Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-12- 2014 to 13-02-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: waxy solid
Details on test material:
- Name of test material (as cited in study report): Guaiacwood oil
- Appearance: Yellow-brown waxy solid
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
Histidine or tryptophan locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner Minimal Plates, Top Agar. - Properly maintained: yes- Periodically checked for viability, spontaneous reversion rate characteristics.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1 (direct plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plateExperiment 2 (pre-incubation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: immiscible in sterile distilled water at 50 mg/mL but fully miscible in dimethyl sulphoxide (DMSO).
Controls
Untreated negative controls:
yes
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
Remarks:
N-ethyl-N-nitro-N-nitrosoguanidine (2, 3 and 5 µg/plate), 9-aminoacridine (80 µg/plate) and 4-nitroquinoline-1-oxide (0.2µg/plate ) without S9 -mix. Benzo(a)pyrene (µg/plate) and 2-Aminoanthracene (1, 2 and 10 µg/plate) with S9-mix
Details on test system and experimental conditions:
DURATION
- Pre-incubation period: with and without S9-mix 20 minutes (prior to exposure in experiment 2 only)
- Exposure duration: 48 hours (experiment 1 and 2)

NUMBER OF REPLICATIONS:
-Test for mutagenicity (exp 1 and 2): in triplicate

DETERMINATION OF MUTAGENICITY
- Method: Evaluate reduction in number of spontaneous revertants and negative effect on the growth of the bacterial background lawn (thinning).

DETERMINATION OF CYTOTOXICITY
- Method: Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Evaluation criteria:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain.

ACCEPTANCE CRITERIA
- All bacterial strains must have demonstrated required characteristics as determined by their respective strains according to Ames et al. (1975)
- Tester strain cultures should exhibit characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- Tester strain culture density should be in the range of 0.9 to 9x10E9 bacteria/mL
- Positive control values must demonstrate intrinsic sensitivity of the test strains and integrity of the S9-mix
- A minimum of four non-toxic test item dose levels is required- No evidence of excessive contamination
Statistics:
No further information provided

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Direct Plate Incorporation Method (exp. 1)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Pre-Incubation Method (exp. 2)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON TEST RESULTS
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method in exp 1 (direct plate method) or exp 2 (pre-incubation method). A small significant increase in WP2uvrA revertant colony frequency was observed in the absence of S9 -mix at 15 µg/plate in the 2nd experiment but is was considered of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual colony counts at 15 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.7 time the concurrent vehicle control.

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: No test item precipitate was observed on the plates after the plate incorporation method at any of the doses tested in either the presence or absence of S9-mix. A light test item precipitate (globular in appearance) was noted under an inverted microscope at 5000 µg/plate after employing the pre-inclubation method (exp 2). This observation did not prevent the scoring of revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Reductions in growth of bacterial background lawns of all tester strains (except WP2uvrA in presence of S9-mix) from 1500 µg/plate (with S9-mix) and 5000 µg/plate (without S9-mix). No weakening of background lawn for E. coli at any test item dose level with S9-mix.
Experiment 2: Reductions in growth of bacterial background lawns of TA 1535 and TA 1537 from 150 µg/plate, TA 100 from 500 µg/plate, and TA98 and WP2uvrA from 5000 µg/plate, all without S9-mix.Reductions in growth of bacterial background lawns ( of TA 100, TA 1535 and TA 1537 from 1500 µg/plate and TA98 from 5000 µg/plate, all with S9-mix. No toxicity was noted for WP2uvrA dosed in the presence of S9-mixThe test item was tested up to the maximum recommended level of 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with or without metabolic activation. Therefore, Guaiacwood oil was considered to be non-mutagenic under the conditions of this test and should not be classified in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP).
Executive summary:

The genotoxicity of the test substance Guaiacwood oil was tested in bacteria strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA according to OECD guideline 471 (Ames test) and under GLP conditions. Two experiments (using the plate incorporation methodology and the pre-incubation methodology) were performed with concentrations of the test substance ranging from 1.5 - 5000 µg/plate, with and without metabolic activation. Negative, vehicle and positive controls were included as well. The frequency of revertant colonies was recorded.

The positive and negative control were valid: all positive control chemicals induced increase in frequency of revertant colonies and the increase observed for the negative control substance was considered acceptable. Cytotoxicity was observed in both experiments by a reduction in growth of the bacterial background lawns, being more apparent in experiment 2 (using the pre-incubation methodology). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in exp. 1 and 2 using different exposure methods.

A small significant increase in WP2uvrA revertant colony frequency was observed in the absence of S9-mix at 15 µg/plate in the 2nd experiment but was considered of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual colony counts at 15 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.7 time the concurrent vehicle control.

Under the conditions of this test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with or without metabolic activation. Therefore, Guaiacwood oil was considered to be non-mutagenic under the conditions of this test and should not be classified in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP).