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Toxicological information

Genetic toxicity: in vitro

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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-referenceopen allclose all
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.

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

Constituent 1
Chemical structure
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 / strainopen allclose all
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 / 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
Controlsopen allclose all
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
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 resultsopen allclose all
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
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.