trans-4-aminocyclohexan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-04-22 to 1999-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC, Directive 92/69/EEC, Annex V of the EEC Directive 67/548/EEC, Part B: methods for the Determination of Toxicity: B.14 "Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay"

Version / remarks:

EEC Publication no. L383 (adopted December 1992)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

trans-4-aminocyclohexan-1-ol

EC Number:

248-492-8

EC Name:

trans-4-aminocyclohexan-1-ol

Cas Number:

27489-62-9

Molecular formula:

C6H13NO

IUPAC Name:

4-aminocyclohexanol

Specific details on test material used for the study:

Description: White PowderBatch 722Purified: Not indicated by sponsor; treated as 100% pureTest substance storage: At room temperature in the dark Expiry date: 2000-04-08 (1 year after receipt of the test substance)

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Histidine mutation: hisD3052/R-factorMutation type: FrameshiftSource: Dr. Bruce N. Ames, university of California at Berkely U.S.A. (Salmonella typhimurium strans); received 12-02-1991, used batch: TA98.150698

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Histidine mutation: hisG46/R-factorMutation type: Base-pair substitutionsSource: Dr. Bruce N. Ames, university of California at Berkely U.S.A. (Salmonella typhimurium strans); received 18-02-1993, used batch: TA100.061098

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

3.10, 33, 100, 333, 1000, 3330 and 5000 μg per plate

Vehicle / solvent:

Milli-Q water

Controls

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle of the test article, beinge Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: Without metabolic Daunomycine and With metabolic activation: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Eigt different doses (with approximately half-log steps) on the test substance were tested in triplicate in strain T98 and T100. The test substance was tested both in the absence and presence of S9-mix in each strain. Top agar in top agar tubes were molten and heated to 45°C. The following solutions were successively to 3mL molten top agar: 0.1mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1mL of a dilution of the test substance in Milli-Q water and either 0.5mL S9-Mix (in case of activation assays) or 0.5mL 0.1M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tubed was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark 38°C for 48h. After this period revertant (histidine independent) colonies were counted.

Rationale for test conditions:

A salmonella tphimurium reverse mutation assay is considered acceptable if it meets the following criteria: a.) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.b.) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. c.) The selected dose range should include a clearly toxic concentration or should exibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5mg/plate.

Evaluation criteria:

No formal hypothesis testing was done A test substance is considered negative (not mutagenic) in the test if: -) The total number of revertants in any tester strain an any concentration is not greater than two times the solvent control value, with or without activation. A test substance is considered positive (mutagenic) in the test if: -) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitate: Trans-4-Aminocyclohexanol did not precipitate in the top agar. Precipitation of Trans-4-Aminocyclohexanol was not observed on the plates at the start or at the end of the incubation period in both tester strains. Toxicity of the test substance: To determine the toxicity of Trans-4-Aminocyclohexanol, the reduction of the bacterial background lawn, the increasde in the size of the microcolonies and the reduction of the HIS*revertant colonies were observed, The defibnitions are stated in Appendix 1. Number of revertant: Both bacterial strains showed negatives responses over the entire dose range, i.e. no dose related, two-fold, increase in the number of revertants. The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the matabolic activatioon system functioned properly.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Trans-4-Aminocyclohexanol is not mutagenic in the Salmonella typhimurium reverse mutation assay.