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Description of key information

An oral diet repeated dose toxicity study was conducted with nopyl acetate according to OECD Guideline No 408 and in compliance with GLP.
The NOAEL (No Observed Adverse Effect Level) for systemic toxicity was determined at 7500 ppm (the highest tested dose level) (the histopathological results of the intermediate and recovery groups are awaited).

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
449 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
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

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP, three groups, each comprising ten male and ten female Sprague Dawley rats (in the main phase), received Nopyl acetate at dietary concentrations of 1500, 3500 or 7500 ppm. A similarly constituted control group received untreated basal diet for the same duration. A further five male and five female Sprague Dawley rats (in the recovery phase) were assigned to each of the control and high dietary concentration groups. All these animals were treated for 13 weeks, followed by a four-week period without treatment to assess the potential for any treatment-related change to recover.


During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by daily visual observation), ophthalmic examination, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken. The mean achieved dosages during the 13 weeks of treatment were 94.2, 213 and 449 mg/kg/day in males and 114, 250 and 545 mg/kg/day in females which received 1500, 3500 or 7500 ppm, respectively. 


 


There were no unscheduled deaths, and the appearance and behavior of the animals were unaffected during the 13 weeks of treatment.


Overall body weight gain during the 13 weeks of treatment for females which received 7500 ppm was reduced when compared to Controls (77% of Controls). Overall body weight gain was similar to Controls during the recovery period for both males and females which received 7500 ppm (R1 to R4).


Overall mean food consumption (Weeks 1-13) was lower than Controls for males which received 7500 ppm (92% of Controls) and remained low during the recovery period (89% of Controls). Statistical significance was also achieved in this group for low food consumption periodically throughout treatment. Statistical significance was achieved in all groups of treated females for low food consumption throughout the treatment period (Weeks 1-13) and for overall mean food consumption (86%, 84% and 81% for females which received 1500, 3500 or 7500 ppm respectively). Conversely, food consumption was increased during recovery for females which received 7500 ppm (125% of Controls).


 


Hematological investigations during Week 13 of treatment revealed lower than Control hemoglobin concentrations for males which received 7500 ppm and a slight dose-related response was apparent, although there was no similar finding in females.  Neutrophil counts for males which received 3500 or 7500 ppm and lymphocyte counts for males which received 7500 ppm were significantly higher than Controls and showed a slight dose-related response. Consequently, white blood cell counts for males which received 7500 ppm were also significantly high when compared to Controls; there were no similar findings in females. Prothrombin times for males and females which received 7500 ppm were significantly lower than Controls. All other inter-group differences from control were either minor or lacked dose-relationship and were considered unrelated to treatment. After 4 weeks of recovery, hemoglobin concentrations, erythrocyte counts, white blood cell counts, neutrophil and lymphocyte counts for males which received 7500 ppm and prothrombin times for males and females at this dose level were similar to Controls.


Biochemical investigation of the blood plasma during Week 13 of treatment revealed significantly increased alkaline phosphatase and alanine aminotransferase enzyme levels in males which received 7500 ppm, females at this dose level showed a slight increase in alkaline phosphatase enzyme levels but did not achieve statistical significance. Aspartate aminotransferase enzyme levels were significantly high in females which received 7500 ppm and a similar trend was seen in males at the same dose level, although males did not achieve statistical significance. Blood bilirubin was slightly lower in males which received 7500 ppm and females which received 3500 or 7500 ppm. Sodium was low in all groups of treated females; potassium was significantly higher than Controls, and calcium was significantly lower than Controls for females which received 7500 ppm; there were no similar findings in males. All other inter-group differences from control were either minor or lacked dose-relationship and were considered unrelated to treatment. This included low creatinine concentrations in females which received 7500 ppm; low glucose concentrations in all groups of treated females; low chloride in females which received 3500 ppm and high phosphatase levels in males which received 7500 ppm.  After 4 weeks of recovery, blood bilirubin and creatinine remained slightly lower in females which received 7500 ppm and blood glucose concentrations were now significantly higher than Controls. Alkaline phosphatase and alanine aminotransferase enzyme levels and blood bilirubin was similar to Controls for males which received 7500 ppm by week 4 of recovery. Alkaline phosphatase enzyme levels, sodium, potassium and calcium concentrations were also similar to Controls in females which received 7500 ppm. All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for animals of this age and strain. Therefore, they were considered not test article related.


 


Week 13 urinalysis revealed increased urinary volumes in males which received 3500 or 7500 ppm when compared to Controls, with an apparent dose-related response. Urinary pH was significantly increased in all groups of treated females and a slight dose-related response was evident; there was no similar findings in males. Males which received 7500 ppm showed a significant increase in total glucose and total chloride and males which received 3500 or 7500 ppm showed an increase in total potassium when compared to Controls. There was no similar findings in females. Both males and females which received 3500 or 7500 ppm showed decreased creatinine concentrations in a dose-related manner. All groups of treated females showed a decrease in urinary glucose and chloride concentrations in a dose-related manner; females which received 3500 or 7500 ppm also showed decreased urinary sodium concentrations in a dose-related manner. There was no similar findings in males. Both males and females which received 3500 or 7500 ppm showed statistically significant changes in urinary total protein, however, males showed an increase, whereas females showed a decrease; therefore, this is considered not to be treatment related. All other inter-group differences from control were either minor or lacked dose-relationship and were considered unrelated to treatment. All other changes from Control after the recovery period were similar to Control for both males and females which received 7500 ppm. This included urinary volume, urinary totals and concentrations of protein, creatinine, glucose and potassium for males and females and urinary pH for females.


 


Changes related to (-)-NOPYL ACETATE were present in the kidneys of males, the liver of males and females and the pancreas of females. Administration of (-)-NOPYL ACETATE for 13 weeks at 7500 ppm to male rats resulted in adverse changes in the kidneys. Kidney weights were higher than controls and hyaline droplet accumulation, with resultant multifocal tubular basophilia, inflammatory cell infiltration and cast formation was present. The hyaline droplet accumulation and subsequent associated pathology were considered to be adverse in the animals affected, as degenerate changes were seen, but are generally considered irrelevant in man. Administration of (-)-NOPYL ACETATE for 13 weeks at 7500 ppm to male and female rats resulted in higher liver weight and centrilobular hypertrophy in the liver. This was considered likely to be an adaptive response. Administration of (-)-NOPYL ACETATE for 13 weeks at 7500 ppm to female rats resulted in decreased acinar secretion in the pancreas. This change is likely to be linked to the decreased food consumption and body weight, therefore, although related to the administration of the test item was secondary and unlikely to be adverse. All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for this animal age and strain. Therefore, they were considered not test article related.


 


In conclusion, dietary administration of Nopyl acetate to Sprague-Dawley rats at concentrations of 1500, 3500 or 7500 ppm for 13 weeks was well tolerated but induced a dose-dependent decrease in bodyweight and bodyweight gains associated with a decrease in food consumption, probably due to the low palatability of the preparations with the test item. Histopathological changes in the kidneys in males associated with high kidney weights, were observed and considered indicative of the species and sex specific alpha 2μ-globulin nephropathy. The hypertrophic changes noted in the livers of males and female are suggestive of an adaptive response to mixed function oxidase induction in the liver. The decreased secretion in the acinar cells of the pancreas in females was considered likely to be a functional response to the decreased body weight and food consumption seen in females.


The NOAEL (No Observed Adverse Effect Level) for systemic toxicity was determined at 7500 ppm (the highest tested dose level) (the histopathological results of the intermediate and recovery groups are awaited).

Justification for classification or non-classification

No adverse effects were observed in the repeated dose toxicity study at 7500 ppm (the highest tested dose). Therefore nopyl acetate is not classified for repeated dose toxicity according to Directive 67/548/EEC and CLP Regulation (EC) No 1272 /2008.