1-(3-chlorophenyl)-4-(3-chloropropyl)piperazinium 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

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

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 peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

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

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the S9 preparation of the liver fractions

Test concentrations with justification for top dose:

0, 1, 3, 10, 33, 100 or 333 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98; -S9), 2-aminoanthracene (all strains, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr
- 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: At least five dose levels of the chemicals were tested, with three plates per dose level.

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:

1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity

Statistics:

Mean and Standard error of mean

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537, TA98

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: The chemical was initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

Remarks on result:

other: No mutagenic potential

Table: Mutation data for the test chemical

Dose (µg/plate)	TA100
	NA		NA		10% HLI		10% HLI		10% RLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	120	5.0	147	4.4	136	4.8	139	7.6	127	11.5	118	7.9
1	142	6.0	135	11.0
3	146	7.6	150	16.2	148	9.7	158	10.8	147	11.1	142	8.6
10	128	13.9	139	7.9	150	12.3	152	13.1	138	7.2	174	5.8
33	129	7.8	137	1.5	13	9.4	158	3.5	136	8.4	167	10.7
100	57s	9.0	144	2.7	149	0.9	138	13.6	96s	9.7	164	4.0
333					104s	10.1	115	7.2	T		70s	13.3
Positive control	410	271	641	13.0	1401	53.4	1088	9.3	601	37.7	423	3.6

 

 

Dose (µg/plate)	TA1535
	NA		NA		10% HLI		10% HLI		10% RLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	21	2.3	28	5.4	8	0.7	15	4.9	9	1.7	16	1.9
1	20	7.8	28	1.3					8	3.3
3	18	3.3	31	6.4	5	1.0	15	5.0	13	2.0	10	2.3
10	23	3.9	31	4.4	7	0.3	8	1.0	16	1.8	11	3.0
33	19	1.5	31	3.5	6	1.5	14	2.0	10	1.9	12	4.2
100	7s	1.9	28	1.8	12	2.2	11	3.3	12	1.9	8	2.2
333					4s	0.6	8	0.9	0s	0.0	5s	2.3
Positive control	406	4.0	564	14.2	309	8.7	477	5.5	163	12.2	157	13.9

 

Dose (µg/plate)	TA1537
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	4	0.3	6	0.9	4	12
1	8	3.3
3	7	1.2	8	2.3	13	0.3
10	6	0.6	8	0.9	14	0.6
33	5	1.2	9	2.0	10	4.2
100	8	2.4	7	0.7	7	0.3
333			9	2.7	2s	1.5
Positive control	126	10.8	341	21.2	98	9.0

 

Dose (µg/plate)	TA98
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	18	1.5	33	3.1	29	9.2
1	24	4.2
3	20	4.3	29	1.8	38	3.2
10	18	4.5	30	2.0	35	2.4
33	20	3.4	34	2.0	46	8.4
100	20	3.2	39	2.4	36	3.3
333			57	3.2	24s	9.0
Positive control	874	21.3	844	41.5	294	21.5

 

Conclusions:

The test chemical did not induce gene mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of0, 1, 3, 10, 33, 100 or 333 µg/plate. Distilled water was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. The test chemical did notinduce gene mutation in theSalmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed for the test chemical evaluate its mutagenic nature

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

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

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The microsomal S-9 fraction was prepared From liver homogenates of Sprague-Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

1, 10, 100, 1000, 5000 mcg/plate

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation assay)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr
- 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: Duplicate

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:

The plates were observed for increase in number of revertants/plate

Statistics:

No data

Species / strain:

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

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:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutation in the Salmonella typhimurium strain TA 1535, TA 1537, TA 100 and TA 98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature. The study was performed as per the plate incorporation assay using Salmonella typhimurium strain TA 1535, TA 1537, TA 100 and TA 98 both in the presence and absence of S9 metabolic activation system at doses of 1, 10, 100, 1000, 5000 mcg/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs at 37°C before the evaluation of the revertant colonies could be made. The test chemical did notinduce gene mutation in theSalmonella typhimurium strain TA 1535, TA 1537, TA 100 and TA 98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Data source

Reference

Reference Type:

review article or handbook

Title:

WoE of gene mutation in vitro toxicity study for CAS no 52605-52-4

Author:

Sustainability Support Services (Europe) AB

Year:

2018

Bibliographic source:

WoE report, Sustainability Support Services (Europe) AB, 2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(3-chlorophenyl)-4-(3-chloropropyl)piperazinium chloride

EC Number:

258-038-0

EC Name:

1-(3-chlorophenyl)-4-(3-chloropropyl)piperazinium chloride

Cas Number:

52605-52-4

Molecular formula:

C13H18Cl2N2.ClH

IUPAC Name:

1-(3-chlorophenyl)-4-(3-chloropropyl)piperazin-1-ium chloride

Details on test material:

- Name of the test chemical: 1-(3-chlorophenyl)-4-(3-chloropropyl) piperazine hydrochloride
- Molecular formula: C13H18Cl2N2ClH
- Molecular weight: 309.6661 g/mol
- Substance tpe: Organic

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

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

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

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

Remarks:

3

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:

Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the S9 preparation of the liver fractions

Test concentrations with justification for top dose:

2. 0, 1, 3, 10, 33, 100 or 333 µg/plate
3. 1, 10, 100, 1000, 5000 mcg/plate

Vehicle / solvent:

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

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

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98; -S9), 2-aminoanthracene (all strains, +S9)

Remarks:

2

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

Remarks:

3

Details on test system and experimental conditions:

2. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr
- 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: At least five dose levels of the chemicals were tested, with three plates per dose level.

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

3. METHOD OF APPLICATION: in agar (plate incorporation assay)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr
- 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: Duplicate

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:

2. 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity

3. The plates were observed for increase in number of revertants/plate

Statistics:

Mean and Standard error of mean

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537, TA98

Remarks:

2

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:

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

Remarks:

3

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:

2. 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: The chemical was initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

3. No data

Remarks on result:

other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 1, 3, 10, 33, 100 or 333 µg/plate. Distilled water was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. The test chemical did notinduce gene mutation in theSalmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In another study, gene mutation toxicity study was performed for the test chemical to evaluate its mutagenic nature. The study was performed as per the plate incorporation assay using Salmonella typhimurium strain TA 1535, TA 1537, TA 100 and TA 98 both in the presence and absence of S9 metabolic activation system at doses of1, 10, 100, 1000, 5000 mcg/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs at 37°C before the evaluation of the revertant colonies could be made. The test chemical did notinduce gene mutation in theSalmonella typhimurium strain TA 1535, TA 1537, TA 100 and TA 98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce gene mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.