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Effects on fertility

Description of key information
Read-across from supporting substance (structural analogue or surrogate): In 13-week dermal subchronic toxicity studies in rats and mice with sodium thioglycolate, 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. 
Effect on fertility: via oral route
Dose descriptor:
NOAEL
20 mg/kg bw/day
Additional information

Studies with NaTG were used as a surrogate to investigate reproduction toxicity of thioglycolic acid and its salts.

2-Generation Study (OECD 416)

The effects of NaTG 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, OECD 416).

Three groups of 25 male and 25 female Sprague-Dawley rats received NaTG daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. NaTG was administered by oral gavage at dose-levels of 10, 20 or 40 mg/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 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 was 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 NaTG 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 by oral gavage at dose levels of 10, 20 or 40 mg/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 was 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 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 was 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/kg/day.

At 40 mg/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/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/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/kg/day groups.

At 40 mg/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.control group), but whereas the control pup deaths were concentrated in four litters, at 40 mg/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 neither in testis, epididymis, prostate, coagulating glands or seminal vesicles of the males nor in the ovaries, oviducts, uterus and vagina of the 40 mg/kg/day of the 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/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/kg/day suggestive of mild hepatotoxicity and especially in dams (4/6) found dead or prematurely sacrificed at time of parturition. NaTG is known to induce fatty liver via an inhibition of theβ-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/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/kg/day, the dose-level of 20 mg/kg/day 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/kg/day was 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 behavior was higher or equal than 40 mg/kg/day.

 

Reproduction/Developmental Screening Study (OECD 421)

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.

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 and one female given 80 mg/kg bw/d were found dead during the pre-mating or mating periods with no clinical signs observed before death and no relevant post-mortem findings. Four females at 80 mg/kg/d and one at 40 mg/kg bw/d were found dead or were prematurely sacrificed because of difficulties to deliver. Ptyalism was observed at 40 and 80 mg/kg bw/d with a dose-related incidence and may be related to the taste of dosing solution. All male and female groups had body weight gains comparable with the controls throughout the study. There were no adverse effects of treatment on mean food consumption, except a slight lowering of food consumption during the lactation period for females given 80 mg/kg bw/d.

The mean number of estrous cycles in each group was between 4 and 5 during the period measured and the mean cycle length was a normal 4 or 5 days. 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 bw/d had a statistically significantly longer gestation period, a non-statistically significantly lower number of corpora lutea and a statistically significantly lower number of implantations and pups. One female had total resorptions and one litter died on day 1 post-partum.

There were no effects of treatment on sperm morphology, motility or counts. The mean absolute seminal vesicle weights were statistically significantly lower for all treated groups compared to the control group, but the mean relative weight was only decreased at 80 mg/kg bw/d. For all treated groups, the absolute weights were in the range of the historical control data. Only at 80 mg/kg bw/d, the relative weight was outside the historical data and was correlated with a slight decrease in secretory content in the seminal vesicles. There were no treatment-related adverse effects in pups based on clinical signs, mean body weight gain and necropsy findings.

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), the NOAEL for 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) 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 which cannot definitively be attributed to maternal condition).

 


Short description of key information:
Thioglycolic acid and its salts are not considered to be reproductive toxicants. The conducted reproduction toxicity studies with sodium thioglycolate can be bridged to other salts of the thioglycolic acid, because in aquous solutions only the organic thioglycolate anion may have the potential to cause reproduction toxicity.

Effects on developmental toxicity

Description of key information
The developmental toxicity of ammonium and sodium thioglycolates has been investigated in standard oral and dermal studies in rats and/or rabbits compliant or comparable to OECD guideline 414, respectively.
When ammonium thioglycolate was administered by gavage, the NOAELs for maternal and embryo-foetal toxicity were 15 and 75 mg/kg bw/d, respectively. No teratogenic effects were observed in all studies.
In the dermal studies with sodium thioglycolate, the NOAELs for maternal toxicity was < 50 mg/kg bw/d in Sprague-Dawley rats and > 65 mg/kg bw/d in New Zealand White rabbits. The developmental toxicity NOAEL was 100 mg/kg bw/d and >65 mg/kg bw/d, respectively.
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
65 mg/kg bw/day
Additional information

The developmental toxicity potential of ammonium thioglycolate by the oral route was evaluated in rats in a study performed according to the OECD Guideline 414 (Gleich, 1998).

Ammonium thioglycolate 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. 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 then 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 and 75 mg/kg bw/d, respectively. No teratogenic effects were observed.

 

The developmental toxicity of sodium thioglycolate by the dermal route was evaluated in pregnant rats and rabbits in studies performed according to a method comparable to the OECD guideline 414 (Tyl, 2001a,b; Tylet al., 2003).

Pregnant Sprague-Dawley rats or 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 19 at dose levels of 0, 50, 100, and 200 mg/kg bw/d for rats orfrom GD 6–29 at 0, 10, 15, 25 or 65 mg/kg bw/d for rabbits.

Twenty-five timed-mated female rats or twenty-four naturally mated female rabbits (in two replicates of 12) 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 or 30, respectively; the following were recorded: maternal clinical condition; body, liver, kidney (rabbits only) 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 or 100% of the live rabbit 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) and all rabbit foetuses were examined for ossified and cartilaginous skeletal alterations.

In rats, 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 and 100 mg/kg bw/d, but not at 200 mg/kg bw/d. Maternal water consumption was significantly increased at 200 mg/kg bw/d and slightly (but not statistically significantly) increased at 50 and 100 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.

The maternal NOAEL could not be identified (<50 mg/kg bw/d), while the developmental toxicity NOAEL was 100 mg/kg bw/d.

In rabbits, 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; for local toxicity at the dosing site, it was below 10 mg/kg bw/d. The NOAEL for developmental toxicity was >65 mg/kg bw/d.

Toxicity to reproduction: other studies

Additional information

At the end of a sub-chronic (90-day) toxicity studies of sodium thioglycolate administered by cutaneous application to F344/N rats and B6C3F1 mice, a special sperm morphology and vaginal cytology evaluation (SMVCE) was performed (Wolfe and Seung, 2003a,b).

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 at 180 and 360 mg/kg bw/d but the reduction was not statistically significant. As the histopathological examination of the testes and epididymis (of rats and mice) performed in these 90-day studies did not show any evidence of damage, this non-significant difference in sperm numbers observed in mice can be dismissed as an incidental finding.

Justification for classification or non-classification

Overall, ammonium and sodium thioglycolate, as well as thioglycolic acid, are not considered to be developmental or reproductive toxicants, excepted at dose levels associated with maternal lethality.