4-ethylbenzaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test according to OECD 471: negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

Safepharm Laboratories Limited, Shardlow Business Park, Shardlow, Derbyshire

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with nominal pore diameter of 4 x 10^-4 microns.
- Final dilution of a dissolved solid, stock liquid or gel: 50mg/ml DMSO

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

E. coli WP2uvrA-

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/ β-naphthoflavone induced rat liver S9 in standard co-factors

Test concentrations with justification for top dose:

In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The assay was performed on strains TA 100 and WP2uvrA-). Based on this assay the following concentrations were selected for the two mutation tests: 15, 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: material immiscible in distilled water

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

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In agar (plate incorporation);
- Cell density at seeding: range of 1 to 9.9 x 10^6 bacteria/mL

DURATION
- Exposure duration: 48h
- Expression time: 48h

SELECTION AGENT
minimal histidine or tryptophan

NUMBER OF REPLICATIONS:
3 in 2 independent experiments.

DETERMINATION OF CYTOTOXICITY
Growth of the bacterial background lawn

OTHER: Numbers of revertant colonies were determined using a Domino colony counter

Evaluation criteria:

The test material should have induced a reproducible, dose-related and statistically significant increase in reverent count in at least one strain of bacteria

Statistics:

Dunnett's method of linear regression

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100, WP2uvrA-

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES
The test material was toxic at 5000 μg/plate to both of the strains of bacteria used (TA100 and WP2uvrA) 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-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

Remarks on result:

other: no mutagenic potential

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test

Executive summary:

In this study, according to the requirements of the OECD TG 471 and GLP principles, the mutagenic potential of the test substance was evaluated.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA¯ were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (Phenobarbitone/ β-naphthoflavone induced rat liver S9). The dose range for the range-finding study was determined in a preliminary toxicity assay and ranged between 0.15 and 5000 µg/plate with or without S9-mix. The experiment was repeated on a separate day using a similar dose range to the range-finding study, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels were included in both experiments to allow for the toxicity of the test material, ensuring there were a minimum of four non-toxic doses. 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. 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. The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test according to OECD 471

In this study, according to the requirements of the OECD TG 471 and GLP principles, the mutagenic potential of the test substance was evaluated (Safepharm 2005).

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA¯ were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (Phenobarbitone/ β-naphthoflavone induced rat liver S9). The dose range for the range-finding study was determined in a preliminary toxicity assay and ranged between 0.15 and 5000 µg/plate with or without S9-mix. The experiment was repeated on a separate day using a similar dose range to the range-finding study, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels were included in both experiments to allow for the toxicity of the test material, ensuring there were a minimum of four non-toxic doses. 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. 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. The test material was considered to be non-mutagenic under the conditions of this test.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.