Bulnesia sarmienti, ext., sapond.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity in vitro: negative (OECD 471)

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine or tryptophan locus

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).

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

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.

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The genotoxicity of Guaiacwood Oil was examined in bacterial strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A (OECD 471, GLP). The concentrations applied were ranging from 1.5 - 5000 µg/plate, with and without metabolic activation. Negative, vehicle and positive controls were determined as valid. No significant increases in the frequency of revertant colonies of biological relevance were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Based on the results of this Ames test, the test item Guaiacwood oil is considered to be non-mutagenic.

Justification for classification or non-classification

Based on the available results, Guaiacwood oil is considered to be non-mutagenic and should not be classified in accordance with to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).