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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-22 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to Section 13]
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Research Institute for Fragrance Materials, Inc. / 130050
- Physical state: Clear colorless liquid
- Date of manufacture: 26 March 2014
- Date of receipt: 09 July 2014
- Expiration date of the lot/batch: 27 September 2015
- Purity test date: 30 June 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light
- Stability: Test substance was considered stable through 27 September 2015
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

None

Conclusions:
Under the test condition, Lavender oil is not mutagenic with and without metabolic activation in S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP). Therefore, the registered substance Clary sage oil is not not considered as mutagenic in bacterial system.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test substance both in the presence and absence of metabolic activation system (10% liver S9-mix) using the plate incorporation method. The first phase, the initial toxicity- mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.

 

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535 and WP2uvrA) with and without metabolic activation

Retest of the initial toxicity-mutation assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 1537) with and without metabolic activation

Confirmatory mutagenicity assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535, TA 1537 and WP2uvrA) with and without metabolic activation

Vehicle (DMSO) and positive control groups were also included in mutagenicity assay.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2uvrA in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains. Due to confluent bacterial growth, tester strain TA1537 was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profile observed (i.e., toxicity at 5000 μg/plate and no precipitate). Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 μg/plate.

 

In the retest of the initial toxicity-mutation assay, no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed at 5000 μg/plate.

 

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains.

 

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA according to the criteria of

the Annex VI of the Regulation (EC) No. 1272/2008 (CLP). Therefore, the registered substance Clary sage oil  is not not considered as mutagenic in bacterial system.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Product name: Lavender oil
Date: 03/27/2014
Number: 130050
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Research Institute for Fragrance Materials, Inc. / 130050
- Physical state: Clear colorless liquid
- Date of manufacture: 26 March 2014
- Date of receipt: 09 July 2014
- Expiration date of the lot/batch: 27 September 2015
- Purity test date: 30 June 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light
- Stability: Test substance was considered stable through 27 September 2015

Method

Target gene:
Histidine and tryptophan
Species / strain
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):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9: S9-mix from the livers of male Sprague-Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535 and WP2uvrA) with and without metabolic activation
Retest of the initial toxicity-mutation assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 1537) with and without metabolic activation
Confirmatory mutagenicity assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535, TA 1537 and WP2uvrA) with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: methyl methanesulfonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
TEST SYSTEM: Salmonella tester strains (TA98, TA100, TA1535 and TA1537) were derived from Dr. Bruce Ames’ cultures; E. coli tester strains (WP2 uvrA) were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 h at 37 ± 2 °C

NUMBER OF REPLICATIONS:
Duplicate plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification.

- OTHER:
- Solubility Test: A solubility test was conducted using sterile water and DMSO to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration up to 50 mg/mL for aqueous solvents and up to 500 mg/mL for organic solvents.
- Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.
- Sterility of the test substance and the vehicle, all test substance dose levels and the vehicle used in the initial toxicity-mutation and confirmatory mutagenicity assays were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.
Evaluation criteria:
The following criteria must be met for the initial toxicity-mutation and the confirmatory mutagenicity assays to be considered valid.
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.
To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10^9 cells/mL.
The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of four non-toxic dose levels is required to evaluate assay data.
A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).
Statistics:
None

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
-Solubility: DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.
- Precipitation: No precipitate was observed.

MUTATION ASSAY
Initial Toxicity-Mutation Assay: In Experiment B1 (Initial Toxicity-Mutation Assay), the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2 uvrA in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains. Due to confluent bacterial growth, tester strain TA1537 was not evaluated for mutagenicity but was retested in Experiment B2 based on the precipitate and toxicity profile observed (i.e., toxicity at 5000 μg/plate and no precipitate). Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 μg/plate.
In Experiment B2 (Retest of the Initial Toxicity-Mutation Assay), no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed at 5000 μg/plate.
Confirmatory Mutagenicity Assay: In Experiment B3 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains.

OTHERS:
Sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test substance both in the presence and absence of metabolic activation system (10% liver S9-mix) using the plate incorporation method. The first phase, the initial toxicity- mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.

 

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535 and WP2uvrA) with and without metabolic activation

Retest of the initial toxicity-mutation assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 1537) with and without metabolic activation

Confirmatory mutagenicity assay: 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 98, TA 100, TA 1535, TA 1537 and WP2uvrA) with and without metabolic activation

Vehicle (DMSO) and positive control groups were also included in mutagenicity assay.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg/plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2uvrA in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains. Due to confluent bacterial growth, tester strain TA1537 was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profile observed (i.e., toxicity at 5000 μg/plate and no precipitate). Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the retest and confirmatory mutagenicity assays was 5000 μg/plate.

 

In the retest of the initial toxicity-mutation assay, no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed at 5000 μg/plate.

 

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. No precipitate was observed. Toxicity was observed beginning at 1500 or at 5000 μg/plate with all Salmonella tester strains.

 

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA according to the criteria of

the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).