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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity, in vitro, Ames-test in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation (according to OECD TG 471, GLP): no mutagenic effect of the test substance was observed (Ciba-Geigy Ltd 1993e). Chromosome aberration assay in V79 cells (according to OECD TG 473, GLP): No chromosome-damaging (clastogenic) effect was observed after treatment with the test substance in the absence and the presence of metabolic activation (CCR 1994). Gene mutation assay in V79 cells (according to OECD TG 476, GLP): non mutagenic (Harlan 2012).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study with well-characterizsed material

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hpprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

25 - 800 µg/ml (4h, without S9)
2.5 - 80 µg/ml (all other incubations)

Vehicle / solvent:

culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Two days (experiment I and experiment II with S9 mix) or three days (experiment II without S9 mix) after sub-cultivation stock cultures
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 7 -9 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 19 days

SELECTION AGENT (mutation assays): 6-thioguanine


NUMBER OF REPLICATIONS: Population doubling time 12 - 14h

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Positive controls validity:

valid

Additional information on results:

Osmolarity: 316 mOsm (at 4 mg/mL)
pH: 7.28
Precipitation occurred at doses of 20 microgramms/ml and above.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL		mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control with medium			-	100.0	100.0	100.0	33.1	1.0	100.0	100.0	100.0	16.6	1.0
Positive control (EMS)	150.0		-	71.3	88.1	118.3	210.0	6.3	72.1	92.3	90.5	219.0	13.2
Test item	25.0	P	-	84.7	108.6	101.2	43.6	1.3	99.1	93.3	95.2	16.5	1.0
Test item	50.0	P	-	80.7	103.5	96.6	28.1	0.8	96.3	96.0	102.5	35.2	2.1
Test item	100.0	P	-	85.7	82.1	105.9	32.0	1.0	97.6	79.6	105.0	39.7	2.4
Test item	200.0	P	-	82.0	92.3	125.1	32.2	1.0	95.7	81.2	97.9	22.2	1.3
Test item	400.0	P	-	91.2	91.4	94.2	13.6	0.4	88.8	97.0	98.9	33.6	2.0
Test item	800.0	P	-	90.9	culture was not continued^#				81.0	culture was not continued^#
Solvent control with medium			+	100.0	100.0	100.0	34.9	1.0	100.0	100.0	100.0	20.1	1.0
Positive control (DMBA)	1.1		+	78.1	101.6	80.3	487.9	14.0	65.2	62.3	97.8	305.9	15.2
Test item	2.5		+	88.8	127.9	132.2	12.0	0.3	108.5	53.8	87.0	19.2	1.0
Test item	5.0		+	81.6	109.0	137.4	17.0	0.5	100.5	99.5	84.6	20.8	1.0
Test item	10.0		+	84.4	122.6	91.2	17.3	0.5	108.5	81.7	76.6	27.3	1.4
Test item	20.0	P	+	88.5	171.1	116.1	10.5	0.3	101.8	60.3	87.8	26.2	1.3
Test item	40.0	P	+	87.5	118.0	101.9	17.4	0.5	102.8	60.8	72.0	32.6	1.6
Test item	80.0	P	+	84.8	culture was not continued^#				103.2	culture was not continued^#
Experiment II / 24 h treatment				culture I					culture II
Solvent control with medium			-	100.0	100.0	100.0	14.8	1.0	100.0	100.0	100.0	12.7	1.0
Positive control (EMS)	150.0		-	115.8	81.0	99.5	455.0	30.8	117.8	73.8	110.2	759.2	59.7
Test item	2.5		-	119.0	98.0	97.6	22.0	1.5	115.4	93.6	100.3	15.0	1.2
Test item	5.0		-	130.2	85.7	99.0	14.4	1.0	110.9	86.0	102.7	15.8	1.2
Test item	10.0		-	119.4	89.2	96.0	9.8	0.7	119.1	103.6	102.4	18.2	1.4
Test item	20.0	P	-	127.3	86.8	96.5	10.6	0.7	125.1	97.2	104.4	17.5	1.4
Test item	40.0	P	-	126.9	92.2	95.9	20.4	1.4	119.6	93.4	97.6	25.9	2.0
Test item	80.0	P	-	123.1	culture was not continued^#				126.6	culture was not continued^#
Experiment II / 4 h treatment
Solvent control with medium			+	100.0	100.0	100.0	8.9	1.0	100.0	100.0	100.0	11.0	1.0
Positive control (DMBA)	1.1		+	38.1	77.2	94.4	736.6	82.5	46.6	66.6	99.2	619.3	56.1
Test item	2.5		+	97.3	104.5	93.8	10.7	1.2	103.2	93.7	98.3	11.6	1.1
Test item	5.0		+	100.7	97.7	95.6	15.3	1.7	95.6	101.8	100.2	22.4	2.0
Test item	10.0		+	93.0	87.2	98.6	13.0	1.5	90.9	93.3	97.7	14.8	1.3
Test item	20.0	P	+	95.9	102.0	96.0	20.7	2.3	99.5	100.4	98.8	25.0	2.3
Test item	40.0	P	+	94.1	142.5	97.4	15.1	1.7	93.4	104.2	114.0	14.4	1.3
Test item	80.0	P	+	94.4	culture was not continued^#				100.6	culture was not continued^#

P = Precipitation; #too many precipitates

Conclusions:

Interpretation of results (migrated information):
negative

The substance did not cause gene mutations in mammalian cells in vitro. Test concentrations were limited by strong precipitation of in the culture medium.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity, in vitro:

To assess the genetic toxicity of the test substance an Ames-test and a chromosomal aberrations assay in V79 cells were performed.

An Ames test with the test substance (purity> 97%) was performed in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A in the presence and absence of a metabolic activation system (cofactor supplemented post-mitochondrial fraction prepared from the livers of male Wistar rats treated with phenobarbital and beta-naphthoflavone; Ciba-Geigy Ltd 1993e). A standard plate test and a preincubation test were performed with up to a top dose of 5000 µg test substance per plate with DMSO as vehicle, respectively. Precipitation of the test substance was found from about 61 μg/plate onward without S9 mix and from about 312 μg/plate onward with S9 mix. No mutagenic effect of the test substance, indicated by an increased mutant frequency was observed in the Salmonella or E. coli strains tested in the absence and presence of metabolic activation. Furthermore, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain with and without S9 -mix.

The study was performed according to OECD guideline 471 and under GLP. It is valid without restriction.

The substance was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in two independent GLP compliant experiments according to OECD guideline 473 (CCR 1994).

The chromosomes were prepared 18 h and 28 h after start of treatment with the test article, which was formulated in DMSO. The

treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two

parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated (18 h: 3 concentrations; 28 h; highest evaluable concentration):

18 h: 300; 3000; 5000 ng/ml 28 h: 5000 ng/ml. The highest concentration used in the experiments (5000 ng/ml) was close to the solubility limit in DMSO. A precipitation of the test article in the culture medium was observed at this concentration (microscopically visible). Toxic effects determined in a pre-test on toxicity (qualitative evaluation of cell density and morphology as indicator for toxicity response) and in the cytogenetic experiments (reduction of the mitotic index) could not be observed up to the highest concentration used (5000 ng/ml). In both independent experiments, there were no biologically or statistically relevant increases in cells with structural aberrations after treatment with the test article at both fixation intervals with and without S9 mix. In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article compared to the frequencies of the controls .

A GLP and OECD guideline 476 compliant study was performed to investigate the potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Harlan 2012). The assay was performed in two independent experiments, using two parallel cultures each. 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 maximum concentration of the pre-experiment was 4000 µg/mL or approximately 10 mM according to the current OECD guideline 476.The test item was suspended in culture medium. 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. Appropriate reference mutagens (and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. The substance is therefore considered to be non mutagenic in mammalian cells in vitro.

Justification for selection of genetic toxicity endpoint
arbitrary, all three studies needed for the assessment.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

Classification, Labelling, 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 mutagenicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive EC 618/2012.