reaction mass of (3R,5R)-3-chloro-5-(trichloromethyl)cyclopentene and (3S,5S)-3-chloro-5-(trichloromethyl)cyclopentene and (3R,4R)-3-chloro-4-(trichloromethyl)cyclopentene and (3S,4S)-3-chloro-4-(trichloromethyl)cyclopentene

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:

2011-04-18 to 2011-05-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive and done to a valid guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of (3R,5R)-3-chloro-5-(trichloromethyl)cyclopentene and (3S,5S)-3-chloro-5-(trichloromethyl)cyclopentene and (3R,4R)-3-chloro-4-(trichloromethyl)cyclopentene and (3S,4S)-3-chloro-4-(trichloromethyl)cyclopentene

EC Number:

939-946-2

Molecular formula:

C6H6Cl4

IUPAC Name:

reaction mass of (3R,5R)-3-chloro-5-(trichloromethyl)cyclopentene and (3S,5S)-3-chloro-5-(trichloromethyl)cyclopentene and (3R,4R)-3-chloro-4-(trichloromethyl)cyclopentene and (3S,4S)-3-chloro-4-(trichloromethyl)cyclopentene

Test material form:

not specified

Specific details on test material used for the study:

EC Number: 939-946-2

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood samples were obtained from a healthy donor not receiving medication. For this study, blood was collected from a female donor (37 years old).
Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to Harlan CCR to initiate cell cultures within 24 h after blood collection. If necessary, the blood was stored before use at 4 °C.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

between 15.4 and 2370 microgram/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 22 h after start of the exposure.
Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks. The culture medium was DMEM:F12 (Dulbecco's modified eagle medium / Ham's F12 medium; mixture 1:1; already supplemented with 15 mM HEPES and 200 mM L-glutamine. The antibiotic solution contained 10,000 U/mL penicillin and 10,000 Ng/mL streptomycin. Additionally, the medium was supplemented with the mitogen Phytohemagglutinin (PHA, final concentration 3 Ng/mL, 10 % FBS (fetal bovine serum), the anticoagulant heparin.
About 72 h after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 NL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 h the cells were spun down by gentle centrifugation for 5 minutes (approx. 900 x g). The supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once as described.
The "saline G" solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400 mg
glucose•H2O 1100 mg
Na2HPO4•2H20 192 mg
KH2PO4 150 mg
pH was adjusted to 7.2
After washing the cells were re-suspended in complete culture medium and cultured until preparation. All cultures were incubated at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

Evaluation criteria:

The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for three values in the presence of S9 mix, where 50 metaphases were scored. Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect, the mitotic index (% cells in mitosis) was determined.
A test item is classified as non-mutagenic if:
the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data
no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data
and either a concentration-related or a significant increase of the number of structuralchromosome aberrations is observed.
However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05)

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

induced structural chromosomal aberrations in human lymphocytes in vitro

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

51.2% at highest evaluated concentration in absence of S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures

Any other information on results incl. tables

TABLE 1     Doses applied in the Chromosome Aberration Assay with Test Item

Preparation interval	Exposure period	Concentrations in µg/mL
Without S9 mix
22 hrs	4 hrs	15.4	26.9	47.2	82.5	144.4	252.7^P	442.9^P	773.9^P	1354.3^P	2370.0^P
With S9 mix
22 hrs	4 hrs	15.4	26.9	47.2	82.5	144.4	252.7^P	442.9^P	773.9^P	1354.3^P	2370.0^P

        Evaluated experimental points are shown in bold characters
^P       Precipitation was observed at the end of treatment

 

TABLE 2     Summary of Results

Summary of results of the chromosomal aberration study with test item

Preparation	Test item	Mitotic indices	Aberrant cells
interval	concentration	in %	in %

in µg/mL

of control

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix
22 hrs	Solvent control^1	100.0	2.5	2.0	0.0
	Positive control^2	62.3	8.5	8.0^S	0.5
	144.4	78.8	2.5	2.5	0.5
	252.7^P	72.4	3.5	2.5	0.0
	1354.3^P	51.2	10.0	10.0^S	2.0
Exposure period 4 hrs with S9 mix
22 hrs	Solvent control^1	100.0	1.0	1.0	0.0
	Positive control^3	65.2	12.5	12.5^S	2.0
	15.4	72.8	11.5	11.0^S	0.5
	26.9^#	89.8	45.0	44.0^S	0.0
	47.2	82.2	16.0	16.0^S	2.0
	82.5^#	102.9	52.0	52.0^S	5.0
	144.4^#	101.6	61.0	61.0^S	4.0

*      Including cells carrying exchanges

^#       Evaluation of 50 metaphases per culture

^P       Precipitation occurred at the end of treatment

^S       Aberration frequency statistically significant higher than corresponding control values

¹       DMSO       0.5 % (v/v)

²       EMS      825.0 µg/mL

³       CPA           7.5 µg/mL

TABLE 3      Toxicity – Main Experiment (Cytotoxicity of Test Item to the Cultures of Human Lymphocytes)

In the Main Experiment the mitotic index in two cultures (1000 cells per culture) was determined.

Concentration (µg/mL)	Exposure time	Preparation interval	Mitotic cells per 1000 cells^*	% of solvent control
Without S9 mix
Solvent control	4 hrs	22 hrs	18.9	100.0
15.4	4 hrs	22 hrs	19.7	104.2
26.9	4 hrs	22 hrs	18.4	97.3
47.2	4 hrs	22 hrs	17.7	93.9
82.5	4 hrs	22 hrs	16.5	87.3
144.4	4 hrs	22 hrs	14.9	78.8
252.7^P	4 hrs	22 hrs	13.7	72.4
442.9^P	4 hrs	22 hrs	12.9	68.4
773.9^P	4 hrs	22 hrs	9.3	49.3
1354.3^P	4 hrs	22 hrs	9.7	51.2
2370.0^P	4 hrs	22 hrs	7.7	40.6
With S9 mix
Solvent control	4 hrs	22 hrs	19.1	100.0
15.4	4 hrs	22 hrs	13.9	72.8
26.9	4 hrs	22 hrs	17.2	89.8
47.2	4 hrs	22 hrs	15.7	82.2
82.5	4 hrs	22 hrs	19.7	102.9
144.4	4 hrs	22 hrs	19.4	101.6
252.7^P	4 hrs	22 hrs	11.5	60.2
442.9^P	4 hrs	22 hrs	13.9	72.8
773.9^P	4 hrs	22 hrs	0.0	0.0
1354.3^P	4 hrs	22 hrs	4.9	25.7
2370.0^P	4 hrs	22 hrs	0.0	0.0

Experimental groups evaluated for cytogenetic damage are shown in bold characters
^*    Mean value of two cultures in %
^P    Precipitation occurred at the end of treatment

TABLE 4               Main Experiment-Mitotic Index;
                              (Preparation Interval 22 hrs with and without S9 Mix)

Treatment	Conc.	S9	Exposure	Polyploid	Mitotic Indices*
group	per mL	mix	period/	cells	Absolute		Mean		%**
			Recovery (hrs)		1	2
Solv. control^#	0.5  %	-	4 / 18 hrs	No polyploidies observed	18.6	19.1	18.9	100.0
Pos. control#^#	825.0       µg	-	4 / 18 hrs	"	11.5	12.0	11.8	62.3
Test item	144.4       µg	-	4 / 18 hrs	"	14.8	14.9	14.9	78.8
"	252.7       µg	-	4 / 18 hrs	"	14.7	12.6	13.7	72.4
"	1354.3       µg	-	4 / 18 hrs	"	11.1	8.2	9.7	51.2
Solv. control^#	0.5  %	+	4 / 18 hrs	No polyploidies observed	16.8	21.4	19.1	100.0
Pos. control^###	7.5       µg	+	4 / 18 hrs	"	10.8	14.1	12.5	65.2
Test item	15.4       µg	+	4 / 18 hrs	"	11.6	16.2	13.9	72.8
"	26.9       µg	+	4 / 18 hrs	"	14.1	20.2	17.2	89.8
"	47.2       µg	+	4 / 18 hrs	"	15.5	15.9	15.7	82.2
"	82.5       µg	+	4 / 18 hrs	"	19.7	19.6	19.7	102.9
"	144.4       µg	+	4 / 18 hrs	"	20.8	18.0	19.4	101.6

^*       The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

^**      For the positive control groups and the test item groups, the relative values of the mitotic index are related to the solvent controls

^#           DMSO

^##         EMS

^###       CPA

TABLE 5       Structural Chromosome Aberrations Main Experiment;
                       (Preparation Interval 22 hrs without S9 Mix:
                       Exposure Period 4 hrs)

Slide	Cells		% Aberrant cells							Aberrations
no.	scored		incl.			excl.		carrying ex-changes		Gaps				Chromatid type								Chromosome type								Other
			gaps*			gaps*				g		ig		b		f		d		ex		ib		if		id		cx		ma		cd
Solvent control: DMSO 0.5 %										Without S9 mix
1		100							0		0		2		0		0		0		0		0		0		0		0		0
2		100							1		0		2		0		0		0		0		0		0		0		0		0
1 + 2		200		2.5	2.0		0.0		1		0		4		0		0		0		0		0		0		0		0		0
Positive control: EMS 825.0 µg / mL
1		100							0		0		6		1		0		0		0		1		0		0		0		0
2		100							1		0		7		2		0		1		0		0		0		0		0		0
1 + 2		200		8.5	8.0		0.5		1		0		13		3		0		1		0		1		0		0		0		0
Test item: 144.4 µg / mL
1		100							0		0		2		0		0		0		0		0		0		0		0		0
2		100							0		0		1		0		0		1		0		1		0		0		0		0
1 + 2		200		2.5	2.5		0.5		0		0		3		0		0		1		0		1		0		0		0		0
Test item: 252.7 µg / mL
1		100							1		0		3		0		0		0		0		0		0		0		0		0
2		100							1		0		2		0		0		0		0		0		0		0		0		0
1 + 2		200		3.5	2.5		0.0		2		0		5		0		0		0		0		0		0		0		0		0
Test item: 1354.3 µg / mL
1		100							0		0		10		0		0		3		0		0		0		0		1		0
2		100							0		0		7		0		0		2		0		2		0		0		0		0
1 + 2		200		10.0	10.0		2.0		0		0		17		0		0		5		0		2		0		0		1		0

*      Including cells carrying exchanges

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

TABLE 6     Structural Chromosome Aberrations Main Experiment;
                       (Preparation Interval 22 hrs with S9 Mix: Exposure Period 4 hrs)

Slide	Cells		% Aberrant cells							Aberrations
no.	scored		incl.			excl.		carrying ex-changes		Gaps				Chromatid type								Chromosome type								Other
			gaps*			gaps*				g		ig		b		f		d		ex		ib		if		id		cx		ma		cd
Solvent control: DMSO 0.5 %										Without S9 mix
1		100							0		0		0		0		0		0		0		0		0		0		0		0
2		100							0		0		2		0		0		0		0		0		0		0		0		0
1 + 2		200		1.0	1.0		0.0		0		0		2		0		0		0		0		0		0		0		0		0
Positive control: CPA 7.5 µg / mL
1		100							0		0		12		0		0		1		1		0		0		0		0		0
2		100							0		0		15		0		0		3		1		0		0		0		0		0
1 + 2		200		12.5	12.5		2.0		0		0		27		0		0		4		2		0		0		0		0		0
Test item: 15.4 µg / mL
1		100							1		0		11		0		0		0		1		0		0		0		0		0
2		100							0		0		9		0		0		1		0		2		0		0		1		0
1 + 2		200		11.5	11.0		0.5		1		0		20		0		0		1		1		2		0		0		1		0
Test item: 26.9 µg / mL**
1		50							1		0		21		1		0		0		0		0		0		0		0		0
2		50							0		0		38		4		0		0		2		0		0		0		7		0
1 + 2		100		45.0	44.0		0.0		1		0		59		5		0		0		2		0		0		0		7		0
Test item: 47.2 µg / mL
1		100							1		0		14		1		0		1		0		1		0		0		0		0
2		100							2		0		13		0		0		3		0		0		0		0		0		0
1 + 2		200		16.0	16.0		2.0		3		0		27		1		0		4		0		1		0		0		0		0
Test item: 82.5 µg / mL**
1		50							2		0		41		0		0		3		0		0		0		0		4		0
2		50							2		0		31		0		0		3		1		0		0		0		3		0
1 + 2		100		52.0	52.0		5.0		4		0		72		0		0		6		1		0		0		0		7		0
Test item: 144.4 µg / mL**
1		50							0		0		53		3		0		2		2		2		0		0		10		0
2		50							0		0		30		1		0		2		0		0		0		0		6		0
1 + 2		100		61.0	61.0		4.0		0		0		83		4		0		4		2		2		0		0		16		0

*      Including cells carrying exchanges

**    Evaluation of 50 metaphases per culture

Abbreviations

g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso-fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

TABLE 7     Biometry

Statistical significance at the five per cent level (p < 0.05) for aberration frequency was evaluated by means of the Fisher’s exact test. Evaluation was performed only for cells carrying aberrations excluding gaps.

Biometry of the Main Experiment

Test group versus solvent control		Preparation interval	Exposure period	S9 mix	p-value
Test group	144.4µg/mL	22 hrs	4 hrs	-	0.376
"	252.7µg/mL	22 hrs	4 hrs	-	0.376
"	1354.3µg/mL	22 hrs	4 hrs	-	0.003^S
"	15.4µg/mL	22 hrs	4 hrs	+	< 0.001^S
"	26.9 µg/mL	22 hrs	4 hrs	+	< 0.001^S
"	47.2 µg/mL	22 hrs	4 hrs	+	< 0.001^S
"	82.5 µg/mL	22 hrs	4 hrs	+	< 0.001^S
"	144.4 µg/mL	22 hrs	4 hrs	+	< 0.001^S
Positive control versus solvent control
EMS	825.0 µg/mL	22 hrs	4 hrs	-	0.003^S
CPA	7.5 µg/mL	22 hrs	4 hrs	+	< 0.001^S

n.c.  Not calculated as the aberration rate is equal or lower than the control rate
^S       Aberration rate is statistically significant higher than the control rate

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation, when tested up to cytotoxic and/or precipitating or the highest evaluable concentration.
The study is considered to be relevant, reliable, adequate for risk assessment, and adequate for classification purposes.

Executive summary:

Study Design:

This in vitro assay was performed to assess the potential of the test item to induce structural chromosomal aberrations in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for three values in the presence of S9 mix, where 50 metaphases were scored. The highest applied concentration in the main study (2370.0 Ng/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (92.9 %) of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and test item precipitation and in accordance with OECD Guideline 473.

Results:

In the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration.

In the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.

Clastogenicity was observed in the absence of S9 mix after treatment with 1354.3 Ng/mL (10.0 % aberrant cells, excluding gaps) and in the presence of S9 mix after treatment with all evaluated concentrations. All values for percent aberrant cells (excluding gaps) are statistically significant and above the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Conclusion:

Under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic and/or precipitating or the highest evaluable concentration