1-fluoro-3-(trans-4-propylcyclohexyl)benzene

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:

24 MAR 2015 - 24 APR 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

Hess. Ministerium für Unwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden (11 April 2013)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-fluoro-3-(trans-4-propylcyclohexyl)benzene

EC Number:

443-100-3

EC Name:

1-fluoro-3-(trans-4-propylcyclohexyl)benzene

Cas Number:

138679-81-9

Molecular formula:

Hill formula: C15H21F CAS formula: C15H21F

IUPAC Name:

1-fluoro-3-[(1s,4r)-4-propylcyclohexyl]benzene

Test material form:

liquid

Method

Target gene:

histidine (S. typhimurium strains), tryptophan (E. coli strain WP2)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: please refer to Table 1

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/p-naphthoflavone induced rat liver
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment, the S9 mix is stored in an ice bath.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment 1: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment 2: 33; 100; 333; 1000; 2500; and 5000 µg/plate (Since no toxic effects were observed in up to 5000 µg/plate in the pre-experiment, 5000 µg/plate was chosen as maximal concentration in experiment 2)

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine, 4-NOPD; methyl methane sulfonate, MMS; 2-aminoanthracene, 2-AA

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:
- Cell density at seeding (if applicable): the bacteria were harvested at the late exponential or early stationary phase (10E8 - 10E9 cells/mL).
- Test substance added in medium; Experiment I: in agar (plate incorporation); Experiment II: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min (Experiment II)
- Exposure duration/duration of treatment: at least 48 hours

FOR GENE MUTATION:
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies: The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager (v.1.21). The counter was connected to a PC with printer to print out the individual values and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation the colonies were partly counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition


METHODS FOR MEASUREMENTS OF GENOTOXICIY
increase in revertant colony numbers

Rationale for test conditions:

In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated in the overlay agar in the test tubes in experiment I from 1000 to 5000 µg/plate with S9 mix and in experiment II from 1000 to 5000 µg/plate with and without S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES (if applicable): In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Ames test:
- Signs of toxicity : No, the plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
- Individual plate counts
- Mean number of revertant colonies per plate and standard deviation
please refer to results tables attached under 'Attached background material'

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Strain without S9 mix with S9 mix
Mean SD Min Max Mean SD Min Max
Solvent control 16 2.84 7 27 18 4.50 7 36
TA 1535 Untreated control 15 3.35 7 27 19 4.99 9 34
Positive control 2248 468.15 803 3722 418 119.81 182 683
Solvent control 10 2.19 6 23 18 4.37 8 32
TA1537 Untreated control 10 2.52 5 22 19 4.64 8 30
Positive control 74 10.30 50 159 300 126.74 77 651
Solvent control 26 4.27 16 46 38 6.29 21 59
TA 98 Untreated control 28 5.15 16 54 41 6.40 21 55
Positive control 326 56.74 211 560 2306 944.29 330 4691
Solvent control 107 20.07 74 184 132 22.97 83 205
TA 100 Untreated control 113 21.29 82 204 142 21.11 82 210
Positive control 1987 376.71 478 2594 2864 946.03 530 4983
Solvent control 52 7.73 34 70 58 7.87 41 77
WP2uvrA Untreated control 52 7.91 32 81 57 7.71 39 74
Positive control 716 259.60 226 1296 354 112.61 180 642

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

A study according to OECD Guideline 471 and following GLP was conducted. This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes in experiment I from 1000 to 5000 µg/plate with S9 mix and in experiment II from 1000 to 5000 µg/plate with and without S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the registered substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.