Potassium (1R,3R)-3-[(1Z)-2-chloro-3,3,3-trifluoroprop-1-en-1-yl]-2,2-dimethylcyclopropanecarboxylate and Potassium (1S,3S)-3-[(1Z)-2-chloro-3,3,3-trifluoroprop-1-en-1-yl]-2,2-dimethylcyclopropanecarboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-09-25 to 1985-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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

Details on test material:

- Physical state: solid
- Colour: white
- Purity test date: 1995-09-05
- Expiration date of the lot/batch: test material used within the stated expiry date (information supplied by sponsor)
- Stability under test conditions: stability and achieved concentrations in the vehicles used were not determined by analysis
- Storage condition of test material: at ambient temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human, one male and one female donor

Details on mammalian cell type (if applicable):

- Blood donors: donor 1 (male), donor 2 (female), both with previously established low incidence of chromosomal aberration in their lymphocytes
- Source of lymphocytes: peripheral human blood, obtained by venipuncture on the day of culture, 0.5 mL per culture (10 mL)
- Mean cell cycle time of lymphocytes: 13.5 hours, determined in test laboratory 1992-09 and 1993-11
- Type and identity of media: RPMI-1640 (Dutch Modification) culture medium, supplemented with 10% fetal bovine serum, 1.0 IU/mL heparin, 100 IU/mL penicillin and 100 microgram/mL streptomycin; stimulated with mitogen: phytohaemagglutinin M Form (PHA)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/naphthoflavone-induced Sprague-Dawley rat liver S9 fraction, 25% w/v homogenate; co-factor stock: glucose-6-phosphate (7.5mM) and NADP (6 mM). 200 microliter of a 1:1 mix of S9 and cofactor stock solution added to each culture (10 mL).

Test concentrations with justification for top dose:

Mitotic index determination without S9 mix, 68 hour sampling time: donor 1: 5000, 1000, 500, 200, 100, 50, 10, 5 microgram/mL, donor 2: 500, 350, 200, 150, 100, 50, 20, 10 microgram/mL
Mitotic index determination with S9 mix, 68 hour sampling time: donor 1 + 2: 1000, 850, 700, 500, 350, 200, 100, 50, microgram/mL
Mitotic index determination without S9 mix, 92 hour sampling time: donor 2: 500, 350, 200, 150 microgram/mL
Mitotic index determination with S9 mix, 92 hour sampling time: donor 2: 1000, 850, 700, 500 microgram/mL
Chromosomal aberration determination without S9 mix, 68 hour sampling time: donor 1: 200, 100, 10 microgram/mL, donor 2: 200, 100, 20 microgram/mL
Chromosomal aberration determination with S9 mix, 68 hour sampling time: donor 1 + donor 2: 1000, 500, 100 microgram/mL
Chromosomal aberration determination without S9 mix, 92 hour sampling time: donor 2: 200 microgram/mL
Chromosomal aberration determination with S9 mix, 92 hour sampling time: donor 2: 1000microgram/mL
All concentrations refer to the weight of the test material, not corrected for purity

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (100 microliter/10 mL culture medium)
- Justification for choice of solvent/vehicle: solubility of test substance and positive control substances

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

performed, but no results reported

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 10 microliter/mL, in duplicate per donor and time point

Positive controls:

yes

Remarks:

0.2 microgram/mL, one culture per donor, 68 hours sampling time only

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

performed, but no results reported

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 10 microliter/mL, in duplicate per donor and time point

Positive controls:

yes

Remarks:

50 microgram/mL, one culture per donor, 68 hours sampling time only

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
INCUBATION TEMPERATURE: 37 °C

DURATION
- Preincubation period: 48 hours, then change to fresh medium
- Exposure duration: without S9-mix: 20 h (24 h for 92-h harvesting time), with S9-mix: 3 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h (for 68-h harvesting time), 44 h (for 92-h harvesting time, with fresh medium at 72 hours total culture time)

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.4 microgram/mL), at 66 or 90 hours of culture

HARVESTING:
- centrifugation (400 g, 5 min) at 68 or 92 hours
- Hypotonic treatment: 0.075 M KCl, 10 min at room temperature
- Fixation: methanol/glacial acetic acid (3:1 v/v), three times

STAIN (for cytogenetic assays): 10% solution of Giemsa stain, buffered to pH 6.8; time: 7 minutes
- Slides mounted in DPX

NUMBER OF REPLICATIONS:
- Two independent tests with lymphocytes from different donors (one male, one female)
- Two cultures per donor, concentration, and harvesting time, in the presence and absence of metabolic activation
- Four slides per culture

NUMBER OF CELLS EVALUATED:
- For mitotic index: 1000 lymphocytes per culture
- For aberration counting: 100 cells in metaphase per culture, i.e. 200 per donor, concentration, and harvesting time

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Chromosomal damage criteria according to: Scott D et al, Metaphase Chromosome Aberration Assays in vitro, in: Kirkland DJ (ed.): Basic Mutagenicity Tests: UKEMS Recommended Procedures; Cambridge University Press, Cambridge 1990
- Determination of polyploidy and endoreplication: Not explicitly reported, probably performed due to guideline requirements
- Determination of osmolality of the culture medium as a function of test substance concentration
- Determination of pH of the culture medium as a function of test substance concentration

Evaluation criteria:

Positive response:
- statistically (p<0.01) and biologically significant increase in the percentage of aberrant cells, compared with the solvent controls

Statistics:

- Calculation of mean percent mitotic index
- Calculation of mean percent aberrant cells (excluding gaps)
- Fisher's exact test (one-sided) to determine statistically significant increases

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: human, one male and one female donor

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: human, one male and one female donor

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

at 68 h sampling time (donors 1+2) and 92 h sampling time (only female donor tested)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The acidic test substance reduced the pH of the culture medium over the concentration range tested, but at the concentrations selected for chromosomal aberration (CA) analysis, these pH reductions were not significant (7.79 in the solvent control vs 7.45 at the highest dose).
- Effects of osmolality: Addition of the test material to the culture medium (up to 5000 microgram/mL) had no significant effect on osmolality (control 447 mmol/kg, highest dose 427 mmol/kg)
- Evaporation from medium: test material is non-volatile
- Water solubility and precipitation: No precipitation observed up to 5000 microgram/mL
- Other confounding effects: none observed

RANGE-FINDING/SCREENING STUDIES: none

COMPARISON WITH HISTORICAL CONTROL DATA: not reported

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Significant reduction of mean mitotic index at 200 microgram/mL (60% in male donor, 46% in female donor), which is the highest concentration used for CA analysis in the absence of metabolic activation (- S9-mix)
- Significant reduction of mean mitotic index at 1000 microgram/mL (58% in male donor, 56% in female donor), which is the highest concentration used for CA analysis in the presence of metabolic activation (+ S9-mix)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: at 68 h sampling time (donors 1+2) and 92 h sampling time (only female donor tested)

Any other information on results incl. tables

Table 1: Chromosomal Aberrations and Mitotic Index (Mean Percentage of Total Cells Analysed)

Metabolic activation	Sampling time (h)	Donor	Treatment		Mean % Aberrant cells §	Aberrations / cell §	Mean % Mitotic index
- S9	68	1	DMSO	10 microliter/mL	1.00	0.010	9.8
			Mitomycin C #	0.2 microgram/mL	29.00**	0.340	5.3
			Test material	200 microgram/mL	1.00	0.010	3.9
				100 microgram/mL	2.50	0.025	7.9
				10 microgram/mL	1.50	0.015	9.2
- S9	68	2	DMSO	10 microliter/mL	1.50	0.015	11.5
			Mitomycin C #	0.2 microgram/mL	23.00**	0.280	6.7
			Test material	200 microgram/mL	3.50	0.035	6.2
				100 microgram/mL	4.50	0.045	8.5
				10 microgram/mL	4.00	0.045	9.8
- S9	92	2	DMSO	10 microliter/mL	2.00	0.025	13.2
			Test material	200 microgram/mL	2.50	0.030	9.3
+ S9	68	1	DMSO	10 microliter/mL	0.00	0.000	14.4
			Cyclophosphamide #	50 microgram/mL	26.00**	0.340	5.9
			Test material	1000 microgram/mL	1.50	0.015	6.0
				500 microgram/mL	1.50	0.015	10.0
				100 microgram/mL	1.00	0.015	10.9
+ S9	68	2	DMSO	10 microliter/mL	4.50	0.045	13.8
			Cyclophosphamide #	50 microgram/mL	29.00**	0.400	6.0
			Test material	1000 microgram/mL	3.50	0.045	6.1
				500 microgram/mL	4.00	0.050	8.3
				100 microgram/mL	1.50	0.015	9.9
+ S9	92	2	DMSO	10 microliter/mL	2.50	0.030	15.1
			Test material	1000 microgram/mL	2.00	0.020	9.8

§ Excluding gaps

# positive control values determined from a single culture

** Statistically significant increase in chromosomal damage, p<0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was found to be non-clastogenic to cultured human lymphocytes in vitro, in the presence or absence of metabolic activation (S9-mix) under the conditions of the assay. The study report is relevant, reliable and adequate for risk assessment, classification and labeling.

Executive summary:

The clastogenic potential of the test substance was assessed according to OECD 473 (chromosome aberration, CA), in human lymphocytes from two donors (male and female), treated in vitro with a range of concentrations of the test material, in the presence and absence of a rat liver-derived metabolic activation system (S9-mix). Cultures were harvested at the standard time of 68 hours after culture initiation, and additional cultures from the female donor were harvested at 92 hours.

Test material concentrations ranged from 5 to 5000 microgramg/mL (with and without S9-mix), to determine mitotic activity, which showed a dose-related reduction under all conditions tested. CA was assessed at three test material concentrations; the highest concentration was chosen to show a significant reduction of the mitotic index: For the 68-hours sampling time, in the absence of S9-mix, cultures treated with 10 or 20 (for male and female donor, respectively), 100, and 200 microgram/mL were selected, whereas in the presence of S9-mix, 100, 500, and 1000 microgram/mL were applied. For the 92-hour sampling (female donor only), 200 and 1000 microgram/mL (without and with S9 -mix) were analyzed. The sensitivity of the test system and the activity of the S9-mix were tested with positive control agents, mitomycin C (- S9 -mix) and cyclophosphamide (+ S9 -mix). Influences of the test material on the pH and osmolality of the culture medium were measured and found to be insignificant.

Both control agents induced highly significant positive results and thus established the validity of the test system. No statistically or biologically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded in the two independent experiments (with lymphocytes from either donor), at any concentration or time point, neither in the presence nor in the absence of S9-mix. It is therefore concluded that, under the conditions of this assay, the test material is not clastogenic to cultured human lymphocytes in vitro.