Cedrus atlantica, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria: not mutagenic (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:

05-03-2015 to 31-03-2015

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)

Qualifier:

according to guideline

Guideline:

other: EPA OCSPP harmonized guideline 870.5100

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Ministry of Economy, Trade and Industry, Ministry of Health, Labour and Welfare, Ministry of Agriculture, Forestry and Fisheries

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Cedarwood Atlas oil

Target gene:

Histidine and 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: 1.5, 5, 15, 50, 150, 500, 5000 µg/plate
Experiment 2: 15, 50, 150, 500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in Acetone at 100 mg/mL

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:

METHOD OF APPLICATION:
Experiment 1: direct plate incorporation methodology (in agar)
Experiment 2: pre-incubation methodology

DURATION
- Preincubation 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

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

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:

no 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
There were no significant increase in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in exp 1 (direct plate method). There were no significant increase in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in exp 2 (pre-incubation method).

TEST-SPECIFIC CONFOUNDING FACTORS
A test item precipitate (light and globular in appearance) was noted under an inverted microscope at 5000 µg/plate. This observation did not prevent the scoring of revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY
In exp 1 (direct plate method) there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence of absence of S9-mix.
In exp 2 (pre-incubation method) a visible reduction of the bacterial background lawns of all Salmonella strains was observed from 1500 µg/plate without S9-mix and at 5000 µg/plate with S9-mix. The toxicity of the test item did not interfere with the test outcome. In the E. coli strain no toxicity was noted.

Remarks on result:

other: all strains/cell types tested

Details on the results can be found in the attached document below.

Conclusions:

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 (direct plate method) and exp 2 (pre-incubation method). Therefore the test item Cedarwood Atlas oil was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The genotoxicity of the test substance Cedarwood Atlas oil was tested in bacteria strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A 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 only in experiment 2 by a reduction in growth of the bacterial background lawns from 1500 µg/plate (-S9) and at 5000 µg/plate (+S9) in the TA strains only, but this did not interfere with the outcome of the test. 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.

Under the conditions of this study, the test item Cedarwood Atlas oil was considered to be non-mutagenic and hence, it does not need to be classified for mutagenicity, according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The genotoxicity of Cedarwood Atlas 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 were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation, indicating that the test substance is not mutagenic under the conditions of this test

Justification for classification or non-classification

Based on the available negative result from this Ames test, Cedarwood Atlas oil shall be considered as non-mutagenic and should not be classified according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).