Fir, Abies balsamea, ext.

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:

18 Oct 2017 - 09 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

in accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines, OPPTS 870.5100, “Bacterial Reverse Mutation Test”, EPA 712-C-98-247

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fir, Abies balsamea, ext.

EC Number:

285-364-0

EC Name:

Fir, Abies balsamea, ext.

Cas Number:

85085-34-3

IUPAC Name:

Fir Balsam Oil

Test material form:

liquid

Details on test material:

Name of test material as cited in study report: Fir Needle Oil Canadian (Fir, Abies Balsamea, ext)

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Botanical source and Lot# 67919
- Expiration date of the lot/batch: 13 septembre 2018
- Purity test date: UVCB, considered 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤ 70 RH%), protected from light, avoid contact with iron.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was dissolved in N,N-Dimethylformamide (DMF) at a concentration of 100 mg/mL. It was then diluted in the same solvent to prepare the required test solutions

Method

Target gene:

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

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented post-mitochondrial S9 fraction (prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats).

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test).

Based on the results of the preliminary tests, a 100 mg/mL stock solution was prepared in DMF. At least nine test concentrations were prepared by successive dilutions of the stock solution, to obtain lower doses. The maximum test concentration was 5000 μg test item/plate.

Examined concentrations in the Initial Mutation Test were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate at Salmonella typhimurium strains with and without metabolic activation. Moreover the examined concentrations were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate at Escherichia coli WP2 uvrA strain with and without metabolic activation.

Examined concentrations in the Confirmatory Mutation Test were 500, 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate at Salmonella typhimurium strains with and without metabolic activation and 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate at Escherichia coli WP2 uvrA strain with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: N,N-Dimethylformamide (DMF)

- Justification for choice of solvent/vehicle: Based on results of a compatibility test, the test item was dissolved in N,N-Dimethylformamide (DMF) at a concentration of 100 mg/mL.
Compatibility test: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). Insolubility was detected at 100 mg/mL concentration using Distilled water. At the same concentration the test item was formed emulsion with DMSO and was soluble (clear solution) using DMF.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD) and 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 h.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: a decrease in the number of revertants and a reduction of the bacterial background lawn

Rationale for test conditions:

According to OECD TG 471

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle / solvent) / positive control and the test plates will be determined by manual counting. Visual examination of the plates will also be performed; precipitation or signs of growth inhibition (if any) will be recorded and reported. Mean values, appropriate standard deviations and mutation factors will be calculated for each concentration level of the test item and for the controls.

Criteria for Validity:
The study is considered valid if:
- the number of revertant colonies of the negative (vehicle / solvent) and positive controls are in the historical control range in all strains of the main tests;
- at least five analyzable concentrations are presented in all strains of the main tests.

Criteria for a Positive Response:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and
Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
A test article is considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Plate incorporation method (Initial Mutation Test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slightly reduced background lawn development

Vehicle controls validity:

valid

Untreated negative controls validity:

other: See vehicle control

Positive controls validity:

valid

Test results 2

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Pre-incubation method (Confirmatory Mutation Test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slightly reduced background lawn development

Vehicle controls validity:

valid

Untreated negative controls validity:

other: See vehicle control

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY:
In the Initial Mutation Test slightly reduced background lawn was observed in all Salmonella typhimurium strains on the plates at 500 and 158.1 μg/plate concentration with and without metabolic activation and in Salmonella typhimurium TA100, TA1535 and TA1537 strains at 500, 158.1 and 50 μg/plate concentration without metabolic activation.
In the Confirmatory Mutation Test reduced/slightly reduced background lawn was observed in all Salmonella typhimurium strains on the plates at 500 and 158.1 μg/plate concentration with metabolic activation and at 500, 158.1, 50 and 15.81 μg/plate concentration without metabolic activation. The same effect was detected in Escherichia coli WP2 uvrA strain without metabolic activation at 5000, 1581 and 500 μg/plate concentrations and slightly reduced background lawn was observed with metabolic activation at 5000 μg/plate concentration.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Fir Needle Oil Canadian (Batch Number: 67919) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli(Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/beta-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method). 

Based on the results of the Compatibility Test, the test item was dissolved in N,N-Dimethylformamide (DMF) at a concentration of 100 mg/mL.Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimuriumTA100 and TA98 in the absence and presence of metabolic activation. 

Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Testwere 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581μg/plate at Salmonella typhimurium strainswith and without metabolic activation.Moreover, the examined concentrations were 5000, 1581, 500, 158.1, 50 15.81, 5 and 1.581 μg/plate at Escherichia coli WP2 uvrA strain with and without metabolic activation.

Examined concentrations in the Confirmatory Mutation Test were 500, 158.1, 50, 15.81, 5, 1.581, 0.5, 0.1581 and 0.05μg/plate at Salmonella typhimurium strains with and without metabolic activation and 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate at Escherichia coli WP2 uvrA strain with and without metabolic activation.

No precipitate was observed in the main testsin all examined strains with and without metabolic activation. 

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in theInitial Mutation Testin all examined Salmonella typhimurium strains with and without metabolic activation and reduced / slightly reduced background lawn development was observed in the Confirmatory Mutation Test in all examined strains with and without metabolic activation at some concentrations.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid. 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Fir Needle Oil Canadian (Batch Number: 67919) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.