(2-hydroxyethyl)ammonium mercaptoacetate

Endpoint summary

Description of key information

No reliable data is available on the repeated dose toxicity of MeaTG.

However, the subchronic toxicity of sodium thioglycolate was evaluated by oral and dermal administrations.

In an oral repeated dose toxicity study (OECD 408), sodium mercaptoacetate was administered by gavage, 7 days/week, for 13 weeks, to male and female Sprague-Dawley rats. Sporadic mortality and fully reversible effects on some haematological and biochemical parameters and histopathological changes in liver were observed at 60 mg/kg bw/d. These effects may be related to the inhibition of the β-oxidation of fatty acids a known mechanism of action of sodium mercaptoacetate. Consequently, based on the effects observed at 60 mg a. i. /kg/day, particularly mortality, hematological and significant blood chemistry changes associated with liver microscopic changes and the limited blood chemistry effects without microscopic adverse changes in the liver observed at 20 mg a. i. /kg/day, the NOAEL of sodium mercaptoacetate was 20 mg a. i. /kg/day, and the NOEL was 7 mg a. i. /kg/day given by daily oral administration (gavage) to rats for 13 weeks. Additional information on the oral repeated dose toxicity of sodium mercaptoacetate is provided by the 2-generation reprotoxicity study (OECD 416) study and the reproduction/developmental screening test (OECD 421). The results of both studies support the NOAEL 20 mg a. i. /kg/day defined in the 13-week toxicity study.

In a repeated dose dermal toxicity (OECD 411), sodium mercaptoacetate was administered via dermal route, 5 days per week, for 13 weeks to male and female Fischer 344 rats and B6C3F1 mice. All animals survived the 13 weeks administration. The only treatment related effect was skin irritation at the site of application. The LOELs for skin irritation were 11.25 and 45 mg/kg bw/d and the NOAELs for systemic toxicity were higher than 180 and 360 mg/kg bw/d in rats and mice, respectively.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

The read-across is a category approach based on the hypothesis that compounds in this category are transformed to a common compound. This approach serves to use existing data on genotoxicity, repeated-dose toxicity, and reproductive toxicity endpoints for substances in this category.
There are no relevant variations in properties among source substances and the same potency is predicted for all target substances. This is Scenario 5 of the RAAF . Substances ATG, MEATG, KTG, CaTG, and NaTG are different inorganic salts of a common acid, thioglycolic acid (TGA; synonym: 2- mercaptoacetic acid). They dissociate rapidly in aqueous media, e.g., the test organism, to the common thioglycolate anion and to their different counter ions. The water solubility of all category members is high, except for CaTG which is only moderately soluble in water.
In the repeated-dose toxicity studies with NaTG, specific toxicity is exerted via the well-investigated inhibition of mitochondrial fatty acid beta-oxidation by the thioglycolate (2-mercaptoacetate) anion 2,3,4. Inhibition of beta-oxidation leads to increased triglycerides and decreased acetyl-CoA in liver, and subsequently reduced gluconeogenesis. The latter presents as hypoglycaemia in NaTGtreated rats, which is aggravated by fasting (Grosdidier, 2011; Report No. 37043 TSR). This mode of action (MoA) is thought to mediate the acute oral toxicity in fasted rats observed with all category members.
It can be predicted with high confidence that the target substances will display the same MoA and lead to the same effects seen with NaTG.
For more detailed information please refer to section 13.2.

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

mortality

Remarks on result:

other: corrected for molecular weight differences

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

mortality

Remarks on result:

other: corrected for molecular weight differences

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on data for NaTG, the subchronic oral NOAEL of MeaTG is assumed to be 27 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

27 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

The read-across is a category approach based on the hypothesis that compounds in this category are transformed to a common compound. This approach serves to use existing data on genotoxicity, repeated-dose toxicity, and reproductive toxicity endpoints for substances in this category.
There are no relevant variations in properties among source substances and the same potency is predicted for all target substances. This is Scenario 5 of the RAAF . Substances ATG, MEATG, KTG, CaTG, and NaTG are different inorganic salts of a common acid, thioglycolic acid (TGA; synonym: 2- mercaptoacetic acid). They dissociate rapidly in aqueous media, e.g., the test organism, to the common thioglycolate anion and to their different counter ions. The water solubility of all category members is high, except for CaTG which is only moderately soluble in water.
In the repeated-dose toxicity studies with NaTG, specific toxicity is exerted via the well-investigated inhibition of mitochondrial fatty acid beta-oxidation by the thioglycolate (2-mercaptoacetate) anion 2,3,4. Inhibition of beta-oxidation leads to increased triglycerides and decreased acetyl-CoA in liver, and subsequently reduced gluconeogenesis. The latter presents as hypoglycaemia in NaTGtreated rats, which is aggravated by fasting (Grosdidier, 2011; Report No. 37043 TSR). This mode of action (MoA) is thought to mediate the acute oral toxicity in fasted rats observed with all category members.
It can be predicted with high confidence that the target substances will display the same MoA and lead to the same effects seen with NaTG.
For more detailed information please refer to section 13.2.

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEL

Effect level:

>= 242 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:

corrected for molecular weight differences

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Remarks on result:

other: corrected for molecular weight differences

Key result

Critical effects observed:

no

Conclusions:

Based on data for NaTG, the subchronic systemic dermal NOAEL of MeaTG in rats is assumed to be greater than or equal to 242 mg/kg bw/day, the highest dose tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

242 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

The read-across is a category approach based on the hypothesis that compounds in this category are transformed to a common compound. This approach serves to use existing data on genotoxicity, repeated-dose toxicity, and reproductive toxicity endpoints for substances in this category.
There are no relevant variations in properties among source substances and the same potency is predicted for all target substances. This is Scenario 5 of the RAAF . Substances ATG, MEATG, KTG, CaTG, and NaTG are different inorganic salts of a common acid, thioglycolic acid (TGA; synonym: 2- mercaptoacetic acid). They dissociate rapidly in aqueous media, e.g., the test organism, to the common thioglycolate anion and to their different counter ions. The water solubility of all category members is high, except for CaTG which is only moderately soluble in water.
In the repeated-dose toxicity studies with NaTG, specific toxicity is exerted via the well-investigated inhibition of mitochondrial fatty acid beta-oxidation by the thioglycolate (2-mercaptoacetate) anion 2,3,4. Inhibition of beta-oxidation leads to increased triglycerides and decreased acetyl-CoA in liver, and subsequently reduced gluconeogenesis. The latter presents as hypoglycaemia in NaTGtreated rats, which is aggravated by fasting (Grosdidier, 2011; Report No. 37043 TSR). This mode of action (MoA) is thought to mediate the acute oral toxicity in fasted rats observed with all category members.
It can be predicted with high confidence that the target substances will display the same MoA and lead to the same effects seen with NaTG.
For more detailed information please refer to section 13.2.

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Dose descriptor:

NOAEL

Effect level:

>= 242 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:

corrected for molecular weight differences

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Remarks on result:

other: corrected for molecular weight differences

Key result

Critical effects observed:

no

Conclusions:

Based on data for NaTG, the subchronic systemic dermal NOAEL of MeaTG in rats is assumed to be greater than or equal to 242 mg/kg bw/day, the highest dose tested.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

63 µg/cm²

Study duration:

subchronic

Species:

rat

Quality of whole database:

adequate and valid

Additional information

The subchronic toxicity of sodium mercaptoacetate was evaluated by dermal and oral administrations.

Oral route

The repeated dose toxicity of sodium mercaptoacetate was evaluated in a key 90-day toxicity study (OECD 408). Supporting information are provided by a 2-generation reproductive toxicity study (OECD 416) and a reproduction/developmental screening test (OECD 421).

In a key OECD Guideline No. 408 study, the potential toxicity of sodium mercaptoacetate was evaluated following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 4-week treatment-free period in order to evaluate the reversibility of any findings. Three groups of male and female Sprague-Dawley rats: 10 per sex for the low- and intermediate‑dose (groups 2 and 3) and 16 per sex for the high-dose (group 4), were treated daily with sodium mercaptoacetate by the oral route (gavage) for 13 weeks, at dose-levels of 7, 20 or 60 mg a. i. /kg/day (a. i. = active ingredient). Sodium mercaptoacetatewas administered as a solution in the vehicle (purified water) under a constant dosage-volume of 5 mL/kg/day. A group of 16 males and16 females received the vehicle alone under the same experimental conditions and acted as a control group (group 1). The concentration of the test item was analyzed before the first treatment and in weeks 4, 8 and 13. At the end of the treatment-period, all the animals were sacrificed, except the last six animals of each sex in groups 1 and 4 which were kept for a 4-week treatment-free period. The animals were checked daily for mortality and clinical signs. In addition, detailed clinical examinations were made in a standard arena once before the treatment period and then once a week until the end of the study. Body weight was recorded once during the pre-treatment period, on the first day of treatment and then once a week until the end of the study. Food consumption was recorded once a week during the treatment and treatment-free periods. Ophthalmology examinations were performed on all animals before the beginning of the treatment period and on the control and high-dose animals at the end of the treatment period. Hematology, blood biochemistry (including analysis of ß‑hydroxybutyrate) and urinalysis investigations were performed on all animals (fasted before blood sampling) at the end of the treatment and treatment-free periods. Functional Observation Battery (FOB), including motor activity, was performed on all animals (except recovery animals) at the end of the treatment period. Animals were sacrificed on completion of the treatment or treatment-free period and were submitted for a full macroscopicpost-mortemexamination. Designated organs were weighed and selected tissue specimens were preserved. Samples of the liver were collected from all males and females, and the glycogen content was evaluated in control and high-dose animals. A microscopic examination was performed on designated tissues from control and high-dose animals sacrificed at the end of the treatment period, and on all macroscopic lesions and the liver (both sexes), kidneys (females only) and heart (males only) of low- and intermediate-dose animals. At the end of the treatment-free period, the liver (both sexes), kidneys (females only) and heart (males only) of surviving control and high‑dose animals were microscopically examined.

The sodium mercaptoacetate concentrations in the administered dosage forms analyzed in weeks 1, 4, 8 and 13 remained within an acceptable range [-1.8% to +7.9%] when compared to the nominal values. At 60 mg a. i. /kg/day, one female was prematurely sacrificed for humane reasons on day 14. Prior to premature sacrifice, this female showed marked hypoglycaemia (1.32 mmol/L), hypoactivity, staggering gait, hunched posture, piloerection, soiled urogenital region, coldness to the touch and thin appearance. The marked hypoglycaemia, which is related to test item treatment, has most likely contributed to the clinical condition that was at the root of the decision to sacrifice this particular female for humane reason. One male given 60 mg a. i. /kg/day was found dead on day 90; no signs of poor clinical condition were observed prior to death. Macroscopically, pale or irregularly colored liver were noted in both animals, which correlated with periportal to diffuse hepatocellular microvaculation at microscopic examination, a test item-related change also observed in animals at the end of the study period. No unscheduled deaths were recorded at 7 or 20 mg a. i. /kg/day. In surviving animals, hypersalivation was noted in almost all males and females given 60 mg a. i. /kg/day, generally from week 2. Piloerection was transiently noted in 3/15 males given 60 mg a. i. /kg/day in week 11 or in week 13. These findings were considered to be non-adverse effects of the test item treatment. There were no clinical signs among test item-treated animals at 7 mg a. i. /kg/day. No relevant test item effects were observed on body weight. Food consumption when compared to controls, was higher in males given 20 mg a. i. /kg/day in week 9 only (+8%, p<0.05) and at 60 mg a. i. /kg/day from week 6 (+5 to +10%, p<0.01). There were no ophthalmological findings of toxicological importance at the end of the treatment period. No test item treatment-related effects were observed during the FOB or on motor activity. Marked leucopenia was noted in animals of both sexes given 60 mg a. i. /kg/day and all the white blood cell types were affected. Higher mean red blood cell count, hemoglobin concentration and packed cell volume were observed in males and females treated at 60 mg a. i. /kg/day when compared to control values. High mean prothrombin time was also noted in males given 60 mg a. i. /kg/day and in females from 20 mg a. i. /kg/day. At the end of the treatment-free period, the hematological parameter disturbances were no longer observed in the high-dose group when compared to controls, suggesting total reversibility of the findings. No hematological changes of toxicological importance were recorded at 7 mg a. i. /kg/day. The bone marrow differential cell count was not affected by the test item treatment. Lower mean glucose level was observed in all test item-treated females (statistically significant at 20 and 60 mg a. i. /kg/day when compared to controls), while hypoglycemia was only seen at 60 mg a. i. /kg/day in the males. Mean chloride level was statistically significantly lower in males and females treated at the high dose-level, with moderately higher urea levels from 20 mg a. i. /kg/day. In males given 60 mg a. i. /kg/day, moderately higher uremia and statistically significant higher creatinine values were observed, when compared to the control values. A statistically significant higher fatty acid level was noted in females given 20 or 60 mg a. i. /kg/day, and in males at 60 mg a. i. /kg/day. Significantly higher aspartate aminotransferase (males only) and alanine aminotransferase (males and females) activities were noted at 60 mg a. i. /kg/day. This change was associated with microscopic liver vacuolation. Significantly lower mean ß-hydroxybutyrate levels were reported in males given 60 mg a. i. /kg/day and in females from 20 mg a. i. /kg/day when compared to control values. A statistically significant higher lactate concentration was also observed in males and females given 60 mg a. i. /kg/day. At the end of the treatment-free period, blood biochemistry parameter disturbances were no longer observed in the high-dose group when compared to controls, suggesting total reversibility of the findings. No blood biochemistry changes were recorded at 7 mg a. i. /kg/day. No relevant test item treatment-related findings were observed in the urinalysis parameters. At microscopic examination, periportal hepatocellular vacuolation was noted in the liver of 2 males given 20 mg a. i. /kg/day and in 4 males and 3 females given 60 mg a. i. /kg/day. This change was not present at the end of the treatment-free period. Microvacuolation in the liver was Oil Red O positive, indicating the presence of neutral lipids and a lipidosis (syn. steatosis) change. Increased Oil Red O positive vacuoles were noted in males treated from 20 mg a. i. /kg/day and in females treated at 60 mg a. i. /kg/day. Tubular vacuolation was observed in the kidneys from females given 60 mg a. i. /kg/day. This correlated with increased urea and creatinine values at clinical examination. Increased absolute and relative liver weights were noted in females treated at 60 mg a. i. /kg/day and correlated microscopically with minimal centrilobular hepatocellular hypertrophy noted in the liver of a few females. Other minor treatment-related changes noted in females treated at 60 mg a. i. /kg/day were observed in the liver. These changes consisted of minimally increased incidence and severity of extramedullary hematopoiesis in the liver. The quantities of glycogen in the livers of controls and rats treated with the test item were very low, probably due to fasting prior to necropsy.

In conclusion, sodium mercaptoacetate was administered by daily oral administration (gavage) to Sprague-Dawley rats at dose‑levels of 7, 20 or 60 mg a. i. /kg/day (a. i. = active ingredient) for 13 weeks. On completion of the treatment period, designated animals were held for a 4-week treatment-free period in order to evaluate the reversibility of any findings. At 60 mg a. i. /kg/day, one female was prematurely sacrificed for humane reasons on day 14and one male was found dead on day 90. Changes, which were also noted in the animals sacrificed on schedule, were found in the kidneys of the female sacrificed for humane reasons, and the liver and thymus of both these animals. The vacuolation/microvacuolation of kidney and liver was considered to be related to treatment with sodium mercaptoacetate. The demise and death of these animals were attributed to treatment with sodium mercaptoacetate. In surviving animals, hypersalivation, piloerection and/or areas of thinned hair were transiently observed in some animals. At laboratory investigations, marked panleucopenia was noted in both sexes (all the white blood cell subtypes were affected). High mean red blood cell count, hemoglobin concentration, packed cell volume and mean prothrombin time were observed in males and females. However, the bone marrow cellularity and number of megakaryocytes were similar to the control values. Hypoglycemia was noted in males and females, associated with high urea (males and females) and creatinine (males only) levels and low chloride levels (male and female). High fat acid level was observed in males and females. High aspartate aminotransferase (males only) and alanine aminotransferase (males and females) activities were noted. Low mean ß‑hydroxybutyrate levels, associated with high lactate concentrations, were reported in males and females. Sodium mercaptoacetate-related changes were noted in the liver of males and females and the kidneys of females. In both organs, there were microvacuolar changes that were considered not to be adverse since they were observed with low incidence and severity. Microvacuolation in the liver was Oil Red O positive, indicating the presence of neutral lipids and a microvesicular lipidosis (syn. steatosis) change. A minimal increase in incidence and severity of extramedullary hematopoiesis was noted in the liver of females. All these changes were not observed at the end of the treatment-free period. At 20 mg a. i. /kg/day, non adverse minimal periportal microvacuolation corresponding to minimally increased severity of lipidosis (syn. steatosis) was noted in two males. In females, low glucose and ß‑hydroxybutyrate levels were noted, associated with high urea and fatty acid concentrations. High mean prothrombin time was also noted in females. At this dose level, no signs of adverse toxic effects were noted. At 7 mg a. i. /kg/day, no changes or signs of toxicity were noted.

Consequently, under the experimental conditions of this study, based on the adverse effects observed at 60 mg a. i. /kg/day, particularly mortality, hematological and significant blood chemistry changes associated with liver microscopic changes, and taking into account the limited blood chemistry effects without microscopic adverse changes in the liver observed at 20 mg a. i. /kg/day, the No Observed Adverse Effect Level (NOAEL) of sodium mercaptoacetate was 20 mg a. i. /kg/day, and the No Observed Effect Level (NOEL) was 7 mg a. i. /kg/day given by daily oral administration (gavage) to rats for 13 weeks.

 

Supporting information is provided by the evaluation of the repeated dose toxicity of sodium mercaptoacetate performed in the frame of a 2-generation reprotoxicity study conducted according to the OECD #416 guideline (Davies, 2010a) and a reproduction/developmental screening test conducted according to the OECD Guideline #421 (Davies, 2010b).

In the OECD #416 study (Davies, 2010a), 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). 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. 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. A complete macroscopic examination was performed, 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.

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 phases. 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 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.

At 40 mg a. i. /kg/day, it is concluded that sodium mercaptoacetate has no effect on non-pregnant, naïve, adult rats. Minimal to moderate periportal heptocellular microvacuolation was observed in pregnant 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 an inhibition of theβ-oxidation of fatty acids. 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 liver effects in males and females observed at 40 mg a. i. /kg/day, the dose-level of 20 mg a. i. /kg/day was considered to be the No Observed Effect Level (NOEL) for parental toxicity.

 

In the OECD #421 study (Davies, 2010b), four groups of 12 male and 12 female Sprague-Dawley rats received sodium mercaptoacetate, 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 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 macroscopicpost-mortemexamination 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.

Two males (weeks 11 and 13) and one female (week 4) given 80 mg/kg/day were found dead during the pre-mating period with no clinical signs observed before death and no relevantpost-mortemfindings. These deaths are considered to be treatment-related. 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 during the pre-mating period; 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 during the pre-mating period. 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 periods. In males, when compared to controls, liver and kidneys weights were slightly but significantly higher. There were no relevant macroscopic or microscopic findings. At 40 mg/kg/day, there were no effects of treatment on body weight and food consumption, and no treatment-related adverse effects on the organ weights and at microscopic examination. There were no effects of treatment at 20 mg/kg/day.

Under these experimental conditions and excluding the mortality of the pregnant females observed at the time of delivery (see same study in section Effects on fertility), the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 40 mg/kg/day (based on deaths at 80 mg/kg/day during the pre-mating treatment period) and the No Observed Effect Level (NOEL) was 20 mg/kg/day.

Dermal route

Summary data, individual data, historical controls data and the standard protocols of two sub-chronic (90-day) toxicity studies of sodium mercaptoacetate administered by cutaneous application toF344/N rats and B₆C₃F₁mice are available on the website of the US National Toxicology Program (NTP) in addition of partial reports (NTP, 2001 and 2003). For the time being, the draft report (TOX-80) is still undergoing a multi-tiered review that includes NTP staff review and external peer review. However, the data available on the NTP website - survival, body weight, hematology, organ weights and histopathology for both species and in addition the clinical chemistry for rats - are sufficiently detailed for a preliminary assessment and justify a reliability 2.

Sodium mercaptoacetate (99% pure) was administered dermally to F344/N rats and B₆C₃F₁mice (10/sex/dose level) following a protocol comparable to the OECD 411 guideline. Rats and mice were given sodium mercaptoacetate in a vehicle of 95% ethanol: deionized water (1:1) by dermal application 5 days per week, for 13 weeks. Doses were applied in a fixed dose volume of 0.5 ml/kg bw to rats or 0.2 ml/kg bw to mice to the center of a shaved dorsal skin areas posterior to the scapulae. Rats were administered 0, 11.25, 22.5, 45, 90, or180 mg sodium mercaptoacetate/kg bw/d and mice 0, 22.5, 45, 90, 180 or 360 mg sodium mercaptoacetate/kg bw/d. In addition, 2 groups of 10 rats per sex/group were dosed with 0 or 180 mg/kg bw/d for 21 days and used for clinical chemistry and haematology estimations on day 4 and day 21 and then disposed of without further examination. Throughout the study, the animals were checked twice a day for mortality and clinical signs. Formal clinical observations were performed and recorded weekly. Body weight and food consumption were recorded weekly. Clinical chemistry and haematology was performed on the main groups of rats at termination, and haematology on the mice at termination. Heart, kidney, liver, lung, spleen, tests, thymus and thyroid/para-thyroid weights were measured at termination, with a complete histopathologic examination inclusive of gross lesions of the high dose and control groups. In addition, a micronucleus chromosome aberration test was performed on the peripheral blood of the mice at termination (results are reported in section Genetic toxicity in vivo), and a special sperm morphology vaginal cytology evaluation (SMVCE) was performed on the controls and three highest dose groups of rats and mice (results are reported in section Toxicity to reproduction). Since there are literature reports that sodium mercaptoacetate inhibits fatty acid oxidation (see section Specific investigations), food consumption and clinical chemistry parameters including 3-hydroxybutyrate, free fatty acids and total cholesterol were also measured.

In rats, all animals survived the 13-week administration. A slight decrease of 8% (p<0.05) and 6% (ns) of the mean body weight gain was observed in males treated with 90 and 180 mg/kg bw/d, respectively. The mean food consumption was not affected by the treatment. Significant clinical observations noted in both sexes were limited to dermal irritation, thickened skin and ulcerations at the site of application (SOA). In males and females, the incidence of dermal irritation at the SOA was 10/10 for all five-treatment groups. Thickening of the skin at the SOA was observed in 1 and 2 males from the 90 and 180 mg/kg bw/d dose groups and in 3, 10 and 10 females from the 45,90 and 180mg/kgbw/ddose groups, respectively. Ulceration at the SOA was observed in 1, 1, 5 and 8 males rats from the 11.25, 22.5, 90 and 180 mg/kg bw/d dose groups and 10 females from each of the 90 and 180 mg/kg bw/d dose groups, respectively. All other clinical observations noted during the study were not considered to be biologically significant. There were slight, but statistically significant, changes (p<0.05) in the relative kidneys weight in males (<+8%) at 90 and 180 mg/kg bw/d and in females (+6%) at 180 mg/kg bw/d, in the absolute and relative liver weights (+10% and +8%) in males at 45 mg/kg bw/d, in the relative spleen weight (+6%) in males at 90 mg/kg bw/d, in the relative testes weight (ca. +7%) at 90 and 180 mg/kg bw/d and in the thyroid/parathyroid relative weight (-20%) in females at 22.5 mg/kg bw/d, but these effects were not dose responsive or considered to be biologically significant. There were limited statistically significant (p<0.05) differences at termination in some blood chemistry (males: total protein (+4%) and albumin (+3%) at 22.5 and 180 mg/kg bw/d, succinate dehydrogenase(SDH) at 45 and 180 mg/kg bw/d (+33% and +31%) and cholesterol at 45 mg/kg bw/d (+8%); females: blood urea nitrogen (BUN) at 180 mg/kg bw/d (+14%)) and hematology (males: neutrophiles at 180 mg/kg bw/d (-24%)) results when compared to the control group, but these effects were not dose responsive nor considered to be biologically significant.

The only treatment related gross and microscopic lesions were at site of application (SOA) with epidermal and sebaceous gland hyperplasia, and hyperkeratosis with severity comparable between all treated groups. There were no other treatment related microscopic lesions. Thus, no target organ for toxicity was identified (other than skin at site of application).

The Lowest-Observed-Effect-Level (LOEL) at the application site was 11.25 mg/kg bw/d based on histopathologic examination. There was no No-Observed-Effect-Level (NOEL) at the application site. The NOAEL for systemic toxicity can be estimated to be higher than 180 mg/kg bw/d.

In mice, all animals survived the 13 weeks administration. Body weights and food consumption were not affected by the treatment in either sex during the course of the study. The only clinical observation related to treatment was skin irritation at site of application in 6/10 males at 360 mg/kg bw/day but not in females. There was a slight increase (8 to 13%, p<0.05) of the absolute and relative heart weight at 180 and 360 mg/kg bw/d male and female, of the absolute kidney weight in females (ca. 10%, p<0.05) at 180 and 360 mg/kg bw/d, and of the absolute (8%, p<0.05) and relative (8 and 11%, p<0.05) liver weight in males at 180 and 360 mg/kg bw/d and in females at all doses (p<0.05) from 45 mg/kg bw/d upwards (12, 10 and 15% for the absolute and 8, 9 and 11% for the relative weight, respectively). Haematology showed no biologically significant changes in males, but in females there was some small decrease, in red blood cells (maximum -8%, p<0.05) at 22.5, 45, 180 and 360 mg/kg bw/d and haemoglobin levels (maximum -6%, p<0.05) at 22.5, 45, and 360 mg/kg bw/d. There were no significant gross lesions. On microscopic histopathology, skin lesions were observed at the SOA in males and females at all dose levels, with the exception of the 22.5 mg/kg bw/d dose group males with minimal to moderate hyperplasia of the epidermis accompanied, in some animals, by sebaceous gland hyperplasia, hyperkeratosis, dermal inflammation and/or parakeratosis. The severity of the changes was comparable between all treatment groups in both the male and female mice. Thus, no target organ for toxicity was identified (other than skin at the SOA).

The Lowest-Observed-Effect-Level (LOEL) at the application site was 45 mg/kg bw/d based on histopathologic examination. The No-Observed-Effect-Level (NOEL) at the application site was 22.5 mg/kg bw/d. The NOAEL for systemic toxicity can be estimated to higher than 360 mg/kg bw/d.

Justification for classification or non-classification

The repeated dose toxicity of sodium mercaptoacetate was evaluated by oral and dermal administration.

By the dermal route, no systemic toxicity was observed in rats and mice up to the highest tested dose levels.

By the oral route, a key 90-day toxicity study (OECD 408) was performed at the dose levels of 7, 20 and 60 mg/kg/day. Supporting information is also provided by a 2-generation reproductive toxicity study (OECD 416) performed at dose levels of 10, 20 and 40 mg/kg/day and a reproduction/developmental screening test (OECD 421) performed at dose levels of 20, 40 and 80 mg/kg/day.

The main effects observed during these three oral studies were sporadic mortalities or premature sacrifice at the dose levels of 80 and 60 mg/kg/day after 2 to 13 weeks of treatment (corresponding to the pre-mating period of the treatment for the reproductive toxicity studies). However, these mortalities was observed at dose levels in the range of the dose levels inducing acute lethality (the acute oral LD50 is 73 and between 50 and 200 mg/kg for mercaptoacetic acid and sodium mercaptoacetates, respectively), indicating that these dose levels exceeded the maximal tolerated dose for a repeated dose toxicity study.

Treatment-related effects on the liver and/or some associated blood chemistry parameters were observed at dose levels of 40 mg/kg/day and upward. It has been demonstrated that mercaptoacetate induced an inhibition of theβ-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 theβ-oxidation pathway. In the repeated dose toxicity studies, the liver effects consisted to minimal to moderate periportal hepatocellular microvacuolation and were associated in the 90-day study with significant decrease in blood glucose and ß‑hydroxybutyrate and increase of fatty acids and lactate in the animals fasted before the blood sampling. These liver effects are consistent with the mechanism of action of inhibition of theβ-oxidation of fatty acids and were fully reversible after a 4-week treatment-free period.

The changes in the heart observed in the 90-day oral rat study (OECD 408) consisted of cardiotoxicity in a single animal which was killed at the terminal sacrifice. The degree of damage was more severe than normally seen in animals of this age but was comparable with that seen in older control rats. The significance of this change in only one rat out of 10 in the high dose group is of doubtful significance, may or may not have been related to treatment, and was thought unlikely to be of a degree sufficient to be a cause of death. Such effects were not seen in the hearts of rats in either the OECD 421 or OECD 416 studies.

To evaluate the new histopathology results from the three potential target organs, liver, heart and kidney from the animals sacrificed in the OECD 416 study in more detail, a personal meeting was held at CIT in March 2010. As a result, the CIT pathologists could finally conclude that only similar mild changes were observed in the OECD 421, 408 and 416 study, and that none of the changes observed in the tissues were of sufficient magnitude to account for the deaths of any of the dams in that study. The pathologists concluded that they had not identified a clear target organ which showed toxicity of a degree sufficient to lead to serious illness or death. No cause of the deaths could be identified (Frank M Sullivan letter to Dr Detlef Schmidt from 28^thSeptember 2010).

The fatty changes and vacuolation observed in the liver in the studies, along with the clinical chemistry changes, suggested that it was possible that important biochemical changes could be induced by sodium mercaptoacetate, especially affecting fat and glucose metabolism. Thus, based on the results of the 90-day OECD 408 study, together with the histopathology results from the OECD 416 multigeneration study, it has not been possible to identify a target organ with evidence of severe toxicity, and so no classification for STOT-RE is indicated by these studies.

The observed mortality occurred at higher dose levels in repeated dose studies were in the range of the acute oral LD50 for mercaptoacetates. Mercaptoacetates are therefore classified for acute lethal effects with Acute Tox 3 - H301: Toxic if swallowed. The fact that rats in oral repeated-dose studies died only after multiple days of dosing rather than after a single dose can be attributed to the fact that the acute oral study featured fasted animals whereas the repeated-dose studies used gavage dosing to non-fasted animals with ad libitum access to feed. On the basis of the above results, and of a review of the literature on mercaptoacetates, it has been suggested that a possible biochemical mechanism which could account for the lethality is the production of hypoglycaemia, especially in fasted animals, of a degree sufficient to result in death. The enhancing effect of fasting on the toxicity of mercaptoacetates has been demonstrated in the study by Grosdidier (2011, Report No. 37043, IUCLID Section 7.9.4). Hypoglycaemia induced by mercaptoacetates is much more pronounced in fasted animals and this is likely to contribute to the difference in acute vs. subchronic toxicity of this compound category.

In conclusion, in animals exposed to up acutely toxic doses of mercaptoacetate, in both 90-day and reproductive toxicity studies, no target tissue has been identified with pathological damage of a degree of severity sufficient to account for the serious toxicity observed. It is thus assumed that a functional or biochemical change has been sufficient to result in death. No target organ has been identified which could lead to classification for a specific target organ systemic toxicity - repeated exposure according to the criteria of Regulation (EC) No 1272/2008. The mortality observed after repeated mercaptoacetate dosing does not represent a separate hazard that would require a specific classification. The lethal effects are sufficiently covered by the proposed classification Acute Tox 3 - H301: Toxic if swallowed.