β,4-dimethylcyclohex-3-ene-1-propan-1-al

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:

04 March-25 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

β,4-dimethylcyclohex-3-ene-1-propan-1-al

EC Number:

229-846-0

EC Name:

β,4-dimethylcyclohex-3-ene-1-propan-1-al

Cas Number:

6784-13-0

Molecular formula:

C11H18O

IUPAC Name:

3-(4-methylcyclohex-3-en-1-yl)butanal

Test material form:

liquid

Method

Target gene:

histidine auxotrophs

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation Test: 0, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate

Vehicle / solvent:

dimethyl sulphoxide

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other:

Details on test system and experimental conditions:

Preliminary Toxicity Test: The test was performed by mixing 0.1 mL of bacterial culture (TA100), 2 mL of molten, trace histidine supplemented, top agar, 0.1 mL of test material formulation, 0.5 mL of S9-mix or phosphate buffer and overlaying onto steril e plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten doses of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colon y counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1: Seven concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporati on method. Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Test - Experiment 2: The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1

Acceptancy Criteria: The reverse mutation assay may be considered valid if the following criteria are met:
• All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2007 and 2008.
• The appropriate characteristics for each tester strain have been confirmed, e.g., rfa cell-wall mutation and pKM101 plasmid R-factor etc.
• All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per mL.
• Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 2007 and 2008 are presented.
• There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test: The test material was toxic at and above 500 µg/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.

Mutation Test: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the 89-mix used in both experiments was shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. The test material caused a visible reduction in the growth of the bacterial background lawn at and above 500 µg/plate both with and without metabolic activation. The test material was, therefore, tested up to the toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of 89-mix. No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

Limoxal was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The study was conducted to determine the mutagenic potential of limoxal in Bacterial Reverse Mutation Test using the direct plate incorporation method in accordance with the OECD Guideline 471 in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 1500 µg/plate in the first experiment.   The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn at and above 500 µg/plate both with and without metabolic activation. The test material was, therefore, tested up to the toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Limoxal was considered to be non-mutagenic under the conditions of this test.