Reaction mass of 1-chlorobut-2-ene and 3-chlorobut-1-ene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Crotyl chloride showed positive results in S. thyphimurium strains TA 100 and TA 1535 with and without metabolic activation. Furthermore, the substance provided negative results in an in vitro gene mutation study in V79 cells (tested up to 250 µg/mL) with and without metabolic activation and in an in vitro chromosome aberration test in human lymphocytes with and without metabolic activation up to cytotoxic concentrations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability:no data
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Arochlor 1254- induced rat liver

Test concentrations with justification for top dose:

7.81, 15.63, 31.25, 62.5, 125 µg/mL (first experiment)
15.63, 31.25, 62.5, 125, 250 µg/mL (second experiment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: appropraite sovent for the test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethyl methanesulfonate without S9 mix and 9,10-Dimethyl-1,2-benzanthracene with S9-mix, respectively

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24h
- Exposure duration: 24h without S9-mix, 4 hours with S9-mix
- Expression time (cells in growth medium): 8 days
- Selection time (if incubation with a selection agent): 8 days (plating efficiency), 12 days (6-Thioguanine)



SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL, 5 replicate plates)



NUMBER OF REPLICATIONS: 3 for Expression time, 5 for selection time


NUMBER OF CELLS EVALUATED: n.a.


DETERMINATION OF CYTOTOXICITY
cloning efficiency

Evaluation criteria:

Test item is negative in the test system, if
- in both independent experiments solvent and positive controls show results within the norm
- test item does not increase the mutation frequency 2fold above the mean of the solvent controls under any condition
- the mutation frequency is always lower than 20 x 10^-6 cells and
- at least 1 x 10^6 cells per conditions have been evaluated

test item is regarded to be positive in the test in case of
- a dose dependent increase of the mutation frequency in both independent exeriments (similar concentrations) to at least 2-fold solvent control
- at least 20x10^-6 both in presence/absence of S9 mix

Solvent control: historical background mutation frequency in the system is reported to be 1-44 mutants per 10^6 survivors (without metabolic activation) and 6-46 mutants per 10^6 survivors (with metabolic activation)
Positive controls: 10fold greater mutation frequency than the solvent control
Mean mutation frequency of the control background data: 14.5 x 10^-6 cells, 20 x 10^-6 cells is taken as cut off

Statistics:

no satisfactory mathematical methods are available

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

125 and 250 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conc (µg/mL)	First experiment - S9		Second experiment –S9		1^stexperiment +S9		2^ndexperiment + S9
	PE	MF	PE	MF	PE	MF	PE	MF
Control (DMSO)	0.95	2.53	0.86	3.37	0.81	1.45	0.92	9.47
7.81	0.92	2.37	--	--	0.83	1.71	--	--
15.63	0.92	2.39	0.8	3.41	0.74	4.06	0.81	3.64
31.25	0.82	7.22	0.77	7.67	0.69	7.74	0.79	5.39
62.5	0.69	17.61	0.72	8.51	0.57	4.07	0.79	6.02
125	0.65	10.49	0.50	17.6	0.41	4.55	0.57	12.38
250	--	--	0.075	16.92	--	--	0.30	17.67
EMS600	0.5	126.67	0.33	607.45	--	--	--	--
EMS700	0.25	208.39	0.37	482.91	--	--	--	--
DMBA 20	--	--	--	--	0.24	229.47	0.23	869.41
DMBA 30	--	--	--	--	0.22	129.66	0.19	620.00

Conclusions:

negative

Under tested conditions, test substance showed no mutagenic effects in V79 cells up to cytotoxic concentrations. All mutations frequencies were below the cut-off value of 20x10^-6. Dose response was not observable in all experiments.

Reference 2

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human blood

Details on mammalian cell type (if applicable):

- Type and identity of media: complete Medium:Chromosome Medium 1A with Phytohemagglutinin (Gibco) and Penicillin/streptomycin (10000 IU/mL); Treatment medium: Ham´s F-10 with 10% FCS
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Arochlor 1254- induced rat liver

Test concentrations with justification for top dose:

15.63, 31.25, 62.5, 125, 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: appropraite sovent for the test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C without S9 mix and cyclophosphamide with S9-mix, respectively

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24h
- Exposure duration: 24h without S9-mix, 4 hours with S9-mix
- Expression time (cells in growth medium): 8 days
- Selection time (if incubation with a selection agent): 8 days (plating efficiency), 12 days (6-Thioguanine)
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL, 5 replicate plates)


NUMBER OF REPLICATIONS: 3 for Expression time, 5 for selection time


NUMBER OF CELLS EVALUATED: n.a.


DETERMINATION OF CYTOTOXICITY
cloning efficiency



METHOD OF APPLICATION: in medium


DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Evaluation criteria:

Test item is positive in the test system, if
-the number of chromosomal aberrations is statistial significantly increased (p< 0.05) compared to solvent control
- increase observed is concentration-dependent
-both duplicate cultures lead to similar results
- increase should not occur in the severely cytotoxic range (mitotic index < 0.25)
- a reproducable increase in the number of cells with chromosomal aberrations

Statistics:

Comparison of the number of chromosomal aberrations of the samples with dose of the solvent control, using the exact test of R.A. Fischer (p <= 0.05)

Species / strain:

lymphocytes: human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

62.5 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Pronounced cytotoxicity was noted at the top concentration of 125 µg/mL in the experiment without metabolic activation for an exposure time of 4 hours or at concentrations of 62.5 and 125 µg/mL medium for an exposure time of 24 hours, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conc (µg/mL)	First experiment 4h exposure - S9		Second experiment 24h–exposure - S9		1^stexperiment 4h-exposure +S9		2^ndexperiment 4h-exposure + S9
	Mitotic index	% cells with aberrations excl. gaps	Mitotic index	% cells with aberrations excl. gaps	Mitotic index	% cells with aberrations excl. gaps	Mitotic index	% cells with aberrations excl. gaps
Control (DMSO)	1.0	0.0	1.0	2.0	1.0	0.5	1.0	0.5
15.63	1.1	0.0	0.95	2.0	--	--	0.9	1.5
31.25	0.97	0.0	0.69	3.0	0.84	0.5	0.94	2.5
62.5	0.42	1.5	0.25	13.0 s.	1.04	0.0	0.79	2.0
125	0.49	0.7#	0.13	9.1 #	0.58	0.0	0.29	13.5 s.
250	--	--	--	17.6	0.04	0.0#	--	--
EMS600	1.13	10.0 s.	0.1	11.5 s.	--	--	--	--
Cyclophosphamide 20	--	--	--	--	0.4	8.5s.	0.48	11.0 s.

#: reduced number of metaphases of sufficient quality for evaluation due to cytotoxicity

s. significantly different from solvent control (p< 0.05)

Conclusions:

negative

Under tested conditions, test substance showed no significant mutagenic effects in a chromosomal aberration test up to cytotoxic concentrations. In cytotox concentrations (Mitotic index below 0.3%) significant increase in chromosomal aberrations were observed but are considered to be due to artefacts in the test system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Phenobarbital (i.p.) and beta-Naphthoflavone (oral) induced rat liver

Test concentrations with justification for top dose:

TA98: 50, 160, 500, 750, 1000 µg/plate
TA100, TA102, TA1535, TA1537: 50, 160, 500, 1600, 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98

Migrated to IUCLID6: 2.5 µg/plate

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA 1535

Migrated to IUCLID6: 5 µg/plate (TA100), 2.5 µg/plate TA1535

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA102

Migrated to IUCLID6: 2.5 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537

Migrated to IUCLID6: 40 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 100 µg/plate 2-Aminofluorene and 2.5 µg/plate 2-Aminoanthracene

Remarks:

all tester strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)


DURATION
- Exposure duration: 72h
-Titers of overnight cultures: > 10^8
- Selection time (if incubation with a selection agent): 72h



SELECTION AGENT (mutation assays): histidine deficient agar


NUMBER OF REPLICATIONS:triplicates, two assays


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth with strains TA98 and TA100

Evaluation criteria:

- negative control in the laboratory historical range for each tester strain
- positive control chemicals produces responses in all tester strains within laboratory historical range
- Mean plate count should be at least two times the concurrent vehicle control group mean in both assays and in a dose dependent response
- selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate
- To ensure that appropriate numbers of bacteria are plated, overnight culture titers must be in excess of 10^8 bacteria/ml

Statistics:

not applicable

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: some values were insignificantly above historical control data, but they are regarded as acceptable


ADDITIONAL INFORMATION ON CYTOTOXICITY: In the pretest with the TA98 tester strain, toxicity and/or reduced background lawn was observed at 1600 and 5000 µg/plate

Remarks on result:

other: strain/cell type: TA 1535

Remarks:

Migrated from field 'Test system'.

Assay 1:

Conc (µg/plate)	TA1535		TA1537		TA 100		TA102
	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Control (water)	0.9	1.1	1.2	0.9	1.0	1.0	1.0	1.0
50	1.6	2.4	0.7	0.8	0.9	1.0	1.2	1.1
160	2.0	2.7	0.8	0.9	1.0	1.1	1.2	1.2
500	2.8	3.1	0.8	0.7	1.1	1.4	1.3	1.5
1600	3.9	6.3	0.8	0.7	1.6	1.6	1.4	1.9
5000	2.0#	5.0#	0.5*	0.8	1.6#	1.3#	0.7#	1.1#
Positive control	30.7		7.2		3.9		4.1
2-Aminofluorene		2.2		3.3		9.2		2.2
2- Aminoanthracene		7.2		5.6		7.5		1.5

 

Assay 2

Conc (µg/plate)	TA1535		TA1537		TA 100		TA102
	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Control (water)	0.8	1.5	1.2	1.1	1.0	1.1	1.1	1.0
25	1.2	1.5	1.1	1.0	1.0	1.1	1.0	1.1
160	1.4	2.2	1.1	1.0	1.2	1.0	1.0	1.4
250	1.6	2.3	0.9	1.0	1.4	1.2	1.0	1.4
1600	2.9	5.2	0.9	0.9	2.6	1.7	1.2	1.6
2500	4.5	6.4	0.6#*	0.8	2.9	2.2	0.2	1.4
Positive control	32.6		6.3		5.5		3.4
2-Aminofluorene		3.5		3.2		10.9		2.6
2- Aminoanthracene		10.6		4.8		7.7		1.8

 

TA98

Conc (µg/plate)	Assay 1		Assay2
	- S9	+ S9	Control (water)	+ S9	- S9
Control (water)	1.4	1.2	50	1.2	0.9
50	1.0	0.9	160	1.1	0.9
160	1.0	1.1	250	1.0	0.8
500	1.3	1.2	500	1.0	0.8
750	1.2	1.1	750	0.7#	0.7
1000	1.1	0.8	2-nitrofluorene	0.9#	0.9
2-nitrofluorene	5.8		2-Aminofluorene	4.5
2-Aminofluorene		56.6	2- Aminoanthracene		41.0
2- Aminoanthracene		20.8	Control (water)		21.7

 

#: reduced background lawn

*: no background lawn, toxicity

Conclusions:

positive

Test substance induced a mutagenic effect in S. typhimurium TA 100 and TA1535 with and without metabolic activation. It is therefore considered to be a bacterial mutagen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Crotyl chloride did neither induce micronuclei in rats in an in vivo MNT nor was it considered to be genotoxic in an in vivo comet assay in rats.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation:
pre-experiment: 10 - 12 weeks [(beginning of treatment); 2 males and 2 females for each pre-test; 6 males and 6 females in total]
main experiment: 8 - 9 weeks [(beginning of treatment); 42 males]
- Housing: group of 7 [main experiment]
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

PEG 400: Polyethylenglycol

Details on exposure:

EXPERIMENTAL DESIGN:
Phase I: 1st pre-experiment (200 mg/kg b.w.); 2nd pre-experiment (100 mg/kg b.w.); 3rd pre-experiment (50 mg/kg b.w.)
Phase II: main micronucleus test [dosing of test group (12.5, 25 and 50 mg/kg b.w.), vehicle control group and positive control group (20 mg/kg b.w. )]

PREPARATION OF DOSING SOLUTIONS:
A stock solution of the test material was prepared in the vehicle. All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. Dosing volume was 10 mL/kg bw.

Duration of treatment / exposure:

44 h (dosing at intervals of 48 h, 24 h and 4 h prior to sacrifice)

Frequency of treatment:

3 times (48 h, 24 h and 4 h prior to sacrifice)

Remarks:

Doses / Concentrations:
50 mg/kg b.w.
Basis:
nominal conc.
test material in vehicle

Remarks:

Doses / Concentrations:
25 mg/kg b.w
Basis:
nominal conc.
test material in vehicle

Remarks:

Doses / Concentrations:
12.5 mg/kg b.w
Basis:
nominal conc.
test material in vehicle

Remarks:

Doses / Concentrations:
20 mg/kg b.w
Basis:
nominal conc.
positive control cyclophosphamide in vehicle

Remarks:

Doses / Concentrations:
0 mg/kg b.w
Basis:
nominal conc.
vehicle control

No. of animals per sex per dose:

Test group: 21
Vehicle control group: 7
Positive control group: 7
(All groups consisted of males only)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA): purity: 97%, dissolved in sterile water

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) in the bone marrow of the rat are the cell population of choice for mammalian cells in vivo. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Three preliminary studies on acute toxicity were performed with two animals per sex and test group under identical conditions (animal strain; vehicle; route, frequency, and volume of administration) as in the mutagenicity study. For genotoxicity investigations it is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly.
In the pre-experiment the animals were treated three times orally with the test item. The second and third treatment intervals were 24 h and 44 h after the first treatment, respectively. The treated animals were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h and 24 h after the first administration, 0-1 h, 2-4 h and 20 h after the second administration and at intervals of around 1 h, 2 h and 4 h after the third administration of the test item.
Four hours after the third administration of the test item, the animals were anaesthetized using CO2 and sacrificed using a guillotine device, followed by bleeding.
After sacrifice of the treated animals a piece of the stomach and liver was prepared and checked for apoptotic or necrotic cells and general evaluability of the prepared slides.
In this study the doses 12.5, 25 and 50 mg/kg b.w. were applied based upon the pre-experimental results.
No sex specific differences were observed. Therefore, the main experiment was performed using male animals only.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of each treatment the animals were weighed and the individual volume to be administered was adjusted to the body weight of the animals. The animals received the test item or the vehicle control three times orally at 48, 24 and 4 hours prior to sacrifice. The positive control substance cyclophosphamide (CPA) was administered once orally at 24 h prior to sacrifice. Seven males were treated per dose group. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h and 24 h after the first administration, 0-1 h, 2-4 h and 20 h after the second administration and 0-1 h, 2 h and 4 h after the third administration of the test item or vehicle. Assessments of induced toxic symptoms are not performed for the positive control group, since it is known that CPA at the indicated doses does not induce toxicity. Sampling of the target tissues was done 48 hours after the first treatment.

DETAILS OF SLIDE PREPARATION:
For bone marrow isolation tibias/femurs were dissected free of surrounding tissue, the epiphyses was cut off and the marrow flushed out with FBS, using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna (Romagna and Staniforth, 1989). Briefly, the cell suspensions were passed through a column consisting of α-Cellulose and Cellulose. The columns were washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. The pellet was re-suspended in a small drop of FBS and spread on slides. The smears were air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei by manual inspection. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides, blinded to the evaluator.

Evaluation criteria:

A test item was classified as mutagenic if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the tested dose levels.
A test item producing neither a dose-related increase in number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the tested dose levels was considered non-mutagenic in this system.
However, both biological and statistical significance were considered together.

Statistics:

Student’s t-test or Mann Whitney rank sum test if normality test failed and thus Student’s t-test could not be used (Krauth, 1971).

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. After treatment with the test item the number of PCEs in the bone marrow was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.

Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. In comparison to the corresponding vehicle control there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei after administration of the test item and with any dose level used.

20 mg/kg b.w. cyclophosphamide administered once orally was used as positive control which showed a substantial and statistically significant increase of induced micronucleus frequency demonstrating the validity of this part of the study.

Test group	Dose 3 times mg/kg b.w.	Sampling time (h)^***	PCEs with micronuclei (%)	Range^*	PCE per 2000 erythrocytes
Vehicle Control	0	4	0.136	0 -6	1037
Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	12.5	4	0.164	2 -6	1031
Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	25	4	0.143	0 -5	1048
Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	50	4	0.157	1 -7	1049
Positive Control	20^**	24	2.086	10 -90	994

*     Range of absolute numbers of micronucleated PCEs

**    Cyclophosphamide was only applied once

***  Sampling time after the last treatment

Conclusions:

negative
In conclusion, it can be stated that under the experimental conditions reported, the test item, up to the maximum dose level tested, did not induce micronuclei as determined by the micronucleus test with bone marrow cells.
Therefore, the test item is considered to be non-genotoxic in the micronucleus part of this study.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, Section 4, No. 489, adopted September 26, 2014, „In Vivo Mammalian Alkaline Comet Assay“

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Minimum Criteria for the acceptance of in vivo alkaline Comet Assay Reports. European Food Safety Authority, EFSA Journal 2012, 10(11): 2977

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

- Age at study initiation:
pre-experiment: 10 - 12 weeks [(beginning of treatment) 2 males and 2 females for each pre-test; 6 males and 6 females in total]
main experiment: 8 - 9 weeks [(beginning of treatment) 42 males]
- Housing: group of 7 [main experiment]
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

PEG 400: Polyethylenglycol

Details on exposure:

EXPERIMENTAL DESIGN:
Phase I: 1st pre-experiment (200 mg/kg b.w.); 2nd pre-experiment (100 mg/kg b.w.); 3rd pre-experiment (50 mg/kg b.w.)
Phase II: main experiment comet assay [dosing of test groups 12.5, 25 and 50 mg/kg b.w.), vehicle control group and positive control group
(25 mg/kg b.w. )]

PREPARATION OF DOSING SOLUTIONS:
A stock solution of the test material was prepared in the vehicle. All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. Dosing volume was 10 mL/kg bw.

Duration of treatment / exposure:

44 h (dosing at intervals of 48 h, 24 h and 4 h prior to sacrifice)

Frequency of treatment:

3 times (48 h, 24 h and 4 h prior to sacrifice)

Remarks:

Doses / Concentrations:
50 mg/kg b.w.
Basis:
other: test material in vehicle

Remarks:

Doses / Concentrations:
25 mg/kg b.w.
Basis:
other: test material in vehicle

Remarks:

Doses / Concentrations:
12.5 mg/kg b.w.
Basis:
other: test material in vehicle

Remarks:

Doses / Concentrations:
25 mg/kg b.w.
Basis:
other: positive control methylmethansulphonate in vehicle

Remarks:

Doses / Concentrations:
0 mg/kg b.w.
Basis:
other: vehicle control

No. of animals per sex per dose:

Test group: 21
Vehicle control group: 7
Positive control group: 7
(All groups consisted of males only)

Control animals:

yes, concurrent vehicle

Positive control(s):

Methylmethansulphonate (MMS): dissolved in 0.9 % NaCl solution

Tissues and cell types examined:

Liver and the stomach were used as target tissues.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Three preliminary studies on acute toxicity were performed with two animals per sex and test group under identical conditions (animal strain; vehicle; route, frequency, and volume of administration) as in the mutagenicity study. For genotoxicity investigations it is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly.
In the pre-experiment the animals were treated three times orally with the test item. The second and third treatment intervals were 24 h and 44 h after the first treatment, respectively. The treated animals were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h and 24 h after the first administration, 0-1 h, 2-4 h and 20 h after the second administration and at intervals of around 1 h, 2 h and 4 h after the third administration of the test item.
Four hours after the third administration of the test item, the animals were anaesthetized using CO2 and sacrificed using a guillotine device, followed by bleeding.
After sacrifice of the treated animals a piece of the stomach and liver was prepared and checked for apoptotic or necrotic cells and general evaluability of the prepared slides.
In this study the doses 12.5, 25 and 50 mg/kg b.w. were applied based upon the pre-experimental results.
No sex specific differences were observed. Therefore, the main experiment was performed using male animals only.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of each treatment the animals were weighed and the individual volume to be administered was adjusted to the body weight of the animals. The animals received the test item or the vehicle control three times orally at 48, 24 and 4 hours prior to sacrifice. The positive control substance methylmethansulphonate (MMS) was administered once orally at 4 h prior to sacrifice. Seven males were treated per dose group. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h and 24 h after the first administration, 0-1 h, 2-4 h and 20 h after the second administration and 0-1 h, 2 h and 4 h after the third administration of the test item or vehicle. Assessments of induced toxic symptoms are not performed for the positive control group, since it is known that MMS at the indicated doses does not induce toxicity. Sampling of the target tissues was done 48 hours after the first treatment.

DETAILS OF SLIDE PREPARATION:
Isolation of cells from the Liver:
A small piece of the left liver lobe was excised and then minced in ice-cold mincing buffer (20 mM EDTA, 10 % DMSO in HBSS pH 7.4-7.6) using fine scissors to obtain a single cell suspension containing hepatocytes. The resulting cell suspension was filtered through a 70 µmcell strainer. The cell suspension was centrifuged at about 10000 rpm.
Isolation of cells from the Stomach:
The stomach was removed and cut open. The main part of the glandular stomach was washed with a syringe containing ice-cold mincing buffer to remove residual food. The glandular stomach was stretched and fixed with needles on a pre-cooled paraffin filled dish, rinsed two times with ice-cold mincing buffer and subsequently kept on ice. The mucosa was scraped with a cell scraper and discarded. After rinsing the glandular stomach with ice-cold mincing buffer, the glandular stomach was covered with additional ice-cold mincing buffer and was then scraped with a cell scraper. The cells on the cell scraper were discarded. The cells on the surface were removed via a pipette and filtered through a 70 µm cell strainer. In addition, the removed cells on the surface of the glandular stomach were resuspended in ice-cold mincing buffer and filtered through a 70 µm cell strainer. An aliquot of the filtered cell suspension was suspended in prewarmed 0.7% agarose.
Preparation of microscopic slides:
Six slides per tissue and per animal were prepared with 10 % cell suspension and 90 % of a 0.7 % (w/v) agarose (low melting point agarose) solution. 100 µL was applied per slide. The slides were cooled before being submerged in lysis buffer.
The following steps of protocol were performed with the slides:
- Lysis: 1 h up to 7 days in Lysis buffer pH 10 at 2 - 8°C in the dark
- Alkaline treatment: 20 min in electrophoresis buffer, pH >= 13 at 2 - 8°C in the dark
- Electrophoresis: 30 min in electrophoresis buffer 25 V, 300 mA, at 2 - 8°C in the dark
- Neutralisation: about 10 min in neutralisation buffer
- Dehydration: approx. 2 min in 99 % ethanol
After dehydration the slides were air-dried and stored protected from dust and light until evaluation.

METHOD OF ANALYSIS:
The DNA of the cells was stained with the fluorescence dye ethidium bromide (20 µg/mL), immediately before evaluation.
All animals per test group, 150 cells per animal and tissue, 50 cells per slide, were evaluated on coded slides with a fluorescence microscope using a 40 x objective. The damage of each nucleus was measured and recorded by an image analysis programme (Comet Assay IV, Perceptive Instruments). Three slides per animal were kept as a reserve for possible re-evaluation.
An increasing extent of DNA migration detected with the Comet assay results in an increase of the median of tail % intensity of one test group compared to the vehicle control. Tail % intensity is expressed as a percentage of the Comet’s total intensity. Additionally, the number of nuclei from apoptotic or necrotic cells (recognised by their cloud like morphology) per 1500 total nuclei was determined per slide.
The following criteria were used for analysis of slides:
- only clearly defined non-overlapping cells were scored
- nuclei from dead/ apoptotic cells were not scored
- cells with unusual staining artefacts were not scored
- all other normal cells, 150/animal and tissue, were scored where possible.
All slides were scored blind (coded). After evaluation the cover-slips were removed and the slides were washed once for about 2 min in absolute ethanol. The slides were air-dried and stored protected from dust and light.

Evaluation criteria:

A test item was classified as mutagenic if it induced either a dose-related increase or a biologically relevant increase in the tail % intensity in a single dose group as compared to the historical control range. Statistical methods were used as an aid in evaluating the results. However, the primary point of consideration was the biological relevance of the results.

Statistics:

Normally distributed data was analysed using a one-tailed student´s t-test.

Sex:

male

Genotoxicity:

negative

Remarks:

liver

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Liver:
As a cytotoxic parameter the dead cell index (dead cells (apoptotic/necrotic cells) per 1500 nuclei per animal) was determined in the Comet assay and did not show any increased cytotoxic effects in the liver cells in comparison to the vehicle control.
For the analysis of the single DNA strand breaks 150 cells per animal (3 slides prepared for each animal, 50 cells evaluated per slide) were evaluated.
The Comet assay on cells of the liver did not reveal any statistically and biologically relevant increase in DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (percentage of DNA in the Comet tail measured as intensity relative to the intensity of the nucleus, % Tail Intensity). Additionally, a clear dose-dependent increase was not observed. Furthermore, the values were in the range of the historical vehicle control data.

Stomach:
As a parameter for cytotoxicity the dead cell index (dead cells (apoptotic/necrotic cells) per 1500 nuclei per animal) was determined in the Comet assay. Compared to vehicle control values, there was a biologically relevant, dose-dependent cytotoxic effect observed, which was highly significant for the high dose group (p<0.005).
For the analysis of the single DNA strand breaks 150 cells per animal (3 slides prepared for each animal, 50 cells evaluated per slide) were evaluated. The relevant parameter for DNA damage is the percentage of DNA in the Comet tail measured as intensity relative to the intensity of the nucleus (% Tail intensity).
A statistically significant and biologically relevant increase in % tail intensity (p<0.001) was observed after treatment with the high dose group. However no clear dose-dependent increase in % tail intensity was observed. The statistically significant increase seen at the top dose is likely to be due to the corrosive properties of the compound. Various tissue damage typical for corrosive and irritative compounds was confirmed by histopathological examination of the glandular and forestomach. Cellular changes in the forestomach included submucosal edema, granulolymphocytic inflammation, mucosal ulceration, erosion and keratinolysis, as well as mucosal hyperplasia, mainly in the high dose group. The mid and low dose groups were affected to a lesser degree. Lesions observed in the glandular stomach consisted mainly of a mild granulolymphocytic inflammatory infiltrate. However this discrepancy when comparing morphological lesions in forestomach and glandular stomach are not uncommon in the rat.
For both tissues, the vehicle controls were in the range to ensure a valid performance of the study. The reference mutagen [MMS, 25 mg/kg b.w. oral] showed a distinct and statistically significant increase of DNA damage as detected by % Tail intensity analysis.

Analysis of Dead Cell Index and % Tail Intensity in cells of the Liver:

Test Group	Dead cells on slides per 1500 cells per animal		Mean of Median (% Tail Intensity)
	Group Mean	SD	Group Mean	SD
Vehicle Control	11.76	3.78	3.95	2.34
12.5 mg/kg b.w. Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	9.62	2.82	3.82	1.74
25 mg/kg b.w. Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	10.0	3.44	3.50	1.62
50 mg/kg b.w. Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	14.38	4.06	5.45	2.46
Positive Control	14.48	7.60	14.40*	2.65

Analysis of Dead Cell Index and % Tail Intensity in cells of the Stomach:

Test Group	Dead cells on slides per 1500 cells per animal		Mean of Median (% Tail Intensity)
	Group Mean	SD	Group Mean	SD
Vehicle Control	11.48	4.41	7.56	4.31
12.5 mg/kg b.w. Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	14.05	7.71	8.38	4.06
25 mg/kg b.w. Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	16.10	9.67	8.05	3.71
50 mg/kg b.w. Reaction mass of (E)-1-chlorobut-2-ene and 3-chlorobut-1-ene (Crotyl chloride)	18.14	7.91	14.05*	3.92
Positive Control	17.48	5.10	23.00*	3.88

 

*p<0.001

Conclusions:

negative
Statistically significant and biologically relevant increases in primary DNA damage in the stomach as determined by the comet assay was observed in the highest dose level. However, no clear dose-dependent increase was observed for the Comet results neither of the liver and stomach. With regard to the results described, the Comet vivo part of this study with the test item is considered to be non-genotoxic for the liver.
For the stomach, a direct genotoxic effect of the substance on stomach cells is not considered, as effects observed are likely to be caused by the corrosive properties of the test material, as confirmed by histopathological examination. However on a long-term scale, an indirect genotoxic effect on the stomach secondary to corrosivity cannot be excluded.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro data are regarded as relevant for humans. 

Justification for classification or non-classification

Based on the outcome of the available studies Crotyl chloride does not have to be classified for mutagenicity.