Bis[(3,4-epoxycyclohexyl)methyl] adipate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 January 1990 to 01 March 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study not conducted in accordance with any specific internationally adopted test guidelines. The PCE:NCE ratio was low due to evaluating only 1000 PCE per animal (the requirement to assess 2000 PCE per animal was included in test guidelines that were adopted later but the study parameters were correct for the time the study was conducted.

Justification for type of information:

Read across to substance with the same functional groups.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The test material was administered by intraperitoneal injection to 18 male and female Swiss Albino mice at each of 3 concentration levels. Bone marrow smears were taken from 5 animals per sex per dose group at 24, 48 and 72 hours after treatment. Any clinical signs of toxicity were recorded and the number of micronucleated polychromatic erythrocytes was noted.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

EC Number:

219-207-4

EC Name:

7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Cas Number:

2386-87-0

Molecular formula:

C14H20O4

IUPAC Name:

7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Test material form:

liquid

Details on test material:

- Physical state: Light yellow liquid
- Storage condition of test material: Stored at ambient temperature, protected from light.

Test animals

Species:

mouse

Strain:

other: Swiss Albino Crl:CD-1 (ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 63 days old
- Weight at study initiation: 19.6 - 35.2g (females) and 27.0 - 37.6g (males)
- Assigned to test groups randomly: yes, under following basis: animals were randomly assigned to treatment groups using the Taussky-Todd overflow procedure to minimize the difference between groups of same sex.
- Housing: Housed individually in hanging stainless steel cages with wire mesh floors,
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 22 °C
- Humidity: 35 - 45 %
- Photoperiod: 12 hour light/dark cycle.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: peanut oil
- Lot/batch no. (if required): 807493B

Details on exposure:

- In order to ensure an adequate survival rate for bone marrow sampling (5 animals/sex/sampling point), an excess number of animals were treated for all concentrations of test material and the vehicle control.
The test material was administered intraperitoneally as a suspension in peanut oil.

- The dosing mixtures were prepared by diluting appropriate amounts of test material with peanut oil to produce 100, 200 and 450 mg/mL emulsions approxiamtely 8 hours before dosing. Dosing mixtures were placed on a magnetic stir plate with a stir bar during withdrawal of the dosing aliquots and analytical samples.

Duration of treatment / exposure:

Single injection

Frequency of treatment:

Single exposure

Post exposure period:

72 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 250 mg/kg bw (total dose)

No. of animals per sex per dose:

18 animals of each sex. However, in the definitive test, 10 animals were used (5 males and 5 females per dose).

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide is known to produce micronuclei in vivo.
- Route of administration: intraperitoneal injection.
- Doses / concentrations: 25 or 40 mg/kg positive control delivered at a rate of 5mL/kg.

Examinations

Tissues and cell types examined:

- Bone marrow
- Polychromatic erythrocytes
- Micronuclei

Details of tissue and slide preparation:

Five male and five female mice from each treatment group and the vehicle control were sacrificed for bone marrow sampling at approximately 24, 48 and 72 hours after the start of treatment. Mice were sacrificed by cervical dislocation and a femur was removed from each animal. The marrow was aspirated from the bone and placed into a 0.5 mL conical bottom beaker containing approximately 0.4 mL foetal bovine serum and centrifuged (~2000 rpm, 5 minutes at room temperature). The supernatant was decanted and the pellet resuspended in the residual serum. Two bone marrow smears were made from each animal. The slides were fixed in methanol and stained with 5 % Giemsa for approximately 20 minutes.

Evaluation criteria:

The test material was considered negative if the highest dose given was the maximum tolerated dose and all dose time-points fell in the negative classification, as outlined by the level of significance achieved following statistical analysis. If no difference was detected at the 5 % level of significance, the results were negative. If a difference was detected at the 5 % level, the results were considered to be positive. If a trend was not observed, the Study Director evaluated the variability observed in the vehicle control and the nature of the statistically significant responses. The results were declared negative, inconclusive, or positive after consideration of all contributing factors.

Statistics:

One-tailed Fischer exact test and binomial approximation tests to determine if there was a statistically significant increase in the frequency of micronucleated cells in the treated group compared to the vehicle control groups. As multiple comparisons were made, Bonferroni corrections were made to adjust the probablilty value required for significance. If a difference was detected the dose response was analysed using the one-tailed Cochran-Armitage test for trend in binomial proportions.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.5 - 4.0 g/kg.
- Solubility: Not documented
- Clinical signs of toxicity in test animals: Decreased motor activity, ataxia, collapse and laboured breathing. On day 1, 2 males and 3 females at 2.5 g/kg and 3 males and 3 females at 4 g/kg died.
- Evidence of cytotoxicity in tissue analysed: Not documented
- Rationale for exposure: The maximum clastogenic effect was likely to be found at doses near the maximum tolerated dose, the lower doses were spaced at approximately half and a quarter of the upper dose.
- Harvest times: Not documented
- High dose with and without activation: Not documented

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not relevant
- Induction of micronuclei (for Micronucleus assay): The ranges for treated groups were 0.6 to 2.0 and 0.2 to 1.8 MN - PCE/1000 for males and females respectively.
- Ratio of PCE/NCE (for Micronucleus assay): Statistically significant decreases in the treatment group PCE/(NCE + PCE) ratios were detected in females at 0.50 and 2.25 g/kg at 48 hours. No statistically significant decreases in PCE/(NCE + PCE) ratios were detected in other treatment groups.
- Appropriateness of dose levels and route: No information provided
- Statistical evaluation: When the data for five animals within a group were pooled and compared to the concurrent vehicle control, small increases in the frequency of MN-PCE were observed. Only the increase observed in males treated with 1.00 g/kg and sampled at 48 hours was statistically significant when analysed with the Fisher exact or binomial approximation test with the Bonferroni correction for multiple comparisons. This increase was not dose-responsive when analysed with the Cochran-Armitage test for trend in binomial proportions. It is likely that the significance of that response is due to the unusually low spontaneous rate (0.0) in the control group for that sampling time; therefore, the response is not considered to be biologically significant.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the test material did not induce micronuclei in bone marrow erythrocytes of mice under the conditions of this test.

Executive summary:

In a study conducted by Wing and Machado (1991), the test material was examined for its ability to induce micronuclei in mouse bone marrow erythrocytes. The test material was diluted in peanut oil and administered intraperitoneally to 18 Swiss Albino mice of each sex. The dose levels used were 0.50, 1.00 and 2.25 g/kg. Bone marrow smears of 5 animals per sex per treatment group were made at approximately 24, 48 and 72 hours after treatment.

Clinical signs of toxicity, including decreased motor activity, collapse, weakness, ataxia and laboured breathing were observed in both sexes at the 2.25 g/kg dose level. Cytotoxicity was noted in females treated with 0.50 and 2.25 g/kg and sampled at 48 hours.

A statistically significant increase in micronucleated polychromatic erythrocytes was noted in males treated with 1.00 g/kg and sampled at 48 hours; however, this increase was not dose-responsive and was not considered to be biologically significant.

Based on the results of this study, the test material did not induce micronuclei in bone marrow erythrocytes of mice under the conditions of this test.