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Description of key information

The potential toxicity of the substance following repeated administration was investigated  according to OECD guideline 422 and EPA 
guideline OPPTS 870.3650 (Takawale, 2010).
The NOAEL by oral route was established to 24 mg/kg bw/day.
No Repeated-dose toxicity studies by inhalation or dermal route were available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
24 mg/kg bw/day
Study duration:
subacute
Species:
rat

Additional information

Two studies by oral route are reported for this endpoint:

The objective of the first study was to determine dose-levels of the substance to be administered in a combined repeated-dose toxicity study with the reproduction/ developmental toxicity screening test (Takawale, 2010b).

The second study (Takawale, 2010a), which was chosen as the key study is the combined repeated dose toxicity study with reproduction /developmental toxicity screening test. This study was performed according to OECD guideline 422 and was conducted in compliance with the principles of Good Laboratory Practices.

No other repeated-dose toxicity studies by inhalation or dermal route are available.

In the main study by oral route (Takawale, 2010a), three groups of 10 males and 10 females Wistar rats received the test substance daily, by gavage, before mating and through mating and, for the females, through gestation until day 3post-partum.The dose-levels were 12, 24 or 48 mg/kg bw/day. Another group of 10 males and 10 females received the vehicle, sterile water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg bw/day.

Animals were examined daily for clinical signs and mortality and detailed clinical observations were performed once before the beginning of the treatment period and then once a week until the end of the study. Body weight was measured weekly, food consumption also except during the mating period. A Functional Observation Battery includingsensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations was performed in the week before treatment and at the end of the study. Blood and urine samples were taken for analysis of hematology, blood biochemistry and urine parameters at the end of the study from randomly selected five males and five females (except urine) from each group.

Males were sacrificed after completion of the mating period on treatment days 29 and 30 and females on day 4post-partum. All animals were subjected to a complete macroscopicpost-mortemexamination with a careful examination of the stomach lining due to the strong irritant / corrosive properties of the test item. A microscopic examination was performed on selected organs of five randomly selected males and five randomely selected females having delivered from the control and high-dose groups, on the non-pregnant females and on all macroscopic lesions. 

There were no treatment related deaths but 5 premature deaths (1 female, 1 female and 2 males and 1 female from the low, mid and high-dose groups respectively) were observed.The macroscopic examinations of the five decedents together with the histopatological findings confirmed that the death was due to misgavaging or regurgitation/aspiration of the test item.

Salivation was observed in all males and females of the high-dose group and in 5 males and 3 females of the mid-dose group. In addition, most of the animals treated at the highest dose had piloerection and respiratory sounds. In the mid-dose group, only 1 male had piloerection while respiratory sounds were noted in 3 males and 1 female respectively. In the low-dose group, respiratory sounds were occasionally observed in few animals as well as in one animal of the control group. No other clinical signs were observed at 12 mg/kg/day.

No relevant differences were observed concerning functional and behavioural examinations in male and female treated groups when compared with controls.

No test item related effects on body weight and body weight change were observed. In males, statistical analysis of the food consumption data revealed no significant effect in treated groups when compared with controls. In females, no effect on food consumption was observed in treated groups during premating and gestation period as compared to controls. However, statistically significant decrease in food consumption in the high-dose group was observed during post natal days 0-4 when compared with controls. There were no effects in females food consumption at 24 or 12 mg/kg/day.

Laboratory investigations showed that females treated at 48 mg/kg bw/day had a statistically significant decrease in haemoglobin and hematocrit when compared with controls. While being in the normal biological range, the decrease in haemoglobin was considered to be treatment-related due to dose dependency. There were no treatment-related effects on hematological parameters in males and no effects on biochemical parameters whatever the sex. No treatment-related effects on organ weights and no particular macroscopic findings were observed.

At microscopic examination, histiocytic infiltration was observed with a dose-related trend at all dose-levels in the ileum of males and females and from the mid-dose level in the jejunum of males. In the absence of any degenerative changes, this observation was considered to be non adverse.In the mesenteric lymph nodes, infiltration with histiocytes were observed in all males whatever the dose and in all females of the mid and high-dose groups and in 2 out 5 females treated at low dose. The grade of the histiocytic infiltration increased in a dose-dependent manner in both sexes being minimal in 5 animals and mild in 2 animals of the low-dose level and mild in 2 animals and moderate in 8 animals of the high-dose level. In the absence of degenerative changes, this observation was considered to be non adverse.

In conclusion, parameters like body weight, neurology, clinical biochemistry, gross necropsy, organ weight remained unaffected up to 48 mg/kg bw/day. At the highest dose-level, most of the animals had piloerection. Salivation and respiratory sounds were also observed but these clinical signs are frequently observed with strong irritant / corrosive substances administered by gavage and were therefore not considered as toxicologically relevant. At the highest dose-level, changes in haematological parameters and decrease in food consumption during the post –natal period were also seen in females. No such findings were noted at the lower dose-levels. Test item-related histopathological changes were observed in small intestine and the mesenteric lymph node in all treated groups.Both males and females had infiltration with histiocytes that probably contained test-item lipid complexes which are poorly degraded by lysosomal enzymes. As no degenerative changes were associated, this effect was therefore considered to be non‑adverse.

Based on the data generated from this study, A No Observed Effect Level (NOEL) could not be established for the parental toxicity due to histopathological findings in small intestine and mesenteric lymph nodes in all treated groups. As no degenerative changes were associated, these findings were therefore considered to be non‑adverse and the No Observed Adverse Effect Level (NOAEL) was considered to be 24 mg/kg/day.

 

Justification for classification or non-classification

According to the criteria laid down in EU regulation (EC) n° 1272/2008 (CLP) and the EU directive 67/548/EEC, the substance is not classified for repeated dose toxicity.