Bicyclo[2.2.1]heptane, 2-ethyl-5-methoxy-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 April 1988 - 17 June 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

403-610-9

EC Name:

-

Cas Number:

122795-41-9

IUPAC Name:

1-ethyl-3-methoxytricyclo[2.2.1.0²,⁶]heptane; 2-ethyl-5-methoxybicyclo[2.2.1]heptane; 2-ethyl-6-methoxybicyclo[2.2.1]heptane

Test material form:

liquid

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited. Marqate, Kent, England
- Age at study initiation: ~ 35 days old (on despatch)
- Weight at study initiation: ~22 and 24 g (on despatch)
- Assigned to test groups randomly: Yes
- Housing: Plastic disposable cage
- Diet: Pelleted Labsure LAD 1 rodent breeding diet ad libitum
- Water: ad libitum
- Acclimation period: ~ 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C
- Air changes: 30 changes per hr
- Photoperiod: Illuminated by artificial light for 12 hours per day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Polyethylene glycol 400
- Concentration of test material in vehicle: Preliminary toxicity test: Phase I: 5, 10, 50, 100 and 500 mg/L. Phase II: 108, 180, 300 and 500 mg/L .
Micronucleus test: 351.9 mg/L

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of EMN were prepared in polyethylene glycol 400 (obtained from FSA Laboratory Supplies) using a Silverson high-speed mixer.

Doses / concentrations

Dose / conc.:

3 519 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Preliminary test: Phase I - 2 mice. Phase II -5 mice
Micronucleus test: 15 mice

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C, obtained from Sigma London Chemical Company Limited (batch number 96F-0547-1). Prepared as a solution in sterile 0.9% saline at a concentration of 0.6 mg/ml
- Route of administration: Oral gavage
- Doses / concentrations: 12 mg/kg

Examinations

Tissues and cell types examined:

Erythrocytes

Details of tissue and slide preparation:

Five males and five females from the negative control and test compound groups were sacrificed 24, 48 and 72 hours after dosing. The positive control group was sacrificed 24 hours after dosing. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone. A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol (>10 minutes) and air-dried. The smears were then stained with 10% Giemsa (prepared by 1 : 9 dilution of standard Gurrts R66 Giemsa (SOH) with distilled water) . After rinsing and differentiation in buffered distilled water (pH 6.8)1 the smears were air-dried and mounted with coverslips using DPX.

The stained smears were examined (under code) by light rnicroscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated normochomatic erythrocytes was also kept.

Evaluation criteria:

If the ratio of the polychrorna to the normochromatic erthrocytes is scored and found to be statistically significantly less than the control value, this is taken as being indicative of toxicity.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
The details of any toxic reactions observed are given in Appendix 2a (Phase I) and Appendix 2b (phase II)

The mortality data from Phase II were subjected to probit analysis which indicated that a dose of 3519 mg/kg bodyweight would be expected to kill approximately 10% of the animals over the 72 hour exposure period (LD50 was estimated as 8390 mg/kg bodyweight). A dosage of 3519 mg EMN per kg bodyweight was chosen for use in the main test.

Micronucleus test:
An initial micronucleus test was performed using a dosage of 5000 mq/kg bodyweight EMN as indicated in the results from Phase I. However excessive mortalities at this dose level resulted in insufficient animals remaining alive after the 72 hour exposure period. Therefore a further toxicity test (Phase II) was performed to establish a more accurate estimate of the LD10.


The results of the second micronucleus test on EMN at the 24, 48 and 72 hour kill times are presented in Tables 2, 3 and 4 respectively. Table 1 gives a summary of the results and the results of statistical analysis. Appendix 4 summarises the vehicle control micronucleated polychromatic erythrocyte counts obtained in previous, unrelated experiments.

Signs and mortalities:
No animals died after treatment with EMN in.the second main study. Clinical signs are detailed in Appendix 3.

Micronucleated polychormatic erythrocyte counts (mnp):
EMN did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three kill times - (P>0.05 using Wilcoxon's sum of ranks test.

Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated polychromatic erythrocytes.

Micronucleated normochromatic erythrocytes (mnn):
EMN did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three kill times.

Ratio of polychromatic to normochromatic erythrocytes (p/n)
Both EMN and mitomycin C failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes (P>0. 05 using Wilcoxon's sum of ranks test.

Any other information on results incl. tables

Table 1: Summary of results - group totals/means for the entire experiment and results of statistical analysis

Kill	Compound	Dosage (mg/kg)	Ratio p/n		Incidence mnp		Incidence mnn
			Mean	P	Mean o/oo	P	Total o/oo
24 hour	Vehicle control   EMN   Mitomycin C	-   3519   12	1.270   1.523   1.052	-   0.685   0.078	1.3   1.1   48.1	-   0.698   <0.001	0.4   0.2   0.0
48 hour	Vehicle control   EMN	-   3519	1.199   1.215	-   0.573	1.4   1.5	-   0.370	0.2   0.0
72 hour	Vehicle control   EMN	-   3519	1.563   1.934	-   0.938	0.9   0.7	-   0.644	0.0   0.6

P - Results of statistical analysis using W11coxon's sum of ranks test (1-sided probabilities)

p/n - Ratio of polychromatic to normochrollatic erythrocytes

mnp - Number of micronucleated polychromatic erythrocytes observed

mnn - Number of micronucleated normochromatic erythrocytes observed

o/oo - Number per thousand cells

Please refer to attached background material for further tables of results and appendices.

Applicant's summary and conclusion

Conclusions:

From the results obtained it is concluded that Neoproxen shows no evidence of mutagenic potential when administered orally in this in vivo test procedure.

Executive summary:

In this assessment of the effect of Neoproxen on the incidence of micronucleated polychromatic erythrocytes in mice, a dosage of 3519 mg/kg bodyweight was administered orally by gavage according to OECD TG 474. A preliminary toxicity test had been carried out to determine the toxicity. At all sampling times the treated mice showed no significant increase in the frequency of micronucleated polychromatic erythrocytes. There was no significant change in the ratio of polychromatic to normochromatic erythrocytes after treatment of the animals. It is concluded from the results obtained that Neoproxen shows no evidence of mutagenic potential in this in vivo test procedure.