3-hydroxy-p-anisaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro. Weight of evidence:

Five Ames tests (all of them similar to OECD 471, non-GLP) for the analogue substance vanillin, which shares the same functional groups with the substance and has comparable values for the relevant molecular properties, were negative with or without metabolic activation. Furthermore, two Chromosomal Aberration tests for vanillin (similar to OECD 473, no GLP) were also negative. No study has been selected, since all genetic toxicity studies were negative and coherent results have been obtained. Based on the available information for the read-across approach, the target substance is deemed to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

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

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

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.

Target gene:

histidine-requiring gene

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.

Positive controls:

yes

Positive control substance:

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

Remarks:

without metabolic activation

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

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

Conclusions:

Based on the read across approach, the target substance is expected to be non-mutagenic with or without metabolic activation.

Executive summary:

A study on the potential mutagenic activity of the analogue substance 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. Based on the read across approach, the target substance is expected to be non-mutagenic with or without metabolic activation.

Reference 3

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Four strains tested: TA104, TA102 and TA100 tested without S9 activation; TA 98 tested with S9 activation.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Aldrich.

Target gene:

hisG428 mutation

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA 104

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

mutagenic response induced by 4NQO

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

10, 8 6, 4 and 2 μmol/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: ascorbic acid

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation): In each experiment, 100 μl of each dose of compound was incorporated in 2 ml top agar and poured onto minimal agar plates. After solidification, a second layer of 2 ml molten top agar, incorporating histidine, biotin, and 100 μl of overnight nutrient broth cultures (Oxoid No. 2) of S. typhirhurium TA104 or TA102 was added.
- For the assessment of antimutagenicity, the second layer of molten top agar incorporated 10% of a solution containing 0.5 mM histidine and 0.5 mM biotin, and undiluted bacterial broth cultures, according to the standard plate incorporation test procedure (Maron and Ames, 1983; see 'attached background material').

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth: For the assessment of survival, the second layer of molten top agar incorporated 10% of a solution containing 0.5 mM biotin and 23.8 mM histidine and bacterial broth cultures diluted 1:106 in ice-cold PBS. Both his+ revertants and survivors were scored after 48 h incubation at 37°C in the dark.
- When evident toxic effects were recorded in mutagenicity plates, further downward dilutions were assayed.

- OTHER: Each dose of compound was assayed in triplicate plates for both mutagenicity and survival, and each compound was assayed in at least three separate experiments until a convincingly reproducible result was attained.

Statistics:

Student's t-test.

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium, other: TA 104

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Vanillin was a powerful inhibitor of the spontaneous mutagenicity in both TA104 (ca. 50% decrease) and TA102 (ca. 40% decrease). For strain TA 102, the dose-response curves tended to flatten at high doses, which suggests a possible saturation of the mechanism(s) involved for the inhibition of spontaneous mutagenicity by the test compound.

Conclusions:

The test item was found to be non-mutagenic.

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 (TA104, TA102 and TA100 without S9 metabolic activation, and TA98 with metabolic activation) were exposed to 10, 8 6, 4 and 2 μmol/plate of test item with or without 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.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium, other: TA 104

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Based on the read across approach, the target substance is expected to be non-mutagenic.

Executive summary:

A study on the potential mutagenic activity of the analogue substance 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 (TA104, TA102 and TA100 without S9 metabolic activation, and TA98 with metabolic activation) were exposed to 10, 8 6, 4 and 2 μmol/plate of test item with or without 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. Based on the read across approach, the target substance is expected to be non-mutagenic.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Two replicates per strain tested.

Principles of method if other than guideline:

Method of Ames, McCann & Yamasaki (1975), see 'attached background material'.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Japan Food Additives Association, Tokyo.
- Purity: checked, no further data.

Target gene:

histidine requiring gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

All these test strains were originally provided by Dr B. N. Ames, University of California, Berkeley, USA.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA 92

Details on mammalian cell type (if applicable):

All these test strains were originally provided by Dr B. N. Ames, University of California, Berkeley, USA.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix.

Test concentrations with justification for top dose:

6 concentrations up to max dose: 10.0 mg/plate.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

untreated.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation. Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating. Duplicate plates were used for each of six different concentrations of the sample. The number of revertant (his +) colonies was scored after incubation at 37°C for 2 days.

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48h.

NUMBER OF REPLICATIONS: 2.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth: preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd).
- Any supplementary information relevant to cytotoxicity: the maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment.

- OTHER: The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co.) pretreated 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip). The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4mM-NADPH, 4mM-NADH, 33mM-KC1, 8 mM-MgCI2, 100 mM-phosphate buffer (pH 7.4) and 3.75 ml S-9 (129 mg protein) in a total volume of 12.5 ml.

Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays were performed.

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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium, other: TA 92

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer was used to determine the maximum dose.

Table 1. Mutagenicity of synthetic food additives in Salmonella/microsome test in vitro.

Additive	CAS	Purity (%)	Max Dose* (mg/plate)	Solvent	Result**
Vanillin	121 -335	-	10.0	DMSO	-

* The maximum dose for positive results represents the dose at which the maximum effect was obtained, while the maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment.

**A negative result indicates that no significant increases in the numbers of revertant colonies were detected in any S. typhimurium strains at the maximum dose.

Conclusions:

The test item was found to be non mutagenic.

Executive summary:

The potential mutagenic activity of the test item was studied using the method of Ames, similar to OECD 471. Five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100, TA98 and TA 92) were exposed to six different concentrations of test item, up to 25 mg/plate, with and without S9 metabolic activation (Kanechlor KC-400-induced rat liver microsome fraction), based on the results of a preliminary citotoxicity study. Vehicle and untreated controls were run in parallel, other substances tested served as positive controls. Under test conditions, the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the test item was found to be non mutagenic.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium, other: TA 92

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Remarks on result:

other: Read-across from analogue.

Conclusions:

Based on the read-across approach, the target substance is expected to be non-mutagenic.

Executive summary:

The potential mutagenic activity of the analogue substance vanillin was studied using the method of Ames, similar to OECD 471. Five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100, TA98 and TA 92) were exposed to six different concentrations of test item, up to 25 mg/plate, with and without S9 metabolic activation (Kanechlor KC-400-induced rat liver microsome fraction), based on the results of a preliminary citotoxicity study. Vehicle and untreated controls were run in parallel, other substances tested served as positive controls. Under test conditions, the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the test item was found to be non mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

100 metaphases observed, no metabolic activation.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Japan Food Additives Association, Tokyo.
- Purity: checked, no further data.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CHL, The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr., as described elsewhere (Ishidate & Odashima, 1977).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

- Test concentrations: 3 different doses up to max dose: 1.0mg/plate. Assay for 24h and 48h.
- Justification: The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiological saline.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Method as previously described [Ishidate & Odashima (1977), see 'Attached background material']: Three different doses, including the 50% inhibition dose of each agent, which was estimated by a growth inhibition test, were prepared and separately added to 3-day-old cultures (about 10^5 cells/6-cm dish). Chromosome preparations were made, at 24 and 48 h. Cells were treated with colcemid (0.2 μg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KCl hypotonic solution for 15 min at 37ºC. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1.5% Giemsa's buffered solution.

DURATION
- Exposure duration: 24-48h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concn. 0.2 μg/ml)

STAIN (for cytogenetic assays): Giemsa solution (1.5%, at pH 6.8; E. Merck)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colcemid (final concn. 0.2 μg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): A hundred well-spread metaphases were observed under the microscope ( x 600 with a nocover objective lens).

DETERMINATION OF CYTOTOXICITY
- Method:relative total growth; cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd)

OTHER EXAMINATIONS:
- Determination of polyploidy: The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate.

Evaluation criteria:

The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0-9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed.

Statistics:

TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/ml).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

For max. dose (1.0 mg/mL), Polyploid: 1.0%, Struct. aberrations: 2.0% (48h).

Table 2. Mutagenicity of synthetic food additives in chromosomal aberration test in vitro.

Additive	CAS	Purity (%)	Max Dose* (mg/plate)	Solvent	Polyploid (%)	Str. Aber.		Result**
						(%)	(h)
Vanillin	121 -33 -5	-	1.0	Physiological saline	1.0	2.0	(48)	-

* The maximum dose for positive results represents the dose at which the maximum effect was obtained, while the maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment.

**A result was considered positive (+) if the total incidence of cells with aberrations (including gaps) was 10.0% or more, equivocal (±) if the incidence was between 5.0 and 9.9%, and negative (-) if the incidence was 4.9% or less. A (P) indicates that polyploidization effects were observed.

Conclusions:

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

Executive summary:

A study to determine the ability of the test item to induce chromosomal aberrrations in Chinese Hamster Lung fibroblasts (CHL) was performed by a method similar to OECD 473. A preliminary test was conducted to determine the maximum dose, which was the dose needed for 50% cell-growth inhibiton (estimated with a cell densitometer). Then, CHL cells were exposed to three concentrations of the test item, up to the maximum dose (1.0 mg/plate) for 24 and 48h, without metabolic activation. Untreated cells and solvent-treated cells served as negative controls (incidence of aberrations < 3.0%); 200 chemicals were tested, the positive results served as controls. A hundred well-spread metaphases were observed under the microscope ( x 600 with a nocover objective lens), and the incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results showed no evidence of the induction of structural aberrations in cultured lymphocytes by the test item under test conditions. Therefore, the test item was not mutagenic.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Conclusions:

Based on the read-across approach, the target substance is expected to be non mutagenic without metabolic activation.

Executive summary:

A study to determine the ability of the analogue substance vanillin to induce chromosomal aberrrations in Chinese Hamster Lung fibroblasts (CHL) was performed by a method similar to OECD 473. A preliminary test was conducted to determine the maximum dose, which was the dose needed for 50% cell-growth inhibiton (estimated with a cell densitometer). Then, CHL cells were exposed to three concentrations of the test item, up to the maximum dose (1.0 mg/plate) for 24 and 48h, without metabolic activation. Untreated cells and solvent-treated cells served as negative controls (incidence of aberrations < 3.0%); 200 chemicals were tested, the positive results served as controls. A hundred well-spread metaphases were observed under the microscope ( x600 with a nocover objective lens), and the incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results showed no evidence of the induction of structural aberrations in cultured lymphocytes by the test item under test conditions. Therefore, the test item was not mutagenic. Based on the read-across approach, the target substance is expected to be non mutagenic without metabolic activation.

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Two strains tested: TA98 and TA 100.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Nakarai Pharmaceutical Co. Ltd., Japan.
- Purity: 90-95%.

Target gene:

histidin requiring gene.

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

CELLS USED
- Source: Dr. S. Ishiwara (Hatano Research Institute, Food and Drug Safety Center, Japan).

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source: Dr. S. Ishiwara (Hatano Research Institute, Food and Drug Safety Center, Japan).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.05 to 1000 μg/plate.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

benzo(a)pyrene

other: aflatoxin B1

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation): The mutagenicity test was conducted in the Salmonella/microsome mutagenicity assay on plates according to the method of Ames (see 'attached background material').

DURATION
- Exposure duration: 48h

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.

- OTHER: Metabolic activation: Rat-liver microsome (S9) from Sprague-Dawley rats treated with Aroclor 1254 by the method of Ames (see 'attached background material').

Evaluation criteria:

For mutagenic potency, a positive result was defined as a reproducible, dose-related increase in the number of revertant colonies per plate, and a greater than 2-fold increase in spontaneous mutation rate was obtained, according to the protocol of Ames (McCann et al., 1975).

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Vanillin did not induce a number of revertants that was over half of the number of spontaenous revertantsof TA98 and TA100 at the indicated dose ranges, either with or without S9 mix.

Conclusions:

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

Executive summary:

The potential mutagenic activity of the test item was studied using the method of Ames, similar to OECD 471. Two histidine requiring strains of Salmonella typhimurium (TA100, TA98) were exposed to various dose levels of test item from 0.05 to 1000 μg/pl, with and without S9 metabolic activation (Rat-liver microsome fraction (S9) from Sprague-Dawley rats treated with Aroclor 1254). Vehicle and untreated controls were run in parallel, AflB1, B[a]P, 2-NF and 4NQO served as positive controls. Under test conditions, the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the test item was found to be non mutagenic.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the read-across approach, the target substance is expected to be non-mutagenic with and without metabolic activation.

Executive summary:

The potential mutagenic activity of the analogue substance vanillin was studied using the method of Ames, similar to OECD 471. Two histidine requiring strains of Salmonella typhimurium (TA100, TA98) were exposed to various dose levels of test item from 0.05 to 1000 μg/pl, with and without S9 metabolic activation (Rat-liver microsome fraction (S9) from Sprague-Dawley rats treated with Aroclor 1254). Vehicle and untreated controls were run in parallel, AflB1, B[a]P, 2-NF and 4NQO served as positive controls. Under test conditions, the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the test item was found to be non mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic with and without metabolic activation.

Reference 11

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

200-500 metaphases observed.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Nakarai Pharmaceutical Co. Ltd., Japan.
- Purity: 90-95%.

Species / strain / cell type:

other:

Remarks:

Chinese Hamster B241 cells.

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. M. Sasaki at Chromosome Research Unit, Hokkaido University, Sapporo, Japan)

MEDIA USED
- Medium: Eagle medium, containing glutamine, sodium bicarbonate, and kanamycin 60ug/ml, with 10% fetal calf serum (Flow Laboratories, Inc.), free from mycoplasma and viruses.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix.

Test concentrations with justification for top dose:

3 doses up to 40 nM, the top dose is that at which maximal frequency of aberration was observed without visible cytotoxicity in the treated cells.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

Source: Nakarai Pharmaceutical Co., Ltd. (Japan)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Exponentially growing cells at one day after seeding were exposed to the test chemical for 24 h, and then incubated another 24 h without the chemical followed by treatment with colchicine (1 x 10e-7 M) for 2-3 h.

DURATION
- Exposure duration: 24h
- Expression time: 24h

STAIN: Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Chromosome samples were prepared by the Giemsa staining method after fixation of the cells with ethanol:acetic acid (3:1). Karyotype analysis was made using the high resolution banding method by Yunis (1981) in synchronized culture with methotrexate and thymidine to consistently obtain a large number of mid and late prophase spreads.

NUMBER OF CELLS EVALUATED: 179 (test 1), 200-500 (test 2).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: The percentage of chromosome aberration was computed by scoring about 200 metaphase spreads, each containing 20-26 chromosomes (mode of chromosome number, 23).

DETERMINATION OF CYTOTOXICITY
- Method:relative total growth.

Evaluation criteria:

The percentage of chromosome aberration was computed by scoring about 200 metaphase spreads, each containing 20-26 chromosomes (mode of chromosome number, 23).

Key result

Species / strain:

other: Chinese Hamster B241 cells.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

> 0.04 nmol/L

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant increase in stuctural and numerical chromosome aberrations, compared to control cells untreated or treated with DMSO alone.

Table 1. Chromosome aberrations in chinese-hamster cells treated with the flavorings.

Compound (nM) (a)	No. cells scored	No. of cells with aberrrations (%)							Total abnormal cells (b)
		Chromatid gap	Chromatid break	Chromosome break	Ring	Dicentric	Chromatid exchange	Others
Vanillin (20)	179	6 (3.4)	5 (2.8)	0	0	0	0	0	11 (6.2)**

In the scoring of the cells, the chromatid was considered broken only if the chromatid fragment distal to an achromatic region was displaced or misaligned. Breaks occurring in the same region of sister chromatids, acentric fragments and chromosome deletion were classified as chromosome breaks. 'Others' include translocation, fragmentation and pulverization.

(a) The final concentration of flavorings at which maximal frequency of aberration was observed without visible citotoxicity in the treated cells.

(b) A portion of cells had multiple type of aberration in one cell. All results are significantly different (P < 0.05* - < 0.001 by Chi-square test) from controls except that marked **.

Table 2. Effects of treatment with flavorings on CH cells (test 2).

Treatment of CH B241 cells		Surviving cells (%) (a)	Frequencies of cells with chromosome aberrations (%)
			Structural	Mumerical (b)
Control
DMSO	(1E-5 dilut.)	100	3.7	3.5
	(1E-5 dilut.)	100	3.2	3.8
Medium		100	3.1	3.8
Vanillin	(5 nM)	98	4.1	4.2
	(20 nM)	99	6.2	6.0
	(40 nM)	97	5.6	6.4
MNNG	(100 nM)	89	18.6**	12.8**
	(1 μM)	68	23.4**	15.0**
	(10 μM)	29	24.3**	16.2**

(a) Survivors of the treated cells were calculatied in comparison with the control (medium) cells 5 days after the treatment with flavoring or DMSO.

(b) Frequency of cells with over 30 chromosomes corresponding to almost 3n to 4n chromosomes. Frequencies of structural and numerical chromosome aberrations were calculated from analysis of about 200 and more than 500 metaphase spreads, respectively, by the Giemsa staining method.

*, **: p < 0.05 and p < 0.001, respectively, by Chi-square test in comparison with control values.

Conclusions:

The test item was found to be non-mutagenic.

Executive summary:

Evaluation of the ability of the test item to induce chromosome aberrations in Chinese hamster (CH) B241 cells was determined by a method similar to OECD 473 (non-GLP). The cells were treated for 24h during their exponential growth period with up to 40 nM test item (dose at which maximal frequency of aberration was observed without visible citotoxicity in the treated cells). Two tests were carried out; negative, solvent and positive controls were run in parallel. Under test conditions, the test item did not induce chromosomal aberrations at any of the concentrations tested and therefore, it was deemed non-mutagenic.

Reference 12

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: Chinese Hamster B241 cells.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

> 0.04 nmol/L

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant increase in stuctural and numerical chromosome aberrations, compared to control cells untreated or treated with DMSO alone.

Conclusions:

Based on the read-across approach, the target substance is expected to be non-mutagenic.

Executive summary:

Evaluation of the ability of the test item to induce chromosome aberrations in Chinese hamster (CH) B241 cells was determined by a method similar to OECD 473 (non-GLP). The cells were treated for 24h during their exponential growth period with up to 40 nM test item (dose at which maximal frequency of aberration was observed without visible citotoxicity in the treated cells). Two tests were carried out; negative, solvent and positive controls were run in parallel. Under test conditions, the test item did not induce chromosomal aberrations at any of the concentrations tested and therefore, it was deemed non-mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Four strains of S. typhimurium (TA 1535, TA1537, TA98, TA100) were tested.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Aldrich. Purity: 99%. The laboratories were supplied with the chemicals as coded Aliquots by Radian Corporation, Austin, TX.

Target gene:

histidine requiring gene

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: All strains were obtained from Dr. Bruce Ames (University of California, Berkeley)
- Methods for maintenance in cell culture if applicable: cells were stored according to the protocol described in the supplement to the methods paper [Ames et al, 1975] which was supplied with the strains. Cultures were kept frozen in liquid nitrogen. Prior to their use for mutagenicity assays, a loopful of a freshly thawed culture was transferred into Oxoid Nutrient Broth #2 (CM67). All cultures were grown overnight for 12-15 h at 37°C on a shaker, and their phenotypes were analyzed as recommended by Ames et a1 [ 1975].

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:

100, 333, 1000, 3333 and 10000 μg/plate.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Preliminary Dose-Setting Experiment: All chemicals were initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. When negative results were obtained in the initial assay, the chemical was retested in all strains with and without activation.
- Preincubation assay: All chemicals were assayed for mutagenicity in the preincubation assay. To each of 13 x 100 mm test tubes maintained at 37ºC were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37ºC for 20 min, at which time 2.5 ml of molten (45ºC) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml of minimal glucose bottom agar in 15 x 100-mm plastic petri dishes. When the top agar had solidified, the plates were inverted and incubated at 37ºC for 48 hr.

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was evidenced by one or more of the following phenomena: appearance of his pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.

- OTHER: Concurrent solvent and positive controls were tested with and without the metabolic activation systems. At least five dose levels of the chemicals were tested, with three plates per dose level. AU assays were repeated (as described above) no less than 1 wk after completion of the initial test.

Evaluation criteria:

The criteria used for data evaluation are summarized as follows: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

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:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item was found to be non-mutagenic.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay, by a method similar to OECD 471, (non-GLP). Four histidine requiring strains of S. typhimurium (TA 1535, TA 98, TA 100 and TA 97) were tested in triplicate at concentrations of 100, 333, 1000, 3333 and 10000 μg/plate of test item, with and without metabolic activation (S-9 fraction prepared from Aroclor 1254 induced male Sprague-Dawley rats or male Syrian hamsters), based on the results of a range-finding test. Vehicle, untreated and positive controls were run in parallel. Under the experimental conditions applied, 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 was deemed non-mutagenic.

Reference 14

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

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:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Read-across from analogue.

Conclusions:

Based on the read-across approach, the target substance is expected to be non-mutagenic.

Executive summary:

The analogue substance vanillin was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay, by a method similar to OECD 471, (non-GLP). Four histidine requiring strains of S. typhimurium (TA 1535, TA 98, TA 100 and TA 97) were tested in triplicate at concentrations of 100, 333, 1000, 3333 and 10000 μg/plate of test item, with and without metabolic activation (S-9 fraction prepared from Aroclor 1254 induced male Sprague-Dawley rats or male Syrian hamsters), based on the results of a range-finding test. Vehicle, untreated and positive controls were run in parallel. Under the experimental conditions applied, 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 was deemed non-mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

All in vitro studies were negative for the analogue substance vanillin, which shares the same functional groups with the substance and has comparable values for the relevant molecular properties. No study has been selected, since all genetic toxicity studies were negative, and coherent results have been obtained for both test substance and analogue substances.

Genetic toxicity in vitro (weight of evidence):

- A bacterial reverse mutation study on the analogue substance vanillin was performed by Florin (1980) using a method similar to OECD 471 (non-GLP). Vanillin did not induce any increase on the number of revertants in Salmonella typhimurium strains tested (TA 98, TA 100, TA 1535 and TA 1537) at any dose, with or without S9 metabolic activation. Therefore, the test item is not mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

- A bacterial reverse mutation study on the analogue substance vanillin was performed by DeFlora (1994) using a method similar to OECD 471 (non-GLP). Vanillin did not induce any increase on the number of revertants in Salmonella typhimurium strains tested (TA104, TA102 and TA100 without S9 metabolic activation, and TA98 with metabolic activation) at any concentration. Therefore, the test item is not mutagenic. Based on the read across approach, the target substance is expected to be non-mutagenic.

- A bacterial reverse mutation study on the analogue substance vanillin was performed by Ishidate (1983) using a method similar to OECD 471 (non-GLP). Vanillin did not induce any increase on the number of revertants in Salmonella typhimurium strains tested (TA1535, TA1537, TA100, TA98 and TA 92) at any dose, with or without S9 metabolic activation. Therefore, the test item is not mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

- A bacterial reverse mutation study on the analogue substance vanillin was performed by Kasamaki (1982) using a method similar to OECD 471 (non-GLP). Vanillin did not induce any increase on the number of revertants in Salmonella typhimurium strains tested (TA100, TA98) at any dose, with or without S9metabolic activation. Therefore, the test item is not mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

- A bacterial reverse mutation study on the analogue substance vanillin was performed by Mortelmans (1986) using a method similar to OECD 471 (non-GLP). Vanillin did not induce any increase on the number of revertants in Salmonella typhimurium strains tested (TA 1535, TA 98, TA 100 and TA 97) at any dose, with or without S9 metabolic activation. Therefore, the test item is not mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

- A chromosome aberration study on the analogue substance vanillin was performed by Ishidate (1983) using a method similar to OECD 473 (non-GLP). Vanillin did not induce chromosomal aberrations in Chinese Hamster Lung fibroblasts (CHL) under test conditions. Therefore, the test item is not mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

- A chromosome aberration study on the analogue substance vanillin was performed by Kasamaki (1982)

using a method similar to OECD 473 (non-GLP). Vanillin did not induce chromosomal aberrations in Chinese Hamster B241 cells under test conditions. Therefore, the test item is not mutagenic. Based on the read-across approach, the target substance is expected to be non-mutagenic.

Justification for classification or non-classification

Based on the available data (negative results in vitro), it is concluded that the substance is not classified for mutagenicity in accordance with CLP Regulation (EC) No.1272/2008.