Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis([1,1'-biphenyl]-4-yl)-2,5-dihydro-

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:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

413-920-6

EC Name:

-

Cas Number:

88949-33-1

Molecular formula:

C30 H20 N2 O2

IUPAC Name:

3,6-bis({[1,1'-biphenyl]-4-yl})-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: Solid
- Lot/batch No.: Z-2281/1,2,4,5
- Expiration date of the lot/batch: April 01, 1997
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

His: Salmonella; Trp: E. Coli

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 3

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

10; 100; 333.3; 1000; and 5000 µg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see: "Details on test system and conditions"

Details on test system and experimental conditions:

POSITIVE CONTROLS:
Without metabolic activation (-S9-mix):
- TA1535, TA 100: sodium azide (SA), 10 µg
- TA1537, TA 98: 4-nitro-o-phenylene-diamine, 4-NOPD 50 µg
- WP2uvrA, WP2 : methylmethanesulfonate (MMS), 10 µl

With metabolic activation (+S9-mix):
- TA1537, : 2-aminoanthracene (2AA), 2.5 µg
- TA1535, TA 1537, TA98 and TA100: 2-aminoanthracene (2AA), 2 µg
- WP2uvrA and WP2: 2-aminoanthracene (2AA), 2 µg

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48 hours. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli were counted.
- COLONY COUNTING:
- Exposure duration: The revertant colonies (histidine independent/ tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A test article is considered as mutagenic if in the strains TA 100, WP2, and its uvrA derivate the number of reversions will be at least twice as high and in the strains TA 1535, TA 1537, and TA 98 it will be at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants isregarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No

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:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix in experiment I

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRECIPITATE: Not mentioned.

TOXICITY: Toxic effects evidenced by a reduction in revertant colony numbers occurred only in strain TA 1535 at 5000 µg/plate without S9 mix in experiment I. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

MUTAGENICITY: No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested. All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Any other information on results incl. tables

Table 1: Number of revertants in the control or after treatment with the test substance

 

First experiment (10 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 1535	no	15	1.1	no	negative
	yes	15	1.2	no	negative
TA 1537	no	6	1.1	no	negative
	yes	6	1.4	no	negative
TA 98	no	20	1.2	no	negative
	yes	33	1.2	no	negative
TA 100	no	78	0.9	no	negative
	yes	88	1.1	no	negative
E. coli WP2 uvrA	no	38	1.2	no	negative
	yes	41	1.1	no	negative
E. coli WP2	no	30	1.6	no	negative
	yes	40	1.2	no	negative
Second experiment (10 - 1000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 1535	no	14	1.1	no	negative
	yes	15	1.2	no	negative
TA 1537	no	11	1.2	no	negative
	yes	15	1.2	no	negative
TA 98	no	24	1.2	no	negative
	yes	41	1.3	no	negative
TA 100	no	90	1.1	no	negative
	yes	91	1.0	no	negative
E. coli WP2 uvrA	no	39	1.4	no	negative
	yes	47	1.1	no	negative
E. coli WP2	no	57	1.1	no	negative
	yes	56	1.1	no	negative

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In a GLP conform study according to OECD guideline 471, the potential of the test substance to induce gene mutations was investigated in two independent experiments using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA and WP2.

In both experiments, the test substance was tested up to a concentration of 5000 µg/plate in the absence and presence of S9-mix. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. The test substance did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strains WP2uvrA and WP2, both in the absence and presence of S9-metabolic activation. These results were confirmed in two independent experiments. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.