9,10-Anthracenedione, 1,4-diamino-, N,N'-bis(4-C7-17-branched alkylphenyl) derivs.

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 the 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 from peer reviewed publication

Qualifier:

according to guideline

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

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:

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 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

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

Test material information

Constituent 1

Reference substance name:

9,10-Anthracenedione, 1,4-diamino-, N,N'-bis(4-C7-17-branched alkylphenyl) derivs.

EC Number:

308-067-0

EC Name:

9,10-Anthracenedione, 1,4-diamino-, N,N'-bis(4-C7-17-branched alkylphenyl) derivs.

Cas Number:

97862-23-2

Molecular formula:

C48H62N2O2

IUPAC Name:

9,10-Anthracenedione, 1,4-diamino-, N,N'-bis(4-C7-17-branched alkylphenyl) derivs.

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 / strain

Species / strain 1

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 2

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

Controls

Controls 1

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

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:

methylmethanesulfonate

Remarks:

For TA100 / 1

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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 results

Test results 1

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

Test results 2

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.