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Description of key information

In the combined repeat-dose/developmental screening study, unspecific toxic effects have been observed in females at the highest dose tested. No toxicological relevant adverse effects were observed in male rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

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

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

The toxic effects on Sprague Dawley rats of both sexes after repeated dosing with the substance, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring, were investigated. A 3-week treatment-free period was allowed in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

The animals received the test item, dissolved in Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v, at a constant volume of 10 mL/kg body weight as indicated in the scheme below.

 

Main groups

Group

Treatment (mg/kg body weight/day)

Number of animals

1

0

10M+10F

2

62.5

10M+10F

3

250

10M+10F

4

1000

10M+10F

 

 Recovery groups

Group

Treatment (mg/kg body weight/day)

Number of animals

5

0

5M+5F

6

1000

5M+5F

 

Main groups

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for approximately 6 weeks. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3post partum. The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology and clinical chemistry), litter data, macroscopic observations, organ weights and histopathological examination.

Body weight, clinical signs and macroscopic observations of pups were also performed.

Recovery groups

Animals of the recovery groups were treated for 4 consecutive weeks and sacrificed after 3 weeks of recovery (Groups 5, 6).

The following parameters were evaluated in these animals: mortality, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), body weight, food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations, organ weights and histopathological examination.

Results

No mortality in parental animals occurred throughout the study.

Staining of different regions of the body surface was the main clinical sign recorded during treatment and recovery period in all treated groups. At observation of the cage tray, green/orange staining was recorded in all treated groups during treatment. Moreover, treated animals of the recovery group (Group 6) showed green staining in the cage tray also during the recovery period. These discolorations were due to the colour of the test item and its properties as textile dyes.

Neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the test item. No relevant differences in motor activity or sensory reactivity to stimuli were noted between control and treated groups.

In males, no relevant differences in body weight and body weight gain were recorded in treated groups when compared to controls. In females, statistically significant decreases in body weight and body weight gain were detected in the high dose group starting from the second week of treatment up to post partum period. No difference in body weight gain was recorded in animals of both sexes during the recovery phase.

Slight decrease in food consumption was recorded in high dose females starting from pairing up to post coitum period. No changes were noted in post partum period. No changes were noted in treated males during treatment. No differences were noted during the recovery period in both sexes.

In males, slight leucopenia was recorded in the high dose group. In addition, slight thrombocytosis was recorded in mid- and high dose groups, with no dose-relation. In females, increase of reticulocytes was detected in the high dose group. These changes were not considered adverse due to the absence of changes in the other blood cell parameters and histopathology. Changes recorded at the coagulation test in mid-dose males and mid- and high dose females were considered of no toxicological relevance. No differences that could be considered treatment-related were detected in the recovery groups, confirming complete reversibility.

In females, relevant changes of liver/cholestasis markers were recorded in 2 out of 5 animals of the high dose group. The above findings could reflect liver impairment/cholestasis, even though changes were not confirmed at histopathological examination. Due to the limited incidence, it is unlikely that changes are treatment-related. In addition, a slight increase of creatinine was recorded in almost all females of the same group. In males, a decrease of liver markers and urea and an increase of total bilirubin and bile acids – which were due to an interference of the dye with the photometric measurement – were the most changes detected in the high dose group. Considering the severity and/or the direction of the findings observed in males were not considered to be suggestive of tissue/organ injury. In the recovery groups, changes recorded during the dosing phase completely recovered.

No significant differences in terminal body weight were noted between controls and treated animals, both in main and recovery groups. The most relevant change noted in the absolute and/or relative organ weight, between treated and control groups, was the increase of thyroid weights in high dose females (main and recovery groups) and the increase of liver weights in high dose males. In addition, a decrease in thymus weights was also detected in high dose females.

The most remarkable changes observed at post mortem examination in treated main group animals, when compared with controls, were reduced size of the thymus in most females dosed at 1000 mg/kg body weight/day and dark staining on the tail, on the dorsum of the skin or on the head. Such staining was considered related to the colour of the test item. In recovery animals, the only relevant changes noted at post mortem examination in treated animals, when compared with controls, were dark staining of the tail.

Treatment-related changes were noted in the thymus of most treated females dosed at > 250 mg/kg body weight/day, sacrificed at the end of treatment period. The incidence of this change was statistically significant in the high dose females with respect to controls. No histopathological changes were noted in the high dose females sacrificed after 3 weeks of recovery period or in females dosed at 62.5 mg/kg body weight/day. The histopathological change detected in the thymus was atrophy, represented by: a minimal to moderate reduction in cortical lymphocytes, shrinkage of the thymic lobules, increased prominence of interlobular septae and an inverse cellular density ratio cortex/medulla. In addition, in some instances, an increased number of apoptotic bodies “tingible body macrophages” was also reported. In control and high dose males, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

 

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 250 mg/kg body weight/day for female rats and 1000 mg/kg body weight/day for male rats.

Justification for classification or non-classification

The NOAEL is 250 mg/kg bw/day (oral). No target organ toxicity or specific toxic effects were observed. No classification applies.