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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 the data from various test chemicals
Cross-referenceopen allclose all
Reason / purpose:
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 from peer reviewed publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
The test substance was tested for mutagenicity in the bacterial reverse mutation assay, using the spot test.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of the test chemical: D and C green 6
- Molecular formula: C28H22N2O2
- Molecular weight: 418.4938 g/mol
- Substance type: Organic
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA1978 and TA98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other:
Cytokinesis block (if used):
Strains TA1535, TA1537 and TA1538 have mutations hisG46, hisC3076 and hisD3052 in their respective histidine operons, resulting in a nutritional requirement for histidine. In addition they possess deletions in the uvr region of the genome removing their capacity for excision repair and contain rfa (deep rough) mutations resulting in the impaired synthesis of the polysaccharide side chains of their surface lipopolysaccharide. Strain TA1978 resembles TA1538 except that it possesses the normal uvr repair system and a mutation in gal E. Strains TA100 and TA98 are derived from TA1535 and TA1538 respectively by insertion of the R factor plasmid pKM101.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 fraction was obtained from Female albino rats
Test concentrations with justification for top dose:
100-200 µg/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:
ethylmethanesulphonate
Remarks:
For TA1535
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For TA100
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
Remarks:
For TA1537
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
TA1538 and TA98.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: as impregnation on paper disk

DURATION
- Preincubation period: No data
- Exposure duration: 72 hrs
- Expression time (cells in growth medium): 72 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: ): 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:
Growth inhibition was noted
Statistics:
No data
Species / strain:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA1978 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 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:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by spot test using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 with and without S9 metabolic activation system. 100-200 µg of the test material dissolved in 10-20µL DMSO/disc was applied to 6mm paper concentration discs and used for mutagenicity testing. From 1 to 4 discs were applied per plate. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitr

Reason / purpose:
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material: Anthraquinone
- Molecular formula: C14H8O2
- Molecular weight: 208.215 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA97, TA98, TA100
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
PCB-induced rat liver S9
Test concentrations with justification for top dose:
0, 5, 10, 50 or 200 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 2 days (48 hrs)
- Expression time (cells in growth medium): 2 days (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: Duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, cell growth was noted

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 a dose dependent increase in the number of revertants/plate
Statistics:
No data
Species / strain:
S. typhimurium, other: TA97, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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

Table: Mutagenic activity on Anthraquinone

Dose

His+ revertants

TA97

TA98

TA100

+S9

-S9

+S9

-S9

+S9

-S9

200

168

206

33

17

111

136

50

170

208

22

27

118

127

10

276

193

46

27

141

 

5

283

182

33

 

152

 

0

210

177

34

32

120

115

 

Conclusions:
The test chemical did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100 with and without PCB induced S9 metabolic activation system at dose levels of 0, 5, 10, 50 or 200µg/plate. The plates were incubated for 48 hrs and the number of dose dependent increase in the revertants was counted. The test chemical did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction and hence it 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 97862-23-2
Author:
Sustainability Support Services (Europe) AB
Year:
2018
Bibliographic source:
WoE report, Sustainability Support Services (Europe) AB, 2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
WoE derived based on the experimental data from various test chemicals
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- IUPAC name: 9,10-Anthracenedione, 1,4-diamino-, N,N'-bis(4-C7-17-branched alkylphenyl) derivs.
- Substance type: Organic
- Physical state: Liquid

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA1978 and TA98
Remarks:
1
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other:
Species / strain / cell type:
S. typhimurium, other: TA97, TA98, TA100
Remarks:
2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Strains TA1535, TA1537 and TA1538 have mutations hisG46, hisC3076 and hisD3052 in their respective histidine operons, resulting in a nutritional requirement for histidine. In addition they possess deletions in the uvr region of the genome removing their capacity for excision repair and contain rfa (deep rough) mutations resulting in the impaired synthesis of the polysaccharide side chains of their surface lipopolysaccharide. Strain TA1978 resembles TA1538 except that it possesses the normal uvr repair system and a mutation in gal E. Strains TA100 and TA98 are derived from TA1535 and TA1538 respectively by insertion of the R factor plasmid pKM101.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 fraction was obtained from Female albino rats
Test concentrations with justification for top dose:
1. 100-200 µg/plate
2. 0, 5, 10, 50 or 200 µg/plate
Vehicle / solvent:
- 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:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
For TA1535 / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For TA100 / 1
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
Remarks:
For TA1537 / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
TA1538 and TA98. / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 / 1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
2
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: as impregnation on paper disk

DURATION
- Preincubation period: No data
- Exposure duration: 72 hrs
- Expression time (cells in growth medium): 72 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: ): 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

2. METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:No data
- Exposure duration:2 days (48 hrs)
- Expression time (cells in growth medium):2 days (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:Duplicate

NUMBER OF CELLS EVALUATED:No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:Yes, cell growth was noted

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. Growth inhibition was noted
2. The plates were observed for a dose dependent increase in the number of revertants/plate
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538, TA1978 and TA98
Remarks:
1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium, other: TA97, TA98, TA100
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce mutation in Salmonella typhimurium strains 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:

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 to determine the mutagenic nature of the test chemical. The study was performed by spot test using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 with and without S9 metabolic activation system. 100-200 µg of the test material dissolved in 10-20µL DMSO/disc was applied to 6mm paper concentration discs and used for mutagenicity testing. From 1 to 4 discs were applied per plate. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce mutation in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA1978 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100 with and without PCB induced S9 metabolic activation system at dose levels of 0, 5, 10, 50 or 200µg/plate. The plates were incubated for 48 hrs and the number of dose dependent increase in the revertants was counted. The test chemical did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.