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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10/03/1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report date:
1978

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Reference from study: Ames, B. N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31, 347-363.
Qualifier:
according to guideline
Guideline:
OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)
Version / remarks:
gen. toxicity in vitro, other (before 2 April 2014); now analogous to EU B.15
Principles of method if other than guideline:
Plate - Test (Agar Incorporation)
Approximately 10e8 cells from an overnight culture of each indicator strain were added to separate test tubes containing 2.0 ml molten agar supplemented with biotin and a trace of histidine.
For non-activation tests, at least 4 dose levels of the test compound were added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates.
In activation tests, at least 4 dose levels of the test chemical were added to the appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 ml containing the 9,000 × g liver homogenate) was added to each of the activation overlay tubes, which were then mixed, and the contents poured over the surface of a minimal agar plate and allowed to solidify.
The plates were incubated for 48 hr at 37°C and scored for the number of colonies growing on each plate.
D4 yeast plates were incubated at 30°C (non-activation) and 37°C (activation) for 3-5 days and then scored.
The concentrations of all chemicals are given in Table 1 (in attached picture).
Positive and solvent controls using both directly active positive chemicals and those that require metabolic activation were run with each assay.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-cyclohexyl-N-methylcyclohexylamine
EC Number:
231-453-4
EC Name:
N-cyclohexyl-N-methylcyclohexylamine
Cas Number:
7560-83-0
Molecular formula:
C13H25N
IUPAC Name:
N-cyclohexyl-N-methylcyclohexanamine
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Abbott Laboratories, department No. 468
A-32512 POLYCAT 12 Lot No. 71-819-AK
- Expiration date of the lot/batch: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

FORM AS APPLIED IN THE TEST
All dilutions of test materials were made in either deionized water or DMSO (This is citation from study, part 2.MATERIALS. It is mentioned also in other section in study. In Table 1 is mentioned as solvent acetone - maybe forgotten setting in computer program)

OTHER SPECIFICS:
colorless liquid

Method

Target gene:
gene for histidine synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Salmonella strains were routinely checked for the his, uvrB, rfa and pKM 101 phenotypes. Only appropriately screened stock cultures were used in chemical evaluations.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
Saccharomyces cerevisiae
Remarks:
D4
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Salmonella strains were routinely checked for the his, uvrB, rfa and pKM 101 phenotypes. Only appropriately screened stock cultures were used in chemical evaluations.
Metabolic activation:
with and without
Metabolic activation system:
supernatant of rat liver and a mixture of cofactors
Test concentrations with justification for top dose:
0.01; 0.1; 1.0; 5.0; 10.0 µL/plate
Plate test procedures do not permit exact quantitation of the number of cells surviving chemical treatment. At low concentrations of the test chemical, the surviving population on the treatment plates is essentially the same as that on the negative control plate. At high concentrations, the surviving population is usually reduced by some fraction. Our protocol normally employs several doses ranging over 2 or 3 log concentrations, the highest of these doses being selected to show slight toxicity as determined by subjective criteria.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
ethylmethanesulphonate
other: QM
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ANTH
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10e8 cells from an overnight culture of each indicator strain were added to separate test tubes containing 2.0 ml molten agar supplemented with biotin and a trace of histidine.

DURATION
- Preincubation period: no
- Expression time (cells in growth medium):
The plates were incubated for 48 hr at 37°C and scored for the number of colonies growing on each plate.
D4 yeast plates were incubated at 30°C (non-activation) and 37°C (activation) for 3-5 days and then scored.

NUMBER OF REPLICATIONS: 1

DETERMINATION OF CYTOTOXICITY
- Method other:
Protocol normally employs several doses ranging over 2 or 3 log concentrations, the highest of these doses being selected to show slight toxicity as determined by subjective criteria.
Evaluation criteria:
Evaluation Criteria for Ames Assay
Because the procedures used to evaluate the mutagenicity of the test chemical are semi-quantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical database. Most datasets are evaluated using the following criteria:
1. Strains TA-1535, TA-1537, and TA-1538
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
2. Strains TA-98, TA-100, and D4
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA-100 and 2-3 times the solvent control value for strains TA-98 and D4 is considered to be mutagenic. For these strains, the dose-response increase should start at approximately the solvent control value.
3. Pattern
Because TA-1535 and TA-100 are both derived from the same parental strain (G-46) and because TA-1538 and TA-98 are both derived from the same parental strain (D3052), there is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain, e.g. TA-1537, responds to a mutagen in non-activation tests, it will generally do so in activation tests (the converse of this relationship is not expected). While similar response patterns are not required for all mutagens, they can be used to enhance the reliability of an evaluation decision.
4. Reproducibility
If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data lose significance.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
Saccharomyces cerevisiae
Remarks:
D4
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 applicable

Any other information on results incl. tables

Recording and Presenting Data

The numbers of colonies on each plate were counted and recorded on printed forms. These raw data were analyzed in a computer program and reported on a printout. The results are presented as revertants (or convertants for D4) per plate for each indicator strain employed in the assay. The positive and solvent controls are provided as reference points.

Applicant's summary and conclusion

Conclusions:
The test compound, A-32512 Polycat-12 Lot-71-819-AK, did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.
Executive summary:

The test compound was examined for mutagenic activity in a series of in-vitro microbial assays employing Salmonella and Saccharomyces indicator organisms. The compound was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats.

The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this compound was from 0.01 µl to 10 µl per plate.

The results of the tests conducted on the compound in the absence of a metabolic activation system were all negative.

The results of the tests conducted on the compound in the presence of a rat liver activation system were all negative.

The test compound, A-32512 Polycat-12 Lot-71-819-AK, did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.