Concrete of Salvia sclarea L. (Lamiaceae) obtained from leaves, flowers and twigs by apolar hydrocarbon solvent extraction with or without purification by molecular distillation

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November to 19 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: LINALOOL CONSORTIUM / EE 93354
- Physical state: Brown waxy solid
- Expiration date of the lot/batch: 01 September 2018
- Purity test date: 27 October 2016

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4 °C in the dark

Target gene:

Histidine and tryptophan

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 TA1535 and TA1537 (with and without S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate. Salmonella strains TA98 and TA100 (with and without S9 mix) and E.coli WP2uvrA (with and without S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Repeat test
Salmonella strain TA100 (with and without S9 mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: In solubility checks performed in-house, the test item was noted as insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully soluble in acetone at 100 mg/mL. Acetone was therefore selected as the vehicle.
- Test substance preparation:
The test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. The test item was confirmed as a UVCB substance; therefore no correction was made for purity. Acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots (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.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

acetone

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

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

acetone

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. Several additional manual counts were required, due to revertant colonies spreading slightly, thus distorting the actual plate count.
- 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.

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 (greasy/globular in appearance) was noted at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

MUTAGENICITY TEST
- The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in both the absence and presence of S9-mix in the first mutation test (plate incorporation method) and consequently the same maximum dose level or 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 stronger toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535 and TA1537), 150 μg/plate (TA98) and 500 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 15 μg/plate (TA100) 150 μg/plate (TA1535), 500 μg/plate (TA1537) and 5000 μg/plate (TA98 and WP2uvrA). 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 (greasy/globular in appearance) was noted at 5000 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). Statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test at 500, 1500 and 5000 μg/plate in both the presence and absence of S9-mix and in the second mutation test at 50 μg/plate in the absence of S9-mix and 500 and 1500 μg/plate in the presence of S9-mix. These increases were considered to have no biological relevance because weakened bacterial background lawns were noted alongside these dose concentrations. Therefore the responses noted in the first and second mutation tests 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 (acetone) 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 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.

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

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 TA1535 and TA1537 (with and without S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate. Salmonella strains TA98 and TA100 (with and without S9 mix) and E.coliWP2uvrA (with and without S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Repeat test: Salmonella strain TA100 (with and without S9 mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.  

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

The vehicle (acetone) 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 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. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in both the absence and presence of S9-mix in the first mutation test (plate incorporation method) and consequently the same maximum dose level or 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 stronger toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535 and TA1537), 150 μg/plate (TA98) and 500 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 15 μg/plate (TA100) 150 μg/plate (TA1535), 500 μg/plate (TA1537) and 5000 μg/plate (TA98 and WP2uvrA). 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 (greasy/globular in appearance) was noted at 5000 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). Statistically significant increases in TA1535 revertant colony frequency were observed in the first mutation test at 500, 1500 and 5000 μg/plate in both the presence and absence of S9-mix and in the second mutation test at 50 μg/plate in the absence of S9-mix and 500 and 1500 μg/plate in the presence of S9-mix. These increases were considered to have no biological relevance because weakened bacterial background lawns were noted alongside these dose concentrations. Therefore the responses noted in the first and second mutation tests 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity test

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Wisher, 2017	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA	-S9 +S9	Up to cytotoxic or highest recommended concentration	-S9 : non mutagenic +S9 : non mutagenic

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no classification is proposed.