Registration Dossier
Registration Dossier
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EC number: 226-999-5 | CAS number: 5590-18-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation test:
The registered chemical i.e. 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) was tested non-mutagenic (negative) in a bacterial reverse mutation test (OECD 471) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
Chromosomal aberrations
The registered chemical i.e. 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) was tested non-clastogenic (negative) in an in vitro cytogenicity test (OECD 473) using human peripheral blood lymphocytes.
Mammalian cell mutagenicity:
The registered chemical i.e. 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) was tested non-mutagenic (negative) in an in vitro mammalian cell gene mutation test (OECD 476) using CHO cells.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- 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:
- The study contains experimental data of the registered substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21July 1997, corrected 26 June 2020
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Purity:99%
- Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced S9 metabolic activation system was used. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to get a final concentration of approximately 10 % v/v in the S9 mix
Composition of S9 mix:
D-glucose-6-phosphate 0.8 g
β-NADP 1.75 g
MgCl2 1.0 g
KCl 1.35 g
Na2HPO4.H2O 6.4 g
NaH2PO4.H2O 1.4 g - Test concentrations with justification for top dose:
- 0.0 (NC), 0.0 (VC), 0.156, 0.313, 0.625, 1.250 and 2.50 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was found insoluble in distilled water. Therefore, the solubility was checked in Dimethyl Sulphoxide (DMSO) and found soluble at 50 mg/mL. Thus, DMSO was chosen as a solvent for pre-experiment, Trial I and Trial II. - Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
- Details on test system and experimental conditions:
- For each strain and dose level, including controls, three plates (triplicate) were used. For the plate incorporation method (Trial I), the following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, negative control, vehicle control, and reference mutagen solution (positive control),
500 µL S9 mix (for the test with metabolic activation) or S9 mix substitution buffer (for the test without metabolic activation),
100 µL Bacterial suspension,
2000 µL Overlay agar
For the preincubation assay (Trial II), 100 µL test solution, 500 µL S9 mix and S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and incubated at 37 °C for 60 minutes. After preincubation, 2.0 mL overlay agar (47°C) was added to each tube. The mixture was poured on minimal agar plates. After solidification, the plates were incubated in an inverted position for 48 hours at 37 °C. - Evaluation criteria:
- A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged as biologically relevant if it was reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative control and vehicle control, the increase was not considered biologically relevant. - Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Results of the solubility test:
The test item was found insoluble in distilled water. Therefore, the solubility was checked in Dimethyl Sulphoxide (DMSO) and found soluble at 50 mg/mL. Thus, DMSO was chosen as a solvent for pre-experiment, Trial I and Trial II.
Results of the precipitation test:
Insolubility was assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. The test item was dissolved in DMSO at 50 mg/mL concentration and checked for precipitation on agar. Precipitation was observed at 5 and 3.5 mg/plate. Slight precipitation was noted at 2.5 mg/plate.
The test item was further assessed for precipitation at 3.5, 3.0 and 2.75 mg/plate concentrations. The results are the follows:
Precipitation was observed at 3.5 mg/plate, 3.0 mg/plate and 2.75 mg/plate concentrations, which was considered to interfere with the colony count. Therefore 2.5 mg/plate was selected as the highest concentration for pre-experiment.
Preliminary cytotoxicity test:
The cytotoxicity test was performed with strains TA98 and TA100. Eight concentrations (i.e. 0.0 (negative control; NC), 0.0 (vehicle control; VC), 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate) with half-log intervals (√10) were tested for toxicity and mutation induction with 3 plates each (triplicates). In TA98 and TA100 tester strains, no reduction in revertant colonies but moderate inhibition of background lawn was observed at 2.5 mg/plate (T8) both in the presence and absence of S9 metabolic activation. No reduction in colony count and diminished background lawn was observed at concentrations of 0.791 – 0.001 mg/plate (T7-T1) neither in the presence (+S9) nor the absence (-S9) of metabolic activation. Based on the results of the pre-experiment, the following doses were selected for the main study trials:0.0 (NC), 0.0(VC), 0.156, 0.313, 0.625, 1.250 and 2.5 mg/plate, both in the absence (-S9) and presence of metabolic activation (+S9). - Remarks on result:
- other: No mutagenic potetial
- Conclusions:
- The registered chemical 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system in study performed according to OECD TG 471.
- Executive summary:
A bacterial reverse mutation assay (OECD TG 471) was performed to investigate the potential of the registered chemical 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments with and without S9 metabolic activation system. The test concentrations were selected based on the results of a pre-liminary cytotoxicity test. The mutagenicity test was performed with the following test substance concentrations: 0 (NC), 0 (VC), 0.156, 0.313, 0.625, 1.250 and 2.5 mg/plate in both trials and in the presence (+S9) and absence of metabolic activation (-S9). Results: No significant increase in the number of revertant colonies, either in the presence or absence of metabolic activation was observed at any concentrations tested as compared to the vehicle control. No trend of an increased number of revertant colonies with increased dosing of the test item was observed. The spontaneous reversion rates in the negative and vehicle controls were within the historical range. Each strain-specific positive control in Trial I and II showed a significant increase in the number of revertant colonies. Conclusion:The registered chemical 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation in study performed according to OECD TG 471.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- 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:
- The study contains experimental data of the registered substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Purity: 98.93%
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 30-32 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-indued liver S9 microsomal fraction
- Test concentrations with justification for top dose:
- 0.0 (NC), 0.0 (VC) 0.062 (T1), 0.125 (T2) and 0.250 (T3) mg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks
DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time: 21 hrs (with and without metabolic activation system- Phase I and II)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer
NUMBER OF REPLICATIONS: No data
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (23 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.
NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations per cytotoxicity experiment based on the solubility, precipitation and pH test of the test item were selected. Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.250 (T1), 0.5 (T2) and 1 (T3) mg/mL of culture media. Cytotoxicity was observed in treated concentrations of 1 (T3), 0.5 (T2) mg/mL both in the absence and in the presence of metabolic activation (1%). In the cytotoxicity experiment, the test concentrations 1 (T3), 0.5 (T2) mg/ mL of culture media showed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence, this concentration was not selected for the main study. Hence, 0.250 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data
- OTHER: No data - Rationale for test conditions:
- No data
- Evaluation criteria:
- A test item can be classified as clastogenic if:
At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
If the increase is dose-related
Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
If there is no dose-related increase
All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.
If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed. - Statistics:
- Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
- Species / strain:
- lymphocytes: Human perpheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- In the cytotoxicity experiment III the highest test concentration 0.016 (T9) mg/ mL of culture media show 47.16 % reduction in absence of metabolic activation and 48.69% in the presence of metabolic activation indicates slight cytotoxicity of test item.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 °C. No significant change in pH was observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was no precipitation observed at 1 mg/mL concentration.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations (0.25, 0.5 and 1 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control.
Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.250 (T1), 0.5 (T2) and 1 (T3) mg/mL of culture media. Cytotoxicity was observed in treated concentrations of 1 (T3), 0.5 (T2) mg/mL both in the absence and in the presence of metabolic activation (1%).
In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.84 (VC), 7.34 (T1), 4.75 (T2), 3.27 (T3) and 8.49 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.28 (NC), 9.93 (VC), 7.55 (T1), 4.90 (T2), 3.60 (T3) and 8.74 (PC).
In the cytotoxicity experiment, the test concentrations 1 (T3), 0.5 (T2) mg/ mL of culture media showed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence, this concentration was not selected for the main study.
Hence, 0.250 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: Please refer table remarks section
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical is not mutagenic at the highest tested concentration of 0.250 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
- Executive summary:
This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0 (NC), 0 (VC), 0.062, 0.125 and 0.250 mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0 (NC), 0 (VC), 0.25, 0.5 and 1.0 mg/mL of culture media based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. The test chemical was tested negative, non-clastogenic at the highest tested concentration of 0.250 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- 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:
- The study provides experimental data on the registered substance.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- Adopted July 29, 2016
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- Purity: 98.8%
- Target gene:
- Hprt gene
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: NCCS, Pune, India
- Suitability of cells: As per recommendations specified in OECD 476
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: CHO cells were cultured in complete RPMI-1640 medium (10 % Fetal bovine serum), 100 units Penicillin/ml, 10 µg Streptomycin/ml, and incubated at 37±2 °C, 5% CO2 in a CO2 incubator. - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital and β-naphthoflavone-induced rat liver microsomal fraction (S9 homogenate) was used.
- Test concentrations with justification for top dose:
- 0 mg/ml (solvent control)
0 mg/ml (negative control)
0.25 mg/ml
0.5 mg/ml
1 mg/ml
2 mg/ml
Justification: No limiting cytotoxicity was observed in the preliminary cytotoxicity assay as the relative survival values were ≥ 65 at 2 mg/ml (-S9, +S9). - Vehicle / solvent:
- Vehicle: DMSO
- Justification for choice of solvent/vehicle:The Test Item was found to be insoluble in distilled water (200 mg/ml) and found to be soluble in dimethyl sulfoxide (200 mg/ml). Hence, dimethyl sulfoxide (DMSO) was selected as a vehicle for the study. - Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Cultures of CHO cells were grown in a complete RPMI-1640 medium; cells at a density of 10 x 106/25 cm2 were used for cytotoxicity measurement and mutation frequency calculation. Before the experiment, spontaneous mutant CHO cells were cleansed by the treatment with 100 μM hypoxanthine, 400 µM aminopterin and 16 μM thymidine (HAT medium) for 3 days at 37 ±2°C in a CO2 incubator.
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: One experiment was performed.
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 x 106 /cm2
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Test substance was added in medium.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): NA
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):7 days
- Selection time (if incubation with a selective agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells):7 days of expression period +9 day mutant selection period =16 days
- Method used: agar or microwell plates for the mouse lymphoma assay: agar plates were used
- Selective agent used: 2x 105cells / 10 ml of cloning medium were seeded with10 µl/ml 6-thioguanine (6TG)in triplicates.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Plating for Cloning efficiency 1 (CE1): 10 ml of cloning media containing 100 cells were dispensed in 60 mm culture plates in triplicates. Plates were incubated at 37±2°C, 5 % CO2, in a CO2 incubator for 7 days.
Plating for expression: 3x105 cells / 5 ml cells were seeded and incubated at 37±2 °C, 5 % CO2 in a CO2 incubator for 7 days to allow phenotypic expression of the induced mutation.
Plating for Cloning efficiency 2 (CE2): At the end of the expression period, cells were trypsinized and counted. Cells at the density of 100 cells / 10 ml of cloning media were plated in 60 mm culture plates in triplicate. The plates were incubated at 37±2 °C, 5 % CO2, in a CO2 incubator for 9 days.
Plating for Mutation Frequency: 2x10 5 cells / 10 ml of cloning media were seeded in the presence of 10 µg/ml of 6-thioguanine (6TG) in triplicate and incubated at 37±2 °C in a CO2 incubator for 9 days for mutation frequency.
- Fixation and staining: At the end of the incubation, cells in the culture plates were fixed with 2.5 % and 10 % of formaldehyde in water for 10 minutes each. After fixation, colonies were stained with 5 % Giemsa stain for 10 minutes, followed by washing with distilled water.
- Colony counting: Colonies in all the plates for CE1, CE2 and Mutation Frequency (MF) was counted manually and recorded in the raw data. - Rationale for test conditions:
- - Solubility:The test Item was found to be insoluble in distilled water (200 mg/ml) and found to be soluble in dimethyl sulfoxide (200 mg/ml).
- Precipitation check:Precipitation check was performed by adding 50 µl of the test Item (200 mg/ml) to 4.950 ml of culture media to attain a concentration of 2 mg/ml. No precipitation was observed at a tested concentration of 2 mg/ml.
- pH check:50 µl of test Item (200 mg/ml) was added to 4.950 ml of complete medium, resulting in a final concentration of 2 mg/ml in the medium. Changes in the pH were measured at 0 and 4th hours.
- Preparation of the test item solution:200.3 mg of test item was dissolved in 1 ml of DMSO to achieve a final concentration of 200 mg/ml. From this stock, subsequent serial dilutions with DMSO were made using spacing factor 2 to obtain concentrations of 1, 0.5 and 0.25 mg/ml.
- Preliminary cytotoxicity assay:Cytotoxicity was assessed at concentrations of 0, 0.125, 0.5, 1 and 2 mg/ml in both presence and absence of metabolic activation using triplicate cultures. - Evaluation criteria:
- Criteria of acceptance of the test:
- The concurrent negative control is considered acceptable for addition to the literature
negative control database.
- Concurrent positive controls induce responses compatible with those generated in the historical positive control database and produce a statistically significant increase compared to the concurrent negative control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested
unless one resulted in positive results.
- Adequate number of cells and concentrations are analyzable.
- The criteria for the selection of top concentration are consistent.
- The spontaneous mutant frequency of vehicle control should be between 5 and 20 x10-6.
Evaluation criteria
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the literature negative control data.
Providing that all acceptability criteria are fulfilled, a test chemical is considered negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) all results are inside the distribution of the literature negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Fisher´s exact test (NCSS statistical software) was used to assess the dose-dependency upon comparing the mutation frequencies of the test-item-treated and control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- RS values were 75.12% (-S9) and 76.96 (+S9) at 2 mg/ml
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:To determine the changes in the pH of the medium, 50 µl of the Test Item (200 mg/ml) was added to 4.950 ml of complete medium, resulting in a final Test Item concentration of 2 mg/ml in the medium. Changes in the pH are as mentioned in any other information on materials and methods
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: The chemical was insoluble in distilled water
- Precipitation and time of the determination: Precipitation of Test Item was performed by adding 50 µl of the Test Item (200 mg/ml) to 4.950 ml of culture media to attain 2 mg/ml. Slight precipitation was observed at a tested concentration of 2 mg/ml.
RANGE-FINDING/SCREENING STUDIES (if applicable):
Preliminary cytotoxicity test:
Based on solubility and precipitation test, the preliminary cytotoxicity testing was performed with concentrations of 0 (VC), 0 (NC), 0.125, 0.25, 0.5, 1 and 2 mg/ml and both in the presence (1 % v/v S9 mix) and absence of S9 metabolic activation system. Cytotoxicity was assessed by the relative survival (RS, that is, cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in negative controls) values. No cytotoxicity (<70% RS ) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation.
Main study:
In the main study, CHO cells were exposed to the test item at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. There was no significant reduction in RS (cytotoxicity) or increase of mutation frequency at any concentrations tested neither in the presence nor in the absence of S9 metabolic activation.No significant reduction in RS (cytotoxicity) and no increase in MF was observed in vehicle control (DMSO) when compared to the negative control (distilled water) either in the presence or absence of S9 metabolic activation. The positive controls (Ethylmethanesulfonate (-S9) and Beno[a]pyrene (+S9) produced statistically significant increases in mutation frequency as compared to the vehicle control.
STUDY RESULTS
- Concurrent vehicle negative and positive control data:
There was no significant reduction in RS (cytotoxicity), and no increase in MF was observed in vehicle control (dimethyl sulfoxide) when compared to the negative control (distilled water) either in the presence or absence of metabolic activation.
The positive controls (Ethylmethanesulfonate and Beno[a]pyrene in absence and presence of metabolic activation, respectively) used in the study produced statistically significant increases in mutation frequency (197.57x10-6 [Ethylmethanesulfonate], 209.32x10-6 [Benzo(a)pyrene] indicating the sensitivity of the test system to specific mutagens and confirmed that the test conditions were appropriate and that the metabolic activation system functioned properly.
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: Kindly refer to the tables as mentioned in any other information on results including tables
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: Prior to treatment (24 hours), CHO cells were prepared with a density of 10 × 106 cells /flask.
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency - Conclusions:
- The registered substance, 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H- IsoIndol-1-one] (CAS no. 5590-18-1) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.
- Executive summary:
A GLP compliant mammalian cell gene mutation assay (OECD TG 476) was conducted to assess the ability of the registered substance, 3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H- IsoIndol-1-one] (CAS no. 5590 -18-1) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese Hamster Ovary (CHO) cells. The test was performed both in the presence and absence of an exogenous metabolic activation system (liver microsomal S9 fraction was obtained from phenobarbital and β-naphthoflavone-injected rats). Test concentrations were selected from preliminary results on cytotoxicity, solubility, and precipitation tests. In the initial cytotoxicity test, CHO cells were exposed to the substance at concentrations of 0 (NC), 0 (VC), 0.125, 0.5, 1 and 2 mg/ml, both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by relative survival (RS), i.e., cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control.
Results: No cytotoxicity (<60% RS) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation. In the gene mutation study, CHO cells were treated for 4 hours at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. Reference mutagens were also included in the test i.e., Ethylmethanesulfonate (EMS, -S9) and Beno(a)pyrene (+S9). In the absence of metabolic activation, relative survival (RS) values were the follows: 97.54% (vehicle control), 94.44% (at 0.25 mg/ml), 87.44% (at 0.5 mg/ml), 79.81% (at 1 mg/ml) and 75.12% (at 2 mg/ml). In the presence of metabolic activation, the RS values were 98.16% (vehicle control), 91.92% (at 0.25 mg/ml), 86.04% (at 0.5 mg/ml), 81.44% (at 1 mg/ml) and 76.96% (at 2 mg/ml). No significant increase in the mutation frequency (MF) was observed neither in absence ( 6.15x10-6, 6.21x10-6, 7.39x10-6and 8.54x10-6at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) nor in the presence of metabolic activation ( 6.02x10-6, 8.68x10-6, 7.08 x10-6and 9.38 x10-6at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) when compared to vehicle control ( 6.35 x10-6, 7.97 x10-6, absence and presence, respectively). No significant reduction in RS values (cytotoxicity) and no increase in mutation frequency was observed in vehicle control (DMSO). The positive controls i.e Ethylmethanesulfonate (-S9) and Beno(a)pyrene (+S9) produced statistically significant increases in mutation frequency (EMS: 197.57x10-6; Beno[a]pyrene 209.32x10-6 p<0.05) as compared to the vehicle control.
Conclusion: The registered substance, 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H- IsoIndol-1-one] (CAS no. 5590-18-1) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.
Referenceopen allclose all
ABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT
Dose (mg/plate) |
R |
Absence of Metabolic Activation (-S9) |
Presence of Metabolic Activation (+S9) |
||
TA100 |
TA 98 |
TA100 |
TA 98 |
||
NC (0.00) |
R1 |
114 |
20 |
116 |
19 |
R2 |
102 |
18 |
120 |
24 |
|
R3 |
116 |
21 |
110 |
22 |
|
VC (0.00) |
R1 |
110 |
24 |
128 |
25 |
R2 |
126 |
20 |
124 |
24 |
|
R3 |
118 |
26 |
114 |
28 |
|
T1 (0.001) |
R1 |
110 |
25 |
120 |
23 |
R2 |
124 |
20 |
124 |
26 |
|
R3 |
118 |
23 |
116 |
24 |
|
T2 (0.003) |
R1 |
122 |
22 |
118 |
25 |
R2 |
110 |
20 |
114 |
20 |
|
R3 |
116 |
23 |
120 |
23 |
|
T3 (0.008) |
R1 |
120 |
23 |
124 |
25 |
R2 |
108 |
21 |
108 |
23 |
|
R3 |
114 |
18 |
112 |
26 |
|
T4 (0.025) |
R1 |
114 |
21 |
112 |
25 |
R2 |
124 |
24 |
122 |
24 |
|
R3 |
112 |
22 |
120 |
26 |
|
T5 (0.079) |
R1 |
114 |
19 |
118 |
21 |
R2 |
112 |
22 |
120 |
25 |
|
R3 |
118 |
20 |
112 |
24 |
|
T6 (0.250) |
R1 |
110 |
22 |
114 |
22 |
R2 |
114 |
23 |
110 |
21 |
|
R3 |
116 |
21 |
122 |
24 |
|
T7 (0.791) |
R1 |
110 |
19 |
116 |
26 |
R2 |
116 |
20 |
112 |
23 |
|
R3 |
120 |
21 |
120 |
23 |
|
T8 (2.5) |
R1 |
104 (+++) |
21 (+++) |
118 (+++) |
23 (+++) |
R2 |
118 (+++) |
23 (+++) |
110 (+++) |
22 (+++) |
|
R3 |
112 (+++) |
20 (+++) |
114 (+++) |
26 (+++) |
|
PC |
R1 |
1192 |
904 |
1184 |
1208 |
R2 |
1232 |
848 |
1328 |
1280 |
|
R3 |
1056 |
888 |
1200 |
1152 |
|
NC = Negative control; VC = Vehicle Control; PC = Positive control
R = Replicate
T = Test concentration (T8: Highest, T1: Lowest)
Background Lawn: +++ = Moderate Inhibition
4-Nitro-o-phenylenediamine [10μg/plate]: TA 98
Sodium azide [10μg/plate]: TA 100,
2-Aminoanthracene [2.5μg/plate]: TA 98, TA 100
TABLE 2 - REVERTANT COUNT IN PLATE
INCORPORATION METHOD
TRIAL I
Dose (mg/plate) |
R |
Presence of Metabolic Activation (+S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
5 |
12 |
24 |
118 |
244 |
R2 |
3 |
11 |
20 |
112 |
228 |
|
R3 |
4 |
13 |
22 |
106 |
240 |
|
VC. (0.00) |
R1 |
7 |
15 |
25 |
120 |
244 |
R2 |
5 |
13 |
24 |
126 |
260 |
|
R3 |
6 |
17 |
26 |
118 |
252 |
|
T1 (0.156) |
R1 |
4 |
15 |
23 |
118 |
252 |
R2 |
7 |
13 |
25 |
114 |
240 |
|
R3 |
6 |
14 |
24 |
120 |
248 |
|
T2 (0.313) |
R1 |
5 |
11 |
22 |
112 |
240 |
R2 |
4 |
15 |
24 |
118 |
248 |
|
R3 |
7 |
14 |
22 |
114 |
232 |
|
T3 (0.625) |
R1 |
4 |
13 |
21 |
120 |
248 |
R2 |
5 |
16 |
22 |
112 |
236 |
|
R3 |
5 |
14 |
23 |
116 |
240 |
|
T4 (1.250) |
R1 |
4 |
13 |
23 |
110 |
244 |
R2 |
5 |
12 |
24 |
116 |
236 |
|
R3 |
4 |
14 |
23 |
120 |
248 |
|
T5 (2.5) |
R1 |
5 |
14 |
21 |
114 |
240 |
R2 |
5 |
12 |
25 |
112 |
248 |
|
R3 |
5 |
14 |
21 |
116 |
228 |
|
PC |
R1 |
168 |
456 |
1188 |
1308 |
1428 |
R2 |
200 |
336 |
1224 |
1164 |
1464 |
|
R3 |
188 |
384 |
1140 |
1212 |
1368 |
|
Dose (mg/plate) |
R |
Absence of Metabolic Activation (-S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
3 |
10 |
19 |
110 |
224 |
R2 |
3 |
13 |
21 |
104 |
240 |
|
R3 |
4 |
11 |
18 |
114 |
232 |
|
VC. (0.00) |
R1 |
5 |
14 |
23 |
124 |
236 |
R2 |
6 |
16 |
22 |
120 |
256 |
|
R3 |
5 |
12 |
25 |
116 |
248 |
|
T1 (0.156) |
R1 |
4 |
13 |
22 |
114 |
232 |
R2 |
4 |
11 |
24 |
120 |
252 |
|
R3 |
6 |
14 |
20 |
112 |
240 |
|
T2 (0.313) |
R1 |
6 |
15 |
21 |
108 |
228 |
R2 |
5 |
10 |
23 |
114 |
244 |
|
R3 |
6 |
14 |
20 |
112 |
236 |
|
T3 (0.625) |
R1 |
5 |
12 |
23 |
114 |
236 |
R2 |
6 |
15 |
20 |
110 |
252 |
|
R3 |
4 |
13 |
24 |
118 |
240 |
|
T4 (1.250) |
R1 |
4 |
14 |
21 |
110 |
248 |
R2 |
4 |
10 |
20 |
112 |
232 |
|
R3 |
4 |
11 |
19 |
116 |
240 |
|
T5 (2.5) |
R1 |
4 |
12 |
20 |
110 |
228 |
R2 |
5 |
14 |
21 |
116 |
248 |
|
R3 |
4 |
11 |
22 |
114 |
236 |
|
PC |
R1 |
172 |
1272 |
848 |
1236 |
1440 |
R2 |
164 |
1188 |
1008 |
1152 |
1608 |
|
R3 |
192 |
1224 |
896 |
1176 |
1536 |
|
NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate
PC= Positive control 2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100, 2- Aminoanthracene [10μg/plate]:TA 102, Sodium azide [10μg/plate]: TA 1535, TA 100,
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate], Methyl methanesulfonate [4μl/plate]: TA 102.
TABLE 3 - REVERTANT COUNT IN PREINCUBATION METHOD TRIAL II
Dose (mg/plate) |
R |
In the Presence of Metabolic Activation (+S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
5 |
14 |
22 |
108 |
236 |
R2 |
4 |
11 |
21 |
114 |
244 |
|
R3 |
4 |
14 |
20 |
110 |
240 |
|
VC. (0.00) |
R1 |
6 |
16 |
25 |
118 |
256 |
R2 |
7 |
17 |
27 |
124 |
272 |
|
R3 |
6 |
15 |
26 |
116 |
268 |
|
T1 (0.156) |
R1 |
5 |
14 |
23 |
110 |
244 |
R2 |
7 |
16 |
25 |
116 |
256 |
|
R3 |
6 |
13 |
22 |
114 |
240 |
|
T2 (0.313) |
R1 |
7 |
15 |
25 |
118 |
232 |
R2 |
4 |
12 |
22 |
112 |
248 |
|
R3 |
5 |
14 |
24 |
114 |
252 |
|
T3 (0.625) |
R1 |
5 |
16 |
22 |
110 |
244 |
R2 |
5 |
14 |
21 |
116 |
240 |
|
R3 |
5 |
15 |
23 |
112 |
236 |
|
T4 (1.250) |
R1 |
4 |
14 |
22 |
118 |
252 |
R2 |
7 |
13 |
21 |
110 |
232 |
|
R3 |
6 |
15 |
25 |
114 |
244 |
|
T5 (2.5) |
R1 |
5 |
16 |
21 |
108 |
248 |
R2 |
6 |
13 |
24 |
116 |
240 |
|
R3 |
4 |
15 |
25 |
112 |
236 |
|
PC |
R1 |
168 |
376 |
972 |
1368 |
1380 |
R2 |
144 |
320 |
1212 |
1404 |
1272 |
|
R3 |
160 |
416 |
1152 |
1320 |
1320 |
|
Dose (mg/plate) |
R |
In the Absence of Metabolic Activation (-S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
3 |
11 |
22 |
110 |
232 |
R2 |
4 |
10 |
18 |
104 |
228 |
|
R3 |
3 |
12 |
21 |
112 |
236 |
|
VC. (0.00) |
R1 |
5 |
15 |
25 |
120 |
242 |
R2 |
7 |
13 |
23 |
114 |
260 |
|
R3 |
6 |
16 |
24 |
118 |
256 |
|
T1 (0.156) |
R1 |
5 |
13 |
21 |
106 |
236 |
R2 |
6 |
15 |
26 |
118 |
252 |
|
R3 |
4 |
14 |
22 |
112 |
244 |
|
T2 (0.313) |
R1 |
6 |
15 |
25 |
114 |
248 |
R2 |
5 |
11 |
23 |
116 |
236 |
|
R3 |
6 |
12 |
20 |
108 |
240 |
|
T3 (0.625) |
R1 |
5 |
12 |
24 |
118 |
240 |
R2 |
4 |
15 |
22 |
112 |
232 |
|
R3 |
4 |
13 |
21 |
110 |
244 |
|
T4 (1.250) |
R1 |
5 |
14 |
24 |
108 |
240 |
R2 |
5 |
12 |
21 |
106 |
232 |
|
R3 |
6 |
13 |
20 |
114 |
236 |
|
T5 (2.5) |
R1 |
6 |
14 |
21 |
114 |
236 |
R2 |
4 |
11 |
23 |
106 |
244 |
|
R3 |
4 |
12 |
22 |
112 |
240 |
|
PC |
R1 |
152 |
840 |
612 |
1140 |
1416 |
R2 |
176 |
1104 |
828 |
1332 |
1344 |
|
R3 |
168 |
924 |
720 |
1284 |
1512 |
|
NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate
PC=
Positive control 2-Aminoanthracene
[2.5μg/plate]: TA 1537, TA1535, TA98, TA100,
2-Aminoanthracene [10μg/plate]:TA 102, Sodium
azide [10μg/plate]: TA 1535, TA 100,
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate], Methyl methanesulfonate [4μl/plate]: TA 102.
TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD TRIALI
Dose (mg/plate) |
In the presence of Metabolic Activation (+S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
4.00 |
1.00 |
12.00 |
1.00 |
22.00 |
2.00 |
112.00 |
6.00 |
237.33 |
8.33 |
VC. (0.00) |
6.00 |
1.00 |
15.00 |
2.00 |
25.00 |
1.00 |
121.33 |
4.16 |
252.00 |
8.00 |
T1 (0.156) |
5.67 |
1.53 |
14.00 |
1.00 |
24.00 |
1.00 |
117.33 |
3.06 |
246.67 |
6.11 |
T2 (0.313) |
5.33 |
1.53 |
13.33 |
2.08 |
22.67 |
1.15 |
114.67 |
3.06 |
240.00 |
8.00 |
T3 (0.635) |
4.67 |
0.58 |
14.33 |
1.53 |
22.00 |
1.00 |
116.00 |
4.00 |
241.33 |
6.11 |
T4 (1.250) |
4.33 |
0.58 |
13.00 |
1.00 |
23.33 |
0.58 |
115.33 |
5.03 |
242.67 |
6.11 |
T5 (2.5) |
5.00 |
0.00 |
13.33 |
1.15 |
22.33 |
2.31 |
114.00 |
2.00 |
238.67 |
10.07 |
PC |
185.33 |
16.17 |
392.00 |
60.40 |
1184.00 |
42.14 |
1228.00 |
73.32 |
1420.00 |
48.50 |
Dose (mg/plate) |
In the Absence of Metabolic Activation (-S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
3.33 |
0.58 |
11.33 |
1.53 |
19.33 |
1.53 |
109.33 |
5.03 |
232.00 |
8.00 |
VC (0.00) |
5.33 |
0.58 |
14.00 |
2.00 |
23.33 |
1.53 |
120.00 |
4.00 |
246.67 |
10.07 |
T1 (0.156) |
4.67 |
1.15 |
12.67 |
1.53 |
22.00 |
2.00 |
115.33 |
4.16 |
241.33 |
10.07 |
T2 (0.313) |
5.67 |
0.58 |
13.00 |
2.65 |
21.33 |
1.53 |
111.33 |
3.06 |
236.00 |
8.00 |
T3 (0.635) |
5.00 |
1.00 |
13.33 |
1.53 |
22.33 |
2.08 |
114.00 |
4.00 |
242.67 |
8.33 |
T4 (1.250) |
4.00 |
0.00 |
11.67 |
2.08 |
20.00 |
1.00 |
112.67 |
3.06 |
240.00 |
8.00 |
T5 (2.5) |
4.33 |
0.58 |
12.33 |
1.53 |
21.00 |
1.00 |
113.33 |
3.06 |
237.33 |
10.07 |
PC |
176.00 |
14.42 |
1228.00 |
42.14 |
917.33 |
82.11 |
1188.00 |
43.27 |
1528.00 |
84.29 |
NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation
PC= Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100
Methyl methanesulfonate [4μl/plate]: TA 102
2-Aminoanthracene [10μg/plate]:TA 102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]
TABLE 5 - MEAN REVERTANT COUNT IN
PREINCUBATIONMETHOD
TRIAL II
Dose (mg/plate) |
Presence of Metabolic Activation (+S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
4.33 |
0.58 |
13.00 |
1.73 |
21.00 |
1.00 |
110.67 |
3.06 |
240.00 |
4.00 |
VC (0.00) |
6.33 |
0.58 |
16.00 |
1.00 |
26.00 |
1.00 |
119.33 |
4.16 |
265.33 |
8.33 |
T1 (0.156) |
6.00 |
1.00 |
14.33 |
1.53 |
23.33 |
1.53 |
113.33 |
3.06 |
246.67 |
8.33 |
T2 (0.313) |
5.33 |
1.53 |
13.67 |
1.53 |
23.67 |
1.53 |
114.67 |
3.06 |
244.00 |
10.58 |
T3 (0.635) |
5.00 |
0.00 |
15.00 |
1.00 |
22.00 |
1.00 |
112.67 |
3.06 |
240.00 |
4.00 |
T4 (1.250) |
5.67 |
1.53 |
14.00 |
1.00 |
22.67 |
2.08 |
114.00 |
4.00 |
242.67 |
10.07 |
T5 (2.5) |
5.00 |
1.00 |
14.67 |
1.53 |
23.33 |
2.08 |
112.00 |
4.00 |
241.33 |
6.11 |
PC |
157.33 |
12.22 |
370.67 |
48.22 |
1112.00 |
124.90 |
1364.00 |
42.14 |
1324.00 |
54.11 |
Dose (mg/plate) |
Absence of Metabolic Activation (-S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
3.33 |
0.58 |
11.00 |
1.00 |
20.33 |
2.08 |
108.67 |
4.16 |
232.00 |
4.00 |
VC (0.00) |
6.00 |
1.00 |
14.67 |
1.53 |
24.00 |
1.00 |
117.33 |
3.06 |
252.67 |
9.45 |
T1 (0.156) |
5.00 |
1.00 |
14.00 |
1.00 |
23.00 |
2.65 |
112.00 |
6.00 |
244.00 |
8.00 |
T2 (0.313) |
5.67 |
0.58 |
12.67 |
2.08 |
22.67 |
2.52 |
112.67 |
4.16 |
241.33 |
6.11 |
T3 (0.635) |
4.33 |
0.58 |
13.33 |
1.53 |
22.33 |
1.53 |
113.33 |
4.16 |
238.67 |
6.11 |
T4 (1.250) |
5.33 |
0.58 |
13.00 |
1.00 |
21.67 |
2.08 |
109.33 |
4.16 |
236.00 |
4.00 |
T5 (2.5) |
4.67 |
1.15 |
12.33 |
1.53 |
22.00 |
1.00 |
110.67 |
4.16 |
240.00 |
4.00 |
PC |
165.33 |
12.22 |
956.00 |
134.88 |
720.00 |
108.00 |
1252.00 |
99.92 |
1424.00 |
84.29 |
NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation
PC= Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100
2-Aminoanthracene [10μg/plate]: TA 102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537 [50μg/plate] TA 98 [10μg/plate]
Methyl methanesulfonate: [4μl/plate]: TA 102
HISTORICAL CONTROL DATA
These data represent the laboratory's historical control data.
Trial I (Plate Incorporation Method) |
|||||||||||||
Strains |
Metabolic Activation |
Treatment |
Mean |
SD |
Max |
Min |
|||||||
TA 1537 |
S9 + |
Negative control |
6 |
2 |
10 |
2 |
|||||||
S9 - |
6 |
2 |
10 |
2 |
|||||||||
S9 + |
Solvent control |
6 |
2 |
10 |
2 |
||||||||
S9 - |
6 |
2 |
10 |
2 |
|||||||||
S9 + |
Positive control |
168 |
38 |
245 |
92 |
||||||||
S9 - |
175 |
43 |
261 |
89 |
|||||||||
TA 1535 |
S9 + |
Negative control |
12 |
3 |
18 |
7 |
|||||||
S9 - |
12 |
3 |
18 |
7 |
|||||||||
S9 + |
Solvent control |
13 |
3 |
18 |
7 |
||||||||
S9 - |
13 |
3 |
18 |
7 |
|||||||||
S9 + |
Positive control |
336 |
211 |
757 |
86 |
||||||||
S9 - |
1200 |
263 |
1726 |
674 |
|||||||||
TA 98 |
S9 + |
Negative control |
24 |
6 |
36 |
11 |
|||||||
S9 - |
23 |
6 |
35 |
11 |
|||||||||
S9 + |
Solvent control |
25 |
6 |
37 |
13 |
||||||||
S9 - |
23 |
5 |
33 |
13 |
|||||||||
S9 + |
Positive control |
1099 |
312 |
1722 |
476 |
||||||||
S9 - |
815 |
284 |
1383 |
248 |
|||||||||
TA 100 |
S9 + |
Negative control |
117 |
28 |
173 |
61 |
|||||||
S9 - |
114 |
26 |
166 |
62 |
|||||||||
S9 + |
Solvent control |
116 |
28 |
172 |
60 |
||||||||
S9 - |
113 |
26 |
165 |
61 |
|||||||||
S9 + |
Positive control |
1488 |
390 |
2268 |
709 |
||||||||
S9 - |
1311 |
298 |
1906 |
715 |
|||||||||
TA 102 |
S9 + |
Negative control |
274 |
42 |
358 |
190 |
|||||||
S9 - |
271 |
55 |
382 |
161 |
|||||||||
S9 + |
Solvent control |
279 |
65 |
409 |
150 |
||||||||
S9 - |
277 |
82 |
442 |
112 |
|||||||||
S9 + |
Positive control |
1648 |
305 |
2258 |
1037 |
||||||||
S9 - |
1896 |
364 |
2624 |
1168 |
|||||||||
Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD
HISTORICAL CONTROL DATA (Contd.)
Trial II (Pre-Incubation Method) |
|||||||||||||
Strains |
Metabolic Activation |
Treatment |
Mean |
SD |
Max |
Min |
|||||||
TA 1537 |
S9 + |
Negative control |
6 |
2 |
10 |
2 |
|||||||
S9 - |
6 |
2 |
10 |
2 |
|||||||||
S9 + |
Solvent control |
6 |
2 |
10 |
3 |
||||||||
S9 - |
6 |
2 |
10 |
2 |
|||||||||
S9 + |
Positive control |
170 |
39 |
249 |
91 |
||||||||
S9 - |
182 |
43 |
268 |
96 |
|||||||||
TA 1535 |
S9 + |
Negative control |
13 |
3 |
18 |
7 |
|||||||
S9 - |
12 |
3 |
18 |
7 |
|||||||||
S9 + |
Solvent control |
13 |
3 |
18 |
8 |
||||||||
S9 - |
13 |
3 |
18 |
7 |
|||||||||
S9 + |
Positive control |
299 |
197 |
694 |
145 |
||||||||
S9 - |
1244 |
260 |
1765 |
724 |
|||||||||
TA 98 |
S9 + |
Negative control |
24 |
6 |
35 |
13 |
|||||||
S9 - |
23 |
5 |
33 |
13 |
|||||||||
S9 + |
Solvent control |
24 |
5 |
35 |
14 |
||||||||
S9 - |
23 |
5 |
32 |
14 |
|||||||||
S9 + |
Positive control |
1269 |
275 |
1819 |
719 |
||||||||
S9 - |
740 |
210 |
1160 |
320 |
|||||||||
TA 100 |
S9 + |
Negative control |
117 |
25 |
166 |
67 |
|||||||
S9 - |
113 |
23 |
159 |
66 |
|||||||||
S9 + |
Solvent control |
116 |
22 |
159 |
73 |
||||||||
S9 - |
112 |
20 |
151 |
73 |
|||||||||
S9 + |
Positive control |
1469 |
347 |
2163 |
775 |
||||||||
S9 - |
1352 |
263 |
1878 |
827 |
|||||||||
TA 102 |
S9 + |
Negative control |
281 |
32 |
345 |
218 |
|||||||
S9 - |
276 |
28 |
331 |
220 |
|||||||||
S9 + |
Solvent control |
281 |
34 |
350 |
212 |
||||||||
S9 - |
276 |
34 |
344 |
207 |
|||||||||
S9 + |
Positive control |
1595 |
287 |
2168 |
1022 |
||||||||
S9 - |
1753 |
248 |
2248 |
1258 |
|||||||||
Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD
Cytotoxicity experiment:
Before conducting the chromosomal aberration study,3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No:5590-18-1)was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.250 (T1), 0.5 (T2) and 1 (T3) mg/mL of culture media. Cytotoxicity was observed in treated concentrations of 1 (T3), 0.5 (T2) mg/mL both in the absence and in the presence of metabolic activation (1%).
In the absence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.84 (VC), 7.34 (T1), 4.75 (T2), 3.27 (T3) and 8.49 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.28 (NC), 9.93 (VC), 7.55 (T1), 4.90 (T2), 3.60 (T3) and 8.74 (PC).
In the cytotoxicity experiment, the test concentrations 1 (T3), 0.5 (T2) mg/ mLof culture mediashowed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence, this concentration was not selected for the main study.
Hence, 0.250 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.
The main study was performed in twoindependentphases;
Phase I
In the experiment, the cultures were exposed to 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No:5590-18-1) for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 11.000 (PC)in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.062 (T1), 0.125 (T2) and 0.250 (T3) mg/mL and positive controls, respectively.
Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.
During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations.The observed mean mitotic indexin the absence of metabolic activation were 10.04, 9.90, 8.84, 8.15, 7.29 and 8.45 andin the presence ofmetabolic activation were 10.17, 9.99, 8.99, 8.35, 7.49 and 8.64 for(NC), 0.0 (VC) 0.062 (T1), 0.125 (T2) and 0.250 (T3) mg/mLand 30 µg/mL(PC)concentrations, respectively.
Phase II
The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were 0.0 (NC), 0.0 (VC) 0.062 (T1), 0.125 (T2) and 0.250 (T3) mg/mLand 30µg/mL(PC)culture both in absence and presence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (VC) 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.667 (VC), 0.667 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.062 (T1), 0.125 (T2) and 0.250 (T3) mg/mL of culture and positive control, respectively.
Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.
The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.
Treatment with test item in the absence and presence of S9 mix, did not show any reduction in mitotic index at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.05, 9.95, 8.95, 8.05, 7.29 and 8.39 andin the presence ofmetabolic activation were 10.22, 9.94, 9.10, 8.54, 7.39 and 8.60 for0.0 (NC), 0.0 (VC) 0.062 (T1), 0.125 (T2) and 0.250 (T3) and 30 µg/mL(PC)concentrations, respectively.
Table 1: Relative Survival – Preliminary Cytotoxicity Assay:Absence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23640000 |
249 |
233 |
237 |
239.67 |
100 |
2.397 |
2.833 |
100.00 |
VC |
Dimethylsulfoxide |
20000000 |
23420000 |
234 |
244 |
237 |
238.33 |
100 |
2.383 |
2.791 |
98.52 |
T1 |
0.125 mg/ml |
20000000 |
23100000 |
231 |
240 |
235 |
235.33 |
100 |
2.353 |
2.718 |
97.39 |
T2 |
0.25 mg/ml |
20000000 |
22840000 |
228 |
235 |
220 |
227.67 |
100 |
2.277 |
2.600 |
93.16 |
T3 |
0.5 mg/ml |
20000000 |
22500000 |
211 |
219 |
210 |
213.33 |
100 |
2.133 |
2.400 |
85.99 |
T4 |
1 mg/ml |
20000000 |
21900000 |
187 |
214 |
190 |
197.00 |
100 |
1.970 |
2.157 |
77.29 |
T5 |
2 mg/ml |
20000000 |
21840000 |
182 |
192 |
187 |
187.00 |
100 |
1.870 |
2.042 |
73.17 |
Key:NC = Negative Control, VC = Vehicle Control, T5-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, ml = milliliter.
Table 2: Relative Survival – Preliminary Cytotoxicity Assay: Presence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23720000 |
251 |
247 |
245 |
247.67 |
100 |
2.477 |
2.937 |
100.00 |
VC |
Dimethylsulfoxide |
20000000 |
23320000 |
240 |
248 |
238 |
242.00 |
100 |
2.420 |
2.822 |
96.06 |
T1 |
0.125 mg/ml |
20000000 |
23300000 |
228 |
234 |
231 |
231.00 |
100 |
2.310 |
2.691 |
95.37 |
T2 |
0.25 mg/ml |
20000000 |
23150000 |
221 |
218 |
226 |
221.67 |
100 |
2.217 |
2.566 |
90.93 |
T3 |
0.5 mg/ml |
20000000 |
22400000 |
210 |
218 |
211 |
213.00 |
100 |
2.130 |
2.386 |
84.54 |
T4 |
1 mg/ml |
20000000 |
22140000 |
209 |
206 |
211 |
208.67 |
100 |
2.087 |
2.310 |
81.86 |
T5 |
2 mg/ml |
20000000 |
21740000 |
189 |
204 |
194 |
195.67 |
100 |
1.957 |
2.127 |
75.38 |
Key: NC = Negative Control, VC = Vehicle Control, T5-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, ml = milliliter.
Table 3: Relative Survival – Main Study : Absence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23480000 |
235 |
244 |
238 |
239.00 |
100 |
2.390 |
2.806 |
100.00 |
VC |
Dimethylsulfoxide |
20000000 |
23260000 |
233 |
239 |
234 |
235.33 |
100 |
2.353 |
2.737 |
97.54 |
T1 |
0.25 mg/ml |
20000000 |
22840000 |
227 |
224 |
228 |
226.33 |
100 |
2.263 |
2.585 |
94.44 |
T2 |
0.5 mg/ml |
20000000 |
22270000 |
217 |
214 |
214 |
215.00 |
100 |
2.150 |
2.394 |
87.47 |
T3 |
1 mg/ml |
20000000 |
21880000 |
198 |
196 |
205 |
199.67 |
100 |
1.997 |
2.184 |
79.81 |
T4 |
2 mg/ml |
20000000 |
21680000 |
197 |
187 |
185 |
189.67 |
100 |
1.897 |
2.056 |
75.12 |
PC |
400 µg/ml |
20000000 |
22140000 |
194 |
204 |
214 |
204.00 |
100 |
2.040 |
2.258 |
82.51 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control(Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Table 4: Relative Survival – Main Study: Presence of metabolic activation
Dose level |
Concentration |
No. of Cells |
No. of colonies |
Mean Colony count |
No. of cells seeded |
CE |
Adjusted CE |
RS |
|||
Before |
After |
R1 |
R2 |
R3 |
|||||||
NC |
Distilled water |
20000000 |
23620000 |
233 |
224 |
229 |
228.67 |
100 |
2.287 |
2.701 |
100.00 |
VC |
Dimethylsulfoxide |
20000000 |
23460000 |
228 |
231 |
219 |
226.00 |
100 |
2.260 |
2.651 |
98.16 |
T1 |
0.25 mg/ml |
20000000 |
22880000 |
214 |
214 |
211 |
213.00 |
100 |
2.130 |
2.437 |
91.92 |
T2 |
0.5 mg/ml |
20000000 |
22620000 |
204 |
203 |
198 |
201.67 |
100 |
2.017 |
2.281 |
86.04 |
T3 |
1 mg/ml |
20000000 |
22180000 |
201 |
189 |
194 |
194.67 |
100 |
1.947 |
2.159 |
81.44 |
T4 |
2 mg/ml |
20000000 |
21860000 |
183 |
186 |
191 |
186.67 |
100 |
1.867 |
2.040 |
76.96 |
PC |
30 µg/ml |
20000000 |
22360000 |
187 |
201 |
211 |
199.67 |
100 |
1.997 |
2.232 |
84.21 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Table 5: Cloning Efficiency(Non selective medium)Main Study: Absence of metabolic activation
Dose level |
Non Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
100 |
221 |
216 |
220 |
219 |
2.19 |
VC |
Dimethylsulfoxide |
100 |
215 |
211 |
204 |
210 |
2.10 |
T1 |
0.25 mg/ml |
100 |
194 |
190 |
185 |
190 |
1.90 |
T2 |
0.5 mg/ml |
100 |
188 |
192 |
184 |
188 |
1.88 |
T3 |
1 mg/ml |
100 |
179 |
180 |
182 |
180 |
1.80 |
T4 |
2 mg/ml |
100 |
176 |
174 |
177 |
176 |
1.76 |
PC |
400 µg/ml |
100 |
174 |
178 |
182 |
178 |
1.78 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control(Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Table 6: Cloning Efficiency(Non selective medium)Main Study: Presence of metabolic activation
Dose level |
Non Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
100 |
219 |
224 |
221 |
221 |
2.21 |
VC |
Dimethylsulfoxide |
100 |
205 |
209 |
213 |
209 |
2.09 |
T1 |
0.25 mg/ml |
100 |
195 |
189 |
197 |
194 |
1.94 |
T2 |
0.5 mg/ml |
100 |
188 |
198 |
190 |
192 |
1.92 |
T3 |
1 mg/ml |
100 |
186 |
191 |
188 |
188 |
1.88 |
T4 |
2 mg/ml |
100 |
178 |
172 |
183 |
178 |
1.78 |
PC |
30 µg/ml |
100 |
180 |
178 |
189 |
182 |
1.82 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Table 7: Cloning Efficiency(Selective medium): Absence of metabolic activation
Dose level |
Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
200000 |
2 |
3 |
3 |
2.67 |
0.00001333 |
VC |
Dimethylsulfoxide |
200000 |
2 |
3 |
3 |
2.67 |
0.00001333 |
T1 |
0.25 mg/ml |
200000 |
2 |
2 |
3 |
2.33 |
0.00001167 |
T2 |
0.5 mg/ml |
200000 |
3 |
2 |
2 |
2.33 |
0.00001167 |
T3 |
1 mg/ml |
200000 |
2 |
3 |
3 |
2.67 |
0.00001333 |
T4 |
2 mg/ml |
200000 |
3 |
2 |
4 |
3.00 |
0.00001500 |
PC |
400 µg/ml |
200000 |
74 |
70 |
67 |
70.33 |
0.00035167 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Table 8: Cloning Efficiency(Selective medium): Presence of metabolic activation
Dose level |
Selective medium |
|
|||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
200000 |
3 |
3 |
2 |
2.67 |
0.00001333 |
VC |
Dimethylsulfoxide |
200000 |
3 |
4 |
3 |
3.33 |
0.00001667 |
T1 |
0.25 mg/ml |
200000 |
2 |
2 |
3 |
2.33 |
0.00001167 |
T2 |
0.5 mg/ml |
200000 |
4 |
2 |
4 |
3.33 |
0.00001667 |
T3 |
1 mg/ml |
200000 |
4 |
2 |
2 |
2.67 |
0.00001333 |
T4 |
2 mg/ml |
200000 |
3 |
3 |
4 |
3.33 |
0.00001667 |
PC |
30 µg/ml |
200000 |
75 |
75 |
79 |
76.33 |
0.00038167 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.
Table 9: Mutation Frequency
Dose level |
Absence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000609 |
6.09 |
VC |
Dimethylsulfoxide |
0.00000635 |
6.35 |
T1 |
0.25 mg/ml |
0.00000615 |
6.15 |
T2 |
0.5 mg/ml |
0.00000621 |
6.21 |
T3 |
1 mg/ml |
0.00000739 |
7.39 |
T4 |
2 mg/ml |
0.00000854 |
8.54 |
PC |
400 µg/ml |
0.00019757 |
197.57 |
Dose level |
Presence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000602 |
6.02 |
VC |
Dimethylsulfoxide |
0.00000797 |
7.97 |
T1 |
0.25 mg/ml |
0.00000602 |
6.02 |
T2 |
0.5 mg/ml |
0.00000868 |
8.68 |
T3 |
1 mg/ml |
0.00000708 |
7.08 |
T4 |
2 mg/ml |
0.00000938 |
9.38 |
PC |
30 µg/ml |
0.00020932 |
209.32 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (absence-Ethylmethanesulfonate, presence –Benao[a]pyrene), T4-T1= Test item concentration from higher to lower, MF = Mutation Frequency, mg = milligram, µg = microgram, ml = milliliter.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial reverse mutation test:
A bacterial reverse mutation assay (OECD TG 471) was performed to investigate the potential of the registered chemical 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments with and without S9 metabolic activation system. The test concentrations were selected based on the results of a pre-liminary cytotoxicity test. The mutagenicity test was performed with the following test substance concentrations: 0 (NC), 0 (VC), 0.156, 0.313, 0.625, 1.250 and 2.5 mg/plate in both trials and in the presence (+S9) and absence of metabolic activation (-S9). Results: No significant increase in the number of revertant colonies, either in the presence or absence of metabolic activation was observed at any concentrations tested as compared to the vehicle control. No trend of an increased number of revertant colonies with increased dosing of the test item was observed. The spontaneous reversion rates in the negative and vehicle controls were within the historical range. Each strain-specific positive control in Trial I and II showed a significant increase in the number of revertant colonies. Conclusion:The registered chemical 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation in study performed according to OECD TG 471.
In vitro cytogenicity test:
This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0 (NC), 0 (VC), 0.062, 0.125 and 0.250 mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0 (NC), 0 (VC), 0.25, 0.5 and 1.0 mg/mL of culture media based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. The test chemical was tested negative, non-clastogenic at the highest tested concentration of 0.250 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions.
In vitro mammalian cell gene mutation study:
A GLP compliant mammalian cell gene mutation assay (OECD TG 476) was conducted to assess the ability of the registered substance, 3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H- IsoIndol-1-one] (CAS no. 5590 -18-1) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese Hamster Ovary (CHO) cells. The test was performed both in the presence and absence of an exogenous metabolic activation system (liver microsomal S9 fraction was obtained from phenobarbital and β-naphthoflavone-injected rats). Test concentrations were selected from preliminary results on cytotoxicity, solubility, and precipitation tests. In the initial cytotoxicity test, CHO cells were exposed to the substance at concentrations of 0 (NC), 0 (VC), 0.125, 0.5, 1 and 2 mg/ml, both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by relative survival (RS), i.e., cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control. Results: No cytotoxicity (<60% RS) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation. In the gene mutation study, CHO cells were treated for 4 hours at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. Reference mutagens were also included in the test i.e., Ethylmethanesulfonate (EMS, -S9) and Beno(a)pyrene (+S9). In the absence of metabolic activation, relative survival (RS) values were the follows: 97.54% (vehicle control), 94.44% (at 0.25 mg/ml), 87.44% (at 0.5 mg/ml), 79.81% (at 1 mg/ml) and 75.12% (at 2 mg/ml). In the presence of metabolic activation, the RS values were 98.16% (vehicle control), 91.92% (at 0.25 mg/ml), 86.04% (at 0.5 mg/ml), 81.44% (at 1 mg/ml) and 76.96% (at 2 mg/ml). No significant increase in the mutation frequency (MF) was observed neither in absence ( 6.15x10-6, 6.21x10-6, 7.39x10-6and 8.54x10-6at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) nor in the presence of metabolic activation ( 6.02x10-6, 8.68x10-6, 7.08 x10-6and 9.38 x10-6at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) when compared to vehicle control ( 6.35 x10-6, 7.97 x10-6, absence and presence, respectively). No significant reduction in RS values (cytotoxicity) and no increase in mutation frequency was observed in vehicle control (DMSO). The positive controls i.e Ethylmethanesulfonate (-S9) and Beno(a)pyrene (+S9) produced statistically significant increases in mutation frequency (EMS: 197.57x10-6; Beno[a]pyrene 209.32x10-6 p<0.05) as compared to the vehicle control. Conclusion: The registered substance, 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H- IsoIndol-1-one] (CAS no. 5590-18-1) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.
Justification for classification or non-classification
The registered substance, i.e. 3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H- IsoIndol-1-one] (CAS no. 5590-18-1) was tested non-mutagenic (negative) in both bacterial and mammalian cells in GLP-compliant and OECD guideline studies performed according to OECD TGs 471 and 476. The substance was also tested non-clastogenic in an in vitro cytogenicity test according to OECD TG 473. Overall, considering the results of the genotoxicity testing battery, the registered substance is regarded to be classified as Not Classified for genetic toxicity according to Regulation EC 1272/2008.
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