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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:
From 22 September to 11 October, 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 471.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 2018-06-19,19&20 / Signed on 2018-09-21.
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
Test material form:
liquid
Details on test material:
- Physical state: Clear, colorless liquid
- Other name: Lavandin Grosso; Lavandin Grosso France; Lavandula x hybrida, Lavandula hybrida oil; lavender, lavandula hybrida, ext.; Huile essentielle de lavandin.
- CAS No.: 91722-69-9
- EINECS-No.: 294-470-6
- Storage condition of test material: To be stored refrigerated (2 to 8°C), in a dry place in tightly - closed containers preferably full. Protect from light.
When stored for more than 18 months, quality should be checked before use. Store under Nitrogen gas.

Method

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
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™). The batch of S-9 used in this study was 3945, which had a protein content of 37 mg/mL.
- method of preparation of S9 mix : according to the method of Ames et al from male Sprague Dawley rats induced with a single dose of Aroclor 1254.
- concentration of S9 in the final culture medium: S9-fraction 10% v/v
- quality controls of S9: enzymatic activity, sterility, metabolic capability (certificate included in the study report)
- other: The S-9 mix was prepared immediately before use and kept on ice.
Test concentrations with justification for top dose:
Cytotoxicity test:1.5, 5, 15, 50, 150, 500, 1500 or 5000 μg/plate in TA98 and WP2 uvrA in the presence and absence of S-9 mix, under the direct plate incorporation method.

Mutagenicity test:
Main test: the doses of Essential Oil of Lavandin Grosso used in Experiment 1, under plate incorporation conditions, were as follows:
• 1.5, 5, 15, 50, 150 or 500 μg/plate in TA1537 in the absence of S-9 mix
• 5, 15, 50, 150, 500 or 1500 μg/plate in TA1535, TA98 and TA100 in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix
• 15, 50, 150, 500, 1500 or 3000 μg/plate in WP2 uvrA in the absence of S-9 mix
• 15, 50, 150, 500, 1500 or 5000 μg/plate in WP2 uvrA in the presence of S-9 mix

Confirmation test: the doses of Essential Oil of Lavandin Grosso used in Experiment 2, under pre-incubation conditions, were as follows:
• 0.5, 1.5, 5, 15, 50 or 150 μg/plate for TA1537 and TA100 in the absence of S-9 mix
• 1.5, 5, 15, 50, 150 or 500 μg/plate for TA1535 and TA98 in the absence of S-9 mix and TA1535, TA1537 and TA100 in the presence of S-9 mix
• 5, 15, 50, 150, 500 or 1500 μg/plate for WP2 uvrA in the absence of S-9 mix and TA98 in the presence of S-9 mix
• 15, 50, 150, 500, 1500 and 5000 μg/plate for WP2 uvrA in the presence of S-9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data. The test item was formulated on the day of use as a solution in dimethylsulphoxide (DMSO), at concentrations up to 50 mg/mL.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-amino-anthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
SOURCE OF TESTER STRAINS: Strains of salmonella typhimurium and E. Coli were obtained from Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™).

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37+/-3 °C
- Exposure duration: 37+/-3 °C for 66 hours

NUMBER OF REPLICATIONS: 3 plates/dose (mutation tests) and 2 plates/dose (range-finder).

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

OTHER:
- After an incubation of about 66 hours at about 37 ºC, the number of colonies per plate was counted.
The number of revertant colonies per plate was counted and recorded by an automatic colony counter. Average plate counts was presented with the mean and the standard deviation for each set of triplicates or diplicates (range-finder) per test item concentration and was used to calculate the ratio of colonies per exposed plate compared to the corresponding negative control.
- Test item solubility: soluble in ethanol
Evaluation criteria:
For the test to be considered positive, the test item would have had to induce a concentration related increase in mean revertants of at least twice the concurrentvehicle control for TA98, TA100 or WP2 uvrA or at least three times the concurrent vehicle control for TA1535 or TA1537. A clear positive response in a single experiment would be sufficient to determine the result, particularly if multiple strains were affected.
As the results met the criteria for a clear negative response, statistical test results were not taken into consideration.
Statistics:
Mean, standard deviation (s.d.), fold increases compared with negative Controls and Dunnett's t-statistic (t) were calculated for each group and bacterial strain.

Results and discussion

Test results
Key result
Species / strain:
bacteria, 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
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was found to be soluble when diluted in ethanol.
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST:
- Range finder: the doses selected for the mutation experiments were based on the toxicity of the test item or were chosen to include the guideline regulatory maximum dose level (5000 μg/plate)
* Cytotoxicity (Experiment 1):
Essential Oil of Lavandin Grosso was analysed up to the limit of toxicity of 150 μg/plate in TA1537 in the absence of S-9 mix, 500 μg/plate in TA1535, TA98 and TA100 in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix, and 1500 μg/plate in WP2 uvrA in both the presence and absence of S-9 mix, under plate incorporation conditions.
There was a reduction (to less than 0.5 times the negative Control value) in the mean colony count in TA100 at 500 μg/plate in the presence of S-9 mix, indicating toxicity of the test item to the bacteria.
* Cytotoxicity (Experiment 2):
Essential Oil of Lavandin Grosso was analysed up to the limit of toxicity of 150 μg/plate in TA1535, TA1537 and TA100 in the absence of S-9 mix, 500 μg/plate in TA98 in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix, and 1500 μg/plate in WP2 uvrA in both the presence and absence of S-9 mix, under pre-incubation conditions.
There was a reduction (to less than 0.5 times the negative Control value) in the mean colony count in WP2 uvrA at 1500 μg/plate in the presence of S-9 mix, indicating toxicity of the test item to the bacteria.

MUTAGENICITY TEST:
- There were no increases in revertant numbers, greater than the defined fold-increases (twice the negative Control value for TA98, TA100 or WP2 uvrA or three times the negative Control value for TA1535 or TA1537), compared with the relevant negative Control values in any strain at any dose level of the test substance, in the presence or absence of S-9 mix.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.



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 WP2uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coliWP2(pKM101) were exposed to the test substance diluted in dimethylsulphoxide (DMSO) at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

Cytotoxicity test: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA98 and WP2 uvrA in the presence and absence of S-9 mix under the direct plate incorporation method. 

Mutagenicity test:

Experiment 1 (direct plate incorporation method):

- 1.5, 5, 15, 50, 150 or 500 μg/plate in TA1537 in the absence of S-9 mix

- 5, 15, 50, 150, 500 or 1500 μg/plate in TA1535, TA98 and TA100 in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix

- 15, 50, 150, 500, 1500 or 3000 μg/plate in WP2 uvrA in the absence of S-9 mix

- 15, 50, 150, 500, 1500 or 5000 μg/plate in WP2 uvrA in the presence of S-9 mix

Experiment 2 ( pre-incubation method):

- 0.5, 1.5, 5, 15, 50 or 150 μg/plate for TA1537 and TA100 in the absence of S-9 mix

- 1.5, 5, 15, 50, 150 or 500 μg/plate for TA1535 and TA98 in the absence of S-9 mix and TA1535, TA1537 and TA100 in the presence of S-9 mix

- 5, 15, 50, 150, 500 or 1500 μg/plate for WP2 uvrA in the absence of S-9 mix and TA98 in the presence of S-9 mix

- 15, 50, 150, 500, 1500 and 5000 μg/plate for WP2 uvrA in the presence of S -9 mix.

Vehicle and positive control groups were also included in mutagenicity tests.

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data.  

There was a marked reduction in the mean colony count in TA100 at 500 μg/plate in the presence of S-9 mix in Experiment 1 and in WP2 uvrA at 1500 μg/plate in the presence of S-9 mix in Experiment 2, indicating toxicity of the test item to the bacteria. No precipitation was evident in either experiment.

There were no increases in revertant numbers, greater than the defined fold-increases, compared with the relevant negative Control values in any strain at any dose level of Essential Oil of Lavandin Grosso, in the presence or absence of S-9 mix, under either plate incorporation

or pre-incubation conditions.

 

Under the test condition, test substance is not mutagenic with and without metabolic activation inS. typhimuriumstrains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.