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EC number: 401-540-3 | CAS number: 84632-65-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 015
- 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
- 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
Constituent 1
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
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