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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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.