Reaction mass of Benzenepropanal, 4-ethyl-α,α-dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

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:

22 Jul 2006 to 22 Aug 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (Ministry of Labor, Official Notification, February 8, 1999) and "III Mutagenicity test" of "Reverse-Mutation Assay in Bacteria" prescribed in "Testing Methods Relating to the New Chemicals Substances" (Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No. 2 (2003.11.13) of the Manufacturing Industries Bureau, METI & No. 031121002 of the Environmental Health Department, MOE (November 21, 2003)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction mass of Benzenepropanal, 4-​ethyl-​α,​α-​dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

EC Number:

916-329-6

Molecular formula:

C13H18O

IUPAC Name:

Reaction mass of Benzenepropanal, 4-​ethyl-​α,​α-​dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

Test material form:

liquid

Details on test material:

As described in 1.2 of dossier

Method

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

- Dose range finding test 1 (without and with S9 mix): 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate
- Dose range finding test 2 (without S9 mix): 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate
- Dose range finding test 2 (with S9 mix): 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate
- Main test (without S9 mix): 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate
- Main test (with S9 mix): 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance was insoluble in distilled water at 50 mg/mL but it was soluble in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL prepared with DMSO was considered to be stable.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates were used for the solvent control and duplicate plate per dose for the test substance treatment and positive control groups.

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

Evaluation criteria:

The test substance was judged positive when the number of revertant colonies increased to twice or more than in the solvent control in a concentration-dependent manner and also the reproducibility of the test results was obtained. In all other cases, it was judged negative.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, 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

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- Dose range finding test 1: The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The bacterial growth inhibition was observed in groups exposed to more than 78.1 µg/plate in all test strains without S9 mix and more than 313 µg/plate in all test strains with S9 mix. The precipitation of the test substance was observed at 5000 µg/plate without S9 mix.
- Dose range finding test 2: The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The bacterial growth inhibition was observed in groups exposed to more than 39.1 µg/plate in TA 100 and TA 1535 without S9 mix and at 78.1 µg/plate in WP2uvrA, TA 98 and TA 1537 without S9 mix and more than 156 µg/plate in all test strains with S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY MAIN TEST:
The bacterial growth inhibition was observed in groups exposed to more than 39.1 µg/plate in TA 100 and TA 1535 without S9 mix and at 78.1 µg/plate in WP2uvrA, TA 98 and TA 1537 without S9 mix and more than 156 µg/plate in all test strains with S9 mix.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella Typhimurium and Escherichia Coli reverse mutation assay performed equivalent to OECD 471.

Executive summary:

The mutagenic activity of the test substance was evaluated in a study equivalent to OECDTG 471 (1997) and according to GLP principles. A pre-incubation assay was performed in the absence and presence of S9 mix. The dose levels were selected based on observed cytotoxicity in two dose range finding studies. The test substance was considered to be toxic between 10 – 200 µg/plate depending on the strain and with and without metabolic activation. Solvent controls and positive controls were included. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.