4-isobutyl-2-methylbenzaldehyde

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

2015

Report date:

2015

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:

other: Testing Methods Concerning New Chemical Substances (Notification 0331 No7. PFSB, MHLW, Japan; No.5 of March 29, 2011, MIB, METI, Japan; No. 110331009, EPB, MOE, Japan; dated March 31, 2011)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-isobutyl-2-methylbenzaldehyde

Cas Number:

73206-60-7

Molecular formula:

C12H16O

IUPAC Name:

4-isobutyl-2-methylbenzaldehyde

Details on test material:

- Name of test material (as cited in study report): 4-Isobutyl-2-methylbenzaldehyde (target) and 2-Isobutyl-4-methylbenzaldehyde (main impurity)
- Other name: IBTAL
- Purity: 98.8%
- Lot No.: 5N24
- Appearance: colorless and transparant liquid
- Expiration date: March 31, 2020
- Storage conditions: stored at room temperature, airtight container, nitrogen sealed (air oxidizability)

Method

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:

Phenobarbital and 5,6-benzoflavone induced SD rats rat liver S9-mix

Test concentrations with justification for top dose:

- Preliminary test: 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate
- Main test I & II, S. typhimurium: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate
- Main test I & II, E. coli, without S9-mix: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate
- Main test I & II, E. coli, 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: The test substance is slightly soluble in water, easily soluble in DMSO and acetone by preliminary information. In the solvent selection test, the test substance was not dissolved or suspended in 50 mg/mL in sterilized distilled water. The substance was dissolved at 50 mg/mL in DMSO. Increases in temperature, discoloration and foaming were not observed when the test substance was mixed with DMSO. According to these results, DMSO was selected as solvent.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 9-aminoacridine hydrochloride monohydrate (9-AA); 2-aminoanthracene (2-AA)

Remarks:

with S9-mix: 2-AA (all stains); without S9 mix: AF-2 (TA 98, TA 100, WP2 uvrA), NaN3 (TA 1535), 9-AA (TA 1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

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

NUMBER OF REPLICATIONS:
- preliminary test: 1 plate per dose
- Main test I: 3 plates per dose
- Main test II: 3 plates per dose

DETERMINATION OF CYTOTOXICITY
- Method: microbial growth inhibition

Evaluation criteria:

ACCEPTABLE CRITERIA
The main tests were accepted as valid if all the following criteria were satisfied.
The preliminary test was accepted as valid if the data by which doses were set in the main tests was provided.
(1) The negative (solvent) control values (mean) and the positive control values (mean) are within the proper ranges calculated on the historical data of the laboratory.
(2) The positive control values (mean) show clear positive responses in the respective tester strains, as evidenced by the number of revertant colonies being greater than 2-fold of the respective negative (solvent) control value (mean)/
(3) There are more than 4 doses showing nog microbial growth inhibition and more than 5 doses applicable to the evaluation.
(4) The result of the sterility test indicates that there is no bacterial contamination.
(5) There are no plates that became invalid for measurement due to contamination or other expectant situations.

JUGDEMENT OF TEST RESULTS
Test substance was judged to have mutagenicity (positive) when the test substance induced a dose-dependent increase in the number of the revertant colonies (mean to a level equal or greater than 2-fold of the negative (solvent) control value (mean value) in any one of the tester strains with or without S9 mix, and when the dose-dependent increase was reproducible. Other results were judged to be negative.

Statistics:

No statistical analysis was performed.

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:

see additional information

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

see additional information

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: precipitation:
Precipitation of the test substance was observed at the following dose levels:
- Preliminary test, without S9-mix: 1250 µg/plate or more (all strains)
- Preliminary test, with S9-mix: 5000 µg/plate (all strains)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Microbial growth inhibition was observed at the following dose levels:
- Preliminary test, without S9-mix: 78.1 µg/plate or more (all strains)
- Preliminary test, with S9-mix: 78.1 µg/plate or more (TA 198, TA 100, TA 1535, TA 1537), 313 µg/plate or more (WP2 uvrA)
- Main test I & II, without S9-mix: 39.1 µg/plate or more (TA 100, TA 1535), 78.1 µg/plate (TA 98, TA 1537, WP2 uvrA)
- Main test I & II, with S9-mix: 78.1 µg/plate (TA 98, TA 100, TA 1535, TA 1537), 156 µg/plate or more (WP2 uvrA)

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 was not mutagenic under the conditions of the study.

Executive summary:

In a GLP compliant bacterial mutagenicity assay (Ames preincubation test), performed according to OECD 471, using four Salmonella typhimurium strains (TA 1535, TA 1537, TA98, and TA100) and one Escherichia coli WP2 uvrA strain, the test substance was evaluated in the presence and absence of rat liver derived metabolic activation system (S9 -mix). In a preliminary study, the substance was tested at 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate with and without S9 mix. Two main experiments were performed in triplicate. The S. typhimurium strains with and without S9 mix and the E. coli without S9 mix were dosed with 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate. The E. coli strains with S9 mix were dosed with 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate.The vehicle used was dimethyl sulphoxide. 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. Precipitation of the test substance was observed at 1250 µg/plate or more in all tester strains without S9 mix, at 5000 µg/plate in all tester strains with S9 mix. Cytotoxicity was observed at 39.1 µg/plate or more (TA 100, TA 1535), 78.1 µg/plate (TA 98, TA 1537, WP2 uvrA) without the use of S9 mix. In combination with S9 mix cytotoxicity was observed at 78.1 µg/plate (TA 98, TA 100, TA 1535, TA 1537), 156 µg/plate or more (WP2 uvrA). An increase in the number of his+ or trp+ revertants was not observed either without S9 mix or after the addition of a metabolizing system. It was therefore concluded that the test substance is not mutagenic under these experimental conditions.