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Description of key information

Oral toxicity study was performed GLP-compliant in SD (Crj:CD) rats by an OECD combined repeated dose and reproductive / developmental toxicity screening test (OECD Guideline 422). The test substance was administered by gavage at doses of 2, 10 and 50 mg/kg bw/day for 45 days to males and from 14 days before mating to day 3 of lactation to females. Based on pathological changes in the liver of both sexes, the NOAEL was considered to be 2 mg/kg bw/day for both sexes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
other information
Study period:
1997
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- males: 42 days
- females: from 14 days before mating to day 3 of lactation
Frequency of treatment:
daily
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: twice a week the first week; weekly afterwards, including during the pregnancy of females. Additionally for females, days 0 and 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption determined in four day blocks during the first week and weekly afterwards.
- Compound intake calculated as mean diet as g food/rat/average over the period of calcul.

HAEMATOLOGY: Yes
- How many animals: all males (13 per dose group)

CLINICAL CHEMISTRY: Yes
- How many animals: all males (13 per dose group)
Sacrifice and pathology:
Terminal killing at day 43 for males and on day 4 of lactation for females.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

The following tissues were prepared for microscopic examination: Heart, thymus, spleen and liver, kidney, adrenal gland, testis, epididymis. These were also weighed.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: Temporary salivation was induced at 10 mg/kg or more groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
Females: One animal died on postpartum day 3 at 50 mg/kg.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: Decrease in body weight gain was observed at 50 mg/kg.
Females: Decrease in body weight gain was observed in 10 mg/kg and more groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: Decrease in food consumption was observed in 15 mg/kg.
Females: Decrease in food consumption was observed in 10 mg/kg and more groups.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males: In blood analysis, there were several changes in 50 mg/kg group, such as an elevation of platelet and white blood cell counts, increases in total protein, albumin, total cholesterol, Ca and inorganic phosphorus, and decreases in the A/G ratio and Cl concentration.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males: Increases in eosinophilic bodies and basophilic changes of the renal tubular epithelial cells were observed in all treatment groups and granular casts in the lower nephrons were observed in 10 mg/kg and more groups. As these pathological changes were observed only in males, accumulation of α2u-macroglobulin is suspected as a cause of male specific renal toxicity. Centrilobular hypertrophy of hepatocyte was observed in 10 and 50 mg/kg groups (±: 4 in 13, +: 9 in 13 for 10 mg/kg, ++: 13 in 13 for 50 mg/kg, compared to no changes in 0 and 2 mg/kg groups).
Females: Centrilobular hypertrophy of hepatocytes was observed in 10 and 50 mg/kg groups (±: 6 in 13, +: 1 in 13 for 10 mg/kg, ±: 1 in 13, +: 11 in 13, ++: 1 in 13 for 50 mg/kg, compared to no changes in 0 and 2 mg/kg groups).
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males: In kidneys, absolute and relative weight was increased in all treatment group and in 10 mg/kg or more groups, respectively. Liver weights significantly increased by 14 and 66 % for absolute weight (14 and 74 % for relative weight) in 10 and 50 mg/kg group, respectively.
Females: In kidneys, absolute and relative weights were increased at 50 mg/kg. Liver weights significantly increased by 43 % for absolute weight (51 % for relative weight) in only 50 mg/kg group.
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males: Increased eosinophilic bodies and basophilic changes of the renal tubular epithelial cells in the kidneys of males treated with the compound at any dose level tested were noted, as well as granular casts in the lower nephrons. Centrilobular hypertrophy of hepatocytes was also detected in the 10 mg/kg or more groups.
Females: Centrilobular hypertrophy of hepatocytes was observed in the 10 mg/kg or more groups.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (nominal)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Critical effects observed:
not specified

Males:

Temporary salivation was induced at 10 mg/kg or more groups. Decrease in body weight gain and food consumption was observed at 50 mg/kg. In kidneys, absolute and relative weight was increased in all treatment group and in 10 mg/kg or more groups, respectively. In addition, increases in eosinophilic bodies and basophilic changes of the renal tubular epithelial cells were observed in all treatment groups and granular casts in the lower nephrons were observed in 10 mg/kg and more groups. As these pathological changes were observed only in males, accumulation of α2u-macroglobulin is suspected as a cause of male specific renal toxicity. Liver weights significantly increased by 14 and 66 % for absolute weight (14 and 74 % for relative weight) in 10 and 50 mg/kg group, respectively. Centrilobular hypertrophy of hepatocyte was observed in 10 and 50 mg/kg groups (±: 4 in 13, +: 9 in 13 for 10 mg/kg, ++: 13 in 13 for 50 mg/kg, compared to no changes in 0 and 2 mg/kg groups). In blood analysis, there were several changes in 50 mg/kg group, such as an elevation of platelet and white blood cell counts, increases in total protein, albumin, total cholesterol, Ca and inorganic phosphorus, and decreases in the A/G ratio and Cl concentration.

Females:

One animal died on postpartum day 3 at 50 mg/kg. Decrease in body weight gain and food consumption was observed in 10 mg/kg and more groups. In kidneys, absolute and relative weights were increased at 50 mg/kg. Liver weights significantly increased by 43 % for absolute weight (51 % for relative weight) in only 50 mg/kg group. However, centrilobular hypertrophy of hepatocytes was observed in 10 and 50 mg/kg groups (±: 6 in 13, +: 1 in 13 for 10 mg/kg, ±: 1 in 13, +: 11 in 13, ++: 1 in 13 for 50 mg/kg, compared to no changes in 0 and 2 mg/kg groups).

Conclusions:
The NOEL for toxicity was suggested to be less than 2 mg/kg/day in males and 2 mg/kg in females by the authors.
Considering the fact that only males were observed with renal changes, it is assumed that alpha-2-macroglobulin is involved in the specific toxicity observed. Then, the NOEL for the test item is 2 mg/kg/day for both sexes, based on liver toxicity.
Executive summary:

A study was conducted to evaluate the repeated dose toxicity and effects of the test substance on the reproductive performance in parental animals and development and growth of F1 pups until day 4 of lactation. The study was conducted according to GLP and to OECD Guidelines for Testing of Chemicals: Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (March 22, 1990).

The study design consisted of 13 male and 13 female Sprague-Dawley (Crj:CD) rats per group, dosed at 0 (vehicle control), 2, 10 and 50 mg/kg for 2 weeks before mating and for 2 weeks after mating, followed by additional 2 weeks after completion of mating in males and throughout the gestation period until day 3 of lactation after parturition in females.

In the males, the test substance induced transient salivation at 10 mg/kg or greater doses and inhibited weight gain and feed consumption at an early stage of the treatment at 50 mg/kg. Necropsy conducted after repeated administration of 42 doses revealed increases in kidney weight at 2 mg/kg or greater doses and increases in liver weight at 10 mg/kg or greater doses. Findings in histopathological examinations included increases in eosinophilic bodies and basophilic renal tubules and the presence of granular casts at 2 mg/kg or greater doses, as well as centrilobular hypertrophy of hepatocytes at 10 mg/kg or greater doses. Hematological examinations of blood samples taken during necropsy showed increases in platelets and WBC at 50 mg/kg. Blood biochemistry revealed increases in the concentrations of total protein, albumin, total cholestrol, calcium and inorganic phosphorus, as well as decreases in the A/G ratio and CI at 50 mg/kg.

In the females, the test substance inhibited weight gain and feed consumption at an early stage of treatment at 10 mg/kg or greater doses. At 50 mg/kg, it also inhibited weight gain and feed consumption during gestation. One animal died 3 days after parturition. Necropsy conducted 4 days after parturition reveales a tendency toward increases in weight of the liver and kidneys. Findings obtained in histopathological examinations suggested centrilobular hypertrophy of hepatocytes at 10 mg/kg or greater doses.

Findings revealed that the test substance had no effects on the copulation index or fertility index at 50 mg/kg or lower doses. The test substance did not affect the length of the gestation period or delivery index in the maternal animals, either. No abnormal parturition was observed. Abnormal nursing behavior was noted in 3 animals from the 50 mg/kg group.

The findings in offspring showed that the test substance had no effects on the parturition index, live pup delivery index, or the sex ratio and body weight on day 0. In the 50 mg/kg group, the offspring viability index and body weight on day 4 of lactation showed a tendency to decrease. No offspring from the test substance groups showed any morphological anomaly.

It was concluded that under the conditions of the present study, the NOEL for parental toxicity of the test substance is slightly lower than 2 mg/kg/day in males and 2 mg/kg/day in females and that the NOEL for reproduction/developmental toxicity is 50 mg/kg/day in males and 10 mg/kg/day in females and offspring.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

Oral toxicity study was performed GLP-compliant in SD (Crj:CD) rats by an OECD combined repeated dose and reproductive / developmental toxicity screening test (OECD Guideline 422). The test substance was administered by gavage at doses of 2, 10 and 50 mg/kg bw/day for 45 days to males and from 14 days before mating to day 3 of lactation to females. In males, temporary salivation was induced in 10 mg/kg bw/day or more groups. Decrease in body weight gain and food consumption was observed at 50 mg/kg bw/day. In kidneys, absolute and relative weight was increased in all treatment groups and in 10 mg/kg bw/day or more groups, respectively. In addition, increases in eosinophilic bodies and basophilic changes of the renal tubular epithelial cells were observed in all treatment groups and granular casts in the lower nephrons were observed in 10 mg/kg bw/day and more groups. Liver weights significantly increased by 14 and 66 % for absolute weight (14 and 74 % for relative weight) in 10 and 50 mg/kg bw/day group, respectively. Centrilobular hypertrophy of hepatocyte was observed in 10 and 50 mg/kg bw/day groups (+/-: 4 in 13, +: 9 in 13 for 10 mg/kg bw/day, ++: 13 in 13 for 50 mg/kg bw/day, compared to no changes in 0 and 2 mg/kg bw/day groups). In blood analysis conducted only in males, several changes were observed only in 50 mg/kg bw/day group. In females, one female died on postpartum day 3 at 50 mg/kg bw/day. Decrease in body weight gain and food consumption was observed in 10 mg/kg bw/day and more groups. In kidneys, absolute and relative weight was increased at 50 mg/kg bw/day. Liver weights significantly increased by 43 % for absolute weight (51 % for relative weight) in only 50 mg/kg bw/day group. However, centrilobular hypertrophy of hepatocytes was observed in 10 and 50 mg/kg bw/day groups (+/-: 6 in 13, +: 1 in 13 for 10 mg/kg bw/day, +/-: 1 in 13, +: 11 in 13, ++: 1 in 13 for 50 mg/kg bw/day, compared to no changes in 0 and 2 mg/kg bw/day groups). As renal pathological changes were observed only in males, accumulation of 2u-macroglobulin is suspected as a cause of male specific renal toxicity. Therefore, based on pathological changes in liver of both sexes, NOAEL was considered to be 2 mg/kg bw/day for both sexes. (MHW Japan, 1997)

The aim of the study of Motoc (1971) was to investigate the toxic effects of the test substance to stomach, liver and kidney following oral administration. The test substance was applied to 50 rats two times per week for 3, 5 and 8 months. According to the authors, the total dose was 2125, 4895 and 7875 mg/animal after 3, 5 and 8 months, respectively. Blood serum was assayed for enzyme activity; stomach, liver and kidneys were examined pathologically. Pathology revealed moderate lesions of the stomach which were most pronounced after 3 months of treatment. Hepatic and renal lesions were observed, too; however, these lesions were less severe than those of the stomach and were at least partially reversible. Serum glycoprotein and albumin levels were decreased. The activities of leucine aminopeptidase, glutamic-oxalacetic transaminase, glucose-6 -phosphate dehydrogenase, aldolase and beta-glucuronidase were increased. According to the authors, the degree of the biochemical changes corresponded with the intensity of the heatic and renal lesions. (Motoc, 1971)

The Toxic Substances Control Act provides additional literature data on the repeated oral dose toxicity in rats and dogs.

Five rats were fed the test substance for 11 days. One rat each died after 3 and 6 daily doses, respectively. Considerable loss of body weight was observed in all rats. Pathology at sacrifice on day 11 revealed peripheral lividity and distended stomachs with slightly red mucosae. Microscopy revealed no significant changes in the organs. (TSCAT, 1984)

The effects of the test substance were investigated in groups of 4 dogs / sex. The animals were fed a diet containing the test substance at concentrations of 50, 150, 300 and 1000 ppm; controls were given unsupplemented diet. The average daily food consumption was 308, 372, 316, 324 and 235 g/kg bw for the males and 354, 337, 368, 314 and 212 g/kg bw for the females of the control, 50 ppm, 150 ppm, 300 ppm and 1000 ppm group, respectively; corresponding to average doses of 0, 16.8, 47.4, 97.2 and 235 mg/kg/d, respectively, for the males and 0, 16.9, 55.2, 94.2 and 212 mg/kg/d, respectively, for the females. One female of the 1000 ppm group died on day 26 of the study; the remaining 7 animals of this group were sacrificed in extremis on day 28 of the study. The animals of this group exhibited body weight losses or no body weight gain and a reduced food consumption. Significant increases in serum alkaline phosphatase, serum glutamic-pyruvic transaminase and serum glutamic-oxalacetic transaminase were found. Relative liver and kidney weights were significantly increased. Histopathology revealed morphologic changes in the liver sections (intracytoplasmatic membranous bodies in the hepatocytes and / or condensations of the cytoplasmatic membranes of many of the animals and focal to multifocal hepatocellular degeneration and necrosis of individual or small groups of hepatocytes scattered throughout the liver lobules of all animals). No deaths or clinical signs of toxicity were observed in the other groups; body weight an food consumption of the dogs fed 50, 150 and 300 ppm of the test substance were similar to the untreated controls. Hematology, blood chemistry and urinanalysis revealed an increase in serum alkaline phosphatase activity of the 300 ppm dogs and a slight increase in blood thiocyanate in the 300 ppm females; however, these alterations were not statistically significant. Relative liver weights were slightly and not statistically significantly increased in 300 ppm animals and in one 150 ppm female. The gross pathologic observations noted for the 50, 150 and 300 ppm group were similar to control and were considered normal. Histopathology revealed treatment-related morphologic changes in the livers (eosinophilic-staining, oval shaped membranous cytoplasmatic bodies in the hepatocytes) of some animals fed 150 and 300 ppm. This change was more frequent in the 300 ppm group than in the 150 ppm group. According to the authors, this finding was regarded to be an adaptive response of the liver cells. No substance-related changes were found in the livers of the 50 ppm and control groups. No substance-related changes of tissues other than liver were observed in the groups fed 50, 150 and 300 ppm. According to the authors, these findings suggested a hepatotoxic effect of the test substance in dogs. (TSCAT, 1974)

Repeated dose toxicity: inhalation

Rusin et al. (1958) examined repeated inhalation toxicity in rats. Five rats were exposed to vapours produced by heating of the test substance to 70 - 80 °C. According to the authors, HCN concentration did not exceed 1.4 - 1.6 mg/m³. One rat died. Morphological changes similar to those observed after intoxication with nitriles and cyanides were found. No further data are given. (Rusin et al., 1958)

The Toxic Substances Control Act provides additional literature data on the repeated inhalation toxicity in rat.

The subacute effects of dusts of the test substance after a total of 10 exposures by inhalation (head-only) were investigated in groups of 10 rats. Two groups were exposed to airborne dust of the test substance; another group was exposed to air only and served as control. According to the authors, the mean dust concentrations were 9.8 mg/m³ (ranging from 6.8 to 13.8 mg/m³) and 79.5 mg/m³ (ranging from 71.8 to 86.8 mg/m³). Five rats/group were sacrificed after the last exposure, the remaining rats were observed for another 14 days without treatment. Urine samples were collected after exposure 9 and recovery day 13; blood samples were collected after exposure 10 and recovery day 14. One high dose rat was sacrificed in extremis after the 4th exposure; however, this death was not substance-related. The surviving treated rats showed slight red ocular and nasal discharge during exposure. Body weights of the high dose group rats were significantly decreased on days 2 to 4 of exposure and returned to normal during the remainder of exposure and post-observation period. In the low dose group, body weights and body weight gains were similar to control. A dose-related increase of the relative liver weights was observed; this finding was reversible within the recovery period. Total serum protein was slightly but not signifcantly increased in the treated groups; urine osmolality was decreased in the high dose group. These clinico-chemical effects were reversible within the 14-day recovery period except the serum finding in the high dose group. Pathology revealed a substance-related liver effect (increased cytoplasmic basophilia of hepatocytes) in high dose group rats which had been sacrificed immediately after termination of exposure. However, this hepatic lesion was not found in the high dose rats after the recovery period. No substance-related pathological lesions were observed in the low dose group at any time. (TSCAT, 1984)

In another study, inhalation exposure of rats to sub-lethal concentrations of the test substance resulted in histopathologic findings (hyaline granular degeneration of the kidney tubules and hypertrophy of the thymic medulla) after a 2 -week post observation period. No further data are given. (TSCAT, 1992)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on the results of these data upon subacute oral exposure in rats, the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.