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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test (OECD TG 471): negative

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:
03 Apr 2017 - 26 Apr 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from Sponsor, B-64530
- Expiration date of the lot/batch: 25 January 2019
- Purity test date: 26 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
-A solubility test was performed based on visual assessment. The stock was heated to 100˚C in a waterbath and homogenous samples were aliquoted and stored at room temperature. One of these aliquots was used to perform the test. The test item was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves until the test item had completely dissolved. Test item concentrations were used within 2 hours after preparation.

OTHER SPECIFICS: UVCB
Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa : deep rough (defective lipopolysaccharide cellcoat) gal : mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: The strain lacks an excision repair system and is sensitive to agents such as UV
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Dose range-finder: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
First and Second Mutation Experiment: 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Suitable solvent, compatible with the used bacterial strains
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DSMO and Saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene (all strains with S9), ICR-191 (TA1537, sithout S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, in agar: plate incorporation.
- Cell density: 1.0 ± 0.1 at 700 nm (10^9 cells/ml), 0.1 ml added to top agar.

DURATION
- Exposure duration:48 ± 4 h

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies.

OTHER:
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
- Exposure temperature 37.0 ± 1.0°C (actual range 34.3 – 39.5°C).
Rationale for test conditions:
The study is designed to comply with the experimental methods indicated in the guidelines
Evaluation criteria:
EVALUATION CRITERIA
-A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
- A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two(2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
-Any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
Key result
Species / strain:
other: TA1535, TA1537, TA98, TA100
Remarks:
Main experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Main experiment I and II
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
First experiment: at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period.
Second experiment: at the start of the incubation period at the concentration of 512 μg/plate and upwards. At the end of the incubation period the test item precipitated at 1600 and 5000 μg/plate in the absence of S9-mix and at 5000 μg/plate in the presence of S9-mix. Except in tester strain TA100 in the absence of S9-mix, where precipitation was already observed at 512 μg/plate.

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 at dose levels of 512 μg/plate and upwards in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: available in study report.
- Negative (solvent/vehicle) historical control data: available in study report.
Conclusions:
Based on the results of this study it is concluded that Cedrol, Cedarwood Texas oil distilled is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay and does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The mutagenic potential of Cedrol, Cedarwood Texas oil distilled was evaluated according to OECDTG 471. In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in tester strain TA100 at dose levels of 512 μg/plate and upwards in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.  Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 5.4 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 5.4 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. Cytotoxicity was observed in all tester strains in the absence and presence of S9 -mix, except in tester strain WP2uvrA in the presence of S9-mix. Cedrol, Cedarwood Texas oil distilled did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Cedrol, Cedarwood Texas oil distilled is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay and does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation study in bacterial cells

The mutagenic potential of Cedrol, Cedarwood Texas oil distilled and/or its  was evaluated according to OECDTG 471. In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in tester strain TA100 at dose levels of 512 μg/plate and upwards in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.  Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 5.4 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 5.4 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or  beyond a precipitating dose level. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix. Cedrol, Cedarwood Texas oil distilled did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Cedrol, Cedarwood Texas oil distilled is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Based on the results of this study Cedrol, Cedarwood Texas oil distilled does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).