Registration Dossier

Administrative data

Description of key information

The NOAEL of the subchronic oral toxicity study in rats was 50 mg/kg bw as assessed in a GLP- and OECD 408 compliant study. Mean liver weights were dose-relatedly increased in males and females of the 50 and 250 mg/kg bw/d treated group. In the livers of animals treated with 250 mg/kg bw/d different microscopic changes were recognized: centrilobular hepatocellular hypertrophy, intracytoplasmic pigment accumulation and (peri)vasculitis.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
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

In a GLP-compliant 90d study following OECD guideline 408, the test item was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 10 (test group 1), 50 (test group 2) and 250 mg/kg bw/d (test group 3) over a period of 3 months. In addition to the required examinations, special attention was given to the reproductive organs of male and female animals.

With regard to clinical examinations, no signs of general systemic toxicity were observed even at a dose level of 250 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained. Regarding clinical pathology, a dysregulation of the liver cell metabolism in male and female rats of test group 3 (250 mg/kg bw/d) was observed. In males, i) prolonged prothrombin time indicating a decreased coagulation factor synthesis and ii) lower cholesterol levels occurred, and in females iii) increased GGT activities were measured. The reason for the increase of inorganic phosphate levels in female animals of test group 3 (250 mg/kg bw/d) could not be elucidated and, thus, were regarded to be adverse. Additionally, in males of the same test group higher incidences of triple phosphate crystals and epithelial and granular casts were found in the urine sediment. This correlated with the histopathologic finding of apha2u-globulin and was regarded as species-specific for rats and not relevant for humans (Hard et al., 1993). No treatment-related effects on sperm parameters were observed even at the high-dose level of 250 mg/kg bw/d.

Regarding pathology, adverse findings were only noted in the livers of male and female animals of test group 3 (250 mg/kg bw/d). Mean liver weights were increased in a dose-related manner in males and females of test groups 2 and 3 (50 and 250 mg/kg bw/d). For both sexes the increase in weight was minimal in test group 2 (50 mg/kg bw/d), while it was considered slight in males and moderate in females of test group 3 (250 mg/kg bw/d). Most livers from males and females of test group 3 (250 mg/kg bw/d) were found to be dark brown discolored, grossly. Treatment-related changes were recognized microscopically in the livers of male and female animals of test groups 2 and 3 (50 and 250 mg/kg bw/d), namely centrilobular hepatocellular hypertrophy and intracytoplasmic pigment accumulation. Centrilobular hepatocellular hypertrophy was observed among females of test group 2 (50 mg/kg bw/d) and in males and females of test group 3 (250 mg/kg bw/d). This hepatocellular hypertrophy is believed to represent the morphologic hallmark of enzyme induction. This change resulted in significant increased mean liver weights in males and females of the mentioned test groups. The mean absolute and relative liver weight increase in females of test group 3 (250 mg/kg bw/d) exceeded 40% and was therefore regarded as adverse. The less severe changes in males of test group 3 (250 mg/kg bw/d) were also considered to be adverse in combination with the aberrant clinical chemistry parameters, i.e. prolonged prothrombin time and lower cholesterol levels. Intracytoplasmic pigment accumulation was noted in the livers of animals of test group 3 (250 mg/kg bw/d). Females were more affected compared to males. The change consisted of a fine brown intracellular pigment located within areas of hypertrophied hepatocytes. Using different staining methods, the pigment was proven to consist of lipofuscin mainly (the pigment was positive for Schmorl and PAS, while the Hall’s and Perl’s staining methods were negative). The presence of lipofuscin was believed to have contributed to the discoloration of the liver, observed grossly. The presence of increased lipofuscin accumulation in relative young animals was considered adverse.

(Peri)vasculitis related to the branches of the hepatic artery was observed at a relative high incidence (30%) in males of the 250 mg/kg bw/d treated group. The vascular change had similar characteristics in all animals with degeneration and fibrinoid necrosis of the vessel wall and a perivascular inflammatory infiltrate (macrophages mainly) and fibroblast reaction. The observed percentage of 30% is comparable to the highest incidence of (peri)vasculitis observed among control male rats; historical control data related to this strain of rats includes incidences of (peri)vasculitis in the liver that varies between 0% and 30% in male control rats, while the lesion was not recorded in control female rats. Because the lesion was also found in a single male and a single female treated with 50 mg/kg bw/d, and a precursor lesion was observed in a single female treated with 250 mg/kg bw/d, a relationship to the treatment with the test item cannot be ruled out. Possibly, the test item aggravated this spontaneous occurring lesion. In a single female of the group treated with 50 mg/kg bw/d an area of slight hepatocellular necrosis was found to be closely related to focal vasculitis.

A slight, statistically significant increase of the mean absolute and relative ovarian weight was present in females treated with 250 mg/kg bw/d. Microscopically, 6 out of 10 females of this group exhibited minimal interstitial cell vacuolation. This interstitial cell vacuolation was at least partial responsible for the increase in ovarian weight. In theory, vacuolation of interstitial cells may be associated with alterations of steroid synthesis leading to lipid accumulation within the cells. This change however is of questionable adversity, since, based on the ovarian morphology, the cyclicity seems not to be disturbed in high-dose females.

The weight changes noted for the adrenal gland in females of the 250 mg/kg bw/d treated group were also not supported by obvious histopathologic changes. Most probably the increase adrenal gland weight can be explained by a diffuse increased cortical cell size that could not be recognized at light microscopic level. Since there were no relevant changes noted in the adrenal glands, the weight changes are considered non-adverse and of no significant toxicologic relevance.

A minimal increase in mean thyroid gland weight was observed in male animals of the 250 mg/kg bw/d treated group, only. This weight change was not supported by convincing histopathologic changes (only one male showed minimal follicular hypertrophy worth mentioning). The only finding that could point to a treatment-related effect is the presence of a slight higher incidence of colloid changes (inspissated colloid) in several high-dose males. Nevertheless, both the changes in thyroid gland weight and histology are considered of uncertain test-item relationship. Although there is a broad variation in the severity of hyaline droplet accumulation in the kidney of male rats in general, for the slight increase in severity observed in male rats of the 250 mg/kg bw/d treated group compared to the control males in this study a relationship to treatment cannot be excluded. The hyaline droplets were proven to consist of alpha2U-globulin mainly.

In conclusion, the oral administration of the test item by gavage to male and female Wistar rats for 3 months caused adverse signs of toxicity in male and female animals at a dose level of 250 mg/kg bw/d taking clinical pathology and pathology findings into account. Therefore, the no observed adverse effect level (NOAEL) was set to 50 mg/kg bw/d for male and female Wistar rats.

In the 28 day oral toxicity study (RCC, Research and Consulting Company, 1997) dose-related increases in the absolute liver weights were recorded in both sexes in all groups (50, 100, 200 mg/kg). Dose levels were chosen based on the results of a five-day screening study. In that study, 1000 mg/kg bw/d caused a marked increase in absolute liver weights: males +31 % and females +49%. In the actual 28 -day study, liver weights were also markedly affected; after the treatment-free recovery period, the liver weights of females of the 200 mg/kg group were statistically significantly higher when compared with the controls.

The following effects were observed for the highest dose group (200 mg/kg): Slight decrease in the glucose level in females and slight increases in the triglyceride and phosphorus levels, and gamma-glutamyl-transferase activity were noted in females. A slight increase in the total cholesterol, phospholipid and potassium level was recorded in males. These findings primarily reflect metabolic adaptation in the liver and are of no toxicological relevance. Liver weights (absolute and/or relative) were higher in animals of both sexes. After the treatment-free recovery period only the liver weights of females were statistically significantly higher when compared with the controls. Centrilobular hepatocellular hypertrophy of the liver was the corresponding finding in all males and females. The severity of this hepatocellular hypertrophy was slight to moderate (except for 1 female rat with grade minimal).

The following effects occurred at the mid dose group (100 mg/kg): Slight increases in the triglyceride and phosphorus level, and gamma-glutamyl-transferase activity were noted in females. Liver weights (absolute and/or relative) were higher in animals of both sexes. Centrilobular hepatocellular hypertrophy of the liver was noted in all males and 4 females. The severity of this hepatocellular hypertrophy was minimal to moderate.

The following effects were observed at the lowest dose group (50 mg/kg): Liver weights (absolute and/or relative) were higher in males at 50 mg/kg. There was no histopathological correlate.

Increased liver weight is a very common effect in toxicity studies. It is usually considered a physiological adaption if it is dose related, reaches a steady state with continued dosing, is reversible after cessation of treatment and is not associated with marked changes in serum enzyme levels or other measures of hepatocyte integrity. The hepatocellular hypertrophy, noted at the termination of the treatment period in the liver of the mid- and high-dose males and females, is considered to be treatment-related. Hepatocellular hypertrophy is regarded as an adaptive process rather than to be a toxic effect. The absence of this finding in the recovery rats is considered to indicate full reversal of this change after the 2-week recovery period.

As no histopathology correlates were observed at 50 mg/kg bw, this is considered the NOAEL. Findings at the higher dose of 200 mg/kg bw bw are consistent with adaptation to metabolism. Considering the degree of liver enlargement and the hypertrophy, findings have to be considered adverse.

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 the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No.1272/2008, classification for repeated dose toxicity is not warranted. No serious effects were noted at the highest dose of 200 mg/kg bw upon subacute exposure.