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 mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

401-540-3

EC Name:

-

Cas Number:

84632-65-5

Molecular formula:

C18H10Cl2N2O2

IUPAC Name:

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

Test material form:

solid: particulate/powder

Method

Target gene:

tk locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 10 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

other: L5178Y TK+/- 3.7.2 C mouse lymphoma

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

3.2; 10; 32; 100; 316 and 1000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: medium (RPMI 5)
- Justification for choice of solvent/vehicle: none required

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 5 medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: 4-nitroquinoline-N-oxide; with S9: Cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Assay 1: 3h; Assay 2: 24 (-S9) and 3 h (+S9)
- Expression time (cells in growth medium): 2 days

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: other: Harmonized relative survival

Evaluation criteria:

Determination of Survival or Viability
From the zero term of the Poisson distribution the probable number of clones/well (P) on microtiter plates in which there are empty wells (EW, without clones) out of a total of wells (TW) is given by:
P = -ln (EW/TW)
The plating efficiency (PE) in any given culture is therefore:
PE = P/1.6
The percentage relative survival (%RS) in each test culture was determined by comparing plating efficiencies in test and vehicle control cultures thus:
% RS = [PE (test)/PE (control)] x 100
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival value for each dose of test item was adjusted as follows:
Harmonised %RS= %RS x (Post treatment cell concentration for dose/Post treatment cell concentration for vehicle control)
All percentage relative survival (%RS) values were adjusted as described above.
Additionally the suspension growth (SG), the relative viability (RV) and relative total growth (RTG) was calculated as follows:
SG = DCG1 x DCG2 (DCG: daily cell growth)
DCG (1 or 2) = cell concentration on day 1 or 2/ 2 x 10^5 (the initial cell concentration)
The RSG (relative suspension growth) was calculated as follows:
% RSG = [SG (test)/SG (control)] x 100
The percentage relative viability (%RV) in each test culture was determined by comparing plating efficiencies in test and vehicle control cultures thus:
% RV = [PE (test)/PE (control)] x 100
The relative total growth was calculated as follows:
RTG (%) = RV x RSG (%)

Determination of Mutant Frequency
It is usual to express mutant frequency (MF) as "mutants per 10^6 viable cells". In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated:
MF = [P (mutant)/2 x 103] x [1.6/P (viable)] x 10^6
= {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 800
The small and large colony mutant frequencies were not calculated.

Statistics:

The heterogeneity of the obtained data was tested. The statistical significance of mutant frequencies (total wells with clones) was carried out using Dunnett’s Test, using TOXSTAT statistical software. The positive control data were compared with the respective vehicle control or untreated control data with 2 Sample t-Test, using TOXSTAT statistical software. The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: highly water insoluble
- Precipitation: yes (in Assay 2 for 10-1000 µg/ml)

RANGE-FINDING/SCREENING STUDIES:
The concentrations applied in the assays (at the 3-hour and 24-hour treatments) were chosen according to the solubility and cytotoxicity results of the pre-experiments. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequency of the negative (vehicle) control cultures were within the expected normal range (50-170 mutants per 10^6 viable cells) in the performed experiments in line with the historical controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary table of the mutagenicity results of Assay 1
Concentration (μg/mL)	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Mutation frequency
Treatment period (hours): 3
Without Exogenous Metabolic Activation (-S9 Mix)
RPMI 5 Medium (Vehicle Control)	629/768	98/768	41/768	88.37
3.2	611/768	108/768	49/768	94.84 n.s.
10	634/768	92/768	42/768	96.21 n.s.
32	619/768	98/768	51/768	104.65 n.s.
100	623/768	99/768	46/768	92.68 n.s.
316	622/768	94/768	52/768	105.91 n.s.
1000	622/768	91/768	55/768	87.48 n.s.
Positive reference control (NQO) (0.1 μg/mL)	184/768	343/768	241/768	787.30 **
Treatment period (hours): 3
With Exogenous Metabolic Activation (+S9 Mix)
RPMI 5 Medium (Vehicle Control)	610/768	119/768	39/768	101.86
3.2	582/768	126/768	60/768	120.08 n.s.
10	618/768	109/768	41/768	92.27 n.s.
32	612/768	90/768	66/768	118.39 n.s.
100	588/768	126/768	54/768	106.99 n.s.
316	578/768	132/768	58/768	114.23 n.s.
1000	597/768	113/768	58/768	120.20 n.s.
Positive reference control (CP) (5 μg/mL)	234/768	300/768	234/768	1392.03 **
NQO : 4-Nitroqinoline-N-oxide; CP : Cyclophosphamide
**: Statistically significantly different compared to the control (2 Sample t-Test; α = 0.01)
n.s: Statistically not significantly different compared to the control (Dunnett’s Test; α = 0.05)

Summary table of the mutagenicity results of Assay 2
Concentration (μg/mL)	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Mutation frequency
Treatment period (hours): 24
Without Exogenous Metabolic Activation (-S9 Mix)
RPMI 5 Medium (Vehicle Control)	618/768	108/768	42/768	100.61
3.2	624/768	109/768	35/768	96.56 n.s.
10	618/768	104/768	46/768	104.97 n.s.
32	629/768	94/768	45/768	93.32 n.s.
100	615/768	117/768	36/768	125.41 n.s.
316	601/768	123/768	44/768	133.88 *
1000	602/768	113/768	53/768	130.86 *
Positive reference control (NQO) (0.1 μg/mL)	189/768	356/768	223/768	927.11 **
Treatment period (hours): 3
With Exogenous Metabolic Activation (+S9 Mix)
RPMI 5 Medium (Vehicle Control)	601/768	124/768	43/768	112.01
3.2	609/768	123/768	36/768	118.20 n.s.
10	620/768	113/768	35/768	96.88 n.s.
32	616/768	107/768	45/768	104.37 n.s.
100	619/768	101/768	48/768	104.52 n.s.
316	589/768	126/768	53/768	124.42 n.s.
1000	624/768	109/768	35/768	94.19 n.s.
Positive reference control (CP) (5 μg/mL)	224/768	325/768	219/768	1161.27 **
NQO : 4-Nitroqinoline-N-oxide; CP : Cyclophosphamide
*: Statistically significantly different compared to the control (Dunnett’s Test; α = 0.05)
**: Statistically significantly different compared to the control (2 Sample t-Test; α = 0.01)
n.s: Statistically not significantly different compared to the control (Dunnett’s Test; α = 0.05)

Applicant's summary and conclusion

Conclusions:

In a study according to OECD Test Guideline 476 (under GLP conditions), the substance did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line).

Executive summary:

The genetic toxicity of the substance was investigated in a Mouse Lymphoma assay using L5178Y TK+/-

3.7.2 C cells according to OECD Test Guideline 476 under GLP conditions with and without metabolic activation (±S9 Mix). Based on the results of the preliminary Solubility and Toxicity Tests and regarding the practical difficulties with the handling, PR 254 was suspended and diluted in RPMI 5 Medium and the RPMI 5 Medium was parallel investigated as vehicle control.

The following concentrations were investigated in the Assay 1: 3-hour treatment (±S9 Mix): 3.2; 10; 32; 100; 316 and 1000 μg/mL; The following concentrations were investigated in the Assay 2: 24-hour treatment (-S9 Mix): 3.2; 10; 32; 100; 316 and 1000 μg/mL;3-hour treatment (+S9 Mix): 3.2; 10; 32; 100; 316 and 1000 μg/mL.

The performed Assays fulfilled the validity criteria regarding the negative control and positive controls as well as in connection with the number of analysable concentration levels (at least four). In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments.

In the performed assays the obtained mutation frequencies (in absence and also in presence of exogenous metabolic activation) did not show dose-related tendencies, did not exceed the relevant GEF thresholds for positive call and remained within the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different (Dunnett’s Test, α = 0.05) from that of the corresponding vehicle control at the 3-hour treatments (±S9 Mix) and the obtained statistical significances at the 24-hour treatment at the concentration levels of 316 and 1000 μg/mL were not considered as biologically relevant.Therefore, the substance is considered to be not mutagenic in this test according to OECD 476