4-methoxybenzyl alcohol

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

Study period:

28 March 2002 and 07 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methoxybenzyl alcohol

EC Number:

203-273-6

EC Name:

4-methoxybenzyl alcohol

Cas Number:

105-13-5

Molecular formula:

C8H10O2

IUPAC Name:

(4-methoxyphenyl)methanol

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix induced by phenobarbitone/p-naphthoflavone in livers of rats

Test concentrations with justification for top dose:

100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: N-ethyl-N'-nitro-N-nitrosoguanidine (TA100 and TA1535), 9-Aminoacridine (TA1537), Mitomycin C (TA102), 4-Nitroquinoline-l-oxide (TA98); -S9: 2-Aminoanthracene (TA100, TA1535, TA1537), Benzo(a)pyrene (TA98), 1,8-Dihydroxyanthraquinone (TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

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

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

Evaluation criteria:

The test material may be considered positive if the following criteria are met:
- The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

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:

valid

Positive controls validity:

valid

Additional information on results:

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

RANGE-FINDING/SCREENING STUDIES: A preliminary study was carried out using both the plate incorporation and pre-incubation methods with the following concentrations: 0,0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100. The test material was non-toxic to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile. Based on the result, the following concentrations of the test susbstance in the main study was used: 100, 333, 1000, 2500 and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: done

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.

Any other information on results incl. tables

Table 1: Spontaneous mutation rates (concurrent negative controls)

Dose µg/plate I/II	TA 100		TA 1535		TA102		TA98		TA1537
	I	II	I	II	I	II	I	II	I	II
Negative control	107	105	34	20	380	357	18	18	11	14

Table 2: Number of revertants (mean number of three plates) without metabolic activation

Dose µg/plate I/II	TA 100		TA 1535		TA102		TA98		TA1537
	I	II	I	II	I	II	I	II	I	II
Solvent Control	133	108	33	31	347	260	18	16	14	13
100	115	95	34	33	339	265	19	12	13	14
333	105	97	34	29	338	256	16	19	17	7
1000	115	101	36	35	327	278	18	12	21	9
2500	109	107	37	39	347	276	16	12	17	11
5000	107	99	39	30	312	240	13	12	18	8
Positive controls
ENNG (3.0)	500	474
ENNG (5.0)			900	205
MMC (0.5)					2414	1886
4NQO (0.2)							168	133
9AA (80)									991	1353

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine, 4NQO: 4-Nitroquinoline-1 -oxide, 9 -AA: 9-Aminoacridine, MMC: Mitomycin C

Table 3: Number of revertants (mean number of three plates) with metabolic activation

Dose µg/plate I/II	TA 100		TA 1535		TA102		TA98		TA1537
	I	II	I	II	I	II	I	II	I	II
Solvent Control	123	90	21	18	371	298	31	33	20	12
100	129	105	16	11	352	267	31	29	16	17
333	117	104	14	20	361	277	28	33	17	17
1000	123	106	15	16	375	281	31	35	19	15
2500	107	96	16	11	392	297	34	29	23	14
5000	113	106	20	13	369	295	35	29	19	16
Positive controls
2-AA (1.0)	1520	1463
2-AA (2.0)			341	267					316	483
DAN (10)					765	922
BP (5)							220	254

2 -AA: 2-Aminoanthracene, BP: Benzo(a)pyrene, DAN: 1,8-Dihydroxyanthraquinone

Applicant's summary and conclusion

Conclusions:

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.

Executive summary:

A bacterial reverse mutation assay (OECD 471) was performed to investigate the mutagenic potential of the test substance in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA100, TA1535, TA102, TA1537 and TA98 with and without metabolic activation. Based on the results of the preliminary study, the following concentrations were chosen for the main study: 100, 333, 1000, 2500 and 5000 µg/plate. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. 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 strains of Salmonella, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. In conclusion, the test material was considered to be non-mutagenic under the conditions of this test.