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Administrative data

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:
26 April to 29 May 2017
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
28 October 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: The Thujone Consortium / MN16 081-1
- Appearance: Clear yellow liquid
- Expiration date of the lot/batch: 06 April 2018
- Purity test date: April 2016

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
Target gene:
Histidine and tryptophan for Salmonella typhimurium and Escherichia coli, respectively
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:
S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male rats dosed with phenobarbital and β-Naphthaflavone
Test concentrations with justification for top dose:
Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
Experiment 2 – Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test formulation: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No correction for purity was required. 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
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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.

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

DURATION
- 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:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system.
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 analysis of data as determined by UKEMS (Mahon et al., 1989).
Key result
Species / strain:
other: TA1535, TA1537, TA98 and TA100
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
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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
- Precipitation: None
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2015 and 2016 of the corresponding Testing Laboratory.

MAIN EXPERIMENTS
Experiment 1 and 2:
- In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 1500 μg/plate in the absence of S9-mix and at 5000 μg/plate in the presence of S9-mix. Consequently the same maximum recommended dose level (5000 μg/plate) of the test item was selected as the maximum dose in the second mutation test. The test item again induced a toxic response in the second mutation test (pre-incubation method) with weakened bacterial background lawns noted in the absence of S9-mix from 500 μg/plate (TA1535 and TA1537) and 1500 μg/plate (TA100, TA98 and WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were noted from 1500 μg/plate (all Salmonella strains) and at 5000 μg/plate (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.
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- There were no 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 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).

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, S9-mix and test item formulation used in both experiments were shown to be sterile.

None

Conclusions:
Under the test conditions, test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA (pKM101) strains.
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 item both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Experiment 2 – Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix 

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

The vehicle (dimethyl sulphoxide) 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 or 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. In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 1500 μg/plate in the absence of S9-mix and at 5000 μg/plate in the presence of S9-mix. Consequently the same maximum recommended dose level (5000 μg/plate) of the test item was selected as the maximum dose in the second mutation test. The test item again induced a toxic response in the second mutation test (pre-incubation method) with weakened bacterial background lawns noted in the absence of S9-mix from 500 μg/plate (TA1535 and TA1537) and 1500 μg/plate (TA100, TA98 and WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were noted from 1500 μg/plate (all Salmonella strains) and at 5000 μg/plate (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. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no 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 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). 

Under the test conditions, test item is not considered as mutagenic in these bacterial systems.

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.