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

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study. GLP accordance not stated in the report. Test article precipitated in soft agar from the lowest concentration used in the Ames assay. No analytical monitoring of test concentration was done.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1981)

Deviations:

yes

Remarks:

no statistical analysis was performed

GLP compliance:

no

Remarks:

not stated in the report

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Purity: Commercial grade
- Lot/batch No.: Op. 11006
- Stability of test article: In the report it was stated that stability was guaranteed by the sponsor.

Method

Target gene:

All strains are histidine auxotrophic mutants.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

One millilitre activation mixture contained 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.

Test concentrations with justification for top dose:

A preliminary toxicity test was carried out with concentrations ranging from 0.08 to 5000 µg/plate suspended in dimethylsulfoxide.
Ames test (without and with microsomal activation): 20, 78, 313, 1250 and 5000 µg/plate suspended in dimethylsulfoxide.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without S9-Mix

Positive control substance:

other: Daunorubicin-HCl (5 and 10 µg/plate)

Remarks:

TA 98

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without S9-Mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

TA 100

Migrated to IUCLID6: 0.125 and 0.25 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without S9-Mix

Positive control substance:

mitomycin C

Remarks:

TA 102

Migrated to IUCLID6: 0.5 and 1 µg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without S9-Mix

Positive control substance:

sodium azide

Remarks:

TA 1535

Migrated to IUCLID6: 2.5 and 5 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without S9-Mix

Positive control substance:

9-aminoacridine

Remarks:

TA 1537

Migrated to IUCLID6: 50 and 100 µg/plate

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

With S9-Mix

Positive control substance:

other: 2-aminoanthracene 5 µg/plate

Remarks:

TA 98, TA 100, TA 1537

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

With S9-Mix

Positive control substance:

other: 2-aminoanthracene 20 µg/plate

Remarks:

TA 102

Controls 8

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

With S9-Mix

Positive control substance:

cyclophosphamide

Remarks:

TA 1535

Migrated to IUCLID6: 250 µg/plate

Details on test system and experimental conditions:

The study consisted of a toxicity pre-screen test followed by the Ames assay (with and without S9-Mix).

OTHER EXAMINATIONS
A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/plate. Thereafter, the Ames test was performed with 20, 78, 313, 1250 and 5000 µg/plate test article (with and without microsomal activation). The substance precipitated in soft agar at and above a concentration of 20 µg/plate.

MUTATION TEST
Mutation assays with and without microsomal activation were performed twice in triplicate per strain and per group (standard plate test).
Each Petri dish contained approximately 20 ml of minimum agar, 0.1 ml of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth) in 2.0 ml of soft agar. Experiments with microsomal activation contained additionally 0.5 ml of an activation mixture, consistent of S9 fraction of liver from Aroclor 1254 induced rats and co-factors. The plates were incubated for about 48 hours at 37 ± 1.5°C in darkness.

EVALUATION CRITERIA
Arithmetic mean of revertant colonies was calculated. The test substance is considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

No statistical analysis

Results and discussion

Test results

Test results 1

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:

other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The substance precipitated in soft agar at and above a concentration of 20 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The number of histidine-prototrophic mutants after treatment with the substance did not vary markedly in comparison to the negative control in any of the experiments.