Registration Dossier
Registration Dossier
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EC number: 401-540-3 | CAS number: 84632-65-5
Toxicological information
Endpoint summary
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Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In a study according to OECD TG 471 (ames test), the substance did not induce any mutagenic effects.
In a GLP-study according to OECD test guideline 487(in vitro micronucleus test) using human peripheral blood lymphocytes both with and without metabolic activation, the registered substance did not exhibit clastogenic or aneugenic activity.
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). The substance did not induce any relevant mutagenic effects.
Link to relevant study records
- 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
- 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:
- tk locus
- 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 - 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.
- 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'.
- 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
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- preliminary test: 0.05, 0,1, 0.2, 0.5, 1, 3, and 5 mg/ml in culture
main test: 0.05, 0,1 and 0.2 mg/m - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Well established solvent for this test - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 1% DMSO
- Positive controls:
- yes
- Positive control substance:
- other: without S9: colchicine; with S9: cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period:
- Exposure duration: preliminary test: 1h; main test: 4h; repeated test: 26h
NUMBER OF REPLICATIONS: duplicate
NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: cytostasis
Cytokinesis was blocked with Cytochalasin B - Evaluation criteria:
- Cells wits more than 2 nuclei, mechanically damaged cells or cells with irregular shape of nuclei were not analysed. Frequency of binucleate cells with micronuclei was calculated as a measure of chromosome damaging potential of the Test item. The result is considered as positive if the Test item increases the average frequency of aberrant cells more than twice of negative control. The criteria for positive results also include, dose-response relationship and reproducibility of the results in Repeated test. It was controlled that the negative and positive control values correspond to limits of historical data.
- Key result
- Species / strain:
- lymphocytes: human peripheral blood
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In a GLP-study according to OECD test guideline 487, the substance did not exhibit clastogenic or aneugenic activity in cultured human peripheral blood lymphocytes and is therefore considered negative.
- Executive summary:
The clastogenicity and aneugenicity potential of the registered substance was determined using Micronucleus Test in vitro (OECD test guideline 487). The GLP study was carried out in human peripheral blood lymphocytes both with and without metabolic activation system in two separate assays. Seven concentrations were used in the Preliminary test: 0.05, 0.1, 0.2, 0.5, 1, 3 and 5 mg/ml of final culture. There was slight cytotoxic effect in all of the tested concentrations with and without metabolic activation.
Three concentrations of the Test item were selected for the Main test: 0.05, 0.1 and 0.2 mg/ml of final culture. These concentrations were chosen on the basis of low solubility of the Test item under physiological conditions. Concentrations used in the Main test were the highest which could be properly evaluated. The cells were treated with a cytokinesis-blocker (Cytochalasin B) and, therefore, arrested in binucleate state, harvested, and the slides were stained. The binucleate cells were analyzed microscopically for the presence of micronuclei. A total of 2000 binucleate cells were examined per one concentration value on coded slides. Concurrent positive (Colchicine, Cyclophosphamide) and negative (Dimethylsulfoxide) controls were included in each experiment. No significant increase in the percentage of binucleate cells with micronuclei over the exposed cell cultures was observed in any of the doses tested in the Main test. Therefore, the Repeated test was performed. Also in the Repeated test no increase of incidence of percentage of aberrant cells was observed. Again, no significant increase in the percentage of binucleate cells was observed. As the registered substance did not exhibit clastogenic or aneugenic activity in cultured human peripheral blood lymphocytes, it is considered negative.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study. GLP accordance not stated in the report. Test article precipitated in soft agar from the lowest concentration used in the Ames assay. No analytical monitoring of test concentration was done.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- (1981)
- Deviations:
- yes
- Remarks:
- no statistical analysis was performed
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- All strains are histidine auxotrophic mutants.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 102
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- One millilitre activation mixture contained 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.
- Test concentrations with justification for top dose:
- A preliminary toxicity test was carried out with concentrations ranging from 0.08 to 5000 µg/plate suspended in dimethylsulfoxide.
Ames test (without and with microsomal activation): 20, 78, 313, 1250 and 5000 µg/plate suspended in dimethylsulfoxide. - Vehicle / solvent:
- - Vehicle used: dimethylsulfoxide (DMSO)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- other: Daunorubicin-HCl (5 and 10 µg/plate)
- Remarks:
- TA 98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- TA 100 Migrated to IUCLID6: 0.125 and 0.25 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- mitomycin C
- Remarks:
- TA 102 Migrated to IUCLID6: 0.5 and 1 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- sodium azide
- Remarks:
- TA 1535 Migrated to IUCLID6: 2.5 and 5 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA 1537 Migrated to IUCLID6: 50 and 100 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With S9-Mix
- Positive control substance:
- other: 2-aminoanthracene 5 µg/plate
- Remarks:
- TA 98, TA 100, TA 1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With S9-Mix
- Positive control substance:
- other: 2-aminoanthracene 20 µg/plate
- Remarks:
- TA 102
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With S9-Mix
- Positive control substance:
- cyclophosphamide
- Remarks:
- TA 1535 Migrated to IUCLID6: 250 µg/plate
- Details on test system and experimental conditions:
- The study consisted of a toxicity pre-screen test followed by the Ames assay (with and without S9-Mix).
OTHER EXAMINATIONS
A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/plate. Thereafter, the Ames test was performed with 20, 78, 313, 1250 and 5000 µg/plate test article (with and without microsomal activation). The substance precipitated in soft agar at and above a concentration of 20 µg/plate.
MUTATION TEST
Mutation assays with and without microsomal activation were performed twice in triplicate per strain and per group (standard plate test).
Each Petri dish contained approximately 20 ml of minimum agar, 0.1 ml of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth) in 2.0 ml of soft agar. Experiments with microsomal activation contained additionally 0.5 ml of an activation mixture, consistent of S9 fraction of liver from Aroclor 1254 induced rats and co-factors. The plates were incubated for about 48 hours at 37 ± 1.5°C in darkness.
EVALUATION CRITERIA
Arithmetic mean of revertant colonies was calculated. The test substance is considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration. - Statistics:
- No statistical analysis
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The substance precipitated in soft agar at and above a concentration of 20 µg/plate. - Conclusions:
- In a study according to OECD TG 471, the substance did not induce any mutagenic effects.
- Executive summary:
The mutagenic potential of the substance was investigated in a bacterial reverse mutation assay according to OECD TG 471. The substance was tested in concentrations of 20, 78, 313, 1250 and 5000 µg/plate suspended in dimethylsulfoxide
in five strains (TA98, TA100, TA102, TA1535, TA1537) with and without metabolic activation (S9).
While the positive controls were clearly mutagenic, the substance did not increase the number of colonies in any strain. Therefore, the substance is considered to be not mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study. GLP accordance not stated in the report. Test article precipitated in soft agar from the lowest concentration used in the Ames assay. No analytical monitoring of test concentration was done.
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.
3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu and under ambient conditions, read-across of toxicological studies from the source to the target form is scientifically justified.
4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’. - Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- (1981)
- Deviations:
- yes
- Remarks:
- no statistical analysis was performed
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- All strains are histidine auxotrophic mutants.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 102
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- One millilitre activation mixture contained 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.
- Test concentrations with justification for top dose:
- A preliminary toxicity test was carried out with concentrations ranging from 0.08 to 5000 µg/plate suspended in dimethylsulfoxide.
Ames test (without and with microsomal activation): 20, 78, 313, 1250 and 5000 µg/plate suspended in dimethylsulfoxide. - Vehicle / solvent:
- - Vehicle used: dimethylsulfoxide (DMSO)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- other: Daunorubicin-HCl (5 and 10 µg/plate)
- Remarks:
- TA 98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- TA 100 Migrated to IUCLID6: 0.125 and 0.25 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- mitomycin C
- Remarks:
- TA 102 Migrated to IUCLID6: 0.5 and 1 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- sodium azide
- Remarks:
- TA 1535 Migrated to IUCLID6: 2.5 and 5 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without S9-Mix
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA 1537 Migrated to IUCLID6: 50 and 100 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With S9-Mix
- Positive control substance:
- other: 2-aminoanthracene 5 µg/plate
- Remarks:
- TA 98, TA 100, TA 1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With S9-Mix
- Positive control substance:
- other: 2-aminoanthracene 20 µg/plate
- Remarks:
- TA 102
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With S9-Mix
- Positive control substance:
- cyclophosphamide
- Remarks:
- TA 1535 Migrated to IUCLID6: 250 µg/plate
- Details on test system and experimental conditions:
- The study consisted of a toxicity pre-screen test followed by the Ames assay (with and without S9-Mix).
OTHER EXAMINATIONS
A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/plate. Thereafter, the Ames test was performed with 20, 78, 313, 1250 and 5000 µg/plate test article (with and without microsomal activation). The substance precipitated in soft agar at and above a concentration of 20 µg/plate.
MUTATION TEST
Mutation assays with and without microsomal activation were performed twice in triplicate per strain and per group (standard plate test).
Each Petri dish contained approximately 20 ml of minimum agar, 0.1 ml of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth) in 2.0 ml of soft agar. Experiments with microsomal activation contained additionally 0.5 ml of an activation mixture, consistent of S9 fraction of liver from Aroclor 1254 induced rats and co-factors. The plates were incubated for about 48 hours at 37 ± 1.5°C in darkness.
EVALUATION CRITERIA
Arithmetic mean of revertant colonies was calculated. The test substance is considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration. - Statistics:
- No statistical analysis
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Cytotoxicity was assessed in a pretest up to 5000 µg/plate. No further details are available.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The substance precipitated in soft agar at and above a concentration of 20 µg/plate. - Conclusions:
- In a study according to OECD TG 471, the substance did not induce any mutagenic effects.
- Executive summary:
The mutagenic potential of the substance was investigated in a bacterial reverse mutation assay according to OECD TG 471. The substance was tested in concentrations of 20, 78, 313, 1250 and 5000 µg/plate suspended in dimethylsulfoxide
in five strains (TA98, TA100, TA102, TA1535, TA1537) with and without metabolic activation (S9).
While the positive controls were clearly mutagenic, the substance did not increase the number of colonies in any strain. Therefore, the substance is considered to be not mutagenic.
- 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
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.
3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu and under ambient conditions, read-across of toxicological studies from the source to the target form is scientifically justified.
4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’. - Reason / purpose for cross-reference:
- reference to same study
- 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:
- tk locus
- 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 - 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.
- 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'.
- 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
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.
3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu and under ambient conditions, read-across of toxicological studies from the source to the target form is scientifically justified.
4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’. - Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- preliminary test: 0.05, 0,1, 0.2, 0.5, 1, 3, and 5 mg/ml in culture
main test: 0.05, 0,1 and 0.2 mg/m - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Well established solvent for this test - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 1% DMSO
- Positive controls:
- yes
- Positive control substance:
- other: without S9: colchicine; with S9: cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period:
- Exposure duration: preliminary test: 1h; main test: 4h; repeated test: 26h
NUMBER OF REPLICATIONS: duplicate
NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: cytostasis
Cytokinesis was blocked with Cytochalasin B - Evaluation criteria:
- Cells wits more than 2 nuclei, mechanically damaged cells or cells with irregular shape of nuclei were not analysed. Frequency of binucleate cells with micronuclei was calculated as a measure of chromosome damaging potential of the Test item. The result is considered as positive if the Test item increases the average frequency of aberrant cells more than twice of negative control. The criteria for positive results also include, dose-response relationship and reproducibility of the results in Repeated test. It was controlled that the negative and positive control values correspond to limits of historical data.
- Key result
- Species / strain:
- lymphocytes: human peripheral blood
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In a GLP-study according to OECD test guideline 487, the substance did not exhibit clastogenic or aneugenic activity in cultured human peripheral blood lymphocytes and is therefore considered negative.
- Executive summary:
The clastogenicity and aneugenicity potential of the registered substance was determined using Micronucleus Test in vitro (OECD test guideline 487). The GLP study was carried out in human peripheral blood lymphocytes both with and without metabolic activation system in two separate assays. Seven concentrations were used in the Preliminary test: 0.05, 0.1, 0.2, 0.5, 1, 3 and 5 mg/ml of final culture. There was slight cytotoxic effect in all of the tested concentrations with and without metabolic activation.
Three concentrations of the Test item were selected for the Main test: 0.05, 0.1 and 0.2 mg/ml of final culture. These concentrations were chosen on the basis of low solubility of the Test item under physiological conditions. Concentrations used in the Main test were the highest which could be properly evaluated. The cells were treated with a cytokinesis-blocker (Cytochalasin B) and, therefore, arrested in binucleate state, harvested, and the slides were stained. The binucleate cells were analyzed microscopically for the presence of micronuclei. A total of 2000 binucleate cells were examined per one concentration value on coded slides. Concurrent positive (Colchicine, Cyclophosphamide) and negative (Dimethylsulfoxide) controls were included in each experiment. No significant increase in the percentage of binucleate cells with micronuclei over the exposed cell cultures was observed in any of the doses tested in the Main test. Therefore, the Repeated test was performed. Also in the Repeated test no increase of incidence of percentage of aberrant cells was observed. Again, no significant increase in the percentage of binucleate cells was observed. As the registered substance did not exhibit clastogenic or aneugenic activity in cultured human peripheral blood lymphocytes, it is considered negative.
Referenceopen allclose all
| 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) |
||||
The number of histidine-prototrophic mutants after treatment with the substance did not vary markedly in comparison to the negative control in any of the experiments.
The number of histidine-prototrophic mutants after treatment with the substance did not vary markedly in comparison to the negative control in any of the experiments.
| 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) |
||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Tested in a study in principle similar to OECD TG 474 (in vivo micronucleus test in vivo), the substance was not mutagenic to Chinese Hamsters.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- GLP compliance was not stated in the report. The test conduct was in principle similar to the OECD 474 (1981). However, with following deviations: Mice or rats are recommended in the guideline; Chinese hamster were used in this study. Additionally, 1000 polychromatic erythrocytes/animal were scored for the incidence of micronuclei instead of 2000 as claimed in the guideline.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- Mice or rats are recommended in the guideline. Chinese hamster were used in this study. Additionally, 1000 polychromatic erythrocytes/animal were scored for the incidence of micronuclei instead of 2000 claimed in the guideline.
Both deviations can be assumed to not have affected the study and its results. - Deviations:
- yes
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- hamster, Chinese
- Strain:
- other: random outbred
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain as stated in the report: Chinese hamster (Cricetulus griseus), random outbred strain
- Source: CIBA-GEIGY LTD. Tierfarm, Sisseln, Switzerland
- Age at study initiation: 6 - 10 weeks (males), 4 - 9 weeks (females)
- Weight at study initiation: 20 - 28 g (females), 23 - 33 g (males)
- Housing: individually
- Diet (ad libitum): standard diet, NAFAG No. 924
- Water (ad libitum): tap water
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23
- Humidity (%): 48 - 51
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle: 0.5 % aqueous solution of sodium carboxymethylcellulose (CMC)
- Concentration of test material in vehicle: 5000 mg/kg bw in 20 ml/kg bw - Duration of treatment / exposure:
- After single oral administration of 5000 mg/kg bw animals were observed for 16, 24 and 48 h.
- Frequency of treatment:
- Single oral administration.
- Post exposure period:
- 16, 24 and 48 h.
- Remarks:
- Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested - No. of animals per sex per dose:
- 16 animals (8 females, 8 males).
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- Animals treated with cyclophosphamide (64 mg/kg bw in 20 ml/kg bw 0.5 % CMC) served as positive control.
- Tissues and cell types examined:
- Bone marrow was harvested from the shafts of both femurs. Normochromatic erythrocytes (NCE) as well as polychromatic erythrocytes (PCE) were counted for determination of the PCE/NCE-ratio. 1000 PCE per animal were used to determine the incidence of micronucleated PCE.
- Details of tissue and slide preparation:
- Bone marrow was harvested from the shafts of both femurs with a pipette and aspirated gently in 0.5 µl rat serum. Small drops of the mixture were transferred on a slide and spread out by pulling it behind a cover glass. The preparations were air-dried and stained in undiluted May-Grünwald solution (2 min) and subsequently in May-Grünwald solution/water 1/1 (2 min), followed by 20 minutes in 40 % Giemsa's. Following rinsing and washing with 55 % methanol and/or water, the slides were air-dried, cleared in Xylene and mounted in Eukitt.
- Statistics:
- The significance of difference was assessed by Chi square - test.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Remarks:
- (Micronucleated PCE cells 0.18 % versus 0.13 % in negative control)
- Toxicity:
- no effects
- Remarks:
- (No significant difference in PCE/NCE-ratio between treated and negative control)
- Vehicle controls validity:
- valid
- Negative controls validity:
- other: see vehicle control
- Positive controls validity:
- valid
- Conclusions:
- Tested in a study in principle similar to OECD TG 474 (in vivo micronucleus test in vivo), the substance was not mutagenic to Chinese Hamsters.
- Executive summary:
Tested in a study in principle similar to OECD TG 474 (in vivo micronucleus test in vivo), the substance was not mutagenic to Chinese Hamsters.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- GLP compliance was not stated in the report. The test conduct was in principle similar to the OECD 474 (1981). However, with following deviations: Mice or rats are recommended in the guideline; Chinese hamster were used in this study. Additionally, 1000 polychromatic erythrocytes/animal were scored for the incidence of micronuclei instead of 2000 as claimed in the guideline.
- Justification for type of information:
- 1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.
3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu and under ambient conditions, read-across of toxicological studies from the source to the target form is scientifically justified. In addition, the zeta-potential of both forms indicated that agglomeration increases with lower pH, which further supports the read-across for studies with oral exposure. The low gastric pH increases agglomeration, which supports the conclusion that both forms are not bioavailable upon oral exposure.
4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’. - Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- Mice or rats are recommended in the guideline. Chinese hamster were used in this study. Additionally, 1000 polychromatic erythrocytes/animal were scored for the incidence of micronuclei instead of 2000 claimed in the guideline.
Both deviations can be assumed to not have affected the study and its results. - Deviations:
- yes
- Principles of method if other than guideline:
- Mice or rats are recommended in the guideline. Chinese hamster were used in this study. Additionally, 1000 polychromatic erythrocytes/animal were scored for the incidence of micronuclei instead of 2000 claimed in the guideline.
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- hamster, Chinese
- Strain:
- other: random outbred
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Strain as stated in the report: Chinese hamster (Cricetulus griseus), random outbred strain
- Source: CIBA-GEIGY LTD. Tierfarm, Sisseln, Switzerland
- Age at study initiation: 6 - 10 weeks (males), 4 - 9 weeks (females)
- Weight at study initiation: 20 - 28 g (females), 23 - 33 g (males)
- Housing: individually
- Diet (ad libitum): standard diet, NAFAG No. 924
- Water (ad libitum): tap water
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23
- Humidity (%): 48 - 51
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle: 0.5 % aqueous solution of sodium carboxymethylcellulose (CMC)
- Concentration of test material in vehicle: 5000 mg/kg bw in 20 ml/kg bw - Duration of treatment / exposure:
- After single oral administration of 5000 mg/kg bw animals were observed for 16, 24 and 48 h.
- Frequency of treatment:
- Single oral administration.
- Post exposure period:
- 16, 24 and 48 h.
- Remarks:
- Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested - No. of animals per sex per dose:
- 16 animals (8 females, 8 males).
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- Animals treated with cyclophosphamide (64 mg/kg bw in 20 ml/kg bw 0.5 % CMC) served as positive control.
- Tissues and cell types examined:
- Bone marrow was harvested from the shafts of both femurs. Normochromatic erythrocytes (NCE) as well as polychromatic erythrocytes (PCE) were counted for determination of the PCE/NCE-ratio. 1000 PCE per animal were used to determine the incidence of micronucleated PCE.
- Details of tissue and slide preparation:
- Bone marrow was harvested from the shafts of both femurs with a pipette and aspirated gently in 0.5 µl rat serum. Small drops of the mixture were transferred on a slide and spread out by pulling it behind a cover glass. The preparations were air-dried and stained in undiluted May-Grünwald solution (2 min) and subsequently in May-Grünwald solution/water 1/1 (2 min), followed by 20 minutes in 40 % Giemsa's. Following rinsing and washing with 55 % methanol and/or water, the slides were air-dried, cleared in Xylene and mounted in Eukitt.
- Statistics:
- The significance of difference was assessed by Chi square - test.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Remarks:
- (Micronucleated PCE cells 0.18 % versus 0.13 % in negative control)
- Toxicity:
- no effects
- Remarks:
- (No significant difference in PCE/NCE-ratio between treated and negative control)
- Vehicle controls validity:
- valid
- Negative controls validity:
- other: see vehicle control
- Positive controls validity:
- valid
- Conclusions:
- Tested in a study in principle similar to OECD TG 474 (in vivo micronucleus test in vivo), the substance was not mutagenic to Chinese Hamsters.
- Executive summary:
Tested in a study in principle similar to OECD TG 474 (in vivo micronucleus test in vivo), the substance was not mutagenic to Chinese Hamsters.
Referenceopen allclose all
One female animal of the control group died during the experiment.
The percentage of micronucleated PCE was 0.13 % in the negative, 0.18 % in the treated group and 3.34 % in the positive control. As expected the positive control was statistically significantly different from negative control.
One female animal of the control group died during the experiment.
The percentage of micronucleated PCE was 0.13 % in the negative, 0.18 % in the treated group and 3.34 % in the positive control. As expected the positive control was statistically significantly different from negative control.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Note that the in vivo study was conducted spontaneously in parallel to the Ames and in vitro micronucleus tests, i.e. there was no specific reasons that triggered the study.
Justification for classification or non-classification
Based on in vitro data from three different genetic toxicity tests and on a supportive in vivo micronucleus test, the registered substance does not need to be classified for genetic toxicity and mutagenicity.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
