Artemisia pallens, 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:

From 2017-02-08 to 2017-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

QA statement signed on 2017-04-20

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Artemisia pallens, ext.

EC Number:

295-155-6

EC Name:

Artemisia pallens, ext.

Cas Number:

91844-86-9

Molecular formula:

not applicable for NCS substances

IUPAC Name:

Essential oil of Artemisia pallens Wall. Ex DC (Asteraceaé) obtained from leaves and stems by steam distillation old name: Essential oil of Artemisia pallens Wall. Ex DC (Compositae) obtained from leaves and stems by steam distillation

Test material form:

liquid

Details on test material:

- Name of test substance: Davana Oil
- CAS number: 8016-03-3 (other identifier)
- Batch No.: R-91187
- Storage: Room temperature (cool, away from heat and sources of ignition)
- Expiration date of the lot/batch: 21 April 2018

Method

Target gene:

Histidine gene for Salmonella and tryptophan gene for E.coli

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):

CELLS USED
- Source of cells: The bacterial strains were acquired by Gentronix Ltd, BioHub at Alderley Park, from the AstraZeneca (AZ) Genetic Toxicology Department, Alderley Park, UK. AZ acquired the strains from the following sources:
*TA1535, TA1537 and TA100 were originally obtained in 1995 by AZ Sweden, from Professor B N Ames, Biochemistry Department, University of California, Berkeley, California, USA.
*TA98 was originally obtained in 2008 by AZ Sweden, from BioReliance Corporation, Rockville, Maryland, USA.
*The E. coli strain was originally obtained in 1995 by AZ Sweden, from Dr S Venitt, Institute of Cancer Research, Royal Marsden Hospital, Surrey, UK.

The bacteria were stored frozen at approximately -196°C after sub-culturing; master cultures
were stored frozen at approximately -196°C after sub-culturing. Before establishing cryopreserved working stock cultures and on the day of the first mutagenicity test, the strains were checked for the following characteristics (phenotype check): absence of contamination; spontaneous revertant frequency; pre-existing revertant numbers; presence of the deep rough (rfa) marker (not uvrA/pKM101); sensitivity to UV light; presence of plasmid pKM101 (TA98, TA100 and uvrA/pKM101).

CULTURE MEDIA
The medium used for the growth of all inoculum cultures was Nutrient Broth No.2 (Oxoid Limited)
supplemented with ampicillin, as required. Test systems were plated onto a minimal glucose agar
medium (Vögel-Bonner agar) prepared by the VWR Scientific Preparation Laboratory, Alderley
Park, UK.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The post-mitochondrial fraction (S9) of a rat liver homogenate was purchased from Molecular Toxicology Inc. (Boone, North Carolina, USA) via TRiNOVA Biochem GmbH, Germany. The S9 was prepared from the livers of male Sprague-Dawley rats treated with Aroclor 1254. Its metabolic capacity was demonstrated by key enzyme assays and the ability to activate reference agents to bacterial mutagens. Furthermore, because of batch to batch variation and to assess suitability for use in this laboratory, each lot of S9 will have undergone in-house testing using two promutagens, cyclophosphamide and benzo[a]pyrene (TA1535 and TA98, respectively). The protein content was adjusted to 30 mg/mL by dilution with 0.1 mol/L phosphate buffer (pH7.4) before incorporation into the S9 mix.
- method of preparation of S9 mix: The S9 fraction was stored deep-frozen in a -80°C freezer. Sufficient vials were removed from frozen, thawed and used; any excess S9 fraction was discarded. The complete activation system (S9 mix) was prepared immediately before use; final concentrations in the S9 mix were as follows:
S9 10% v/v
Phosphate buffer (0.1 mol/L, pH 7.4) 100 mmol/L
Magnesium chloride 8 mmol/L
Potassium chloride 33 mmol/L
Nicotinamide adenine dinucleotide phosphate 4 mmol/L
Glucose-6-phosphate 5 mmol/L
- concentration of S9 in the final culture medium: 10%
- quality controls of S9: sterility, metabolic capability

Test concentrations with justification for top dose:

- Experiment 1 (+/- S9): 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate (up to maximum recommended concentration) for plate incorporation method.
- Experiment 2: 16, 50, 160, 500, 1600 and 5000 µg/plate (up to maximum recommended concentration) for plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: A preliminary assessment of solubility of the test item in the preferred solvent (vehicle), dimethyl sulphoxide (DMSO), was conducted. As solubility of the test item in DMSO was satisfactory, it was used as the solvent for the mutagenicity testing.
- The test item was administered in solvent, dimethyl sulphoxide (DMSO) at a volume of 100 μL per plate, within four hours of formulation for mutagenicity test 1 and within three hours of formulation for mutagenicity test 2.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

In the presence of S9-mix

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene: 2 µg/plate for TA98, TA100, TA1537 and TA1535 and 20 µg/plate for uvrA/pKM101

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

In the absence of S9-mix

Positive control substance:

2-nitrofluorene

sodium azide

other: 9-Aminoacridine.HCl and Potassium dichromate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: In all cases there were three plates in the solvent (DMSO) control, test item and positive control groups.
- Number of independent experiments: Two independent plate incorporation mutagenicity tests were performed using Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, in both the presence and absence of S9 mix.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation): both experiments

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Briefly, frozen aliquots of the bacterial strains (-70°C or lower) were incubated overnight (8 to 10 hours) at 37°C in an orbital incubator (120 rpm) in nutrient broth. For strains TA98, TA100 and uvrA/pKM101, the nutrient broth was supplemented with ampicillin at 25 µg/mL.
- Exposure duration/duration of treatment: 48-72 hours (3 days at 37°C)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
After incubation at 37°C for 3 days, all plates were examined both macroscopically and microscopically for evidence of cytotoxicity, precipitates and any other effect relevant to the interpretation of the test. Microscopic examination was used to check the condition of the histidine/tryptophan-requiring microcolonies that make up the ‘background lawns’.
Revertant (his+, trp+) colony numbers were scored using a Sorcerer Colony Counter (Perceptive Instruments, UK). In cases where the mean incidence of revertant colonies on the solvent control plates was 20 or less (typically with strains TA1535 and TA1537), or if cytotoxicity or precipitate interfered with image analysis, the plates were scored manually. All counts were manually entered into the results sheet.

CHECK
- Sterility Controls: Plates containing each component of the test system except the bacteria were included to check against possible accidental contamination of the system.
- Viability Determination: Confirmation that adequate numbers of viable bacteria were exposed to the test item was obtained from viability determinations for each strain performed concurrently with the mutagenicity test.

Rationale for test conditions:

Tested up to maximum recommended concentration or up to cytoxicity.

Evaluation criteria:

TOXICITY
A dose of the test item was judged to be toxic to a bacterial strain if the formation of
microcolonies (background lawn) was reduced, or a decrease in the number of revertant colonies
was seen.

MUTAGENICITY
A test item was considered to be mutagenic if the following criteria were satisfied:
1. For TA1535 or TA1537, the mean number of revertant colonies of one or more doses of the test item, with or without metabolic activation was equal to or greater than 2 times the concurrent solvent control mean value and the relevant historical mean value; for any other strain, the mean number of revertant colonies was equal to or greater than 2 times the concurrent solvent control mean value in the presence of one or more doses of the test item, with or without metabolic activation.
2. There was a dose-related increase in the number of revertant colonies.
3. Any increase was reproducible.

Statistics:

Not reported

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 500 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 500 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 500 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 500 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1600 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above a dose of 160 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above a dose of 160 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above a dose of 160 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above a dose of 160 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

FORMULATION ANALYSIS
In accordance with the regulatory test guidelines, no analyses of the stability of the test item in administered formulations or dilutions were undertaken as fresh solutions were prepared on the day of use and each mutagenicity test was dosed within four hours of formulation for mutagenicity test 1 and within three hours of formulation for mutagenicity test 2.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitate was seen at and above a dose of 500 μg per plate in both the presence and absence of S9 mix on the day of dosing, and at and above 1600 μg per plate in both the presence and absence of S9 mix on the day of scoring.
- controls: The mean solvent control (DMSO) revertant numbers were within the acceptable ranges for each of the indicator strains. The results obtained with positive control agents demonstrated the mutability of the individual strains and, where appropriate, the ability of the S9 mix to metabolise 2-aminoanthracene. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The sterility controls were all free from contamination and the viability determination confirmed that adequate numbers of viable bacteria of each strain were exposed to the test item. Phenotypes of bacterial indicator strains were confirmed.

STUDY RESULTS
Experiment 1 (plate incorporation):
Based on current OECD guidelines, the dose range selected was 1.6 to 5000 μg per plate in both the presence and absence of S9 mix.
Precipitate was seen at and above a dose of 500 μg per plate in both the presence and absence of S9 mix on the day of dosing, and at and above 1600 μg per plate in both the presence and absence of S9 mix on the day of scoring.
Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen at and above a dose of 160 μg per plate for the four S. typhimurium strains in the absence of S9 mix, at and above 500 μg per plate for the four S. typhimurium strains in the presence of S9 mix, and at and above 1600 μg per plate for the E. coli strain in both the presence and absence of S9 mix. Due to excessive cytotoxicity, plates could not be scored for numbers of revertant colonies at 5000 μg per plate for the four S. typhimurium strains in the presence and absence of S9 mix.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.

Experiment 2 (plate incorporation):
Based on findings in mutagenicity test 1, the dose range selected was 16 to 5000 μg per plate in both the presence and absence of S9 mix.
Precipitate was seen at and above a dose of 500 μg per plate in both the presence and absence of S9 mix on the day of dosing, and at and above 1600 μg per plate in both the presence and absence of S9 mix on the day of scoring.
Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen at and above a dose of 160 μg per plate for the four S. typhimurium strains in the absence of S9 mix, at and above 500 μg per plate for the four S. typhimurium strains in the presence of S9 mix, and at and above 1600 μg per plate for the E. coli strain in both the presence and absence of S9 mix. Due to excessive cytotoxicity, plates could not be scored for numbers of revertant colonies at 5000 μg per plate for the four S. typhimurium strains in the presence and absence of S9 mix.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.

HISTORICAL CONTROL DATA
- Positive historical control data: cf. attached document
- Negative (solvent/vehicle): cf. attached document

Any other information on results incl. tables

See tables of result attached in the "attached background material" section.

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli the test item did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test, it was concluded that Davana Oil was not mutagenic using a plate incorporation method for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.

Executive summary:

In a reverse gene mutation assay in bacteria, Salmonella typhimurium LT2 bacteria strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA/pKM101 as indicator organisms were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors), according to the OECD TG 471 guideline and the methods of Maron and Ames, 1983, Venitt et al., 1984, Mortelmans and Zeiger, 2000 and Mortelmans and Riccio, 2000.

 

A preliminary assessment of solubility was performed to determine suitable dose levels of Davana Oil for the mutagenicity tests. 
The dose ranges used were:
Mutagenicity test 1: 1.6 to 5000 μg per plate
Mutagenicity test 2: 16 to 5000 μg per plate

 

The mean solvent control (DMSO) revertant numbers were within the acceptable ranges for each of the indicator strains. The results obtained with positive control agents demonstrated the mutability of the individual strains and, where appropriate, the ability of the S9 mix to metabolise 2-aminoanthracene. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The sterility controls were all free from contamination and the viability determination confirmed that adequate numbers of viable bacteria of each strain were exposed to the test item. Phenotypes of bacterial indicator strains were confirmed.

The test item showed evidence of cytotoxicity to the bacteria. The minimum dose showing evidence of cytotoxicity was 160 μg per plate. The maximum dose level scored for revertant colonies was 5000 μg per plate. The minimum dose at which precipitate was seen on the day of scoring was 1600 μg per plate.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.

 

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, at any of the dose levels used either with or without metabolic activation (S9-mix).

Under the conditions of this test, it was concluded that Davana Oil was not mutagenic using a plate incorporation method for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.