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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study with well-characterizsed material, read across substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

416-250-2

EC Name:

-

Cas Number:

84632-59-7

Molecular formula:

C26 H28 N2 O2

IUPAC Name:

3,6-Bis-(4-tert-butyl-phenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione

Test material form:

solid: particulate/powder

Details on test material:

Batch: 0005694917
Description Yellow solid (powder)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 14 December 2021

Method

Target gene:

hpprt

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

				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

Applicant's summary and conclusion

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.