Benzonitrile, 3,3'-(2,3,5,6-tetrahydro-3,6-dioxopyrrolo[3,4-c]pyrrole-1,4-diyl)bis-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several in vivo and in vitro assays were performed to assess the genotoxic potential of the test item. The substance and a structural analogue did not induce mutagenicity in bacterial or mammalian cells. The test substance has shown no evidence of polyploidy, dastogenic activity was observed in the absence of S-9 mix, but not in its presence. When applied to mice or rats, the test item failed to show a clastogenic potential and did not induce DNA repair. In summary, the test compound is not considered to have a genotoxic potential.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-06-23 to 1993-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver (S9)

Test concentrations with justification for top dose:

Test I (±S9): 0 (solvent control), 5000, 2500, 1250, 652, 312,5 µg / plate
Test II (±S9): 0 (solvent control), 5000, 2500, 1250, 652, 312,5 µg / plate

Vehicle / solvent:

- Vehicle used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

-S9: N-ethyl-N-nitro-N-nitrosoguanidine (TA 1535; TA 100, WP2 uvrA), 9-Aminoacridine (TA 1537), 2-Nitrofluorene (TA 1538; TA 98), +S9: 2-Aminoanthracene (all six strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 3 days

NUMBER OF REPLICATIONS: three plates per concentration

DETERMINATION OF CYTOTOXICITY
- other: mean number of revertant colonies (compared between treated plates and solvent control plates)

Evaluation criteria:

a) If treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

b) If treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in the paragraphs above, the following approach is taken in order to resolve the issue of the material's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the material is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989).

Statistics:

No

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:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

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

Positive controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test material formed a suspension in the solvents tested, which were: water, dimethyl sulphoxide, ethanol, methanol, acetone, tetrahydrofuran, hexane and dimethyl formamide. The test material was only soluble in hexane, which is not an suitable solvent for the Ames test. Therefore, water was used as the suspending agent to formulate the test material for the mutation assay.
- Precipitation: no data, but as the test item was applied as a suspension in water, precipitation - at least at the highest test condentration - can be assumed.

RANGE-FINDING/SCREENING STUDIES:
Four concentrations (±S9: 5000, 500, 50, 5 µg / plate) of test substance were assessed for toxicity using the six tester strains. The test item was not toxic towards the tester strains but some contamination was observed on the S. typhimurium plates. It is assued that the increase in revertants observed with tester strain TA 98 at 5000 /µg/plate in the preliminary toxicity assay was a result of the contamination.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genotoxicity tests in vitro

The material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The test item was applied at concentrations of 312.5 μg - 5000 μg/plate in presence and absence of a metabolic activation system (S9 mix). The test item was applied as a suspension in water, precipitation - at least at the highest test condentration - can be assumed.. A bacteriotoxic effect was not observed. A relevant increase in the number of his+ or trp+ revertants was also not observed in both experiments either without S9 mix or after the addition of a metabolizing system.

Two studies were performed to assess the ability of the test item to induce chromosomal aberrations in human lymphocytes cultured in vitro. Cultured human lymphocytes were exposed to the test substance concentrations up to 5000 ug/ml both in the presence and absence of S-9 mix at . At the 18 hour harvest in the absence of S-9 mix, the test material caused a large statistically significant increase in the number of metaphase figures with chromosomal aberrations at the highest dose level, 2500 /ug/ml. This positive result was confirmed in a repeat test. In the course of the second study, the test item was tested in a mammalian cell line derived from Chinese hamster ovary tissue at concentrations up to 156.3 ug/ml. The cells were incubated with the test compound both with and without supplementary metabolic activation (rat S-9 mix). In both the presence and absence of S-9 mix at all harvest times, the test compound did not cause any substantial increase in the proportion of cells with chromosomal aberrations. It is concluded that the test substance, has shown no evidence of polyploidy. Furthermore, it is concluded that the test material has shown evidence of dastogenic activity in the absence of S-9 mix, but not in its presence.

read across to CAS 84632 -59 -7

The potential of the test item to induce mutagenicity in mammalien cells was not examined. Reliable data on a structural analogue are available. Both substances are pigments and share high similarity in structure and are of low solubilitx in water (< 0.1 mg/l). The analogue has a slightly higher molecular weight which gives an additional safety margin.

This study was performed to investigate the potential of the analogue to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The concentration range of the main experiments was limited by precipitation of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.

Genotoxicity tests in vivo

This first in vivo study was designed to assess the potential induction of micronuclei by the test substance in bone marrow cells of mice. Mice were treated with a single acute oral administration of the test substance by intragastric gavage at a dosage of 5000 mg/kg. The negative control group received the vehicle, aqueous 1 % methylcellulose. Bone marrow smears were obtained from five male and five female animals in the negative control and test substance groups at each of 3 sampling times; these being 24. 48 or 72 hours after dosing. At all sampling times, mice treated with the test item did not show any significant increase in the frequency of micronucleated polychromatic erythrocytes. There was no significant decrease in the ratio of polychromatic to normochromatic erythrocytes after treatment of the animals.

The test item was assessed for induction of DNA repair in hepatocytes following acute oral administration to male rats at dosages of 600 and 2000 mg/kg bodyweight. A negative control group was treated with the vehicle, aqueous 1% methylcellulose and a positive control group was treated with dimethylnitrosamine at 4 mg/kg (for the 2 hour expression) or 2-acetylaminofluorene at 50 mg/kg (for the 14 hour expression). Hepatocytes were isolated by enzymatic dissociation at 2 or 14 hours after exposure of the animals to the test substance. The test material did not cause any substantial increases in either the gross nuclear grain count or the net nuclear grain count at any dose level at either sampling time.

In summary, the test compound is not considered to have a genotoxic potential.

Justification for selection of genetic toxicity endpoint
Arbitrary, test with highest sensitivity.

Justification for classification or non-classification

 Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.