3-hydroxy-p-anisaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

4 strains tested.

Principles of method if other than guideline:

The study followed the method of Ames (see 'attached background material').

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Vanillin

EC Number:

204-465-2

EC Name:

Vanillin

Cas Number:

121-33-5

Molecular formula:

C8H8O3

IUPAC Name:

4-hydroxy-3-methoxybenzaldehyde

Test material form:

solid

Details on test material:

OTHER SPECIFICS:
Details on properties of test surrogate or analogue material:
- Name of test material (as cited in study report): vanillin
- Smiles notation (if other than submission substance): COc1cc(C=O)ccc1O
- InChl (if other than submission substance): InChI=1/C8H8O3/c1-11-8-4-6(5-9)2-3-7(8)10/h2-5,10H,1H3

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
All compounds have been checked for purity using thin-layer chromatography, gas chromatography and NMR; those containing more than 3% impurity have been purified by means of liquid chromatography, recrystallization or distillation. The structures of some of the compounds studied were confirmed by ['H]- and [13C]NMR, e.g. the substitution pattern of multisubstituted aromatic substances and the branching pattern of methylsubstituted long chain alkyl derivatives.

Method

Target gene:

histidine-requiring gene

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: obtained from Dr Bruce N. Ames (University of California, Berkeley, USA).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

3 μmol/plate (456 μg/plate)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol.

Controls

Controls 1

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation): Initially, cultures were grown in Difco nutrient broth. Since this medium is suspected to have a weak mutagenic activity [Ames, B.N., pers. comm.], it was substituted for Oxoid nutrient broth No. 2 in later experiments. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 μmol histidine and 0.05 μmol biotin. Plates used for viable counts contained 10 μmol histidine (and 0.05μmol biotin). The experiments were carried out essentially as described by Ames.

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: at least 2.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: In absence of a background lawn of bacteria on the plates (indicating toxicity)the test was repeated with a lower concentration of the substance. Substances giving an uncertain result in the spot testswere tested quantitatively at 4 concentration levels (0.03, 0.3, 3 and 30 umol/plate unless otherwise indicated).

- OTHER: Preparation of S-9 fractions: Aroclor 1254 (S-9A) or 3-methylcholanthrene (S-9M) (both suspended in corn oil) were used as inducing agents. Aroclor induction was performed on male Sprague--Dawley rats (~200 g), by giving each rat a single i.p. injection of 500 mg/kg 5 days before decapitation, while 3-methylcholanthrene induction was accomplished by giving a daily injection i.p. of 20 mg/kg during 3 days before decapitation. The S-9 fraction was prepared by centrifugation of a liver homogenate at 9000 g for 10 min. Aliquots of the supernatant S-9 were stored at -70°C. The activity of the S-9 preparations was tested using 2-aminoanthracene.

Evaluation criteria:

Evaluation: determination of viable count, measuring of number of spontaneous revertants, and determination of rfa-mutation by crystal violet inhibition. For strains TA 98 and TA 100, presence of the plasmid pKM 101 was checked by resistance to ampicillin.

Statistics:

All values are calculated averages from the results of at least 2 experiments.

Results and discussion

Test results

Key result

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:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Confounding effects: none reported

Applicant's summary and conclusion

Conclusions:

The test item was found to be non-mutagenic with or without metabolic activation.

Executive summary:

A study on the potential mutagenic activity of vanillin was performed using the Bacterial Reverse Mutation Assay as described by Ames, method similar to OECD 471 (non-GLP). Four histidine-requiring mutants of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) were exposed to the test item with and without S9 metabolic activation (liver fraction from Aroclor 1254 or methylcholanthrene induced rats). Under test conditions, the test item did not induce any increase on the number of revertants in any of the strains tested, with or without metabolic activation. Therefore, the test item is not mutagenic.