(4E)-4-(3,3,4-trimethylcyclopentylidene)butanal

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:

2019-08-28 to 2019-10-10

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:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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 2

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)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-4-(3,3,4S-trimethylcyclopentylidene)butanal

Molecular formula:

C12 H20 O

IUPAC Name:

(4E)-4-(3,3,4S-trimethylcyclopentylidene)butanal

Constituent 2

Reference substance name:

(4E)-4-(3,3,4R-trimethylcyclopentylidene)butanal

Molecular formula:

C12 H20 O

IUPAC Name:

(4E)-4-(3,3,4R-trimethylcyclopentylidene)butanal

Test material form:

liquid

Details on test material:

Appearance: Very pale yellow liquid

Method

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:
British Industrial Biological Research Association,
Trinova Biochem GmbH

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Moltox. Lot No. 4123 and the protein level was adjusted to 20 mg/mL
- method of preparation of S9 mix:
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
- 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): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (up to maximum recommended concentration).
- Experiment 1 (- S9 / repeat for TA 100): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate (up to cytotoxicity).
- Experiment 2:
TA100 (absence of S9): 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate (up to cytotoxicity)
Remaining strains (absence and presence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate (up to maximum recommended concentration).

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test item was only partially miscible 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.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

In the presence of S9-mix

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

In the absence of S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) [Test 1] ; preincubation [Test 2]

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48-72 hours

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

Rationale for test conditions:

Tested up to maximum recommended concentration or up to cytoxicity.

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.

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.

Results and discussion

Test results

Test results 1

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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

True negative controls validity:

not applicable

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No test item precipitate was observed

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
Experiment 1 (plate incorporation):
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 for all of the bacterial strains except TA100 dosed in the absence of metabolic activation (S9-mix) which was tested up to the toxic limit in a repeat experiment.
Toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure in the absence of metabolic activation (S9-mix) to all tester strains initially from 15 µg/plate to TA100, 150 µg/plate to TA1535, TA98 and TA1537 and 500 µg/plate to WP2uvrA. In the presence of metabolic activation (S9-mix), toxicity was noted from 150 µg/plate to TA100, TA98 and TA1537 and 500 µg/plate to TA1535 and WP2uvrA.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
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 (pre-incubation):
The maximum dose level of the test item in the second experiment was toxic limit. A stronger toxic response was noted after employing pre-incubation methodology with visible reductions in the growth of the bacterial background lawns or reductions in revertant counts observed with test item exposure in the absence of metabolic activation (S9-mix) to all tester strains from 5 µg/plate to TA100, 15 µg/plate to TA1537 and 50 µg/plate to WP2uvrA, TA1535 and TA98. In the presence of metabolic activation (S9-mix), toxicity was noted from 50 µg/plate to TA98 and TA1537 and 150 µg/plate to TA1535, TA100 and WP2uvrA. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix). 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).
- Individual plate counts and Mean number of revertant colonies per plate and standard deviation are in the attached document.

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

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, the test item was considered to be non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were exposed to the test item diluted DMSO using both the Ames plate incorporation and pre-incubation methods at up to nine 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 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 µg/plate. However, the test item induced excessive toxicity to Salmonella strain TA100 dosed in the absence of S9 and this part of Experiment 1 was repeated using an amended range of 0.15 and 150 µg/plate. The experiment was repeated on a separate day (pre-incubation method) 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 0.005 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. Nine test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

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. 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 for all of the bacterial strains except TA100 dosed in the absence of metabolic activation (S9-mix) which was tested up to the toxic limit in a repeat experiment. Toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure to all tester strains initially from 15 and 150 µg/plate, in the absence and presence of metabolic activation (S9-mix), respectively. Based on the results of Experiment 1, the toxic limit of the test item was employed as the maximum dose in the second mutation test (pre-incubation method). Toxicity to all of the bacterial strains was again noted in the second experiment, with weakened bacterial background lawns noted initially from 5 and 50 µg/plate, in the absence and presence of metabolic activation (S9-mix), respectively. No test item precipitate 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 (plate incorporation method). Similarly, no 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).

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, the test item was considered to be non-mutagenic.

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.