Ocimum basilicum, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

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:

The study was conducted between 28 August 2017 - 21 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix from male Sprague Dawley rats injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

- Dose range finding test (reported as part of experiment 1):
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate

- Experiment 1
TA 98, TA1535 and TA1537 (without and with S9): 5.4, 17, 52, 164, 512 and 1600 µg/plate
- Experiment 1A:
TA 98 and TA 1535 (without and with S9): 1600 and 5000 µg/plate

- Experiment 2:
TA 98, TA 100, TA 1535, TA 1537 and WP2uvrA (without and with S9): 17, 52, 164, 512, 1600 and 5000 µg/plate

For each experiment, doses were determined based on the results of preceeding experiments.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the substance was dissolved in dimethyl sulfoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Remarks:

For more details on positive controls, see 'other information on materials and methods' section.

Details on test system and experimental conditions:

Two independent experiments were performed, at first a direct plate assay and secondly a pre-incubation assay.

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period (experiment 2 only): 30 ± 2 minutes
- Exposure duration (both experiments): 48 ± 4 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

INTERPRETATION:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

ACCEPTABILITY CRITERIA:
- The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared
against relevant historical control data generated at the test facility.
- The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
- No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 µg/plate in experiment 1A and at and above 512 µg/plate in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1600 µg/plate in experiment 1 and at and above 512 µg/plate in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 µg/plate in experiment 1A and at and above 512 µg/plate in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1600 and 5000 µg/plate in experiment 1 and at and above 512 in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only at and above 1600 µg/plate in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Since in experiment 1 no cytotoxicity was observed in tester strains TA98 and TA1535, an additional experiment was performed (1A) to assess the cytotoxicity of these strains at concentrations of 1600 and 5000 μg/plate.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In both experiments, precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate. No precipitation was observed at the end of the incubation period.

HISTORICAL CONTROL DATA (see table 2 and 3 in 'any other informationon results'):
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1:
Cytotoxicity was observed in all strains, with and without S9, except for strain WP2uvrA.
TA1537: 1600 µg/plate
TA100: 1600 and 5000 µg/plate

EXPERIMENT 1A:
TA1535: 5000 µg/plate
TA98: 5000 µg/plate

EXPERIMENT 2:
Cytotoxicity was observed in all strains, with and without S9.
TA1535: ≥ 512 µg/plate
TA1537: ≥ 512 µg/plate
TA98: ≥ 512 µg/plate
TA100: ≥ 512 µg/plate
WP2uvrA ≥ 1600 µg/plate

Table 2 Historical data of solvent controls

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 - 36	3 - 32	3 – 20	3 – 23	8 - 41	9 - 55	66 - 161	63 - 160	10 – 59	9 - 69
Mean	11	11	6	7	16	23	105	105	25	31
SD	4	4	3	3	5	7	19	20	7	8
n	2057	2039	1950	1931	2023	2083	2027	2033	1739	1745

SD = Standard deviation; n = Number of observations

Table 3 Historical data of positive controls

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	125 - 1381	78 - 1058	55 – 1324	55 – 1051	410 – 1995	250 - 1977
Mean	839	220	736	382	1369	929
SD	153	112	331	150	310	345
n	2065	1967	1740	1933	1920	2014

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	537 – 1848	408 - 2651	93 – 1951	93 - 1359
Mean	908	1330	1128	422
SD	178	324	484	151
n	2007	2020	1679	1728

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between May 2015 and May 2017.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD TG 471 and according to GLP principles. The test was performed in two independent experiments (one direct plate and one pre-incubation test) up to and including 5000 µg/plate, in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in a dose range finding test (reported as part of experiment 1). Adequate positive controls and a solvent control were included. The substance did not precipitate on the plates.

In the first experiment cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in strains TA100 and TA1537 (1600 µg/plate) in the absence and presence of S9-mix. Since no cytotoxocity was observed in strains TA98 and TA1535, these were tested in an additional experiment (1A) to assess the cytotoxicity at dose levels of 1600 and 5000 µg/plate. In this additional experiment strains TA98 and TA1535 showed cytotoxicity at a dose level of 5000 µg/plate. In the second experiment, cytotoxicity was observed in all Salmonella strains at and above 512 µg/plate and above, with and without S9, and in tester strain WP2uvrA at and above 1600 µg/plate and above, with and without S9. No mutagencity was observed in any of the experiments, in all tester strains, with and without S9. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test

The mutagenic activity of the substance was evaluated in accordance with OECD TG 471 and according to GLP principles. The test was performed in two independent experiments (one direct plate and one pre-incubation test) up to and including 5000 µg/plate, in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in a dose range finding test (reported as part of experiment 1). Adequate positive controls and a solvent control were included. The substance did not precipitate on the plates.

In the first experiment cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in strains TA100 and TA1537 (1600 µg/plate) in the absence and presence of S9-mix. Since no cytotoxocity was observed in strains TA98 and TA1535, these were tested in an additional experiment (1A) to assess the cytotoxicity at dose levels of 1600 and 5000µg/plate. In this additional experiment strains TA98 and TA1535 showed cytotoxicity at a dose level of 5000 µg/plate. In the second experiment, cytotoxicity was observed in all Salmonella strains at and above 512 µg/plate and above, with and without S9, and in tester strain WP2uvrA at and above 1600 µg/plate and above, with and without S9. No mutagencity was observed in any of the experiments, in all tester strains, with and without S9. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Based on the results of the available data, the substance does not have to be classified for genotoxicity in according to EU CLP Regulation (EC) No. 1272/2008 and its amendments.