[No public or meaningful name is available]

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 August 23 to October 10, 2018

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:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 21 August 2018 / signed on 19 November 2018)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(±)-1-[(1RS,2SR,4SR)-BICYCLO[2.2.1]HEPT-2-YL]-5-HEXEN-2-ONE

Cas Number:

1352216-65-9

Molecular formula:

C13H20O1

IUPAC Name:

(±)-1-[(1RS,2SR,4SR)-BICYCLO[2.2.1]HEPT-2-YL]-5-HEXEN-2-ONE

Constituent 2

Reference substance name:

(±)-1-[(1RS,2RS,4SR)-BICYCLO[2.2.1]HEPT-2-YL]-5-HEXEN-2-ONE

Cas Number:

1352216-63-7

Molecular formula:

C13H20O1

IUPAC Name:

(±)-1-[(1RS,2RS,4SR)-BICYCLO[2.2.1]HEPT-2-YL]-5-HEXEN-2-ONE

Test material form:

liquid

Details on test material:

Appearance: Colourless liquid

Specific details on test material used for the study:

- Storage condition of test material: Approximately 4 °C in the dark under nitrogen
- Formulated concentrations were adjusted to allow for the stated water/impurity content (4%) of the test item.

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:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Experiment 1 (Pre-incubation method):
TA1535 [absence of S9-mix] & all strains [with S9-mix] : 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
TA 100, TA98, TA1537 & E.coli Wp2uvrA [absence S-9 mix] : 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate.

Experiment 2 (Pre-Incubation Method):
All tester strains (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
TA100 and TA1535 (presence of S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
WP2uvrA (presence of S9 mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
TA98 and TA1537 (presence of S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 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 DMSO at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the vehicle.

Controls

Controls 1

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

Controls 2

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; (preincubation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 37 ± 3 °C for 20 minutes (with shaking)
- Exposure duration/duration of treatment: 37 ± 3 °C for between 48 and 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: background growth inhibition

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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
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).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Possibility of evaporation from medium: The test item was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls. For volatile substances, the pre-incubation method may increase exposure of the bacteria to the test item in comparison to the standard plate incorporation method.
- Water solubility: Test item was immiscible in sterile distilled water at 50 mg/mL
- Precipitation and time of the determination: No precipitate of the test item was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- Signs of toxicity
Experiment 1: The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. However, the test item induced excessive toxicity to tester strains TA100, TA98 and TA1537 and E.coli strain WP2uvrA dosed in the absence of S9-mix and, therefore, part of the experiment was repeated using an amended dose range of 0.05 to 150 μg/plate.
The test item induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA100, TA98, TA1537and WP2uvrA) and 150 μg/plate (TA1535). In the presence of S9-mix, weakened bacterial background lawns were noted from 150 μg/plate (TA100 and TA1535), 500 μg/plate (TA98 and TA1537) and 1500 μg/plate (WP2uvrA).
Experiment 2: The maximum dose level of the test item in the second experiment was 5000 μg/plate or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.
The test item induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100) and 50 μg/plate (TA1535, TA98, TA1537and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted from 150 μg/plate (TA100, TA1535 and TA1537), 500 μg/plate (TA98) and at 5000 μg/plate (WP2uvrA).
- Individual plate counts / Mean number of revertant colonies per plate and standard deviation (cf. attached document):
Experiment 1: There were no 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).
Experiment 2: No biologically relevant 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). A statistically significant value was noted (WP2uvrA at 15 μg/plate in the absence of S9-mix; not identified as statistically significant on Table 4, as per the requirements of the Study Plan), however this response was not dose-related, not reproducible, and was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Cf. attached document.

Applicant's summary and conclusion

Conclusions:

Under the test condition, the test item was not mutagenic in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA- at any of the dose levels used either with or without metabolic activation.

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 TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames pre-incubation method at 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). The original dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. However, the test item induced excessive toxicity to tester strains TA100, TA98 and TA1537 and E.coli strain WP2uvrA dosed in the absence of S9-mix and, therefore, part of the experiment was repeated using an amended dose range of 0.05 to 150 μg/plate. Experiment 2 was performed on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and ranged between 0.05 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve four non-toxic dose levels and to achieve the toxic limit of the test item. Both experiments were performed using the pre-incubation methodology because the volatility of the test item was considered to be incompatible with the plate incorporation method.

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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Depending on bacterial strain type and presence or absence of S9-mix, the maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate or the toxic limit. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawn of all of the tester strains, initially from 50 and 150 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively.

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) or the toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item once again induced a toxic response with a visible reduction in the growth of the bacterial background lawns noted to all of the tester strains, initially from 15 and 150 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively.

No precipitate of the test item was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

There were no 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.

Similarly, no biologically relevant 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. One statistically significant value was noted (WP2uvrA at 15 μg/plate in the absence of S9-mix; not identified as statistically significant on Table 4, as per the requirements of the Study Plan), however this response was not dose-related, not reproducible, and was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

 

Under the test condition, the test item was not mutagenic in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA- at any of the dose levels used either with or without metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.