Registration Dossier
Registration Dossier
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EC number: 267-636-0 | CAS number: 67905-17-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation test
The registered substance was tested non-mutagenic (negative) both in the presence and absence of S9 metabolic activation in Ames test using Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains. The test was performed according to OECD TG 471 and in compliance with OECD Principles of Good Laboratory Practice.
In vitro mammalian chromosomal aberration test:
The registered substance i.e. N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1 anthryl)amino]phenyl]acetamide (CAS: 67905-17-3), was tested non-clastogenic (negative ) both in the presence and absence of S9 metabolic activation using primary culture of human lymphocytes. The test was performed according to OECD test guideline 473 (adopted on July 29, 2016) and in compliance with OECD Principles of Good Laboratory Practice.
In vitro mammalian cell gene mutation test:
The registered substance i.e., N-{4-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]phenyl}acetamide (CAS No. 67905-17-3) was tested non-mutagenic (negative) in an in vitro mammalian cell gene mutation test both in the presence and absence of S9 metabolic activation using cultured CHO cells. The test was performed according to OECD 476 and in compliance with OECD Principles of Good Laboratory Practice.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27-10-2004 - 19-11-2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from study report
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Qualifier:
- according to guideline
- Guideline:
- other: EC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Substance Control Law (JSCL) Test Guideline III. 1 Gene Mutation Test With Bacteria
- Principles of method if other than guideline:
- Salmonella/microsome mutagenicity test (Ames test) (plate incorporation) was performed to assess the mutagenic nature of the test chemical.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine for Salmonella typhimurium strains and tryptophan for E.coli strains
- 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:
- other: Salmonella typhimurium strains TA98: hisD3052 rfa uvr B+R TA100: hisG46 rfa uvr B+R Ta1535: hisG46 rfa uvrB TA1537: hisC3076 rfa uvrB Escherichia coli WP2uvrA: pKM101
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- other: pKM101
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 metabolic activation system was prepared from liver of male Sprague Dawley rat homogenate
- Test concentrations with justification for top dose:
- Plate incorporation assay:
With: 0, 50, 160, 500, 1600 or 5000 µg/plate
Without: 0, 50, 160, 500, 1600 or 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solube in DMSO - Untreated negative controls:
- yes
- Remarks:
- Untreated control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- sodium azide
- other: 2-nitrofluorene (TA98, -S9), 2-aminoanthracene (All strains, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): No data
DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: Triplicate
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data
NUMBER OF CELLS EVALUATED: No data
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes
- Any supplementary information relevant to cytotoxicity: Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occuring mutants compared to the corresponding solvent control value
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- 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:
- The test chemical was considered positive if-
- it produces atleast 2-fold increase in the mean number of revertants/plate of atleast one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose related increase in the mean number of revertants per plate of at least one of the test strains over the mean number of revertants per plate of the appropriate vehicle in atleast two or three concentrations of the test item at complete bacterial background lawn
If the aboce critera is not achieved, it is considered to show no mutagenic activity in the system - Statistics:
- No data
- Key result
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Visible precipitation was observed on plates at 160 µg/plate and above
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: No data
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: No data
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 registered substance was tested non-mutagenic (negative) both in the presence and absence of S9 metabolic activation in Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains. The test was performed according to OECD TG 471 and in compliance with OECD Principles of Good Laboratory Practice.
- Executive summary:
Salmonella/microsome mutagenicity test (Ames test) was performed to assess the mutagenic nature of theregistered substance. The study was conducted according to the plate incorporation method and in the presence and absence of S9 metabolic activation system using Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains. The test chemical was dissolved in DMSO and used at concentrationsof 0, 50, 160, 500, 1600 or 5000 µg/plate. The plates were incubated for 48 hrs and observed for revertant colonies. Concurrent solvent(Dimethyl sulfoxide),negative controland positive control substances were also included in the study. The test chemical did not induce a relevant or dose-dependent increase in the number of revertants colonies at any concentrations tested either in the presence or absence of S9 metabolic activation system using Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains.
- 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 Chromosomal Aberration Test)
- Version / remarks:
- Adopted on July 29, 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Molecular Weight: 372.37
Molecular Formula: C22H16N2O4
Manufacture Date: June 14, 2019
Storage Condition: Room Temperature (20 to 30oC) - Species / strain / cell type:
- lymphocytes:
- Remarks:
- Human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Human blood
- Sex, age and number of blood donors: Blood was obtained from a healthy donor being in the range of 25 to 34 years of age
- Whether whole blood or separated lymphocytes were used: Whole blood.
- Whether blood from different donors were pooled or not: Not pooled.
- Mitogen used for lymphocytes: Phytohaemagglutinin
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system.
The S9 fraction was obtained from Aroclor 1254-induced rat.
- source of S9:Aroclor 1254- induced S9 was procured from Defence Research and Development Establishment, Nagpur, India.
- method of preparation of S9 mix: Appropriate quantity of S9 microsomal fraction was mixed with a cofactor solution, which contained 0.80 g of D-glucose-6-phosphate, 1.00 g of MgCl2, 1.35 g of KCl, 6.40 g of Na2HPO4, 1.40 g of NaH2PO4.H2O, 1.75 g of β-NADP in 500 mL of RO water. The S9 mix used was freshly prepared during the test.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 % v/v in Phase I, and 2 % v/v in Phase II of the experiment.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The efficiency of the S9 metabolic activation system was tested. - Test concentrations with justification for top dose:
- Test concentrations without S9 mix:
0 mg/ml (negative control)
0 mg/kg (vechicle control)
0.5 mg/ml
1.0 mg/ml
2 mg/ml
Test concentrations with S9 mix:
0 mg/ml (NC)
0 mg/ml (VC)
0.25 mg/ml
0.5 mg/ml
1 mg/ml
Justification:
Test concentrations were selected based on solubility, precipitation and pH checks and a preliminary cytotoxicity test. Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control. The test concentration which yielded 55±5% cytotoxicity, i.e., reduction in MI to 45±5% of the concurrent negative control was selected as the highest test concentration. In the preliminary cytotoxicity test, the cytotoxicity was 50.76% at 2 mg/ml in the absence of S9 metabolic activation. In the presence of S9 mix, 1 mg/ml of test substance concentration induced 50.32 % of cytotoxicity. Hence, 2 mg/ml and 1 mg/ml were selected as the highest test concentrations in the absence and presence of S9 metabolic activation, respectively. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO at 200 mg/ml. - Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cell culture used: Primary culture of human blood lymphocytes was used. Blood was drawn from a healthy volunteer by venous puncture using a heparinised syringe. Cells were stimulated to divide by adding a mitogen (phytohemagglutinin, PHA) to the culture medium for 48 hours.
- Cell culture preparation: Blood cultures were set up in bulk within 24 hours after collection in cell culture flasks (e.g., 150 cm²). Cell culture flasks contained the followings (for 10 culture tubes):
65.7 ml RPMI - 1640 7.5 ml Fetal Bovine Serum
1.00 ml Phytohaemagglutinin 0.5 ml Heparin solution
4.50 ml Whole Blood 0.8 ml Antibiotic Solution
Culture flasks were incubated at 37°C in a humidified atmosphere with 5 % CO2 for 48 hours.
-Treatment: The treatment of cultures with the test item was made in two independent phases (Phase I and Phase II).
Proliferating cells were treated with three different test substance concentrations and with negative (distilled water) and vehicle (DMSO) and positive controls and both in the presence ((Phase I - 1 % and Phase II - 2 % v/v) and absence of S9 metabolic activation.
The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in medium without serum mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in a complete medium mixed with phosphate buffer for the treatment in presence and the absence of metabolic activation system, respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes. Negative control tubes were treated with 80 µL of distilled water; vehicle control tubes were treated with 80 µL of DMSO; treatment groups were treated with 80 µL of respective test item stock solution. The cultures (duplicates) were incubated at 37°C for 4 hours (Phase I) or 24 hours (Phase II).
For Phase I, after incubation, cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. Next, the supernatant with the dissolved test item was discarded, and the cells were resuspended in Phosphate Buffer Saline (PBS). The washing procedure in PBS was repeated, then cells were resuspended in a complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37°C for 1.5 normal cell cycle lengths (23 hours). The cultures were harvested at the end of incubation 24 hours after treatment.
- Copy of cultures used (single, duplicate, triplicate): Duplicate cultures were used for each test concentration and control.
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration, duration and period of cell exposure.: Colcemide (0.3 µg/ml) was added 3 hours before harvesting.
- Methods of slide preparation and staining technique used, including the stain used (for cytogenetic assays): 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 and labelled. Two slides were made from each sample. The cells were stained with 5 % fresh Giemsa stain then mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A minimum of 1000 cells were counted in different fields of slide per culture, and the number of metaphases was recorded for mitotic index calculation. Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverisation, 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. Only metaphases with 46 ± 2 centromere regions were included in the analysis. To describe a cytotoxic effect, the mitotic index (% cells in mitosis) were determined.
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Mitotic Index
- Any supplementary information relevant to cytotoxicity:
To evaluate the toxicity of the test item, a preliminary cytotoxicity assay was performed both in the presence and absence of the S9 metabolic activation system. Cytotoxicity was assessed at the concentrations 0.0 (NC), 0.0 (VC), 0.5, 1.0 and 2.0 mg/ml of culture media based on the results of solubility, precipitation and pH tests. Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control 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 vehicle 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 vehicle control
- If there is no dose-related increase
- All results are inside the historical vehicle control range
Statistical significance was confirmed by means of the non-parametric Mann-Whitney Test. However, both biological and statistical significance were considered together. - Statistics:
- Statistical significance at the p<0.05 was evaluated by means of the non-parametric Mann-Whitney test.
- Key result
- Species / strain:
- lymphocytes:
- Remarks:
- Human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In Phase I, the cytotoxicity (%) was 51.21% (at 2 mg/ml) and 50.56 % (at 1 mg/ml) in the absence and presence of S9 mix. In Phase II, the cytotoxicity was 53.66% (at 2 mg/ml) and 54.73% (at 1 mg/ml) without and with S9 mix.
- 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: The pH of the test item in the culture medium was assessed at 0 h and 4 hours after incubation at 37 ± 2°C. No significant change in pH was observed at 0 and 4 hours when compared with negative controls.
- Solubility: Insoluble in water at 200 mg/ml. Soluble in dimethyl sulfoxide at 200 mg/ml.
- Precipitation: Slight precipitation was observed at 2 mg/ml. This slight precipitation did not interfere with scoring, and hence 2 mg/ml was selected as the top dose for the cytotoxicity experiment.
CYTOTOXICITY
RANGE-FINDING/SCREENING STUDIES (if applicable):
To evaluate the cytotoxicity of the test substance, a preliminary cytotoxicity test was performed both in the presence and absence of S9 metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.5, 1.0 and 2.0 mg/ml of culture media. In the absence of S9 mix, the mean mitotic index (MI) observed was 9.99 (NC), 9.84 (VC), 6.44 (at 0.5 mg/ml), 5.08 (at 1.0 mg/ml), 4.84 (at 2.0 mg/ml) and 9.27 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.10 (NC), 9.88 (VC), 6.94 (at 0.5 mg/ml), 4.91 (at 1.0 mg/ml), 4.47 (at 2.0 mg/ml) and 8.87 (PC). The test substance at 2.0 mg/ml produced a 50.76% decrease of MI compared to the vehicle control and in the absence of S9 metabolic activation. In the presence of S9 mix, the test substance produced 50.32% and 54.74% decreases in MI compared to the vehicle control at 1.0 and 2.0 mg/ml, respectively. Based on the observations of the pre-test, 2.0 mg/ml was chosen as the highest test concentration in the absence of S9 metabolic activation, and 1 mg/ml was selected in the presence of S9 metabolic activation.
CHROMOSOME ABERRATION (CA) TEST: Tabular data on percent of aberrant cells are presented in section “Any other information on results including tables”.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Historical control data are presented in section “Any other information on results including tables”.
- Negative (solvent/vehicle) historical control data: Historical control data are presented in section “Any other information on results including tables”. - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The registered substance i.e. N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1 anthryl)amino]phenyl]acetamide (CAS: 67905-17-3), was tested non-clastogenic (negative) both in the presence and absence of S9 metabolic activation using primary culture of human lymphocytes. The test was performed according to OECD test guideline 473 (adopted on July 29, 2016) and in compliance with OECD Principles of Good Laboratory Practice.
- Executive summary:
The registered substance, i.e. N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino] phenyl]acetamide (CAS: 67905-17-3) was tested in a mammalian chromosome aberration test according to OECD 473 (adopted on July 29, 2016).The test was performed to assess the ability of the substance to induce structural chromosomal aberrations in primary cultures of human peripheral blood lymphocytes. The experiment was conducted in two phases (Phase I and II) and both in the presence and absence of a metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction derived from Aroclor 1254-induced rats was used in both phases of the experiment.Dimethyl sulfoxide (DMSO) was selected as the vehicle for the test substance. A preliminary cytotoxicity test, was performed to select test concentrations. In this pre-test, proliferating cells were exposed to 0.0 (NC), 0.0 (VC), 0.5, 1.0 and 2.0 mg/ml of test substance with and without S9 metabolic activation (1% v/v). Cytotoxicity was determined by the mitotic index. At 2. 0 mg/ml and in the absence of metabolic activation (-S9), the test substance produced a 50.76% decrease of the mitotic index when compared to the vehicle control (DMSO). In the presence of metabolic activation (+S9), 50.32% and 54.74% reductions of the mitotic index were noted at 1.0 and 2.0 mg/ml compared to the vehicle.The concentration that yielded 55±5% cytotoxicity (i.e. reduction in the mitotic index to 45±5%) of the concurrent vehicle control was chosen as the highest test concentration.Hence, 2.0 mg/ml and 1.0 mg/ml were selected as the highest test concentrations in the absence and presence of S9 metabolic activation for the main test, respectively.Results:In Phase I of the main test, cells were treated with 0 (NC), 0 (VC), 0.5, 1.0, 2.0 mg/ml and 0 (NC), 0 (VC), 0.25, 0.5, 1.0 mg/ml for 4 hours without and with S9 metabolic activation (1% v/v), respectively.The mean percentage of aberrant cells was 0.333 (NC), 0.667 (VC), 0.333 (at 0.5 mg/ml), 0.667 (at 1.0 mg/ml), 0.667 (at 2.0 mg/ml), 10.000 (PC) and 0.333 (NC), 0.333 (VC), 0.333 (at 0.25 mg/ml), 0.333 (at 0.5 mg/ml), 0.667 (at 1.0 mg/ml) and 9.000 (PC) in the absence and presence of S9 metabolic activation, respectively. In addition, the mean mitotic indices were the follows: 10.03 (NC), 9.94 (VC), 6.85 (at 0.5 mg/ml), 5.22 (at 1.0 mg/ml), 4.85 (at 2.0 mg/ml), 8.19 (PC) and 10.16 (NC), 9.89 (VC), 7.10 (at 0.25 mg/ml), 6.80 (at 0.5 mg/ml), 4.89 (at 1.0 mg/ml) and 8.58 (PC) in the absence and presence of S9 metabolic activation, respectively. In Phase II of the main test, cells were treated with 0 (NC), 0 (VC), 0.5, 1.0, 2.0 mg/ml and 0 (NC), 0 (VC), 0.25, 0.5, 1.0 mg/ml for 24 hours in the absence and presence of S9 metabolic activation (2% v/v) respectively. The mean percentage of aberrant cells were 0.333(NC), 0.333(VC), 0.333 (at 0.5 mg/ml), 0.333 (at 1.0 mg/ml), 0.667(at 2.0 mg/ml), 10.000 (PC) and 0.333 (NC), 0.333 (VC), 0.333 (at 0.25 mg/ml), 0.333 (at 0.5 mg/ml),0.667 (at 1.0 mg/ml) and 10.667 (PC) in the absence and presence of S9 metabolic actibation (2% v/v), respectively.Furthermore, the mean mitotic indices were the follows: 9.99 (NC), 9.69 (VC), 6.10 (at 0.5 mg/ml), 4.91 (at 1.0 mg/ml), 4.49 (at 2.0 mg/ml), 8.85 (PC) and 10.00 (PC), 9.94 (VC), 6.75 (at 0.25 mg/ml), 4.70 (at 0.5 mg/ml), 4.50 (at 1.0 mg/ml), and 8.79 (PC) without and with S9 metabolic activation (2% v/v), respectively. There was no statistically significant increase in percent aberrant cells in cultures treated with the vehicle (DMSO) neither in the presence nor in the absence of S9 metabolic activation in either phases of the experiment. Conclusion:The registered substance i.e. N-[4-[(9,10- dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl] acetamide (CAS: 67905-17-3) did not induce structural chromosal aberration in primary cultures of human lymphocytes when it was tested up to 1 mg/ml in the presence (1% and 2%) and 2 mg/ml in the absence of S9 metabolic activation.
- 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 contains experimental data of 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
- 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:
- Appearance: Dark blue Powder
Batch Number: B-1/20
Purity: 98.7%
Manufactured by: Parshwnath Dye Chem Industries Pvt. Ltd.
Manufacturing date: 22-01-2021
Expiry Date: 21-01-2025
Storage condition: Room Temperature (20 to 30C)
Purity: 98.7% - Target gene:
- hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Chinese Hamster Ovary (CHO) cell line,
- Source: NCCS, Pune, India
- Absence of Mycoplasma contamination: Yes.
- Periodically checked for karyotype stability: Yes
- Periodically ‘cleansed’ of spontaneous mutants: Yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cell were cultured in complete RPMI-1640 medium (10 % Fetal bovine serum), 100 units Penicillin/ml, 10 µg Streptomycin/ml at 37±2 °C, 5% CO2, in a CO2 incubator. - Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor-supplemented S9 microsomal fraction was used as metabolic activation system. S9 was obtained from phenobarbital and β-naphthoflavone-induced rat lliver.
Details of the S9 cofactor mix:
Glucose-6-phosphate (180 mg/ml): 1%
NADP (25 mg/ml): 1%
Potassium chloride (150 mM): 1%
S9 Fraction (ml):2%
Final Volume (ml): 5 ml
S9 Mix: 40% - Test concentrations with justification for top dose:
- Test concentrations: 0.0 (NC), 0.0 (VC), 0.03125, 0.0625, 0.125 and 0.25 mg/ml.
Justification:
The highest test concentration was selected based on the results of a preliminary cytotoxicity test and solubility, precipitation and pH checks. The test substance was insoluble in water but was soluble in dimethyl sulphoxide (DMSO) at 100 mg/ml. The substance formed precipitation at 1 and 0.5 mg/ml. Slight precipitation was observed at 0.25 and 0.125 mg/ml, which did not interfere with scoring. The test substance did not affect the pH of the medium after 4 hours of incubation. In the pre-test, the cytotoxicity was determined by the 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. At 0.25 mg/ml, the RS was 63.98% and 63.32% in the absence and presence of S9 metabolic activation, respectively. - Vehicle / solvent:
- The test substance was soluble in dimethyl sulphoxide (DMSO) at 100 mg/ml. Hence, DMSO was selected as a vehicle for the test substance.
- Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulphoxide
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates
- Number of independent experiments:
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 × 106 cells/ flask
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: 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): After treatment, cells were sub-cultured for 7 days to determine cytotoxicity and allow phenotypic expression. After expression, cells were seeded in a medium with and without selective medium for 9 days to detect mutant colonies and cell viability.
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):
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 6-thioguanine, 10 µg/ml
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Plating for cloning efficiency (CE) 1: 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 in new culture flasks from respective treated cultures 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 (CE) 2: Cells at a 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, 5 % CO2 in a CO2 incubator for 9 days for mutation frequency (MF) determination.
- Criteria for small (slow-growing) and large (fast-growing) colonies: Not observed.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g., background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was determined by relative survival (RS).
- Any supplementary information relevant to cytotoxicity: If the maximum test concentration is based on cytotoxicity, the highest concentration should aim to achieve between 20 and 10% RS.
METHODS FOR MEASUREMENTS OF GENOTOXICIY: Colonies in all the plates for CE1 (before expression), CE2 (after expression) and mutation frequency (MF) determinations were counted manually and recorded in the raw data. The mutant frequency was presented for 10-6 cells. - Evaluation criteria:
- The test substance is considered to be clearly positive if in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically 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 were outside the distribution of the laboratory negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly 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 laboratory negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Statistical analysis was performed to assess a possible dose-dependent increase of mutation frequency using Fisher's Exact Test (NCSS statistics software). The mutation frequency of the Test Item-treated group was compared to the solvent 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:
- At 0.25 mg/ml the relative survival was 64.09% and 65.55% in the absence and presence of S9 metabolic activation, respectively.
- 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: 50 µl of the test substance formulation was added to 4.95 ml of complete medium, resulting in a final test substance concentration of 0.25 mg/ml in the medium. pH values were checked at 0 and 4th hour. The test substance did not affect the pH in the medium after 4-hour incubation.
- Solubility: The test substance was insoluble in water at 200 mg/ml. The substance was soluble in dimethyl sulphoxide at 100 mg/ml.
- Precipitation and time of the determination: Precipitation was observed at the tested concentrations of 1 and 0.5 mg/ml. Slight precipitation was noted at the tested concentrations of 0.25 and 0.125 mg/ml.
RANGE-FINDING/SCREENING STUDIES (if applicable):
Cytotoxicity was tested at 0 (NC), 0 (VC), 0.015625, 0.03125, 0.0625, 0.125 and 0.25 mg/ml in both in the presence and absence of S9 metabolic activation system (1 % v/v S9 mix) using triplicates.
- Results: In the absence of metabolic activation, the relative survival values were 97.23% (vehicle control), 98.91% (at 0.015625 mg/ml), 93.24% (at 0.03125 mg/ml), 82.52% (at 0.0625 mg/ml), 75.15% (at 0.125 mg/ml) and 63.97% (at 0.25 mg/ml). In the presence of metabolic activation, the relative survival values were 98.49% (vehicle control), 97.88%, (at 0.015625 mg/ml), 91.71% (at 0.03125 mg/ml), 83.81% (at 0.0625 mg/ml), 74.80% (at 0.125 mg/ml) and 63.32% (at 0.25 mg/ml).
GENE MUTATION STUDY RESULTS
CHO cells were exposed to negative, vehicle, different concentrations of the test substance, and positive control for 4 hours (short term exposure) in the absence and presence of metabolic activation.
- Results: No significant increase in the mutation frequency (MF) either in absence (7.02x10-6, 6.90 x10-6, 9.09 x10-6 and 9.69 x10-6 at 0.03125 mg/ml, 0.0625 mg/ml, 0.125 mg/ml and 0.25 mg/ml, respectively) or presence of metabolic activation (6.90 x10-6, 8.71x10-6, 8.99x10-6 and 10.38x10-6 at 0.25 mg/ml, 0.03125 mg/ml, 0.0625 mg/ml, 0.125 mg/ml and 0.25 mg/ml, respectively) was observed when compared to vehicle control (7.55 x10-6, 8.40 x 10-6, absence and presence of S9, respectively).
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to the section “Any other information on results including tables”.
- Negative (solvent/vehicle) historical control data: Please refer to the section “Any other information on results including tables”. - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The registered substance i.e., N-{4-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]phenyl}acetamide (CAS No. 67905-17-3) was tested non-mutagenic (negative) in an in vitro mammalian cell gene mutation test both in the presence and absence of S9 metabolic activation using cultured CHO cells. The test was performed according to OECD 476 and in compliance with OECD Principles of Good Laboratory Practice.
- Executive summary:
The test chemical, i.e., N-{4-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]phenyl} acetamide (CAS No. 67905-17-3) was tested in an in vitro mammalian cell gene mutation test according to OECD test guideline 476. The test was performed to assess the ability of the substance to cause gene mutation in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented S9 microsomal fraction, derived from the liver of phenobarbital and β-naphthoflavone-induced rat, was used as a metabolic activation system. Dimethyl sulfoxide was selected as a vehicle for the substance. Test concentrations were chosen based on the solubility, precipitation and pH checks and a preliminary cytotoxicity test. The test substance formed precipitation at 0.5 and 1 mg/ml, which was considered to interfere with scoring and slight precipitation at 0.25 mg/ml to 0.125 mg/ml in the culture medium. Therefore the cytotoxicity test was performed with concentrations of 0.0 (NC), 0.0 (VC), 0.015625, 0.03125, 0.0625, 0.125, and 0.25 mg/ml both in the presence and absence of S9 metabolic activation using triplicates.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. In the pre-test, no limiting cytotoxicity (10-20% RS) was observed; the highest test concentration, 0.25 mg/ml, produced >60% RS both in the presence and absence of metabolic activation (at 0.25 mg/ml RS: 63.97% without S9 mix; RS: 63.32% with S9 mix). Since the test substance formed precipitation at 0.5-1 mg/ml, which interfered with scoring, 0.25 mg/ml was selected as the maximum test concentration in the main test. Three lower concentrations were included in the assay using spacing factor 2. In the gene mutation test, CHO cells were exposed to the test substance at 0.0 (NC), 0.0 (VC), 03125, 0.0625, 0.125 and 0.25 mg/ml for 4 hours both in the presence and absence of S9 metabolic activation using triplicate cultures. Positive control substances (Ethylmethanesulfonate without S9 mix, Benzo[a]pyrene with S9 mix) were also included in the test.Results:In the absence of metabolic activation the relative survival values were 98.69% (VC), 92.24% (at 0.03125 mg/ml), 85.75% (at 0.0625 mg/ml), 77.64% (at 0.125 mg/ml), 64.09% (at 0.25 mg/ml) and 93.61% (PC, 400 µg/ml Ethyl methanesulfonate). In the presence of metabolic activation, the relative survival values were 98.60% (VC), 90.37% (at 0.03125 mg/ml), 82.90% (at 0.0625 mg/ml), 77.45% (at 0.125 mg/ml) 65.55% (at 0.25 mg/ml) and 97.64% (PC, 30 µg/ml Benzo[a]pyrene). No significant increase in the mutation frequency (MF) either in absence (7.02x10-6, 6.90 x10-6, 9.09 x10-6 and 9.69 x10-6 at 0.03125 mg/ml, 0.0625 mg/ml, 0.125 mg/ml and 0.25 mg/ml, respectively) or presence of metabolic activation (6.90 x10-6, 8.71x10-6, 8.99x10-6and 10.38x10-6at 0.25 mg/ml, 0.03125 mg/ml, 0.0625 mg/ml, 0.125 mg/ml and 0.25 mg/ml, respectively) was observed when compared to vehicle control (7.55 x10-6, 8.40 x 10-6, absence and presence of S9, respectively).No significant reduction in the relative survival (cytotoxicity) or increase in mutation frequency 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 used in the study produced statistically significant increases in mutation frequency, 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.Conclusion:The registered substance, i.e., N-{4-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]phenyl}acetamide (CAS No. 67905-17-3) did not induce gene mutation at the locus of hypoxanthine-guanine phosphoribosyltransferase (Hprt) in CHO cells up to the concentration of 0.25 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system.
Referenceopen allclose all
Table: Results of the mutagenicity test for the test chemical
TA100 |
||||||||
S9 |
Dose levelsµg/plate |
Number of revertants/plate |
Mean |
SD |
Ratio dose/control |
p |
||
Plate 1 |
Plate 1 |
Plate 3 |
||||||
+ |
2-Aminoanthracene |
1703 |
1512 |
1460 |
1558.3 |
128.0 |
12.7 |
|
+ |
Negative control |
124 |
128 |
119 |
123.7 |
4.5 |
- |
|
+ |
DMSO |
111 |
134 |
122 |
122.3 |
11.5 |
- |
|
+ |
50 |
135 |
116 |
126 |
125.7 |
9.5 |
1.0 |
|
+ |
160 |
137 |
130 |
151 |
139.3 |
10.7 |
1.1 |
P |
+ |
500 |
135 |
125 |
127 |
129.0 |
5.3 |
1.1 |
P |
+ |
1600 |
129 |
127 |
139 |
131.7 |
6.4 |
1.1 |
P |
+ |
5000 |
122 |
104 |
110 |
112.0 |
9.2 |
0.9 |
p |
- |
Sodium azide |
586 |
539 |
620 |
581.7 |
40.7 |
5.4 |
|
- |
Negative control |
123 |
115 |
119 |
119.0 |
4.0 |
- |
|
- |
DMSO |
109 |
119 |
98 |
108.7 |
10.5 |
- |
|
- |
50 |
109 |
109 |
104 |
107.3 |
2.9 |
1.0 |
|
- |
160 |
109 |
103 |
130 |
114.0 |
14.2 |
1.0 |
P |
- |
500 |
109 |
98 |
109 |
105.3 |
6.4 |
1.0 |
P |
- |
1600 |
103 |
108 |
115 |
108.7 |
6.0 |
1.0 |
P |
- |
5000 |
109 |
98 |
79 |
95.3 |
15.2 |
0.9 |
P |
P: Precipitated
TA1535 |
||||||||
S9 |
Dose levelsµg/plate |
Number of revertants/plate |
Mean |
SD |
Ratio dose/control |
p |
||
Plate 1 |
Plate 1 |
Plate 3 |
||||||
+ |
2-Aminoanthracene |
440 |
439 |
456 |
445.0 |
9.5 |
47.7 |
|
+ |
Negative control |
4 |
7 |
10 |
7.0 |
3.0 |
- |
|
+ |
DMSO |
10 |
9 |
9 |
9.3 |
0.6 |
- |
|
+ |
50 |
8 |
8 |
4 |
6.7 |
2.3 |
0.7 |
|
+ |
160 |
11 |
7 |
5 |
7.7 |
3.1 |
0.8 |
P |
+ |
500 |
11 |
5 |
4 |
6.7 |
3.8 |
0.7 |
P |
+ |
1600 |
7 |
10 |
8 |
8.3 |
1.5 |
0.9 |
P |
+ |
5000 |
12 |
6 |
7 |
8.3 |
3.2 |
0.9 |
p |
- |
Sodium azide |
193 |
1889 |
143 |
175.0 |
37.8 |
32.8 |
|
- |
Negative control |
13 |
6 |
11 |
10.0 |
3.6 |
- |
|
- |
DMSO |
6 |
4 |
6 |
5.3 |
1.2 |
- |
|
- |
50 |
7 |
4 |
5 |
5.3 |
1.5 |
1.0 |
|
- |
160 |
6 |
7 |
5 |
6.0 |
1.0 |
1.1 |
P |
- |
500 |
10 |
7 |
3 |
6.7 |
3.5 |
1.3 |
P |
- |
1600 |
6 |
8 |
5 |
6.3 |
1.5 |
1.2 |
P |
- |
5000 |
8 |
5 |
10 |
7.7 |
2.5 |
1.4 |
P |
P: Precipitated
TA1537 |
||||||||
S9 |
Dose levelsµg/plate |
Number of revertants/plate |
Mean |
SD |
Ratio dose/control |
p |
||
Plate 1 |
Plate 1 |
Plate 3 |
||||||
+ |
2-Aminoanthracene |
99 |
102 |
93 |
98.0 |
4.6 |
5.9 |
|
+ |
Negative control |
19 |
17 |
12 |
16.0 |
3.6 |
- |
|
+ |
DMSO |
18 |
16 |
16 |
16.7 |
1.2 |
- |
|
+ |
50 |
28 |
22 |
17 |
22.3 |
5.5 |
1.3 |
|
+ |
160 |
20 |
23 |
26 |
23.0 |
3.0 |
1.4 |
P |
+ |
500 |
24 |
24 |
20 |
22.7 |
2.3 |
1.4 |
P |
+ |
1600 |
19 |
23 |
24 |
22.0 |
2.6 |
1.3 |
P |
+ |
5000 |
14 |
17 |
27 |
19.3 |
6.8 |
1.2 |
p |
- |
9-aminoacridine |
270 |
222 |
153 |
215.0 |
58.8 |
23.0 |
|
- |
Negative control |
12 |
15 |
8 |
11.7 |
3.5 |
- |
|
- |
DMSO |
7 |
9 |
12 |
9.3 |
2.5 |
- |
|
- |
50 |
13 |
11 |
16 |
13.3 |
2.5 |
1.4 |
|
- |
160 |
9 |
11 |
8 |
9.3 |
1.5 |
1.0 |
P |
- |
500 |
8 |
7 |
12 |
9.0 |
2.6 |
1.0 |
P |
- |
1600 |
15 |
13 |
10 |
12.7 |
2.5 |
1.4 |
P |
- |
5000 |
10 |
13 |
11 |
11.3 |
1.5 |
1.2 |
P |
P: Precipitated
TA98 |
||||||||
S9 |
Dose levelsµg/plate |
Number of revertants/plate |
Mean |
SD |
Ratio dose/control |
p |
||
Plate 1 |
Plate 1 |
Plate 3 |
||||||
+ |
2-Aminoanthracene |
1054 |
1120 |
1076 |
1083.3 |
33.6 |
54.2 |
|
+ |
Negative control |
18 |
23 |
23 |
21.3 |
2.9 |
- |
|
+ |
DMSO |
19 |
21 |
20 |
20.0 |
1.0 |
- |
|
+ |
50 |
21 |
18 |
14 |
17.7 |
3.5 |
0.9 |
|
+ |
160 |
28 |
27 |
21 |
25.3 |
3.9 |
1.3 |
P |
+ |
500 |
30 |
21 |
28 |
26.3 |
4.7 |
1.3 |
P |
+ |
1600 |
32 |
26 |
34 |
30.7 |
4.2 |
1.5 |
P |
+ |
5000 |
20 |
34 |
34 |
29.3 |
8.1 |
1.5 |
p |
- |
2-nitrofluorene |
301 |
299 |
301 |
300.3 |
1.2 |
26.5 |
|
- |
Negative control |
12 |
14 |
9 |
11.7 |
2.5 |
- |
|
- |
DMSO |
14 |
13 |
7 |
11.3 |
3.8 |
- |
|
- |
50 |
14 |
14 |
8 |
12.0 |
3.5 |
1.1 |
|
- |
160 |
8 |
14 |
13 |
11.7 |
3.2 |
1.0 |
P |
- |
500 |
16 |
13 |
12 |
13.7 |
2.1 |
1.2 |
P |
- |
1600 |
13 |
4 |
12 |
9.7 |
4.9 |
0.9 |
P |
- |
5000 |
7 |
11 |
10 |
9.3 |
2.1 |
0.8 |
P |
P: Precipitated
E.coli WP2uvrA |
||||||||
S9 |
Dose levelsµg/plate |
Number of revertants/plate |
Mean |
SD |
Ratio dose/control |
p |
||
Plate 1 |
Plate 1 |
Plate 3 |
||||||
+ |
2-Aminoanthracene |
125 |
97 |
88 |
103.3 |
19.3 |
7.0 |
|
+ |
Negative control |
15 |
18 |
11 |
14.7 |
3.5 |
- |
|
+ |
DMSO |
16 |
11 |
17 |
14.7 |
3.2 |
- |
|
+ |
50 |
17 |
19 |
17 |
17.7 |
1.2 |
1.2 |
|
+ |
160 |
16 |
15 |
15 |
15.3 |
0.6 |
1.0 |
P |
+ |
500 |
15 |
11 |
18 |
14.7 |
3.5 |
1.0 |
P |
+ |
1600 |
14 |
16 |
14 |
14.7 |
1.2 |
1.0 |
P |
+ |
5000 |
16 |
13 |
12 |
13.7 |
2.1 |
0.9 |
p |
- |
9-NQO |
529 |
578 |
572 |
559.7 |
26.7 |
31.7 |
|
- |
Negative control |
17 |
17 |
23 |
19.0 |
3.5 |
- |
|
- |
DMSO |
26 |
14 |
13 |
17.7 |
7.2 |
- |
|
- |
50 |
21 |
15 |
15 |
17.0 |
3.5 |
1.0 |
|
- |
160 |
18 |
14 |
17 |
16.3 |
2.1 |
0.9 |
P |
- |
500 |
15 |
20 |
20 |
18.3 |
2.9 |
1.0 |
P |
- |
1600 |
19 |
17 |
15 |
17.0 |
2.0 |
1.0 |
P |
- |
5000 |
12 |
19 |
18 |
16.3 |
3.8 |
0.9 |
P |
P: Precipitated
Mitotic index - Cytotoxicity test
Treatment |
R |
Mitotic Index (%) |
|||||||
Absence of Metabolic Activation (-S9) |
Presence of Metabolic Activation (1% S9) |
||||||||
Mitotic Index |
Mean |
SD |
Percent Reduction |
Mitotic Index |
Mean |
SD |
Percent Reduction |
||
NC |
R1 |
9.99 |
9.99 |
0.00 |
- |
9.91 |
10.10 |
0.28 |
- |
R2 |
9.99 |
10.30 |
|||||||
VC |
R1 |
9.69 |
9.84 |
0.20 |
- |
9.77 |
9.88 |
0.15 |
- |
R2 |
9.98 |
9.98 |
|||||||
T1 |
R1 |
6.60 |
6.44 |
0.23 |
34.55 |
6.48 |
6.94 |
0.65 |
29.72 |
R2 |
6.27 |
7.40 |
|||||||
T2 |
R1 |
4.89 |
5.08 |
0.27 |
48.34 |
4.76 |
4.91 |
0.20 |
50.32 |
R2 |
5.27 |
5.05 |
|||||||
T3 |
R1 |
4.45 |
4.84 |
0.55 |
50.76 |
4.62 |
4.47 |
0.21 |
54.74 |
R2 |
5.23 |
4.32 |
|||||||
PC |
R1 |
9.37 |
9.27 |
0.14 |
5.72 |
9.16 |
8.87 |
0.41 |
10.15 |
R2 |
9.17 |
8.58 |
|||||||
Key: R = Replicate, NC = Negative control, VC = Vehicle control, PC = Positive control, SD = Standard Deviation
Summary of mitotic index
Mitotic Index (%) |
|||||
Phase I |
|||||
Treatment |
Absence of Metabolic Activation (-S9) |
Treatment |
Presence of Metabolic Activation (+S9) (1%) |
||
Mean |
SD |
Mean |
SD |
||
NC |
10.03 |
0.06 |
NC |
10.16 |
0.11 |
VC |
9.94 |
0.06 |
VC |
9.89 |
0.15 |
T1 (0.5 mg/mL) |
6.85 |
0.36 |
T1 (0.25 mg/mL) |
7.10 |
0.42 |
T2 (1.0 mg/mL) |
5.22 |
0.55 |
T2 (0.5 mg/mL) |
6.80 |
0.57 |
T3 (2.0 mg/mL) |
4.85 |
0.29 |
T3 (1.0 mg/mL) |
4.89 |
0.03 |
PC |
8.19 |
0.12 |
PC |
8.58 |
0.13 |
Mitotic Index (%) |
|||||
Phase II |
|||||
Treatment |
Absence of Metabolic Activation (-S9) |
Treatment |
Presence of Metabolic Activation (+S9) (2%) |
||
Mean |
SD |
Mean |
SD |
||
NC |
9.99 |
0.01 |
NC |
10.00 |
0.15 |
VC |
9.69 |
0.14 |
VC |
9.94 |
0.06 |
T1 (0.5 mg/mL) |
6.10 |
0.14 |
T1 (0.25 mg/mL) |
6.75 |
0.63 |
T2 (1.0 mg/mL) |
4.91 |
0.15 |
T2 (0.5 mg/mL) |
4.70 |
0.14 |
T3 (2.0 mg/mL) |
4.49 |
0.07 |
T3 (1.0 mg/mL) |
4.50 |
0.13 |
PC |
8.85 |
0.07 |
PC |
8.79 |
0.13 |
Key: NC = Negative control, VC = Vehicle control PC = Positive control, MI = Mitotic Index, - S9 = Absence of metabolic activation, + S9 = Presence of metabolic activation.
Summary of percent aberrant cells
Percent Aberrant Cells |
|||||
Phase I |
|||||
Treatment |
Absence of Metabolic Activation (-S9) |
Treatment |
Presence of Metabolic Activation (+S9) (1%) |
||
Mean |
SD |
Mean |
SD |
||
NC |
0.333 |
0.471 |
NC |
0.333 |
0.471 |
VC |
0.667 |
0.000 |
VC |
0.333 |
0.471 |
T1 (0.5 mg/mL) |
0.333 |
0.471 |
T1 (0.25 mg/mL) |
0.333 |
0.471 |
T2 (1.0 mg/mL) |
0.667 |
0.000 |
T2 (0.5 mg/mL) |
0.333 |
0.471 |
T3 (2.0 mg/mL) |
0.667 |
0.000 |
T3 (1.0 mg/mL) |
0.667 |
0.000 |
PC |
10.000 |
0.943 |
PC |
9.000 |
1.414 |
Percent Aberrant Cells |
|||||
Phase II |
|||||
Treatment |
Absence of Metabolic Activation (-S9) |
Treatment |
Presence of Metabolic Activation (+S9) (2%) |
||
Mean |
SD |
Mean |
SD |
||
NC |
0.333 |
0.471 |
NC |
0.333 |
0.471 |
VC |
0.333 |
0.471 |
VC |
0.333 |
0.471 |
T1 (0.5 mg/mL) |
0.333 |
0.471 |
T1 (0.25 mg/mL) |
0.333 |
0.471 |
T2 (1.0 mg/mL) |
0.333 |
0.471 |
T2 (0.5 mg/mL) |
0.333 |
0.471 |
T3 (2.0 mg/mL) |
0.667 |
0.000 |
T3 (1.0 mg/mL) |
0.667 |
0.000 |
PC |
10.000 |
0.943 |
PC |
10.667 |
1.886 |
Key: NC = Negative Control, VC = Vehicle control, SD = Standard Deviation, PC = Positive Control
Induvidual observation of slides for mitotic index and chromosome aberrations
Phase I in the absence of S9 metabolic activation (-S9)
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
|
NC |
R1 |
10.07 |
- |
0 |
0.00 | |
R2 |
9.99 |
1 fragments |
1 |
0.67 | ||
VC |
R1 |
9.98 |
1 fragment |
1 |
0.67 | |
R2 |
9.89 |
1 fragment |
1 |
0.67 | ||
T1 (0.5 mg/mL) |
R1 |
6.60 |
1 ctb |
1 |
0.67 | |
R2 |
7.11 |
- |
0 |
0.00 | ||
T2 (1.0 mg/mL) |
R1 |
5.61 |
1 csb |
1 |
0.67 | |
R2 |
4.83 |
1 ctg, 1 fragment |
2 |
0.67 | ||
T3 (2.0 mg/mL) |
R1 |
4.65 |
1 ctb, 1 fragment |
2 |
0.67 | |
R2 |
5.06 |
1 ctb, 1 csg |
2 |
0.67 | ||
PC |
R1 |
8.10 |
2 ctb, 2 cte, 2 ctg, 3 csb, 2 cse, 3 csg, 1 DC, 10 fragments |
25 |
9.33 | |
R2 |
8.28 |
2 ctb, 2 cte, 3 ctg, 2 csb, 1 cse, 2 csg, 1 DC, 1 AC, 14 fragments |
28 |
10.67 |
Phase I in the presence of S9 metabolic activation (+S9)
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
NC |
R1 |
10.08 |
1 fragments |
1 |
0.67 |
R2 |
10.24 |
- |
0 |
0.00 |
|
VC |
R1 |
9.99 |
1 fragment |
1 |
0.67 |
R2 |
9.78 |
- |
0 |
0.00 |
|
T1 (0.25 mg/mL) |
R1 |
7.40 |
1 DC |
1 |
0.67 |
R2 |
6.80 |
- |
0 |
0.00 |
|
T2 (0.5 mg/mL) |
R1 |
6.40 |
1 csg, 1 fragment |
2 |
0.67 |
R2 |
7.20 |
- |
0 |
0.00 |
|
T3 (1.0 mg/mL) |
R1 |
4.87 |
1 ctb, 1 fragment |
2 |
0.67 |
R2 |
4.91 |
1 ctb |
1 |
0.67 |
|
PC |
R1 |
8.67 |
2 ctb, 1 cte, 2 ctg, 1 csb, 1 cse, 3 csg, 1 DC, 1 AC, 9 fragments |
21 |
8.00 |
R2 |
8.49 |
1 ctb, 2 cte, 3 ctg, 1 csb, 2 cse, 3 csg, 1 DC, 1 AC, 10 fragments |
24 |
10.00 |
Key:MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric, AC = Acentriccte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control, PC = Positive Control
Phase II in the absence of S9 metabolic activation (-S9)
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
NC |
R1 |
9.98 |
1 ctb |
1 |
0.67 |
R2 |
9.99 |
- |
0 |
0.00 |
|
VC |
R1 |
9.79 |
- |
0 |
0.00 |
R2 |
9.59 |
1 fragment |
1 |
0.67 |
|
T1 (0.5 mg/mL) |
R1 |
6.00 |
1 cte |
0 |
0.00 |
R2 |
6.19 |
1 cse, 1 DC |
2 |
0.67 |
|
T2 (1.0 mg/mL) |
R1 |
4.81 |
- |
0 |
0.00 |
R2 |
5.02 |
1 cse |
1 |
0.67 |
|
T3 (2.0 mg/mL) |
R1 |
4.44 |
1 csg, 1 fragment |
2 |
0.67 |
R2 |
4.54 |
1 csb, 1 csg |
2 |
0.67 |
|
PC |
R1 |
8.80 |
3 ctb, 2 cte, 3 ctg, 1 csb, 1 cse, 4 csg, 1 DC, 12 fragements |
27 |
9.33 |
R2 |
8.90 |
2 ctb, 2 cte, 3 ctg, 3 csb, 2 cse, 3 csg, 1 DC, 8 fragments |
24 |
10.67 |
Phase II in the presence of S9 metabolic activation (+S9)
Treatment |
Culture No. |
Mitotic Index |
Frequencies of Aberration |
Total No. of Aberration |
Percentage of Aberrated Cells |
|
NC |
R1 |
10.10 |
- |
0 |
0.00 |
|
R2 |
9.89 |
1 fragment |
1 |
0.67 |
||
VC |
R1 |
9.98 |
2 fragments |
2 |
0.67 |
|
R2 |
9.89 |
- |
0 |
0.00 |
||
T1 (0.25 mg/mL) |
R1 |
6.30 |
- |
0 |
0.00 |
|
R2 |
7.19 |
1 ctb, 1 ctg |
2 |
0.67 |
||
T2 (0.5 mg/mL) |
R1 |
4.60 |
- |
0 |
0.00 |
|
R2 |
4.80 |
1 cse |
1 |
0.67 |
||
T3 (1.0 mg/mL) |
R1 |
4.59 |
1 ctb |
1 |
0.67 |
|
R2 |
4.40 |
1 cte, 1 DC |
2 |
0.67 |
||
PC |
R1 |
8.88 |
2 ctb, 2 cte, 2 ctg, 2 csb, 2 cse, 3 csg, 1 DC, 9 fragments |
23 |
9.33 |
|
R2 |
8.70 |
2ctb, 2 cte, 2 ctg, 2 csb, 2 cse, 4 csg, 1 DC, 1AC, 10 fragments |
26 |
12.00 |
Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric, cte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control, PC = Positive Control.
HISTORICAL DATA :
HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) |
||||||||||
S.No. |
Study No. |
Vehicle |
Phase I |
Phase II |
||||||
Absence of S9 |
Presence of S9 |
Absence of S9 |
Presence of S9 |
|||||||
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
|||
1 |
1151 |
DMSO |
0.000 |
9.000 |
0.000 |
10.500 |
0.000 |
9.000 |
0.000 |
8.000 |
2 |
1333 |
DMSO |
0.000 |
8.000 |
0.000 |
7.500 |
0.500 |
8.500 |
0.500 |
9.000 |
3 |
2060 |
DMSO |
0.500 |
8.000 |
0.000 |
7.000 |
1.500 |
6.500 |
0.000 |
9.000 |
4 |
2450 |
DMSO |
0.000 |
10.000 |
0.000 |
10.500 |
0.000 |
11.500 |
0.000 |
12.000 |
5 |
2452 |
DMSO |
0.000 |
10.000 |
0.000 |
8.500 |
0.000 |
9.500 |
0.000 |
8.500 |
6 |
3000 |
PBS |
0.000 |
7.500 |
0.000 |
8.500 |
0.000 |
11.000 |
0.000 |
10.000 |
7 |
3313 |
DMSO |
0.000 |
8.000 |
0.000 |
10.500 |
0.500 |
9.500 |
0.000 |
9.500 |
8 |
3422 |
DMSO |
0.000 |
9.000 |
0.500 |
10.000 |
1.000 |
9.500 |
1.000 |
8.500 |
9 |
3665 |
RPMI |
0.500 |
8.500 |
0.000 |
7.500 |
0.000 |
8.500 |
0.500 |
8.000 |
10 |
3801 |
Sodium Phosphate Buffer |
1.500 |
9.500 |
1.000 |
9.000 |
1.000 |
9.500 |
0.500 |
9.500 |
11 |
3862 |
DMSO |
1.500 |
9.500 |
1.000 |
9.000 |
1.000 |
9.500 |
0.500 |
9.500 |
12 |
4792 |
PBS |
0.500 |
7.500 |
0.500 |
8.500 |
0.500 |
8.500 |
0.000 |
8.000 |
13 |
4938 |
DMSO |
0.500 |
8.500 |
1.000 |
8.500 |
0.500 |
8.000 |
1.000 |
8.000 |
14 |
5123 |
DMSO |
0.333 |
9.000 |
0.667 |
8.667 |
0.333 |
9.667 |
0.333 |
9.000 |
15 |
5739 |
Ethyl alcohol |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
10.667 |
16 |
5824 |
PBS |
0.333 |
10.000 |
0.333 |
11.000 |
0.333 |
9.333 |
0.333 |
10.000 |
17 |
6461 |
PBS |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
9.000 |
0.333 |
10.000 |
18 |
6196 |
RPMI |
0.333 |
11.000 |
0.333 |
10.000 |
0.333 |
10.667 |
0.333 |
10.000 |
19 |
6121 |
DMSO |
0.667 |
8.667 |
0.667 |
9.667 |
0.667 |
9.667 |
0.667 |
9.333 |
HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) |
||||||||||
S.No. |
Study No. |
Vehicle |
Phase I |
Phase II |
||||||
Absence of S9 |
Presence of S9 |
Absence of S9 |
Presence of S9 |
|||||||
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
|||
20 |
6678 |
DMSO |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
10.667 |
21 |
6687 |
DMSO |
0.333 |
11.333 |
0.333 |
11.333 |
0.333 |
12.333 |
0.333 |
12.000 |
22 |
6221 |
DMSO |
0.333 |
9.667 |
0.333 |
10.667 |
0.333 |
9.667 |
0.333 |
10.333 |
23 |
6834 |
DMSO |
0.333 |
10.333 |
0.333 |
11.333 |
0.333 |
11.333 |
0.333 |
10.667 |
24 |
6759 |
PBS |
0.667 |
10.667 |
0.000 |
10.000 |
0.333 |
12.000 |
0.333 |
11.333 |
25 |
6430 |
DMSO |
0.333 |
9.000 |
0.333 |
10.000 |
0.667 |
9.667 |
0.667 |
9.667 |
26 |
7703 |
DMSO |
0.333 |
10.000 |
0.333 |
10 |
0.333 |
10.333 |
0.333 |
10.333 |
27 |
7576 |
RPMI |
0.333 |
10.000 |
0.333 |
10.667 |
0.333 |
10.333 |
0.333 |
10.333 |
28 |
7572 |
DMSO |
0.667 |
10.333 |
0.667 |
10 |
0.667 |
9.667 |
0.333 |
10 |
29 |
7574 |
Ethyl alcohol |
0.333 |
10.333 |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.333 |
30 |
7434 |
DMSO |
0.667 |
10.000 |
0.333 |
10.667 |
0.333 |
9.667 |
0.333 |
11.000 |
31 |
7708 |
DMSO |
0.333 |
9.667 |
0.333 |
10.333 |
0.333 |
9.333 |
0.333 |
9.667 |
32 |
7263 |
DMSO |
0.333 |
10.667 |
0.333 |
10.333 |
0.667 |
10.667 |
0.667 |
9.667 |
33 |
8072 |
DMSO |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.333 |
0.333 |
10.333 |
34 |
4825 |
DMSO |
0.667 |
9.000 |
0.667 |
9.333 |
0.333 |
10.000 |
0.667 |
9.000 |
35 |
8112 |
DMSO |
0.333 |
9.667 |
0.333 |
10.000 |
0.333 |
10.667 |
0.333 |
10.333 |
36 |
8142 |
DMSO |
0.333 |
10.000 |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.333 |
37 |
8091 |
DMSO |
0.333 |
10.333 |
0.333 |
10.333 |
0.333 |
10.000 |
0.333 |
10.000 |
38 |
8174 |
RPMI |
0.333 |
11.000 |
0.333 |
10.000 |
0.333 |
9.667 |
0.333 |
10.667 |
39 |
7657 |
DMSO |
0.333 |
10.333 |
0.333 |
11.333 |
0.333 |
10.000 |
0.333 |
11.000 |
HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H) |
||||||||||
S.No. |
Study No. |
Vehicle |
Phase I |
Phase II |
||||||
Absence of S9 |
Presence of S9 |
Absence of S9 |
Presence of S9 |
|||||||
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
Vehicle Control |
Positive Control |
|||
40 |
8176 |
DMSO |
0.333 |
11.333 |
0.333 |
8.667 |
0.333 |
10.667 |
0.333 |
10.000 |
41 |
8541 |
DMSO |
0.667 |
9.667 |
0.333 |
10.000 |
0.667 |
9.667 |
0.333 |
10.000 |
42 |
8064 |
DMSO |
0.333 |
10.667 |
0.667 |
9.667 |
0.333 |
10.333 |
0.333 |
10.000 |
43 |
8486 |
DMSO |
0.333 |
10.000 |
0.333 |
10.333 |
0.333 |
10.333 |
0.667 |
11.333 |
44 |
8660 |
RPMI |
0.333 |
11.000 |
0.333 |
10.000 |
0.333 |
10.333 |
0.333 |
10.000 |
45 |
8722 |
DMSO |
0.667 |
10.000 |
0.667 |
10.667 |
0.667 |
9.333 |
0.667 |
10.666 |
46 |
8670 |
DMSO |
0.333 |
10.000 |
0.333 |
11.000 |
0.333 |
10.667 |
0.667 |
10.000 |
47 |
8680 |
DMSO |
0.333 |
10.333 |
0.667 |
10.333 |
0.333 |
10.000 |
0.333 |
10.000 |
48 |
9845 |
DMSO |
0.333 |
10.000 |
0.333 |
11.000 |
0.333 |
10.669 |
0.333 |
10.667 |
Mean |
|
0.392 |
9.750 |
0.374 |
9.858 |
0.422 |
9.882 |
0.374 |
9.934 |
|
SD |
|
0.308 |
0.952 |
0.259 |
1.005 |
0.279 |
0.988 |
0.225 |
0.954 |
|
Mean + 2SD |
|
1.009 |
11.654 |
0.892 |
11.868 |
0.980 |
11.857 |
0.824 |
11.843 |
|
Mean - 2SD |
|
-0.224 |
7.846 |
-0.144 |
7.847 |
-0.136 |
7.907 |
-0.077 |
8.025 |
|
Preliminary cytotoxicity assay - Relative survival (-S9 mix)
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 |
22800000 |
238 |
230 |
227 |
231.67 |
100 |
2.317 |
2.641 |
100.00 |
VC |
Dimethyl sulfoxide |
20000000 |
22460000 |
226 |
229 |
231 |
228.67 |
100 |
2.287 |
2.568 |
97.23 |
T1 |
0.015625 mg/ml |
20000000 |
22280000 |
232 |
227 |
225 |
228.00 |
100 |
2.280 |
2.540 |
98.91 |
T2 |
0.03125 mg/ml |
20000000 |
21900000 |
218 |
223 |
215 |
218.67 |
100 |
2.187 |
2.394 |
93.24 |
T3 |
0.0625 mg/ml |
20000000 |
21440000 |
204 |
198 |
191 |
197.67 |
100 |
1.977 |
2.119 |
82.52 |
T4 |
0.125 mg/ml |
20000000 |
20420000 |
187 |
188 |
192 |
189.00 |
100 |
1.890 |
1.930 |
75.15 |
T5 |
0.25 mg/ml |
20000000 |
19440000 |
171 |
170 |
166 |
169.00 |
100 |
1.690 |
1.643 |
63.97 |
Preliminary cytotoxicity assay - Relative survival (+ S9 mix)
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 |
22860000 |
231 |
240 |
235 |
235.33 |
100 |
2.353 |
2.690 |
100.00 |
VC |
Dimethyl sulfoxide |
20000000 |
22740000 |
234 |
235 |
230 |
233.00 |
100 |
2.330 |
2.649 |
98.49 |
T1 |
0.015625 mg/ml |
20000000 |
22580000 |
228 |
224 |
237 |
229.67 |
100 |
2.297 |
2.593 |
97.88 |
T2 |
0.03125 mg/ml |
20000000 |
22120000 |
220 |
219 |
220 |
219.67 |
100 |
2.197 |
2.430 |
91.71 |
T3 |
0.0625 mg/ml |
20000000 |
21840000 |
203 |
211 |
196 |
203.33 |
100 |
2.033 |
2.220 |
83.81 |
T4 |
0.125 mg/ml |
20000000 |
20860000 |
188 |
190 |
192 |
190.00 |
100 |
1.900 |
1.982 |
74.80 |
T5 |
0.25 mg/ml |
20000000 |
19620000 |
175 |
168 |
170 |
171.00 |
100 |
1.710 |
1.678 |
63.32 |
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.
Relative survival - Main study (- S9)
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 |
22840000 |
228 |
239 |
235 |
234.00 |
100 |
2.340 |
2.672 |
100.00 |
VC |
Dimethyl sulfoxide |
20000000 |
22800000 |
231 |
227 |
236 |
231.33 |
100 |
2.313 |
2.637 |
98.69 |
T1 |
0.03125 mg/ml |
20000000 |
21980000 |
224 |
219 |
221 |
221.33 |
100 |
2.213 |
2.432 |
92.24 |
T2 |
0.0625 mg/ml |
20000000 |
21640000 |
210 |
203 |
214 |
209.00 |
100 |
2.090 |
2.261 |
85.75 |
T3 |
0.125 mg/ml |
20000000 |
20340000 |
202 |
199 |
203 |
201.33 |
100 |
2.013 |
2.048 |
77.64 |
T4 |
0.25 mg/ml |
20000000 |
18240000 |
188 |
186 |
182 |
185.33 |
100 |
1.853 |
1.690 |
64.09 |
PC |
400 µg/ml |
20000000 |
22140000 |
224 |
230 |
215 |
223.00 |
100 |
2.230 |
2.469 |
93.61 |
Relative survival - Main study (+ S9)
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 |
22660000 |
224 |
230 |
234 |
229.33 |
100 |
2.293 |
2.598 |
100.00 |
VC |
Dimethyl sulfoxide |
20000000 |
22540000 |
230 |
221 |
231 |
227.33 |
100 |
2.273 |
2.562 |
98.60 |
T1 |
0.03125 mg/ml |
20000000 |
22120000 |
211 |
215 |
202 |
209.33 |
100 |
2.093 |
2.315 |
90.37 |
T2 |
0.0625 mg/ml |
20000000 |
21240000 |
205 |
201 |
194 |
200.00 |
100 |
2.000 |
2.124 |
82.90 |
T3 |
0.125 mg/ml |
20000000 |
20180000 |
190 |
194 |
206 |
196.67 |
100 |
1.967 |
1.984 |
77.45 |
T4 |
0.25 mg/ml |
20000000 |
18660000 |
184 |
180 |
176 |
180.00 |
100 |
1.800 |
1.679 |
65.55 |
PC |
30 µg/ml |
20000000 |
22040000 |
219 |
235 |
227 |
227.00 |
100 |
2.270 |
2.502 |
97.64 |
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.
Cloning efficiency - Main study (non-selective medium) without S9 mix
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 |
205 |
194 |
196 |
198 |
1.98 |
VC |
Dimethyl sulfoxide |
100 |
202 |
205 |
189 |
199 |
1.99 |
T1 |
0.03125 mg/ml |
100 |
194 |
190 |
186 |
190 |
1.90 |
T2 |
0.0625 mg/ml |
100 |
189 |
196 |
195 |
193 |
1.93 |
T3 |
0.125 mg/ml |
100 |
184 |
188 |
178 |
183 |
1.83 |
T4 |
0.25 mg/ml |
100 |
168 |
172 |
176 |
172 |
1.72 |
PC |
400 µg/ml |
100 |
165 |
179 |
176 |
173 |
1.73 |
Cloning efficiency - Main study (non-selective medium) with S9 mix
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 |
205 |
209 |
196 |
203 |
2.03 |
VC |
Dimethyl sulfoxide |
100 |
201 |
198 |
196 |
198 |
1.98 |
T1 |
0.03125 mg/ml |
100 |
192 |
198 |
190 |
193 |
1.93 |
T2 |
0.0625 mg/ml |
100 |
186 |
196 |
192 |
191 |
1.91 |
T3 |
0.125 mg/ml |
100 |
186 |
189 |
181 |
185 |
1.85 |
T4 |
0.25 mg/ml |
100 |
178 |
172 |
180 |
177 |
1.77 |
PC |
30 µg/ml |
100 |
177 |
172 |
188 |
179 |
1.79 |
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.
Cloning efficiency - Main study (selective medium) without S9 mix
Dose level |
Selective medium |
||||||
Concentration |
No. of cells seeded |
No. of colonies |
Mean No. of colonies |
CE |
|||
R1 |
R2 |
R3 |
|||||
NC |
Distilled water |
200000 |
4 |
2 |
2 |
2.67 |
0.00001333 |
VC |
Dimethyl sulfoxide |
200000 |
4 |
2 |
3 |
3.00 |
0.00001500 |
T1 |
0.03125 mg/ml |
200000 |
3 |
2 |
3 |
2.67 |
0.00001333 |
T2 |
0.0625 mg/ml |
200000 |
4 |
2 |
2 |
2.67 |
0.00001333 |
T3 |
0.125 mg/ml |
200000 |
3 |
4 |
3 |
3.33 |
0.00001667 |
T4 |
0.25 mg/ml |
200000 |
4 |
3 |
3 |
3.33 |
0.00001667 |
PC |
400 µg/ml |
200000 |
82 |
86 |
74 |
80.67 |
0.00040333 |
Cloning efficiency - Main study (selective medium) with S9 mix
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 |
2 |
4 |
3.00 |
0.00001500 |
VC |
Dimethyl sulfoxide |
200000 |
3 |
3 |
4 |
3.33 |
0.00001667 |
T1 |
0.03125 mg/ml |
200000 |
2 |
4 |
2 |
2.67 |
0.00001333 |
T2 |
0.0625 mg/ml |
200000 |
3 |
3 |
4 |
3.33 |
0.00001667 |
T3 |
0.125 mg/ml |
200000 |
4 |
2 |
4 |
3.33 |
0.00001667 |
T4 |
0.25 mg/ml |
200000 |
3 |
4 |
4 |
3.67 |
0.00001833 |
PC |
30 µg/ml |
200000 |
88 |
81 |
89 |
86.00 |
0.00043000 |
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.
Mutation frequency
Dose level |
Absence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000672 |
6.72 |
VC |
Dimethyl sulfoxide |
0.00000755 |
7.55 |
T1 |
0.03125 mg/ml |
0.00000702 |
7.02 |
T2 |
0.0625 mg/ml |
0.00000690 |
6.90 |
T3 |
0.125 mg/ml |
0.00000909 |
9.09 |
T4 |
0.25 mg/ml |
0.00000969 |
9.69 |
PC |
400 µg/ml |
0.00023269 |
232.69 |
Dose level |
Presence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000738 |
7.38 |
VC |
Dimethyl sulfoxide |
0.00000840 |
8.40 |
T1 |
0.03125 mg/ml |
0.00000690 |
6.90 |
T2 |
0.0625 mg/ml |
0.00000871 |
8.71 |
T3 |
0.125 mg/ml |
0.00000899 |
8.99 |
T4 |
0.25 mg/ml |
0.00001038 |
10.38 |
PC |
30 µg/ml |
0.00024022 |
240.22 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Absence of metabolic activation- Ethylmethanesulfonate, Presence of metabolic activation- Benzo[a]pyrene), T4-T1= Test item concentration from higher to lower, MF = Mutation Frequency, mg = milligram, µg = microgram, ml = milliliter.
Historical Control Data
Mutation Frequency (10-6) |
NC |
VC |
PC |
|||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Mean |
7.03 |
6.76 |
7.56 |
7.78 |
213.85 |
222.44 |
SD |
0.80 |
1.02 |
1.25 |
0.79 |
19.08 |
12.43 |
Min |
6.09 |
6.02 |
6.35 |
6.51 |
197.57 |
209.32 |
Max |
8.21 |
8.43 |
8.9 |
8.7 |
236.11 |
240.46 |
Key: - NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Absence of metabolic activation- Ethylmethanesulfonate, Presence of metabolic activation- Benzo[a]pyrene), SD = Standard Deviation, Min = Minimum, Max = Maximum, - S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation. Number of studies = 06.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Bacterial reverse mutation test:
Study 1:
Salmonella/microsome mutagenicity test (Ames test) was performed to assess the mutagenic nature of theregistered substance. The study was conducted according to the plate incorporation methodandin the presence and absence of S9 metabolic activation system using Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains. The test chemical was dissolved in DMSO and used at concentrationsof 0, 50, 160, 500, 1600 or 5000 µg/plate. The plates were incubated for 48 hrs and observed for revertant colonies. Concurrent solvent(Dimethyl sulfoxide),negative controland positive control substanceswere also included in the study. The test chemical did notinducea relevant or dose-dependent increase in the number of revertants colonies at any concentrations tested either in the presence or absence of S9 metabolic activation system using Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA tester strains.
Study 2:
Salmonella/microsome mutagenicity test (Ames test) was performed to assess the mutagenic nature of the test chemical. The study was performed according to the preincubation method and in the presence and absence of S9 metabolic activation system using Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA. The test substance was dissolved in dimethyl sulfoxide (DMSO).In the main experiment, the bacterial cells were exposed to 0 (NC), 0 (VC), 50, 160, 500, 1600 and 5000µg/plate along with positive control substances and with and without S9 mix. The plates were preincubated for 20-30 mins and incubated for 48 hrs to observe for revertant colonies. Concurrent solvent (DMSO) and negative control chemicals were also included in the study. In TA 98, a slight increase in the mean revertant counts was observed at precipitation concentrations of 500 and 1600µg/plate in the presence of S9 metabolic activation. However, individual values overlapped with the historical solvent and negative control data, and no clear dose-dependency occurred, and the dose/control mutation rations were only 1.6 and 2.0. Therefore the increase in revertant colonies observed in TA 98 was considered equivocal. In addition, in TA 1537, an incomplete background lawn was observed at all test concentrations with and without the S9 mix. Therefore a repeat experiment with TA 1537 tester strain was performed with concentrations of 0 (NC), 0 (VC), 0.5, 1.6, 5, 16, 50 and 160µg/plate and 0 (NC), 0 (VC), 50, 160, 500, 1600 and 5000µg/plate in the presence and absence of S9 metabolic activation, respectively. In TA 1537, no increase in the number of revertants was observed at any concentrations tested both with and without S9 metabolic activation. A second repeat experiment with TA 98 was performed with concentrations of 0 (NC), 0(VC), 500, 1000, 1600, 3000 and 5000µg/plate with S9 mix. Incomplete background lawn and precipitation was detected at 50 and 160µg/plate (-S9) and 160 and 5000µg/plate (+S9). In addition, there was no increase in revertant counts at any of the concentrations tested in the presence of S9 mix in TA 98. Precipitation and incomplete background lawn was noted at≥500 µg/plate. In the second repeated experiment with TA 98, bacterial cells were exposed to 0 (NC), 0 (VC), 16, 50, 160, 500, 1000, 1600, 3000, 5000 µg/plate in the presence of S9 mix.There was no relevant and dose-dependent increase in revertant counts at any doses tested. Slight increases in the mean revertant counts were noted at 500, 1600 and 3000 µg/plate. The dose/control ratios were 1.9, 1,7 and 1.5 at 500, 1600 and 3000 µg/plate, respectively. However, the mean revertant count at the precipitating dose 500 µg/plate was within the historical negative control range. Cytotoxicity and precipitation were observed ≥160 µg/plate. In summary, the slight increase in revertants observed in TA98 was not considered to be biologically relevant. In conclusion, the test chemical did not cause a biological relevant mutagenic effect in Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA strains in the presence and absence of S9 metabolic activation system.
Study 3:
An Amestest was performed to determine the mutagenic nature of the registered substance (CAS 67905-17-3) using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli WP2uvrA tester strains. The test substance was dissolved in DMSO and used at six concentrations within the range of 0.8 -2500 µg/plate. The doses for the main study were based on the preliminary cytotoxicity study conducted with concentrations of 4-10000 µg/plate. The test substance proved to be toxic at≥2500 µg/plate. Visible precipitation on the plates was observed at 500 µg/plate. Thinning of the bacterial lawn was noted at 2500 - 10000 µg/plate. Based on this experiment, 2500 µg/plate was chosen as the highest dose for the main study. Concurrent solvent and positive control plates were also included in the study. Control plates without mutagen showed that the spontaneous revertant colonies were similar to that described in the literature. All the positive control chemicals gave the expected increase in the number of revertant colonies. In the absence of S9 metabolic activation system, the test chemical did not cause a significant increase in revertant colonies with any of the tester strains. In the presence of S9 metabolic activation system, a relatively small but dose-dependent increase in the number of colonies was obtained with TA 1538 and TA 98. In a second experiment, these results were confirmed. Hence, it was concluded that the test substance was mutagenic in TA 98 and TA 1538 and in the presence of S9 metabolic activation.
In vitro mammalian chromosomal aberration test:
The registered substance, i.e. N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino] phenyl]acetamide (CAS: 67905-17-3) was tested in a mammalian chromosome aberration test according to OECD 473 (adopted on July 29, 2016).The test was performed to assess the ability of the substance to induce structural chromosomal aberrations in primary cultures of human peripheral blood lymphocytes. The experiment was conducted in two phases (Phase I and II) and both in the presence and absence of a metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction derived from Aroclor 1254-induced rats was used in both phases of the experiment.Dimethyl sulfoxide (DMSO) was selected as the vehicle for the test substance. A preliminary cytotoxicity test, was performed to select test concentrations. In this pre-test, proliferating cells were exposed to 0.0 (NC), 0.0 (VC), 0.5, 1.0 and 2.0 mg/ml of test substance with and without S9 metabolic activation (1% v/v). Cytotoxicity was determined by the mitotic index. At 2. 0 mg/ml and in the absence of metabolic activation (-S9), the test substance produced a 50.76% decrease of the mitotic index when compared to the vehicle control (DMSO). In the presence of metabolic activation (+S9), 50.32% and 54.74% reductions of the mitotic index were noted at 1.0 and 2.0 mg/ml compared to the vehicle.The concentration that yielded 55±5% cytotoxicity (i.e. reduction in the mitotic index to 45±5%) of the concurrent vehicle control was chosen as the highest test concentration.Hence, 2.0 mg/ml and 1.0 mg/ml were selected as the highest test concentrations in the absence and presence of S9 metabolic activation for the main test, respectively.Results:In Phase I of the main test, cells were treated with 0 (NC), 0 (VC), 0.5, 1.0, 2.0 mg/ml and 0 (NC), 0 (VC), 0.25, 0.5, 1.0 mg/ml for 4 hours without and with S9 metabolic activation (1% v/v), respectively.The mean percentage of aberrant cells was 0.333 (NC), 0.667 (VC), 0.333 (at 0.5 mg/ml), 0.667 (at 1.0 mg/ml), 0.667 (at 2.0 mg/ml), 10.000 (PC) and 0.333 (NC), 0.333 (VC), 0.333 (at 0.25 mg/ml), 0.333 (at 0.5 mg/ml), 0.667 (at 1.0 mg/ml) and 9.000 (PC) in the absence and presence of S9 metabolic activation, respectively. In addition, the mean mitotic indices were the follows: 10.03 (NC), 9.94 (VC), 6.85 (at 0.5 mg/ml), 5.22 (at 1.0 mg/ml), 4.85 (at 2.0 mg/ml), 8.19 (PC) and 10.16 (NC), 9.89 (VC), 7.10 (at 0.25 mg/ml), 6.80 (at 0.5 mg/ml), 4.89 (at 1.0 mg/ml) and 8.58 (PC) in the absence and presence of S9 metabolic activation, respectively. In Phase II of the main test, cells were treated with 0 (NC), 0 (VC), 0.5, 1.0, 2.0 mg/ml and 0 (NC), 0 (VC), 0.25, 0.5, 1.0 mg/ml for 24 hours in the absence and presence of S9 metabolic activation (2% v/v) respectively. The mean percentage of aberrant cells were 0.333(NC), 0.333(VC), 0.333 (at 0.5 mg/ml), 0.333 (at 1.0 mg/ml), 0.667(at 2.0 mg/ml), 10.000 (PC) and 0.333 (NC), 0.333 (VC), 0.333 (at 0.25 mg/ml), 0.333 (at 0.5 mg/ml),0.667 (at 1.0 mg/ml) and 10.667 (PC) in the absence and presence of S9 metabolic actibation (2% v/v), respectively.Furthermore, the mean mitotic indices were the follows: 9.99 (NC), 9.69 (VC), 6.10 (at 0.5 mg/ml), 4.91 (at 1.0 mg/ml), 4.49 (at 2.0 mg/ml), 8.85 (PC) and 10.00 (PC), 9.94 (VC), 6.75 (at 0.25 mg/ml), 4.70 (at 0.5 mg/ml), 4.50 (at 1.0 mg/ml), and 8.79 (PC) without and with S9 metabolic activation (2% v/v), respectively. There was no statistically significant increase in percent aberrant cells in cultures treated with the vehicle (DMSO) neither in the presence nor in the absence of S9 metabolic activation in either phases of the experiment.Conclusion:The registered substance i.e. N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl) amino]phenyl]acetamide (CAS: 67905-17-3) did not induce structural chromosal aberration in primary cultures of human lymphocytes when it was tested up to 1 mg/ml in the presence (1% and 2%) and 2 mg/ml in the absence of S9 metabolic activation.
In vitro mammalian cell gene mutation test:
The test chemical, i.e., N-{4-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]phenyl} acetamide (CAS No. 67905-17-3) was tested in an in vitro mammalian cell gene mutation test according to OECD test guideline 476. The test was performed to assess the ability of the substance to cause gene mutation in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented S9 microsomal fraction, derived from the liver of phenobarbital and β-naphthoflavone-induced rat, was used as a metabolic activation system. Dimethyl sulfoxide was selected as a vehicle for the substance. Test concentrations were chosen based on the solubility, precipitation and pH checks and a preliminary cytotoxicity test. The test substance formed precipitation at 0.5 and 1 mg/ml, which was considered to interfere with scoring and slight precipitation at 0.25 mg/ml to 0.125 mg/ml in the culture medium. Therefore the cytotoxicity test was performed with concentrations of 0.0 (NC), 0.0 (VC), 0.015625, 0.03125, 0.0625, 0.125, and 0.25 mg/ml both in the presence and absence of S9 metabolic activation using triplicates.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. In the pre-test, no limiting cytotoxicity (10-20% RS) was observed; the highest test concentration, 0.25 mg/ml, produced >60% RS both in the presence and absence of metabolic activation (at 0.25 mg/ml RS: 63.97% without S9 mix; RS: 63.32% with S9 mix). Since the test substance formed precipitation at 0.5-1 mg/ml, which interfered with scoring, 0.25 mg/ml was selected as the maximum test concentration in the main test. Three lower concentrations were included in the assay using spacing factor 2. In the gene mutation test, CHO cells were exposed to the test substance at 0.0 (NC), 0.0 (VC), 03125, 0.0625, 0.125 and 0.25 mg/ml for 4 hours both in the presence and absence of S9 metabolic activation using triplicate cultures. Positive control substances (Ethylmethanesulfonate without S9 mix, Benzo[a]pyrene with S9 mix) were also included in the test.Results:In the absence of metabolic activation the relative survival values were 98.69% (VC), 92.24% (at 0.03125 mg/ml), 85.75% (at 0.0625 mg/ml), 77.64% (at 0.125 mg/ml), 64.09% (at 0.25 mg/ml) and 93.61% (PC, 400 µg/ml Ethyl methanesulfonate). In the presence of metabolic activation, the relative survival values were 98.60% (VC), 90.37% (at 0.03125 mg/ml), 82.90% (at 0.0625 mg/ml), 77.45% (at 0.125 mg/ml) 65.55% (at 0.25 mg/ml) and 97.64% (PC, 30 µg/ml Benzo[a]pyrene). No significant increase in the mutation frequency (MF) either in absence (7.02x10-6, 6.90 x10-6, 9.09 x10-6 and 9.69 x10-6 at 0.03125 mg/ml, 0.0625 mg/ml, 0.125 mg/ml and 0.25 mg/ml, respectively) or presence of metabolic activation (6.90 x10-6, 8.71x10-6, 8.99x10-6and 10.38x10-6at 0.25 mg/ml, 0.03125 mg/ml, 0.0625 mg/ml, 0.125 mg/ml and 0.25 mg/ml, respectively) was observed when compared to vehicle control (7.55 x10-6, 8.40 x 10-6, absence and presence of S9, respectively).No significant reduction in the relative survival (cytotoxicity) or increase in mutation frequency 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 used in the study produced statistically significant increases in mutation frequency, 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. Conclusion:The registered substance, i.e., N-{4-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]phenyl}acetamide (CAS No. 67905-17-3) did not induce gene mutation at the locus of hypoxanthine-guanine phosphoribosyltransferase (Hprt) in CHO cells up to the concentration of 0.25 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system.
Justification for classification or non-classification
The test results from experiments determining mutagenic and/or genotoxic effects provided unambiguous, relevant and reliable evidence that the registered substance, i.e.,N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1 anthryl)amino]phenyl]acetamide (CAS: 67905-17-3)does not induce gene mutation in bacterial and mammalian somatic cells or structural chromosomal aberration in mammalian cells in vitro. Hence, the registered substance N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1 anthryl)amino]phenyl]acetamide (CAS: 67905-17-3) is classified as Non-classified for germ cell mutagenicity according to the Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP).
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