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Key value for chemical safety assessment

Effects on fertility

Additional information

From the available in vitro and in vivo data (cf. chapter toxicokinetics), it can be reasonably concluded that 2-mercaptoethanol is metabolized under formation of 2-mercaptoacetate in the mammalian organism. Thus, due to animal welfare reasons (Article 25) and according to Annex XI, chapter 1.2. of the REACh regulation, the data requirements according to Annex X regarding “Toxicity to reproduction” were fulfilled by using available data on 2-mercaptoethanol and sodium 2-mercaptoacetate (CAS 367-51-1) on the basis of a weight of evidence approach:

In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD TG422), groups of 10 male and 10 female rats were gavaged once daily with 0, 15, 50, or 75 mg/kg (0, 0.192, 0.640, or 0.960 mmol/Kg bw/d) 2 -mercaptoethanol. Males were treated 5 weeks before mating, during mating and post-mating period until sacrifice after approximately 7 weeks. Females were treated 5 weeks before mating, during mating and pregnancy and lactation until day 21 post partum inclusive except at the mid and high dose when treatment was interrupted from days 19 and 20 post coitum until delivery due to toxic effects; all females were sacrificed on day 21 post partum. There wer no treatment-related effects on mating and fertility parameters at any dose level. Seminology in males revealed no effects on sperm count, motility and morphology. The estrous cycle, mating and fertility indices, and pre-coital time were not affected, and no treatment-related effects were detected in reproductive organs on macro- or microscopic examination. Maternal toxicity was evident with deaths of pregnant females at 50 and 75 mg/kg bw/day (six dead or sacrificed on post coitum (PC) days 19 -23 at 75 mg/kg bw/day, three dead on PC day 21 or day 2 post partum at 50mg/kg bw/day). Surviving females in the mid- and high dose groups showed higher body weight gain (52 and 67%) and food consumption (14%; statistically significant in the high-dose group) during the premating period and lactation, while those in the top-dose group had reduced body weight gain in last week of pregnancy (-47%). Effects on body weight gain and food consumption were considered by the investigators to be treatment related. At 75mg/kg bw/day the duration of gestation was increased (22.3 days v 21.5 days in controls) and the number of live born pups/littter was significantly decreased due to one dam with only 1 live born pup. The number of females with liv-born pups was reduced in the mid- and high-dose groups (7/10 and 4/10 versus 8/9 in controls; statistically significant at the high dose). Gestation indices were given as 78% and 40% in the mid- and high dose groups compared to 100% in the low-dose group and the controls. There was no effect on sex ratio. The primary treatment-related effect on reproduction in the TG422 study was prolonged labor and dystocia at dose levels of 50 and 75mg/kg/day. Certain aliphatic thiol compounds, including 2 -mercaptoethanol, have been shown to act as antagonists to the neuropeptide oxytocin, blocking the contractile response of oxytocin on the rat uterus (Martin and Schild, 1965). Oxytocin is secreted primarily by the posterior pituitary gland and is critical to the normal progress of parturition and sustaining sufficient uterine contractions during labor to expel the fetus(es) and to ligate severed blood vessels within the contracted myometrium after the placenta separates, thus preventing hemorrhage. It is possible that oral administration of 2 -mercaptoethanol to pregnant rats in the TG422 study was sufficient to disrupt the normal oxytocin-mediated progression of parturition by diminishing uterine contractions and prolonging labor. Slightly higher pup body weights were noted at birth int he 75mg/kg/day group, probably as a result of the slight increase in the length of pregnancy. However, despite these higher pup weights at birth, mean pup body weight at 75mg/kg was lower than control values throughout the remainder of the lactation period as a result of significatntly lower pup body weight gain beginning on postnatal day 4. It is not clear whether the effects on pup body weight and survival at 75mg/kg are the result of direct exposure to the test article in utero or via the milk production. Oxytoxin is known to play an important role, not only in parturition, but also in milk production during lactation. Given the potential antagonistic effects of 2 -mercaptoethanol on oxytocin, it is possible that maternal exposure to 2 -mercaptoethanol during lactation may hinder milk production and consequently pup growth and viability. In addition to the effects on parturition and pup body weight, mean live litter size was significantly (p<0.5) lower in the 75mg/kg/day group compared to the control group (10.0 versus 14.9 pups). The small litter size at 75mg/kg was primarily attributed to one dam (out of 4 surviving dams) that delivered a single pup. This reduction in mean live litter size correlated with higher post-implantation loss and decreased pup survival at the same dose level. However, the small number of surviving dams/litters available for evaluation at 75mg/kg (n=3 or 4 dams) is a potential major confounding factor in establishing a relationship between these endpoints (live litter size and pup survival) and test article administration. No apparent effects on mating or fertility indices or on male reproductive parameters were observed at any dose level evaluated.

As a result of the number of pregnant females found dead or euthanized in extremis at 50 and 75mg/kg due to dystocia and prolonged labor, the number of pups/litter available for evaluation was limited, especially at 75mg/kg. The NOAEL for maternal and reproductive toxicity was determined to be 15 mg/kg bw (0.192 mmol/Kg bw/d).

In a reproduction/developmental screening test according to OECD Guideline 421 performed with sodium mercaptoacetate (sodium thioglycolate, CAS 367-51-1), SD rats received the test substance by oral gavage administration at dose-levels of 0, 20, 40 or 80 mg/kg bw/d (0, 0.175, 0.351 or 0.701 mmol/Kg bw/d. 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 were considered to be treatment-related. Three out of 11 surviving females given 80 mg/kg/day were found dead on day 23 post-coitum, all having delivered pups, although one female had one fetus in the vagina and still had 11 dead fetuses in the uterine horns at necropsy. Another pregnant female with dead and live fetuses in the uterine horns was sacrificed on day 23 post-coitum because of poor clinical condition. At 80 mg/kg/day, one additional female was prematurely sacrificed on day 1 post-partum because all the pups were dead and another female was found dead on day 2 post-partum. One female given 40 mg/kg/day was found dead on day 22 post-coitum, pregnant with dead fetuses in the uterine horns. Ptyalism was observed at 40 and 80 mg/kg/day with a dose-related incidence and may be related to the taste of the test item. Females given 80 mg/kg/day had a significantly longer gestation period, a nonsignificantly lower number of corpora lutea and a significantly lower number of implantations and pups. 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 postpartum. 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. 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 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). 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.

At 40 mg/kg/day, one pregnant female with dead fetuses in the uterine horns was found dead on day 22 post-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. 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 (0.175mmol/kg bw/d, based on deaths at 40 and 80 mg/kg/day). 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 (0.175mmol/kg bw/d). The NOEL for toxic effects on progeny was set at 40 mg/kg/day, based on the dead litter at 80 mg/kg/day.

In a subsequent two-generation reproductive toxicity study according to OECD Guideline 416, SD rats received sodium mercaptoacetate (sodium thioglycolate, CAS 367-51-1) by oral gavage administration at dose-levels of 0, 10, 20 or 40 mg/kg bw/d (0, 0.088, 0.175 or 0.351 mmol/kg bw/d). There were no effects of treatment at 10 or 20 mg /kg bw. At 40 mg /kg bw, 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. The found dead females had no microscopic findings which could explain the deaths but one female had hemorrhage of a mesometrial triangle. 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. Minimal to moderate periportal hepatocellular microvacuolation was observed at 40 mg /kg bw in 2/25 males and 6/25 females, and in 4/6 prematurely sacrificed/found dead females suggesting liver toxicity at this dose-level. Female plasma fatty acid concentration was statistically significantly decreased.

There were no effects of treatment on F1 pups in the 10 or 20 mg /kg bw groups. At 40 mg /kg bw, the pups had a higher 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 pups in the control group), but whereas the control pup deaths were concentrated in four litters, at 40 mg /kg bw 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 high dose females.

The female F2 pups had a slightly lower mean body weight at the end of lactation 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 bw, it is concluded that the test item has no effect on non-pregnant, 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 bw suggestive of hepatotoxicity and especially in dams found dead or prematurely sacrificed at time of parturition. Sodium thioglycolate is known to induce fatty liver via an inhibition of the βoxidation of fatty acids.

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 bw, the dose-level of 20 mg /kg bw (0.175mmol/kg bw/d) 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 bw were secondary to the severe and lethal effects observed in the pregnant dams at that dose level. The NOEL for males fertility and female mating behaviour was higher or equal than 40 mg /kg bw.

Reference Martin, P.J., and Schild, H.O. (1965). The antagonism of disulphide polypeptides by thiols. Brit. J. Pharmacol. 25:418 -431.

Effects on developmental toxicity

Description of key information

OECD TG 414 in rats: NOAEL >= 25 mg/kg/d (BASF SE, 2013)

OECD TG 414 in rabbits (sodium mercaptoacetate, CAS 367 -51 -1): NOAEL >= 65 mg/kg bw

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
65 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Additional information

From the available in vitro and in vivo data (cf. chapter toxicokinetics), it can be reasonably concluded that 2-mercaptoethanol is metabolized under formation of 2-mercaptoacetate in the mammalian organism. Thus, due to animal welfare reasons (Article 25) and according to Annex XI, chapter 1.2. of the REACh regulation, the data requirements according to Annex X regarding "Developmental toxicity / teratogenicity" (incl. second species) were fulfilled by using available data on 2 -mercaptoethanol and sodium 2-mercaptoacetate (CAS 367-51-1) on the basis of a weight of evidence approach.

2 -Mercaptoethanol was tested for its prenatal toxicity in Wistar rats according to OECD TG 414 and GLP. The test substance was administered as a solution in isotonic saline solution to groups of 25 of time-mated Wistar rats by gavage at doses of 5, 15 and 25 mg/kg body weight/day (mg/kg bw/d) (0.064, 0.192 and 0.320 mmol/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (isotonic saline solution) in parallel.

The high dose was selected based on signs of toxicity noted at a doselevel of 50 mg/kg bw/d in a previously conducted OECD 422 study (test facility CIT, laboratory study number 24847 RSR, 2004) and in a maternal toxicity range-finding study (BASF project 10RO234/04R034) which preceded this definitive prenatal developmental toxicity study. In the OECD 422 combined repeated dose with the reproductive/developmental screening test, 2 out of 10 Sprague-Dawley dams in the 50 mg/kg bw/d group died immediatley prior to or during parturition with another death occurring during early lactation. Although the OECD Guideline 414 study design does not continue the dose administration through the period immediatley prior to and during parturition, the presence of this toxicity was pertinent in the selection of dose levels for the definitive prenatal developmental toxicity study. In the maternal toxicity range-finding study, 10 pregnant Wistar rats were administered the test substance by oral gavage from gestational day (GD) 6 through GD 19. One dam died on GD 18 and one dam was sacrificed in moribund condition on GD 20, showing signs of piloerection, semiclosed eyelids, apathy, and hypothermia. While it is possible that the mortality observed and the clinical signs were due to gavage errors in administering the test substance (as they were not observed in the OECD 422 study at the 50 mg/kg and higher dose levels), a 50mg/kg bw/d was considered to be potentially lethal to the dams in the OECD 414 study. The selected high dose for the present study represented half of this lethal dose. This approved procedure of decreasing a lethal dose by a factor of two to become the high dose in a subsequent regulatory study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 ("how to avoid unecessary testing on animals", chapter 4 "animal welfare": ECHA-10 -B-17 -EN, 2010) which is in compliance with EU Directive 86/609/EEC on animal protection. the oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle over a 9 -day period in a refrigerator (in the dark). Generally, clinical observations revealed no toxicologically relevant difference between the animals receiving 5, 15 or 25 mg/kg bw/d 2 -mercaptoethanol and controls. Concerning clinical pathology and organ weights of dams no treatment related, adverse effects were observed up to a dose of 25 mg/kg bw/d. No differences of toxicological relevance between the control and the treated groups (5, 15 or 25 mg/kg bw/d) were determined for any reproductive parameters, sucha s conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test compound on sex distribution of the fetuses was noted at any dose.

In the assessment of all fetal external, soft tissue and skeletal observations no evidence for toxicologically relevant adverse effects of the test substance was determined on fetal morphlogy at any dose.

Taken together, under the conditions of this prenatal developmental toxicity study, the oral administration of 2 -Mercaptoethanol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6 -19) at a dose of 5, 15 and 25 mg/kg bw/d caused neither maternal toxicity nor prenatal developmental toxicity.

In conclusion, the no observed adverse effect level (NOAEL) of 2 -Mercaptoethanol for maternal and prenatal developmental toxicity is at least 25 mg/kg bw/d (0.320 mmol/kg bw/d).

The developmental toxicity of sodium mercaptoacetate (sodium thioglycolate, CAS 367-51-1) was evaluated in pregnant rabbits in a study performed according to a method comparable to the OECD guideline 414 (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 (0, 0.088, 0.131, 0.219 or 0.570 mmol/kg bw/d).

No maternal deaths were associated with sodium thioglycolate treatment. Clinical observations were almost exclusively limited to effects of treatment at 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.

Thus, the maternal NOAEL for systemic toxicity was at or above 65 mg/kg bw/d (0.570 mmol/kg bw/d); for local toxicity at the dosing site, it was below 10 mg/kg bw/d (0.088 mmol/kg bw/d). The NOAEL for developmental toxicity was at or above 65 mg/kg bw.

Justification for classification or non-classification

Thiols, such as 2-mercaptoethanol and 2-mercaptoacetate, have been known to produce specific pharmacological antagonism of oxytocin by breaking its disulphide bonds (Martin and Schild, 1965). Oxytocin is a mammalian neuropeptide secreted primarily by the posterior pituitary gland and is critical to the normal progress of parturition and sustaining sufficient uterine contractions during Iabor to expel the fetus(es) and to ligate severed blood vessels within the contracted myometrium after the placenta separates, thus preventing hemorrhage. The dose-related mortality and morbidity in the pregnant female rats after administration of 2-mercaptoethanol (≥ 50 mg/kg bw/day) or sodium 2-mercaptoacetate (≥ 40 mg/kg bw/day) was not a gender-specific effect or the result of other reported treatment-related effects, but most likely a manifestation of delivery related difficulties at the time of parturition. In general, the classification criteria for reproductive toxicity according to Regulation EC 1272/2008 (CLP) are met.

Repeated oral administration of 2-mercaptoethanol resulted in clinical symptoms (excessive salivation, decreased body weight in males) and effects on the liver (organ weight increased and vacuolated liver cells in both genders) and the heart (degenerative cardiomyopathy in females at ≥ 50 mg/kg bw/day, in males at 75 mg/kg bw/day). Thus, 2-mercaptoethanol is classified for Specific Target Organ Toxicity (STOT RE cat.2; target organs: liver, heart) based on the effects observed at ≥ 50 mg/kg bw/day. In conclusion, the mortality and morbidity in the pregnant female rats due to delivery related difficulties at the time of parturition is not an isolated effect and was accompanied by additional systemic toxicity which requires classification for Specific Target Organ Toxicity. According to chapter 3.7.2.2.1. of Regulation EC 1272/2008 (CLP), classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction. Therefore, classification with category 2 for reproductive toxicity (H361f) is considered the most appropriate in line with the criteria laid down in Regulation EC 1272/2008 (CLP).