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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Menthol was investigated in the Salmonella/microsome test (Ames test). Result: negative, no evidence of mutagenic activity of menthol was seen (with and without mutagenic activation). Additional, menthol was evaluated as negative in a cytogenetic assay and also in a CHO/HGPRT test. In an in-vivo micronucleus assay no indication for a mutagenic effect was found.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sufficient documented for evaluation
Principles of method if other than guideline:
Ames test
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
DL-Menthol, CAS 15356-70-4 = CAS 89-78-1

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2 batches test:
Lot No. 4-HTP-6
Lot No. Nl 1-26-74-2054

Identity confirmed
Batch 1:
2 impurites:
0.3 %
1.3 %
Batch 2:
0.2 % Impurity
Target gene:
no data
Species / strain / cell type:
other: S. typhimurium TA 97, TA 98, TA 100, TA 1535
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 4-amino-o-phenylenediamin, 2-aminoanthracene
Details on test system and experimental conditions:
IUCLID4 Type: Ames test
Key result
Species / strain:
other: S. typhimurium TA 97, TA 98, TA 100, TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: S. typhimurium TA 97, TA 98, TA 100, TA 1535

DL-menthol was negative in the Salmonella microsome test

Conclusions:
Interpretation of results: negative
Executive summary:

Three hundred chemicals inclusive DL-menthol were tested for mutagenicity, under code, in Salmonella typhimurium, using a preincubation protocol. All tests were performed in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters.

DL-menthol was negative in the test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sufficient documented for evaluation
Principles of method if other than guideline:
chromosome aberration test in chinese hamster ovary cells
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
DL-Menthol, CAS 15356-70-4 = CAS 89-78-1

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2 batches test:
Lot No. 4-HTP-6
Lot No. Nl 1-26-74-2054

Identity confirmed
Batch 1:
2 impurites:
0.3 %
1.3 %
Batch 2:
0.2 % Impurity
Target gene:
no data
Species / strain / cell type:
other: Chinese hamster ovary cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
up to 5 mg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: mitomycin c, cyclophosphamide
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: Chinese hamster ovary cells

In the aberration assay DL-menthol was tested up to toxic or near-toxic levels as evidenced by the reduction of cell confluence at the highest doses.

Conclusions:
Interpretation of results: negative
Executive summary:

In the chromosome aberration assay without activation cells were exposed to the test chemical for 8 hours. The test chemical was washed off, and the cells were treated with 0.1 µg/ml Colcemid for 2 -2.5 houts. With metabolic activation, the cells were exposed to the test chemical plus the metabolic activation mixture for 2 hours., washed and then treated with Colcemid for 2.-2.5 hours.

DL-menthol was negative in the chromosome aberration test with and without metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sufficient documented for evaluation
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
mouse lymphoma cell test in L5178 Y cells
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2 batches test:
Lot No. 4-HTP-6
Lot No. Nl 1-26-74-2054

Identity confirmed
Batch 1:
2 impurites:
0.3 %
1.3 %
Batch 2:
0.2 % Impurity


SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2 batches test:
Lot No. 4-HTP-6
Lot No. Nl 1-26-74-2054

Identity confirmed
Batch 1:
2 impurites:
0.3 %
1.3 %
Batch 2:
0.2 % Impurity
Target gene:
no data
Species / strain / cell type:
other: L5178Y Mouse lymphoma cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
up to 200 µg/ml
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
no data
Details on test system and experimental conditions:
IUCLID4 Type: Mouse lymphoma assay
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: other: L5178Y Mouse lymphoma cells

DL-menthol was negative in the L5178Y mouse lymphoma cell test.

Conclusions:
Interpretation of results: negative
Executive summary:

A mouse lymphoma cell test in L5178 Y cells was performed for the national toxicology program (NTP).

DL-menthol was negative in the L5178Y mouse lymphoma cell test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Menthol was investigated in the Salmonella/microsome test (Ames test). Result: negative, no evidence of mutagenic activity of menthol was seen (with and without mutagenic activation). Additional, menthol was evaluated as negative in a cytogenetic assay and also in a CHO/HGPRT test. In an in-vivo micronucleus assay no indication for a mutagenic effect was found.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: sufficient documented for evaluation
Principles of method if other than guideline:
DL-menthol was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
DL-Menthol, CAS 15356-70-4 = CAS 89-78-1
Species:
mouse
Strain:
B6C3F1
Sex:
male
Route of administration:
intraperitoneal
Duration of treatment / exposure:
one injection per day on 3 consecutive days
Frequency of treatment:
one injection per day
Post exposure period:
24 hours following the final exposure
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 or 6 animals/dose
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
mouse bone marrow
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
3/6 (number of animals surviving tratment over number of animals treated)
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid

The initial test was negative to 1000 mg/kg and was not repeated. 
Survival:    0 mg/kg   5/5
           250 mg/kg   5/5
           500 mg/kg   5/5
          1000 mg/kg   3/6

Conclusions:
Interpretation of results: negative
Executive summary:

Menthol was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following final exposure. Menthol was negative in this test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In all relevant (key-studies) in vitro genetic toxicity assays (Ames test, cytogenetic test, CHO/HGPRT test) and the in-vivo micronucleus test, menthol was negative.

Justification for classification or non-classification

Menthol was negative in all relevant test systems. Therefore a classification according to CLP classification criteria (Regulation (EC) No 1272/2008) is no justified.