2,9-bis[4-(phenylazo)phenyl]anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Dec 2011 - 19 Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Principles of method if other than guideline:

The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines.

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,9-bis[4-(phenylazo)phenyl]anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

EC Number:

221-264-5

EC Name:

2,9-bis[4-(phenylazo)phenyl]anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Cas Number:

3049-71-6

Molecular formula:

C48H26N6O4

IUPAC Name:

2,9-bis[4-(phenyldiazenyl)phenyl]isoquino[4',5',6':6,5,10]anthra[2,1,9-def]isoquinoline-1,3,8,10(2H,9H)-tetrone

Details on test material:

Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.

Specific details on test material used for the study:

- Physical state: solid, red
- Storage condition of test material: room temperature
- Lot/batch No.:35613-116A
- Expiration date of the lot/batch: 25 Oct 2013

Method

Target gene:

his, trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix and uninduced hamster liver S-9 mix

Test concentrations with justification for top dose:

33 μg - 5 000 μg/plate (SPT)
33 μg - 5 000 μg/plate (PIT)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with rat S9 (SPT): 2-aminoanthracene (all strains) - with hamster S9 (PIT): 2-aminoanthracene (all strains), Congo red (TA98), benzidine (TA98)

Remarks:

with S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (TA1535, TA100), 4-nitro-o-phenylendiamine (TA98), 9-aminoacridine (TA1537), 4-nitroquinoline-N-oxide (E. coli WP2 uvrA)

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); Prival preincubation
The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from 333 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the Ames standard plate test depending on the strain and test conditions from 2500 μg/plate onward. In the Prival preincubation assay weak bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at 5000 μg/plate.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (Ames standard plate test and Prival preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant

increase in the number of revertant colonies within the range of the historical positive control data or above.

Results of Experiment I (Standard Plate Test with and without rat liver S-9 mix):

	TA98		TA100		TA1535		TA1537		WP2 uvrA
concentration	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	20	22	79	84	11	14	6	7	47	48
33	19	23	76	82	11	15	6	7	45	51
100	19	24	79	78	13	15	6	8	44	53
333	21	21	87	81	10	15	6	6	43	49
1000	20	25	86	75	12	16	6	8	19	49
2500	17	19	74	78	11	14	5	5	46	52
5000	20	16	73	70	14	14	4	5	47	47
MNNG			675		1176
2-AA		864		790		174		123		237
AAC							363
NOPD	531
4-NQO									608

Controls:

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)

2 -AA: 2-aminoanthracene (2.5 µg/plate for TA 98, 100, 1535, 1537; 60 µg/plate for WP2 uvrA)

AAC: 9-aminoacridine (100 µg/plate)

NOPD: 4-nitro-o-phenylendiamnine (10 µg/plate)

4 -NQO: 4-nitroquinoline-N-oxide (5 µg/plate)

Results of Experiment II (Pre-incubation Test with and without hamster liver S-9 mix):

	TA98		TA100		TA1535		TA1537		WP2 uvrA
concentration	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	28	40	120	133	16	18	8	10	51	51
33	29	36	125	1217	16	17	8	11	45	52
100	26	38	113	130	16	16	8	13	49	46
333	26	31	123	130	18	20	8	9	50	46
1000	26	34	119	117	16	17	6	10	50	47
2500	26	31	118	115	19	16	7	9	39	57
5000	21	37	105	102	14	13	7	10	49	38
MNNG			854		740
2-AA		852		1695		742		142		252
AAC							478
NOPD	708
4-NQO									235
CONGOR.		639
BENZID.		655

Controls:

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)

2 -AA: 2-aminoanthracene (10 µg/plate)

AAC: 9-aminoacridine (100 µg/plate)

NOPD: 4-nitro-o-phenylendiamnine (10 µg/plate)

4 -NQO: 4-nitroquinoline-N-oxide (5 µg/plate)

CONGOR: Congo red (0.3 µmol/plate)

benzid: benizidine (0.3 µmol/plate)

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic substance in the bacterial reverse mutation test (Ames standard plate test and Prival preincubation assay) in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay (Ames standard plate test and Prival preincubation test). The Prival preincubation test is a modification of the standard Ames reverse mutation assay, in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines .This test is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds such as the test compound. Both assays were performed in the presence and absence of a metabolic activation system at a concentration range of 33 – 5000 µg/plate. Precipitation of the test substance was found from a concentration of 333 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the Ames standard plate test depending on the strain and test conditions from 2500 μg/plate onward. In the Prival preincubation assay weak bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at 5000 μg/plate. According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (Ames standard plate test and Prival preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Ames standard plate test and in the Prival preincubation test in the absence and the presence of metabolic activation.