1-Butanone, 3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-, rel-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 March 2004 to 13 May 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (Inspected on 2002-12-02)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(3S)-3-(dodecylthio)-1-[(1S,2R)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone

Molecular formula:

C25 H46 O S

IUPAC Name:

(3S)-3-(dodecylthio)-1-[(1S,2R)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone

Constituent 2

Reference substance name:

(3R)-3-(dodecylthio)-1-[(1S,2R)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone

Molecular formula:

C25 H46 O S

IUPAC Name:

(3R)-3-(dodecylthio)-1-[(1S,2R)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone

Constituent 3

Reference substance name:

(3S)-3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone

Molecular formula:

C25 H46 O S

IUPAC Name:

(3S)-3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone

Constituent 4

Reference substance name:

(3R)-3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone

Molecular formula:

C25 H46 O S

IUPAC Name:

(3R)-3-(dodecylthio)-1-[(1R,2S)-2,6,6-trimethyl-3-cyclohexen-1-yl]-1-butanone

Test material form:

liquid

Details on test material:

- Physical state: white solid or Colourless liquid (depending on temperature)

Specific details on test material used for the study:

- Storage condition of test material: room temperature under nitrogen in the dark

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/beta-naphthoflavone induced rat liver S9.

Test concentrations with justification for top dose:

Preliminary cytotoxicity / range-finding test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. Main tests: 0, 50, 150, 500, 1500 and 5000 µg/plate (the maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (dried on molecular sieve)
- Justification for choice of solvent/vehicle: to improve water solubility of the test substance. Well known solvent/vehicle not reacting with the test substance. Volume of vehicle/solvent in the medium: not a requirement of the test guideline.
Vehicle/solvent controls (negative control) were tested in the current study.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/2

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

mitomycin C

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See Table 7.6.1/2

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene; 1,8-Dihydroxyanthraquinone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plate per dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (Preliminary cytotoxicity test)

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control 2003 & 2004).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per ml.
4. 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.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.

Rationale for test conditions:

Experiment 1 and 2 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).

Evaluation criteria:

EVALUATION CRITERIA: The test material may be considered positive in this test system if the following criteria are met: the test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression if necessary

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: Test material vapour pressure (0.0000075 Pa at 25 °C) is too low to expect a significant effect of evaporation on test results.
- Water solubility: Test substance was solubilized in DMSO to improve solubility
- Precipitation: observed at 1500 & 5000 µg/plate
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity/range-finding test was carried out to determine the toxicity of the test material and to select the appropriate dose levels for use in the main test (See in "Remarks on results including tables and figures"). No Cytotoxicity was observed for TA100 strain (no reduction in the background number of revertants per plate was observed for each concentration tested).

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. The comparison was made with the historical control ranges for 2002 and 2003 of the corresponding Testing Laboratory (See Tables of results - Appendix 1 in "Background attached material").

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material was non-toxic to the bacterial background lawns of the strains of bacteria used.

Any other information on results incl. tables

1.) Preliminary Cytotoxicity Test:

The test material not toxic to the strain of Salmonella used (TA100). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) Metabolic Activation (S9)	Strain	Dose (μg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	86	81	77	87	77	96	87	85	75	74P	87P
+	TA100	115	63	118	125	118	98	86	96	92	78P	105P
P: Precipitate

 

2.) Mutation Test:

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (See Tables of results in “Background attached material”) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 (See Tables of results in “Background attached material”).

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No toxicologically 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.

A small statistically significant increase in revertant colony frequency was observed in tester strain TAI00 at 1500 µg/plate (without metabolic activation) in experiment 1. However, the plate counts were within the acceptable range for this particular tester strain, there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.28 times that of the concurrent solvent control. Therefore, the response was considered to of no biological or toxicological significance.

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-rnix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA 1537, TA98, TA100 & TA102).

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 473, and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98, TA100 and TA 102 were exposed to the test material diluted in DMSO both in the presence and absence of mammalian metabolic activation (Phenobarbitone/beta-naphthoflavone induced rat liver S9).

The dose range was determined in a preliminary toxicity assay and was 50 to 5000 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 (acetone) control plates gave counts of revertant colonies generally 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No toxicologically 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.

A small statistically significant increase in revertant colony frequency was observed in tester strain TA100 at 1500 µg/plate (without metabolic activation) in experiment 1. However, the plate counts were within the acceptable range for this particular tester strain, there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.28 times that of the concurrent solvent control. Therefore, the response was considered to of no biological or toxicological significance.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98, TA100 & TA102).

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.