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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13th November 2015 - 27th May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Deviations:

no

GLP compliance:

yes

Type of assay:

transgenic rodent mutagenicity assay

Test material

Test material information

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Daicel Corporation, Lot No.CELP-FB-008
- Purity: 96%

Test animals

Species:

mouse

Strain:

other: MutaMouse

Remarks:

CD2-LacZ80/HazfBR (MutaTM Mouse) [SPF]

Details on species / strain selection:

CD2-LacZ80/HazfBR mice are commonly used transgenic animals, and animals of this strain are readily available in in vivo gene mutation assays

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Laboratory Animals, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: 24.4-28.1 g
- Housing: group (2-3/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0-24.6
- Humidity (%): 40-61
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 November 2015 To: 22 December 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: based on solubility of the test material
- Concentration of test material in vehicle: 25-100 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Corn oil (lot No. WEF2972), the vehicle to prepare the test substance formulations, was used.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Post exposure period:

3 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Intermediate dose group

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group

No. of animals per sex per dose:

6 males (5 males per group evaluated)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylnitrosurea
- Route of administration: intraperitoneal
- Doses / concentrations: 100 mg/kg bw/d

Examinations

Tissues and cell types examined:

Liver, forestomach, nasal epithelium, germ cells

Details of tissue and slide preparation:

The liver, stomach, nasal cavity, testes and vas deferens/cauda epididymis were removed from the animals at termination.

Liver: Two samples were prepared from the left lateral lobe using a biopsy trephine. The samples were separately placed in microtubes and frozen in liquid nitrogen. The other lobes were stored frozen in liquid nitrogen.

Stomach: The greater curvature of the stomach was incised and the stomach contents removed by washing with physiological saline. The forestomach was stored frozen in liquid nitrogen; the glandular stomach was discarded.

Nasal tissue: The nasal cavity was incised and the mucous membrane from the nasal cavity removed and stored frozen in liquid nitrogen.

Germ cells: The seminiferous tubules and vas deferens/cauda epididymis were cut and placed in a Petri dish containing 2 mL cold Dulbecco’s phosphate-buffered saline (PBS). The germ cells that migrated into the PBS were filtered using a cell strainer (pore size 40 µm). About 0.5 mL of the resulting cell suspension was put into each of three microtubes and stored frozen in liquid nitrogen.

Evaluation criteria:

The results were evaluated as positive when the mutation frequency in the test substance-treated group was significantly different from that in the negative control group, and the increase was dose-dependent.

Statistics:

Liver, forestomach and germ cells

Data on the mutant frequency from the negative control group and each treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant using Bartlett’s test), Dunnett’s multiple comparison test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and each test substance treated group. If there was no homogeneity (significant using Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyse the differences.
The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant using F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t-test (two-sided, significance level of 0.05) was performed to analyse the differences.

Nasal tissue
As genomic DNA was extracted from the pooled nasal tissue of each test group, χ2 test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and each test substance-treated group or the positive control group. If significant difference was observed, dose dependency was analyzed by the Cochran-Armitage trend test (one-sided significance level: 2.5%).

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Germ cells

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: There was no effect of treatment on mutation frequency in the male germ cells. 

Test results 2

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Liver

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: An increase in mutation frequency seen at 1000 mg/kg bw/d does not meet the laboratory’s criterion for a positive response

Test results 3

Key result

Sex:

male

Genotoxicity:

ambiguous

Remarks:

Stomach

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The result for the stomach at 1000 mg/kg bw/d is positive according to the laboratory's evaluation criterion, but is not considered to be of clear relevance to the risk assessment

Test results 4

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Nasal tissue

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: There was no effect of treatment on mutation frequency in the nasal tissue

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 125-1000 mg/kg bw/d (7 days)
- Clinical signs of toxicity in test animals: none

Any other information on results incl. tables

Nasal tissue

There was no effect of treatment on mutation frequency in the nasal tissue. The study report concludes that the results show an absence of mutagenicity in the nasal tissue.

Forestomach

An increase in mutation frequency was seen in the forestomach of mice administered 1000 mg/kg bw/d. This was statistically significant and met the laboratory’s criterion for a positive response, in that it exceeded the ‘acceptable range’, defined by the laboratory as the historical control mean value ± three standard deviations. However the mean mutation frequency for the forestomach at 1000 mg/kg bw/d (78.5 x10^-6) only marginally exceeds the ‘acceptable range’ defined by the laboratory (mutation frequency of 15.6-78.0 x10^-6) and clearly lies within the background range (mutation frequency 31.1-84.7 x10^-6).

Liver

An increase in mutation frequency was seen in the liver of mice administered 1000 mg/kg bw/d (mutation frequency 78.2 x10^-6); this increase was statistically significant but did not meet the laboratory’s criterion for a positive response as it lies within the ‘acceptable range’ (mutation frequency of 0.6-99.6 x10^-6). The value is also clearly within the background range (mutation frequency of 31.1-84.7 x10^-6). 

Germ cells

There was no effect of treatment on mutation frequency in the male germ cells. The study report concludes that the results show an absence of mutagenicity in germ cells.

Mutation frequencies

Tissue	Mutation frequency (/10e6)
	Dose level (mg/kg bw/d)				+ control
	0	250	500	1000	ENU	Background range	Acceptable range
Nasal tissue	53.7	40.6	50.2	54.3	215.7*	-	-
Liver	48.2	62.0	61.2	78.2*	143.8*	16.6 - 95.0	0.6 - 99.6
Forestomach	49.1	52.2	54.9	78.5	624.7*	31.1 - 84.7	15.6 -78.0
Germ cells	32.6	33.7	40.3	42.4	82.1*	12.2 - 83.5	-

*significantly different to the concurrent control group (p<0.05)

Acceptable range defined as historical mean +/- 3sd

Applicant's summary and conclusion

Conclusions:

The study report concludes a positive response for CEL2021P based on a marginal (but statistically significant) response seen in the stomach at the highest dose level of 1000 mg/kg bw/d. The conclusion is based on the mean mutation value for the stomach marginally exceeding the laboratory's 'acceptable range', although the mean mutation frequency is clearly within the background control range for this tissue. The result of this study is therefore not considered to be sufficiently convincing to trigger classification of the substance.

Executive summary:

A somatic and germ cell mutation assay was performed with the substance 7-oxabicyclo [4.1.0] hept-3-ylmethyl-7-oxabicyclo [4.1.0] heptane-3-carboxylate; (CEL2021P) using male transgenic mice (MutaMouse). The study was conducted to assess the potential of the test material to induce gene mutations in the liver, stomach, nasal cavity and germ cells, using the lacZ gene as a mutation reporter gene. The dose levels of CEL2021P used in the main study were based on the results of a dose range-finding study in which CD2F1/Slc mice were gavaged for 7 days with 0, 125, 250, 500, or 1000 mg/kg bw/d CEL2021P in corn oil. No signs of toxicity were observed in any of the dose groups. A dose level of 1000 mg/kg bw/d was therefore selected as the high dose level for the main study.

 

The test material was administered by gavage (in corn oil) to groups of six male transgenic mice orally for 28 consecutive days. A control group was treated with the vehicle alone and a positive control group was administered ENU by intraperitoneal injection at 100 mg/kg bw/d for two days. Three days following administration of the final dose, the liver, stomach, nasal cavity, testes and vas deferens/cauda epididymis were removed. Mutation frequencies in the liver, stomach, nasal tissue and germ cells were determined for five mice per group.

 

The mutation frequencies in the nasal tissue and germ cells of all groups treated with CEL2021P did not show any statistically significant increase compared to the vehicle control group. A slight (but statistically significant) increase in the mean mutation frequency was seen in the liver of mice administered 1000 mg/kg bw/d; however the value was within the background control range and was also within the laboratory’s acceptable range (defined as the mean of the background control range ±3sd). A slight (but statistically significant) increase in the mean mutation frequency was seen in the stomach of mice administered 1000 mg/kg bw/d. This value was within the background control range, but marginally exceeded the laboratory’s acceptable range and was therefore considered by the laboratory to represent a positive response. The mutation frequencies in the liver, stomach, nasal tissue and germ cells of the positive control group were statistically significantly increased compared with the negative control group, demonstrating the sensitivity of the assay.

 

The study report concludes that CEL2021P induces gene mutations in the stomach of transgenic mice under the conditions of this study. This interpretation of a marginal response which is clearly within the background control range, is considered to be questionable.