Registration Dossier
Registration Dossier
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 229-851-8 | CAS number: 6786-83-0
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, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (CAS Number: 6786-83-0) tested non-mutagenic (negative) in Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102 with and without S9 metabolic activation system. 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, (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) was tested non-clastogenic (negative) in mammalian cells up to the concentration of 0.00625 mg/ml either in the presence or absence of S9 metabolic activation system in CHO cells. The test was performed according to OECD TG 473 and in compliance with GLP.
In vitro gene mutation test in mammalian cells:
The registered substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol [CAS No.: 6786-83-0] was tested non-mutagenic (negative) in mammalian cells up to 0.015 mg/mll both in the presence and absence of S9 metabolic activation. The test was performed according to OECD TG 476 and in compliance with GLP.
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: 21st July 1997, Corrected: 26th June2020
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Name of test material: (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]methanol
- Molecular formula: C33H33N3O
- SMILES: CN(C)C1=CC=C(C=C1)C(C2=CC=C(C=C2)N(C)C)(C3=CC=C(C4=CC=CC=C43)NC5=CC=CC=C5)O
- Physical state: Solid
- Purity: 99.62%
- Apperance: Blackish powder
- Batch Number: KCP/FS/742/20
- Expiry Date: 21.08.2021
- Storage condition: Room Temperature (20 to 30 C) - Target gene:
- Histidine operon
- 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:
- S9 mix [Cofactors (Cofactor ingredients: D-glucose-6-phosphate, β-NADP, magnesium chloride, potassium chloride, sodium phosphate and Liver homogenate] was prepared prior to use in the study. The post-mitochondrial fraction (liver homogenate) was used at the concentration of 10 % (v/v) in the S9 mix.
- Test concentrations with justification for top dose:
- 0 (solvent control), 0.0625, 0.125, 0.25, 0.5 and 1 mg/plate.
Justification for the top dose: The highest test concentration was selected based on solubility, precipitation checks, and a preliminary cytotoxicity test. The test substance was soluble in DMSO up to 10 mg/ml, giving the final test concentration of 1 mg/ml. No precipitation that can interfere with scoring was observed at 1 mg/ml. A reduction in revertant counts and moderate inhibition of the background lawn was observed at 1 mg/m. Hence, 1 mg/ml was selected as the highest test dose for the reverse mutation test. - Vehicle / solvent:
- dimethyl sulfoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Trial1 (plate incorporation) in agar, Trial 2 (preincubation) in medium
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
SELECTION AGENT (mutation assays): Top agar was prepared with 0.6 % (w/v) bacto agar and 0.6 % (w/v) sodium chloride and was supplemented with 10 % (v/v) of 0.5 mM histidine/biotin solution for selection of histidine revertants
NUMBER OF REPLICATIONS: Triplicates - Evaluation criteria:
- A Test Item is considered as a mutagen if a biologically relevant increase in the mean number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed. A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. A dose-dependent increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent experiment. A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent experiment.
- Statistics:
- The colonies were counted manually. Then, the mean number of revertant colonies and the standard deviation within the plates for each concentration were compared to the spontaneous reversion rates of the control. Microsoft Office Excel-based calculations were used for descriptive statistical analysis.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- A decrease in mean revertant counts was observed at 1 mg/ml both with and without 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:
- For each tester strain, the frequency of the spontaneous revertant colonies in the vehicle control was within the acceptable range of historical data of the lab. In addition, the positive controls used in the study produced significant increases in the mean number of revertant colonies in all of the tester strains when compared to the control data, thus confirming the validity of the test system to detect specific mutagens in the presence and absence of a metabolic activation system.
- Remarks on result:
- other: No mutagenic potential was observed
- Conclusions:
- The registered substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (CAS Number: 6786-83-0; EC Number: 229-851-8) tested non-mutagenic (negative) in Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102 with and without S9 metabolic activation system. The test was performed according to OECD TG 471 and in compliance with OECD Principles of Good Laboratory Practice.
- Executive summary:
The mutagenicity of the test substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (CAS Number: 6786-83-0; EC Number: 229-851-8) was tested according to OECD 471 using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102. The test was performed as two independent experiments according to the plate incorporation method (Trial I) and the preincubation method (Trial II), both in the presence and absence of rat liver microsomal activation (S9 mix). Concentrations used in the mutagenicity assay were selected based on a preliminary cytotoxicity assay. This preliminary cytotoxicity assay was performed using concentrations of 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2 and 1 mg/plate, with and without metabolic activation (10 % v/v S9 mix) using TA98 and TA100 strains. A slight reduction in the revertant counts and moderate inhibition of the background lawn was observed at 1mg/plate, and no reduction in the revertant count and/or inhibition in the background lawn was noted at concentrations from 0.0000128 to 0.2 mg/plate when compared to the vehicle control data. Based on the observations of the pre-test, the following doses were applied in the mutagenicity test: 0.0 (NC), 0.0 (VC), 0.0625, 0.125, 0.25, 0.5 and 1mg/plate, both in the presence and absence of metabolic activation, along with the negative, vehicle and positive controls. Trial I was performed according to the plate incorporation method. The test substance, together with the negative, vehicle and positive controls, were tested in triplicates. The Test Item doses were selected using a spacing factor of 2. There was slight precipitation, a slight reduction in revertant colony count and a moderate inhibition in the background lawn at 1 mg/plate. Furthermore, there was no increase in the number of revertant colonies and/or diminution of the background lawn at concentrations of 0.0625 - 0.5 mg/plate, neither in the presence nor the absence of metabolic activation compared to the vehicle control data. Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method; the substance was tested in all the tester strains in triplicates. There was slight precipitation, a slight reduction in revertant colony count and a moderate inhibition in the background lawn at 1 mg/plate. There was no increase in the number of revertant colonies and/or diminution of the background lawn at concentrations of 0.0625 - 0.5 mg/plate, neither in the presence nor in the absence of metabolic activation when compared to the vehicle control data. The numbers of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains compared to the vehicle control data confirming the validity of the mutagenicity test. Based on the results of this study, it is concluded that(4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) is non-mutagenic as it does not induce gene mutations (point and frameshift) in the histidine operon by base-pair changes or frameshifts, in the presence or the absence of metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102).
- 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: 29 July 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KCP/FS/742/20
- Purity: 99.62%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 to 30oC)
OTHER SPECIFICS
-Solubility: The Test Item was found to be soluble up to 10 mg/ml in dimethyl sulfoxide.
-Precipitation of Test Item was performed by adding 50 µl of the Test Item solution (10 mg/ml) to 4.950 ml of culture media to attain 0.1 mg/ml. No precipitation was observed at a tested concentration of 0.1 mg/ml. - 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: CHO Cell lines are a commonly used test system for in vitro chromosomal aberration tests. The CHO cell line is recommended in OECD 473 (29th July 2016), and it meets the requirements of most regulatory agencies..
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), and maintained at 37±2 °C, 5% CO2, in a CO2 incubator - Metabolic activation:
- with and without
- Metabolic activation system:
- - source of S9
: A combination of phenobarbitone and β-naphthoflavone-induced rat liver microsomal enzymes (S9 homogenate) prepared in-house was used for the assay
- method of preparation of S9 mix: Prior to treatment freshly prepared S9 mix was used, an appropriate quantity of S9 fraction was thawed and mixed with co-factor solution to obtain a final concentration of 1% v/v as mentioned below. Cofactor solution: Glucose-6-phosphate [180 mg/ml], NADP [25 mg/ml] and Potassium chloride [150 mM] ) to obtain a final concentration of 1% v/v.Type and composition of metabolic activation system:
- concentration or volume of S9 mix and S9 in the final culture medium : 1 v/v% - Test concentrations with justification for top dose:
- Test concentrations: 0.0 (VC), 0.0 (NC), 0.0015625, 0.003125 and 0.00625 mg/ml
Justification: The test concentrations were selected based on the solubility, precipitation and pH checks, as well as a preliminary cytotoxicity test. The substance was soluble in DMSO at a 0.1 mg/ml concentration. No precipitation was observed at the concentration of 0.1 mg/ml. The preliminary cytotoxicity assay was performed with test substance concentrations of 0.0 (NC), 0.0 (VC), 0.00625, 0.0125, 0.025, 0.05, 1.0 mg/ml. Excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item concentrations from ≥0.0125 mg/ml, both in the presence and absence of metabolic activation. RICC decreased by 55±5% compared to vehicle control at 0.00625 mg/ml in the presence and absence of metabolic activation. Hence, 0.00625 mg/ml was selected as the highest test concentration. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: The substance was insoluble in distilled water and was soluble in dimethyl sulfoxide up to 10 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:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Single cultures were used.
- Number of independent experiments: Phase I (4-hour treatment, without S9), Phase II (4hrs treatment with S9), Phase III (24 hrs treatment without S9)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 × 10^6 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: 4hrs (Phase I-II), 24 hrs (Phase III)
- Harvest time after the end of treatment (sampling/recovery times): Recovery period: 20 hrs (Phase I-II), Harvest time: 24 hrs (Phase I-III)
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Two hours before cell harvest, colchicine (final concentration of 1 µg/ml) was added to cultures and incubated at 37 ±2 °C, 5 % CO2.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Each cell suspension obtained after harvesting was dropped on a clean chilled slide using the hanging drop method and kept for drying on a slide warmer. Duplicate slides were prepared from each culture.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 300 well-spread metaphases per concentration (single culture) were analysed using 100x magnification for the incidence of structural aberrations.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). Cells that contain the number of centromeres equal to the number 2n ± 2 were scored.
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: The cytotoxicity was evaluated based on the reduction in the Relative Increase in Cell Counts (RICC). The concentration, which yielded 55±5% cytotoxicity, i.e., reduction in RCC to 45±5% of the concurrent vehicle control, was selected as the highest test concentration. Two lower concentrations using spacing factor 2 were selected as middle and low test concentrations. - Evaluation criteria:
- The substance was considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a significant increase compared with the concurrent negative/vehicle control,
- The increase is dose-related when evaluated with an appropriate trend test,
- Any of the results are outside of the distribution of the laboratory historical negative/vehicle control database
When all these criteria were met, the Test Item was then considered to induce chromosomal aberrations in cultured mammalian cells in this test system.
The substance was considered clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a significant increase compared with the concurrent negative/vehicle control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- The results are inside the distribution of the laboratory historical negative control database. - Statistics:
- Statistical analysis was performed to assess a possible dose-dependent increase of aberrant cell frequencies using Fisher’s Exact Test (NCSS statistics software). The percentage of aberrant cells from 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:
- Cytotoxicity was 55.79%, (without S9, Phase I), 54.96% (with S9, Phase II) and 51.24 % (without S9, in Phase III) observed at 0.00625 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:
- Solubility and precipitation check: The substance was soluble in dimethyl sulfoxide at 10 mg/ml concentration. No precipitation was observed at the concentration of 0.1 mg/ml.
pH check: The substance at concentration of 0.1 mg/ml did not induce alteration of the pH of medium at 0 and 4 hours incubation.
Preliminary cytotoxicity test: In the preliminary cytotoxicity assay, excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of ≤40% of the concurrent vehicle control data) was observed for the Test Item concentrations from ≥0.0125 mg/ml, both in the presence and absence of metabolic activation. RICC decreased by 55±5% compared to vehicle control at 0.00625 mg/ml both in the presence and absence of metabolic activation - Remarks on result:
- other: No mutagenic potetial was observed
- Conclusions:
- The registered substance, (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) did not induce chromosomal aberration up to 0.00625 mg/ml either in the presence or absence of S9 metabolic activation system in CHO cells. The test was performed according to OECD TG 473 and in compliance with GLP.
- Executive summary:
The potential of (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) to induce structural chromosomal aberration was tested according to OECD TG 473 with and without metabolic activation in cultured Chinese Hamster Ovary (CHO) cells. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation. The S9 fraction was obtained from phenobarbitone and β-naphthoflavone-injected rats. The substance was insoluble in distilled water but was soluble in dimethyl sulfoxide up to 10 mg/ml. Test concentrations were chosen based on a preliminary cytotoxicity experiment. In this pre-test, CHO cells were exposed to 0.0 (VC), 0.0 (NC), 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/ml for 4 hours in the presence (1 % v/v S9 mix) and absence of metabolic activation system. Excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.0125 mg/ml, both in the presence and absence of metabolic activation. RICC decreased by 55±5% compared to vehicle control at 0.00625 mg/ml both in the presence and absence of metabolic activation. Hence, Phase I (short term[4hrs]exposure in the absence of metabolic activation), Phase II (short term[4 hrs]exposure in the presence of metabolic activation) and Phase III (continuous[24 hrs]exposure in the absence of metabolic activation) were conducted with the Test Item at the concentrations of 0.0015625, 0.003125 and 0.00625 mg/ml, along with vehicle (DMSO), negative (Distilled water) and concurrent positive (Methyl methanesulfonate, 20µg/ml, without S9 mix;Benzo (a) pyrene, 30µg/ml with S9 mix)controls. At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations. Breaks (chromatid and chromosomal), gaps(chromatid and chromosomal), rings and fragments, and dicentric chromosomes were found as structural chromosome aberrations. Gaps were recorded and reported separately but not included in the total aberration frequency.Results:In Phase I experiment, CHO cells were exposed to the substance, vehicle, negative and positive controls for 4 hours (short term exposure) in the absence of metabolic activation. The average RICC values were 98.85 % (vehicle control), 71.04 % (at 0.0015625 mg/ml), 61.00 % (at 0.003125 mg/ml) and 44.21 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells was observed at 0.0015625 mg/ml (mean % aberrant cells: 0.67%), 0.003125 mg/ml (mean % aberrant cells: 0.67%), or at 0.00625 mg/ml (mean % aberrant cells: 0.33%) compared to the vehicle control data (mean % aberrant cells 0.33 %). In Phase II experiment, cultures were exposed to the substance, vehicle, negative and positive controls for 4 hours (short term exposure) in the presence of metabolic activation (1 % v/v S9 mix). The average RICC values were 97.73 % (vehicle control), 74.42 % (at 0.0015625 mg/ml), 65.27 % (at 0.003125 mg/ml) and 45.04 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells was observed at 0.0015625 mg/ml (mean % aberrant cells: 0.67%), 0.003125 mg/ml (mean % aberrant cells: 0.33%), or at 0.00625 mg/ml (mean % aberrant cells: 0.00%) compared to the vehicle control data (mean % aberrant cells: 0.00 %). In Phase III, cultures were exposed to the substance, vehicle, negative and positive control for 24 hours (continuous exposure) without metabolic activation. The average RICC values were 92.65 % (vehicle control), 73.76 % (at 0.0015625 mg/ml), 63.64 % (at 0.003125 mg/ml) and 48.76 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells was observed at 0.0015625 mg/ml (mean % aberrant cells: 0.00%), 0.003125 mg/ml (mean % aberrant cells: 1.00%), or at 0.00625 mg/ml (mean % aberrant cells: 0.67%) compared to the vehicle control data (mean % aberrant cells 0.33 %). In all phases of the study, no significant reduction in RICC (cytotoxicity) and no increase in percent aberrant cells were observed in vehicle control (DMSO) either in the presence or absence of metabolic activation, when compared to the negative control (distilled water). The positive controls (-S9: methyl methanesulfonate, +S9: Benzo[a]pyrene) used in the study produced statistically significant increases in the percent of cells with structural chromosome aberrations (Phase I: 6.67%; p<0.0001, Phase II: 7.33%; p<0.0001, Phase III: 7.67%; p<0.0001) 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, (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) did not induce chromosomal aberration up to the highest concentration of 0.00625mg/ml, either in the presence or absence of S9 metabolic activation in CHO cells.
- 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:
- OECD 476 (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:
- SOURCE OF TEST MATERIAL
- Lot/batch number of test material: KCP/FS/439/21
- Expiration date of the lot/batch: 09-11-2022
- Purity: >90%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 to 30oC)
OTHER SPECIFICS
-Solubility:The Test Item was found to be soluble up to 50 mg/ml in dimethyl formamide.
-Precipitation of Test Item was performed by adding 50 µl of Test Item formulation (50 mg/ml) to 4.95 ml of culture media to attain 0.5 mg/ml. Slight precipitation was observed at the tested concentrations of 0.5 mg/ml, which was considered not to interfere with the conduct of the study. - Target gene:
- hypoxanthine-guanine phosphoribosyltransferase (Hprt)
- 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: CHO cell lines are commonly used for in vitro mammalian cell gene mutation assays because of the stable karyotype, relatively low spontaneous mutant frequency and sensitivity to various chemical mutagens.
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:
- Type and composition of metabolic activation system:
- source of S9 : A combination of phenobarbital and β-naphthoflavone-induced rat liver microsomal enzymes (S9 homogenate) prepared at the test facility.
- method of preparation of S9 mix: 2 ml of S9 fraction was thawed and mixed with 1 ml of 180 mg/ml Glucose-6-phosphate, 1 ml of 25 mg/ml NADP and 1 ml of 150 mM Potassium chloride (cofactor solution) to obtain a concentration of 40% v/v S9 in Cofactor mix.
- concentration or volume of S9 mix and S9 in the final culture medium : A volume of 2.5 ml S9 cofactor mix (40%) was added to 100 ml of culture medium to achieve 1 % v/v S9 in the culture medium. - Test concentrations with justification for top dose:
- Test concentrations: 0 (Negative control), 0 (Vehicle control), 0.001875, 0.00375, 0.0075 and 0.015 mg/ml
Justification: Test concentrations were selected based on the solubility, precipitation and pH checks as well as a preliminary cytotoxicity test. The test substance was soluble in dimethylformamide up to 50 mg/ml. Slight precipitation was observed at 0.5 mg/ml under the actual testing conditions, which did not interfere with the conduct of the study. The initial cytotoxicity test was performed with concentrations of 0.0 (NC), 0.0 (VC), 0.03125, 0.0625, 0.125, 0.25, and 0.5 mg/ml in the presence and absence of S9 metabolic activation system. Complete cytotoxicity (determined by the Relative Survival [RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in vehicle control]) was observed at tested concentrations of 0.03125-0.5 mg/ml both in the presence and absence of metabolic activation. Hence, the preliminary cytotoxicity test was repeated with the concentrations of 0.0 (NC), 0.0 (VC), 0.001875, 0.00375, 0.0075, 0.015 and 0.03 mg/ml. The concentration of 0.03 mg/ml was cytotoxic in the absence and presence of metabolic activation. Limited cytotoxicity (within the recommended range of 10-20% RS) was observed at 0.015 mg/ml. Hence, in the main test, the following test concentrations were selected: 0.001875, 0.00375, 0.0075 and 0.015 mg/ml, with and without the S9 metabolic activation system. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethylformamide
- Justification for choice of solvent/vehicle: Based on the solubility of the test item, dimethylformamide was used as a vehicle.
- Justification for percentage of solvent in the final culture medium: The Test Item was found to be insoluble in distilled water, but was soluble in dimethylformamide up to 50 mg/ml.
- Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethylformamide
- 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
- Number of independent experiments: One
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 x 10^6 cells
- Test substance added in medium: Complete RPMI-1640 medium (10 % Fetal bovine serum)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:4 hours at 37°C
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 day (7-day expression time and and 9-day culturing on selective medium)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: 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 vehicle control. The maximum test concentration that yielded 20 and 10% RS value were selected as the highest test concnetration.
- Any supplementary information relevant to cytotoxicity: Limited cytotoxicity was observed up to the highest tested concentration, i.e. 0.015 mg/ml, either in the absence (RS: 18.73%) or presence (RS: 17.45%) of metabolic activation. - Evaluation criteria:
- The test chemical was considered to be clearly positive if in any of the experimental conditions examined:
a)at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control,
b) the increase was 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 were met, the test chemical was then considered able to induce gene mutations in cultured mammalian cells in this test system.
The test chemical was considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibited a significant increase compared with the concurrent negative control,
b) all results were inside the distribution of the laboratory negative control data.
The test chemical was 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 TestItem-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:
- Limited cytotoxicity was observed at 0.015 mg/ml, either in the absence (RS: 18.73%) or presence (RS: 17.45%) of metabolic activation.
- 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 formulation was added to 4.95 ml of complete medium, resulting in a final Test Item concentration of 0.5 mg/ml in the medium. There was no alteration of the pH after 0 and 4 hrs incubation.
- Water solubility: Test chemical was insoluble in distilled water, but was soluble in dimethylformamide up to 50 mg/ml.
- Precipitation: Slight precipitation was observed at the tested concentrations of 0.5 mg/ml, which was considered not to interfere with the conduct of the study.
RANGE-FINDING/SCREENING STUDIES (if applicable):
Test concentrations were selected based on a preliminary cytotoxicity test.
The initial cytotoxicity test was performed with concentrations of 0.0 (NC), 0.0 (VC), 0.03125, 0.0625, 0.125, 0.25, and 0.5 mg/ml in the presence and absence of S9 metabolic activation system. Complete cytotoxicity (determined by the Relative Survival [RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in vehicle control]) was observed at tested concentrations of 0.03125-0.5 mg/ml both in the presence and absence of metabolic activation. Hence, the preliminary cytotoxicity test was repeated with the concentrations of 0.0 (NC), 0.0 (VC), 0.001875, 0.00375, 0.0075, 0.015 and 0.03 mg/ml. The concentration of 0.03 mg/ml was cytotoxic in the absence and presence of metabolic activation. Limited cytotoxicity (within the recommended range of 10-20% RS) was observed at 0.015 mg/ml. Hence, in the main test, the following test concentrations were selected: 0.001875, 0.00375, 0.0075 and 0.015 mg/ml, with and without the S9 metabolic activation system..
STUDY RESULTS
- Concurrent vehicle negative and positive control data: Data available. For tabular data refer to the section “Any additional information on results including tables”.
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: For tabular data, refer to the section “Any additional information on results including tables”.
- Genotoxicity results:
o Number of cells treated and sub-cultures for each culture: 10 x 10^6 cell/flask
o Number of cells plated in selective and non-selective medium: 2x10^5cells / 10 ml of cloning media were seeded in the presence of 10 µg/ml of 6-thioguanine (6TG) in triplicates. 100 cells / 10 ml of cloning media and then plated in 60 mm culture plates in triplicate to determine the cloning efficiency.
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: For tabular data, refer to the section “Any additional information on results incl 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: Available but not shown.
- Negative (solvent/vehicle) historical control data: Available but not shown. - Remarks on result:
- other: No mutagenic potential observed
- Conclusions:
- The registered substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol [CAS No.: 6786-83-0] was tested non-mutagenic (negative) in mammalian cells both in the presence and absence of S9 metabolic activation. The test was performed according to OECD TG 476 and in compliance with GLP.
- Executive summary:
The potential of the registered substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol [CAS No.: 6786-83-0] to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus was tested in the presence and absence of an exogenous metabolic activation system using cultured Chinese Hamster Ovary (CHO) cells. The study was performed as per OECD Test Guideline No. 476 (Adopted: 29 July 2016). Cofactor-supplemented S9 liver microsomal fraction was used as an exogenous metabolic activation system. Dimethylformamide was selected as a solvent for the test substance during the experiment. Test concentrations were determined based on a preliminary cytotoxicity test. In the initial cytotoxicity test, the CHO cells were exposed to test item concentrations of 0 (VC), 0 (NC), 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/ml in culture medium, both in the presence and absence of a metabolic activation system (1 % v/v S9 mix). Cytotoxicity was determined by the Relative survival (RS[RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in vehicle control]) values. Complete cytotoxicity was observed at tested concentrations of 0.03125-0.5 mg/ml both in the presence and absence of metabolic activation. Hence, the preliminary cytotoxicity test was repeated using the following concentrations: 0 (VC), 0 (NC), 0.001875, 0.00375, 0.0075, 0.015 and 0.03 mg/ml both in the presence and absence of a metabolic activation system (1 % v/v S9 mix). In the absence of metabolic activation, the RS values were 100% (negative control), 95.90% (vehicle control), 92.66% (at 0.001875 mg/ml), 74.95% (at 0.00375 mg/ml), 52.11% (at 0.0075 mg/ml), 18.73% (at 0.015 mg/ml) and 0.00% (at 0.03 mg/ml). In the presence of metabolic activation, the RS values were 100% (negative control), 96.13% (vehicle control), 89.99%, (at 0.001875 mg/ml), 66.90% (at 0.00375 mg/ml), 51.04% (at 0.0075 mg/ml), 17.45% (at 0.015 mg/ml) and 0.00% (at 0.03 mg/ml). Thus, complete cytotoxicity was observed at 0.03 mg/ml in the absence and presence of metabolic activation. Whereas at the reaming tested concentrations, limited cytotoxicity (>17% RS)was observed at 0.015 mg/ml, in the absence (RS: 18.73%) or presence (RS: 17.45%) of the S9 metabolic activation system. Hence, the gene mutation study was conducted with substance concentrations of 0.0 (NC), 0.0 (VC), 0.001875, 0.00375, 0.0075 and 0.015 mg/ml, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with and positive controls (Ehtylmethanesulfonate: 400 µg/ml without S9mix;Benzo (a) pyrene: 30µg/ml, with S9 mix).Results:In the absence of metabolic activation, the RS values were 100% (negative control), 95.55% (vehicle control), 90.74% (at 0.001875 mg/ml), 73.01% (at 0.00375 mg/ml), 52.88% (at 0.0075 mg/ml), 18.78% (at 0.015 mg/ml) and 89.75% (at 400 µg/ml-positive control [Ehtylmethanesulfonate]). In the presence of metabolic activation, the RS values were 100% (negative control), 96.66% (vehicle control), 87.11% (at 0.001875 mg/ml), 67.73% (at 0.00375 mg/ml), 50.55% (at 0.0075 mg/ml) 18.51% (at 0.015 mg/ml) and 88.01% (30 µg/ml-positive control[Benzo[a]pyrene]). No significant increase in the mutation frequency (MF) either in absence(9.68x10-6, 9.07 x10-6, 10.17 x10-6and 10.52 x10-6at 0.001875 mg/ml, 0.00375 mg/ml, 0.0075 mg/ml and 0.015 mg/ml, respectively) or presence of metabolic activation (9.68 x10-6, 9.11x10-6, 9.56x10-6and 10.58x10-6at 0.001875 mg/ml, 0.00375 mg/ml, 0.0075 mg/ml and 0.015 mg/ml, respectively) was observed when compared to vehicle control (8.73 x10-6, 8.00 x10-6, absence and presence of S9, respectively). The positive controls (Ethylmethanesulfonate and Beno[a]pyrene in the absence and presence of metabolic activation, respectively) used in the study produced statistically significant increases in mutation frequency (234.56 x10-6, p<0.0001 [Ethylmethanesulfonate], 237.50 x10-6, p<0.0001 [Benzo(a)pyrene] in the absence and presence of metabolic activation, respectively).Conclusion:The Substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol [CAS No.: 6786-83-0], did not induce gene mutation at the locus of hypoxanthine-guanine phosphoribosyltransferase (Hprt) in CHO cells up to 0.5 mg/ml, either in the presence or absence of S9 metabolic activation system.
Referenceopen allclose all
TABLES
Table1 Mean Revertant Colony Count – Preliminary Cytotoxicity Assay
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
||||||
- S9 |
+ S9 |
- S9 |
+ S9 |
|||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (Distilled water) |
22.00 (NI) |
2.00 |
20.33 (NI) |
0.58 |
101.33 (NI) |
3.06 |
101.00 (NI) |
6.24 |
VC (Dimethyl sulfoxide) |
20.67 (NI) |
2.08 |
21.00 (NI) |
2.65 |
100.67 (NI) |
6.11 |
99.00 (NI) |
3.00 |
T1 (0.0000128) |
22.00 (NI) |
1.73 |
21.67 (NI) |
1.15 |
95.67 (NI) |
1.53 |
96.67 (NI) |
3.06 |
T2 (0.000064) |
20.00 (NI) |
2.00 |
21.33 (NI) |
2.31 |
100.67 (NI) |
2.08 |
97.33 (NI) |
1.53 |
T3 (0.00032) |
20.33 (NI) |
2.31 |
21.67 (NI) |
1.15 |
95.67 (NI) |
2.89 |
94.33 (NI) |
1.53 |
T4 (0.0016) |
20.33 (NI) |
1.15 |
22.00 (NI) |
2.00 |
95.00 (NI) |
2.65 |
94.67 (NI) |
2.08 |
T5 (0.008) |
21.33 (NI) |
1.53 |
19.67 (NI) |
3.06 |
97.33 (NI) |
4.93 |
93.00 (NI) |
3.46 |
T6 (0.04) |
19.67 (NI) |
2.52 |
21.33 (NI) |
0.58 |
95.67 (NI) |
3.51 |
88.00 (NI) |
5.29 |
T7 (0.2) |
19.33 (NI) |
1.53 |
18.67 (NI) |
0.58 |
91.00 (NI) |
5.00 |
88.00 (NI) |
3.46 |
T8 (1.0) |
15.00 (MI) |
1.00 |
16.67 (MI) |
1.15 |
86.00 (MI) |
2.00 |
82.00 (MI) |
1.73 |
PC |
333.67 (NI) |
13.58 |
323.33 (NI) |
8.33 |
684.00 (NI) |
5.00 |
673.67 (NI) |
11.24 |
Key: NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T8 = Test Item concentration from lower to higher, NI = No inhibition, MI = Moderate inhibition.
Positive Controls:
2-Nitrofluorene |
: |
TA98 (absence of metabolic activation) |
Sodium azide |
: |
TA100 (absence of metabolic activation) |
Benzo[a]pyrene |
: |
TA98 and TA100 (presence of metabolic activation) |
Table2 Mean Revertant Colony Count in Trial I(Plate Incorporation Method)
Absence of metabolic activation |
Presence of metabolic activation (+S9 10% v/v S9 Mix) |
|||||||||||||||||||
Test Item Concentration (mg/plate) |
TA 1535 |
TA 1537 |
TA 102 |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
TA 98 |
TA 100 |
||||||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (Distilled water) |
12.67 |
1.53 |
7.00 |
1.00 |
240.00 |
8.19 |
20.33 |
2.52 |
102.33 |
4.51 |
14.33 |
1.15 |
5.67 |
0.58 |
236.67 |
6.81 |
21.67 |
3.21 |
94.67 |
1.15 |
VC (Dimethyl sulfoxide) |
13.00 |
1.00 |
5.33 |
0.58 |
241.67 |
4.04 |
21.00 |
2.65 |
97.67 |
4.73 |
12.33 |
0.58 |
6.33 |
1.15 |
233.00 |
6.24 |
20.67 |
2.08 |
102.67 |
4.04 |
T1(0.625) |
12.67 |
1.15 |
6.67 |
1.15 |
237.67 |
8.62 |
21.33 |
2.89 |
104.33 |
2.52 |
12.33 |
1.15 |
5.67 |
0.58 |
230.67 |
6.11 |
20.67 |
2.31 |
97.00 |
5.57 |
T2 (0.125) |
11.67 |
1.15 |
6.67 |
0.58 |
237.67 |
7.23 |
21.67 |
2.52 |
99.33 |
5.51 |
13.33 |
2.08 |
7.33 |
1.15 |
236.67 |
5.69 |
18.33 |
1.15 |
98.67 |
3.06 |
T3 (0.25) |
13.33 |
1.15 |
6.00 |
2.00 |
231.67 |
4.93 |
19.33 |
1.15 |
98.33 |
4.51 |
12.67 |
1.53 |
6.33 |
1.15 |
232.33 |
7.37 |
20.67 |
1.15 |
96.00 |
2.65 |
T4 (0.5) |
12.67 |
2.08 |
6.33 |
0.58 |
229.67 |
6.66 |
21.00 |
2.00 |
102.67 |
4.04 |
13.33 |
0.58 |
6.00 |
2.00 |
235.33 |
7.09 |
18.67 |
1.15 |
95.33 |
2.52 |
T5 (1.0) |
10.33 |
0.58 |
5.67 |
1.15 |
198.33 |
12.66 |
16.67 |
1.15 |
82.00 |
2.00 |
10.00 |
1.00 |
5.00 |
1.73 |
193.67 |
11.93 |
16.00 |
1.00 |
84.67 |
4.73 |
PC |
329.00 |
16.09 |
191.00 |
4.36 |
1606.00 |
27.84 |
329.00 |
15.87 |
682.33 |
9.71 |
315.00 |
12.29 |
196.00 |
10.44 |
1627.67 |
9.61 |
323.33 |
8.33 |
701.67 |
14.50 |
Key: NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T5 = Test Item concentration from lower to higher.
Positive Controls:
2-Nitrofluorene |
: |
TA98 (absence of metabolic activation) |
|||
Sodium azide |
: |
TA100 and TA1535 (absence of metabolic activation) |
|||
9-Aminoacridine |
: |
TA1537 (absence of metabolic activation) |
|||
Mitomycin-C |
: |
TA102(absence of metabolic activation) |
|||
Benzo[a]pyrene |
: |
TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation) |
Table3 Mean Revertant Colony Count in Trial II(Preincubation Method)
Absence of metabolic activation |
Presence of metabolic activation (+S9 10% v/v S9 Mix) |
|||||||||||||||||||
Test Item Concentration (mg/plate) |
TA 1535 |
TA 1537 |
TA 102 |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
TA 98 |
TA 100 |
||||||||||
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
|
NC (Distilled water) |
12.33 |
1.53 |
7.33 |
1.15 |
239.00 |
7.00 |
21.7 |
3.2 |
99.33 |
6.81 |
13.67 |
0.58 |
6.33 |
1.53 |
233.00 |
7.00 |
21.33 |
1.53 |
100.67 |
3.21 |
VC (Dimethyl sulfoxide) |
12.00 |
1.73 |
6.33 |
1.15 |
239.67 |
9.45 |
20.0 |
2.0 |
101.33 |
7.02 |
12.67 |
1.15 |
5.33 |
0.58 |
236.67 |
7.09 |
21.33 |
0.58 |
97.33 |
2.89 |
T1(0.625) |
12.33 |
1.53 |
6.67 |
1.15 |
238.00 |
8.72 |
20.0 |
3.0 |
95.33 |
2.08 |
13.33 |
0.58 |
6.67 |
0.58 |
235.00 |
10.15 |
21.67 |
3.21 |
91.67 |
2.65 |
T2 (0.125) |
12.67 |
2.31 |
6.00 |
1.73 |
233.33 |
6.51 |
20.3 |
2.3 |
98.33 |
4.04 |
13.67 |
1.53 |
7.67 |
0.58 |
232.67 |
4.04 |
20.67 |
2.08 |
102.00 |
3.51 |
T3 (0.25) |
12.67 |
0.58 |
5.33 |
0.58 |
234.67 |
2.31 |
22.0 |
2.0 |
94.33 |
2.52 |
12.33 |
1.15 |
5.67 |
1.53 |
236.00 |
7.00 |
22.00 |
1.73 |
96.00 |
4.36 |
T4 (0.5) |
14.33 |
1.53 |
6.67 |
1.15 |
230.00 |
12.12 |
19.0 |
1.0 |
92.67 |
3.21 |
12.67 |
1.15 |
5.67 |
2.08 |
234.33 |
7.57 |
20.00 |
3.00 |
95.00 |
0.58 |
T5 (1.0) |
10.67 |
1.15 |
5.00 |
1.44 |
194.33 |
8.50 |
15.7 |
1.5 |
81.67 |
3.79 |
9.33 |
1.15 |
3.33 |
1.15 |
185.67 |
6.66 |
14.67 |
0.58 |
82.00 |
3.21 |
PC |
323.33 |
10.02 |
204.33 |
11.68 |
1590.33 |
15.63 |
325.7 |
10.6 |
700.67 |
14.57 |
313.00 |
6.56 |
201.00 |
13.45 |
1630.00 |
18.68 |
329.67 |
17.21 |
713.67 |
6.51 |
Key: NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation,T1-T5 = Test Item concentration from lower to higher.
Positive Controls:
2-Nitrofluorene |
: |
TA98 (absence of metabolic activation) |
Sodium azide |
: |
TA100 and TA1535 (absence of metabolic activation) |
9-Aminoacridine |
: |
TA1537 (absence of metabolic activation) |
Mitomycin-C |
: |
TA102 (absence of metabolic activation) |
Benzo[a]pyrene |
: |
TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation) |
Table4 Fold Increase
Trial I - Plate Incorporation Method |
Trial II – Preincubation Method |
|||||||||||||||||||
Test Item Concentration (mg/plate) |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
TA 102 |
||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
NC (Distilled water) |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
VC (Dimethyl sulfoxide) |
1.03 |
0.95 |
0.95 |
1.08 |
1.03 |
0.86 |
0.76 |
1.12 |
1.01 |
0.98 |
0.92 |
1.00 |
1.02 |
0.97 |
0.97 |
0.93 |
0.86 |
0.84 |
1.00 |
1.02 |
T1(0.625) |
1.02 |
1.00 |
1.07 |
0.94 |
0.97 |
1.00 |
1.25 |
0.89 |
0.98 |
0.99 |
1.00 |
1.02 |
0.94 |
0.94 |
1.03 |
1.05 |
1.05 |
1.25 |
0.99 |
0.99 |
T2 (0.125) |
1.03 |
0.89 |
1.02 |
0.96 |
0.90 |
1.08 |
1.25 |
1.16 |
0.98 |
1.02 |
1.02 |
0.97 |
0.97 |
1.05 |
1.06 |
1.08 |
0.95 |
1.44 |
0.97 |
0.98 |
T3 (0.25) |
0.92 |
1.00 |
1.01 |
0.94 |
1.03 |
1.03 |
1.13 |
1.00 |
0.96 |
1.00 |
1.10 |
1.03 |
0.93 |
0.99 |
1.06 |
0.97 |
0.84 |
1.06 |
0.98 |
1.00 |
T4 (0.5) |
1.00 |
0.90 |
1.05 |
0.93 |
0.97 |
1.08 |
1.19 |
0.95 |
0.95 |
1.01 |
0.95 |
0.94 |
0.91 |
0.98 |
1.19 |
1.00 |
1.05 |
1.06 |
0.96 |
0.99 |
T5 (1.0) |
0.79 |
0.77 |
0.84 |
0.82 |
0.79 |
0.81 |
1.06 |
0.79 |
0.82 |
0.83 |
0.78 |
0.69 |
0.81 |
0.84 |
0.89 |
0.74 |
0.79 |
0.63 |
0.81 |
0.78 |
PC |
15.67 |
15.65 |
6.99 |
6.83 |
25.31 |
25.54 |
35.81 |
30.95 |
6.65 |
6.99 |
16.28 |
15.45 |
6.91 |
7.33 |
26.94 |
24.71 |
32.26 |
37.69 |
6.64 |
6.89 |
Key: NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, T1-T5 = Test Item concentration from lower to higher.
Table5 S9 Efficiency Check- Summary
Summary of S9 efficiency check |
||||
|
TA100 |
TA1535 |
||
Mean |
SD |
Mean |
SD |
|
VC Dimethyl Sulfoxide (-S9) |
100.67 |
6.11 |
13.00 |
1.00 |
VC Dimethyl Sulfoxide (+S9) |
99.00 |
3.00 |
12.33 |
0.58 |
PC Benzo[a]pyrene (-S9) |
96.33 |
2.08 |
13.67 |
0.58 |
PC Benzo[a]pyrene (+S9) |
673.67 |
11.24 |
315.00 |
12.29 |
Key: VC
= Vehicle control, PC = Positive control, -S9 = Absence of metabolic
activation, +S9 = Presence of metabolic activation, SD = Standard
Deviation.
Appendix1: Relative Increase in Cell Counts – Preliminary Cytotoxicity Assay
Dose Level |
Conc. (mg/ml) |
Absence of Metabolic activation |
Presence of Metabolic activation |
||||||
Cell count |
RICC |
% Cytotoxicity |
Cell count |
RICC |
% Cytotoxicity |
||||
Starting |
Final |
Starting |
Final |
||||||
NC |
Distilled water |
1000000 |
3650000 |
100.00 |
0.00 |
1000000 |
3680000 |
100.00 |
0.00 |
VC |
DMSO |
1000000 |
3620000 |
98.87 |
1.13 |
1000000 |
3650000 |
98.88 |
1.12 |
T1 |
0.00625 |
1000000 |
2140000 |
43.51 |
56.49 |
1000000 |
2210000 |
45.66 |
54.34 |
T2 |
0.0125 |
1000000 |
1280000 |
10.69 |
89.31 |
1000000 |
1320000 |
12.08 |
87.92 |
T3 |
0.025 |
1000000 |
0 |
0.00 |
100.00 |
1000000 |
0 |
0.00 |
100.00 |
T4 |
0.05 |
1000000 |
0 |
0.00 |
100.00 |
1000000 |
0 |
0.00 |
100.00 |
T5 |
0.1 |
1000000 |
0 |
0.00 |
100.00 |
1000000 |
0 |
0.00 |
100.00 |
Key: NC = Negative Control, VC = Vehicle Control, Conc. = Concentration, mg = milligram, ml = milliliter, RICC = Relative Increase in Cell Counts, % = percentage.
Appendix 2: Relative Increase in Cell Counts- Main Study
Dose Level |
Conc.
|
Phase I -Absence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
NC |
Distilled water |
1000000 |
3620000 |
100.00 |
0.00 |
VC |
DMSO |
1000000 |
3590000 |
98.85 |
1.15 |
T1 |
0.015625 mg/ml |
1000000 |
2840000 |
71.04 |
28.96 |
T2 |
0.003125 mg/ml |
1000000 |
2580000 |
61.00 |
39.00 |
T3 |
0.00625 mg/ml |
1000000 |
2145000 |
44.21 |
55.79 |
PC |
20 µg/ml |
1000000 |
2560000 |
60.23 |
39.77 |
Dose Level |
Conc. |
Phase II -Presence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
NC |
Distilled water |
1000000 |
3640000 |
100.00 |
0.00 |
VC |
DMSO |
1000000 |
3580000 |
97.73 |
2.27 |
T1 |
0.015625 mg/ml |
1000000 |
2920000 |
74.42 |
25.58 |
T2 |
0.003125 mg/ml |
1000000 |
2684000 |
65.27 |
34.73 |
T3 |
0.00625 mg/ml |
1000000 |
2162000 |
45.04 |
54.96 |
PC |
30 µg/ml |
1000000 |
2540000 |
59.69 |
40.31 |
Dose Level |
Conc. |
Phase III -Absence of Metabolic activation |
|||
Cell count |
RICC |
% Cytotoxicity |
|||
Starting |
Final |
||||
NC |
Distilled water |
1000000 |
3612000 |
100.00 |
0.00 |
VC |
DMSO |
1000000 |
3420000 |
92.65 |
7.35 |
T1 |
0.015625 mg/ml |
1000000 |
2785000 |
73.76 |
26.24 |
T2 |
0.003125 mg/ml |
1000000 |
2540000 |
63.64 |
36.36 |
T3 |
0.00625 mg/ml |
1000000 |
2180000 |
48.76 |
51.24 |
PC |
20 µg/ml |
1000000 |
2560000 |
64.46 |
35.54 |
Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control, Conc. = Concentration, mg = milligram, ml = milliliter, µg = microgram, RICC = Relative Increase in Cell Counts, % = percentage.
Appendix 3:Individual Data on Chromosome Aberrations-Phase I:Absence of metabolic activation (short term)
Dose level & Concentration |
No. of Metaphases |
Frequencies of Aberration |
Total No of Aberrant cells |
||||||
with gap |
without gap |
||||||||
NC (Distilled wate) |
300 |
- |
0 |
0 |
|||||
VC (DMSO) |
300 |
1 Ctb |
1 |
1 |
|||||
T1 0.0015625 mg/ml |
300 |
2 fragments |
2 |
2 |
|||||
T2 0.003125 mg/ml |
300 |
1 ring, 1 fragment |
2 |
2 |
|||||
T3 0.00625 mg/ml |
300 |
1 Ctg, 1 Ctb |
2 |
1 |
|||||
PC 20 µg/ml |
300 |
1 Csg, 9 Ctb, 3 fragment, 2 ring, 3 exchange, 4 dic |
21 |
20 |
|||||
Key: NC = Negative control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control (methyl methanesulfonate), mg = milligram, µg = microgram, ml = milliliter, T3- T1 = Test Item concentration from higher to lower, Ctg = Chromatid gap, Csg = Chromosome gap, Ctb = Chromatid break, Csb = Chromosome break, dic = dicentric.
Appendix 4:Individual Data on Chromosome Aberrations- Phase II:Presence ofmetabolic activation (short term)
Dose level & Concentration |
No. of Metaphases |
Frequencies of Aberration |
Total No of Aberrant cells |
||||||
with gap |
without gap |
||||||||
NC 0 mg/ml |
300 |
- |
0 |
0 |
|||||
VC 0 mg/ml |
300 |
- |
0 |
0 |
|||||
T1 0.0015625 mg/ml |
300 |
1 Ctb, 1 dic |
2 |
2 |
|||||
T2 0.003125 mg/ml |
300 |
1 Ctb |
1 |
1 |
|||||
T3 0.00625 mg/ml |
300 |
- |
0 |
0 |
|||||
PC 30 µg/ml |
300 |
9 Ctb, 8 fragment, 3 ring, 2 exchange, 3 dic, 1 minute |
22 |
22 |
|||||
Key: NC = Negative control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control (methyl methanesulfonate), mg = milligram, µg = microgram, ml = milliliter, T3-T1 = Test Item concentration from higher to lower, Ctg = Chromatid gap, Csg = Chromosome gap, Ctb = Chromatid break, Csb = Chromosome break, dic = dicentric.
Appendix 5: Individual Data on Chromosome Aberrations- Phase III:Absenceof metabolic activation (Continuous)
Dose level & Concentration |
No. of Metaphases |
Frequencies of Aberration |
Total No of Aberrant cells |
|
with gap |
without gap |
|||
NC 0 mg/ml |
300 |
- |
0 |
0 |
VC 0 mg/ml |
300 |
1 Ctb |
1 |
1 |
T1 0.0015625 mg/ml |
300 |
- |
0 |
0 |
T2 0.003125 mg/ml |
300 |
1 ring, 1 Csb, 1 fragment |
3 |
3 |
T3 0.00625 mg/ml |
300 |
1 Ctb, 1 dic |
2 |
2 |
PC 20 µg/ml |
300 |
9 Ctb, 1 Csb, 5 fragment, 1 ring, 2 exchange, 7 dic |
23 |
23 |
Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control (methyl methanesulfonate), mg = milligram, µg = microgram, ml = milliliter, T3-T1 = Test Item concentration from higher to lower, Ctg = Chromatid gap, Csg = Chromosome gap, Ctb = Chromatid break, Csb = Chromosome break, dic = dicentric.
Appendix 6: Summary Dataon Chromosome Aberrations - Phase I
Dose Level |
Concentration
|
Absence of metabolic activation |
||
Total No. of Aberrant cells without gap |
Percent aberrant cells |
|
||
NC |
Distilled water |
0 |
0.00 |
|
VC |
DMSO |
1 |
0.33 |
|
T1 |
0.015625 mg/ml |
2 |
0.67 |
|
T2 |
0.003125 mg/ml |
2 |
0.67 |
|
T3 |
0.00625 mg/ml |
1 |
0.33 |
|
PC |
20 µg/ml* |
20 |
6.67 |
|
Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control, mg = milligram, ml = milliliter, µg = microgram,* = Statistical significant increase in % aberrant cell (p<0.05).
Appendix 7:Summary Data on Chromosome Aberrations - Phase II
Dose Level |
Concentration
|
Presence of metabolic activation |
||
Total No. of Aberrant cells without gap |
Percent aberrant cells |
|
||
NC |
Distilled water |
0 |
0.00 |
|
VC |
DMSO |
0 |
0.00 |
|
T1 |
0.015625 mg/ml |
2 |
0.67 |
|
T2 |
0.003125 mg/ml |
1 |
0.33 |
|
T3 |
0.00625 mg/ml |
0 |
0.00 |
|
PC |
30 µg/ml* |
22 |
7.33 |
|
Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control, mg = milligram, ml = milliliter, µg = microgram,* = Statistical significant increase in % aberrant cell (p<0.05).
Appendix 8: SummaryData on Chromosome Aberrations - Phase III
Dose Level |
Concentration |
Absence of metabolic activation |
||
Total No. of Aberrant cells without gap |
Percent aberrant cells |
|
||
NC |
Distilled water |
0 |
0.00 |
|
VC |
DMSO |
1 |
0.33 |
|
T1 |
0.015625 mg/ml |
0 |
0.00 |
|
T2 |
0.003125 mg/ml |
3 |
1.00 |
|
T3 |
0.00625 mg/ml |
2 |
0.67 |
|
PC |
20 µg/ml* |
23 |
7.67 |
|
Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control, mg = milligram, ml = milliliter, µg = microgram,* = Statistical significant increase in % aberrant cell (p<0.05).
Appendix1: 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 |
23300000 |
- |
- |
- |
- |
- |
- |
- |
- |
VC |
Dimethylformamide |
20000000 |
22820000 |
- |
- |
- |
- |
- |
- |
- |
- |
T1 |
0.03125 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
T2 |
0.0625 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
T3 |
0.125 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
T4 |
0.25 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
T5 |
0.5 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
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.
Appendix2: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 |
22980000 |
- |
- |
- |
- |
- |
- |
- |
- |
VC |
Distilled water |
20000000 |
22640000 |
- |
- |
- |
- |
- |
- |
- |
- |
T1 |
0.03125 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
T2 |
0.0625 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
T3 |
0.125 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
T4 |
0.25 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
T5 |
0.5 mg/ml |
20000000 |
0 |
- |
- |
- |
- |
- |
- |
- |
- |
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.
Appendix 3: Relative Survival – Repeated 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 |
23650000 |
235 |
218 |
230 |
227.67 |
100 |
2.277 |
2.692 |
100.00 |
VC |
Dimethylformamide |
20000000 |
23260000 |
225 |
215 |
226 |
222.00 |
100 |
2.220 |
2.582 |
95.90 |
T1 |
0.001875 mg/ml |
20000000 |
22820000 |
215 |
203 |
211 |
209.67 |
100 |
2.097 |
2.392 |
92.66 |
T2 |
0.00375 mg/ml |
20000000 |
21620000 |
175 |
182 |
180 |
179.00 |
100 |
1.790 |
1.935 |
74.95 |
T3 |
0.0075 mg/ml |
20000000 |
16680000 |
165 |
156 |
163 |
161.33 |
100 |
1.613 |
1.346 |
52.11 |
T4 |
0.015 mg/ml |
20000000 |
8662000 |
116 |
105 |
114 |
111.67 |
100 |
1.117 |
0.484 |
18.73 |
T5 |
0.03 mg/ml |
20000000 |
0 |
0 |
0 |
0 |
0.00 |
100 |
0.000 |
0.000 |
0.00 |
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.
Appendix 4:Relative Survival – Repeated 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 |
23820000 |
224 |
236 |
232 |
230.67 |
100 |
2.307 |
2.747 |
100.00 |
VC |
Dimethylformamide |
20000000 |
23440000 |
215 |
226 |
235 |
225.33 |
100 |
2.253 |
2.641 |
96.13 |
T1 |
0.001875 mg/ml |
20000000 |
22420000 |
216 |
211 |
209 |
212.00 |
100 |
2.120 |
2.377 |
89.99 |
T2 |
0.00375 mg/ml |
20000000 |
19240000 |
185 |
193 |
173 |
183.67 |
100 |
1.837 |
1.767 |
66.90 |
T3 |
0.0075 mg/ml |
20000000 |
16540000 |
158 |
163 |
168 |
163.00 |
100 |
1.630 |
1.348 |
51.04 |
T4 |
0.015 mg/ml |
20000000 |
8862000 |
108 |
98 |
106 |
104.00 |
100 |
1.040 |
0.461 |
17.45 |
T5 |
0.03 mg/ml |
20000000 |
0 |
0 |
0 |
0 |
0.00 |
100 |
0.000 |
0.000 |
0.00 |
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.
Appendix 5: RelativeSurvival – 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 |
23440000 |
232 |
224 |
229 |
228.33 |
100 |
2.283 |
2.676 |
100.00 |
VC |
Dimethylformamide |
20000000 |
23140000 |
221 |
224 |
218 |
221.00 |
100 |
2.210 |
2.557 |
95.55 |
T1 |
0.001875 mg/ml |
20000000 |
21820000 |
216 |
210 |
212 |
212.67 |
100 |
2.127 |
2.320 |
90.74 |
T2 |
0.00375 mg/ml |
20000000 |
19280000 |
190 |
197 |
194 |
193.67 |
100 |
1.937 |
1.867 |
73.01 |
T3 |
0.0075 mg/ml |
20000000 |
16660000 |
168 |
162 |
157 |
162.33 |
100 |
1.623 |
1.352 |
52.88 |
T4 |
0.015 mg/ml |
20000000 |
8140000 |
122 |
113 |
119 |
118.00 |
100 |
1.180 |
0.480 |
18.78 |
PC |
400 µg/ml |
20000000 |
21480000 |
219 |
214 |
208 |
213.67 |
100 |
2.137 |
2.295 |
89.75 |
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.
Appendix 6: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 |
23320000 |
230 |
221 |
221 |
224.00 |
100 |
2.240 |
2.612 |
100.00 |
VC |
Dimethylformamide |
20000000 |
23020000 |
218 |
224 |
216 |
219.33 |
100 |
2.193 |
2.525 |
96.66 |
T1 |
0.001875 mg/ml |
20000000 |
21180000 |
203 |
209 |
211 |
207.67 |
100 |
2.077 |
2.199 |
87.11 |
T2 |
0.00375 mg/ml |
20000000 |
18720000 |
186 |
180 |
182 |
182.67 |
100 |
1.827 |
1.710 |
67.73 |
T3 |
0.0075 mg/ml |
20000000 |
15820000 |
162 |
154 |
168 |
161.33 |
100 |
1.613 |
1.276 |
50.55 |
T4 |
0.015 mg/ml |
20000000 |
8524000 |
110 |
111 |
108 |
109.67 |
100 |
1.097 |
0.467 |
18.51 |
PC |
30 µg/ml |
20000000 |
20960000 |
210 |
215 |
211 |
212.00 |
100 |
2.120 |
2.222 |
88.01 |
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.
Appendix 7: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 |
214 |
221 |
211 |
215 |
2.15 |
VC |
Dimethylformamide |
100 |
213 |
206 |
211 |
210 |
2.10 |
T1 |
0.001875 mg/ml |
100 |
186 |
190 |
192 |
189 |
1.89 |
T2 |
0.00375 mg/ml |
100 |
179 |
182 |
190 |
184 |
1.84 |
T3 |
0.0075 mg/ml |
100 |
184 |
181 |
176 |
180 |
1.80 |
T4 |
0.015 mg/ml |
100 |
176 |
177 |
170 |
174 |
1.74 |
PC |
400 µg/ml |
100 |
165 |
174 |
179 |
173 |
1.73 |
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.
Appendix 8: CloningEfficiency(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 |
220 |
218 |
217 |
218 |
2.18 |
VC |
Dimethylformamide |
100 |
211 |
198 |
216 |
208 |
2.08 |
T1 |
0.001875 mg/ml |
100 |
189 |
184 |
195 |
189 |
1.89 |
T2 |
0.00375 mg/ml |
100 |
186 |
179 |
184 |
183 |
1.83 |
T3 |
0.0075 mg/ml |
100 |
181 |
168 |
174 |
174 |
1.74 |
T4 |
0.015 mg/ml |
100 |
178 |
168 |
174 |
173 |
1.73 |
PC |
30 µg/ml |
100 |
168 |
174 |
178 |
173 |
1.73 |
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
Appendix 9: 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 |
4 |
4 |
2 |
3.33 |
0.00001667 |
VC |
Dimethylformamide |
200000 |
3 |
5 |
3 |
3.67 |
0.00001833 |
T1 |
0.001875 mg/ml |
200000 |
4 |
4 |
3 |
3.67 |
0.00001833 |
T2 |
0.00375 mg/ml |
200000 |
3 |
4 |
3 |
3.33 |
0.00001667 |
T3 |
0.0075 mg/ml |
200000 |
4 |
3 |
4 |
3.67 |
0.00001833 |
T4 |
0.015 mg/ml |
200000 |
4 |
3 |
4 |
3.67 |
0.00001833 |
PC |
400 µg/ml |
200000 |
88 |
76 |
79 |
81.00 |
0.00040500 |
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.
Appendix 10: Cloning Efficiency(Selective medium)Phase I: 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 |
4 |
3 |
3.33 |
0.00001667 |
VC |
Dimethylformamide |
200000 |
4 |
2 |
4 |
3.33 |
0.00001667 |
T1 |
0.001875 mg/ml |
200000 |
3 |
5 |
3 |
3.67 |
0.00001833 |
T2 |
0.00375 mg/ml |
200000 |
4 |
4 |
2 |
3.33 |
0.00001667 |
T3 |
0.0075 mg/ml |
200000 |
3 |
3 |
4 |
3.33 |
0.00001667 |
T4 |
0.015 mg/ml |
200000 |
3 |
5 |
3 |
3.67 |
0.00001833 |
PC |
30 µg/ml |
200000 |
82 |
80 |
85 |
82.33 |
0.00041167 |
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.
Appendix11: Mutation Frequency: Absence of metabolic activation
Dose level |
Absence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000774 |
7.74 |
VC |
Dimethylformamide |
0.00000873 |
8.73 |
T1 |
0.001875 mg/ml |
0.00000968 |
9.68 |
T2 |
0.00375 mg/ml |
0.00000907 |
9.07 |
T3 |
0.0075 mg/ml |
0.00001017 |
10.17 |
T4 |
0.015 mg/ml |
0.00001052 |
10.52 |
PC |
400 µg/ml* |
0.00023456 |
234.56 |
Dose level |
Presence of metabolic activation |
||
Concentration |
Mutation Frequency |
MF x 10-6 |
|
NC |
Distilled water |
0.00000763 |
7.63 |
VC |
Dimethylformamide |
0.00000800 |
8.00 |
T1 |
0.001875 mg/ml |
0.00000968 |
9.68 |
T2 |
0.00375 mg/ml |
0.00000911 |
9.11 |
T3 |
0.0075 mg/ml |
0.00000956 |
9.56 |
T4 |
0.015 mg/ml |
0.00001058 |
10.58 |
PC |
30 µg/ml* |
0.00023750 |
237.50 |
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, * = Stastically significant increse in mutation frequency.
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:
The mutagenicity of the test substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (CAS Number: 6786-83-0; EC Number: 229-851-8) was tested according to OECD 471 using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102. The test was performed as two independent experiments according to the plate incorporation method (Trial I) and the preincubation method (Trial II), both in the presence and absence of rat liver microsomal activation (S9 mix). Concentrations used in the mutagenicity assay were selected based on a preliminary cytotoxicity assay. This preliminary cytotoxicity assay was performed using concentrations of 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2 and 1 mg/plate, with and without metabolic activation (10 % v/v S9 mix) using TA98 and TA100 strains. A slight reduction in the revertant counts and moderate inhibition of the background lawn was observed at 1mg/plate, and no reduction in the revertant count and/or inhibition in the background lawn was noted at concentrations from 0.0000128 to 0.2 mg/plate when compared to the vehicle control data. Based on the observations of the pre-test, the following doses were applied in the mutagenicity test: 0.0 (NC), 0.0 (VC), 0.0625, 0.125, 0.25, 0.5 and 1mg/plate, both in the presence and absence of metabolic activation, along with the negative, vehicle and positive controls. Trial I was performed according to the plate incorporation method. The test substance, together with the negative, vehicle and positive controls, were tested in triplicates. The Test Item doses were selected using a spacing factor of 2. There was slight precipitation, a slight reduction in revertant colony count and a moderate inhibition in the background lawn at 1 mg/plate. Furthermore, there was no increase in the number of revertant colonies and/or diminution of the background lawn at concentrations of 0.0625 - 0.5 mg/plate, neither in the presence nor the absence of metabolic activation compared to the vehicle control data. Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method; the substance was tested in all the tester strains in triplicates. There was slight precipitation, a slight reduction in revertant colony count and a moderate inhibition in the background lawn at 1 mg/plate. There was no increase in the number of revertant colonies and/or diminution of the background lawn at concentrations of 0.0625 - 0.5 mg/plate, neither in the presence nor in the absence of metabolic activation when compared to the vehicle control data. The numbers of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains compared to the vehicle control data confirming the validity of the mutagenicity test. Based on the results of this study, it is concluded that(4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) is non-mutagenic as it does not induce gene mutations (point and frameshift) in the histidine operon by base-pair changes or frameshifts, in the presence or the absence of metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102).
In vitro mammalian chromosome aberration test:
The potential of (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) to induce structural chromosomal aberration was tested according to OECD TG 473 with and without metabolic activation in cultured Chinese Hamster Ovary (CHO) cells. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation. The S9 fraction was obtained from phenobarbitone and β-naphthoflavone-injected rats. The substance was insoluble in distilled water but was soluble in dimethyl sulfoxide up to 10 mg/ml. Test concentrations were chosen based on a preliminary cytotoxicity experiment. In this pre-test, CHO cells were exposed to 0.0 (VC), 0.0 (NC), 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/ml for 4 hours in the presence (1 % v/v S9 mix) and absence of metabolic activation system. Excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.0125 mg/ml, both in the presence and absence of metabolic activation. RICC decreased by 55±5% compared to vehicle control at 0.00625 mg/ml both in the presence and absence of metabolic activation. Hence, Phase I (short term[4hrs]exposure in the absence of metabolic activation), Phase II (short term[4 hrs]exposure in the presence of metabolic activation) and Phase III (continuous[24 hrs]exposure in the absence of metabolic activation) were conducted with the Test Item at the concentrations of 0.0015625, 0.003125 and 0.00625 mg/ml, along with vehicle (DMSO), negative (Distilled water) and concurrent positive (Methyl methanesulfonate, 20µg/ml, without S9 mix; Benzo (a) pyrene, 30µg/ml with S9 mix)controls. At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations. Breaks (chromatid and chromosomal), gaps(chromatid and chromosomal), rings and fragments, and dicentric chromosomes were found as structural chromosome aberrations. Gaps were recorded and reported separately but not included in the total aberration frequency.Results:In the Phase I experiment, CHO cells were exposed to the substance, vehicle, negative and positive controls for 4 hours (short term exposure) in the absence of metabolic activation. The average RICC values were 98.85 % (vehicle control), 71.04 % (at 0.0015625 mg/ml), 61.00 % (at 0.003125 mg/ml) and 44.21 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells was observed at 0.0015625 mg/ml (mean % aberrant cells: 0.67%), 0.003125 mg/ml (mean % aberrant cells: 0.67%), or at 0.00625 mg/ml (mean % aberrant cells: 0.33%) compared to the vehicle control data (mean % aberrant cells 0.33 %). In the Phase II experiment, cultures were exposed to the substance, vehicle, negative and positive controls for 4 hours (short term exposure) in the presence of metabolic activation (1 % v/v S9 mix). The average RICC values were 97.73 % (vehicle control), 74.42 % (at 0.0015625 mg/ml), 65.27 % (at 0.003125 mg/ml) and 45.04 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells was observed at 0.0015625 mg/ml (mean % aberrant cells: 0.67%), 0.003125 mg/ml (mean % aberrant cells: 0.33%), or at 0.00625 mg/ml (mean % aberrant cells: 0.00%) compared to the vehicle control data (mean % aberrant cells: 0.00 %). In Phase III, cultures were exposed to the substance, vehicle, negative and positive control for 24 hours (continuous exposure) without metabolic activation. The average RICC values were 92.65 % (vehicle control), 73.76 % (at 0.0015625 mg/ml), 63.64 % (at 0.003125 mg/ml) and 48.76 % (at 0.00625 mg/ml). No significant increase in mean percent aberrant cells was observed at 0.0015625 mg/ml (mean % aberrant cells: 0.00%), 0.003125 mg/ml (mean % aberrant cells: 1.00%), or at 0.00625 mg/ml (mean % aberrant cells: 0.67%) compared to the vehicle control data (mean % aberrant cells 0.33 %). In all phases of the study, no significant reduction in RICC (cytotoxicity) and no increase in percent aberrant cells were observed in vehicle control (DMSO) either in the presence or absence of metabolic activation, when compared to the negative control (distilled water). The positive controls (-S9: methyl methanesulfonate, +S9: Benzo[a]pyrene) used in the study produced statistically significant increases in the percent of cells with structural chromosome aberrations (Phase I: 6.67%; p<0.0001, Phase II: 7.33%; p<0.0001, Phase III: 7.67%; p<0.0001) 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, (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) did not induce chromosomal aberration up to 0.00625mg/ml, either in the presence or absence of S9 metabolic activation in CHO cells.
In vitro gene mutation test in mammalian cells:
The potential of the registered substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol [CAS No.: 6786-83-0] to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus was tested in the presence and absence of an exogenous metabolic activation system using cultured Chinese Hamster Ovary (CHO) cells. The study was performed as per OECD Test Guideline No. 476 (Adopted: 29 July 2016). Cofactor-supplemented S9 liver microsomal fraction was used as an exogenous metabolic activation system. Dimethylformamide was selected as a solvent for the test substance during the experiment. Test concentrations were determined based on a preliminary cytotoxicity test. In the initial cytotoxicity test, the CHO cells were exposed to test item concentrations of 0 (VC), 0 (NC), 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/ml in culture medium, both in the presence and absence of a metabolic activation system (1 % v/v S9 mix). Cytotoxicity was determined by the Relative survival (RS[RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in vehicle control]) values. Complete cytotoxicity was observed at tested concentrations of 0.03125-0.5 mg/ml both in the presence and absence of metabolic activation. Hence, the preliminary cytotoxicity test was repeated using the following concentrations: 0 (VC), 0 (NC), 0.001875, 0.00375, 0.0075, 0.015 and 0.03 mg/ml both in the presence and absence of a metabolic activation system (1 % v/v S9 mix). In the absence of metabolic activation, the RS values were 100% (negative control), 95.90% (vehicle control), 92.66% (at 0.001875 mg/ml), 74.95% (at 0.00375 mg/ml), 52.11% (at 0.0075 mg/ml), 18.73% (at 0.015 mg/ml) and 0.00% (at 0.03 mg/ml). In the presence of metabolic activation, the RS values were 100% (negative control), 96.13% (vehicle control), 89.99%, (at 0.001875 mg/ml), 66.90% (at 0.00375 mg/ml), 51.04% (at 0.0075 mg/ml), 17.45% (at 0.015 mg/ml) and 0.00% (at 0.03 mg/ml). Thus, complete cytotoxicity was observed at 0.03 mg/ml in the absence and presence of metabolic activation. Whereas at the reaming tested concentrations, limited cytotoxicity (>17% RS)was observed at 0.015 mg/ml, in the absence (RS: 18.73%) or presence (RS: 17.45%) of the S9 metabolic activation system. Hence, the gene mutation study was conducted with substance concentrations of 0.0 (NC), 0.0 (VC), 0.001875, 0.00375, 0.0075 and 0.015 mg/ml, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with and positive controls (Ehtylmethanesulfonate: 400 µg/ml without S9mix;Benzo (a) pyrene: 30µg/ml, with S9 mix).Results:In the absence of metabolic activation, the RS values were 100% (negative control), 95.55% (vehicle control), 90.74% (at 0.001875 mg/ml), 73.01% (at 0.00375 mg/ml), 52.88% (at 0.0075 mg/ml), 18.78% (at 0.015 mg/ml) and 89.75% (at 400 µg/ml-positive control [Ehtylmethanesulfonate]). In the presence of metabolic activation, the RS values were 100% (negative control), 96.66% (vehicle control), 87.11% (at 0.001875 mg/ml), 67.73% (at 0.00375 mg/ml), 50.55% (at 0.0075 mg/ml) 18.51% (at 0.015 mg/ml) and 88.01% (30 µg/ml-positive control[Benzo[a]pyrene]). No significant increase in the mutation frequency (MF) either in absence(9.68x10-6, 9.07 x10-6, 10.17 x10-6and 10.52 x10-6at 0.001875 mg/ml, 0.00375 mg/ml, 0.0075 mg/ml and 0.015 mg/ml, respectively) or presence of metabolic activation (9.68 x10-6, 9.11x10-6, 9.56x10-6and 10.58x10-6at 0.001875 mg/ml, 0.00375 mg/ml, 0.0075 mg/ml and 0.015 mg/ml, respectively) was observed when compared to vehicle control (8.73 x10-6, 8.00 x10-6, absence and presence of S9, respectively). The positive controls (Ethylmethanesulfonate and Beno[a]pyrene in the absence and presence of metabolic activation, respectively) used in the study produced statistically significant increases in mutation frequency (234.56 x10-6, p<0.0001 [Ethylmethanesulfonate], 237.50 x10-6, p<0.0001 [Benzo(a)pyrene] in the absence and presence of metabolic activation, respectively).Conclusion:The Substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol [CAS No.: 6786-83-0], did not induce gene mutation at the locus of hypoxanthine-guanine phosphoribosyltransferase (Hprt) in CHO cells up to 0.5 mg/ml, either in the presence or absence of S9 metabolic activation system.
Justification for classification or non-classification
The registered substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol [CAS No.: 6786-83-0], did not induce gene mutation by base-pair exchange and/or frameshift in five Salmonella Typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) in the presence and absence of liver S9 microsomal activation. The substance was tested non-clastogenic in mammalian cells as the exposure of Chinese Hamster Ovary (CHO) cells to the substance did not raise the percent of cells with structural chromosomal aberrations in either the presence or the absence of S9 metabolic activation. Additionally, the substance tested non-mutagenic (negative) in cultured mammalian cells as it did not produce point mutation at the locus of hypoxanthine-guanine phosphoribosyltransferase (Hprt) in CHO cells either in the presence or absence of S9 metabolic activation. Hence, the registered substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol [CAS No.: 6786-83 -0] is classified as Non-Classified for Germ cell mutagenicity according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
