5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only 2-Aminoanthracene as positive control with metabolic activation)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

EC Number:

253-256-2

EC Name:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

Cas Number:

36888-99-0

Molecular formula:

C16H9N5O6

IUPAC Name:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dipyrimidine-2,4,6(1H,3H,5H)-trione

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: yellow powder
- Analytical purity: technically pure
- Storage condition of test material: +4°C

Specific details on test material used for the study:

- Physical state: yellow powder
- Analytical purity: technically pure
- Storage condition of test material: +4°C

Method

Target gene:

his, trp

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98 and E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Remarks:

control for sterility

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, MNNG, 4-nitro-o-phenylendiamine, 9-aminoacridine chloride monohydrate, N-ethyl-N'-nitro-N-nitrosoguanidin (see details below)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation)

DURATION
- Preincubation period:
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 4 in the first experiment, 2 in the second experiment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: Negative control: Parallel with each experiment with and without S-9 mix, a negative control (solvent control, sterility control) is
carried out for each tester strain in order to determine the spontaneous mutation rate.

Positive controls
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix:
10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1538, TA 1537 and TA 1535
60 µg 2-aminoanthracene (dissolved in DMSO) for E. coli WP2 uvrA
without S-9 mix
5 µg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strains TA 98 and TA 1538
100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537
10 µg N-ethyl-N´-nitro-N-nitrosoguanidin (ENNG) (dissolved in DMSO) for the strain E. coli WP2 uvrA

Evaluation criteria:

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

Statistics:

The data were not statistically analyzed.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Standard plate test (20 - 5000 µg/plate): 1. Exp.
Strain	Metabolic activation system	Replicates	Mean revertants in Controls	Maximum revertant factor	Dose dependency	Assessment
TA 98	no	4	18	1.1	no	negative
	yes	4	42	1.0	no	negative
TA 1537	no	4	8	1.0	no	negative
	yes	4	12	1.0	no	negative
TA 1538	no	4	10	1.2	no	negative
	yes	4	28	0.9	no	negative
TA 1535	no	4	18	0.9	no	negative
	yes	4	13	1.1	no	negative
TA 100	no	4	110	1.0	no	negative
	yes	4	106	1.0	no	negative
WP2 uvr A	no	4	37	1.0	no	negative
	yes	4	37	1.1	no	negative

Standard plate test (20 - 5000 µg/plate): 2.Exp.
Strain	Metabolic activation system	Replicates	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	2	21	1.3	no	negative
	yes	2	35	1.1	no	negative
TA 1537	no	2	10	0.9	no	negative
	yes	2	8	1.1	no	negative
TA 1538	no	2	13	1.0	no	negative
	yes	2	24	1.1	no	negative
TA 1535	no	2	20	1.1	no	negative
	yes	2	24	1.1	no	negative
TA 100	no	2	120	1.0	no	negative
	yes	2	122	1.3	no	negative
WP2 uvr A	no	2	31	1.1	no	negative
	yes	2	35	1.3	no	negative

Incomplete solubility of the test substance in DMSO from about 500 µg/plate onward.

No bacteriotoxic effect was observed.

Mutagenicity: An increase in the number of his + or trp+ revertants was not observed either without S-9 mix or after the addition of a metabolizing system.

Applicant's summary and conclusion

Conclusions:

Pigment Yellow 139 was not mutagenic in bacteria.