Essential oil of Copaiba obtained from the exudate of Copaifera (Fabaceae) trees by distillation

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-05-217 - 31-05-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Copaiba obtained from the exudate of Copaifera (Fabaceae) trees by distillation

EC Number:

946-913-6

Molecular formula:

Not applicable for UVCB Substance

IUPAC Name:

Essential oil of Copaiba obtained from the exudate of Copaifera (Fabaceae) trees by distillation

Test material form:

liquid

Remarks:

pale yellow liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor, batch S-71699
- Expiration date of the lot/batch: 14 February 2019
- Purity test date: 14 February 2017

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor, batch S-71699
- Expiration date of the lot/batch: 14 February 2019
- Purity test date: 14 February 2017

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

Method

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain

Species / strain 1

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 meta bolism 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 2

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: 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

Controls

Untreated negative controls:

yes

Remarks:

The negative control was dimethyl sulfoxide, the vehicle of the test item.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and Saline (= physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

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, without 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.
- Exposure duration:

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.

NUMBER OF REPLICATIONS: triplicate

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

Rationale for test conditions:

The study is designed to comply with the experimental methods indicated in the guidelines.

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA1537 in the presence of S9-mix at the highest tested concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA1537 in the absence of S9-mix and TA100 in the absence and presence of S-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Experiment 1 precipitation of Copaiba balsam oil on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period. Experiment 2: Precipitation of Copaiba balsam oil on the plates was observed at the start of the incubation period at concentrations of 2800 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period.

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.

HISTORICAL CONTROL DATA
- Positive historical control data: available in study report.
- Negative (solvent/vehicle) historical control data: available in study report.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Copaibla balsam oil 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 Copaiba balsam oil was evaluated according to guidline OECDTG 471. Copaiba balsam oil 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 5% (v/v) S9-mix : 52, 164, 512, 1600 and 5000 μg/plate Precipitation of Copaiba balsam oil on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the presence of S9-mix at the highest tested concentration. In a second experiment, the test item was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the absence and presence of 10% (v/v) S9-mix. Precipitation of Copaiba balsam oil on the plates was observed at the start of the incubation period at concentrations of 2800 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in testerstrain TA1537 in the absence and presence of S9-mix and in tester strain TA100 in the presence of S-mix. Copaiba balsam oil 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. 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 Copaiba balsam oil 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).