Cyclopropanecarboxylic acid, 3-[(1Z)-2-chloro-3,3,3-trifluoro-1-propen-1-yl]-2,2-dimethyl-, (1R,3R)-rel-

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:

09 October 2017 to 30 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of (1R,3R)-3-[(1Z)-2-chloro-3,3,3-trifluoroprop-1-en-1-yl]-2,2-dimethylcyclopropanecarboxylic acid and (1S,3S)-3-[(1Z)-2-chloro-3,3,3-trifluoroprop-1-en-1-yl]-2,2-dimethylcyclopropanecarboxylic acid

EC Number:

614-283-9

Cas Number:

68127-59-3

Molecular formula:

C9H10ClF3O2

IUPAC Name:

reaction mass of (1R,3R)-3-[(1Z)-2-chloro-3,3,3-trifluoroprop-1-en-1-yl]-2,2-dimethylcyclopropanecarboxylic acid and (1S,3S)-3-[(1Z)-2-chloro-3,3,3-trifluoroprop-1-en-1-yl]-2,2-dimethylcyclopropanecarboxylic acid

Test material form:

solid

Specific details on test material used for the study:

Storage conditions: At room temperature
The dose were not adjusted for the purity of the test material.

Method

Target gene:

The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively.

Species / strain

Species / strain / cell type:

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

Remarks:

E.coli WP2 pKM101 and WP2 uvrA pKM101

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The bacterial strains TA1535, TA1537, TA98, TA100, WP2 uvrA pKM101, and WP2pKM101 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany). Strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% (MERCK, D-64293 Damrstadt) in liquid nitrogen.
- Suitability of cells: Regular checking of the properties of the S.typhimurium and E.coli strains regarding the membrane permeability and ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontanous mutation rates are performed by the testing laboratory according to

Ames , B.N., McCann J. and Yamasaki E. (1977), Methodes for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, In: Klbey B.J. et al. (Eds.) "Handbook of Mutagenicity Test Procedures" Elsevi er, Amsterdam, 1-17;
Maron D.M., Ames, B.N. (1983), Revised methods for the Salmonella mutagenicity test, Mutation Res. 113, 173-215
Mortelamans K., Riccio ES. (2000), The bacterial tryptophan reverse mutation assay with Escherichia coli WP2, Mutation Res. 455 61-69

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
preculture nutrient medium: 8 g Nutrient Broth and 5 g NaCl
overlay agar for Salmonella strains: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine x HCl x H2O, 12.2 mg Biotin
overlay agar for Escherichia coli strains: 7.0 g Agar Agar, 6.0 g NaCl and 10.2 mg Tryptophan

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal activation (S9 mix)

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test substance tested was 3 - 5000 μg/plate. The pre-experiment is reported as Experiment I since the acceptability criteria of the assay were met. Since cytotoxic effects were observed in Experiment I, a minimum of six concentrations were tested in Experiment II. 5000 μg/plate was chosen as the maximal concentration in experiment II. The test substance was tested at the following concentrations:
Pre-experiment / Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II.
Strains TA 1535 and TA 98: 33; 100; 333; 1000; 2500; and 5000 μg/plate
The remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:DMSO (DMSO, purity 99%) was chosen because of its solubilitsation properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenyl-diamine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I was performed as a plate incorporation assay. Since negative result was obtained in this experiment, experimetn II was performed as a pre-incubation assay. According to the direct plate incorportation and pre-incubation method the bacteria are exposed to the test substance with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.
in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density: The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10e8 -10e9 cells/mL).
- Number of Replicates: Each concentration, including the controls, was tested in triplicate.
DURATION
- Preincubation period: 60 minutes at 37°C

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Cytotoxicity of the test substance results in a reduction of the number of spontanous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.

S9 MIX
Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The amount of S9 supernatant was 10% v/v in the S9 mix. Co-factors 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

Evaluation criteria:

Evaluation of results: A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration 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 concentration 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.

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:

no 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:

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 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

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 7

Key result

Species / strain:

E. coli WP2

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 8

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

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Strain TA1135: mean/SD 1130/143.1 (without S9 mix) and 388/58.2 (with S9 mix
Strain TA1537: mean/SD 82/12.7 (without S9 mix) and 191/60.8 (with S9 mix)
Strain TA98: mean/SD 378/73.7 (without S9 mix) and 3949/77.8 (with S9 mix)
Strain TA 100: mean/SD 1966/293.2 (without S9 mix) and 3798/830.4 (with S9 mix)
Strain WP2 pKM 101: mean/SD 3776/466.8 (without S9 mix) and 1355/498.3 (with S9 mix)
Strain SP2 uvrA pKM 101: Mean/SD 3364/637.1 (without S9 mix) and 2123/229.1 (with S9 mix)

- Negative (solvent/vehicle) historical control data:
Strain TA1135: mean/SD 12/2.5 (without S9 mix) and 12/2.5 (with S9 mix
Strain TA1537: mean/SD 10/2.2 (without S9 mix) and 13/3.5 (with S9 mix)
Strain TA98: mean/SD 25/4.4 (without S9 mix) and 34/6.2 (with S9 mix)
Strain TA 100: mean/SD 156/26.0 (without S9 mix) and 148/32.3 (with S9 mix)
Strain WP2 pKM 101: mean/SD 207/23.5 (without S9 mix) and 231/26.3 (with S9 mix)
Strain SP2 uvrA pKM 101: Mean/SD 334/33.0 (without S9 mix) and 378/37.7 (with S9 mix)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduction of the number of revertants (below the induction factor of 0.5)

Any other information on results incl. tables

Table 1: Cytotoxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentration (ug/plate)

	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	5000	/	5000	/
TA 98	/	/	/	5000
TA 100	/	5000	5000	2500 -5000
WP2 pKM101	5000	5000	5000	5000
WP2 uvrA pKM101	5000	5000	/	5000

Table 2: Historical data

These data represent the laboratory´s historical control data from November 2014 until November 2016 representing approx. 600 experiments (WP2 pKM 101 and WP2 uvrA pKM 101 the historical data are based on approx. 100 experiments).

Strain		Without S9 mix				With S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent Control	12	2.5	6	25	12	2.5	7	26
	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positiv control	1130	143.1	334	1816	388	58.2	176	688
TA 1535	Solvent Control	10	2.2	6	19	13	3.5	7	30
	Untreated control	11	2.7	5	21	14	4.0	7	31
	Positiv control	82	12.7	43	157	191	60.8	83	434
TA 98	Solvent Control Untreated control Positiv control	25	4.4	13	43	34	6.2	15	58
		27	4.9	12	43	37	6.5	11	57
		378	73.7	211	627	3949	771.8	360	6586
TA 100	Solvent Control Untreated control Positiv control	156 223 3776	26.0 23.6 293.2	78 79 498	209 217 2767	148 172 3798	32.3 25.4 830.4	73 85 536	208 218 6076
WP2 pKM 101	Solvent Control Untreated control Positiv control	207 223 3776	23.5 22.7 466.8	171 166 2796	302 314 5156	231 230 1355	26.3 24.4 498.3	194 202 1055	332 334 4068
WP2 uvrA pKM 101	Solvent Control Untreated control Positiv control	334 347 3364	33.0 33.2 637.1	242 283 1981	392 412 4828	378 392 2123	37.7 36.2 229.1	276 296 1708	464 483 2782

Mean = mean value of revertants/plate SD = standard deviation

Min = minimal value/Max = maximal value

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity tests and under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test material is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

Study Design

This study was performed to investigate the potential of the test material to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA pKM101 and WP2 pKM101.

Results

In Experiment I the plates incubated with the test item showed normal background growth in all strains with and without metabolic activation. In Experiment II reduced background growth was observed in strains TA 1535, TA 1537 and TA 100 with and without metabolic activation.Cytotoxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in strains TA 1537, TA 98, TA 100, WP2 pKM101, and WP2 uvrA pKM101. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with CA3514 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no observed 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, which showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity tests and under the experimental conditions reported, thte test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test material is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.