conclusions:

Topically applied sodium thioglycolate resulted in toxicity to the dam at 200 mg/kg/day (expressed as reduced body weights and weight gain, increased relative water consumption, observations at the dosing site and one death). Sodium thioglycolate at 200 mg/kg/day also resulted in significantly decreased male and female foetal body weights per litter, with no other evidence of embryofoetal toxicity and no evidence of treatment-related teratogenicity. Based on the study findings, a no observed adverse effect level (NOAEL) could be identified at 100 mg/kg/day (80.7 mg/kg bw/d as mercaptoacetic acid) for maternal and developmental toxicity.

Executive summary:

The developmental toxicity of sodium thioglycolate was evaluated in pregnant rats in a study performed according to a method comparable to the OECD guideline # 414. Pregnant Sprague-Dawley rats were exposed topically, 6 hr/day, to sodium thioglycolate (99% pure) in vehicle (95% ethanol:distilled water, 1:1) from gestational day (gd) 6 through 19 at dose levels of 0, 50, 100, and 200 mg/kg bw/d. Twenty-five timed-mated female rats were assigned to each group and monitored at regular intervals throughout gestation for clinical signs (including dosing site condition), feed and water intake, and body weight. At necropsy on gestational day 20; the following were recorded: maternal clinical condition; body, liver and gravid uterine weights; and pregnancy status. The number of ovarian corpora lutea and uterine implantations (resorbed, dead, or live foetuses) was recorded. All live foetuses were counted, weighed, and examined for external alterations, including cleft palate. Approximately 50% of the live rat foetuses per litter were sexed internally and examined for visceral alterations. These foetuses were decapitated and the heads fixed, decalcified, and examined for soft tissue craniofacial alterations. All foetal carcasses were eviscerated (and rat foetuses not scheduled for a visceral examination were sexed internally), macerated, and stained with alizarin red S and alcian blue. Intact rat foetuses (those not decapitated) were examined for ossified and cartilaginous skeletal alterations.

Sodium thioglycolate treatment was associated with one maternal death at 200 mg/kg/d (only clinical observations at the dosing site preceded death). Other effects, dependent on dose and exposure duration, included body weight and weight gain reductions, changes in relative feed and water intake, and discoloration and slight erythema at the application site. Feed consumption was significantly increased above the control at 50 (5.4%) and 100 (6.1%) mg/kg bw/d, but not at 200 mg/kg bw/d (2.2%). Maternal water consumption was significantly increased at 200 mg/kg bw/d (12.7%) and slightly (but not statistically significantly) increased at 50 (3.2%) and 100 (5.1%) mg/kg bw/d. Maternal body weights and body weight changes were decreased only at 200 mg/kg bw/d. At scheduled necropsy, there were no macroscopic indications of organ toxicity. In addition, organ weights and gravid uterine weights were equivalent across groups; maternal body weight was significantly reduced at 200 mg/kg/d. Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. A significant, dose-related, upward trend was present for % foetuses with skeletal variations per litter, but there were no significant pairwise comparisons to the vehicle control group value and the % were in the range of the historical control data . Body weights of male and female foetuses per litter at 200 mg/kg bw/d were significantly lower than control values. Based on the study findings, a no observed adverse effect level (NOAEL) could be identified at 100 mg/kg/day (80.7 mg/kg bw/d as mercaptoacetic acid) for maternal and developmental toxicity.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test animals:        
- Source: Covance Research Products (Denver, PA)         
- Age: no data         
- Body weight at GD 0: 2.-876- 4.327 kg
- Acclimatation period : 3 days         
- Diet: Purina Certified Rabbit Chow (No. 5322), ad libitum         
- Water: Tap water, ad libitum        
- Housing: singly housed in stainless-steel cages with mesh flooring.         

Environmental conditions:        
- Temperature: 64.1-74.41F         
- Humidity: 41.6-86.5%         
- Air changes: no data         
- Photoperiod: Alternating 12-hour light and dark cycles

Route of administration:

dermal

Vehicle:

other: 95%  ethanol:distilled water, 1:1

Details on exposure:

0.5 ml/kg of test material in vehicle or vehicle alone was applied  directly to a 3x3 inch shaved area on the dorsum of the rat with a glass  syringe fitted with a 16-gauge, stainless-steel, ball-tipped dosing  needle. The formulation was allowed to remain on the animal for a minimum  of 6 hr/treatment day. At the end of each 6-hr period, the application  site was wiped with warmwater-soaked gauze to remove residual vehicle or  sodium thioglycolate.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prepared weekly based on stability information and were verified to be  within 85.1-111.7% of target.

Duration of treatment / exposure:

gestation days 6-29

Frequency of treatment:

daily

Duration of test:

necropsy on GD 30

Dose / conc.:

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

Remarks:

8.1 mg/kg bw/d as mercaptoacetic acid

Dose / conc.:

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

Remarks:

12.1 mg/kg bw/d as mercaptoacetic acid

Dose / conc.:

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

Remarks:

20.2 mg/kg bw/d as mercaptoacetic acid

Dose / conc.:

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

Remarks:

52.5 mg/kg bw/d as mercaptoacetic acid

No. of animals per sex per dose:

24 (2 replicates of 12)

Control animals:

yes, concurrent vehicle

Details on study design:

Range-finding study:
Dose selection was based on a topical developmental toxicity screen in pregnant rabbits (NTP, 2000). Naturally mated female New Zealand White rabbits (8/group) were dosed topically with sodium thioglycolate at 25, 50, 100, 150, and 175 mg/kg/day or vehicle control (ethanol 95%: distilled water, 1: 1) for a minimum of 6 hours per day on gestational days (gd) 6 through 29. Dose volume was 0.5 ml/kg. Eight time-mated rabbits were assigned to each group and monitored throughout gestation for clinical signs, food intake, and body weight. At necropsy (gd 30), the following were recorded: maternal clinical condition; body, liver, paired kidney and gravid uterine weights; and pregnancy status. The number of resorbed, dead, or live fetuses was also recorded. Live fetuses were weighed and examined for external morphological abnormalities, including cleft palate.
At 100, 150 and 175 mg/kg/day, excessive maternal toxicity (morbidity and mortality) resulted in termination of surviving does in these groups by gd 15. No maternal deaths or severe toxicity occurred at 25 or 50 mg/kg/day. There was a significant reduction in body weight gain for gd 6-9 for the 150 and 175 mg/kg/day groups, but no reduced body weight or weight gain during gestation (gd 0 to 30), treatment or for any other time interval for does in the 25 and 50 mg/kg/day groups. Feed consumption was not affected by treatment. Edema and erythema at the dosing site were seen in all treatment groups until sacrifice with dose-related increases in incidence and severity. Moderate to severe erythema was seen at >= 50 mg/kg/day. Other signs of toxicity included clinical weight loss (defined as >=150 g in any weigh period) and lethargy at 100-175 mg/kg/day. There were no effects of treatment at scheduled necropsy for body weight, gravid uterine weight, and liver or kidney weights at 25 or 50 mg/kg/day. Prenatal viability and embryo/fetal growth were not statistically affected. However, the number of live fetuses per litter showed a significant downward trend across dose groups. Percent nonlive implants per litter increased from 1.1 % in the controls to 25.3% at 50 mg/kg/day. However, this parameter was not statistically significant (due to small sample sizes and large variance). There was no effect of treatment on fetal external morphology.
Based on the maternal mortality and morbidity, the maximal tolerated dose for the main developmental toxicity study was between 50 and 100 mg/kg/day.

That study indicated that sodium thioglycolate was maternally lethal (>=50% of the does) at 100, 150, and 175 mg/kg/day, but not adverse at 50 mg/kg/day. According to the OECD TG 414, the highest dose should be chosen with the aim to induce some developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering. Because of the steep dose-response curve in the screening study, a dose of 65 mg/kg/day was chosen as the top dose and anticipated to result in moderate maternal and developmental toxicity. If excessive toxicity occurred at 65 mg/kg/day, 50 mg/kg/day was also chosen to be available as a potential high dose for the second replicate, if necessary. Doses for the 30 and 50 mg/kg/day groups, however, were inadvertently formulated as 15 and 25 mg/kg/day (i.e., 50% of the original target doses) for the first replicate. Because of the dose-related increase in erythema and oedema at the dosing site seen at these lower doses, the second replicate also employed doses of 0, 10, 15, 25, and 65 mg/kg/day.

Maternal examinations:

Twelve naturally mated females were assigned to each group in a two replicate design for a total of twenty-four does/group and monitored at regular intervals throughout gestation for clinical signs (including  dosing site condition), feed intake, and body weight.
At necropsy on  gestational day 30,  maternal clinical  condition; body, liver, kidney (paired) weights were recorded.

Ovaries and uterine content:

At necropsy on  gestational day 30, gravid uterine weights; and  pregnancy status were recorded:. The number of ovarian corpora lutea and uterine  implantations (resorbed, dead, or live foetuses) was recorded. 

Fetal examinations:

All live  foetuses were counted, weighed, and examined for external alterations,  including cleft palate. One hundred percent of the live foetuses per  litter were sexed internally and examined for visceral alterations.  Approximately 50% of the foetuses were decapitated and the heads fixed,  decalcified, and examined for soft tissue craniofacial alterations.  All  foetal carcasses were eviscerated, macerated, and stained with alizarin  red S and alcian blue. All foetuses were examined for ossified and  cartilaginous skeletal alterations.

Statistics:

The unit for statistical measurement was the pregnant female or the  litter. Quantitative continuous data (e.g., maternal body weights, foetal  body weights, feed consumption, etc.) were compared among treatment  groups, per species, by parametric statistical tests whenever Bartlett  test for homogeneity of variance was not significant.  General linear models (GLM) procedures were applied to the ANOVA and the  tests for linear trend. Before GLM analysis, an arc sine-square root  transformation was carried out on all litter-derived percentage data  (e.g., % resorptions/litter, % malformations/litter, % variations/litter)  (Snedecor and Cochran, 1967). For litter-derived percentage data, the  ANOVA was weighted according to litter size. When a significant (p <  0.05) main effect for dose occurred, Dunnett's multiple comparison test  (Dunnett, 1955, 1964) was used to compare each treatment group to the  control group for that measure. A one-tailed (i.e., Dunnett's test) was used for all pairwise comparisons  to the vehicle control group, except that a two-tailed test was used for  maternal body and organ weight parameters, maternal feed consumption,  fetal body weight, and percent males per litter. Data for any measure that showed a significant (p < 0.05) dose X  replicate interaction in a two-way (dose X replicate) ANOVA were further  evaluated for dose effects within each replicate using a one-way ANOVA,  test for linear trend, and Dunnett's test. The data are presented pooled  across the two replicates for the rabbit. All nominal scale measures were analyzed by w2 for Independence for  differences among treatment groups (Snedecor and Cochran, 1967) and by  the Cochran-Armitage test for linear trend on proportions (Cochran, 1954;  Armitage, 1955; Agresti, 1990). The alpha level for each statistical  comparison was 0.05, and the significance levels for trend tests and  pairwise comparisons were reported as p < 0.05 or p < 0.01. necropsy.

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical observations were almost exclusively limited to effects of treatment on the dosing site.
Soft feces and diarrhea were observed in few animals without any relationship to treatrnent as was alopecia late in gestation when the females began pulling their fur in preparation for delivery

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Very slight erythema in few females during the first two days of dosing at greater than or equal to 15 mg/kg per day occurred and progressed to include does at 10 mg/kg per day after 4 days of dosing. Moderate to severe erythema and oedema occurred at doses greater than or equal to 15 mg/kg per day and oedema occurred in all dose groups by gestational day 16. Petechia at the dosing site and dry skin also was noted in the high dose group as early as gestational day 9 but was also seen in one doe at 25 mg/kg per day.

Skin related clinical signs at the dosing site appeared by the afternoon of the first day of dosing (gd 6) for does in the 15, 25 and 65 mg/kg/day groups (3-5 does in each group). On gd 8 all groups exhibited very slight erythema with well defined to severe erythema occurring in the 25 and 65 mg/kg/day groups. By gd 9. petechia (pin-point subcutaneous hemorrhages) were seen in 50% of the does and edema in 10/24 does in the high dose group with dry skin beginning to occur. This dose response for skin reactions at the dosing site continued throughout the remainder of the dosing period with the incidence of erythema and the degree of erythema becoming progressively more severe. The majority of does at doses >= 10 mg/kg/day exhibited very slight erythema on gd days 8-18 but beginning on day 19 the majority of does in all dose groups exhibited well-defined to moderate to severe erythema throughout the remainder of the dosing period. Dry/flaky skin was seen at all doses with brown patches occurring only at 65 mg/kg/day at the dosing site. One control doe exhibited very slight erythema on gd 19-25 and slight edema on gd 25-30.

Mortality:

no mortality observed

Description (incidence):

One female in the 25 mg/kg per day group delivered early and one doe in the 65 mg/kg per day group were removed due to a pre-existing condition (trichobezoar or hairball in the stomach).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight and body weight gain were equivalent across dose groups for all intervals measured except for body weight gain for gestational days 12-15. There was a trend for decreased body weight gain for this interval, which was significant in the high dose group when compared to controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Absolute feed consumption per rabbit (grams per day) was significantly reduced from gestational days 9-12 in the 10 mg/kg per day group (Replicate II only) and for gestational days 15-18 at 65 mg/kg per day (Replicate II). Relative feed consumption (grams per kilogram per day) was significantly decreased for gestational days 6-9 (65 mg/kg per day, Replicate II), gestational days 9-12 (10 and 65 mg/kg per day, Replicate II), gestational day 12-15 at 15 mg/kg per day (Replicate I), gestational days 15-18 for 10 and 65 mg/kg per day (Replicate II), and 18-21 at 25 mg/kg per day (Replicate II). Maternal feed consumption in grams per day as well as in grams per kg per day for gestational days 29-30 was significantly reduced at 15 mg/kg per day for Replicate I only. Therefore, there were no consistent treatment related reductions in feed consumption.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absoiute weights of the gravid uterus, liver and paired kidneys were similar across the groups. Also, no differences were identified in the relative weights of the liver and kidneys calculated as % of the sacrifice weigh

Gross pathological findings:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 65 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

52.5 mg/kg bw/d as mercaptoacetic acid

Abnormalities:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 65 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

52.5 mg/kg bw/d as mercaptoacetic acid

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Topically applied sodium thioglycolate resulted in toxicity to the doe at all dose groups but only at the dosing site; no systemic toxicity was seen at any dose group. There was no evidence of embryo/foetal toxicity in any dose group or treatment related teratogenicity. The maternal NOAEL for systemic toxicity was at or above 65 mg/kg/day; for localized toxicity at the dosing site, it was below 10 mg/kg/day. The NOAEL for developmental toxicity was >65 mg/kg/day (52.5 mg/kg bw/d as mercaptoacetic acid).

Executive summary:

The developmental toxicity of sodium thioglycolate was evaluated in pregnant rabbits in a study performed according to a method comparable to the OECD guideline # 414. Pregnant New Zealand White Rabbits were exposed topically, 6 hr/day, to sodium thioglycolate (99% pure) in vehicle (95% ethanol:distilled water, 1:1) from gestational day (GD) 6 through 29 at dose levels of 0, 10, 15, 25 or 65 mg/kg bw/d (equivalent to 0, 8.1, 12.1, 20.2 and 52.5 mg/kg bw/d as mercaptoacetic acid). The dose levels were selected on the basis of a range finding study showing maternal mortality at dose levels >= 100 mg/kg/day. Twenty-four naturally mated female rabbits (in two replicates of 12) were assigned to each group andmonitored at regular intervals throughout gestation for clinical signs (including dosing site condition), feed and water intake, and body weight. At necropsy on gestational day 30; the following were recorded: maternal clinical condition; body, liver, kidney and gravid uterine weights; and pregnancy status. The number of ovarian corpora lutea and uterine implantations (resorbed, dead, or live foetuses) was recorded. All live foetuses were counted, weighed, and examined for external alterations, including cleft palate. Heads fixed, decalcified, and examined for soft tissue craniofacial alterations. All foetal carcasses were eviscerated (and rat foetuses not scheduled for a visceral examination were sexed internally), macerated, and stained with alizarin red S and alcian blue. Intact rat foetuses (those not decapitated) andall rabbit foetuseswere examined for ossified and cartilaginous skeletal alterations.

No maternal deaths were associated with sodium thioglycolate treatment. One female in the 25 mg/kg bw/d group delivered early and one doe in the 65 mg/kg bw/d group was removed due to a preexisting condition (trichobezoar or hairball in the stomach). Clinical observations were almost exclusively limited to effects of treatmentat the dosing site (skin erythema) in all groups. Maternal body weight and body weight gain were equivalent across dose groups for all intervals measured except for body weight gain for gestational days 12-15. There was a trend for decreased body weight gain for this interval, which was significant in the high dose group when compared to controls. There were no consistent treatment related reductions in feed consumption. At scheduled necropsy, there was no effect of treatment on terminal maternal body weight. In addition, organ weights and gravid uterine weights were equivalent across groups. Prenatal viability was unaffected by maternal exposure to sodium thioglycolate. The incidences of foetal external, visceral, and skeletal alterations were also unaffected. Body weights of male and female foetuses per litter and percent males and females per litter were equivalent across dose groups. The maternal NOAEL for systemic toxicity was at or above 65 mg/kg bw/d (52.5 mg/kg bw/d as mercaptoacetic acid); for local toxicity at the dosing site, it was below 10 mg/kg bw/. The NOAEL for developmental toxicity was at or above 65 mg/kg bw/d (52.5 mg/kg bw/d as mercaptoacetic acid).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

52.5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

63.3 mg/kg bw/day

Study duration:

subacute

Species:

rat

Toxicity to reproduction: other studies

Description of key information

In 13-week dermal subchronic toxicity studies in F344 rats and B6C3F1 mice with sodium mercaptoacetate, no treatment-related effects on sperm density and motility, caudal epididymal sperm, spermatid head counts in the testes and testis weights, as well as oestrous cycles, were observed up to dose levels of 180 and 360 mg/kg bw/d, respectively.

Additional information

At the end of the sub-chronic (90-day) toxicity studies of sodium mercaptoacetate administered by cutaneous application to F344/N rats and B₆C₃F₁mice (as reported in the "repeated dose toxicity" section), a special sperm morphology and vaginal cytology evaluation (SMVCE) was performed (Wolfe and Seung, 2003). Sperm density and motility, caudal epididymal sperm, spermatid head counts in the testes and testis weights, as well as of vaginal cytology, to allow examination of oestrous cycles, were examined in rats and mice for the controls and three top dose groups.

No differences were observed between the control and treated groups in the female oestrous cycles, in length of cycle, stages, number of females cycling and number of females with regular cycles in either rats or mice. No differences were observed between controls and treated males in sperm parameters or in testicular spermatid counts, or testes and epididymal weights in either rats or mice. However, in mice, the number of sperm per mg cauda, and per total cauda were decreased by 16% and 24% respectively at 180 mg/kg bw/d, and by 26% and 20% at 360 mg/kg bw/d but the reduction was not statistically significant (p<0.11). As the histopathological examination of the testes and epididymis (of rats and mice) performed in the NTP 90-day studies did not show any evidence of damage, this non-significant difference in sperm numbers observed in mice can be dismissed as a chance finding.

Mode of Action Analysis / Human Relevance Framework

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 (Grosdidier, 2011), 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. This could explain why significant effects on blood glucose, fatty acids, lactate, ß-hydroxybutyrate and/or acetoacetate was observed in the 90-day oral study (Rousseau, 2010) whereas no effect was observed in the 2-generation study (Davies 2010b). In the 90-day study, the animals were fasted overnight before the blood sampling, 5-6 hours after the treatment, in the 2-generation study, the animals were not fasted before blood sampling.

In the reproduction/developmental screening OECD 421 study (Davies, 2010a) and 2-generation OECD 416 reprotoxicity study (Davies, 2010b), mortalities of the pregnant females at the time of delivery and of some pups during lactation were observed at the dose levels of 80 and 40 mg/kg bw/day, respectively. From the known mechanism of action of mercaptoacetate and from the results of the study in fasted and non fasted rats, it can be hypothized that 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. An indirect confirmation of the mechanism is provided by the F1 pregnant females from the 2-generation study, when treatment was stopped on gestation day 19, the delivery passed without problem in all females. 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 40 mg/kg bw/day and higher in the screening and 2-generation reproductive toxicity studies.

Therefore, considering the lack of reproductive toxicity at the non toxic dose level of 20 mg/kg bw/day in the 2 -generation study, and the lack of developmental toxicity in the developmental toxicity studies, no classification is warranted for reproductive and developmental toxicity under CLP and DSD.