(2,4,6-trioxo-1,3,5-triazine-1,3,5(2H,4H,6H)-triyl)triethane-2,1-diyl tris(3-mercaptopropionate)

Endpoint summary

Description of key information

NOAEL = 20 mg/kg bw/d (13 wk repeated dose toxicity study, oral, rat, NaTG)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and source substances have similar toxicological properties because:
- all substances are small organic molecules;
- they share structural similarities with common functional groups: one or more thiol and/or thioether group(s) and carboxylic acid (as free acid, salt or ester);
- the metabolism (i.e. ester hydrolysis) leads to comparable products (sulfur-containing core structure in its acid form and alcohols of differing chains lengths)
- covalent protein binding via S-S bonds may be a common mode of action and or chelation of divalent cations via sulfur

The substances were assigned to subgroups according to their main structural features (see Table 1); further justification for subgrouping based on toxicological properties is given below:
- TGA family: Thioglycolic acid, its salts and esters
- 3-MPA family: 3-Mercaptopropionic acid, its salts and esters
- TLA family: Thiolactic acid and its salts
- Intramolecular-S family: Thiodiglycolic acid or Dithiodiglycolic acid and its esters, Thiodipropionic acid or Dithiodipropionic acid and its esters, Methylene bis(butyl thioglycolate)
- Mercaptanes: Thioglycerol, Bis(2-mercaptoethyl) sulfide, 4-Mercaptomethyl-3,6-dithia-1,8-octanedithiol

The acids and salts will dissociate to the respective Thioglycolate or 3-Mercaptopropionate or Thiolactate and the corresponding cation.
In case of the esters, the metabolism expected to occur is ester hydrolysis resulting in the corresponding acid and alcohol.

It was demonstrated, that PETMP and 3-MPA strongly bind to plasma proteins (e.g. via S-S bond to cysteine) in vitro, which is well known for substances containing free SH-groups (Bruno Bock, 2014). Strong protein binding is also expected to occur with the other substances assessed within this paper. The members of the intramolecular-S family are an exception, as they do not contain free SH-groups – protein binding may be less relevant for this family.

Overall, based on close structural similarities, a read-across from the existing repeated dose and reproduction toxicity studies is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see cross-reference -> supporting information

3. ANALOGUE APPROACH JUSTIFICATION
see cross-reference -> supporting information

4. DATA MATRIX
see cross-reference -> supporting information

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read-across: supporting information

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Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Executive summary:

Across the whole group of substances, there were some differences in the type of effects and effect levels: Mainly unspecific toxicity was observed in the 3-MPA, TLA, intramolecular-S and mercaptan family.

 

In the oral repeated dose toxicity studies conducted with NaTG, 2-EHTG, and Di-2-EHDTDG, mild liver toxicity was observed consisting of e.g. hypertrophy, vacuolation, and lipidosis.

 

Comparing these effects to the literature, in vivo and in vitro studies with liver mitochondria suggested that the most probable mechanism of toxicity of TGA and 3-MPA is the inhibition of the β‑oxidation of fatty acids. TGA (syn. 2-mercaptoacetate) is a substrate for acetyl-CoA synthase, the resulting compound, 2-mercaptoacetyl-CoA may inhibit the long chain acyl-CoA dehydrogenase. Consequently, the concentration of long-chain fatty acids or acyl-CoA will increase in the mitochondria. Consequently, they will be esterified in the liver to form triglycerides leading to lipidosis (Bauché, 1981; Bauché, 1982; Bauché, 1983).This is in line with the effects observed in the available studies.

 

Nevertheless, the effects observed in the studies described above were not sufficient to explain the mortality observed at dose levels of 40 mg/kg bw/d and higher (NaTG).

The changes in blood fatty acid levels and lipidosis in liver were likely to be the result of changes in the biochemistry of the animals and reflected physiological, rather than pathological changes induced by the test material. Changes were observed in the clinical chemistry with high transaminases which also indicated toxicity to the liver, but these changes were very high in only one animal.

 

Thus, as no specific target organ with evidence of severe toxicity has been identified, a classification for specific target organ toxicity after repeated exposure is not warranted.

 

For better comparison, the NO(A)ELs have been recalculated on the basis of S-content, which is assumed to be the main driver for toxicity. Additionally, an adjustment for differences in study duration was made (subacute and chronic studies were normalized to subchronic):

 

Overall comparison of NO(A)ELs(general toxicity)

Family	Substance (study)	NO(A)EL [mg/kg bw/d]	% S in molecule	Related to S-content	Adjustment for study duration
TGA	NaTG (13 wk NOAEL, OECD TG 408, oral: gavage)	20	30.7	6.1	6.1
	NaTG (ca 90 - 111 d NOAEL, OECD TG 421, oral: gavage, general toxicity)	20	30.7	6.1	6.1
	NaTG (NOAEL, OECD TG 416, oral: gavage)	20	30.7	6.1	6.1
	GMT (4 wk NOEL, OECD TG 422, oral: gavage)	50	21.1	10.5	3.5
	2-EHTG (6-7 wk NOAEL, OECD TG 421, oral: gavage, general toxicity)	50	17.1	8.6	2.9
	2-EHTG (4 wk NOAEL, OECD TG 407, oral: diet, general toxicity)	170	17.1	29.1	9.7
3-MPA	MMP (4 wk NOAEL, OECD TG 422, oral: gavage)	50	29.1	14.6	4.9
	PETMP (13 wk NOAEL, OECD TG 408, oral: gavage)	50	7.2	3.6	3.6
TLA	TLA (4 wk NOAEL, OECD TG 407, oral:gavage)	150	33.0	49.5	16.5
Intra-molecular-S	E12 (13 wk NOAEL, OECD TG 408, oral: gavage)	350	6.8	23.8	23.8
	E18 (2 yr NOAEL, oral: diet)	1125	5.1	57.6	115.2
	Di-2-EHDTDG (4 wk NOAEL, OECD TG 407, oral: gavage)	200	9.3	18.7	6.2
Mercaptans	DMPT (4 wk NOAEL, OECD TG 407, oral: gavage)	50	13.5	6.7	3.3

 

After adjustment to S-content and study duration, the NOAELs of most of the substances were within the same order of magnitude, with the exception of TLA, E12 and E18, which were less toxic. Using the lowest NOAEL obtained in the 13 wk repeated dose toxicity study conducted with NaTG is considered to be an appropriate starting point for DNEL derivation. Remaining uncertainties due to read-across will be taken care of by applying appropriate assessment factors.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available key studies are reliable or reliable with restrictions (Klimisch 1 – 2) and were conducted according to or similar to guidelines.

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

Additional information

For the assessment of repeated dose toxicity, data are available from NaTG, GMT, 2-EHTG, MMP, PETMP, TLA, E12, E18, Di-2-EHDTDG and DMPT. A justification for read-across is attached to IUCLID section 13.

 

Studies with NaTG

NaTG was administered daily, for 14 days, by the oral route (gavage), to Sprague-Dawley rats at dose-levels of 15/100/150, 30, 60 or 75 mg/kg/day (Arkema, 2010b). Following absence of toxicity, the 15 mg/kg/day dose-level was increased, on day 8 of dosing, to 100 mg/kg/day and was further increased, on day 11 of dosing, to 150 mg/kg/day. Due to mortalities at 150 mg/kg/day, the remaining animals in the group were not dosed on day 14. The dose-level of 150 mg/kg/day resulted in mortality, reduced body weight gain (males) or body weight loss (females) and reduced food consumption. There were no group mean effects of treatment on body weight performance or food consumption at dose-levels of 15/100, 30, 60 or 75 mg/kg/day. Four animals given 75 mg/kg/day had ptyalism at the end of the study and one male given 30 mg/kg/day and one male given 75 mg/kg/day had low body weight gains between day 7 to day 11 or day 1 to day 4, respectively. Macroscopic abnormalities were observed in the liver at 30 (females), 60 (males), 75 and 15/100/150 (females) mg/kg/day and in the kidneys in females given 60 or 75 mg/kg/day and in males given 60 or 15/100/150 mg/kg/day. 150 mg/kg/day is considered to exceed the maximum tolerated dose because mortality occurred after 3 days of treatment.

 

In a 13 wk oral study with NaTG (0, 7, 20, 60 mg/kg bw/d) in rat (Bruno Bock, 2010) 2 animals (1 m, 1 f) of the high dose group (60 mg/kg bw/d) died or were sacrificed prematurely. In the high dose increased absolute and relative liver weights were noted correlating with minimal centrilobular hepatocellular hypertrophy. Further, higher values of liver transaminases (ALAT and ASAT) were observed, periportal hepatocellular vacuolation, lipidosis, tubular vacuolation in the kidneys correlated with increased urea and creatinine values.

LOAEL=60 mg/kg bw/d; NOAEL = 20 mg/kg bw/d; NOEL=7 mg/kg bw/d

 

In a 13 wk dermal study with NaTG (0, 11.25, 22.5, 45, 90, 180 mg/kg bw/d in rat; 0, 22.5, 45, 90, 180, 360 mg/kg bw/d in mice) in either mouse or rat no mortality occurred (NTP, 2003). Increases in absolute and relative liver weights and relative kidney, spleen and testes weights were observed in rat, which were without histopathological correlates. In mice absolute and relative liver and heart weights were increased without histopathological correlates. The only histopathogical observation was at the site of application (dermal hypertrophy or hyperplasia).

NOAEL(rat, systemic) >/= 180 mg/kg bw/d; LOAEL(local) =11.25 mg/kg bw/d, no NOAEL determined

NOAEL(mouse, systemic) >/= 360 mg/kg bw/d; NOAEL(local) =22.5 mg/kg bw/d

 

In the reproduction/developmental toxicity screening study with NaTG (Bruno Bock, 2010b) (0, 20, 40, 80 mg/kg bw/d) increased liver and kidney weights were observed in males dosed with 80 mg/kg bw/d (high dose), as well as an increased glycogen content. The NOAEL for general parental toxicity was 20 mg/kg bw/d based on mortality at 40 and 80 mg/kg bw/d.

 

In the 2-generation reproduction toxicity test with NaTG (Arkema, 2010) (0, 10, 20, 40 mg/kg bw/d) minimal to moderate periportal hepatocellular microvacuolation was observed at 40 mg/kg bw/d (high dose) in 2/25 males and 6/25 females, and in 4/6 prematurely sacrificed or found dead females suggesting mild liver toxicity at this dose-level. Furthermore, female plasma fatty acid concentration was statistically significantly decreased. The NOEL for general parental toxicity was 20 mg/kg bw/d.

 

Study with GMT

GMT was tested in a combined repeated dose/reproduction/developmental toxicity screening test (Thioesters Association, 2007a) (0, 15, 50, 150 mg/kg bw/d). In the high dose group (150 mg/kg bw/d) 5 females died or were killed in extremis. The cause of death was, however, not evident from histopathological examination. Females of the high dose group showed reduced food consumption, body weight and body weight gain (not statistically significant). No other treatment related effects were noted. The NOEL for systemic toxicity was 50 mg/kg bw/d.

 

Studies with 2-EHTG

2-EHTG was tested in a 7 d (Bibra, 1992) (0, 150, 200, 250 mg/kg bw/d) and in a 28 d (Bibra, 1988) (0, 0.05, 0.1, 0.2% in diet) repeated dose toxicity study in rats. In the 7 d study, 6/10 of the high dose animals (250 mg/kg bw/d) and 4/10 of the mid dose animals (200 mg/kg bw/d) died. The main histopathological effect was microvesiculation in the liver, possibly due to fat accumulation. The 7 d NOAEL was 150 mg/kg bw/d.

No adverse effects were noted in the 28 d dietary study (0, 0.05, 0.1 or 0.2% 2-EHTG in diet) up to the highest dose level. NOAEL >/= 168 (males) / 173 (females) mg/kg bw/d

 

2-EHTG in corn oil was administered orally by gavage to three groups of five Crl:CD (SD) rats/sex/dose once daily for 14 days (Thioesters Association, 2005b). Dosage levels were 10, 50 and 150 mg/kg/day administered at a dosage volume of 4 mL/kg. A concurrent control group composed of five animals/sex received the vehicle on a comparable regimen. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded daily. On study day 14, each surviving animal was subjected to a gross necropsy and selected organs were weighed.

One female in the 150 mg/kg/day group was found dead on study day 2. Decreased defecation was observed in three females in the 150 mg/kg/day group.

Body weight losses and associated slight decrease in feed consumption were observed in the 150 mg/kg/day group males and females during the first week of dose administration. Nevertheless, when the overall treatment period (study days 0-14) was evaluated, mean male and female body weight gains in the 150 mg/kg/day groups were found to be only slightly lower (not statistically significant) than the respective control groups.

Increased absolute and relative (to final body weight) liver weights were observed in the 50 mg/kg/day (males) and the 150 mg/kg/day (males and females) groups. Absolute and relative kidney weights were increased in the 50 and 150 mg/kg/day males compared to control group values. Absolute and relative thymus gland and thyroid/parathyroid gland weights in the 150 mg/kg/day males and females were slightly reduced compared to the control animals.

 

In the reproduction/developmental toxicity screening study with 2-EHTG (0, 10, 50, 150 mg/kg bw/d) (Thioesters Association, 2005) 3 males and 3 females were found dead or were euthanized in extremis in the high dose (150 mg/kg bw/d).

At the same dose level reduced body weight and/or body weight gain was observed in males, though food consumption was similar to controls. In the prematurely deceased animals pale liver correlated with hepatocellular vacuolization was observed. Relative liver and kidney weights were increased at this dose level in the surviving animals. The NOAEL for general parental toxicity was 50 mg/kg bw/d.

 

Study with MMP

MMP was tested in a combined repeated dose/reproduction/developmental toxicity screening test (0, 25, 50, 100 mg/kg bw/d) (Thioesters Association, 2007b). In the mid and high dose, relative liver weights were dose dependently increased in males. In the absence of a histopathological correlation this was considered to be of no adverse character. Furthermore at 100 mg/kg/day, a minimal to slight hyperplasia of the forestomach squamous epithelium was noted in males and females. No other test-item related adverse effects were noted. The NOAEL for general toxicity was 50 mg/kg bw/d.

 

Studies with PETMP

In the 14 d dose range finding study with PETMP (0, 50, 200, 800 mg/kg bw/d) (Bruno Bock, 2015a), all males and all females but one of the high dose group (800 mg/kg bw/d) died before scheduled necropsy. Clinical signs included sedation, weakened condition and ruffled fur. Reduced food consumption and lower mean body weights were noted. In the mid dose, transient sedation was noted. There were no effects on food consumption or body weights. There were no substance related adverse effects on organ weights or macroscopical findings. In the low dose, no clinical signs were evident and there were no effects on food consumption or body weights. There were no organ weight differences of toxicological relevance or macroscopical findings.

 

In the 13 wk repeated dose study with PETMP (Bruno Bock, 2015b) (0, 12.5, 50, 200 mg/kg bw/d) no test item-related deaths were noted, no differences in mean food consumption or body weights, weekly observations (weeks 1 - 13) or functional observational battery (week 13), no differences of toxicological relevance in the fore- and hind limb grip strength values, no test item-related differences in the ophthalmoscopy, no test item related effects on hematology or urine parameters. There were no test item-related macroscopic findings. There were differences in blood biochemistry (higher levels of sodium, potassium and chloride in mid and high dose males, and higher potassium and chloride levels in high dose females). Test item-related microscopic findings were recorded in the stomach of high dose animals (slight forestomach erosion, slight forestomach ulceration, minimal or moderate forestomach squamous hyperplasia). Minimal forestomach squamous hyperplasia was also noted in one low dose female and two mid dose females. The NOAEL was 50 mg/kg bw/d.

 

Study with TLA

TLA was tested in a subacute toxicity study (Bruno Bock, 2000) according to OECD TG 407 (28 d + 14 d recovery) (0, 15, 150, 500/250 mg/kg bw/d). Five treatment-related deaths occurred in the high dose (500 mg/kg/day, reduced to 250 mg/kg/day from day 7), clinical signs consisted of clonic convulsion, distended abdomen, dehydration, pallor of the extremities, hunched posture, lethargy, pilo-erection, decreased respiratory rate, gasping, laboured and noisy respiration and tiptoe gait, which persisted also after reduction to 250 mg/kg bw/d. Laboured and noisy respiration were still observed during the first week of the treatment-free period.

Reduced bodyweight gain with individual weight loss and reduced food consumption was observed in the 500 mg/kg bw/d group; after reduction to 250 mg/kg/d, bodyweight normalised and was similar to controls. Effects on hematology and blood biochemistry were noted in high dose animals, which were at least partially reversible. Absolute and relative liver and kidney weight were increased in high dose animals and still present in recovery females

Gross pathology and histopathology revealed changes in the gastro intestinal tract:

Stomach: Mucosal erosion / submucosal inflammatory cell infiltrates in the stomach as well as acanthosis, hyperkeratosis, inflammatory cell infiltrates and focal ulceration in the forestomach.

The effects observed were most likely a result of gastric irritation which in turn produced blood chemical changes resulting in a so-called protein losing enteropathy

No treatment-related effects were observed in the low and mid dose. NOEL = 150 mg/kg bw/d

 

Studies with E12, E18 and DTDPA

Oral gavage administration of E12 to rat at dose levels of between 125 and 1000 mg/kg/day for four weeks was not associated with any signs of toxicity. Consequently, dose levels of 125, 350 and 1000 mg/kg/day were recommended for the 13 week study (BASF, 1993).

 

In a 13 week repeated dose toxicity study with E12 (0, 125, 350, 1000 mg/kg bw/d) (Thioesters Association, 2001a)no unscheduled deaths, no treatment related clinical signs, no effects on body weight gain and food consumption and hematology were observed. A reversible elevation in serum cholesterol in the high dose females and a reversible elevation of ALAT + ASAT activities in all high dose animals were noted.

Minor differences in the weight of the major organs were considered of no toxicological significance in the absence of microscopic lesions.

Treatment related microscopic lesions were seen in the heart of high dose animals. The lesion was described as small foci of degenerated or necrotic fibers associated with minimal to moderate mononuclear cell infiltration. This association suggested early or ongoing myocarditis. These lesions were not present in recovery animals.

NOAEL = 350 mg/kg bw/d, NOEL = 125 mg/kg bw/d

 

In a 2 yr chronic toxicity study (Thioesters Association, 2001b) with E18 (0, 0.5, 1, 3% in diet) only minor effects on the weight development were observed. At the end of the study, 3, 2, 7 and 2 of 20 rats died from the control, 0.5, 1.0 and 3.0% groups, respectively. No characteristic gross pathology was evident from the autopsies performed on the respective experimental groups. NOAEL = 3% in diet, corresponding to approx. 1125 mg/kg bw/d.

 

E12,E18 andDTDPA have been evaluated as antioxidants for fats (WHO, 1962): up to 3% in diet (corresponding to approx. 1500 mg/kg bw/d) did not cause adverse effects in the rat.

 

Study with Di-2-EHDTDG

In a 28 d toxicity study + 2 week recovery period (0, 10, 50, 200, 800 mg/kg bw/d) with Di-2-EHDTDG (Ciba-Geigy, 1992) 2/10 males and 5/10 females died in the 800 mg/kg bw/d dose group. Due to mortality and marked signs of toxicity, the 800 mg/kg bw/d dose group was terminated. Body weight, food and water consumption were reduced in this group. At necropsy fatty change of perilobular region in liver, hyperplasia/ulceration of non-glandular stomach, atrophy of testis and thymus were observed. Minor reversible hematological findings (minimal increase in white blood cell count in males and females) at 200 mg/kg bw/d. No other adverse effects were noted. NOEL = 50 mg/kg bw/d, NOAEL = 200 mg/kg bw/d.

 

Study with DMPT

DMPT was tested in a 4 week toxicity study + 2 week recovery in rats (0, 10, 50, 200 mg/kg bw/d) (Mitsui Toatsu Chemicals Incorporated, 1991). No mortality occurred. Clinical signs including salivation, stained fur, hair-loss, lack of grooming were present in high dose animals.Body weight gain and food efficiency were low. Hematologiocal changes were observed in high dose animals:slightly high erythrocyte count, low mean cell volume and mean cell hemoglobin in males; high platelet count in males and females; longer activated partial thromboplastin time in males and longer prothrombin time in males and females. Prothrombin time was also longer in mid dose males. At the end of the recovery period high erythrocyte count, low mean cell volume and mean cell haemoglobin were still evident. Effects to blood biochemistry parameters were noted in high dose animals: low alkaline phosphatase and high erythrocyte acetylcholinesterase activities and low total cholesterol concentration in males and high alanine amino-transferase activity in males and females. Alkaline phosphatase activity was also low in males receiving 50 mg/kg/day. At the end of the recovery period high erythrocyte acetylcholinesterase and alkaline phosphatase activities were evident.

Absolute and relative liver weights were increased in high dose females. There were no toxicologically relevant macroscopic changes. Histopathology revealed slight centriacinar hepatocytic fatty vacuolation in 3/10 females. NOAEL = 50 mg/kg bw/d.

 

Summary

Across the whole group of substances, there were some differences in the type of effects and effect levels: Mainly unspecific toxicity was observed in the 3-MPA, TLA, intramolecular-S and mercaptan family.

 

In the oral repeated dose toxicity studies conducted with NaTG, 2-EHTG, and Di-2-EHDTDG, mild liver toxicity was observed consisting of e.g. hypertrophy,vacuolation, and lipidosis.

 

Comparing these effects to the literature,in vivoandin vitrostudies with liver mitochondria suggested that the most probable mechanism of toxicity of TGA and 3-MPA is the inhibition of the β‑oxidation of fatty acids. The thioglycolate also known as 2-mercaptoacetate anion is a substrate for acetyl-CoA synthase, the resulting compound, 2-mercaptoacetyl-CoA may inhibit the long chain acyl-CoA dehydrogenase. Consequently, the concentration of long-chain fatty acids or acyl-CoA will increase in the mitochondria. Consequently, they will be esterified in the liver to form triglycerides leading to lipidosis (Bauché, 1981; Bauché, 1982; Bauché, 1983).This is in line with the effects observed in the available studies.

 

Nevertheless, the effects observed in the studies described above were not sufficient to explain the mortality observed at dose levels of 40 mg/kg bw/d and higher (NaTG).

The changes in blood fatty acid levels and lipidosis in liver were likely to be the result of changes in the biochemistry of the animals and reflected physiological, rather than pathological changes induced by the test material. Changes were observed in the clinical chemistry with high transaminases which also indicated toxicity to the liver, but these changes were very high in only one animal.

 

Thus, as no specific target organ with evidence of severe toxicity has been identified, a classification for specific target organ toxicity after repeated exposure is not warranted.

 

For better comparison, the NO(A)ELs have been recalculated on the basis of S-content, which is assumed to be the main driver for toxicity. Additionally, an adjustment for differences in study duration was made (subacute and chronic studies were normalized to subchronic).

 

Overall comparison of NO(A)ELs (general toxicity)

Family	Substance (study)	NO(A)EL [mg/kg bw/d]	% S in molecule	Related to S-content [mg/kg bw/d]	Adjustment for study duration
TGA	NaTG (13 wk NOAEL, OECD TG 408, oral: gavage)	20	30.7	6.1	6.1
	NaTG (ca 90 - 111 d NOAEL, OECD TG 421, oral: gavage, general toxicity)	20	30.7	6.1	6.1
	NaTG (NOAEL, OECD TG 416, oral: gavage)	20	30.7	6.1	6.1
	GMT (4 wk NOEL, OECD TG 422, oral)	50	21.1	10.5	3.5
	2-EHTG (6-7 wk NOAEL, OECD TG 421, oral: gavage, general toxicity)	50	17.1	8.6	2.9
3-MPA	MMP (4 wk NOAEL, OECD TG 422, oral: gavage)	50	29.1	14.6	4.9
	PETMP (13 wk NOAEL, OECD TG 408, oral: gavage)	50	7.2	3.6	3.6
TLA	TLA (4 wk NOAEL, OECD TG 407, oral:gavage)	150	33.0	49.5	16.5
Intra-molecular-S	E12 (13 wk NOAEL, OECD TG 408, oral: gavage)	350	6.8	23.8	23.8
	E18 (2 yr NOAEL, oral: feed)	1125	5.1	57.6	115.2
	Di-2-EHDTDG (4 wk NOAEL, OECD TG 407, oral: gavage)	200	9.3	18.7	6.2
Mercaptans	DMPT (4 wk NOAEL, OECD TG 407, oral: gavage)	50	13.5	6.7	3.3

 

After adjustment to S-content and study duration, the NOAELs of most of the substances were within the same order of magnitude, with the exception of TLA, E12 and E18, which were less toxic. Therefore, using the lowest NOAEL obtained in the 13 wk repeated dose toxicity study conducted with NaTG is considered to be an appropriate starting point for DNEL derivation for the whole group of substances described in Table 1. Remaining uncertainties due to read-across will be taken care of by applying appropriate assessment factors.

References

 

Arkema, 2010. Two-generation reproduction toxicity study by oral route (gavage) in rats. Unpublished report. CIT 35047 RSR

 

Arkema, 2010b.Sodium Thioglycolate, 2-week range-finding toxicity study by oral route (gavage) in rats. Unpublished report. CIT 30720 TSR

 

Bauché F. et al. 1981. 2-Mercaptoacetate administration depresses the β-oxidation pathway through an inhibition of long-chain acyl-CoA dehydrogenase activity. Biochem. J. (1981) 196, 803-809

 

Bauché F. et al. 1982. Effects of 2-mercaptoacetate in isolated liver mitochondria in vitro. Biochem. J. (1982) 206, 53-59

 

Bauché F. et al. 1983. Inhibition in vitro of acyl-CoA dehydrogenases by 2-mercaptoacetate in rat liver mitochondria. Biochem. J. (1983) 215, 457 – 464

 

BASF, 1993. 4 week oral (gavage) dose range-finding study in the rat. Unpublished report. Pharmakon Europe 380/572

 

Bibra, 1988. A 28-day study in rats with thioglycolic acid, 2-ethylhexyl ester including investigation of hepatic peroxisomal activity. Unpublished report. Bibra 689/1/88

 

Bibra, 1992. A 7-day toxicity study with thioglycolic acid-2-ethylhexyl ester. Unpublished report. Bibra 932/3/92

 

Bruno Bock, 2000. Thiolactic acid 98/99%: Twenty-eight day repeated dose oral (gavage) toxicity study in the rat.Unpublished report. SPL 1158/028.

 

Bruno Bock, 2010. 13-week toxicity study by oral route (gavage) in rats followed by a 4-week treatment-free period. Unpublished report. CIT 34814

 

Bruno Bock, 2010b. Reproduction/developmental toxicity screening test by oral route (gavage) in rats. Unpublished report. CIT 30721 RSR

 

Bruno Bock, 2015a.PETMP: 14-Day Oral Toxicity (Gavage) Study in the Wistar Rat.Unpublished report. HarlanD89713

 

Bruno Bock, 2015b.PETMP: 13-Week Oral (Gavage) Toxicity Study in the Wistar Rat with a 4-Week Recovery Period.Unpublished report. HarlanD89724

 

Ciba-Geigy, 1992. 28 days subactute, oral toxicity study in rats (gavage). Unpublished report. Ciba-Geigy 894135

 

Mitsui Toatsu Chemicals Incorporated, 1991. GST: Four-week toxicity and reversibility study following oral administration to rats. Unpublished report. LSR 90/MT0050/1286

 

NTP, 2003. Thirteen-week subchronic dermal toxicity study of sodium thioglycolate (NaT) in Fisher 344 rats and B6C3F1 mice. Unpublished report. BioReliance 98007.03/99-98007.04

 

Thioesters Association, 2001a. IUCLID data set for 3,3’-thiodipropionic acid, didodecyl ester, submission to US EPA

 

Thioesters Association, 2001b. IUCLID data set for 3,3’-thiodipropionic acid, dioctadecyl ester, submission to US EPA

 

Thioesters Association, 2005. 2-Ethylhexyl mercaptoacetate [CAS No. 7659-86-1]: a reproduction/developmental toxicity screening study in rats. Unpublished report. WIL-528002

 

Thioesters Association, 2005b. A 14-day dose range-finding study of 2-ethylhexyl mercaptoacetate in rats. Unpublished report.WIL-528001

 

Thioesters Association, 2007a. Glyceryl thioglycolate 80%: oral (gavage) combined repeat dose toxicity study with reproduction/developmental toxicity screening test in the rat. Unpublished report. SPL 2207/0003

 

Thioesters Association, 2007b.Methyl 3-Mercaptopropionate:Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat. Unpublished report.RCC A74698

 

WHO, 1962. Evaluation of a number of antimicrobials and antioxidants. Sixth report of the Joint FAO/WHO Expert Committee on Food Additives.

 

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.