2,9-dimethylanthra[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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Non-GLP and no E.coli strain tested, otherweise fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,9-dimethylanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

EC Number:

226-866-1

EC Name:

2,9-dimethylanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Cas Number:

5521-31-3

Molecular formula:

C26H14N2O4

IUPAC Name:

2,9-dimethylisoquino[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:

- Analytical purity: 100%
- Storage condition of test material: room temperature
- Lot/batch No.: 90-025

Method

Target gene:

his+

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 µg/plate (standard plate test and preincubation test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix; all strains

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

Remarks:

without S9 mix; TA 1535 and TA 100

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitri-o-phenylenediamine

Remarks:

without S9 mix; TA 98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix; TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorporation method:
- Exposure duration: ca. 48 hours at 37°C
Preincubation method:
- Preincubation period: 20 minutes at 37°C
- Exposure duration: ca. 48 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
reduced his background growth

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity
No bacteriotoxic effect (reduced his background growth) was observed.

Solubility
Incomplete solubility of test substance in DMSO was observed from about 100 µg/plate onward.

Any other information on results incl. tables

Standard Plate Test:

	TA 1535		TA 100		TA 1537		TA 98
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	18	16	95	108	9	19	24	39
20	16	19	99	118	10	15	24	35
100	20	21	111	112	10	12	24	41
500	23	19	80	82	10	13	17	37
2500	20	20	88	85	8	10	13	39
5000	25	19	77	85	7	11	22	38
2-AA		281		1250		283		1474
MNNG	2423		1508
AAC					488
NPD							1328

Preincubation Test:

	TA 1535		TA 100		TA 1537		TA 98
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	14	14	124	123	11	12	28	42
20	16	18	111	86	11	12	25	31
100	19	18	118	94	11	14	19	29
500	19	16	85	79	10	14	17	30
2500	14	20	83	76	9	12	16	28
5000	14	14	75	81	7	10	14	31
2-AA		192		1161		123		794
MNNG	959		995
AAC					493
NPD							855

Controls:

2-AA: 2 aminoanthracene (10 µg/plate)

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

AAC: aminoacridine (100 µg/plate)

NPD: 4-nitro-o-phenylendiamine (10 µg/plate)

Applicant's summary and conclusion

Conclusions:

Under the conditions chosen, the test substance was not mutagenic in the reverse bacteria mutation assay.

Executive summary:

The test article was tested in the Ames reverse mutation assay (according to OECD guideline 471, no GLP) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 20 to 5000 µg/plate in the standard plate test and in the preincubation test (vehicle: DMSO) with and without metabolic activation. An increase in the number of his+ revertants was not observed in the standard plate test or in the preincubation test either without S-9mix or after the addition of S-9 mix. No bacteriotoxic effect (reduced his background growth) was observed. Incomplete solubility of test substance in DMSO was observed from about 100 µg/plate onward. Under the conditions tested, the test substance does not induce point mutations in bacteria.