Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From May 2008 to September 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: RA study
Justification for type of information:
Refer to the section 13 for details on the read across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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 substance 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 substance 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 substance 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.
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 substance 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
Remarks:
Doses / Concentrations:
7, 20 and 60 mg/kg bw/d
Basis:
actual ingested
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 substance 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 substance 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
Description (incidence and severity):
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 substance 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 substance.
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 substance 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 substance-related.
Mortality:
mortality observed, treatment-related
Description (incidence):
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 substance 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.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gains throughout the study were similar between test substance-treated groups and controls. No treatment-related effects on the body weight and the body weight gain were observed during the treatment and the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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 substance treatment.
No test substance treatment-related effects were observed on food consumption in females at any dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
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 substance treatment-related.
No other abnormalities were observed at the end of treatment period at the ophthalmological examination.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
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 substance 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 substance 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.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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 substance-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 substance 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 findings:
no effects observed
Description (incidence and severity):
The urinalysis parameters were unaffected by the treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
FUNCTIONAL OBSERVATION BATTERY:
No test substance treatment-related effects were observed during the FOB or on motor activity.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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 substance 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.
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 substance 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.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
BONE-MARROW DIFFERENTIAL CELL COUNT: Table 4
The bone marrow cellularity and number of megakaryocytes were similar in the control and test substance-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 substance treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Details on results:
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.
Key result
Dose descriptor:
LOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: mortality and reversible haematological, blood chemistry and liver microscopic changes
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:
other: limited blood chemistry effects without microscopic changes in the liver
Key result
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:
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:
Based on the results of the read across 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 NOAEL of the test substance 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.
Executive summary:

A study was conducted to determine the potential toxicity of the read across substance, sodium thioglycolate, to rats according to OECD Guideline 408 and EU Method B.26 of Council Regulation (EC) No 440/2008, in compliance with GLP. The test substance was administered by daily oral administration (gavage) to Sprague-Dawley rats at doselevels of 7, 20 or 60 mg a.i./kg/day (a.i.) 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 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 the test substance. The demise and death of these animals were attributed to treatment with the test substance. 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 ßhydroxybutyrate levels, associated with high lactate concentrations, were reported in males and females. The test substance-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 ßhydroxybutyrate 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. Based on the results of the read across 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 NOAEL of the test substance 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 (Rousseau, 2010).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From December 03, 1999 to July 04, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-7 weeks
- Weight at study initiation: Males - 134-179 g and females - 124-156 g
- Fasting period before study: No
- Housing: housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 December, 1999 To: 05 January, 2000
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test substance was administered daily, for up to 28 d, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg bw/day of distilled water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the test samples were done once per week for the entire duration of the study. The test substance formulations were diluted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/mL. Standard solutions of test substance were prepared in methanol at a nominal concentration of 0.1 mg/mL. For homogeneity determination, the test substance formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container. Sampling was performed in triplicate. For stability determination, the test substance formulations were sampled and analysed initially and then after storage at ambient temperature in the light for four hours. For analysis of concentrations, the test substance formulations were sampled and analysed immediately after preparation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Animals treated with 500 mg/kg bw/day showed a marked deterioration in
health and it was decided to stop dosing on Day 6. Dosing restarted on Day 7 at the revised dose level of 250 mg/kg bw/day and continued to the remainder of the study period.
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses for the main study were selected based on a 8-day dose range finding toxicity study in rats at 0, 150 and 500 mg/kg bw/day. No abnormalities were observed in this study and hence the doses for the main study were selected to be 0, 50, 150 and 500 mg/kg bw/day.
- Rationale for animal assignment (if not random): Randomised
- Rationale for selecting satellite groups: Based on standard practices, the recovery groups were selected to be vehicle control and high dose group.
- Post-exposure recovery period in satellite groups: 14 d
Observations and examinations performed and frequency:
Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all surviving non-recovery group animals at the end of the treatment period and for all surviving recovery group animals at the end of the treatment-free period.
Sacrifice and pathology:
At the end of the dosing period, all animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs developed in the high dose animals on Day 2 of the study with a number of animals showing a severe deterioration in condition. Clinical signs among these animals included clonic convulsion, distended abdomen, dehydration, pallor of the extremities, hunched posture, lethargy, pilo-erection, decreased respiratory rate, gasping, laboured and noisy respiration and tiptoe gait. Following reduction of the dose level to 250 mg/kg bw/day on Day 7 clinical observations persisted throughout the treatment period and included distended abdomen, dehydration, hunched posture, pilo-erection, decreased, gasping,laboured and noisy respiration, increased salivation after dosing, red/brown staining of the mouth and snout, tiptoe gait. No clinical signs were observed in other dose levels.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were five treatment-related deaths during the study. One high dose male and two high dose female animals were killed in extremis on Day 5 of the study following a severe deterioration in condition. One high dose male was killed in extremis on Day 8 whilst a further male from this dose group died prior to treatment on Day 11. No mortality was observed from any other dose levels.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain was reduced at the high dose level during the first week of study and later recovered as the high dose was reduced to 250 mg/kg bw/day. No change in body weight was observed in other dose levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Similar to the effect on body weight gain, reduction in food consumption was observed at the high dose level which resolved from week 2.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Reductions in haemoglobin, haematocrit, erythrocyte count and mean corpuscular volume have been observed at the high dose and recovery groups. No effects were observed in other groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Reductions in total plasma protein, triglyceride, cholesterol and albumin were observed at the high dose. No changes in other groups.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Open-field assessments conducted during the study confirmed the clinical signs observed in high dose animals (500/250 mg/kg bw/day) throughout the study period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increase in the absolute and relative weight of liver and kidney were observed at the high dose and recovery group. There were no treatment-related effects in other groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The toxicologically significant macroscopic abnormalities were confined to gastro-intestinal tract changes and small and/or pale spleen among the animals which died from the high dose. No abnormalities were reported from the other two groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Conclusions:
Under the study conditions, the repeat dose toxicity NOAEL of the substance in rats was determined to be 150 mg/kg bw/day.
Executive summary:

A study was conducted to determine the repeat dose toxicity of the test substance in rats according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. The test substance was administered as an aqueous solution by oral gavage to groups of five male and five female Sprague-Dawley rats for 28 d, at dose levels of 0, 15, 150 and 500 mg/kg bw/day (incorporating a correction factor for 98.4% purity). Due to a deterioration in the health of the animals at 500 mg/kg bw/day, the highest dose level (including recovery group animals) was reduced to 250 mg/kg/day from Day 7. Treatment-related mortality, clinical signs, reduction in body weight gain and food consumption, reduction in red blood cell parameters and changes in clinical chemistry were observed at the high dose only. At gross pathology, treatment-related lesions in the stomach were observed in the high dose animals which died during the study. Under the study conditions, the repeat dose toxicity NOAEL of the substance in rats was determined to be 150 mg/kg bw/day (Cormack, 2000).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
other: biochemistry
Organ:
other: limited blood chemistry effects without microscopic changes in the liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Study 1:

A study was conducted to determine the repeat dose toxicity of the test substance in rats according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. The test substance was administered as an aqueous solution by oral gavage to groups of five male and five female Sprague-Dawley rats for 28 d, at dose levels of 0, 15, 150 and 500 mg/kg bw/day (incorporating a correction factor for 98.4% purity). Due to a deterioration in the health of the animals at 500 mg/kg bw/day, the highest dose level (including recovery group animals) was reduced to 250 mg/kg/day from Day 7. Treatment-related mortality, clinical signs, reduction in body weight gain and food consumption, reduction in red blood cell parameters and changes in clinical chemistry were observed at the high dose only. At gross pathology, treatment-related lesions in the stomach were observed in the high dose animals which died during the study. Under the study conditions, the repeat dose toxicity NOAEL of the substance in rats was determined to be 150 mg/kg bw/day (Cormack, 2000).

 

Study 2:

A study was conducted to determine the potential toxicity of the read across substance, sodium thioglycolate, to rats according to OECD Guideline 408 and EU Method B.26, in compliance with GLP. The test substance was administered by daily oral administration (gavage) to Sprague-Dawley rats at doselevels of 7, 20 or 60 mg a.i./kg/day 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 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 the test substance. The demise and death of these animals were attributed to treatment with the test substance. 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 ßhydroxybutyrate levels, associated with high lactate concentrations, were reported in males and females. The test substance-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 ßhydroxybutyrate 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. Based on the results of the read across 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 NOAEL of the test substance 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 (Rousseau, 2010).

Justification for classification or non-classification

Based on the results of a 90 day repeated dose oral toxicity study (OECD Guideline 422) with the read across substance, sodium thioglycolate, and a 28 day repeated dose oral toxicity study (OECD Guideline 408) with the test substance itself, the test substance does not require classification for this endpoint according to EU CLP (EC 1272/2008) criteria.