N-ethyl-2-(isopropyl)-5-methylcyclohexanecarboxamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the Chinese hamster fibroblast cell line CHL and human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Link to relevant study records

Reference

Reference 1

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 from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

2. Histidine
3. Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

2

Details on mammalian cell type (if applicable):

No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

3

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data available

Metabolic activation:

with and without

Metabolic activation system:

(The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared)

Test concentrations with justification for top dose:

2. 0.00, 6.4, 32, 160, 800 µg/plate
3. 0, 6.4, 32, 160 or 800 µg/plate

Vehicle / solvent:

2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material soluble in DMSO

3.- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-anthramine for all strains with S9 metabolic activation

Remarks:

2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-anthramine (+S9; all strains)

Remarks:

3

Details on test system and experimental conditions:

2. METHOD OF APPLICATION:
Standard plate
DURATION
NUMBER OF REPLICATIONS: 5 plate
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

3.METHOD OF APPLICATION: No data

NUMBER OF REPLICATIONS: Each dosage was tested on 5 parallel plates and all the tests were performed on two separate occasions.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

Rationale for test conditions:

2. No data available
3. No data available

Evaluation criteria:

2. The histidine-revertant (his') colonies arising on the plates were counted
3. The plates were observed for increase in the number of revertants/plate

Statistics:

2. Mean number of revertants for n mumber of plates for each dose level was calculated to be the square value of mean(y) of squared roots. Standard error of mean was calculated

3.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 squared root s2/n where s2 is the pooled variance of all individual square root values

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535 ,TA 1537

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Remarks:

DMSO

Untreated negative controls validity:

valid

Remarks:

DMSO

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA1537, TA1535, TA100, TA98

Remarks:

3

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest dose of 800 µg/plate showed a toxic effect on all bacterial strains

Vehicle controls validity:

valid

Remarks:

DMSO

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

2. No data available

3. TEST-SPECIFIC CONFOUNDING FACTORS

RANGE-FINDING/SCREENING STUDIES: The toxicity of the substances was tested in the tester strains at 10-7 dilutions. Based on the results obtained, the doses were selected for the main study

ADDITIONAL INFORMATION ON CYTOTOXICITY: Test chemical did not induce gene mutation in Salmonella typhimuruim strains TA1537, TA1535, TA100, TA98 and TA97 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce mutation in the Salmonella typhimurium strains TA98, TA100, TA1535, TA 1537 and TA97 both in the presence and absence of S9 metabolic actuvation system and hence is not likely to be mutagenic under the conditions of this study.

Executive summary:

Data available for the read across and structurally and functionally similar chemicals were reviewed to determine the mutagenic nature of the chemical. The studies are as mentioned below:

Salmonella Mutagenicity Tests of test chemical was performed in Salmonella strainTA98, TA100, TA1535, and TA 1537. Both in the presence and absence of S9 metabolic activation system.

 The test chemical was dissolved in DMSO to made dose concentration of 0.00, 6.4, 32, 160, 800 µg/plate in 5 parallel plates. The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared. However 2-anthramine served positive control with S9 fraction for all strain of Salmonella typhimurium and sodium azide (TA1535 and TA 100), 2-nitrofluorene (TA 1538 and TA98) served as positive control without S9 fraction.

 Mutagenicity evaluated by counting histidine-revertant (his') colonies arising on these plates. As test chemical did not produce mutation in Salmonella strainTA98, TA100, TA1535, and TA 1537,Therefore it is considered to be negative for gene mutation in vitro.

 

In another study, Ames assay was performed to determine the mutagenic nature of test chemical. The test chemical was studies for its mutagenic nature using Salmonella typhimuruim strains TA1537, TA1535, TA100, TA98 and TA97 with and without S9 metabolic activation system. The test chemical was mixed with DMSO and used at dose level of 0, 6.4, 32, 160 or 800 µg/plate. Concurrent solvent and negative control chemicals were also included in the study. Test chemical at the highest dose of 800µg/plate showed a toxic effect on all bacterial strains. However, menthol did not induce gene mutation in Salmonella typhimuruim strains TA1537, TA1535, TA100, TA98 and TA97 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the various read across and structurally and functionally similar chemicals and applying the weight of evidence approach, the chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA97, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration 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 from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration

Target gene:

5. No data
6. No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Remarks:

5

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum
Essential 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 / cell type characteristics:

not specified

Species / strain / cell type:

lymphocytes: Human

Remarks:

6

Details on mammalian cell type (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 / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

5. At three different doses with 0.2 mg/mL being the maximum dose concentration
6. 0, 0.1, 1 or 10 mM

Vehicle / solvent:

5. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The chemical was soluble in ethanol

6.- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

yes

Remarks:

Untreated cells served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

5

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

6

Details on test system and experimental conditions:

5. METHOD OF APPLICATION: in medium

DURATION
- 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 data

SELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

6. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Rationale for test conditions:

5. No data
6. No data

Evaluation criteria:

5. 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 than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.
6. The metaphase cells were observed for chromosomal aberrations

Statistics:

5. No data
6. Chi-square test

Species / strain:

S. typhimurium, other: Chinese hamster fibroblast cell line CHL

Remarks:

5

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Species / strain:

lymphocytes: Human

Remarks:

6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Additional information on results:

50 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 data

RANGE-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 densitometer

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

6. 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 data

RANGE-FINDING/SCREENING STUDIES: 10mM-menthol was chosen as the highest concentration
because higher concentrations significantly
affected the growth of human lymphocytes in
phytohaemagglutinin-stimulated cultures

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL 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 data

RANGE-FINDING/SCREENING STUDIES: 10mM-menthol was chosen as the highest concentration
because higher concentrations significantly
affected the growth of human lymphocytes in
phytohaemagglutinin-stimulated cultures

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and 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.

Executive summary:

Data available from various read across and structurally and functionally similar chemicals were reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Chromosomal aberration study was performed to determine the mutagenic nature of 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%. 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.

 

In another study, In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of 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, 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.

Based on the observations made, the read acrossand structurally and functionally similar chemicals did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available from various read across test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Ames assay:

Data available for the read across chemicals were reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of Aroclor™ induced rat liver (S9).

The test chemical was examined for its ability to cause mutagenic changes when tested in five strains of the bacteria Salmonella typhimurium, specifically, TA 1535, TA 1537, TA97, TA 98 and TA 100 through the preincubation assay method. Preliminary dose range finding study was performed initially to set the doses for the main study. The test was conducted both in the presence and absence of metabolic activation using male rat and hamster liver derived S-9 mix at dose levels of 0, 33, 100, 333, 1000, 3333 or 10000 ug/plate. The test was repeated and atleast three plates were used at each dose level. The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In another study, the test chemical was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test using four strains of the bacteria Salmonella typhimurium, specifically TA 98, TA 100, TA 1535 and TA 1537. Spot test was performed for the chemical at dose levels of 0.03, 0.3, 3 and 30 μmol/plate. The study was conducted both in the presence and absence of metabolic activation using S9 mix from Aroclor 1254 or methylcholanthrene induced rats. The test chemical is not mutagenic in the bacterium Salmonella typhimurium LT-2 strains TA 98, TA 100, TA1535 and TA37 with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

Based on the data available for the various test chemicals and applying the weight of evidence approach, the test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In vitro mammalian chromosome aberration study:

Data available from various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Chromosomal aberration study was performed to determine the mutagenic nature of 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%. 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.

 

In another study, In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of 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, 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.

Based on the observations made, test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and 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.

Based on the data available and applying weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.