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Description of key information

Key study: Cyclacet 90-day rat NOAEL ≥1500 mg/kg bw/day females (OECD 408)

Supporting study: Cyclacet systemic toxicity in Reproductive/Developmental Toxicity screening study (OECD TG 421) >=1000 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
The 90-day oral repeated dose toxicity study is adequate for covering this endpoint.

Additional information

Below the executive summaries are presented of Cyclacet being tested in the 90 -day repeated dose toxicity study and in the Reproductive/Developmental Toxicity Screening study. Beside these also a short summary of the repeated dose of Verdox ( EC no. 243-718-1 Cas no. 20298-69-5 on the ECHA website) is presented to support the read across between Cyclacet and Verdox for systemic toxicity considering the effect on hydrocarbon nephropathy and developmental toxicity.

Cyclacet 90 -day repeated dose study

Introduction and method

In a sub-chronic repeated dose toxicity study performed according to OECD 408 and under GLP conditions, rats were orally exposed to Cyclacet for 90 days via the food. The dose levels were 0, 200, 2000, 6000, and 20000 ppm, corresponding to 0, 15.3, 154.9, 464.1, and 1504.6 mg/kg bw/day, respectively. Clinical signs, functional observations, body weight change, dietary intake, and water consumption were monitored during the study. During week 7 and at the end of the study, haematology, blood chemistry and urinalysis were evaluated. Furthermore, oestrus cycle assessment was performed on female animals between week 6 and 7 and week 12 and 13. Opthalmoscopic examination was performed at the start and end of the study in control and high-dose animals. At the end of the study, all animals were subjected to gross necropsy examination and histopathological examination of tissues was performed on animals from control and high-dose. Furthermore, sperm assessment was performed on males at necropsy.

Results

Clinical signs:Reduced overall body weight was observed in male and female animals in the highest dose group, correlated with reduced food consumption and adverse effects on food efficiency, indicating decreased food palatability rather than an adverse effect of the test substance.

Haematology: No effects were found.

Blood biochemistry: Blood chemical investigations showed reductions in chloride and sodium concentration of 1 -5%, which is considered minimal. The minimal decrease in ASAT and ALAT levels is not toxicological relevant. The reduction and increase in bile acids in males and females, respectively, is not considered toxicologically relevant either. The increases in cholesterol in males treated with either 20000 or 6000 ppm are most likely linked to the liver weight findings and the histopathological effects seen in this organ and can be considered of an adaptive nature.

Organ weights and histopathological findings: Relative liver weight is increased in males only up to 17% with slight adaptive and non-adverse centrilobular to midzonal hepatocellular hypertrophy in males only. Increase in adrenal weights, both absolute and relative to terminal body weights detected in males treated with 20000 ppm were correlated with histopathological findings of the adrenal gland. Microscopic examinations revealed increased incidence in vacuolation of the zona fasciculata in all treated males. This finding may be regarded as an adaptive effect occurring as a stress response to the physiological changes observed. Macroscopic examinations also revealed effects on the kidney such as an increase in kidney weights and hyaline droplets nephropathy in all treated males. The hyaline droplets can be directly linked to accumulation of the alpha-2-microglobulin, which is unique to the mature male rat, therefore, this finding is adverse for the male rat but has no relevance for other species including man. No other treatment-related effects were observed.

Conclusion: Under the conditions of the test no toxicologically significant effects were observed both in males and females. Therefore the NOAEL is at the highest dose tested, 20000 ppm or 1500 mg/kg bw/day.

Cyclacet tested in Reproscreen study (OECD TG 421)

In this reproduction / developmental toxicity screening test there was no systemic toxicity seen and the NOAEL of >= 1000 mg/kg bw was derived, see for details the reproductive toxicity section.

Verdox 90 -day study is summarised here to support the read across to Cyclacet from Verdox on developmental toxicity

A sub-chronic (13-week) repeated-dose toxicity study was performed according to OECD TG 408 and GLP principles (2017).Animals were given intended concentrations of 800, 2500 and 7500 mg/kg in diet based on the dose range finder (DRF) for a >=10 weeks Repeated dose / Reproscreen study (extended OECD TG 422). In the DRF study the palatability of the substance at >= 7700 mg/kg diet (nominal 500 mg/kg and 1000 mg/kg bw) was influencing the body weight (gain) and food consumption. Based on these effects the maximum dose was set to 7500 mg/kg diet. In addition, significant and dose related increased relative liver weights were seen at the 7700 and 15400 mg/kg diet (increase at least of 11 and 5% for males and females, respectively at 7700 and 20 and 17%, for males and females, respectively, at 15400 mg/kg diet. This relative liver weight increase indicates that metabolic capacity is increased close to the maximum non-adverse level at 7500 mg/kg diet.

Analysis of the diets: Analysis for the actual concentration of the test substance in the experimental diets showed some loss of the test substance during preparation and/ or during storage for 24 hours in the animal room. Therefore, the test substance concentrations in the test diets were corrected. The achieved dietary concentrations were 629, 2003 and 7054 mg/kg diet for the low-dose, mid-dose and high-dose groups, respectively. Compensated for the actual levels achieved, the overall intake was 37, 118 and 423 mg/kg body weight/day for males, and 43, 133 and 457 mg/kg body weight/day for females, respectively.

Clinical signs: The administration of the test substance was well tolerated at all dose levels, and did not induce any relevant changes in general condition, feed or water intake, neurobehavioral observations, ophthalmoscopy, hematology, clinical chemistry, urinalysis, or in macroscopic examination at necropsy.

Body weightswere decreased in the high-dose group in both sexes, but the differences with the control group were only slight (within 10%).

Heamatology: No adverse effects were seen.

Clinical biochemistry: No adverse effects were seen.

Organ effects: The relative weight of the liver was slightly increased (12%) in high-dose males. This finding was not accompanied by pathology and is anticipated as an adaptive response. Furthermore, the relative weight of the kidneys was increased in males of the mid- and high-dose group (12% and 24%, respectively). Renal pathology was observed in male rats of all dose groups, but not in females. Immunohistochemical staining forα2-urinary globulin showed that the observed pathological changes were mediated byα2-urinary globulin which is specific for male rats. The incidence and/or severity of the effects aggravated with increasing dose levels and the findings were already present in low-dose males. These findings were accompanied by an increase in the relative weight of the kidneys in male animals of the mid-dose and high-dose group and by an increase of epithelial cells in the urinary sediment in low- and high-dose males, which resulted in increased kidney weight. It should be noted thatα2-urinary globulin-mediated renal pathology, observed in this study at 37 mg/kg bw (629 ppm and a LOAEL for male rats) is not considered relevant for humans and that no other major effects were noted in this study. Therefore, the human relevant NOAEL should be set at >=7054 mg/kg diet, corresponding to a test substance intake of >=423 mg /kg body weight/day based on the absence of human relevant effects at the highest dose tested in female rats. This value is additional supported by the absence of human relevant effects in male rats at the highest dose tested. The absolute and relative weights of the uterus were increased in high-dose females (up to 77%). In relation, a relatively high number of females in the high-dose group macroscopically showed a swollen uterus (2 controls versus 7 high-dose rats) which is ascribed to random variation in the estrous stage. Therefore the increase in uterus weight is considered a chance finding. This is further supported by the absence of any relevant histopathology.

Conclusion: No human relevant adverse effects were seen at >=423 mg/kg bw.

Justification for classification or non-classification

Based on the information above resulting in no adverse effects up to the limit dose >=1000 mg/kg bw, the test substance does not need to be classified for repeated dose toxicity according to EU CLP (EC 1272/2008 and its amendments).