Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Not enough documentation to ensure the total compliance with guideline OECD 474 Only male mice tested, no rationale given for this choice.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Only male tested and no documentation to support such choice. Globally limited documentation.
Principles of method if other than guideline:
Same principle as OECD 474, but not enough documentation available to certify a total compliance.
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material declared to be "D,L-Menthol", which does not match the CAS Number given (see above)

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NATIONAL TOXICOLOGY PROGRAM - TACONIC FARMS
- Age at study initiation: BETWEEN 9 AND 14 WEEKS
- Weight at study initiation: WITHIN 2 g RANGE OF MEAN WEIGHT BETWEEN 25 AND 33 g
- Assigned to test groups randomly: YES, JUST BEFORE SEUTHANASIA

NO MORE DETAILS MENTIONNED

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The solvent used is Corn Oil (from GIBCO, Grand Island, NY), because of the water insolubility of Menthol.
Details on exposure:
Intraperitoneal injections (0.4ml) of test chemical (or control) on 3 consecutive days.
Duration of treatment / exposure:
3 consecutive days.
Frequency of treatment:
Daily injections.
Post exposure period:
Mice euthanized with CO2 24 hours after the last injection.
Doses / concentrations
Remarks:
Doses / Concentrations:
0; 250; 500 and 1000 mg/kg, based on a preliminary dose determination study taking into account mortality, solubility in solvent and depression in the percentage of bone marrow polychromatic erythrocytes)
Basis:
nominal conc.
No. of animals per sex per dose:
5 to 7 male mice per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Substances used as positive control: 12-dimethylbenzanthracene (57-97-6); mitomycin C (50-07-7);
- Route of administration: INTRAPERITONEAL

Examinations

Tissues and cell types examined:
Bone marrow smears were examined;
2000 polychromatic erythrocytes per animal were scored
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Two slides per mouse were prepared.

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were fixed in absolute methanol, and stained with acridine orange.

METHOD OF ANALYSIS:
Slides were evaluated at 1000X magnification, using epi-illuminated fluorescence microscopy (450-490 nm excitation, 520 nm emission), for the number of Micronucleated-Polychromatic Erythrocytes (over a total of 2000 Polycrhomatic erythrocytes.
Evaluation criteria:
Significant increase in Micronucleated-Polychromatic Erythrocytes compared to negative control.
Statistics:
The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data [JLS, 1990].
The level of significance was set at an alpha level of 0.05.
To determine whether a specific treatment resulted in a significant increase in Micronucleated-Polychromatic Erythrocytes (MN-PCE), the number of MN-PCE were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability.
In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives.
The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data.
Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chisquared test which incorporated the calculated variance inflation factor for the study.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
only for the highest dose (1000 mg/kg)
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
See table below

Any other information on results incl. tables

Results summary:

Micronucleus Induction Toxicity
Trend P value Dose
(mg/kg)
MN-PCE/Total PCE
Number of animal scored Pair-wise comparison
α = 0.05
Survival/Total Mice % PCE
Menthol 0.374 0
250
500
1000
2.90 ± 0.43
3.60 ± 0.58
2.20 ± 0.34
3.67 ± 0.60
5
5
5
3

0.1922
0.8368
0.2025
5/5
5/5
5/5
3/6
54.5
64.2
56.7
51.8
Solvent control 2.38 ± 0.93
Positive Control
  - DMBA
  - Mitomycin c

7.93 ± 1.69
6.85 ± 2.26

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of male B6C3F1 mice.