Methoxycarbonyloxycyclooct-4-ene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methoxycarbonyloxycyclooct-4-ene

EC Number:

401-620-8

EC Name:

Methoxycarbonyloxycyclooct-4-ene

Cas Number:

87731-18-8

Molecular formula:

C10H16O3

IUPAC Name:

(1R,3aS,6aS)-octahydropentalen-1-yl methyl carbonate; (1S,3aS,6aS)-octahydropentalen-1-yl methyl carbonate; (3Z)-cyclooct-3-en-1-yl methyl carbonate; (4Z)-cyclooct-4-en-1-yl methyl carbonate; bicyclo[3.2.1]octan-8-yl methyl carbonate

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Charles River CD strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 28 ± 1 day
- Weight at study initiation: 63 to 75 gram
- Housing: group-housed with five per cage
- Diet: Labsure LAD 1 Diet, ad libitum (except for overnight prior to sacrifice)
- Water: tap water, ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21.8 (mean minimum and mean maximum)
- Humidity (%): 50.1-57.4 (mean minimum and mean maximum)
- Air changes (per hr): 19
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 23 April 1986 To: 21 May 1986

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Amount of vehicle: 5 ml/kg/day:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The determination of concentrations of cyclooctenyl methyl carbonate in test solution prepared for day 1 of the study was performed. Method used was gas-liquid chromatography.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dosage levels of the test substance were selected on the basis of the following acute data:
Acute oral toxicity in rats:
LD 50 (male} 4.2 (3.4 to 5.5} g/kg bodyweight.
LD 50 (female} 2.4 (1.9 to 2.9} g/kg bodyweight.
LD 50 (male and female combined} 3.2 (2.6 to 3.8} g/kg bodyweight.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The quantity of food consumed by each group of rats was measured at weekly intervals throughout the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 4
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: all
- Parameters as described in the guideline of 1995 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4
- Animals fasted: Yes
- How many animals: all
- Parameters as described in the guideline of 1995 were examined. Bile acids as requested according to OECD 407 (2008) were not measured).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Weighed: adrenals, kidneys, liver, ovaries, testes (with epididymes)

Statistics:

All statistical analyses were carried out separately for males and females.
Bodyweight data were analysed using weight gains.
The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of values different from the mode was analysed by appropriate methods.
Otherwise:
(ii) Bartlett's test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
(iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
(iv) Analyses of variance were followed by Student's 't' test and Williams' test for a dose-related response, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the 't' test and Williams' test (Shirley's test).
Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, the final bodyweight was used as a covariate in an attempt to allow for differences in bodyweight which might influence the organ weights.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no signs of ill health. Salivation was noted in two animals (m+f) of the highest dose group on day 25.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal died after blood sampling, this death was found to be related to an anaesthetic accident.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in bodyweight gain between rats receiving cyclooctenyl methyl carbonate and the controls during the study (See Table 1 in the attached report: page 96)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

As a result of an error, food consumption of male animals was not recorded during Week 1 of the study. Otherwise, food consumed by animals of all groups was measured at weekly intervals. Throughout the study, food consumption measured in animals treated with cyclooctenyl methyl carbonate was similar to that of the controls (see Table 2 in the attached report, pg. 97)

Haematological findings:

no effects observed

Description (incidence and severity):

No changes were recorded that were attributable to treatment with cyclooctenyl methyl carbonate. Statistically significantly lower total WBC and lymphocyte counts were recorded for all male groups receiving cyclooctenyl methyl carbonate in comparison with the controls (p<0.05 or 0.01). The levels of treated animals were however within limits of the normal ranges, thus the effects were not considered to be adverse. An increase in RBC count {P<0.05) amongst female animals of the high dosage group (500 mg/kg/day) was not considered to be of toxicological importance (see Table 3 in the attached report, page 98 and 99).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly higher total protein levels (p<0.01) were recorded in female rats of the high (500 mg/kg/day) and intermediate (100 mg/kg/day) dosage groups,. This effect appeared to be attributable to higher globulin levels amongst rats of these two groups (high p<0.01; intermediate P<0.05) in comparison with the
controls and was considered to be related to treatment with cyclooctenyl methyl carbonate. Higher BUN levels were also recorded in females of the high (p<0.01) and intermediate {P<0.05) dosage groups than in the controls. However, these changes were small in magnitude and, as all individual values were within the normal range, apparent increases in BUN levels were not considered to be of toxicological importance. Statistically significantly higher sodium levels were recorded in all female rats (p<0.01; no dose dependency) and in male rats at 100 and 500 mg/kg bw (p<0.05).
Statistically significantly lower chloride levels were recorded in male rats at 100 and 500 mg/kg bw (p<0.05 and p<0.01 resp.).

ALT and AST levels amongst male rats of all groups receiving cyclooctenyl methyl carbonate were statistically significantly lower than those of the controls (all p<0.01). However, all individual values were within the normal range and these apparent changes which were small in magnitude were, therefore, considered to have arisen by chance. Lower phosphorus levels (p<0.05) in female rats of the high dosage group were considered to have arisen by chance and, therefore, to be of no toxicological importance (See Table 4 in the attached report, page 100 and 101).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher adjusted liver weights were recorded for female animals of the high dosage group receiving cyclooctenyl methyl carbonate, 500 mg/kg/day in comparison with those receiving corn oil alone +12.5%).
Adjusted kidney weights of female rats in all groups receiving cyclooctenyl methyl carbonate were also significantly greater than those of the controls (low and intermediate dosage groups P<0.05; high dosage group P<0.01, +23% at the high dose) (See table 5 in the attached report, page 102 and 103.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes in the livers or kidneys of female rats were observed at post mortem examination.
Pallor of the kidneys was recorded amongst male rats receiving cyclooctenyl methyl carbonate at the high (500 mg/kg/day) and intermediate (100 mg/kg/day) dosage levels. This effect was absent in animals treated at the low (20 mg/kg/day) dosage level and in the controls.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Moderate cortical tubular epithelial eosinophilic inclusions in the kidneys of all male rats receiving 500 mg/kg/day and minimal eosinophilic inclusions in all males receiving 100 mg/kg/day and 20 mg/kg/day were seen. These changes were not seen in any of the female animals at any dosage level or in either male or female control group animals. The eosinophilic inclusions detected in the renal cortical tubules of male rats from treated groups is consistent with the so-called light hydrocarbon nephropathy.
In the present study, the inclusions were not associated with any evidence of degenerative changes. The inclusions appear to be a specific response of the male rat (See Table 7 in the attached report, page 105).

Effect levels

Key result

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

The mean concentrations of Violiff test solution were analysed during the study. Mean results were within 4% of nominal concentrations. Its chemical stability in corn oil, stored at ambient temperature, was confirmed over a period of 4 hours.

Applicant's summary and conclusion

Conclusions:

In a 28-day oral repeated dose toxicity study with rats a NOAEL of 500 mg/kg bw/day is established, the highest dose tested. The dose was selected based on acute oral toxicity at 2000 mg/kg bw.

Executive summary:

Violiff was tested in a 28-day oral repeated dose toxicity study comparable to the OECD TG 407, according to GLP principles with rats. Five male and five female rats were orally exposed to the substance at 0, 20, 100 and 500 mg/kg bw/ day. The doses were selected based on the acute oral toxicity study with the lowest LD50 for females at 2400 mg/kg bw and 2 females were dead at 2000 mg/kg bw.

Results: No treatment-related clinical effects were seen, no effects were detected on body weight gain or food consumption. No treatment related effects were seen on heamatology in males and females. In males slight decreases in WBC counts were seen (ca 20%) which were not dose related and not considered to be toxicologically relevant. No adverse effects on biochemistry were seen. The slight decrease in GPT and GOT, which parameters can be related to e.g. liver function, are not considered to toxicologically relevant because increases may be indicative of concern but not decreases.

Organ and related effects: In males and females there were no or minimal effects on liver, kidney, and adrenal weights, macroscopic or microscopic effects. In males no effects on testes was seen and in females there were no effects on ovaries.  In males moderate cortical tubular epithelial eosinophilic inclusions were observed in the kidneys of all male rats receiving 500 mg/kg/day and minimal eosinophilic inclusions in all males receiving 100 mg/kg/day and 20 mg/kg/day. However, since eosinophilic inclusions in the kidney are considered to be related to light hydrocarbon nephropathy which is specific for the male rat this effect will not be considered for deriving the NOAEL for humans. The NOAEL is set at 500 mg/kg bw/ day  the highest dose tested because the effects seen are considered minimal, adaptive in nature and/or not relevant for humans.