Cypress, Cupressus funebris, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD TG 471): not mutagenic

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:

26-SEPT-2016 until 16-OCT-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European
Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD principles of Good Laboratory Practice, EC Council Directive 2004/10/EC

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot /batch No.of test material: Sponsor Batch: 1002515317
- Expiration date of the lot/batch: until 31 August 2017 (expiry date)
- Purity/Composition: 100.0% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: until 31 August 2017 (expiry date)

OTHER SPECIFICS:
Test item: 207805/A
Identification: Cedarwood Oil China
Appearance:Pale yellow to yellow liquid
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Essential oil of Chamaecyparis funebris (Cupressaceae) obtained from the wood by steam distillation
CAS Number: 85085-29-6
Molecular structure: UVCB
Molecular formula: 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

Details on mammalian cell type (if applicable):

-

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

Details on mammalian cell type (if applicable):

-

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

-

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1: 17, 52, 164, 512, 1600 and 5000 μg/plate (Based on a dose range finding test with the strains TA100 and WP2uvrA, with and without 5% (v/v) S9-mix.)
Experiment 2: 86, 154, 275, 492, 878 and 1568 μg/plate (Based on the results of the first mutation assay)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item could not be dissolved in water or dimethyl sulfoxide. The test item was soluble in ethanol. Therefore ethanol was used as solvent in this project.

Untreated negative controls:

no

Remarks:

Solvent control was the only negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene (all strains; with metabolic activation)

Remarks:

Solvents used for pos control: Saline, DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat) preincubation

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

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background
lawn or a microcolony formation

Rationale for test conditions:

Recommended test system in international guidelines (e.g.
OECD, EC).

Evaluation criteria:

Test is considered negative if:
a. The total number of revertants in the tester strain TA100 or WP2uvrA < 2 times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is < than 3 times the concurrent vehicle control.
b. The negative response should be reproducible in at least one follow-up experiment.

Test is considered positive if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA > 2 times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than 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.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest tested concentration in experiment 1 5000µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

only vehicle control used

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest tested concentration (5000µg/plate in Experiment 1) (1537 µg/plate, in absence of S9 mix in experiment 2)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

only vehicle control used

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

only vehicle contro used

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest tested concentration in experiment 2 (1568 µg/plate) with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

only vehicle control used

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Remarks:

only vehicle control used

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: A solubility test was performed. The test item could not be dissolved in water or dimethyl sulfoxide. The test item was soluble in ethanol. Therefore ethanol was used as solvent in this
project.
- Precipitation: The test item precipitated on the plates at dose levels of 1568, 1600 and 5000 μg/plate, except in tester strains TA1537 and TA98 in the absence of S9-mix, where precipitation was already observed at 512 μg/plate.

RANGE-FINDING/SCREENING STUDIES:
Cedarwood Oil China was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.
Based on the results of the dose range finding test, the following dose range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 μg/plate. No increase in the number of revertants was observed upon treatment with Cedarwood Oil China under all conditions tested.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive/negative historical control data: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Conclusions:

Based on the results of this study it is concluded that Cedarwood Oil China is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, and should not be classified according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The genotoxicity of the test substance Cedarwood Chinese 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. The test was performed in two independent experiments in the presence and absence of S9-mix.

A dose range finding test for Cedarwood Chinese oil, was performed in in the strains TA100 and WP2uvrA, with and without S9 metabolic activation (1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate). Precipitate was found at 1600 and 5000 µg/plate. Cytotoxicity was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed.

Subsequently, Cedarwood Chinese oil was tested in the tester strains TA1535, TA1537 and TA98 at a concentration range of 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9 metabolic activation. Negative, vehicle and positive controls were included. Cytotoxicity was observed in tester strains TA1535 and TA1537 at the highest tested concentration. Precipitation was observed at 1600 and 5000 μg/plate, except in tester strains TA1537 and TA98 in the absence of S9-mix, where precipitation was already observed at 512 μg/plate.

In a follow-up experiment, a concentration range of 86 to 1568 μg/plate was used, in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 1568 μg/plate. Cytotoxicity was observed in tester strain TA1537 in the absence of S9-mix and TA100 in the absence and presence of S9-mix.

All the negative and strain-specific positive controls were within the laboratory historical control data. No significant increases were observed in the frequency of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) or in the number of revertant (Trp+) colonies in the tester strain WP2uvrA, in the absence or presence of S9-metabolic activation. Under the conditions of this study, the test item Cedarwood Chinese 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)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The genotoxicity of the test substance Cedarwood Chinese 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. The test was performed in two independent experiments in the presence and absence of S9-mix. A dose range finding test for Cedarwood Chinese oil, was performed in in the strains TA100 and WP2uvrA, with and without S9 metabolic activation (1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate). Precipitate was found at 1600 and 5000 µg/plate. Cytotoxicity was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed. Subsequently, Cedarwood Chinese oil was tested in the tester strains TA1535, TA1537 and TA98 at a concentration range of 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9 metabolic activation. Negative, vehicle and positive controls were included. Cytotoxicity was observed in tester strains TA1535 and TA1537 at the highest tested concentration. Precipitation was observed at 1600 and 5000 μg/plate, except in tester strains TA1537 and TA98 in the absence of S9-mix, where precipitation was already observed at 512 μg/plate.

In a follow-up experiment, a concentration range of 86 to 1568 μg/plate was used, in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 1568 μg/plate. Cytotoxicity was observed in tester strain TA1537 in the absence of S9-mix and TA100 in the absence and presence of S9-mix.

All the negative and strain-specific positive controls were within the laboratory historical control data. No significant increases were observed in the frequency of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) or in the number of revertant (Trp+) colonies in the tester strain WP2uvrA, in the absence or presence of S9-metabolic activation. Under the conditions of this study, the test item Cedarwood Chinese oil was considered to be non-mutagenic.

Justification for classification or non-classification

Based on the available data, Cedarwood oil Chinese does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).