Chlorocyclopentane

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:

2018-05-25 to 2018-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

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:

Chlorocyclopentane

EC Number:

213-212-5

EC Name:

Chlorocyclopentane

Cas Number:

930-28-9

Molecular formula:

C5H9Cl

IUPAC Name:

chlorocyclopentane

Test material form:

liquid

Details on test material:

- Name as stated in the report: JNJ-64259884-AAA (Cyclopentylchloride)
- Physical state: Liquid
- Appearance: clear to light yellow liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16CD0746
- Expiration date of the lot/batch: 2018-11-01 (retest date)
- Manufacture date: 2016-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied in this study.

Method

Target gene:

Histidine locus (histidine-requiring S. typhimurium strains); Tryptophan locus (tryptophan-requiring E. coli strains)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Aroclor 1254 induced rat liver metabolic activation system)

Test concentrations with justification for top dose:

The maximum final concentration for the dose range finding test was based on the solubility of the test item in DMSO (highest recommended concentration).
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 and WP2uvrA with and without 5% (v/v) S9-mix

The top doses for the mutation experiments were selected based on the toxicity observed in the dose range finding test.
Mutation experiment 1: 52, 164, 512, 1600 and and 5000 μg/plate in TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix;
Mutation experiment 2: 492, 878, 1568, 2800 and 5000 µg/plate in TA1535, TA1537, TA98, TA100 and WP2uvrA with and without 10% (v/v) S9-mix;
Mutation experiment 3: 492, 878, 1568, 2800 and 5000 µg/plate in TA1535 with 10% (v/v) S9-mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item was soluble at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9-mix; 5 μg/plate (TA1535)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9-mix; 2.5 μg/plate (TA1537)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9-mix; 10 μg/plate (TA98)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9-mix; 650 μg/plate (TA100)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9-mix; 10 μg/plate (WP2uvrA)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.5 μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5 μg/plate (TA1537 with 10% S9-mix), 1 μg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2 μg/plate (TA100 with 10% S9-mix), 15 μg/plate (WP2uvrA with 5 and 10% S9-mix)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2 + 1 repeat experiment with one strain

METHOD OF APPLICATION: in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 ± 2°C.
The following solutions were successively added to 3 ml molten top agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains
- 0.1 ml of a dilution of the test item in DMSO and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time (if incubation with a selection agent): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium histidine-requiring strains); Tryptophan (E. coli tryptophan-requiring strains)

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle 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 vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up 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 above, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml.
- Precipitation:
Mutation experiment 1: not observed at the start or at the end of the incubation period in any tester strain.
Mutation experiment 2: at the start of the incubation period at the concentrations of 2800 and 5000 μg/plate and no precipitate was observed at the end of the incubation period.
Mutation experiment 3: at the start of the incubation period at the concentrations of 2800 and 5000 μg/plate and no precipitate was observed at the end of the incubation period

RANGE-FINDING/SCREENING STUDIES: The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. The dose range finding test results are reported as a part of mutation experiment 1.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The vehicle control values were within the laboratory historical control data ranges, except the response for TA98 in the first experiment (absence of S9-mix). Since the mean number of revertant colonies showed a slightly lower number of revertant colonies (7 revertant colonies) when compared against relevant historical control data (8 revertant colonies), the validity of the test was considered to be not affected.
The 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.

- Signs of toxicity:
Mutation experiment 1: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strains TA1535 and TA100 (absence and presence of S9-mix) and TA98 (presence of S9-mix) at the dose level of 5000 μg/plate.
Mutation experiment 2: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strains TA98 and TA100 in the absence of S9-mix at the highest tested concentration.
Mutation experiment 3: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

- Genotoxicity results
Mutation experiment 1: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
Mutation experiment 2: In tester strain TA1535 in the presence of S9-mix, the test item induced an up to 5.1-fold increase in the number of revertant colonies compared to the vehicle control. The increases observed were above the laboratory historical control data ranges and more than three-fold the concurrent solvent control. In all other tester strains, no increase in the number of revertants was observed.
Mutation experiment 3: The test item induced an up to 7.1-fold increase in the number of revertant colonies compared to the vehicle control. The increases observed were above the laboratory historical control data ranges and more than three-fold the concurrent solvent control.

Any other information on results incl. tables

Repeat experiments:

To verify the mutagenic response observed in tester strain TA1535 in the presence of S9-mix, an additional third experiment was performed. In the additional third experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strain TA1535 in the presence of 10% (v/v) S9-mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of the results:
positive with metabolic activation in the tester strain TA1535, negative with metabolic activation in the tester strains TA100, TA1537, TA98 and WP2uvrA, negative without metabolic activation

In the absence of S9-mix, all bacterial strains showed negative responses over the entire dose range, i.e. no dose-related increase in the number of revertants.
In the presence of 10 % (v/v) S9-mix, the test item induced up to 7.1-fold, dose related increases in the number of revertant colonies compared to the vehicle control in tester strain TA1535. The increases observed were in two independently repeated experiments, above the laboratory historical control data range and more than threefold the concurrent controls, therefore these increases are considered biologically relevant.

Based on the results of this study it is concluded that the test item is mutagenic in the tester strain TA1535 of the Salmonella typhimurium reverse mutation assay in the presence of S9-mix. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA100, TA1537 or TA98) or the Escherichia coli reverse mutation assay.