Sodium mercaptoacetate

Endpoint summary

Description of key information

The repeated dose toxicity of sodium mercaptoacetate has been 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:

experimental study

Adequacy of study:

key study

Study period:

May-2008 - Sep-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain and Sanitary status: Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).
- Source: Charles River Laboratories France, l’Arbresle, France.
- Age on the first day of treatment: 6 weeks old.
- Weight at study initiation: 208 g (range: 180 g to 237 g) for the males and 165 g (range: 145 g to 182 g) for the females
- Acclimatation period: at least 9 days before the beginning of the treatment period
- Housing: two rats of the same sex and group in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm)
- Diet (ad libitum): SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water (filtered with a 0.22 µm)

ENVIRONMENTAL CONDITIONS
· temperature : 22 ± 2°C,
· relative humidity : 50 ± 20%,
· light/dark cycle : 12 h/12 h (07:00 - 19:00),
· ventilation : approximately 12 cycles/hour of filtered, non-recycled air

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Preparation of dosing solutions:
The test item was mixed with the vehicle and administered as a solution. The low and intermediate concentrations (1.4 and 4 mg a.i/mL) were prepared by dilution of the high concentration (12 mg a.i/mL) with vehicle. The test item dosage formulations were prepared under nitrogen atmosphere for up to 9 days, according to known stability data (11 days), stored at +4°C and under nitrogen atmosphere prior to use and delivered in brown flasks. On day 1, distribution of the test item dosage forms prepared for administration on days 1 to 3 was not performed under nitrogen atmosphere, but nitrogen was added to the flasks prior to storage at +4°C (see § Study plan adherence).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

High Performance Liquid Chromatography with Ultra-Violet detection method. The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1, 4, 8 and 13 was determined.
The test item concentrations in the administered dosage forms analyzed in weeks 1, 4, 8 and 13 remained within an acceptable range of [-1.8% to +7.9%] of variation when compared to the nominal values.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 (dose levels 7 and 20 mg/kg bw/d)
16 (dose level 60 mg/kg bw/d)

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: 4 weeks
- Dose selection rationale:
The dose-levels were determined in agreement with the Sponsor, based on the results of a previous dose range-finding toxicity study (CIT/Study No. 30720 TSR) and on the results of a reproduction/developmental screening test (OECD 421 CIT/Study No. 30721 RSR).
During the dose range-finding toxicity study (CIT/Study No. 30720 TSR), the test item 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. The dose-level of 150 mg/kg/day, given from day 11, resulted in mortality (1/6 males and 3/6 females) and reduced body weight gain (males) or body weight loss (females) which coincided with a reduced food consumption, and was consequently considered to exceed the Maximum Tolerated Dose (MTD). There were no effects of treatment on body weight or food consumption at 15 mg/kg/day (given from days 1 to 7), 100 mg/kg/day (given from day 8 to 10), 30, 60 or 75 mg/kg/day. At 75 mg/kg/day, 2/6 males and 2/6 females showed hypersalivation at the end of the study, and one male given 30 mg/kg/day and one male given 75 mg/kg/day showed low body weight gains from day 7 to day 11 or from 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 of females given 60 or 75 mg/kg/day and of males given 60 or 15/100/150 mg/kg/day. In females, relative uterus weight was -9 to -17% lower than control mean values at all dose-levels. No histopathology was conducted.

During the OECD 421 study (CIT/Study No. 30721 RSR), the test item was administered daily by oral gavage to male and female Sprague Dawley rats for 10 weeks before mating, during mating, during gestation and until day 5 post partum, at dose-levels of 20, 40 or 80 mg/kg/day. The dose level of 80 mg/kg/day was considered to be higher than the MTD for a dosing period of 13 weeks or more. The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 20 mg/kg/day (based on deaths at 40 and 80 mg/kg/day). The No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 20 mg/kg/day (based on deaths at 40 and 80 mg/kg/day). The NOEL for toxic effects on progeny was 40 mg/kg/day (based on the dead litter at 80 mg/kg/day which cannot be definitively attributed to maternal condition).
Consequently, the dose-levels of 7, 20, and 60 mg of active sodium thioglycolate/kg/day were selected for the present study.

- Post-exposure recovery period in satellite groups: 4 weeks

Observations and examinations performed and frequency:

MORBIDITY AND MORTALITY:
Each animal was checked for mortality or signs of morbidity at least twice a day during the treatment period, including weekends and public holidays.

GENERAL CLINICAL OBSERVATION:
Each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.

DETAIL CLINICAL OBSERVATION:
Detailed clinical examinations were performed for all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study. During week 11, this examination was included in the detailed clinical observation of the Functional Observation Battery (FOB) for all animals except recovery animals.Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic and tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also recorded.

BODY WEIGHT:
The body weight of each animal was recorded once before group allocation, on the first day of treatment, then once a week until the end of the study.

FOOD CONSUMPTION:
The quantity of food consumed by the animals in each cage was recorded once a week, over a 7 day period, during the study.Food consumption was calculated per animal and per day. If one of the two animals in the same cage dies, the number of days for which that animal has been present in the cage is taken into consideration for the calculation of food consumption.

OPHTALMOLOGY:
Ophthalmological examinations were performed on all animals, before the beginning of the treatment period and on control and high-dose animals on one occasion at the end of the treatment period.

LABORATORY INVESTIGATIONS:
The following parameters were determined for all animals except recovery animals at the end of the treatment period (5 to 6 hours after the last treatment) and for recovery animals at the end of the treatment-free period. Prior to blood sampling and during urine collection, the animals were deprived of food for an overnight period of at least 14 hours.
- Hematology: erythrocytes (RBC), hemoglobin (Hb), mean cell volume (MCV), packed cell volume (PCV), mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), thrombocytes (PLT), leucocytes (WBC), differential white cell count with cell morphology, prothrombin time (PT).
- Blood biochemistry: sodium, potassium, chloride, calcium, inorganic phosphorus (I. PHOS), urea, creatinine (Creat), total bilirubin (Tot.Bil), alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), total proteins (Prot), albumin (Alb), albumin/globulin ratio (A/G), lactate (Lact), glucose (Glic), free fatty acids (Fat ac) triglycerides (Trig), total cholesterol (Chol) and ß-hydroxybutyrate(OHBut).
- Urinalysis: volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, cytology of sediment, appearance, color
- Bone marrow: Bone marrow smears were prepared from the femoral bone of all animals sacrificed prematurely or on completion of the treatment or treatment-free period. The bone marrow differential cell count was determined for control and high-dose males and females (groups 1 and 4) and low- and intermediate-dose males (groups 2 and 3) at the end of the treatment period and for recovery males (groups 1 and 4) on completion of the treatment-free period.

Sacrifice and pathology:

PATHOLOGY:
- Macroscopic post-mortem examination: a complete macroscopic post-mortem examination was performed on all animals.
- Organ weights: according to Table 1.
- Microscopic examination:
A microscopic examination was performed on:
. all the tissues listed in the Tissue Procedure Table for animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period and for all animals that died or were sacrificed prematurely,
. all the macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment period.
Based upon the microscopic results of the high-dose group, selected tissues from the low- and intermediate dose animals (groups 2 and 3) sacrificed at the end of the treatment period and from recovery animals (groups 1 and 4) sacrificed on completion of the treatment-free period were examined as follows:
. the liver of all low- and intermediate-dose males and females and of all recovery males and females,
. the kidneys of all low- and intermediate-dose females and of all recovery females,
. the heart of all low- and intermediate-dose males and of all recovery males.
Moreover, microscopic examination on liver was performed after Oil Red O stain in all the animals.

Other examinations:

- Evaluation of liver glycogen content:
One sample of the liver (right lateral lobe, and papillary process of the caudate lobe of the liver if necessary: approximately 3 g) of each animal sacrificed at the end of the treatment period was removed and immediately snap-frozen in liquid nitrogen.
The liver samples were kept at -80°C until shipment to the Principal Investigator for the determination of glycogen (based on the blood biochemistry results). The determination of glycogen was performed in the liver samples of control and high dose animals (groups 1 and 4).

Statistics:

Citox software was used to perform the statistical analysis of body weight, food consumption, hematology, blood biochemistry and urinalysis data. PathData software (version 6.2b5) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY:
On day 14 (week 2), the decision was taken to prematurely sacrifice one female given 60 mg a.i./kg/day (R23675) due to poor clinical condition. Hypoactivity, staggering gait, hunched posture, piloerection, soiled urogenital region, coldness to the touch and thin appearance were noted prior to death. Examination of biochemical parameters revealed marked hypoglycaemia (glucose: 1.32 mmol/L. The mean female control value in week 13 was 8.09 mmol/L) which was related to test item treatment and most likely contributed to the poor clinical condition. Black foci were noted in the stomach in the female, with no microscopic correlates.
One male given 60 mg a.i./kg/day (R23585) was found dead on day 90 (week 13). No signs of poor clinical condition were observed prior to death. Only hypersalivation (recorded as ptyalism) had been noted from week 6. Macroscopically, irregularly colored liver was noted in this animal, which correlated microscopically to slight periportal hepatocellular microvacuolation corresponding to marked diffuse Oil Red O-positive vacuoles (lipids), a change noted only in animals treated with sodium thioglycolate. Microscopically, the heart of this animal had moderate vacuolated myocardium characterized by cardiomyocyte vacuolation and interstitial vacuolation, however, these changes are common post-mortem related artifacts and hence cannot be easily related to changes noted in the animals sacrificed on schedule. In addition, this animal presented a minimal subacute degenerative cardiomyopathy.
No unscheduled deaths occurred at 7 or 20 mg a.i./kg/day.

CLINICAL SIGNS: Table 2
Hypersalivation (recorded as ptyalism) was noted in almost all the 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 13. Areas of thinned hair were observed in some males from 20 mg a.i./kg/day and in females from all groups. These findings were considered to be non adverse effects of the test item treatment. These signs resolved during the treatment-free period in all but two females previously treated at 60 mg a.i./kg/day.

Abnormal growth of teeth was observed in 3/15 males and 1/15 females given 60 mg a.i./kg/day. Since this change is encountered in untreated animals and since it was seen at low incidence in treated animals, it was considered to bear no relationship with treatment with the test item.
Alopecia was observed in some males in control and high-dose groups (two and six animals respectively) and in females from control to high-dose group (four, one, three and four animals, respectively). Since this sign was also observed in the control group, no test item relationship can be established.
Other clinical signs, such as reflux at dosing, ear enlargement or scabs (on the head, neck, cheeks, back or tail) were observed with a low incidence in isolated animals. These findings were not dose-related and were observed in some control animals. The incidence and distribution were similar to those usually observed in animals of this strain and age in the laboratory and consequently these clinical signs were considered not to be treatment or test item-related.

FUNCTIONAL OBSERVATION BATTERY:
No test item treatment-related effects were observed during the FOB or on motor activity. BODY WEIGHT: No treatment-related effects on the body weight and the body weight gain were observed during the treatment and the recovery period.

BODY WEIGHT
Body weight and body weight gains throughout the study were similar between test item-treated groups and controls.

FOOD CONSUMPTION:
When compared to control values, statistically significantly higher food consumption was noted 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). Food consumption remained higher in the males during the treatment-free period. This effect was considered to be related to the test item treatment.
No test item treatment-related effects were observed on food consumption in females at any dose level.

OPHTHALMOLOGY
At the end of treatment period one female given 60 mg a.i./kg/day (R23669) showed a chorioretinopathy. This finding, observed in a single animal, can be commonly found in untreated rats of this strain and age, and was thus considered not to be test item treatment-related.
No other abnormalities were observed at the end of treatment period at the ophthalmological examination.

HAEMATOLOGY: Table 3
A marked decrease in total white blood cell count (-54% in males and -59% in females) was observed in animals treated at 60 mg a.i./kg/day, when compared with controls. All the white blood cells subtypes (especially lymphocytes and eosinophils) were affected and this was considered to be related to the test item treatment at the highest dose-level.
Statistically significantly 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 with control values.
A statistically significantly higher mean prothrombin time was also noted at 60 mg a.i./kg/day in males and females when compared with controls. A small increase was also noted at 20 mg a.i./kg/day in females, which was statistically, but not biologically, significant from controls.
These hematological findings were considered to be test item treatment-related.
At the dose-level of 7 mg a.i./kg/day, no relevant abnormalities were noted.
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.

BONE-MARROW DIFFERENTIAL CELL COUNT: Table 4
The bone marrow cellularity and number of megakaryocytes were similar in the control and test item-treated groups.
When compared to control males, the M/E ratio was statistically significantly lower than in high dose control males. This finding was due to statistically significantly higher total erythroïd elements when compared to controls. As this variation was slight, and in the absence of any relevant microscopic findings in this tissue, the bone marrow differential cell count was considered not to have been affected by the test item treatment.

BLOOD CHEMISTRY: Table 5
Indication of poor liver function was observed at 60 mg a.i./kg/day (statistically significantly increased urea concentration and alanine aminotransferase activity).
A statistically significantly lower mean glucose concentration was observed in test item-treated females from 20 mg a.i./kg/day and in males at 60 mg a.i./kg/day. Mean chloride concentration was statistically significantly lower in high-dose males and females, with moderately higher (1.6 fold increase) urea levels from 20 mg a.i./kg/day in females, in a context of normal creatinine values. In males given 60 mg a.i./kg/day, moderately higher (1.7-fold increase) uremia and statistically significant higher creatinine values were observed when compared to control values.
A statistically significant higher fat acid level was also noted at 60 mg a.i./kg/day in males and from 20 mg a.i./kg/day in females when compared to control values.
Statistically significantly higher aspartate aminotransferase (males only - 2.3-fold increase) and alanine aminotransferase (males and females) activities were noted at 60 mg a.i./kg/day. Alanine aminotransferase levels increases were 2.9-fold in males (values close to what is considered as adverse levels) and marginal (1.4-fold increase) in females.
Statistically significant 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.
All these findings were considered to be test item treatment-related.
At the dose-level of 7 mg a.i./kg/day, no relevant abnormalities were noted.
At the end of the treatment-free period, when compared to controls, no blood biochemistry parameter disturbances were observed in the high-dose group, thus suggesting total reversibility of the findings.

URINALYSIS:
The urinalysis parameters were unaffected by the treatment.

ORGAN WEIGHTS: Table 6
Higher absolute and relative mean liver weights were recorded in sodium thioglycolate treated females at 60 mg a.i./kg/day and these correlated with the microscopic changes noted at microscopic examination. No differences in liver weights were observed in the recovery groups. Other differences, particularly the ones noted in uterus at the end of the treatment-free period, were considered to be fortuitous and to bear no relationship with the treatment. The cause of this statistically significant change was uterus horn dilation by serous contents, which is a normal feature of the proestrus stage of the estrous cycle in rats and hence was considered to be due to chance.

MACROSCOPIC OBSERVATIONS:
- Unscheduled deaths
Irregularly colored liver was noted in the found-dead male and correlated with periportal microvacuolation in the liver, a change which was also noted in rats at the end of the treatment period. This change could be attributed to test item treatment. Pale discoloration was noted in the liver and black foci were observed in the stomach of the female sacrificed for humane reasons. Pale colored liver could also be attributed to treatment with sodium thioglycolate and correlated with diffuse microvacuolation microscopically. Black foci in the stomach were without microscopic correlates and most likely correlated with minute mucosal hemorrhage, a non-specific agonal change.
- Scheduled sacrifice
Marked lobular pattern (synonym: accentuated lobular pattern) were noted in the liver of 2/9 males treated at 60 mg a.i./kg/day at the end of the treatment period and these correlated with the periportal hepatocellular microvacuolation noted at microscopic examination.
Small thymus (described as "reduced in size") was found in two males and a single female treated at 60 mg a.i./kg/day. Since these thymic changes were not correlated with reduced thymic absolute or relative weights, they were considered of no toxicological importance.
The other necropsy findings noted in treated rats at the end of the treatment and the treatment-free periods were considered to be part of the background changes that may occasionally or commonly be found in untreated rats of this strain and age.

MICROSCOPIC EXAMINATIONS:
- Unscheduled deaths
In the found dead male, the liver had periportal hepatocellular microvacuolation, which corresponded to marked periportal Oil Red O positive micro-vacuoles (lipids) throughout the parenchyma, which correlated with irregularly colored liver noted at necropsy and was attributed to test item treatment. No lesion described above could account for the cause of death of this male animal.
Significant changes were noted in the liver and kidney of the female sacrificed for humane reasons and consisted of slight basilar, microvesicular proximal tubule vacuolation and slight diffuse periportal hepatocellular microvacuolation, corresponding to marked diffuse Oil Red O-positive vacuoles (lipids). These changes, which were also observed in treated animals at the end of the treatment period, were attributed to the treatment with sodium thioglycolate. No lesion described above could account for the cause of death of this female animal.
- Scheduled sacrifice
Moderately vacuolated myocardium was noted in the single male R23579 treated at 60 mg a.i./kg/day sacrificed on schedule along with an increased severity of degenerative cardiomyopathy. These changes were characterized by multifocal coalescing areas of fibroplasias, Anitschkoff cells, basophilic mononuclear cells, single cell necrosis of individual cardiomyocytes and vacuolation of cardiomyoytes. 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.
In heart of other animals, minimal to slight vacuolation were also observed in control and treated males and in one high-dose female. This lesion was considered not to be related to treatment in view of the low magnitude and its occurrence in one control male.
These findings were not seen in male rats at the end of the treatment-free period. Minimal focal degenerative cardiomyopathy was observed in 2/9 females given 60 mg a.i./kg/day but not in controls. Since this change was minimal, focal and with a pattern similar to what is found in untreated males, the relationship of this finding to treatment was considered to be unlikely.
Minimal to slight proximal tubule vacuolation was noted in the kidney of 5/9 females treated at 60 mg a.i./kg/day. These vacuoles were small (microvesicular pattern) and located at the basal pole of the tubular cells (see Table 7). This change was not observed in females treated at 60 mg a.i./kg/day at the end of the treatment-free period. In the context of an absence of indications of adversity of renal biomarkers such as creatinine in females, it is considered to be non-adverse.
Minimal to slight periportal hepatocellular microvacuolation, along with minimal single cell necrosis in the most affected male rat, were noted in the liver of 4/9 males treated at 60 mg a.i./kg/day (see text table 7). When slight, the change was characterized by densely packed microvacuoles within the cytoplasm, eliciting a change of texture of hepatocytes visible from low magnification (i.e. 20x). When minimal, it was present in a distribution and severity slightly above what may be occasionally seen in untreated controls. Minimal periportal hepatocellular microvacuolation was noted in 2/10 males treated at 20 mg a.i./kg/day and 3/9 females treated at 60 mg a.i./kg/day. This finding correlated with the accentuated lobular pattern noted in a few males at necropsy. These micro vacuoles, which were Oil Red O positive, corresponded to neutral lipids and were indicative of lipidosis (synonym: steatosis). The slight microvesicular lipidosis correlated with moderate increase in ALAT (2.9-fold). Higher severity of Oil Red O positive micro-vacuoles were noted in males treated from 20 mg a.i./kg/day and females treated at 60 mg a.i./kg/day (see text table 7). In addition, minimal centrilobular hepatocellular hypertrophy was noted in the liver of a few females and correlated with increased absolute and relative liver weights in the females. This change was considered to be a metabolically adaptive, non adverse change. These changes were no longer present at the end of the treatment-free period. In particular, lipidosis (syn. steatosis) was similar between control and treated groups as judged by Oil Red O staining of neutral lipids (see Table 8).
A minimal increase in incidence and severity of extramedullary hematopoiesis (EMH) was noted in the liver of females treated at 60 mg a.i./kg/day (see Table 9). This change was not observed in the liver of females treated at 20 mg a.i./kg/day or below. It was no longer present in the liver of females at the end of the treatment-free period. The marginally increased incidence and severity of extramedullary hematopoiesis, also seen in the spleen from females given 60 mg a.i./kg/day, was not considered to be related to treatment because of the small magnitude of the change and the lack of hematological change compatible with a significant red blood cell loss.
An increase in slight cortical atrophy (4/9 animals with grade 2 vs. 1/10 in controls) was noted in the thymus of males treated at 60 mg a.i./kg/day and correlated with small thymus at necropsy. Since it was without thymus weight correlate, it was not considered to be of any toxicological relevance.

EVALUATION OF LIVER GLYCOGEN CONTENT
The quantities of glycogen in the livers of both controls and treated rats were very low. Indeed, 28 out of 38 rat livers had a quantity of glycogen per gram of liver below the limit of detection of 0.05 mg (i.e. 1000 times less than in rat livers taken from animals having access to food, i.e. 48 mg per gram of liver). One male and one female controls had measurable levels of 0.57 and 2.11 mg/g respectively, and six males and two females in the treated groups had measurable levels of 0.34-15.23 mg/g in the males and 5.02 and 8.29mg/g. This difference from expected valued could be due to the fact that the rats coming from the study were fasted for 14 hours before autopsy.
However it could be noticed that for some treated rats the quantity of glycogen was higher than that measured in the control groups, however this quantity remains very weak.

Dose descriptor:

LOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

haematology

histopathology: non-neoplastic

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

haematology

histopathology: non-neoplastic

mortality

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects

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

Table 2. Number of affected surviving principal animals with test item treatment-related clinical signs

Sex	Male				Female
Dose-level (mg a.i./kg/day)	0	7	20	60	0	7	20	60
Ptyalism				16				15
Piloerection				3
Thinning of hair			1	1	1	1	2	6

Table 3. Selected hematology parameters (mean and SD values)

Sex	Male				Female
Dose-level (mg a.i./kg/day)	0	7	20	60	0	7	20	60
WBC (G/L)	10.79 1.805	9.72 2.912 (-10%)	10.12 1.133 (-6%)	4.96** 2.148 (-54%)	6.10 1.096	6.24 1.402 (+2%)	5.72 1.823 (-6%)	2.51** 1.085 (-59%)
. Neutrophils (G/L)	1.57 0.508	1.21 0.331 (-23%)	1.59 0.385 (+1%)	1.18 0.488 (-25%)	0.73 0.288	0.64 0.138 (-12%)	0.76 0.217 (+4%)	0.43** 0.137 (-41%)
. Eosinophils (G/L)	0.11 0.024	0.12 0.043 (+9%)	0.16 0.077 (+45%)	0.06* 0.033 (-45%)	0.11 0.054	0.10 0.038 (-9%)	0.08 0.023 (-27%)	0.03** 0.018 (-73%)
. Basophils (G/L)	0.03 0.010	0.02 0.014 (-33%)	0.03 0.005 (0%)	0.01** 0.005 (-67%)	0.01 0.007	0.01 0.007 (0%)	0.01 0.007 (0%)	0.00 0.005 (-100%)
.Lymphocytes and large      unstained cells(G/L)	8.72 1.969	8.12 2.803 (-7%)	7.90 1.342 (-9%)	3.47** 1.576 (-60%)	5.05 0.906	5.30 1.337 (+5%)	4.61 1.714 (-9%)	1.98** 1.032 (-61%)
. Monocytes (G/L)	0.37 0.069	0.25 0.102 (-32%)	0.43 0.143 (+16%)	0.24 0.215 (-35%)	0.21 0.077	0.19 0.061 (-10%)	0.27 0.162 (+29%)	0.07** 0.051 (-67%)
Red Blood Cells (T/L)	8.57 0.311	8.81 0.351 (+3%)	8.61 0.376 (+0%)	9.07** 0.279 (+6%)	8.02 0.243	8.01 0.252 (-0%)	8.29 0.339 (+3%)	8.40* 0.469 (+5%)
Hemoglobin (g/dL)	15.5 0.49	15.8 0.58 (+2%)	15.6 0.52 (+1%)	16.5** 0.50 (+6%)	15.0 0.33	15.2 0.39 (+1%)	15.4 0.41 (+3%)	15.9** 0.64 (+6%)
Packed Cell Volume (L/L)	0.45 0.013	0.46 0.022 (+2%)	0.45 0.017 (0%)	0.47* 0.012 (+4%)	0.42 0.013	0.42 0.014 (0%)	0.43 0.014 (+2%)	0.44* 0.022 (+5%)
Prothrombin Time (s)	16.0 1.00	15.0 2.09 (-6%)	15.7 0.55 (-2%)	20.0** 1.42 (+25%)	15.3 1.11	15.5 0.35 (+1%)	16.8 * 0.71 (+10%)	20.2** 2.75 (+32%)

Statistically significant from controls: *: p<0.05, **: p<0.01.

In brackets: percentage difference from controls.

Table 4. Mean total myeloid and erythroid elements and mean myeloid/erythroid ratio noted in the bone marrow smears at the end of the treatment period(mean and SD values)

Sex	Male				Female
Dose-level (mg a.i./kg/day)	0	7	20	60	0	60
Cellularity(incidence of grade 3)^a	10/10	10/10	10/10	9/9	10/10	8/9
Megakaryocytes (mean grade)	4	4	4	4	4	4
Total myeloid elements (%)	38.79 2.591	38.12 3.035	38.84 3.151	35.60 2.573	36.04 4.057	34.80 3.389
Total erythroid elements (%)	36.18 5.076	36.94 2.472	38.86 2.580	41.83** 3.639	40.99 4.804	43.23 4.473
Myeloid/Erythroid ratio (M/E)	1.09 0.176	1.04 0.101	1.00 0.092	0.86** 0.118	0.89 0.173	0.82 0.145
variation from controls	na	-5%	-8%	-21%	na	-8%

^a: grade 3 for cellularity is the highest (almost all fields entirely covered with cells).

Statistically significant from controls: **: p<0.01.

na: not applicable.

Table 5. Selected blood biochemistry parameters (mean and SD values)

Sex	Male				Female
Dose-level (mg a.i./kg/day)	0	7	20	60	0	7	20	60
Chloride (mmol/L)	104.9 1.24	105.7 1.22 (+1%)	104.7 0.57 (0%)	101.7** 1.07 (-3%)	105.1 1.46	105.2 0.85 (0%)	104.5 1.25 (-1%)	100.7** 1.35 (-4%)
Glucose (mmol/L)	8.29 1.283	7.47 0.480 (-10%)	8.23 0.855 (-1%)	5.88** 0.764 (-29%)	8.09 1.122	7.45 0.638 (-8%)	6.71* 0.756 (-17%)	5.47** 1.831 (-32%)
Urea (mmol/L)	4.4 0.34	4.6 0.51 (+5%)	4.4 0.61 (0%)	7.5** 0.97 (+70%)	4.3 0.41	4.7 0.42 (+9%)	6.9** 1.25 (+60%)	6.9** 1.21 (+60%)
Creatinine (µmol/L)	41 2.3	43 2.1 (+5%)	42 2.6 (+2%)	47** 3.5 (+15%)	48 3.3	49 2.7 (+2%)	48 1.8 (0%)	47 2.6 (-2%)
Triglycerides (mmol/L)	0.57 0.286	0.46 0.204 (-19%)	0.62 0.355 (+9%)	0.39 0.152 (-32%)	0.38 0.176	0.32 0.083 (-16%)	0.26* 0.046 (-32%)	0.29 0.063 (-24%)
Fatty acid (mmol/L)	0.52 0.076	0.53 0.074 (+2%)	0.49 0.086 (-6%)	1.22** 0.350 (+135%)	0.50 0.116	0.53 0.093 (+6%)	0.68* 0.174 (+36%)	1.39** 0.327 (+178%)
ASAT (IU/L)	76 15.1	80 32.4 (+5%)	77 11.1 (+1%)	176** 93.0 (+132%)	83 18.1	78 23.7 (-6%)	87 16.7 (+5%)	103 32.9 (+24%)
ALAT (IU/L)	43 15.0	37 6.6 (-14%)	45 9.6 (+5%)	126** 96.2 (+193%)	35 7.2	36 10.4 (+3%)	40 5.6 (+14%)	49* 14.7 (+40%)
Lactate (mmol/L)	1.57 0.262	1.48 0.198 (-6%)	1.64 0.459 (+4%)	2.82** 0.885 (+80%)	1.93 0.685	1.68 0.450 (-13%)	1.54 0.271 (-20%)	4.00** 1.589 (+107%)
ß‑hydroxybutyrate (µmoL/L)	302.46 161.420	279.10 93.195 (-8%)	190.81 63.877 (-37%)	39.20** 32.276 (-87%)	383.78 201.934	387.90 93.076 (+1%)	82.93** 61.089 (-78%)	61.88** 44.428 (-84%)

Statistically significant from controls: *: p<0.05, **: p<0.01.

In brackets: percentage difference from controls.

Conclusions:

Thioglycolate is known to inhibit the mitochondrial beta-oxidation of fatty acids in liver resulting in a greater conversion of the latter into triglycerides that accumulated in the liver, as a result, ketogenesis was inhibited (Bauché et al., 1977, 1981, 1982 and 1983). The changes observed in the blood chemistry parameters (decreased blood glucose and ß-hydroxybutyrate and increased lactate and fatty acids) and in the liver (microvesicular lipidosis) are consistent with the mode of action of the compound. In the kidney of the female, subtle microvacuolar changes were noted in the proximal convoluted tubules of the kidney, and again this change could correspond to mitochondrial changes.
However, within the experimental conditions of this study, the cause of death observed in 1/10 males and 1/10 females treated at high dose (60 mg a.i./kg/day) could not be attributed to any single lesion. It is considered that the morphological changes cannot be solely responsible for the death observed in these animals.
At 60 mg a.i./kg/day, one female was prematurely sacrificed for humane reasons on day 14 and 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 thioglycolate. The demise and death of these animals were attributed to treatment with sodium thioglycolate. 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 fatty acid levels were 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 thioglycolate-related changes were noted in the liver of males and females and in 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 the limited blood chemistry effects without microscopic changes in the liver observed at 20 mg a.i./kg/day, the No Observed Adverse Effect Level (NOAEL) of sodium thioglycolate 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.

The significance of the moderate vacuolation together with moderate degenerative cardiomyopathy in only one rat (R23579) 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.

Executive summary:

The potential toxicity of sodium thioglycolate 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. The basic protocol was in compliance with OECD Guideline No. 408 and method B.26 of Council Regulation (EC) No 440/2008. 

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 thioglycolate 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 thioglycolate was 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 analysed 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. Haematology, blood biochemistry (including analysis of β‑hydroxybutyrate) and urinalysis investigations were performed on all animals 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 macroscopic post-mortem examination. 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 test item concentrations in the administered dosage forms analyzed in weeks 1, 4, 8 and 13remained 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 coloured 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 countwas 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/dayin 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 males given 20 mg a.i./kg/day and in males and 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.

 

Sodium thioglycolate 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 thioglycolate. The demise and death of these animals were attributed to treatment with sodium thioglycolate.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 thioglycolate-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 theywere 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, haematological and significant blood chemistry changes associated with liver microscopic changes and the limited blood chemistry effects without microscopic changes in the liver observed at 20 mg a.i./kg/day, the No Observed Adverse Effect Level (NOAEL) of sodium thioglycolate 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 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:

experimental study

Adequacy of study:

key study

Study period:

Dec 2002 - Mar 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: According to the standard protocol posted on the NTP website 

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sigma Chemical Company (Columbus, OH), lot #88H1166
- Purity, including information on contaminants, isomers, etc.: ca 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored under a headspace of inert gas at less than or equal to −20°C, protected from light, in amber glass bottles
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: confirmed for at least 10 days

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Type of coverage:

open

Vehicle:

other: 95 % Ethanol in deionized water (1:1, v/v)

Details on exposure:

Treatment: After a 10- to 14-day quarantine period, animals are assigned at random  to treatment groups. The study includes five treatment groups each  administered a different concentrations of the test chemicals plus a  control group. Each group contains 10 animals per sex per species. The  animals receive the subject chemical by dermal route of exposure.  Controls receive vehicle alone. Animals are exposed five times per week,  weekdays only, for 90 days after which they are sacrificed with no recovery period. All animals are housed individually. 

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks, 24 h/d

Frequency of treatment:

five times per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Positive control:

not appropriate

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
Animals are weighed individually on day one on test, after seven days,  and at weekly periods thereafter. 

DETAILED CLINICAL OBSERVATIONS: Yes
Animals are observed twice daily, at  least six hours apart (before 10:00 AM and after 2:00 PM), including  holidays and weekends, for moribundity and death. Animals found moribund  or showing clinical signs of pain or distress are humanely euthanized.  Formal clinical observations are performed and recorded weekly. 

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
Blood is collected from both sexes of "special study" rats, at days 4 and  22 and from the core study rats at the end of the study. These are  processed for haematology and clinical chemistry determinations. Blood is  collected from core study mice at the end of the study for haematology  determinations.

-Haematology:
Erythrocyte count,  Mean corpuscular volume,  Haemoglobin,  Packed cell volume,  Mean corpuscular haemoglobin,  Mean corpuscular haemoglobin concentration,  Erythrocyte morphologic assessment,  Leukocyte count,  Leukocyte differential,  Reticulocyte count,  Platelet count and morphologic assessment 
-Clinical chemistry:
Sorbitol dehydrogenase (SDH),  Alkaline Phosphatase (ALP),  Creatine Kinase (CK),  Creatinine,  Total Protein,  Albumin,  Urea Nitrogen (BUN),  Total Bile Acids,  Alanine Aminotransferase (ALT)

OTHER:
Sperm Morphology and Vaginal Cytology Evaluations (SMVCE) (see section  7.8.3): SMVCE are conducted on core study rats and mice. Mortality, body weight  changes and clinical signs of toxicity are used to determine the 3 dose  levels used for the SMVCE evaluations.

Sacrifice and pathology:

GROSS PATHOLOGY AND HISTOPATHOLOGY
Organ weights:  Liver, thymus, right kidney, right testis, heart, and lungs weights are  recorded from all animals surviving until the end of the study. 
A complete necropsy is performed on all treated and control animals that  either die or are sacrificed. All tissues required for complete  histopathology are trimmed, embedded, sectioned and stained with  hematoxylin and eosin for histopathologic evaluation.

HISTOPATHOLOGY:
A complete histopathologic evaluation inclusive of gross lesions is done  on all control animals, all animals in the highest dose group with at  least 60% survivors at the time of sacrifice, and all animals in higher  dose groups inclusive of early deaths and survivors. Chemical-related  lesions (target organs) are identified, and these organs plus gross  lesions are examined for all lower doses. Only those tissues designated  as target tissues and gross lesions are evaluated in lower doses to a  no-effect-level. A complete histopathologic evaluation is performed on  all natural death/moribund sacrifice animals in lower dose groups.

Tissues examined histopathologically:  Adrenal glands,  Brain (3 sections including frontal cortex and basal ganglia, parietal  cortex and thalamus, and cerebellum and pons), Clitoral glands,  Esophagus,  Eyes,  Femur, including diaphysis with marrow cavity and epiphysis (femoral  condyle with epiphyseal cartilage plate, articular cartilage and  articular surface),  Gallbladder (mouse),  Gross lesions,  Harderian glands,  Heart and aorta,  Intestine, large (cecum, colon, rectum),  Intestine, small (duodenum, jejunum, ileum), Kidneys,  Liver (2 sections including left lateral lobe and median lobe),  Lungs and mainstem bronchi,  Lymph nodes  - mandibular and mesenteric - inguinal, gluteal, internal iliac (chronic studies only, if lesion  observed, not merely discolouration),  Mammary gland with adjacent skin,  Muscle, thigh (only if neuromuscularsigns were present),  Nasal cavity and nasal turbinates (3 sections),  Ovaries,  Pancreas,  Parathyroid glands,  Pituitary gland,  Preputial glands,  Prostate,  Salivary glands,  Seminal vesicle,  Skin: site of application (topical studies),  Spinal cord and sciatic nerve (if neurologic signs were present),  Spleen, Stomach (forestomach and glandular),  Testes with epididymus,  Thymus,  Thyroid glands,  Tissue masses and regional lymph nodes,  Trachea,  Urinary bladder,  Uterus

Statistics:

yes

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

1.Survival
All Core Study male and female rats survived until terminal sacrifice.

2.Clinical observations
Significant observations noted in both sexes were limited to dermal  irritation at the site of application (SOA), thickened skin at the SOA  and ulcerations at the SOA.
In the males, the incidence of dermal irritation at the SOA was 10/10 for  all five test article treatment groups. Thickening of the skin at the SOA  was observed in 1/10 and 2/10 rats from the 90.0 and 180.0 mg/kg dose  groups, respectively. Ulceration at the SOA was observed in 1/10, 1/10,  5/10 and 8/10 rats from the 11.25, 22.5, 90.0 and 180.0 mg/kg dose  groups, respectively. All other observations noted during the study were  not considered to be biologically significant.
In the females, the incidence of dermal irritation at the SOA was also  10/10 for all five treatment groups. Thickening of the skin at the SOA  was observed in 3/10 rats from the 45.0 mg/kg dose group and in 10/10  rats each from the 90.0 and 180.0 mg/kg dose groups. Ulceration at the  SOA was observed in 10/10 rats from each of the 90.0 and 180.0 mg/kg dose  groups. All other observations noted during the study were not considered  to be biologically significant.

3.Body Weights
Statistical analysis of the Core Study final (terminal) body weights  revealed statistically significant (p<0.05) decreased values in the male  group treated with 90.0 mg/kg when compared to the vehicle control. No  statistically significant differences in body weights were noted for the  female rats when the test article groups were compared to the vehicle  control group.

4.Organ Weights
a.Absolute Organ Weights
The absolute liver weights of the male rats treated with 45.0 mg/kg were significantly (+10%; p<=0.05) increased when compared to the vehicle  control group. There were no other statistically significant differences  in any other absolute organ weights in the male rats. There were no  statistically significant differences in any absolute organ weights in  the female rats.

b.Organ to Body Weight Ratios
The relative kidney and testes weights of the male rats treated with 90.0  (+7.6% and 6.7%, respectively) and 180.0 mg/kg (+8.2 and 7.6%,  respectively) were significantly (p<=0.05) increased when compared to the  vehicle control group. The relative liver weights for the male rats  treated with 45.0 mg/kg were significantly (+8.5%; p<=0.05) increased  when compared to the vehicle control group. The relative spleen weights  of the male rats treated with 90.0 mg/kg were significantly (+6.0%;  p<=0.05) increased when compared to the vehicle control group. The  relative kidney weights of the female rats treated with 180.0 mg/kg were  significantly (+6.6%; p<=0.05) increased when compared to the vehicle  control group. The relative thyroid/parathyroid weights of the female  rats treated with 22.5 mg/kg were significantly (-20%; p<=0.05) decreased  when compared to the vehicle control group. There were no other  statistically significant differences in any relative organ weight for  the male or female rats.

5.Clinical Pathology
There were limited statistically significant (p<0.05) differences in the  chemistry and haematology results when compared to the control group, but  these were not dose responsive nor considered to be biologically  significant.

6.Anatomic Pathology
a.Gross Lesions
For the males, abnormal gross necropsy findings were limited to skin  accumulation (no mass noted) at the SOA in 1/10 rats from the 11.25, 45.0  and 180.0 mg/kg dose groups, a nodule on the thoracic inlet of the  thoracic cavity in 1/10 rats from both the 11.25 and 22.5 mg/kg dose  groups, a mass on the median lobe of the liver in 1/10 rats from the  180.0 mg/kg dose group and a nodule on the liver in 1/10 rats from the  22.5 mg/kg dose group. In addition, one male from the 180.0 mg/kg dose  group had retained the right testis in its abdominal region. There were  no other significant abnormal gross lesions noted in any of the male  treatment groups. These findings were not considered to be test article  related or biologically significant.
For the females, abnormal gross necropsy findings were limited in number.  In the vehicle control group, one female rat was noted as having a focus  on the left kidney. In the 11.25 mg/kg test article treatment group, four  females were noted as having a nodule on the liver, one with a nodule on  the pancreas, one rat with a nodule on the thoracic cavity and one rat  with dilated uterine horns: One rat from the 22.5 mg/kg dose group had a  cyst on the left ovary. There were two and three rats from the 45.0 and  90.0 mg/kg treatment groups, respectively, noted as having a nodule on  the liver. In the high dose treatment group (180.0 mg/kg), there were two  animals with nodules on the liver, two animals with nodules in the  thoracic cavity, one rat with enlarged mediastinal lymph nodes and one  rat with dilated uterine horns. These findings were not considered to be  test article related or biologically significant. Findings which may be  contributed to the treatment of NaT were limited to the 180.0 mg/kg  treatment group where three rats were noted as having an irritation on  the SOA.

b.Microscopic Pathology Repeated dermal administration of Sodium Thioglycolate (NaT) for thirteen  weeks (excluding weekends) resulted in test article related microscopic  changes at the site of application (SOA) in both male and female rats at  all treatment doses. Changes in the skin SOA revealed minimal to mild  hyperplasia of the epidermis accompanied, in many animals, by sebaceous  gland hyperplasia and hyperkeratosis. The severity of the changes was  comparable between all treatment groups in both the male and female rats.  A NOEL was not reached in female or male rats.

Microscopic evaluation of the other tissues required by the protocol  revealed a few findings which were observed either in small numbers  and/or in both control and treated animals. And, all of these changes are  commonly observed in F344 rats. For these reasons, these changes were  considered incidental findings.
[NOTE: The pathologist used the following criteria for severity scoring  of the epidermal hyperplasia; minimal 2-3 cell layers thick, mild 4-6  cell layers thick, moderate 7-8 cell layers thick and marked >9 cell  layers thick (at the thickest point).]

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 180 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

LOAEL

Remarks:

Local effects

Effect level:

11.25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Key result

Critical effects observed:

no

Female rats organ weight summary table

Dose Group (mg/kg)

Body Wt (Sac)(g)

Heart Wt (g)

%Heart/Body

Liver Wt (g)

%Liver/Body

Lung Wt (g)

%Lungs/Body

R Kidney Wt (g)

%R Kidney/Body

Spleen Wt (g)

%Spleen/Body

Thyroid Wt (g)

%Thyroid/Body

Day 93

0

177.3 ± 2.98

0.65 ± 0.013

0.37 ± 0.004

5.60 ± 0.173

3.16 ± 0.068

1.053 ± 0.0289

0.59 ± 0.008

0.70 ± 0.014

0.392 ± 0.0041

0.44 ± 0.007

0.25 ± 0.002

0.026 ± 0.0009

0.014 ± 0.0006

11.25

185.4 ± 2.56

0.65 ± 0.016

0.35 ± 0.005

5.59 ± 0.123

3.01 ± 0.048

1.001 ± 0.0427

0.54 ± 0.020

0.73 ± 0.009

0.391 ± 0.0025

0.45 ± 0.009

0.24 ± 0.004

0.026 ± 0.0010

0.014 ± 0.0006

22.5

184.5 ± 2.74

0.66 ± 0.008

0.36 ± 0.007

6.04 ± 0.156

3.27 ± 0.057

0.978 ± 0.0219

0.53 ± 0.010

0.72 ± 0.010

0.389 ± 0.0044

0.44 ± 0.006

0.24 ± 0.004

0.023 ± 0.0007

0.012 ± 0.0004

45

185.9 ± 2.75

0.65 ± 0.011

0.35 ± 0.004

5.90 ± 0.153

3.17 ± 0.048

1.101 ± 0.0468

0.59 ± 0.027

0.72 ± 0.020

0.389 ± 0.0086

0.45 ± 0.012

0.24 ± 0.005

0.024 ± 0.0013

0.013 ± 0.0007

90

179.9 ± 3.54

0.67 ± 0.016

0.37 ± 0.008

5.94 ± 0.141

3.31 ± 0.066

1.059 ± 0.0414

0.59 ± 0.018

0.75 ± 0.021

0.415 ± 0.0090

0.44 ± 0.012

0.25 ± 0.007

0.026 ± 0.0009

Organ Weights Summary Table in male rats

(Mean ± SEM)

Dose Group (mg/kg)

Body Wt (Sac)(g)

Heart Wt (g)

%Heart/Body

Liver Wt (g)

%Liver/Body

Lung Wt (g)

%Lungs/Body

R Kidney Wt (g)

%R Kidney/Body

R Testis Wt (g)

%R Testis/Body

Spleen Wt (g)

%Spleen/Body

Thyroid Wt (g)

%Thyroid/Body

Day 93

0

334.5 ± 3.86

0.97 ± 0.014

0.29 ± 0.003

11.22 ± 0.270

3.35 ± 0.048

1.532 ± 0.0627

0.46 ± 0.019

1.14 ± 0.021

0.340 ± 0.0044

1.442 ± 0.0248

0.432 ± 0.0089

0.72 ± 0.010

0.21 ± 0.002

0.026 ± 0.0015

0.008 ± 0.0004

11.25

333.7 ± 4.16

0.98 ± 0.022

0.29 ± 0.005

11.90 ± 0.264

3.57 ± 0.062

1.502 ± 0.0512

0.45 ± 0.014

1.20 ± 0.026

0.359 ± 0.0065

1.468 ± 0.0227

0.440 ± 0.0042

0.72 ± 0.012

0.21 ± 0.003

0.026 ± 0.0010

0.008 ± 0.0004

22.5

332.0 ± 7.07

0.95 ± 0.018

0.29 ± 0.004

11.71 ± 0.341

3.52 ± 0.062

1.541 ± 0.0849

0.46 ± 0.019

1.17 ± 0.024

0.352 ± 0.0054

1.386 ± 0.0247

0.418 ± 0.0075

0.72 ± 0.017

0.22 ± 0.004

0.027 ± 0.0011

0.008 ± 0.0004

45

339.4 ± 4.77

0.97 ± 0.010

0.29 ± 0.003

12.36 ± 0.292

3.64 ± 0.046

1.518 ± 0.0535

0.45 ± 0.016

1.18 ± 0.019

0.348 ± 0.0053

1.419 ± 0.0286

0.418 ± 0.0067

0.72 ± 0.016

0.21 ± 0.003

0.030 ± 0.0049

0.009 ± 0.0014

90

312.1 ± 8.54

0.95 ± 0.023

0.31 ± 0.005

10.46 ± 0.349

3.35 ± 0.074

1.451 ± 0.0448

0.47 ± 0.012

1.14 ± 0.023

0.366 ± 0.0041

1.435 ± 0.0300

0.461 ± 0.0082

0.71 ± 0.016

0.23 ± 0.004

0.028 ± 0.0018

0.009 ± 0.0007

180

319.3 ± 5.89

0.93 ± 0.019

0.29 ± 0.004

11.26 ± 0.261

3.53 ± 0.066

1.497 ± 0.0268

0.47 ± 0.008

1.17 ± 0.024

0.368 ± 0.0048

1.482 ± 0.0282

0.465 ± 0.0081

0.72 ± 0.016

0.23 ± 0.003

0.027 ± 0.0011

0.008 ± 0.0004

Hematology Summary Table in male rats

(Mean ± SEM)

Dose Group (mg/kg)

Erythrcyt (10^6/uL)

Hgb (g/dL)

HCT (Automated) (%)

MCV (fL)

MCH (pg)

MCHC (g/dL)

Retics (10^6/uL)

Platelet (10^3/uL)

Leukocytes (10^3/uL)

Neut (10^3/uL)

Lymph (10^3/uL)

Mono (10^3/uL)

EOS (10^3/uL)

Basophils (10^3/uL)

Day 4

0

6.79 ± 0.115

13.40 ± 0.228

40.3 ± 0.70

59.25 ± 0.250

19.71 ± 0.058

33.25 ± 0.033

6.790 ± 6.3157

594.38 ± 26.679

10.613 ± 0.6269

1.18 ± 0.065

8.550 ± 0.5125

0.645 ± 0.0619

0.034 ± 0.0032

0.228 ± 0.0272

11.25

6.87 ± 0.099

13.53 ± 0.175

40.8 ± 0.53

59.44 ± 0.294

19.72 ± 0.101

33.18 ± 0.086

0.540 ± 0.0181

560.00 ± 39.746

9.267 ± 0.5191

1.03 ± 0.084

7.437 ± 0.3901

0.580 ± 0.0488

0.022 ± 0.0036

0.189 ± 0.0182

22.5

6.70 ± 0.062

13.18 ± 0.122

39.7 ± 0.39

59.22 ± 0.324

19.64 ± 0.053

33.21 ± 0.090

0.526 ± 0.0253

530.56 ± 25.532

10.133 ± 0.4269

1.17 ± 0.075

8.011 ± 0.4151

0.614 ± 0.0411

0.047 ± 0.0231

0.289 ± 0.0648

45

6.75 ± 0.088

13.21 ± 0.193

39.7 ± 0.55

58.78 ± 0.222

19.58 ± 0.092

33.23 ± 0.078

0.526 ± 0.0233

591.00 ± 15.200

9.467 ± 0.4304

1.05 ± 0.053

7.679 ± 0.3323

0.550 ± 0.0610

0.027 ± 0.0041

0.183 ± 0.0213

90

6.62 ± 0.131

12.99 ± 0.231

39.1 ± 0.74

59.11 ± 0.200

19.64 ± 0.071

33.28 ± 0.072

0.454 ± 0.0250

561.78 ± 30.219

10.333 ± 0.6727

1.13 ± 0.068

8.326 ± 0.5376

0.620 ± 0.0624

0.032 ± 0.0066

0.224 ± 0.0306

180

6.90 ± 0.127

13.55 ± 0.257

41.0 ± 0.76

59.38 ± 0.183

19.68 ± 0.067

33.15 ± 0.060

0.528 ± 0.0228

569.50 ± 29.019

10.788 ± 0.4951

1.33 ± 0.189

8.556 ± 0.4194

0.689 ± 0.0282

0.025 ± 0.0038

0.203 ± 0.0230

Day 22

0

7.71 ± 0.209

15.18 ± 0.393

45.6 ± 1.20

59.11 ± 0.200

19.72 ± 0.060

33.32 ± 0.064

0.297 ± 0.0262

522.44 ± 36.472

15.089 ± 2.0608

1.27 ± 0.142

12.442 ± 1.7536

0.987 ± 0.1496

0.048 ± 0.0092

0.323 ± 0.0401

11.25

7.42 ± 0.086

14.63 ± 0.169

43.9 ± 0.53

59.20 ± 0.200

19.73 ± 0.091

33.34 ± 0.079

0.307 ± 0.0153

563.70 ± 18.524

15.520 ± 3.0618

1.47 ± 0.262

12.694 ± 2.5321

0.905 ± 0.2006

0.059 ± 0.0109

0.390 ± 0.0828

22.5

7.45 ± 0.151

14.68 ± 0.286

44.1 ± 0.91

58.90 ± 0.180

19.72 ± 0.053

33.36 ± 0.083

0.278 ± 0.0182

534.10 ± 31.043

18.050 ± 3.1819

1.60 ± 0.247

14.848 ± 2.6952

1.090 ± 0.1804

0.073 ± 0.0133

0.456 ± 0.0953

45

7.45 ± 0.082

14.72 ± 0.113

44.2 ± 0.36

59.30 ± 0.213

19.78 ± 0.088

33.36 ± 0.072

0.318 ± 0.0104

512.30 ± 20.314

16.740 ± 4.6934

1.61 ± 0.472

13.853 ± 4.0386

0.785 ± 0.1583

0.059 ± 0.0135

0.443 ± 0.1542

90

7.54 ± 0.108

14.87 ± 0.192

44.6 ± 0.56

59.30 ± 0.153

19.73 ± 0.075

33.37 ± 0.079

0.301 ± 0.0120

545.30 ± 14.062

14.410 ± 2.2688

1.42 ± 0.206

11.944 ± 1.9392

0.714 ± 0.0967

0.056 ± 0.0120

0.287 ± 0.0404

180

7.38 ± 0.103

14.58 ± 0.208

43.8 ± 0.64

59.30 ± 0.213

19.78 ± 0.090

33.32 ± 0.076

0.301 ± 0.0197

519.00 ± 17.491

16.770 ± 3.2436

1.47 ± 0.265

14.046 ± 2.7995

0.885 ± 0.1514

0.039 ± 0.0071

0.346 ± 0.0533

Day 93

0

9.13 ± 0.056

15.66 ± 0.130

45.3 ± 0.34

49.70 ± 0.213

17.17 ± 0.062

34.54 ± 0.078

0.200 ± 0.0114

511.60 ± 13.123

10.910 ± 0.3854

2.88 ± 0.178

7.087 ± 0.2720

0.591 ± 0.0522

0.122 ± 0.0087

0.228 ± 0.0256

11.25

9.10 ± 0.105

15.62 ± 0.197

45.1 ± 0.55

49.40 ± 0.163

17.18 ± 0.057

34.64 ± 0.075

0.216 ± 0.0170

503.60 ± 12.751

11.080 ± 0.3797

3.03 ± 0.211

6.865 ± 0.3349

0.739 ± 0.0562

0.148 ± 0.0061

0.288 ± 0.0179

22.5

9.19 ± 0.116

15.83 ± 0.205

45.8 ± 0.56

49.90 ± 0.180

17.26 ± 0.058

34.56 ± 0.109

0.221 ± 0.0067

518.30 ± 13.856

10.600 ± 0.4487

2.57 ± 0.221

7.083 ± 0.3183

0.602 ± 0.0525

0.117 ± 0.0118

0.222 ± 0.0224

45

9.04 ± 0.107

15.45 ± 0.194

44.6 ± 0.55

49.30 ± 0.153

17.08 ± 0.049

34.60 ± 0.058

0.211 ± 0.0087

525.50 ± 12.172

10.990 ± 0.5098

2.76 ± 0.138

7.203 ± 0.3873

0.610 ± 0.0577

0.141 ± 0.0219

0.295 ± 0.0668

90

9.26 ± 0.083

15.90 ± 0.146

46.1 ± 0.44

50.00 ± 0.149

17.16 ± 0.054

34.46 ± 0.076

0.232 ± 0.0125

524.80 ± 15.867

10.770 ± 0.3821

2.98 ± 0.139

6.784 ± 0.2742

0.606 ± 0.0611

0.132 ± 0.0178

0.259 ± 0.0316

180

9.15 ± 0.081

15.79 ± 0.148

45.4 ± 0.44

49.70 ± 0.153

17.24 ± 0.062

34.73 ± 0.088

0.209 ± 0.0077

502.90 ± 13.825

9.750 ± 0.4143

2.19 ± 0.191

6.769 ± 0.3326

0.474 ± 0.0309

0.097 ± 0.0070

0.209 ± 0.0247

Abbreviations:

NA: Not Available,SEM: Standard Error of Means,V: Vehicle Control,Erythrcyt: Erythrocytes,Hgb: Hemoglobin,HCT: Hematocrit,MCV: Mean Corpuscular Volume,MCH: Mean Corpuscular Hemoglobin,MCHC: Mean Corpuscular Hemoglobin Concentration,Retics: Reticulocytes,Platelet: Platelets,Leukocytes: Leukocytes,Neut: Neutrophils,Lymph: Lymphocytes,Mono: Monocytes,EOS: Eosinophils,Basophils: Basophils, CL = Sample clotted

Conclusions:

The Lowest-Observed-Effect-Level (LOEL) at the application site was 11.25 mg/kg 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.

Executive summary:

The potential subchronic dermal toxicity of sodium mercaptoacetate was evaluated according to a NTP protocol. Sodium mercaptoacetate was applied once daily in Sprague-Dawley rats (10 Males and 10 Females) skin, at the dose-levels of 11.25, 22.5, 45.0, 90.0, and 180.0  mg/kg bw/d during 90 days, 5 days a week. A control group was tested with vehicle (ethanol in water). No satellite group was tested for reversibility or persistence occurrence of toxic effects.

Body weights were recorded on day one on test, weekly and prior necropsy. Animals were observed twice daily for morbidity and mortality. Blood was collected from both sexes of "special study" rats, at days 4 and 22 and from the core study rats at the end of the study. These was processed for haematology and clinical chemistry determinations. Blood was collected from core study mice at the end of the study for haematology determinations. All animals were examined for gross pathology, and organs were weighted and submitted to histopathology.

No death were reported in any treated group. Significant observations noted in both sexes were limited to dermal irritation at the site of application (SOA), thickened skin at the SOA and ulcerations at the SOA. There were only limited statistically significant differences in the body weight, organ weight, chemistry and haematology results. No gross lesions finding were considered to be test article related or biologically significant. Repeated dermal administration of Sodium Thioglycolate (NaTG) for thirteen weeks (excluding weekends) resulted in test article related microscopic changes at the site of application (SOA) in both male and female rats at all treatment doses. Changes in the skin SOA revealed minimal to mild hyperplasia of the epidermis accompanied, in many animals, by sebaceous gland hyperplasia and hyperkeratosis. The severity of the changes was comparable between all treatment groups in both the male and female rats.

The Lowest-Observed-Effect-Level (LOEL) at the application site was 11.25 mg/kg 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

180 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:

experimental study

Adequacy of study:

key study

Study period:

Dec 2002 - Mar 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: According to the standard protocol posted on the NTP website 

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sigma Chemical Company (Columbus, OH), lot #88H1166
- Purity, including information on contaminants, isomers, etc.: ca 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored under a headspace of inert gas at less than or equal to −20°C, protected from light, in amber glass bottles
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: confirmed for at least 10 days

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Type of coverage:

open

Vehicle:

other: 95 % Ethanol in deionized water (1:1, v/v)

Details on exposure:

Treatment: After a 10- to 14-day quarantine period, animals are assigned at random  to treatment groups. The study includes five treatment groups each  administered a different concentrations of the test chemicals plus a  control group. Each group contains 10 animals per sex per species. The  animals receive the subject chemical by dermal route of exposure.  Controls receive vehicle alone. Animals are exposed five times per week,  weekdays only, for 90 days after which they are sacrificed with no recovery period. All animals are housed individually. 

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks, 24 h/d

Frequency of treatment:

five times per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Positive control:

not appropriate

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
Animals are weighed individually on day one on test, after seven days,  and at weekly periods thereafter. 

DETAILED CLINICAL OBSERVATIONS: Yes
Animals are observed twice daily, at  least six hours apart (before 10:00 AM and after 2:00 PM), including  holidays and weekends, for moribundity and death. Animals found moribund  or showing clinical signs of pain or distress are humanely euthanized.  Formal clinical observations are performed and recorded weekly. 

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
Blood is collected from both sexes of "special study" rats, at days 4 and  22 and from the core study rats at the end of the study. These are  processed for haematology and clinical chemistry determinations. Blood is  collected from core study mice at the end of the study for haematology  determinations.

-Haematology:
Erythrocyte count,  Mean corpuscular volume,  Haemoglobin,  Packed cell volume,  Mean corpuscular haemoglobin,  Mean corpuscular haemoglobin concentration,  Erythrocyte morphologic assessment,  Leukocyte count,  Leukocyte differential,  Reticulocyte count,  Platelet count and morphologic assessment 
-Clinical chemistry:
Sorbitol dehydrogenase (SDH),  Alkaline Phosphatase (ALP),  Creatine Kinase (CK),  Creatinine,  Total Protein,  Albumin,  Urea Nitrogen (BUN),  Total Bile Acids,  Alanine Aminotransferase (ALT)

OTHER:
Sperm Morphology and Vaginal Cytology Evaluations (SMVCE) (see section  7.8.3): SMVCE are conducted on core study rats and mice. Mortality, body weight  changes and clinical signs of toxicity are used to determine the 3 dose  levels used for the SMVCE evaluations.

Sacrifice and pathology:

GROSS PATHOLOGY AND HISTOPATHOLOGY
Organ weights:  Liver, thymus, right kidney, right testis, heart, and lungs weights are  recorded from all animals surviving until the end of the study. 
A complete necropsy is performed on all treated and control animals that  either die or are sacrificed. All tissues required for complete  histopathology are trimmed, embedded, sectioned and stained with  hematoxylin and eosin for histopathologic evaluation.

HISTOPATHOLOGY:
A complete histopathologic evaluation inclusive of gross lesions is done  on all control animals, all animals in the highest dose group with at  least 60% survivors at the time of sacrifice, and all animals in higher  dose groups inclusive of early deaths and survivors. Chemical-related  lesions (target organs) are identified, and these organs plus gross  lesions are examined for all lower doses. Only those tissues designated  as target tissues and gross lesions are evaluated in lower doses to a  no-effect-level. A complete histopathologic evaluation is performed on  all natural death/moribund sacrifice animals in lower dose groups.

Tissues examined histopathologically:  Adrenal glands,  Brain (3 sections including frontal cortex and basal ganglia, parietal  cortex and thalamus, and cerebellum and pons), Clitoral glands,  Esophagus,  Eyes,  Femur, including diaphysis with marrow cavity and epiphysis (femoral  condyle with epiphyseal cartilage plate, articular cartilage and  articular surface),  Gallbladder (mouse),  Gross lesions,  Harderian glands,  Heart and aorta,  Intestine, large (cecum, colon, rectum),  Intestine, small (duodenum, jejunum, ileum), Kidneys,  Liver (2 sections including left lateral lobe and median lobe),  Lungs and mainstem bronchi,  Lymph nodes  - mandibular and mesenteric - inguinal, gluteal, internal iliac (chronic studies only, if lesion  observed, not merely discolouration),  Mammary gland with adjacent skin,  Muscle, thigh (only if neuromuscularsigns were present),  Nasal cavity and nasal turbinates (3 sections),  Ovaries,  Pancreas,  Parathyroid glands,  Pituitary gland,  Preputial glands,  Prostate,  Salivary glands,  Seminal vesicle,  Skin: site of application (topical studies),  Spinal cord and sciatic nerve (if neurologic signs were present),  Spleen, Stomach (forestomach and glandular),  Testes with epididymus,  Thymus,  Thyroid glands,  Tissue masses and regional lymph nodes,  Trachea,  Urinary bladder,  Uterus

Statistics:

yes

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

1.Survival
All Core Study male and female rats survived until terminal sacrifice.

2.Clinical observations
Significant observations noted in both sexes were limited to dermal  irritation at the site of application (SOA), thickened skin at the SOA  and ulcerations at the SOA.
In the males, the incidence of dermal irritation at the SOA was 10/10 for  all five test article treatment groups. Thickening of the skin at the SOA  was observed in 1/10 and 2/10 rats from the 90.0 and 180.0 mg/kg dose  groups, respectively. Ulceration at the SOA was observed in 1/10, 1/10,  5/10 and 8/10 rats from the 11.25, 22.5, 90.0 and 180.0 mg/kg dose  groups, respectively. All other observations noted during the study were  not considered to be biologically significant.
In the females, the incidence of dermal irritation at the SOA was also  10/10 for all five treatment groups. Thickening of the skin at the SOA  was observed in 3/10 rats from the 45.0 mg/kg dose group and in 10/10  rats each from the 90.0 and 180.0 mg/kg dose groups. Ulceration at the  SOA was observed in 10/10 rats from each of the 90.0 and 180.0 mg/kg dose  groups. All other observations noted during the study were not considered  to be biologically significant.

3.Body Weights
Statistical analysis of the Core Study final (terminal) body weights  revealed statistically significant (p<0.05) decreased values in the male  group treated with 90.0 mg/kg when compared to the vehicle control. No  statistically significant differences in body weights were noted for the  female rats when the test article groups were compared to the vehicle  control group.

4.Organ Weights
a.Absolute Organ Weights
The absolute liver weights of the male rats treated with 45.0 mg/kg were significantly (+10%; p<=0.05) increased when compared to the vehicle  control group. There were no other statistically significant differences  in any other absolute organ weights in the male rats. There were no  statistically significant differences in any absolute organ weights in  the female rats.

b.Organ to Body Weight Ratios
The relative kidney and testes weights of the male rats treated with 90.0  (+7.6% and 6.7%, respectively) and 180.0 mg/kg (+8.2 and 7.6%,  respectively) were significantly (p<=0.05) increased when compared to the  vehicle control group. The relative liver weights for the male rats  treated with 45.0 mg/kg were significantly (+8.5%; p<=0.05) increased  when compared to the vehicle control group. The relative spleen weights  of the male rats treated with 90.0 mg/kg were significantly (+6.0%;  p<=0.05) increased when compared to the vehicle control group. The  relative kidney weights of the female rats treated with 180.0 mg/kg were  significantly (+6.6%; p<=0.05) increased when compared to the vehicle  control group. The relative thyroid/parathyroid weights of the female  rats treated with 22.5 mg/kg were significantly (-20%; p<=0.05) decreased  when compared to the vehicle control group. There were no other  statistically significant differences in any relative organ weight for  the male or female rats.

5.Clinical Pathology
There were limited statistically significant (p<0.05) differences in the  chemistry and haematology results when compared to the control group, but  these were not dose responsive nor considered to be biologically  significant.

6.Anatomic Pathology
a.Gross Lesions
For the males, abnormal gross necropsy findings were limited to skin  accumulation (no mass noted) at the SOA in 1/10 rats from the 11.25, 45.0  and 180.0 mg/kg dose groups, a nodule on the thoracic inlet of the  thoracic cavity in 1/10 rats from both the 11.25 and 22.5 mg/kg dose  groups, a mass on the median lobe of the liver in 1/10 rats from the  180.0 mg/kg dose group and a nodule on the liver in 1/10 rats from the  22.5 mg/kg dose group. In addition, one male from the 180.0 mg/kg dose  group had retained the right testis in its abdominal region. There were  no other significant abnormal gross lesions noted in any of the male  treatment groups. These findings were not considered to be test article  related or biologically significant.
For the females, abnormal gross necropsy findings were limited in number.  In the vehicle control group, one female rat was noted as having a focus  on the left kidney. In the 11.25 mg/kg test article treatment group, four  females were noted as having a nodule on the liver, one with a nodule on  the pancreas, one rat with a nodule on the thoracic cavity and one rat  with dilated uterine horns: One rat from the 22.5 mg/kg dose group had a  cyst on the left ovary. There were two and three rats from the 45.0 and  90.0 mg/kg treatment groups, respectively, noted as having a nodule on  the liver. In the high dose treatment group (180.0 mg/kg), there were two  animals with nodules on the liver, two animals with nodules in the  thoracic cavity, one rat with enlarged mediastinal lymph nodes and one  rat with dilated uterine horns. These findings were not considered to be  test article related or biologically significant. Findings which may be  contributed to the treatment of NaT were limited to the 180.0 mg/kg  treatment group where three rats were noted as having an irritation on  the SOA.

b.Microscopic Pathology Repeated dermal administration of Sodium Thioglycolate (NaT) for thirteen  weeks (excluding weekends) resulted in test article related microscopic  changes at the site of application (SOA) in both male and female rats at  all treatment doses. Changes in the skin SOA revealed minimal to mild  hyperplasia of the epidermis accompanied, in many animals, by sebaceous  gland hyperplasia and hyperkeratosis. The severity of the changes was  comparable between all treatment groups in both the male and female rats.  A NOEL was not reached in female or male rats.

Microscopic evaluation of the other tissues required by the protocol  revealed a few findings which were observed either in small numbers  and/or in both control and treated animals. And, all of these changes are  commonly observed in F344 rats. For these reasons, these changes were  considered incidental findings.
[NOTE: The pathologist used the following criteria for severity scoring  of the epidermal hyperplasia; minimal 2-3 cell layers thick, mild 4-6  cell layers thick, moderate 7-8 cell layers thick and marked >9 cell  layers thick (at the thickest point).]

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 180 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

LOAEL

Remarks:

Local effects

Effect level:

11.25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Key result

Critical effects observed:

no

Female rats organ weight summary table

Dose Group (mg/kg)

Body Wt (Sac)(g)

Heart Wt (g)

%Heart/Body

Liver Wt (g)

%Liver/Body

Lung Wt (g)

%Lungs/Body

R Kidney Wt (g)

%R Kidney/Body

Spleen Wt (g)

%Spleen/Body

Thyroid Wt (g)

%Thyroid/Body

Day 93

0

177.3 ± 2.98

0.65 ± 0.013

0.37 ± 0.004

5.60 ± 0.173

3.16 ± 0.068

1.053 ± 0.0289

0.59 ± 0.008

0.70 ± 0.014

0.392 ± 0.0041

0.44 ± 0.007

0.25 ± 0.002

0.026 ± 0.0009

0.014 ± 0.0006

11.25

185.4 ± 2.56

0.65 ± 0.016

0.35 ± 0.005

5.59 ± 0.123

3.01 ± 0.048

1.001 ± 0.0427

0.54 ± 0.020

0.73 ± 0.009

0.391 ± 0.0025

0.45 ± 0.009

0.24 ± 0.004

0.026 ± 0.0010

0.014 ± 0.0006

22.5

184.5 ± 2.74

0.66 ± 0.008

0.36 ± 0.007

6.04 ± 0.156

3.27 ± 0.057

0.978 ± 0.0219

0.53 ± 0.010

0.72 ± 0.010

0.389 ± 0.0044

0.44 ± 0.006

0.24 ± 0.004

0.023 ± 0.0007

0.012 ± 0.0004

45

185.9 ± 2.75

0.65 ± 0.011

0.35 ± 0.004

5.90 ± 0.153

3.17 ± 0.048

1.101 ± 0.0468

0.59 ± 0.027

0.72 ± 0.020

0.389 ± 0.0086

0.45 ± 0.012

0.24 ± 0.005

0.024 ± 0.0013

0.013 ± 0.0007

90

179.9 ± 3.54

0.67 ± 0.016

0.37 ± 0.008

5.94 ± 0.141

3.31 ± 0.066

1.059 ± 0.0414

0.59 ± 0.018

0.75 ± 0.021

0.415 ± 0.0090

0.44 ± 0.012

0.25 ± 0.007

0.026 ± 0.0009

Organ Weights Summary Table in male rats

(Mean ± SEM)

Dose Group (mg/kg)

Body Wt (Sac)(g)

Heart Wt (g)

%Heart/Body

Liver Wt (g)

%Liver/Body

Lung Wt (g)

%Lungs/Body

R Kidney Wt (g)

%R Kidney/Body

R Testis Wt (g)

%R Testis/Body

Spleen Wt (g)

%Spleen/Body

Thyroid Wt (g)

%Thyroid/Body

Day 93

0

334.5 ± 3.86

0.97 ± 0.014

0.29 ± 0.003

11.22 ± 0.270

3.35 ± 0.048

1.532 ± 0.0627

0.46 ± 0.019

1.14 ± 0.021

0.340 ± 0.0044

1.442 ± 0.0248

0.432 ± 0.0089

0.72 ± 0.010

0.21 ± 0.002

0.026 ± 0.0015

0.008 ± 0.0004

11.25

333.7 ± 4.16

0.98 ± 0.022

0.29 ± 0.005

11.90 ± 0.264

3.57 ± 0.062

1.502 ± 0.0512

0.45 ± 0.014

1.20 ± 0.026

0.359 ± 0.0065

1.468 ± 0.0227

0.440 ± 0.0042

0.72 ± 0.012

0.21 ± 0.003

0.026 ± 0.0010

0.008 ± 0.0004

22.5

332.0 ± 7.07

0.95 ± 0.018

0.29 ± 0.004

11.71 ± 0.341

3.52 ± 0.062

1.541 ± 0.0849

0.46 ± 0.019

1.17 ± 0.024

0.352 ± 0.0054

1.386 ± 0.0247

0.418 ± 0.0075

0.72 ± 0.017

0.22 ± 0.004

0.027 ± 0.0011

0.008 ± 0.0004

45

339.4 ± 4.77

0.97 ± 0.010

0.29 ± 0.003

12.36 ± 0.292

3.64 ± 0.046

1.518 ± 0.0535

0.45 ± 0.016

1.18 ± 0.019

0.348 ± 0.0053

1.419 ± 0.0286

0.418 ± 0.0067

0.72 ± 0.016

0.21 ± 0.003

0.030 ± 0.0049

0.009 ± 0.0014

90

312.1 ± 8.54

0.95 ± 0.023

0.31 ± 0.005

10.46 ± 0.349

3.35 ± 0.074

1.451 ± 0.0448

0.47 ± 0.012

1.14 ± 0.023

0.366 ± 0.0041

1.435 ± 0.0300

0.461 ± 0.0082

0.71 ± 0.016

0.23 ± 0.004

0.028 ± 0.0018

0.009 ± 0.0007

180

319.3 ± 5.89

0.93 ± 0.019

0.29 ± 0.004

11.26 ± 0.261

3.53 ± 0.066

1.497 ± 0.0268

0.47 ± 0.008

1.17 ± 0.024

0.368 ± 0.0048

1.482 ± 0.0282

0.465 ± 0.0081

0.72 ± 0.016

0.23 ± 0.003

0.027 ± 0.0011

0.008 ± 0.0004

Hematology Summary Table in male rats

(Mean ± SEM)

Dose Group (mg/kg)

Erythrcyt (10^6/uL)

Hgb (g/dL)

HCT (Automated) (%)

MCV (fL)

MCH (pg)

MCHC (g/dL)

Retics (10^6/uL)

Platelet (10^3/uL)

Leukocytes (10^3/uL)

Neut (10^3/uL)

Lymph (10^3/uL)

Mono (10^3/uL)

EOS (10^3/uL)

Basophils (10^3/uL)

Day 4

0

6.79 ± 0.115

13.40 ± 0.228

40.3 ± 0.70

59.25 ± 0.250

19.71 ± 0.058

33.25 ± 0.033

6.790 ± 6.3157

594.38 ± 26.679

10.613 ± 0.6269

1.18 ± 0.065

8.550 ± 0.5125

0.645 ± 0.0619

0.034 ± 0.0032

0.228 ± 0.0272

11.25

6.87 ± 0.099

13.53 ± 0.175

40.8 ± 0.53

59.44 ± 0.294

19.72 ± 0.101

33.18 ± 0.086

0.540 ± 0.0181

560.00 ± 39.746

9.267 ± 0.5191

1.03 ± 0.084

7.437 ± 0.3901

0.580 ± 0.0488

0.022 ± 0.0036

0.189 ± 0.0182

22.5

6.70 ± 0.062

13.18 ± 0.122

39.7 ± 0.39

59.22 ± 0.324

19.64 ± 0.053

33.21 ± 0.090

0.526 ± 0.0253

530.56 ± 25.532

10.133 ± 0.4269

1.17 ± 0.075

8.011 ± 0.4151

0.614 ± 0.0411

0.047 ± 0.0231

0.289 ± 0.0648

45

6.75 ± 0.088

13.21 ± 0.193

39.7 ± 0.55

58.78 ± 0.222

19.58 ± 0.092

33.23 ± 0.078

0.526 ± 0.0233

591.00 ± 15.200

9.467 ± 0.4304

1.05 ± 0.053

7.679 ± 0.3323

0.550 ± 0.0610

0.027 ± 0.0041

0.183 ± 0.0213

90

6.62 ± 0.131

12.99 ± 0.231

39.1 ± 0.74

59.11 ± 0.200

19.64 ± 0.071

33.28 ± 0.072

0.454 ± 0.0250

561.78 ± 30.219

10.333 ± 0.6727

1.13 ± 0.068

8.326 ± 0.5376

0.620 ± 0.0624

0.032 ± 0.0066

0.224 ± 0.0306

180

6.90 ± 0.127

13.55 ± 0.257

41.0 ± 0.76

59.38 ± 0.183

19.68 ± 0.067

33.15 ± 0.060

0.528 ± 0.0228

569.50 ± 29.019

10.788 ± 0.4951

1.33 ± 0.189

8.556 ± 0.4194

0.689 ± 0.0282

0.025 ± 0.0038

0.203 ± 0.0230

Day 22

0

7.71 ± 0.209

15.18 ± 0.393

45.6 ± 1.20

59.11 ± 0.200

19.72 ± 0.060

33.32 ± 0.064

0.297 ± 0.0262

522.44 ± 36.472

15.089 ± 2.0608

1.27 ± 0.142

12.442 ± 1.7536

0.987 ± 0.1496

0.048 ± 0.0092

0.323 ± 0.0401

11.25

7.42 ± 0.086

14.63 ± 0.169

43.9 ± 0.53

59.20 ± 0.200

19.73 ± 0.091

33.34 ± 0.079

0.307 ± 0.0153

563.70 ± 18.524

15.520 ± 3.0618

1.47 ± 0.262

12.694 ± 2.5321

0.905 ± 0.2006

0.059 ± 0.0109

0.390 ± 0.0828

22.5

7.45 ± 0.151

14.68 ± 0.286

44.1 ± 0.91

58.90 ± 0.180

19.72 ± 0.053

33.36 ± 0.083

0.278 ± 0.0182

534.10 ± 31.043

18.050 ± 3.1819

1.60 ± 0.247

14.848 ± 2.6952

1.090 ± 0.1804

0.073 ± 0.0133

0.456 ± 0.0953

45

7.45 ± 0.082

14.72 ± 0.113

44.2 ± 0.36

59.30 ± 0.213

19.78 ± 0.088

33.36 ± 0.072

0.318 ± 0.0104

512.30 ± 20.314

16.740 ± 4.6934

1.61 ± 0.472

13.853 ± 4.0386

0.785 ± 0.1583

0.059 ± 0.0135

0.443 ± 0.1542

90

7.54 ± 0.108

14.87 ± 0.192

44.6 ± 0.56

59.30 ± 0.153

19.73 ± 0.075

33.37 ± 0.079

0.301 ± 0.0120

545.30 ± 14.062

14.410 ± 2.2688

1.42 ± 0.206

11.944 ± 1.9392

0.714 ± 0.0967

0.056 ± 0.0120

0.287 ± 0.0404

180

7.38 ± 0.103

14.58 ± 0.208

43.8 ± 0.64

59.30 ± 0.213

19.78 ± 0.090

33.32 ± 0.076

0.301 ± 0.0197

519.00 ± 17.491

16.770 ± 3.2436

1.47 ± 0.265

14.046 ± 2.7995

0.885 ± 0.1514

0.039 ± 0.0071

0.346 ± 0.0533

Day 93

0

9.13 ± 0.056

15.66 ± 0.130

45.3 ± 0.34

49.70 ± 0.213

17.17 ± 0.062

34.54 ± 0.078

0.200 ± 0.0114

511.60 ± 13.123

10.910 ± 0.3854

2.88 ± 0.178

7.087 ± 0.2720

0.591 ± 0.0522

0.122 ± 0.0087

0.228 ± 0.0256

11.25

9.10 ± 0.105

15.62 ± 0.197

45.1 ± 0.55

49.40 ± 0.163

17.18 ± 0.057

34.64 ± 0.075

0.216 ± 0.0170

503.60 ± 12.751

11.080 ± 0.3797

3.03 ± 0.211

6.865 ± 0.3349

0.739 ± 0.0562

0.148 ± 0.0061

0.288 ± 0.0179

22.5

9.19 ± 0.116

15.83 ± 0.205

45.8 ± 0.56

49.90 ± 0.180

17.26 ± 0.058

34.56 ± 0.109

0.221 ± 0.0067

518.30 ± 13.856

10.600 ± 0.4487

2.57 ± 0.221

7.083 ± 0.3183

0.602 ± 0.0525

0.117 ± 0.0118

0.222 ± 0.0224

45

9.04 ± 0.107

15.45 ± 0.194

44.6 ± 0.55

49.30 ± 0.153

17.08 ± 0.049

34.60 ± 0.058

0.211 ± 0.0087

525.50 ± 12.172

10.990 ± 0.5098

2.76 ± 0.138

7.203 ± 0.3873

0.610 ± 0.0577

0.141 ± 0.0219

0.295 ± 0.0668

90

9.26 ± 0.083

15.90 ± 0.146

46.1 ± 0.44

50.00 ± 0.149

17.16 ± 0.054

34.46 ± 0.076

0.232 ± 0.0125

524.80 ± 15.867

10.770 ± 0.3821

2.98 ± 0.139

6.784 ± 0.2742

0.606 ± 0.0611

0.132 ± 0.0178

0.259 ± 0.0316

180

9.15 ± 0.081

15.79 ± 0.148

45.4 ± 0.44

49.70 ± 0.153

17.24 ± 0.062

34.73 ± 0.088

0.209 ± 0.0077

502.90 ± 13.825

9.750 ± 0.4143

2.19 ± 0.191

6.769 ± 0.3326

0.474 ± 0.0309

0.097 ± 0.0070

0.209 ± 0.0247

Abbreviations:

NA: Not Available,SEM: Standard Error of Means,V: Vehicle Control,Erythrcyt: Erythrocytes,Hgb: Hemoglobin,HCT: Hematocrit,MCV: Mean Corpuscular Volume,MCH: Mean Corpuscular Hemoglobin,MCHC: Mean Corpuscular Hemoglobin Concentration,Retics: Reticulocytes,Platelet: Platelets,Leukocytes: Leukocytes,Neut: Neutrophils,Lymph: Lymphocytes,Mono: Monocytes,EOS: Eosinophils,Basophils: Basophils, CL = Sample clotted

Conclusions:

The Lowest-Observed-Effect-Level (LOEL) at the application site was 11.25 mg/kg 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.

Executive summary:

The potential subchronic dermal toxicity of sodium mercaptoacetate was evaluated according to a NTP protocol. Sodium mercaptoacetate was applied once daily in Sprague-Dawley rats (10 Males and 10 Females) skin, at the dose-levels of 11.25, 22.5, 45.0, 90.0, and 180.0  mg/kg bw/d during 90 days, 5 days a week. A control group was tested with vehicle (ethanol in water). No satellite group was tested for reversibility or persistence occurrence of toxic effects.

Body weights were recorded on day one on test, weekly and prior necropsy. Animals were observed twice daily for morbidity and mortality. Blood was collected from both sexes of "special study" rats, at days 4 and 22 and from the core study rats at the end of the study. These was processed for haematology and clinical chemistry determinations. Blood was collected from core study mice at the end of the study for haematology determinations. All animals were examined for gross pathology, and organs were weighted and submitted to histopathology.

No death were reported in any treated group. Significant observations noted in both sexes were limited to dermal irritation at the site of application (SOA), thickened skin at the SOA and ulcerations at the SOA. There were only limited statistically significant differences in the body weight, organ weight, chemistry and haematology results. No gross lesions finding were considered to be test article related or biologically significant. Repeated dermal administration of Sodium Thioglycolate (NaTG) for thirteen weeks (excluding weekends) resulted in test article related microscopic changes at the site of application (SOA) in both male and female rats at all treatment doses. Changes in the skin SOA revealed minimal to mild hyperplasia of the epidermis accompanied, in many animals, by sebaceous gland hyperplasia and hyperkeratosis. The severity of the changes was comparable between all treatment groups in both the male and female rats.

The Lowest-Observed-Effect-Level (LOEL) at the application site was 11.25 mg/kg 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.

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.