Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical is not mutagenic to the Salmonella typhimurium TA1537, TA1535, TA100, TA 97, TA98 and TA102 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration:

The test chemical is not mutagenic to the Chinese hamster fibroblast cell line CHL and human lymphocytes in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Based on the data summarized, (+)-neomenthol (CAS no 2216-52-6) is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of 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:
data from handbook or collection of data
Remarks:
Experimental data of read across substances
Justification for type of information:
Weight of evidence approach based on structurally similar chemicals
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
2.
Principles of method if other than guideline:
Based on two gene toxicity study 1. To evaluate the mutagenic potential of test chemical in Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97and TA98.2. Bacterial Reverse Mutation Test of test chemical in Salmonella typhimurium Tester Strains by Plate incorporation method, was conducted as per OECD guideline No. 471.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
1. Histidine2. Histidine
Species / strain:
other: TA1537, TA1535, TA100, TA 97and TA98
Remarks:
1.
Details on mammalian cell lines (if applicable):
not specified
Additional strain characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
2.
Details on mammalian cell lines (if applicable):
not specified
Additional strain characteristics:
other: Tester Strains Genotypes Tester Strains his Mutation Additional Mutations Plasmid Repair LPS TA98 hisD3052 uvrB rfa pKM101 TA100 hisG46 uvrB rfa PKM101 TA1535 hisG46 uvrB rfa - TA1537 hisC3076 uvrB rfa - TA102 hisG428 - rfa pKM101 and pAQ1
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 - induced rat liver Microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
1. 0, 6.4, 32,160 or 800 μg/plate2. Trial 1: 0, 312.5, 625, 1250, 2500 and 5000 μg/plate Trial 2: 0, 128, 320, 800, 2000 and 5000 μg/plate
Vehicle:
1. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO2. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: Based on the solubility test dimethyl sulfoxide (Make: Sigma,Lot no.: BCBR0695V for preliminary cytotoxicity assay and Fischer scientific, Lot no.: 1967260517 for Main assay) was selected as a vehicle for the study.
Negative controls:
not specified
Remarks:
1.
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: For all strains, 2-anthramine with S9 served as a positive control.
Negative controls:
not specified
Remarks:
2.
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
mitomycin C
other: 2- Aminoanthracene (TA1537, TA1535, TA102, TA100 and TA98; Without S9)
Details on test system and conditions:
1. METHOD OF APPLICATION: in agar- Cell density at seeding (if applicable): no dataDURATION- Preincubation period: No data- Exposure duration: No data- Expression time (cells in growth medium): No data- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: Each dosage was tested on 5 parallel plates and all the tests were performed on two separate occasions.METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No dataNUMBER OF CELLS EVALUATED: No dataNUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No dataCRITERIA FOR MICRONUCLEUS IDENTIFICATION: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data- Any supplementary information relevant to cytotoxicity: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole orfragmented chromosomes (if applicable): No data- OTHER: No data2. METHOD OF APPLICATION: in agar (plate incorporation)- Cell density at seeding (if applicable): No dataDURATION- Preincubation period: No data- Exposure duration: 48 hrs- Expression time (cells in growth medium): 48 hrs- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: TriplicateMETHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No dataNUMBER OF CELLS EVALUATED: No dataNUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No dataCRITERIA FOR MICRONUCLEUS IDENTIFICATION: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, bacterial cell growth wasobserved- Any supplementary information relevant to cytotoxicity: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole orfragmented chromosomes (if applicable): No data- OTHER: No data
Rationale for test conditions:
2. The Bacterial reverse mutation assay is considered acceptable if the following criteria are met:- Regular background growth in the solvent control- The positive control substances must produce a significant increase in revertant colony frequencies- The spontaneous reversion rates in the solvent control must be in the range of the historical data.
Evaluation criteria:
1. The mean number of revertants for n plates at each dose level was calculated. The test was considered to be positive if mean number of revertants for n plates at each dose level was greater than control.2. Criteria for a Positive response:• Tester Strains TA98, TA100 and TA102 : For a test item to be considered positive, it must produce at least a 2–fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response (minimum of 2 to 3 concentrations) to in creasing concentrations of the test item.• Tester Strains TA1537 and TA1535 : For a test item to be considered positive, it must produce at least a 3–fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertantsper plate must be accompanied by a dose response (minimum of 2 to 3 concentrations) to increasing concentrations of the test item.Criteria For a Negative Response:A test item for which the results do not meet the above criteria is considered non-mutagenic in this test.
Statistics:
1. Comparisons of the number of revertants per plate were done as t-tests after a square-root transformation of each number had been performed to give homogeneity of variance. The mean number of revertants for n plates at each dose level was calculated to be the squared value of the mean (y) of the square roots. The standard error of the mean was calculated as 2 y~[' s2/n where s 2 is the pooled variance of all individual square root values2. No formal hypothesis testing was performed to analyse the data.
Species / strain:
other: TA1537, TA1535, TA100, TA 97 and TA98
Remarks:
1.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenic effect were observed.
Species / strain:
other: TA98, TA100, TA1535, TA1537 and TA102
Remarks:
2.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical is not mutagenic to the Salmonella typhimurium TA1537, TA1535, TA100, TA 97, TA98 and TA102 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Ames test

Study 1:

Genetic toxicity in vitro study for the test chemical was assessed for its possible mutagenic potential. For this purpose Salmonella/mammalian-microsome test was performed on Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97 and TA98. The test material was exposed to the bacterial strain at the concentration of 0, 6.4, 32, 160 or 800 µg/plate in the presence and absence of S9. The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate. DMSO was used as solvent control. Positive controls were also used for all strain respectively. The test was performed by standard plate method. The toxicity of the substances was tested in the tester strains at 10-7 dilutions. On the basis of the results obtained, the following concentrations of the test substance were selected for testing: 800, 160, 32 or 6.4 µg per plate. Each dosage was tested on 5 parallel plates. The mean number of revertants for n plates at each dose level was calculated. No mutagenic effects were observed in all strain except strain TA1537. Contrary to these results, test chemical induced an increased number of revertants in the strain TA1537 (dose levels of 6.4 and 32 µg per plate). But no effects were observed at the dose level of 160 or 800 µg/plate in strain TA1537 without S9. On the basis of observations made, the test chemical was considered to be non mutagenic in all strain at highest dose concentration and in the presence of S9 too in all strain. Hence the test material cannot be classified as gene mutant in vitro.

Study 2:

Bacterial Reverse Mutation Test of test chemical in Salmonella typhimurium Tester Strains by Plate incorporation method. This study was performed as per OECD guideline No. 471. Based on the solubility test, dimethyl sulfoxide was selected as a vehicle for the test item in the study. The Study was performed to evaluate the mutagenic potential of test chemical using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102 in Trial I (with 5 % v/v S9 mix and without metabolic activation) and Trial II (10% v/v S9 mix) along with vehicle (DMSO) and positive control in triplicates. Following concentrations were used for the respective trials: Trial I: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate and Trial II: 0, 128, 320, 800, 2000 and 5000 µg/plate. Trial I was performed at five test concentrations (factor 2) both in the presence (5 % v/v S9 mix) and absence of metabolic activation system along with vehicle and the positive control. There was no increase in the number of revertant colonies up to the tested concentration of 5000 µg/plate both in the presence (5% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control. Trial II was conducted to confirm the negative results observed in Trial I. For the negative confirmation, the test item concentration was modified with a spacing factor of 2.5 and concentration of metabolic activation (S9 fraction) was increased to 10% v/v. Trial II was conducted with all the tester strains along with vehicle and positive control only in the presence of metabolic activation system. There was no increase in the number of revertant colonies up to the tested concentration of 5000 µg/plate in the presence (10 % v/v S9 mix) of metabolic activation, when compared to the vehicle control. The spontaneous revertant colonies of the vehicle control were within the acceptable range of historical control data of all the tester strains. The positive controls used in the study exhibited significant increase in the mean number of revertant colonies as compared to vehicle control respective to their strains, indicating the sensitivity of the test system to specific mutagens. On the basis of the results of this study, it is concluded that test chemical is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence or absence of metabolic activation system in all the five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.

The test chemical is not mutagenic to the Salmonella typhimurium TA1537, TA1535, TA100, TA 97, TA98 and TA102 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of 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:
data from handbook or collection of data
Remarks:
Experimental data of read across substances
Justification for type of information:
Weight of evidence approach based on structurally similar chemicals
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1
Principles of method if other than guideline:
WoE based on two gene toxicity studies.1. Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical2. In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: Chromosome aberration assay
Test material information:
Composition 1
Species / strain:
other: Chinese hamster fibroblast cell line CHL
Remarks:
1.
Details on mammalian cell lines (if applicable):
- Type and identity of media: MinimumEssential Medium (MEM; GIBCO) supplemented by 10% calf serum- Properly maintained: yes by 4 day passages- Periodically checked for Mycoplasma contamination: No data available- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available
Additional strain characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
not specified
Metabolic activation system:
not specified
Species / strain:
lymphocytes: Human
Remarks:
2.
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin- Properly maintained: yes- Periodically checked for Mycoplasma contamination: No data- Periodically checked for karyotype stability: No data- Periodically "cleansed" against high spontaneous background: No data
Additional strain characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
The post-mitochondrial fraction (S9) was derived from the liver of a male Sprague-Dawley rat induced with Aroclor 1254
Test concentrations with justification for top dose:
1. At three different doses with 0.2 mg/mL being the maximum dose concentration2. 0, 0.1, 1 or 10 mM
Vehicle:
1. - Vehicle(s)/solvent(s) used: Ethanol- Justification for choice of solvent/vehicle: The chemical was soluble in ethanol2. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Negative controls:
yes
Remarks:
Untreated cells served as negative control
Solvent controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
1.
Negative controls:
not specified
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
2.
Details on test system and conditions:
1. METHOD OF APPLICATION: in mediumDURATION- Preincubation period: No data- Exposure duration: 24 and 48 hrs- Expression time (cells in growth medium): 24 and 48 hrs- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)SPINDLE INHIBITOR (cytogenetic assays): ColcemidSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: No dataNUMBER OF CELLS EVALUATED: 100 well spread metaphasesDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: Yes- Determination of endoreplication: No data- Other: No dataOTHER: No data
Rationale for test conditions:
The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results were considered to be negative if the incidence was less than4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.2. The metaphase cells were observed for chromosomal aberrations
Evaluation criteria:
1.
Species / strain:
other: Chinese hamster fibroblast cell line CHL
Remarks:
1.
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
not specified
Remarks on result:
other: No mutagenic potential
Species / strain:
lymphocytes: Human
Remarks:
2.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenic potential
Additional information on results:
1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometerCOMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No data2. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: 10mM test chemical was chosen as the highest concentrati on because higher concentrations significantly affected the growth of human lymphocytes in phytohaemagglutinin-stimulated culturesCOMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No dataTEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: 10mM-menthol was chosen as the highest concentration because higher concentrations significantly affected the growth of human lymphocytes inphytohaemagglutinin-stimulated culturesCOMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Conclusions:
The test chemical is not mutagenic to the Chinese hamster fibroblast cell line CHL and human lymphocytes in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Chromosome aberration:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of (+)-neomenthol (CAS no 2216-52-6) when administered to Chinese hamster fibroblast cell line CHL and human lymphocytes.

Study 1:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.2 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence is not likely to classify as a gene mutant in vitro.

Study 2:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using lymphocytes isolated from the heparinized peripheral blood samples of 12 male and 12 female adult human non-smoking volunteers both with and withoutS9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0, 0.1, 1.0 or 10 mM. About 0.5-1.0 x 106isolated lymphocytes were cultured in RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin. Concurrent solvent and positive control chemicals were also included in the study. All cultures were incubated in the dark at 37°C for 72 hr. Following 1 hr of exposure of the cells to colchicine the slides were prepared. Chromosomal aberrations were scored in 100 metaphase cells from each donor and tested for statistical significance by the chi-square test. The combined percentage structural aberration rate for males and females in the solvent (DMSO) control was 1.76. Lymphocyte cultures treated with 10mM test chemical alone (10mM), had a rate of 2.11. This difference was statistically insignificant. The presence of S-9 in the culture did not influence the aberration frequency. Cultures grown in the presence of MMC showed a several-fold increase in chromosomal aberration frequency, thus validating the experimental conditions used. Based on these considerations, the test chemical did not induce chromosomal aberrations in human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

The test chemical is not mutagenic to the Chinese hamster fibroblast cell line CHL and human lymphocytes in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Based on the data summarized, (+)-neomenthol (CAS no 2216-52-6) is not likely to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene toxicity – in-vitro

Data available for the test chemical and read across chemicals was reviewed to determine the in- vitro gene toxicity of (+)-neomenthol (CAS no 2216-52-6). The studies are as mentioned below:

Ames test:

Study 1:

Genetic toxicity in vitro study for the test chemical was assessed for its possible mutagenic potential. For this purpose Salmonella/mammalian-microsome test was performed on Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97 and TA98. The test material was exposed to the bacterial strain at the concentration of 0, 6.4, 32, 160 or 800 µg/plate in the presence and absence of S9. The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate. DMSO was used as solvent control. Positive controls were also used for all strain respectively. The test was performed by standard plate method. The toxicity of the substances was tested in the tester strains at 10-7 dilutions. On the basis of the results obtained, the following concentrations of the test substance were selected for testing: 800, 160, 32 or 6.4 µg per plate. Each dosage was tested on 5 parallel plates. The mean number of revertants for n plates at each dose level was calculated. No mutagenic effects were observed in all strain except strain TA1537. Contrary to these results, test chemical induced an increased number of revertants in the strain TA1537 (dose levels of 6.4 and 32 µg per plate). But no effects were observed at the dose level of 160 or 800 µg/plate in strain TA1537 without S9. On the basis of observations made, the test chemical was considered to be non mutagenic in all strain at highest dose concentration and in the presence of S9 too in all strain. Hence the test material cannot be classified as gene mutant in vitro.

Study 2:

Bacterial Reverse Mutation Test of test chemical in Salmonella typhimurium Tester Strains by Plate incorporation method. This study was performed as per OECD guideline No. 471. Based on the solubility test, dimethyl sulfoxide was selected as a vehicle for the test item in the study. The Study was performed to evaluate the mutagenic potential of test chemical using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102 in Trial I (with 5 % v/v S9 mix and without metabolic activation) and Trial II (10% v/v S9 mix) along with vehicle (DMSO) and positive control in triplicates. Following concentrations were used for the respective trials: Trial I: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate and Trial II: 0, 128, 320, 800, 2000 and 5000 µg/plate. Trial I was performed at five test concentrations (factor 2) both in the presence (5 % v/v S9 mix) and absence of metabolic activation system along with vehicle and the positive control. There was no increase in the number of revertant colonies up to the tested concentration of 5000 µg/plate both in the presence (5% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control. Trial II was conducted to confirm the negative results observed in Trial I. For the negative confirmation, the test item concentration was modified with a spacing factor of 2.5 and concentration of metabolic activation (S9 fraction) was increased to 10% v/v. Trial II was conducted with all the tester strains along with vehicle and positive control only in the presence of metabolic activation system. There was no increase in the number of revertant colonies up to the tested concentration of 5000 µg/plate in the presence (10 % v/v S9 mix) of metabolic activation, when compared to the vehicle control. The spontaneous revertant colonies of the vehicle control were within the acceptable range of historical control data of all the tester strains. The positive controls used in the study exhibited significant increase in the mean number of revertant colonies as compared to vehicle control respective to their strains, indicating the sensitivity of the test system to specific mutagens. On the basis of the results of this study, it is concluded that test chemical is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence or absence of metabolic activation system in all the five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.

The test chemical is not mutagenic to the Salmonella typhimurium TA1537, TA1535, TA100, TA 97, TA98 and TA102 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of (+)-neomenthol (CAS no 2216-52-6) when administered to Chinese hamster fibroblast cell line CHL and human lymphocytes.

Study 1:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.2 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence is not likely to classify as a gene mutant in vitro.

Study 2:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using lymphocytes isolated from the heparinized peripheral blood samples of 12 male and 12 female adult human non-smoking volunteers both with and withoutS9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0, 0.1, 1.0 or 10 mM. About 0.5-1.0 x 106isolated lymphocytes were cultured in RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin. Concurrent solvent and positive control chemicals were also included in the study. All cultures were incubated in the dark at 37°C for 72 hr. Following 1 hr of exposure of the cells to colchicine the slides were prepared. Chromosomal aberrations were scored in 100 metaphase cells from each donor and tested for statistical significance by the chi-square test. The combined percentage structural aberration rate for males and females in the solvent (DMSO) control was 1.76. Lymphocyte cultures treated with 10mM test chemical alone (10mM), had a rate of 2.11. This difference was statistically insignificant. The presence of S-9 in the culture did not influence the aberration frequency. Cultures grown in the presence of MMC showed a several-fold increase in chromosomal aberration frequency, thus validating the experimental conditions used. Based on these considerations, the test chemical did not induce chromosomal aberrations in human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

The test chemical is not mutagenic to the Chinese hamster fibroblast cell line CHL and human lymphocytes in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Based on the data summarized, (+)-neomenthol (CAS no 2216-52-6) is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data summarized, (+)-neomenthol (CAS no 2216-52-6) is not likely to classify as a gene mutant in vitro.