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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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:
other: GLP and OECD Guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test compound: Sodium thioglycolate
Source: Bruno Bock Chemische Fabrik GmbH & Co KG
Batch: 22785
Content of active ingredient (iodometric): 46,1 % in water

Test animals

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

Administration / exposure

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
Doses / concentrations
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 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

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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
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
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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 andmethod 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 thioglycolatewas 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 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 test item concentrations in the administered dosage forms analyzed inweeks 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 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 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) toSprague-Dawley ratsat dose‑levels of7, 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, 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.