3,4-dimethylbenzaldehyde

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA (TSCA OPPTS harmonised guidelines

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Remarks:

SafePharm Laboratories Ltd., Shardlow Business park, London Road, Shardlow, Derby, DE72 2GD, UK

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dimethylbenzaldehyde

EC Number:

227-770-2

EC Name:

3,4-dimethylbenzaldehyde

Cas Number:

5973-71-7

Molecular formula:

C9H10O

IUPAC Name:

3,4-dimethylbenzaldehyde

Test material form:

other: liquid

Details on test material:

- Name of test material: 3,4-dimethylbenzaldehyde
- Stability under test conditions: Stable under normal temperatures and pressure
- Storage condition of test material: Approximately 4°C in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (only TA 100 and WP2uvrA).
Mutation test experiment 1 (Range-finding test): 15, 50, 150, 500, 1500, 5000 µg/plate.
Mutation test experiment 2 (Main test): 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The test material was fully miscible in DMSO at 50 mg/mL in solubility checks performed in-house.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation: N-ethyl-N-nitro-N-nitrosoguanidine: E.coli, TA100, TA 1535; 9-aminoacridine: TA 1537; 4-nitroquinoline-N-oxide: TA 98

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene (2AA)

Remarks:

With metabolic activation: 2AA used with all strains except TA98 where benzopyrene was used.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: effects on the growth of the bacterial bakground lawn

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.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

PRELIMINARY TOXICITY TEST: The test material was toxic at 5000 µg/plate to both of the strains of bacteria used. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains at 5000 µg/plate both in the presence and absence of S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance is considered to be non-mutagenic under the conditions of this test.

Executive summary:

In a GLP compliant bacterial mutagenicity assay (Ames plate incorporation method), performed according to OECD 471, using four Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli WP2uvrA strain, the test substance was evaluated in the presence and absence of rat liver derived metabolic activation system (S9-mix). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate. The experiment was repeated on a separate day using an amended dose range of 50 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test material formulations. Experiments were performed in triplicate. 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 or 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 to all of the tester strains at 5000 µg/plate both in the presence and absence of S9. 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. In conclusion, the test material was considered to be non-mutagenic under the conditions of this test.