Lavender, Lavandula hybrida, 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:

24 November to 16 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 471 without any deviation.

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on July 05, 2016 / Signed on October 28, 2016)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Concrete of Lavandula Hybrida (Lamiaceae) obtained from the flowering plant by organic solvent extraction

EC Number:

946-009-1

Cas Number:

91722-69-9

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

Concrete of Lavandula Hybrida (Lamiaceae) obtained from the flowering plant by organic solvent extraction

Test material form:

other:

Remarks:

described as a dark green/brown very viscous pasty liquid

Details on test material:

Identification : lavandin concrete

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: LINALOOL CONSORTIUM / 1002865766
- Physical state: Dark green/brown viscous pasty liquid
- Expiration date of the lot/batch: 31 December 2017
- Purity test date: 20 October 2016

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

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 rats induced with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method:
Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with and without S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method:
Salmonella strains TA100 and TA1535 (without S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Salmonella strains TA98 and TA1537 (without S9 mix); TA1535 (with S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate;
Salmonella strains TA 98, TA 100, TA 1537, E. coli WP2uvrA (with S9 mix) and E. coli WP2uvrA (without S9 mix): 15, 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: In solubility checks performed in-house, the test item was noted as insoluble in dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and in acetone at 100 mg/mL but was fully soluble in tetrahydrofuran at 200 mg/mL. Tetrahydrofuran was therefore selected as the vehicle.
- Test substance preparation:
The test item was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 20 minutes at 40 °C on the day of each experiment. The test item was confirmed as a UVCB substance; therefore no correction was made for purity. Tetrahydrofuran is toxic to the bacterial cells at and above 50 μL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 μL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
- Bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

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

DURATION
- Preincubation period: 37 °C ± 3 °C for 20 minutes (with shaking)
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
- All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 μg/plate because of test item precipitation.
- The sterility controls were performed in triplicate as follows: Top agar and histidine/biotin or tryptophan in the absence of S9-mix; Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

Rationale for test conditions:

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: None
- Precipitation: A test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

MUTAGENICITY TEST
- There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was initially employed in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test item induced significant toxicity as weakened bacterial background lawns and substantial reductions in the revertant colony frequency of a number of tester strains to the extent where a number of strains required repeat analysis employing an amended test item dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 μg/plate) or the toxic limit of the test item was employed in the second mutation test. Results from the second mutation test showed that the test item induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 μg/plate (TA100 and TA1535) and 1500 μg/plate (TA98 and TA1537). In the presence S9-mix, weakened bacterial background lawns were initially noted from 150 μg/plate (TA1535) and 1500 μg/plate (TA100, TA98 and TA1537). No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
- There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 5000 μg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship and the individual revertant colony counts at 5000 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control. Further statistically significant increases in TA1535 revertant colony frequency was observed in the presence of S9-mix at 150 and 500 μg/plate in the second mutation test. These increases were also considered to have no biological relevance because weakened bacterial background lawns were noted alongside these dose concentrations. Therefore the responses noted in the second mutation test would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and present as non-revertant colonies.
- The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. Two revertant colony counts for TA1535 (vehicle control dosed in the presence of S9-mix in the second mutation test) were just above the maximum vehicle and untreated control historical control levels. These counts were still considered acceptable as the other vehicle and untreated control counts were within expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9-mix. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

HISTORICAL CONTROL DATA
- See "Attached background material" section for Historical data

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Any other information on results incl. tables

See the "Attached background material" for information on tables of results

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.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test substance both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method:

Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with and without S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method:

Salmonella strains TA100 and TA1535 (without S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.

Salmonella strains TA98 and TA1537 (without S9 mix); TA1535 (with S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate;

Salmonella strains TA 98, TA 100, TA 1537, E. coli WP2uvrA (with S9 mix) and E. coli WP2uvrA (without S9 mix): 15, 50, 150, 500, 1500 and 5000 μg/plate.

 

Negative, vehicle (tetrahydrofuran) and positive control groups were also included in mutagenicity tests.

 

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies generally 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was initially employed in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test item induced significant toxicity as weakened bacterial background lawns and substantial reductions in the revertant colony frequency of a number of tester strains to the extent where a number of strains required repeat analysis employing an amended test item dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 μg/plate) or the toxic limit of the test item was employed in the second mutation test. Results from the second mutation test showed that the test item induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 μg/plate (TA100 and TA1535) and 1500 μg/plate (TA98 and TA1537). In the presence S9-mix, weakened bacterial background lawns were initially noted from 150 μg/plate (TA1535) and 1500 μg/plate (TA100, TA98 and TA1537). No toxicity was noted to E. coli strain WP2uvrAat any test item dose level in either the absence or presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

 

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 5000 μg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship and the individual revertant colony counts at 5000 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control. Further statistically significant increases in TA1535 revertant colony frequency was observed in the presence of S9-mix at 150 and 500 μg/plate in the second mutation test. These increases were also considered to have no biological relevance because weakened bacterial background lawns were noted alongside these dose concentrations. Therefore the responses noted in the second mutation test would be due to additional histidine being available to His^-bacteria allowing these cells to undergo several additional cell divisions and present as non-revertant colonies.

 

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.