2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3-trimethyl-3H-indolium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from J-check, 2017

Data source

Reference

Reference Type:

other: J-check

Title:

Reverse Mutation Test of chlorocyclohexane on Bacteria

Author:

National Institute of Technology and Evaluation

Year:

2017

Bibliographic source:

Japan Chemicals Collaborative Knowledge Database

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Bacterial reverse mutation test was performed to determine the mutagenic nature of Chlorocyclohexane

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Chlorocyclohexane

EC Number:

208-806-6

EC Name:

Chlorocyclohexane

Cas Number:

542-18-7

Molecular formula:

C6H11Cl

IUPAC Name:

Chlorocyclohexane

Details on test material:

- Name of test material: Chlorocyclohexane
- Molecular formula: C6H11Cl
- Molecular weight: 118.606 g/mol
- Substance type: Organic

Specific details on test material used for the study:

- Name of test material: Chlorocyclohexane
- Molecular formula: C6H11Cl
- Molecular weight: 118.606 g/mol
- Substance type: Organic
- Physical state: Slightly pale yellow from colorless liquid
- Purity: 99.7%
- Impurities (identity and concentrations): 0.3%

Method

Target gene:

Histidine for Salmonella typhimurium and tryptophan for E. coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

SD male rat liver, induced by phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate (TA100, TA1535 strains),
9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA98, TA1537, WP2uvrA/pKM101 strains)

+S9 mix: 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA100, TA1535, TA98, TA1537 strains),
9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (WP2uvrA/pKM101 strain)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: S9 mix: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA 100, TA98 and WP2 uvrA/pKM101); +S9 mix: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

In any strain(s) tested with or without S9 mix, when the mean number of revertant colonies per plate increased twice more than that of the negative control and when the increase was shown to be dose-related and reproducible, the chemical was judged mutagenic.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

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: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Concentration: 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 μg/plate with and without S9

COMPARISON WITH HISTORICAL CONTROL DATA: No data

Remarks on result:

other: No mutagenic effect were observed.

Any other information on results incl. tables

 Table: Mutagenic nature of Chlorocyclohexane

Metabolic activation	Dose (μg/plate)	TA100	TA1535	Wp2uvrA	TA98	TA1537
S9 mix (-)	DMSO	123	15	65	17	16
	1.22	118	16	69	26	21
	4.88	111	15	82	16	16
	19.5	100	19	64	24	22
	78.1	94*	16*	68	18	18
	313	0*	0*	59*	20*	8*
	1250	0*	0*	0*	0*	0*
	5000	0*	0*	0*	0*	0*
S9 (+)	DMSO	133	16	104	24	19
	1.22	117	10	104	24	25
	4.88	107	14	98	24	16
	19.5	112	16	92	31	25
	78.1	88	17	84	31	22
	313	90*	7*	77*	24*	12*
	1250	0*	0*	56*	0*	0*
	5000	0*	0*	0*	0*	0*
S9 (-)		AF-2	NaN3	AF-2	AF-2	9-AA
	Dose	0.01	0.5	0.0005	0.1	80
	Revertants/plate	623	560	640	837	389
S9 (+)		2-AA	2-AA	2-AA	2-AA	2-AA
	Dose	1	2	2	0.5	2
	Revertants/plate	959	212	600	423	154

 

Metabolic activation	Dose (μg/plate)	TA100	TA1535	Wp2uvrA	TA98	Ta1537
S9 mix (-)	DMSO	103 91 100	9 12 13	94 86 85	22 16 22	11 13 13
	1.22	108 102 92	6 9 8	-	-	-
	4.88	129 93 88	11 10 11	-	-	-
	9.77	116 96 95	6 11 7	84 74 98	28 22 28	11 11 15
	19.5	92 95 94	8 9 9	117 106 93	17 24 16	12 8 17
	39.1	94 106 104	11 6 8	109 88 84	17 16 23	13 14 12
	78.1	108* 96* 111*	13* 10* 7*	102 87 81	22 22 24	16 13 13
	156	112* 94* 107*	9* 10* 8*	101 93 70	17 29 25	18 16 16
	313	-	-	59* 79* 51*	17* 15* 22*	9* 10* 14*
S9 (+)	DMSO	96 103 113	10 10 13	116 130 103	24 25 30	15 13 11
	9.77	81 115 133	13 8 11	108 117 108	25 18 29	13 14 15
	19.5	108 95 108	9 8 6	115 110 108	19 25 27	23 19 11
	39.1	103 120 120	7 7 14	100 100 93	26 26 30	19 22 17
	78.1	109 116 98	11 14 9	117 110 105	29 29 22	19 21 11
	156	119* 112* 95*	8* 11* 11*	93 109 108	28 24 24	19 15 15
	313	106* 87* 100*	8* 6* 3*	81* 114* 76*	22* 24* 27*	15* 10* 15*
	625	-	-	49* 50* 69*	-	-
S9 (-)		AF-2	NaN3	AF-2	AF-2	9-AA
	Dose	0.01	0.5	0.0005	0.1	80
	Revertants/plate	692 587 644	570 635 630	1226 1219 1038	907 894 986	444 381 452
S9 (+)		2-AA	2-AA	2-AA	2-AA	2-AA
	Dose	1	2	2	0.5	2
	Revertants/plate	1446 1280 1324	230 193 222	880 674 604	441 461 396	205 226 175

 

Metabolic activation	Dose (μg/plate)	TA100	TA1535	Wp2uvrA	TA98	Ta1537
S9 mix (-)	DMSO	95 113 105	10 8 10	62 75 75	19 15 13	18 13 9
	1.22	103 97 102	10 10 10	-	-	-
	4.88	112 102 94	14 11 10	-	-	-
	9.77	110 104 114	8 9 9	88 59 76	18 23 19	16 9 8
	19.5	113 104 108	8 10 14	75 86 73	19 18 13	9 11 13
	39.1	110 117 99	14 9 14	67 78 74	19 18 16	10 12 19
	78.1	85* 86* 82*	7* 13* 6*	84 73 80	24 21 16	9 15 13
	156	85* 88* 87*	7* 7* 9*	80 61 72	15 18 15	10 11 15
	313	-	-	68* 78* 59*	13* 17* 3*	9* 7* 10*
S9 (+)	DMSO	101 111 121	13 14 11	83 110 114	25 18 25	19 17 17
	9.77	111 115 117	9 15 13	87 9 101	22 23 32	17 17 12
	19.5	123 111 118	9 9 16	99 89 92	24 24 23	17 16 15
	39.1	114 129 101	9 15 11	90 115 84	23 20 32	15 18 18
	78.1	129 126 121	11 15 15	93 84 82	18 25 26	19 19 11
	156	119* 110* 110*	11* 11* 9*	93 108 102	27 22 24	22 13 16
	313	86* 91* 84*	7* 13* 8*	99* 97* 8*	24* 24* 24*	11* 15* 18*
	625	-	-	58* 68* 44*	-	-
S9 (-)		AF-2	NaN3	AF-2	AF-2	9-AA
	Dose	0.01	0.5	0.0005	0.1	80
	Revertants/plate	688 672 726	554 597 568	1230 976 853	984 805 767	328 310 350
S9 (+)		2-AA	2-AA	2-AA	2-AA	2-AA
	Dose	1	2	2	0.5	2
	Revertants/plate	1669 1290 1398	200 248 293	876 714 667	379 372 430	188 189 190

 

 

Applicant's summary and conclusion

Conclusions:

Chlorocyclohexane did not induce gene mutation in S. typhimurium TA 1535, TA 1537, TA 98, TA100 and E. coli WP2 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Bacterial reverse mutation test was performed to determine the mutagenic nature of Chlorocyclohexane. The study was performed as per the preincubation protocol using S. typhimurium TA 1535, TA 1537, TA 98, TA100 and E. coli WP2 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate (TA100, TA1535 strains), 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA98, TA1537, WP2uvrA/pKM101 strains) without S9 and 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA100, TA1535, TA98, TA1537 strains), 9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (WP2uvrA/pKM101 strain) with S9. The plates were preincubated for 20 mins at 37°C and the exposure duration was 48 hrs. The plates were observed for dose-related and reproducible doubling of the mean number of revertant colonies per plate than that of the negative control. Chlorocyclohexane did not induce gene mutation in S. typhimurium TA 1535, TA 1537, TA 98, TA100 and E. coli WP2 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.