Benzamide, N,N-dibutyl-4-[4-(4-chlorophenyl)-2,3,5,6-tetrahydro-3,6-dioxopyrrolo[3,4-c]pyrrol-1-yl]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 17 January 2013 to 25 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented guideline and GLP study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

K-36

IUPAC Name:

K-36

Constituent 2

Reference substance name:

N,N-dibutyl-4-[4-(4-chlorophenyl)-3,6-dioxo-2H,3H,5H,6H-pyrrolo[3,4-c]pyrrol-1-yl]benzamide

Cas Number:

1391764-61-6

Molecular formula:

C27H28ClN3O3

IUPAC Name:

N,N-dibutyl-4-[4-(4-chlorophenyl)-3,6-dioxo-2H,3H,5H,6H-pyrrolo[3,4-c]pyrrol-1-yl]benzamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Metabolic activation system:

from commercially available rat liver S9 induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

9.34, 18.7, 37.3, 74.7, 149, 299, 598, 1200, 2390, 4780 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: 1% CMC was selected because it is available for treatment of highest dose of the test substance (up to 10 mM).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 6 hours (short-term treatment) or 24 hours (long-term treatment)
- Expression time (cells in growth medium): 18 hours (short-term treatment) or 0 hour (long-term treatment)
- Fixation time: 2 hours before harvesting of the cells

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATES: 2

NUMBER OF CELLS EVALUATED: 100 cells per slide (200 per dose level)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

If the following criteria for the frequency of the cells with structural aberrations (excluding gap) were met, the potentiality of the test substance to induce structural aberrations was judged to be equivocal (from 5 to 9%) or positive (10% or more):
1) 5% or more
2) statistically significant increase in a dose-dependent manner as compared with the negative control
For thenumerical aberrations, the potentiality of the test substance to induce numerical aberrations was judged to be positive in cases where the frequency of cells with polyploidy was statistically significantly increased in a dose-dependent manner as compared with the negative control group.

Statistics:

not performed

Results and discussion

Test results

Species / strain:

mammalian cell line, other: CHL/IU

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH, Effects of osmolality, Evaporation from medium, Water solubility: no data
- Precipitation: the precipitates of the test substance were observed at the dose levels of 299 µg/mL or more in all treatment methods.

COMPARISON WITH HISTORICAL CONTROL DATA: no data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Treatment time	Concentration (µg/mL)	Relative cell growth (%)	Number of cells showing structural aberrations (SA)		Number of cells showing numerical aberrations (NA)		Final judgment
			observed	Total %	observed	Total %	SA	NA
6 hr S9 mix (-)	0	100	200	1.0	200	0.3
	37.5	81.0	Not observed		Not observed		-	-
	75.0	88.1	200	2.5	200	1.0
	150	88.5	200	2.0	200	0.5
	300	80.5	200	2.0	200	0.5
	MMC 0.05	-	200	26.0	200	0.5	+	-
6 hr S9 mix (+)	0	100	200	2.0	200	0.3
	37.5	106.8	Not observed		Not observed	0.0	-	-
	75.0	97.4	200	0.0	200	0.3
	150	106.8	200	1.5	200	0.0
	300	107.3	200	2.0	200	0.3
	MMC 0.05	-	200	28.5	200	1.0	+	-
24 hr	0	100	200	2.0	200	0.8
	37.5	104.7	200	1.5	200	0.3	-	-
	75.0	93.3	200	1.0	200	0.3
	150	100.0	200	0.5	200	0.0
	300	83.1	Not observed due to excessive precipitation
	MMC 0.05	-	200	30.5	200	0.0	+	-

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions, K-36 does not show mutagenic activity in the mammalian cell chromosomal aberration test with CHL/IU cells.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration assay) performed according to OECD guideline 473 and GLP, CHL/IU cells were exposed to K-36 in 1% CMC, at concentrations of 9.34 to 4780 µg/mL with and without metabolic activation. K-36 was tested up to precipitating concentrations and the dose-levels selected for metaphase analysis were 37.5, 75, 150 and 300 µg/mL.

The precipitates of the test substance were observed at the dose levels of 299 µg/mL or more in all treatment methods.

Positive controls induced the appropriate response.
There was no evidence of chromosome aberration induced over background. In all treatment methods, no celle growth inhibition was observed and the IC50 values exceeded 4780 µg/mL.

Therefore, K-36 is considered to be non-clastogenic in this chromosome aberration test when tested up to precipitating concentrations.

This study is classified as acceptable. This study satisfies the requirement for OECD guideline 473 for in vitro cytogenetic mutagenicity data.