2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals (1997), Guideline under the Japanese Industrial Safety and Health Law (1988, 1997)

GLP compliance:

yes

Remarks:

Japanese GLP on Industrial Chemicals (1984, 1988) GLP under the Japanese Industrial Safety and Health Law (1988)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

EC Number:

278-388-8

EC Name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Cas Number:

76199-85-4

Molecular formula:

C16H11N5O4

IUPAC Name:

2-cyano-N-methyl-2-[3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)-2,3-dihydro-1H-isoindol-1-ylidene]acetamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): C.I. Pigment Yellow 185
- Physical state: yellow powder
- Analytical purity: 99.3 %
- Lot/batch No.: 98-3107;
- Storage condition of test material: room temperature

Method

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strain).

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:

S9-mix from the liver of male Sprague-Dawley rats which were intraperitoneally injected with phenobarbital (PB) at 30, 60, 60 and 60 mg/kg every 24 hours, and 5,6-benzoflavone at 80 mg/kg at the third PB injection.

Test concentrations with justification for top dose:

5000, 2500, 1250, 625, 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: A solvent selection test indicated that the test substance was insoluble in water for injection (abbrev. DW; Otsuka Pharmaceutical Factory) and DMSO, but uniformly suspended in DMSO at 50 mglmL. No heat, decoloration or foaming was observed when mixing with DMSO. Therefore, DMSO was selected as the solvent.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide, sodium azide, N-ethyl-N' -nitro- N-nitrosoguanidine, 9-aminoacridine hydrochloride, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2

OTHER
- Negative control: dimethylsulfoxide DMSO

Positive controls
- without S9 mix:
• 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2): 0.01 µg/plate (TA 100)
• sodium azide (NaN3): 0.5 µg/plate (TA 1535)
• N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 µg/plate (E.coli WP2uvrA)
• 9-aminoacridine hydrochloride (9-AA): 80 µg/plate (TA 1537)

- with S9 mix:
2-aminoanthracene (2-AA):
• 0.5 µg/plate (TA 98)
• 1 µg/plate (TA 100)
• 2 µg/plate (TA 1535 and TA 1537)
• 10 µg/plate (E.coli WP2uvrA)

Evaluation criteria:

The test substance was judged to be mutagenic (or positive) when the mean number of revertant colonies at any concentration increased two or more times than that of the corresponding negative control for at least one tester strain either in the presence or absence of S9 mix, and when the number of revertant colonies increased dose-dependently and reproducibly.

Statistics:

The data was not analyzed statistically.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 19.5 µg/plate and over in the absence of S9 mix and at 78.1 µg/plate in the presence of S9 mix, precipitates were observed on the agar plates in the dose-finding and main test.

RANGE-FINDING/SCREENING STUDIES:
- A dose-finding test was conducted at 5000, 1250, 313, 78.1, 19.5, 4.88, and 1.22 µg/plate, and it showed the number of revertant colonies was smaller than twice that of the corresponding negative (solvent) control in all the tester strains regardless of the presence or absence of S9 mix. Microbial toxicity was not observed in any of the tester strains regardless of the presence or absence of S9 mix. According to these results, the main test was conducted.

Any other information on results incl. tables

Standard plate test (313 - 5000 µg/plate)
Strain	Metabolic activation system	Mean revertants in Controls	Maximum revertant factor	dose dependency	Assessment
TA 100	no	110	1,0	no	negative
	yes	105	1,1	no	negative
TA 1535	no	11	1	no	negative
	yes	12	1,1	no	negative
WP2uvrA	no	31	1,1	no	negative
	yes	39	1,0	no	negative
TA 98	no	21	1,0	no	negative
	yes	27	1,1	no	negative
TA 1537	no	10	1	no	negative
	yes	10	1,8	no	negative

In the test, the number of revertant colonies was smaller than twice that of the corresponding negative (solvent) control in all the tester strains regardless of the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

The results suggest that the test substance is not mutagenic under the conditions of this study.

Executive summary:

In a GLP conform study conducted similar to OECD guideline 471, the potential of the test substance (purity: 99.3 weight-%) to induce gene mutations was investigated in Salmonella typhimurium strains TA 100, TA 1535, TA1537 and TA 98, and in Escherichia coli WP2uvrA. The test was performed with phenobarbital and benzoflavone induced rat liver S9-mix and without microsomal activation at a dose range up to 5000 µg/plate. At 19.5 µg/plate, and over that dose in the absence of S9 mix, and at 78.1 µg/plate in the presence of S9 mix, precipitates were observed on the agar plates. Microbial toxicity was not observed in any of the tester strains regardless of S9 mix being present or absent. 

The number of revertant colonies was smaller than twice that of the corresponding negative (solvent) control in all the tester strains regardless of the presence or absence of S9 mix. These results suggest that the test substance is not mutagenic under the conditions of the study.