N-ethyl-2-(isopropyl)-5-methylcyclohexanecarboxamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from WHO Food addtivies series

Data source

Reference

Reference Type:

review article or handbook

Title:

Repeated dose oral toxicity study of the test chemical

Author:

Abott et al

Year:

2006

Bibliographic source:

WHO FAS

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

A 22 week study was carried out on groups of 15 Sprague-Dawley (CFY) male/female rats with intake of the test chemical through gavage route to measure its effect on repeated exposure.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

N-ethyl-2-(isopropyl)-5-methylcyclohexanecarboxamide

EC Number:

254-599-0

EC Name:

N-ethyl-2-(isopropyl)-5-methylcyclohexanecarboxamide

Cas Number:

39711-79-0

Molecular formula:

C13H25NO

IUPAC Name:

N-ethyl-2-(isopropyl)-5-methylcyclohexanecarboxamide

Details on test material:

- Name of test material: N-ethyl-2-isopropyl-5-methylcyclohexanecarboxamide
- Molecular formula: C13H25NO
- Molecular weight: 211.346 g/mol
- Smiles notation : C1[C@@H](CC[C@@H](C(C)C)[C@@H]1C(NCC)=O)C
- InChl : VUNOFAIHSALQQH-UHFFFAOYSA-N
- Substance type: Organic
- Physical state: Solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CFY

Details on species / strain selection:

Not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not specified

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Not specifed

Vehicle:

not specified

Details on oral exposure:

Not specifed

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not specifed

Duration of treatment / exposure:

22 weeks

Frequency of treatment:

Not specifed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 other: mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 other: mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

725 other: mg/kg bw/day

No. of animals per sex per dose:

3 groups of 15 Sprague-Dawley (CFY) rats of each sex

0 mg/Kg/day: 15 males and 15 females
100 mg/Kg/day: 15 males and 15 females
300 mg/Kg/day: 15 males and 15 females
725 mg/Kg/day: 15 males and 15 females

Control animals:

yes, concurrent vehicle

Details on study design:

Not specified

Positive control:

Not specified

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study period
- Cage side observations checked in table [No.?] were included. Clinical signs and behavioral changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Throughout the study period

BODY WEIGHT: Yes
- Time schedule for examinations: Throughout the study period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes - Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. Blood clotting times

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. Plasma glucose and serum sodium levels, serum alkaline phosphatase activity and total serum protein levels

URINALYSIS: Yes
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Not specified

Statistics:

Not specified

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

After initial dosing, but not thereafter, several males were lethargic and had mild hypothermia, ataxia and lachrymation, and two females at the highest dose were semi-comatose. Throughout the study, mild transient salivation was noted in rats at the highest dose and to a lesser extent in those at 300 mg/kg bw per day. At the end of the second week and for the remainder of the study, rats at the highest dose and a few at the intermediate dose showed reduced grooming activity. Several rats had mild sialodacryoadenitis during weeks 8-12.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male and one female at the highest dose died at week 4; however, the deaths were probably the result of an intubation error. Another male at the highest dose was killed because of ocular haemorrhage after orbital blood sampling. One male at the intermediate dose was found dead during week 12 with signs of lung congestion. An additional 11 treated rats died during the last 3 weeks of the study, probably due to blood sampling error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced body-weight gain was also reported in females at the highest dose, and rats at the highest dose continued to show lower weight gain during the last 9 weeks.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant variations were found in food consumption between test and control rats; however, during the first 3 months, males at the two higher doses had a dose-related reduction in growth rate.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The reduced body-weight gain observed at the highest dose was accompanied by a reduction in feed use efficiency from week 5 of the study.

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormalities of the eye were reported.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The only variations in haematological parameters reported were in males at weeks 12, 13, 15 and 22, which showed increased blood clotting times in thrombo tests in comparison with controls; however, this effect was thought to be a result of tissue fluid contamination, and therefore additional blood samples were collected by cardiac puncture at weeks 21 and 22. At week 21, the thrombo test values for rats at the two lower doses were below the upper normal limit; although the values for animals at the highest dose remained above the normal limits of variation, the values were markedly reduced from previous weeks. At week 22, a further improvement was noted in thrombotest values in blood collected by cardiac puncture in comparison with values in blood obtained from the orbital sinus.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant variations in clinical chemistry at week 6 were limited to a reduction in serum alkaline phosphatase activity in animals at the highest dose in comparison with controls; however, the values were within the normal limits of variation. Clinical chemistry parameters examined at week 12 revealed the following statistically significant changes: increased plasma glucose and serum sodium levels, decreased serum alkaline phosphatase activity in males at the highest dose and increased total serum protein levels in both sexes. In an extension of the study, serum protein was also increased in rats of each sex at the two lower doses.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At weeks 6 and 7, urine samples from controls and animals at the two higher doses contained leukocytes; however, none were seen at week 12. Epithelial cells were reported in urine samples taken at weeks 6 and 7, but not at week 12. Females at the highest dose had a dose-related increase in specific gravity with no change in urine volume at weeks 7 and 12, whereas males had an increase in volume output with no change in specific gravity. An increased urinary volume was observed in males at the intermediate dose at week 13. Additionally, reducing substances were found in the urine of females with increasing frequency over the test period.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dose-related variations in organ weights were limited to significant increases in the absolute and relative liver weights of females in all test groups in comparison with controls ,and significantly elevated relative liver weights in males at the highest dose. All other variations in organ weights were considered to be incidental or to reflect body weight differences and, as such, were not deemed to be toxicologically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The results of gross examinations were unremarkable

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Although histopathological examination revealed some changes in cardiac and pulmonary tissues and in the livers in all groups of animals, the changes were typical of this strain of laboratory rat and considered not to be related to treatment

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Not specified

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

300 other: mg/Kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No significant effects were noted at the mentioned dose level

2

Dose descriptor:

LOAEL

Effect level:

100 other: mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

urinalysis

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) for the test chemical to Sprague Dawley male rat was considered to be 300 mg/kg bw/day while to female rat it was considered to be <100 mg/kg bw/day after repeated exposure via oral route.

Executive summary:

In a 22-week study, groups of 15 Sprague-Dawley (CFY) rats of each sex were given the test chemical by gavage at a dose of 0, 100, 300 or 725 mg/kg bw. The parameters examined were mortality, Clinical signs, body weight, food efficiency, Urinalysis, Haematology, Biochemistry, organ weight, Opthalmoscopy, Gross pathology and histopathology. After initial dosing, but not thereafter, several males were lethargic and had mild hypothermia, ataxia and lachrymation, and two females at the highest dose were semi-comatose. Throughout the study, mild transient salivation was noted in rats at the highest dose and to a lesser extent in those at 300 mg/kg bw per day. At the end of the second week and for the remainder of the study, rats at the highest dose and a few at the intermediate dose showed reduced grooming activity. Several rats had mild sialodacryoadenitis during weeks 8-12. One male and one female at the highest dose died at week 4; however, the deaths were probably the result of an intubation error. Another male at the highest dose was killed because of ocular haemorrhage after orbital blood sampling. One male at the intermediate dose was found dead during week 12 with signs of lung congestion. An additional 11 treated rats died during the last 3 weeks of the study, probably due to blood sampling error. Significantly reduced body-weight gain was also reported in females at the highest dose, and rats at the highest dose continued to show lower weight gain during the last 9 weeks. No significant variations were found in food consumption between test and control rats; however, during the first 3 months, males at the two higher doses had a dose-related reduction in growth rate. The reduced body-weight gain observed at the highest dose was accompanied by a reduction in feed use efficiency from week 5 of the study. The only variations in haematological parameters reported were in males at weeks 12, 13, 15 and 22, which showed increased blood clotting times in thrombo tests in comparison with controls; however, this effect was thought to be a result of tissue fluid contamination, and therefore additional blood samples were collected by cardiac puncture at weeks 21 and 22. At week 21, the thrombo test values for rats at the two lower doses were below the upper normal limit; although the values for animals at the highest dose remained above the normal limits of variation, the values were markedly reduced from previous weeks. At week 22, a further improvement was noted in thrombotest values in blood collected by cardiac puncture in comparison with values in blood obtained from the orbital sinus. Statistically significant variations in clinical chemistry at week 6 were limited to a reduction in serum alkaline phosphatase activity in animals at the highest dose in comparison with controls; however, the values were within the normal limits of variation. Clinical chemistry parameters examined at week 12 revealed the following statistically significant changes: increased plasma glucose and serum sodium levels, decreased serum alkaline phosphatase activity in males at the highest dose and increased total serum protein levels in both sexes. In an extension of the study, serum protein was also increased in rats of each sex at the two lower doses. At weeks 6 and 7, urine samples from controls and animals at the two higher doses contained leukocytes; however, none were seen at week 12. Epithelial cells were reported in urine samples taken at weeks 6 and 7, but not at week 12. Females at the highest dose had a dose-related increase in specific gravity with no change in urine volume at weeks 7 and 12, whereas males had an increase in volume output with no change in specific gravity. An increased urinary volume was observed in males at the intermediate dose at week 13. Additionally, reducing substances were found in the urine of females with increasing frequency over the test period. Dose-related variations in organ weights were limited to significant increases in the absolute and relative liver weights of females in all test groups in comparison with controls ,and significantly elevated relative liver weights in males at the highest dose. All other variations in organ weights were considered to be incidental or to reflect body weight differences and, as such, were not deemed to be toxicologically significant. The results of gross examinations were unremarkable. Although histopathological examination revealed some changes in cardiac and pulmonary tissues and in the livers in all groups of animals, the changes were typical of this strain of laboratory rat and considered not to be related to treatment. Based on the observations made, no observed adverse effect level (NOAEL) for the test chemical to Sprague Dawley male rat was considered to be 300 mg/kg bw/day while to female rat it was considered to be <100 mg/kg bw/day after repeated exposure via oral route.