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EC number: 202-213-6 | CAS number: 93-04-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation test in vitro:
Bacterial reverse mutation test:
The registered substance, 2-methoxynapthalene (CAS No. 93-04-9), was tested non-mutagenic (negative) in a bacterial reverse mutation test both in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471 and GLP.
In vitro mammalian chromosome aberration test:
The registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), tested non-clastogenic (negative) in primary cultures of human peripheral blood lymphocytes in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and GLP.
In vitro mammalian gene mutation test:
An in vitro gene mutataion test with the registered substance (CAS 93-04-9) according to OECD TG 476 and GLP is ongoing. This results will be submitted at a later timepoint.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Data waiving:
- other justification
- Justification for data waiving:
- other:
- 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
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- 29. July 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Purity: 99.06%
Molecular Weight:158.2 g/mol
Molecular Formula: C11H10O
Manufacture Date: June 29, 2018
Expiry Date: June 29, 2023 - Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Human lymphocytes were collected from the blood. Blood samples were obtained from a healthy donor in the range of 25-29 years of age.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor-supplemented liver S9 microsomal fraction was used. The S9 fraction was obtained from Aroclor 1254-induced rat.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, which resulted in a final concentration in the S9 mix of approximately 1 % (v/v) and 2 % (v/v) for Phase I and Phase II, respectively.
The cofactor solution contained the following quantity of chemicals in 500 mL of Reverse Osmosis (RO) water:
D-glucose-6-phosphate: 0.80 g
MgCl2: 1.00 g
KCl:1.35 g
Na2HPO4: 6.40 g
NaH2PO4.H2O: 1.40 g
β-NADP: 1.75 g - Test concentrations with justification for top dose:
- Test concentrations: 0.0 (NC: distilled water), 0.0 (VC: DMSO), 0.0006 mg/ml, 0.0012 mg/ml, 0.0024 mg/ml
Justifications:
Based on the solubility, precipitation, and pH tests, three test concentrations, 0.125, 0.063 and 0.031 mg/ml in culture media, were selected for evaluation. Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control. The test concentration 0.0024 mg/mL produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and presence of metabolic activation (+S9), respectively. - Vehicle / solvent:
- Dimethyl sulfoxide was used as vehicle.
- 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:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicates were used.
- Number of independent experiments: Two (Phase I-II)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): NA
- 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 hrs (Phase I), 24 hrs (Phase II)
- Harvest time after the end of treatment (sampling/recovery times): 24 hrs (Phase I-II)
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.:Three hours before cell harvesting, 240 µL of colcemid® (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each culture tube/flask and continued incubation at 37 °C.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure. NA
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Microscopic slides with the mitotic metaphase spreads were prepared by dropping the cell suspension onto a clean pre-chilled microscope slide. Two slides were made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): NA
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): NA
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, 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.
- 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: Cytotoxicity was demonstrated by calculating the Mitotic Index (MI).
- Any supplementary information relevant to cytotoxicity: - Evaluation criteria:
- A test item is 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-dependent when evaluated with the appropriate trend test,
- any of the results are outside the historical vehicle control range.
A test item is 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-dependent increase.
- all results are inside the historical vehicle control range. - Statistics:
- Statistical significance was confirmed using the non-parametric Mann-Whitney test. However, both biological and statistical significance were considered together.
- Key result
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In Phase I, the concentration of 0.0024 mg/ml produced 55.74% and 52.16% without and with S9 mix, respectively. In Phase II, cytotoxicity was 54.67% and 41.22% at 0.0024 mg/ml without and with S9 mix, 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: The pH of the culture medium (with the test item added) was measured following 0 and 4 hours of exposure at 37°C. No significant changes in pH were observed at 0 or 4 hours compared to the vehicle.
- Data on osmolality:NA
- Possibility of evaporation from medium: NA
- Water solubility: The test substance was insoluble in water.
- Precipitation and time of the determination: Precipitation was observed at concentrations of 2.0, 1.0, 0.5, and 0.25 mg/ml at 0th hour. Slight precipitation was observed at 0.125 mg/mL test concentration at the 0th hour.
- Definition of acceptable cells for analysis:
- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES (if applicable):
STUDY RESULTS
- Concurrent vehicle negative and positive control data: See in section “Any other information on results incl. tables”
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any: See in section “Any other information on results incl. tables”
Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: See in section “Any other information on results incl. tables”
o For lymphocytres in primary cultures: mitotic index (MI):
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps: Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, 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.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps: See in section “Any other information on results incl. tables”
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen: See in section “Any other 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: Yes. See in section “Any other information on results incl. tables”
- Negative (solvent/vehicle) historical control data: Yes. See in section “Any other information on results incl. tables” - Remarks on result:
- other: Non-clastogenic (negative)
- Conclusions:
- The registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), tested non-clastogenic (negative) in primary cultures of human peripheral blood lymphocytes in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and GLP.
- Executive summary:
The clastogenic potential of the registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), was assessed in human peripheral blood lymphocytes in the presence and absence of an exogenous metabolic activation system in an OECD 473 study. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was derived from the Aroclor 1254-induced rats. The test concentrations were selected based on the solubility, precipitation and pH checks and a preliminary cytotoxicity test. The test substance was soluble in Dimethyl Sulfoxide (DMSO) up to 200 mg/ml to give the final treatment concentration of 2 mg/ml. No significant changes in pH were observed at 0 or 4 hours when compared with the vehicle control. Precipitation was observed at concentrations of 2.0, 1.0, 0.5, and 0.25 mg/ml. Slight precipitation was observed at 0.125 mg/m test concentration at the 0th hour, which was judged not to interfere with the conduct of the test. Hence, the concentration of 0.125 mg/ml was selected as the high dose for the cytotoxicity experiment.
In the cytotoxicity test, cells were exposed to test item concentrations of 0.0 (NC: distilled water), 0.0 (VC: DMSO), 0.125, 0.063 and 0.031 mg/ml in culture media in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control data. All the tested concentrations at the initial cytotoxicity experiment were cytotoxic both in the presence and absence of the S9 metabolic activation system. Next, the cytotoxicity was assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.015 mg/ml of culture media in a second cytotoxicity experiment (Experiment II). In cytotoxicity experiment (II), all tested concentrations were cytotoxic both in the presence and absence of the S9 metabolic activation system. Cytotoxicity was then assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml in culture media in the third cytotoxicity experiment (Experiment III). The test concentration of 0.0024 mg/ml produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and the presence of metabolic activation (+S9), respectively. The dose level that results in approximately 55 ± 5 % reduction in the mitotic index was selected as the highest test concentration in the main test. Hence, 0.0024 mg/ml was chosen as the highest test item concentration for the main study, both in the presence and absence of metabolic activation. In the CA test, proliferating peripheral blood cells (whole blood) were used for the test, and the treatment of cultures with the test item was conducted in two independent phases. In the Phase I experiment, cells were exposed to test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml along with positive controls (EMS: 600 µg/ml without S9 mix and CPA: 30 600 µg/ml with S9 mix) for 4 hours both in the presence and absence of S9 metabolic activation system (1 v/v %) using duplicates. In the Phase II experiment, cells in duplicate cultures were treated with 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml for 4 hours in the presence of the S9 metabolic activation system (2 v/v%) or 24 hours in the absence of S9 metabolic activation. Cells were harvested 24 hrs after treatments in both experiements. A minimum of 1000 cells was counted in different fields of slide per culture, and the number of metaphases was recorded for Mitotic Index (MI) calculation. Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides. Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, 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. Results: In the cytotoxicity test (Experiment III), the test concentration 0.0024 mg/ml produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and the presence of metabolic activation (+S9), respectively. The test concentration 0.0012 mg/ml produced 42.00 % and 44.16 % decreases in the mitotic index compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The test concentration 0.0006 mg/ml produced a 33.13 % and 31.67 % decrease in the mitotic index (cytotoxicity) compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively.
In Phase I experiment, the cultures were exposed to the test substance for 4 hours both in the presence (+S9) and absence (-S9) of the metabolic activation system (1%). The mean percentages of aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 ( at 0.0006 mg/ml), 0.667 (at 0.0012 mg/ml), 0.667 (at 0.0024 mg/ml) and 11.000 (PC: 600 µg/mL EMS) in the absence of metabolic activation. In the presence of metabolic activation (1 v/v%), the mean percentage of aberrant cells were 0.333 (NC), 0.333 (VC), 0.000 (at 0.0006 mg/ml), 0.333 (at 0.0012 mg/ml), 0.667 (at 0.0024 mg/ml) and 10.333 (PC: 30 µg/ml CPA). Treatment with positive control substances caused significant increases in the percent of aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were deemed biologically significant. A 55.74% and 52.16 % of reduction in the mitotic index, i.e., cytotoxicity, was observed at the tested concentration of 0.0024 mg/ml compared to vehicle control (VC) in the absence (-S9) and presence (+S9) of metabolic activation system, respectively. The observed mean mitotic index in the absence of metabolic activation were 9.87 (NC), 9.66 (VC), 6.87 (at 0.0006 mg/ml), 5.57 (at 0.0012 mg/ml), 4.28 (at 0.0024 mg/ml) and 8.37 (PC: 600 µg/mL EMS). In the presence of metabolic activation (1 v/v%), the mean mitotic index was 9.53 (NC), 8.66 (VC), 5.59 (at 0.0006 mg/ml), 4.92 (at 0.0012 mg/ml), 4.14 (at 0.0024 mg/ml) and 7.69 (PC: 30 µg/mL CPA). The Phase II experiment was performed to confirm the negative results obtained in Phase I in the presence and absence of S9 metabolic activation. In the absence of metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 (at 0.0006 mg/ml), 0.333 (at 0.0012 mg/ml), 0.333 (at 0.0024 mg/ml) and 10.667 (PC: 600 µg/mL EMS). In the presence of the metabolic activation, the mean percent aberrant cells were 0.667 (NC), 0.333 (VC), 0.000 (at 0.0006 mg/ml), 1.000 (at 0.0012 mg/ml ), 0.333 (at 0.0024 mg/ml) and 11.000 (PC: 30 µg/mL CPA). Treatment with positive control substances in the absence and presence of metabolic activation caused a significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were deemed biologically significant. A reduction of 54.67 and 41.22 in the mitotic index was observed at the tested concentration of 0.0024 mg/ml compared to the vehicle control (VC) in the absence (-S9) and presence (+S9) of the metabolic activation system, respectively. The observed mean mitotic index in the absence (-S9) of metabolic activation were 9.35 (NC), 9.12 (VC), 6.38 (at 0.0006 mg/ml), 5.82 (at 0.0012 mg/ml), 4.14 (at 0.0024 mg/ml) and 8.12 (PC: 600 µg/mL EMS). In the presence (+S9) of metabolic activation, the mean mitotic index was 8.71 (NC), 8.31 (VC), 7.12 (at 0.0006 mg/ml), 5.38 (at 0.0012 mg/ml), 4.89 (at 0.0024 mg/ml) and 7.62 (PC: 30 µg/mL CPA). No significant increase in the percent of aberrant cells was observed in the vehicle control groups (Dimethyl sulfoxide) in Phase I and Phase II when compared to the negative control (distilled water). The increased frequency of aberrant cells observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system suitability of the methods and conditions employed in the experiment. Conclusion: The registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), is non-clastogenic in human peripheral lymphocytes both in the presence (1% and 2%) and in the absence of metabolic activation.
- 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
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Adopted on July 21st 1997, corrected June 26th 2020
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Purity: 99.06%
Manufacture Date: June 29, 2018
Expiry Date: June 29, 2023 - Target gene:
- Histidine gene
- 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:
- Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction derived from Aroclor 1254-injected rats.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to get a final concentration of approximately 10 % v/v in the S9 mix.
Cofactor solution contains the following quantity of chemicals in 500 mL of distilled water.
D-glucose-6-phosphate: 0.8 g
β-NADP: 1.75 g
MgCl2: 1.0 g
KCl: 1.35 g
Na2HPO4.H2O: 6.4 g
NaH2PO4.H2O: 1.4 g - Test concentrations with justification for top dose:
- Test concentrations: 0.0 (NC, Distilled water), 0.0 (VC, DMSO), 0.039, 0.078, 0.156, 0.313 and 0.625 ug/plate
Justification:
Test concentrations were selected based on a preliminary cytotoxicity test. In this pre-test, bacterial cells were exposed to test item concentrations of 0.0 (NC), 0.0 (VC), 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 µg/plate together with positive control substances. Complete inhibition of the bacterial background lawn was observed at ≥1.25 µg/plate, while at 0.625 µg/plate, moderate inhibition of the background lawn was noted in TA98 and TA100 testers both in the presence and absence of S9 metabolic activation. - Vehicle / solvent:
- Dimethyl sulphoxide was used as a vehicle.
- Untreated negative controls:
- yes
- Remarks:
- Distilled water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-o-phenylenediamine, 2-Aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: Two (Trial I-II)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1-2 x 109 cells/mL
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Trial I: plate incorporation, Trial II: preincubation
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min
- Exposure duration/duration of treatment: 48 hrs
- Harvest time after the end of treatment (sampling/recovery times): 48 hrs
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 reduction in the number of revertant counts and/or inhibition of the bacterial background lawn growth.
- Any supplementary information relevant to cytotoxicity:
A pre-experiment was performed with strains TA98 and TA100. Eight concentrations (i.e. 0.0 (negative control; NC), 0.0 (vehicle control; VC), 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate) with half-log intervals (√10) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in the pre-experiment were the same as described below for Trial I. - Evaluation criteria:
- The Substance is considered as a mutagen, if a biologically relevant increase in the 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 vehicle/solvent control is observed.
A dose-dependent increase is considered biologically relevant if the threshold of biological significance is exceeded at more than one concentration. An increase exceeding the threshold of biological significance at only one concentration was judged as biologically relevant if it is reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold of biological significance is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and vehicle control, the increase is not considered biologically relevant. - Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.
- 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:
- Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.
- 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:
- Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.
- 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:
- Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.
- 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 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Inhibition of the bacterial background lawn is noted at 0.625 ug/plate.
- 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: No data
- Data on osmolality: No data
- Possibility of evaporation from medium:NA
- Water solubility: Insoluble
- Precipitation and time of the determination: Precipitation was observed at 5 and 3.75 mg/plate concentrations, which was considered to interfere with the colony count. At 2.5 mg/plate concentration, slight precipitation was observed, which was considered to be non-interfering with colony count.
- Definition of acceptable cells for analysis: Regular background growth in the negative and solvent (vehicle) control.
- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES (if applicable):
STUDY RESULTS
- Concurrent vehicle negative and positive control data: Raw data is shown in section “Any other information on results incl, tables”.
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any: No data
- Any other criteria: e.g. GEF for MLA
Ames test:
- Signs of toxicity: Toxicity was noted as inhibition of the bacterial background lawn.
- Individual plate counts: Raw data is shown in section “Any other information on results incl, tables”.
- Mean number of revertant colonies per plate and standard deviation: Raw data is shown in section “Any other 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. Raw data is shown in section “Any other information on results incl, tables”.
- Negative (solvent/vehicle) historical control data: Available . Raw data is shown in section “Any other information on results incl, tables”. - Remarks on result:
- other: Non-mutagenic
- Conclusions:
- The registered substance, 2-methoxynapthalene (CAS No. 93-04-9), was tested non-mutagenic (negative) in a bacterial reverse mutation test both in the presence and absence of S9 metabolic activation system. The test was performed according to OECD TG 471 and GLP.
- Executive summary:
The mutagenic potential of the registered substance, i.e., 2-methoxynapthalene (CAS No. 93-04-9), was tested according to OECD TG 471 and GLP using Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. The test was performed in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction was used, and the S9 fraction was obtained from Aroclor 1254-induced rats. Test concentrations were selected based on the solubility, precipitation, and preliminary cytotoxicity tests. The Substance was insoluble in water and was soluble in Dimethyl sulphoxide up to 50 mg/ml. Insolubility was assessed as precipitation in the final mixture under the actual test conditions and was evident to the unaided eye. The test item was dissolved in DMSO at 50 mg/mL concentration and was checked for precipitation on agar. Precipitation was observed at 5 and 3.75 mg/plate concentrations, which was considered to interfere with the colony count. At 2.5 mg/plate concentration, slight precipitation was observed, which was considered non-interfering with colony count. Therefore 2.5 mg/plate was selected as the highest concentration for the pre-experiment. In the preliminary cytotoxicity test, bacterial cells of TA 98 and TA100 were exposed to test concentrations of 0.0 (NC, distilled water), 0.0 (VC, DMSO), 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 µg/plate in the presence and absence of S9 metabolic activation. Complete inhibition of the bacterial background lawn was observed at ≥1.25 µg/plate, while at 0.625 µg/plate, moderate inhibition of the background lawn was noted in TA98 and TA100 testers both in the presence and absence of S9 metabolic activation. Thus, the main test was performed with the flowing test item concentrations: 0.0 (VC), 0.0 (NC), 0.039, 0.078, 0.156, 0.313 and 0.625 µg/plate both in the presence and absence of S9 metabolic activation system using five Salmonella Typhimurium tester strains. For each strain and dose level, including negative, vehicle and positive controls, three plates (triplicate) were tested. The following positive control substances were used: sodium azide (TA 1535, TA100, without S9 mix), 4-Nitro-o-phenylenediamine (TA1537, TA98, without S9 mix), Methyl methanesulfonate (TA102 without S9), 2-Aminoanthracene (TA98, TA100, TA1535, TA1537 and TA102 wit S9 mix). Trial I was performed according to the plate incorporation method with five test concentrations along with the negative (Distilled water), vehicle (DMSO), and positive controls, and using three strains, i.e., TA1537, TA1535 and TA102.For TA98 and TA100 tester strains, the revertant colony counts were directly incorporated in Trial I from the pre-experiment up to the five concentrations (0.625 mg/plate to 0.039 mg/plate). Trial II was performed according to the preincubation method with all five tester strains along with the negative, vehicle, and positive controls. Results: No significant increase in the number of revertant colonies was observed following the treatment with the test substance up to the concentration of 0.625 µg/plate either in the presence or absence of S9 metabolic activation in all five tester strains in both trials (Trial I-II) when compared to the vehicle control. No trend of an increased number of revertant colonies with increased dosing of the test item was observed. Each strain-specific positive control showed a significant increase in the number of revertant colonies both in the presence and absence of metabolic activation, thus confirming the validity of the assay. Conclusion: The registered substance, i.e., 2-methoxynapthalene (CAS No. 93-04-9), did not induce gene mutation within the histidine operon by base-pair exchange or frameshift in Salmonella Typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) either in the presence or absence of S9 metabolic activation system.
Referenceopen allclose all
Table 1: Mitotix index - Cytotoxicity experiment III
Treatment | R | Mitotic Index (%) | |||||||
Absence of Metabolic Activation (-S9) | Presence of Metabolic Activation (1% S9) | ||||||||
Mitotic Index | Mean | SD | Percent Reduction vs. NC | Mitotic Index | Mean | SD | Percent Reduction vs. NC | ||
NC (0.0 mg/mL) | R1 | 9.89 | 9.73 | 0.23 | - | 9.85 | 9.55 | 0.43 | - |
R2 | 9.57 | 9.24 | |||||||
VC (0.0 mg/mL) | R1 | 9.66 | 9.62 | 0.06 | - | 9.75 | 9.55 | 0.28 | - |
R2 | 9.58 | 9.35 | |||||||
T7 | R1 | 6.69 | 6.43 | 0.36 | 33.13 | 6.29 | 6.53 | 0.34 | 31.67 |
R2 | 6.18 | 6.77 | |||||||
T8 | R1 | 5.38 | 5.58 | 0.28 | 42.00 | 5.18 | 5.33 | 0.22 | 44.16 |
R2 | 5.78 | 5.49 | |||||||
T9 | R1 | 3.89 | 3.98 | 0.14 | 58.59 | 4.30 | 4.20 | 0.14 | 56.08 |
R2 | 4.08 | 4.10 | |||||||
PC | R1 | 7.99 | 8.14 | 0.21 | 15.37 | 8.48 | 8.24 | 0.35 | 13.76 |
R2 | 8.29 | 7.99 |
Table 2: Summary of mitotic index
Mitotic Index (%) | |||||||
Phase I | |||||||
Treatment | Absence of Metabolic Activation (-S9) | Treatment | Presence of Metabolic Activation (+S9) (1%) | ||||
Mean | SD | Percentage reduction vs. VC | Mean | SD | Percentage reduction vs. VC | ||
NC | 9.87 | 0.13 | - | NC | 9.53 | 0.20 | - |
VC | 9.66 | 0.28 | 2.12 | VC | 8.66 | 0.56 | 9.17 |
T1 (0.0006 mg/mL) | 6.87 | 0.14 | 28.94 | T1 (0.0006 mg/mL) | 5.59 | 0.00 | 35.41 |
T2 (0.0012 mg/mL) | 5.57 | 0.14 | 42.35 | T2 (0.0012 mg/mL) | 4.92 | 0.36 | 43.13 |
T3 (0.0024 mg/mL) | 4.28 | 0.13 | 55.74 | T3 (0.0024 mg/mL) | 4.14 | 0.08 | 52.16 |
PC | 8.37 | 0.58 | 13.40 | PC | 7.69 | 0.56 | 11.19 |
Mitotic Index (%) | |||||||
Phase II | |||||||
Treatment | Absence of Metabolic Activation (-S9) | Treatment | Presence of Metabolic Activation (+S9) (2%) | ||||
Mean | SD | Percentage reduction vs. VC | Mean | SD | Percentage reduction vs. VC | ||
NC | 9.35 | 0.15 | - | NC | 8.71 | 0.06 | - |
VC | 9.12 | 0.35 | 2.42 | VC | 8.31 | 0.06 | 4.52 |
T1 (0.0006 mg/mL) | 6.38 | 0.15 | 30.05 | T1 (0.0006 mg/mL) | 7.12 | 0.35 | 14.29 |
T2 (0.0012 mg/mL) | 5.82 | 0.07 | 36.16 | T2 (0.0012 mg/mL) | 5.38 | 0.29 | 35.26 |
T3 (0.0024 mg/mL) | 4.14 | 0.07 | 54.67 | T3 (0.0024 mg/mL) | 4.89 | 0.44 | 41.22 |
PC | 8.12 | 0.1 | 10.97 | PC | 7.62 | 0.21 | 8.34 |
Table 3: 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 | 0.333 | 0.471 | VC | 0.333 | 0.471 |
T1 (0.0006 mg/mL) | 0.667 | 0.943 | T1 (0.0006 mg/mL) | 0.000 | 0.000 |
T2 (0.0012 mg/mL) | 0.667 | 0.000 | T2 (0.0012 mg/mL) | 0.333 | 0.471 |
T3 (0.0024 mg/mL) | 0.667 | 0.000 | T3 (0.0024 mg/mL) | 0.667 | 0.000 |
PC | 11.000 | 1.414 | PC | 10.333 | 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 | 0.667 | 0.000 |
VC | 0.333 | 0.471 | VC | 0.333 | 0.471 |
T1 (0.0006 mg/mL) | 0.667 | 0.943 | T1 (0.0006 mg/mL) | 0.000 | 0.000 |
T2 (0.0012 mg/mL) | 0.333 | 0.471 | T2 (0.0012 mg/mL) | 1.000 | 0.471 |
T3 (0.0024 mg/mL) | 0.333 | 0.471 | T3 (0.0024 mg/mL) | 0.333 | 0.471 |
PC | 10.667 | 0.943 | PC | 11.000 | 1.414 |
Table 4: Induvidual observations for slides for mitotic index and chromosme aberrations
Phase I [In the Presence of Metabolic Activation, (1% S9)]
Treatment | Culture No. | Mitotic Index | Frequencies of Aberration | Total No. of Aberration | Number of aberrated cells | Percentage of Aberrated Cells |
NC | R1 | 9.67 | 1 cse | 1 | 1 | 0.67 |
R2 | 9.39 | - | 0 | 0 | 0.00 | |
VC | R1 | 8.26 | - | 0 | 0 | 0.00 |
R2 | 9.05 | 1 cte, 1 csb | 2 | 1 | 0.67 | |
T1 (0.0006 mg/mL) | R1 | 5.59 | - | 0 | 0 | 0.00 |
R2 | 5.59 | - | 0 | 0 | 0.00 | |
T2 (0.0012 mg/mL) | R1 | 4.67 | 1 cse | 1 | 1 | 0.67 |
R2 | 5.17 | - | 0 | 0 | 0.00 | |
T3 (0.0024 mg/mL) | R1 | 4.20 | 1 ctb, 1 cte | 2 | 1 | 0.67 |
R2 | 4.09 | 1 cse | 1 | 1 | 0.67 | |
PC | R1 | 7.29 | 5 ctb, 5 cte, 2 ctg, 5 csb, 5 cse, 2 AC, 04 fragments | 26 | 17 | 11.33 |
R2 | 8.08 | 4 ctb, 4 cte, 3 ctg, 4 csb, 3 cse, 2 csg, 2 DC, 2 AC, 06 fragments | 25 | 14 | 9.33 |
Phase I [In the Absence of Metabolic Activation, (-S9)]
Treatment | Culture No. | Mitotic Index | Frequencies of Aberration | Total No. of Aberration | Number of aberrated cells | Percentage of Aberrated Cells |
NC | R1 | 9.96 | - | 0 | 0 | 0.00 |
R2 | 9.78 | 1 cse | 1 | 1 | 0.67 | |
VC | R1 | 9.46 | 1 csb | 1 | 1 | 0.67 |
R2 | 9.86 | - | 0 | 0 | 0.00 | |
T1 (0.0006 mg/mL) | R1 | 6.97 | - | 0 | 0 | 0.00 |
R2 | 6.77 | 1 ctb, 1cse | 2 | 2 | 1.33 | |
T2 (0.0012 mg/mL) | R1 | 5.47 | 1 cse | 1 | 1 | 0.67 |
R2 | 5.67 | 1 csb | 1 | 1 | 0.67 | |
T3 (0.0024 mg/mL) | R1 | 4.18 | 1 cse | 1 | 1 | 0.67 |
R2 | 4.37 | 1 cte, 1 csb | 2 | 1 | 0.67 | |
PC | R1 | 8.77 | 6 ctb, 8 cte, 4 csb, 4 cse, 1 csg, 3 AC, 01 fragments | 26 | 18 | 12.00 |
R2 | 7.96 | 4 ctb, 5 cte, 3 csb, 3 cse, 3 csg, 3 AC, 04 fragments | 22 | 15 | 10.00 |
Phase II [In the Absence of Metabolic Activation (-S9)]
Treatment | Culture No. | Mitotic Index | Frequencies of Aberration | Total No. of Aberration | Number of aberrated cells | Percentage of Aberrated Cells |
NC | R1 | 9.24 | 1cse | 1 | 1 | 0.67 |
R2 | 9.45 | - | 0 | 0 | 0.00 | |
VC | R1 | 8.87 | - | 0 | 0 | 0.00 |
R2 | 9.37 | 1 cte | 1 | 1 | 0.67 | |
T1 (0.0006 mg/mL) | R1 | 6.27 | - | 0 | 0 | 0.00 |
R2 | 6.49 | 1 ctb, 1 cse | 2 | 2 | 1.33 | |
T2 (0.0012 mg/mL) | R1 | 5.77 | 1 cse | 1 | 1 | 0.67 |
R2 | 5.88 | - | 0 | 0 | 0.00 | |
T3 (0.0024 mg/mL) | R1 | 4.19 | - | 0 | 0 | 0.00 |
R2 | 4.08 | 1 cte | 1 | 1 | 0.67 | |
PC | R1 | 8.19 | 5 ctb, 6 cte, 2 ctg, 5 csb, 4 cse, 1DC, 3 AC, 04 fragments | 28 | 17 | 11.33 |
R2 | 8.05 | 4 ctb, 5 cte, 4 csb, 3 cse, 1 csg, 1 DC, 3 AC, 02 fragments | 22 | 15 | 10.00 |
Phase II [In the Presence of Metabolic Activation (2% S9)]
Treatment | Culture No. | Mitotic Index | Frequencies of Aberration | Total No. of Aberration | Number of aberrated cells | Percentage of Aberrated Cells |
NC | R1 | 8.75 | 1 csb | 1 | 1 | 0.67 |
R2 | 8.67 | 1 cse | 1 | 1 | 0.67 | |
VC | R1 | 8.36 | - | 0 | 0 | 0.00 |
R2 | 8.27 | 1 csb | 1 | 1 | 0.67 | |
T1 (0.0006 mg/mL) | R1 | 6.88 | - | 0 | 0 | 0.00 |
R2 | 7.37 | - | 0 | 0 | 0.00 | |
T2 (0.0012 mg/mL) | R1 | 5.59 | 1 cse | 1 | 1 | 0.67 |
R2 | 5.17 | 1 cte, 1 csb | 2 | 2 | 1.33 | |
T3 (0.0024 mg/mL) | R1 | 4.58 | 1 cte | 1 | 1 | 0.67 |
R2 | 5.19 | - | 0 | 0 | 0.00 | |
PC | R1 | 7.77 | 6 ctb, 4 cte, 3 ctg, 4 csb, 3 cse, 2 csg, 3 AC, 09 fragments | 29 | 18 | 12.00 |
R2 | 7.47 | 5 ctb, 4 cte, 2 ctg, 5 csb, 4 cse, 1 csg , 2 AC, 06 fragments | 26 | 15 | 10.00 |
Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric, AC = Acentric, 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 | Dimethyl sulfoxide | 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 |
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 | 10.000 | 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 | 10.000 | 0.667 | 9.667 | 0.333 | 10.000 |
29 | 7574 | Dimethyl sulfoxide | 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 | 0.333 | 10.667 | 0.333 | 9.667 |
33 | 8072 | DMSO | 0.333 | 10.333 | 0.333 | 10.000 | 0.333 | 10.333 | 0.333 | 10.333 |
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 | |||
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 |
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 | 8658 | DMSO | 0.333 | 10.000 | 0.333 | 11.000 | 0.333 | 10.667 | 0.667 | 10.000 |
49 | 9845 | DMSO | 0.333 | 10.000 | 0.333 | 11.000 | 0.333 | 10.667 | 0.333 | 10.667 |
50 | 9861 | DMSO | 0.333 | 10.667 | 0.333 | 10.333 | 0.333 | 10.333 | 0.667 | 10.000 |
51 | 9862 | DMSO | 0.667 | 10.000 | 0.333 | 9.000 | 0.333 | 10.000 | 0.333 | 10.667 |
52 | 9911 | DMSO | 0.333 | 10.667 | 0.333 | 11.333 | 0.333 | 9.333 | 0.333 | 10.000 |
53 | 9925 | DMSO | 0.333 | 11.333 | 0.333 | 11.000 | 0.333 | 11.333 | 0.333 | 11.000 |
54 | 10049 | DMSO | 0.333 | 10.000 | 0.333 | 11.000 | 0.333 | 9.667 | 0.667 | 11.000 |
55 | 9939 | DMSO | 0.333 | 9.667 | 0.333 | 11.000 | 0.333 | 8.000 | 0.333 | 9.000 |
56 | 10679 | DMSO | 0.333 | 11.667 | 0.333 | 10.667 | 0.333 | 13.333 | 0.333 | 15.000 |
57 | 10807 | DMSO | 0.333 | 10.333 | 0.667 | 11.000 | 0.333 | 9.000 | 0.333 | 10.667 |
58 | 10858 | RPMI | 0.667 | 10.333 | 0.667 | 11.000 | 0.333 | 11.000 | 0.333 | 10.667 |
59 | 10859 | DMSO | 0.333 | 10.667 | 0.667 | 10.000 | 0.667 | 10.333 | 0.333 | 10.000 |
Mean | 0.395 | 9.887 | 0.384 | 10.0012 | 0.411 | 9.955 | 0.384 | 10.082 | ||
SD | 0.275 | 0.938 | 0.242 | 0.998 | 0.254 | 1.071 | 0.213 | 1.114 | ||
Mean + 2SD | 0.945 | 11.764 | 0.868 | 12.005 | 0.919 | 12.096 | 0.810 | 12.310 | ||
Mean - 2SD | -0.155 | 8.010 | -0.100 | 8.012 | -0.098 | 7.813 | -0.043 | 7.854 |
Table 1: Revertant counts in the preliminary cytotoxicity test
Dose (mg/plate) | R | Absence of Metabolic Activation (-S9) | Presence of Metabolic Activation (+S9) | ||
TA100 | TA 98 | TA100 | TA 98 | ||
NC (0.00) | R1 | 106 | 21 | 120 | 20 |
R2 | 112 | 18 | 106 | 22 | |
R3 | 114 | 20 | 108 | 20 | |
VC (0.00) | R1 | 118 | 24 | 116 | 26 |
R2 | 126 | 23 | 120 | 24 | |
R3 | 112 | 25 | 124 | 25 | |
T1 (0.020) | R1 | 110 | 24 | 120 | 22 |
R2 | 118 | 21 | 114 | 25 | |
R3 | 112 | 22 | 116 | 23 | |
T2 (0.039) | R1 | 120 | 23 | 112 | 25 |
R2 | 106 | 22 | 118 | 21 | |
R3 | 118 | 24 | 108 | 22 | |
T3 (0.078) | R1 | 108 | 24 | 112 | 24 |
R2 | 118 | 20 | 110 | 23 | |
R3 | 114 | 21 | 120 | 20 | |
T4 (0.156) | R1 | 116 | 22 | 108 | 25 |
R2 | 104 | 19 | 110 | 23 | |
R3 | 118 | 25 | 118 | 24 | |
T5 (0.313) | R1 | 116 | 24 | 120 | 22 |
R2 | 112 | 26 | 110 | 20 | |
R3 | 114 | 20 | 116 | 24 | |
T6 (0.625) | R1 | 108 (+++) | 22 (+++) | 112 (+++) | 23 (+++) |
R2 | 114 (+++) | 23 (+++) | 122 (+++) | 25 (+++) | |
R3 | 112 (+++) | 18 (+++) | 110 (+++) | 25 (+++) | |
T7 (1.25) | R1 | 116 (++) | 24 (++) | 112 (++) | 22 (++) |
R2 | 112 (++) | 21 (++) | 120 (++) | 23 (++) | |
R3 | 110 (++) | 23 (++) | 116 (++) | 24 (++) | |
T8 (2.5) | R1 | 118 (++) | 23 (++) | 112 (++) | 24 (++) |
R2 | 106 (++) | 20 (++) | 118 (++) | 23 (++) | |
R3 | 112 (++) | 21 (++) | 110 (++) | 24 (++) | |
PC | R1 | 1280 | 816 | 1328 | 1288 |
R2 | 1160 | 912 | 1240 | 1456 | |
R3 | 1304 | 848 | 1376 | 1360 |
NC=Negative control; VC = Vehicle Control; PC=Positive control
R = Replicate, T= Test concentration (T8: Highest, T1: Lowest)
Background Lawn: (+++) = Moderate Inhibition; (++) = Complete Inhibition
4-Nitro-o-phenylenediamine [10μg/plate]: TA 98, Sodium azide [10μg/plate]: TA 100,
2-Aminoanthracene [2.5μg/plate]: TA 98, TA 100
Table 2: Revertant counts in Trial I (Plate incorporation assay)
Dose (mg/plate) | R | Presence of Metabolic Activation (+S9) | ||||
TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | ||
NC (0.00) | R1 | 5 | 12 | 20 | 120 | 240 |
R2 | 4 | 11 | 22 | 106 | 232 | |
R3 | 5 | 15 | 20 | 108 | 244 | |
VC. (0.00) | R1 | 6 | 14 | 26 | 116 | 264 |
R2 | 6 | 17 | 24 | 120 | 252 | |
R3 | 7 | 16 | 25 | 124 | 256 | |
T1 (0.039) | R1 | 5 | 15 | 25 | 112 | 256 |
R2 | 6 | 16 | 21 | 118 | 240 | |
R3 | 4 | 13 | 22 | 108 | 236 | |
T2 (0.078) | R1 | 6 | 14 | 24 | 112 | 232 |
R2 | 7 | 15 | 23 | 110 | 256 | |
R3 | 5 | 14 | 20 | 120 | 248 | |
T3 (0.156) | R1 | 5 | 13 | 25 | 108 | 236 |
R2 | 6 | 12 | 23 | 110 | 256 | |
R3 | 5 | 16 | 24 | 118 | 232 | |
T4 (0.313) | R1 | 4 | 13 | 22 | 120 | 244 |
R2 | 7 | 11 | 20 | 110 | 232 | |
R3 | 6 | 15 | 24 | 116 | 252 | |
T5 (0.625) | R1 | 5 | 13 | 23 | 112 | 232 |
R2 | 3 | 13 | 25 | 122 | 252 | |
R3 | 6 | 14 | 25 | 110 | 236 | |
PC | R1 | 164 | 368 | 1288 | 1328 | 1440 |
R2 | 172 | 328 | 1456 | 1240 | 1560 | |
R3 | 156 | 448 | 1360 | 1376 | 1392 |
Dose (mg/plate) | R | Absence of Metabolic Activation (-S9) | ||||
TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | ||
NC (0.00) | R1 | 4 | 14 | 21 | 106 | 232 |
R2 | 4 | 10 | 18 | 112 | 224 | |
R3 | 3 | 12 | 20 | 114 | 248 | |
VC. (0.00) | R1 | 5 | 15 | 24 | 118 | 256 |
R2 | 7 | 13 | 23 | 126 | 260 | |
R3 | 5 | 16 | 25 | 112 | 240 | |
T1 (0.039) | R1 | 5 | 15 | 23 | 120 | 240 |
R2 | 5 | 12 | 22 | 106 | 236 | |
R3 | 4 | 13 | 24 | 118 | 252 | |
T2 (0.078) | R1 | 5 | 13 | 24 | 108 | 232 |
R2 | 5 | 15 | 20 | 118 | 248 | |
R3 | 6 | 11 | 21 | 114 | 236 | |
T3 (0.156) | R1 | 5 | 14 | 22 | 116 | 244 |
R2 | 4 | 15 | 19 | 104 | 236 | |
R3 | 4 | 12 | 25 | 118 | 232 | |
T4 (0.313) | R1 | 6 | 12 | 24 | 116 | 248 |
R2 | 4 | 13 | 26 | 112 | 240 | |
R3 | 5 | 12 | 20 | 114 | 236 | |
T5 (0.625) | R1 | 5 | 14 | 22 | 108 | 232 |
R2 | 3 | 13 | 23 | 114 | 248 | |
R3 | 4 | 11 | 18 | 112 | 228 | |
PC | R1 | 184 | 1260 | 816 | 1280 | 1536 |
R2 | 160 | 1140 | 912 | 1160 | 1404 | |
R3 | 176 | 1284 | 848 | 1304 | 1608 |
NC = Negative Control, VC = Vehicle Control, T = Test concentration (T5: Highest, T1: Lowest), R = Replicate, PC = Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100, 2- Aminoanthracene [10μg/plate]:TA 102, Sodium azide [10μg/plate]: TA 1535, TA 100, 4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate], Methyl methanesulfonate [4μl/plate]: TA 102.
Table 3: Revertant counts in Trial II (Preincubation assay)
Dose (mg/plate) | R | In the Presence of Metabolic Activation (+S9) | ||||
TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | ||
NC (0.00) | R1 | 4 | 12 | 24 | 106 | 240 |
R2 | 5 | 14 | 21 | 116 | 236 | |
R3 | 4 | 11 | 22 | 112 | 248 | |
VC. (0.00) | R1 | 7 | 15 | 27 | 124 | 272 |
R2 | 6 | 13 | 28 | 118 | 256 | |
R3 | 6 | 17 | 24 | 120 | 260 | |
T1 (0.039) | R1 | 5 | 13 | 26 | 110 | 256 |
R2 | 7 | 15 | 24 | 120 | 240 | |
R3 | 5 | 14 | 23 | 116 | 248 | |
T2 (0.078) | R1 | 6 | 13 | 25 | 108 | 252 |
R2 | 4 | 16 | 21 | 116 | 260 | |
R3 | 6 | 12 | 24 | 114 | 244 | |
T3 (0.156) | R1 | 5 | 15 | 24 | 114 | 256 |
R2 | 4 | 13 | 25 | 110 | 240 | |
R3 | 6 | 16 | 23 | 120 | 248 | |
T4 (0.313) | R1 | 5 | 13 | 27 | 114 | 236 |
R2 | 5 | 16 | 23 | 108 | 252 | |
R3 | 4 | 14 | 24 | 118 | 244 | |
T5 (0.625) | R1 | 4 | 13 | 22 | 110 | 248 |
R2 | 5 | 14 | 24 | 114 | 256 | |
R3 | 4 | 12 | 23 | 118 | 236 | |
PC | R1 | 180 | 424 | 1296 | 1452 | 1344 |
R2 | 204 | 320 | 1392 | 1512 | 1488 | |
R3 | 168 | 368 | 1356 | 1332 | 1416 |
Dose (mg/plate) | R | In the Absence of Metabolic Activation (-S9) | ||||
TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | ||
NC (0.00) | R1 | 5 | 13 | 19 | 110 | 240 |
R2 | 3 | 13 | 22 | 102 | 228 | |
R3 | 4 | 12 | 21 | 108 | 232 | |
VC. (0.00) | R1 | 6 | 16 | 23 | 116 | 260 |
R2 | 7 | 13 | 26 | 122 | 244 | |
R3 | 5 | 15 | 25 | 120 | 252 | |
T1 (0.039) | R1 | 5 | 14 | 22 | 110 | 248 |
R2 | 5 | 15 | 26 | 116 | 240 | |
R3 | 5 | 12 | 24 | 114 | 252 | |
T2 (0.078) | R1 | 7 | 16 | 23 | 106 | 236 |
R2 | 5 | 12 | 21 | 112 | 240 | |
R3 | 5 | 14 | 25 | 118 | 244 | |
T3 (0.156) | R1 | 5 | 14 | 21 | 116 | 244 |
R2 | 4 | 13 | 24 | 108 | 232 | |
R3 | 4 | 16 | 23 | 114 | 248 | |
T4 (0.313) | R1 | 4 | 15 | 24 | 112 | 236 |
R2 | 3 | 11 | 22 | 106 | 248 | |
R3 | 5 | 13 | 27 | 114 | 252 | |
T5 (0.625) | R1 | 4 | 15 | 22 | 114 | 248 |
R2 | 6 | 12 | 25 | 108 | 240 | |
R3 | 4 | 13 | 23 | 112 | 236 | |
PC | R1 | 156 | 1152 | 852 | 1380 | 1404 |
R2 | 184 | 1380 | 708 | 1284 | 1608 | |
R3 | 172 | 1284 | 972 | 1308 | 1452 |
NC = Negative Control, VC = Vehicle Control, T = Test concentration (T5: Highest, T1: Lowest), R= Replicate
PC = Positive control 2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100,
2-Aminoanthracene [10μg/plate]:TA 102, Sodium azide [10μg/plate]: TA 1535, TA 100,
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate], Methyl methanesulfonate [4μl/plate]: TA 102.
Table 4: The mean revertant counts in Trial I (Plate incorporation assay)
Dose (mg/plate) | In the presence of Metabolic Activation (+S9) | |||||||||
TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | ||||||
MEAN | SD | MEAN | SD | MEAN | SD | MEAN | SD | MEAN | SD | |
NC (0.00) | 4.67 | 0.58 | 12.67 | 2.08 | 20.67 | 1.15 | 111.33 | 7.57 | 238.67 | 6.11 |
VC (0.00) | 6.33 | 0.58 | 15.67 | 1.53 | 25.00 | 1.00 | 120.00 | 4.00 | 257.33 | 6.11 |
T1 (0.039) | 5.00 | 1.00 | 14.67 | 1.53 | 22.67 | 2.08 | 112.67 | 5.03 | 244.00 | 10.58 |
T2 (0.078) | 6.00 | 1.00 | 14.33 | 0.58 | 22.33 | 2.08 | 114.00 | 5.29 | 245.33 | 12.22 |
T3 (0.156) | 5.33 | 0.58 | 13.67 | 2.08 | 24.00 | 1.00 | 112.00 | 5.29 | 241.33 | 12.86 |
T4 (0.313) | 5.67 | 1.53 | 13.00 | 2.00 | 22.00 | 2.00 | 115.33 | 5.03 | 242.67 | 10.07 |
T5 (0.625) | 4.67 | 1.53 | 13.33 | 0.58 | 24.33 | 1.15 | 114.67 | 6.43 | 240.00 | 10.58 |
PC | 164.00 | 8.00 | 381.33 | 61.10 | 1368.00 | 84.29 | 1314.67 | 68.97 | 1464.00 | 86.53 |
Dose (mg/plate) | In the Absence of Metabolic Activation (-S9) | |||||||||
TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | ||||||
MEAN | SD | MEAN | SD | MEAN | SD | MEAN | SD | MEAN | SD | |
NC (0.00) | 3.67 | 0.58 | 12.00 | 2.00 | 19.67 | 1.53 | 110.67 | 4.16 | 234.67 | 12.22 |
VC (0.00) | 5.67 | 1.15 | 14.67 | 1.53 | 24.00 | 1.00 | 118.67 | 7.02 | 252.00 | 10.58 |
T1 (0.039) | 4.67 | 0.58 | 13.33 | 1.53 | 23.00 | 1.00 | 114.67 | 7.57 | 242.67 | 8.33 |
T2 (0.078) | 5.33 | 0.58 | 13.00 | 2.00 | 21.67 | 2.08 | 113.33 | 5.03 | 238.67 | 8.33 |
T3 (0.156) | 4.33 | 0.58 | 13.67 | 1.53 | 22.00 | 3.00 | 112.67 | 7.57 | 237.33 | 6.11 |
T4 (0.313) | 5.00 | 1.00 | 12.33 | 0.58 | 23.33 | 3.06 | 114.00 | 2.00 | 241.33 | 6.11 |
T5 (0.625) | 4.00 | 1.00 | 12.67 | 1.53 | 21.00 | 2.65 | 111.33 | 3.06 | 236.00 | 10.58 |
PC | 173.33 | 12.22 | 1228.00 | 77.15 | 858.67 | 48.88 | 1248.00 | 77.15 | 1516.00 | 103.46 |
NC = Negative Control, VC = Vehicle Control, T = Test concentration (T5: Highest, T1: Lowest), SD = Standard Deviation
PC = Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100
Methyl methanesulfonate [4μl/plate]: TA 102
2-Aminoanthracene [10μg/plate]:TA 102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]
Table 5: The mean revertant counts in Trial II (preincubation assay)
Dose (mg/plate) | Presence of Metabolic Activation (+S9) | |||||||||
TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | ||||||
MEAN | SD | MEAN | SD | MEAN | SD | MEAN | SD | MEAN | SD | |
NC (0.00) | 4.33 | 0.58 | 12.33 | 1.53 | 22.33 | 1.53 | 111.33 | 5.03 | 241.33 | 6.11 |
VC (0.00) | 6.33 | 0.58 | 15.00 | 2.00 | 26.33 | 2.08 | 120.67 | 3.06 | 262.67 | 8.33 |
T1 (0.039) | 5.67 | 1.15 | 14.00 | 1.00 | 24.33 | 1.53 | 115.33 | 5.03 | 248.00 | 8.00 |
T2 (0.078) | 5.33 | 1.15 | 13.67 | 2.08 | 23.33 | 2.08 | 112.67 | 4.16 | 252.00 | 8.00 |
T3 (0.156) | 5.00 | 1.00 | 14.67 | 1.53 | 24.00 | 1.00 | 114.67 | 5.03 | 248.00 | 8.00 |
T4 (0.313) | 4.67 | 0.58 | 14.33 | 1.53 | 24.67 | 2.08 | 113.33 | 5.03 | 244.00 | 8.00 |
T5 (0.625) | 4.33 | 0.58 | 13.00 | 1.00 | 23.00 | 1.00 | 114.00 | 4.00 | 246.67 | 10.07 |
PC | 184.00 | 18.33 | 370.67 | 52.05 | 1348.00 | 48.50 | 1432.00 | 91.65 | 1416.00 | 72.00 |
Dose (mg/plate) | Absence of Metabolic Activation (-S9) | |||||||||
TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | ||||||
MEAN | SD | MEAN | SD | MEAN | SD | MEAN | SD | MEAN | SD | |
NC (0.00) | 4.00 | 1.00 | 12.67 | 0.58 | 20.67 | 1.53 | 106.67 | 4.16 | 233.33 | 6.11 |
VC (0.00) | 6.00 | 1.00 | 14.67 | 1.53 | 24.67 | 1.53 | 119.33 | 3.06 | 252.00 | 8.00 |
T1 (0.039) | 5.00 | 0.00 | 13.67 | 1.53 | 24.00 | 2.00 | 113.33 | 3.06 | 246.67 | 6.11 |
T2 (0.078) | 5.67 | 1.15 | 14.00 | 2.00 | 23.00 | 2.00 | 112.00 | 6.00 | 240.00 | 4.00 |
T3 (0.156) | 4.33 | 0.58 | 14.33 | 1.53 | 22.67 | 1.53 | 112.67 | 4.16 | 241.33 | 8.33 |
T4 (0.313) | 4.00 | 1.00 | 13.00 | 2.00 | 24.33 | 2.52 | 110.67 | 4.16 | 245.33 | 8.33 |
T5 (0.625) | 4.67 | 1.15 | 13.33 | 1.53 | 23.33 | 1.53 | 111.33 | 3.06 | 241.33 | 6.11 |
PC | 170.67 | 14.05 | 1272.00 | 114.47 | 844.00 | 132.18 | 1324.00 | 49.96 | 1488.00 | 106.66 |
NC = Negative Control, VC = Vehicle Control, T = Test concentration (T5: Highest, T1: Lowest), SD = Standard Deviation
PC = Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100
2-Aminoanthracene [10μg/plate]: TA 102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537 [50μg/plate] TA 98 [10μg/plate]
Methyl methanesulfonate: [4μl/plate]: TA 102
Table 6: Historical control data
Trial I (Plate Incorporation Method) | ||||||
Strains | Metabolic Activation | Treatment | Mean | SD | Max | Min |
TA 1537 | S9 + | Negative control | 6 | 2 | 10 | 2 |
S9 - | 6 | 2 | 10 | 2 | ||
S9 + | Solvent control | 6 | 2 | 10 | 2 | |
S9 - | 6 | 2 | 10 | 2 | ||
S9 + | Positive control | 168 | 38 | 245 | 92 | |
S9 - | 175 | 43 | 261 | 89 | ||
TA 1535 | S9 + | Negative control | 12 | 3 | 18 | 7 |
S9 - | 12 | 3 | 18 | 7 | ||
S9 + | Solvent control | 13 | 3 | 18 | 7 | |
S9 - | 13 | 3 | 18 | 7 | ||
S9 + | Positive control | 336 | 211 | 757 | 86 | |
S9 - | 1200 | 263 | 1726 | 674 | ||
TA 98 | S9 + | Negative control | 24 | 6 | 36 | 11 |
S9 - | 23 | 6 | 35 | 11 | ||
S9 + | Solvent control | 25 | 6 | 37 | 13 | |
S9 - | 23 | 5 | 33 | 13 | ||
S9 + | Positive control | 1099 | 312 | 1722 | 476 | |
S9 - | 815 | 284 | 1383 | 248 | ||
TA 100 | S9 + | Negative control | 117 | 28 | 173 | 61 |
S9 - | 114 | 26 | 166 | 62 | ||
S9 + | Solvent control | 116 | 28 | 172 | 60 | |
S9 - | 113 | 26 | 165 | 61 | ||
S9 + | Positive control | 1488 | 390 | 2268 | 709 | |
S9 - | 1311 | 298 | 1906 | 715 | ||
TA 102 | S9 + | Negative control | 274 | 42 | 358 | 190 |
S9 - | 271 | 55 | 382 | 161 | ||
S9 + | Solvent control | 279 | 65 | 409 | 150 | |
S9 - | 277 | 82 | 442 | 112 | ||
S9 + | Positive control | 1648 | 305 | 2258 | 1037 | |
S9 - | 1896 | 364 | 2624 | 1168 |
Trial II (Pre-Incubation Method) | ||||||
Strains | Metabolic Activation | Treatment | Mean | SD | Max | Min |
TA 1537 | S9 + | Negative control | 6 | 2 | 10 | 2 |
S9 - | 6 | 2 | 10 | 2 | ||
S9 + | Solvent control | 6 | 2 | 10 | 3 | |
S9 - | 6 | 2 | 10 | 2 | ||
S9 + | Positive control | 170 | 39 | 249 | 91 | |
S9 - | 182 | 43 | 268 | 96 | ||
TA 1535 | S9 + | Negative control | 13 | 3 | 18 | 7 |
S9 - | 12 | 3 | 18 | 7 | ||
S9 + | Solvent control | 13 | 3 | 18 | 8 | |
S9 - | 13 | 3 | 18 | 7 | ||
S9 + | Positive control | 299 | 197 | 694 | 145 | |
S9 - | 1244 | 260 | 1765 | 724 | ||
TA 98 | S9 + | Negative control | 24 | 6 | 35 | 13 |
S9 - | 23 | 5 | 33 | 13 | ||
S9 + | Solvent control | 24 | 5 | 35 | 14 | |
S9 - | 23 | 5 | 32 | 14 | ||
S9 + | Positive control | 1269 | 275 | 1819 | 719 | |
S9 - | 740 | 210 | 1160 | 320 | ||
TA 100 | S9 + | Negative control | 117 | 25 | 166 | 67 |
S9 - | 113 | 23 | 159 | 66 | ||
S9 + | Solvent control | 116 | 22 | 159 | 73 | |
S9 - | 112 | 20 | 151 | 73 | ||
S9 + | Positive control | 1469 | 347 | 2163 | 775 | |
S9 - | 1352 | 263 | 1878 | 827 | ||
TA 102 | S9 + | Negative control | 281 | 32 | 345 | 218 |
S9 - | 276 | 28 | 331 | 220 | ||
S9 + | Solvent control | 281 | 34 | 350 | 212 | |
S9 - | 276 | 34 | 344 | 207 | ||
S9 + | Positive control | 1595 | 287 | 2168 | 1022 | |
S9 - | 1753 | 248 | 2248 | 1258 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Genetic Toxicity / In Vitro:
Bacterial reverse mutation test:
The mutagenic potential of the registered substance, i.e., 2-methoxynapthalene (CAS No. 93-04-9), was tested according to OECD TG 471 and GLP using Salmonella Typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. The test was performed in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction was used, and the S9 fraction was obtained from Aroclor 1254-induced rats. Test concentrations were selected based on the solubility, precipitation, and preliminary cytotoxicity tests. The Substance was insoluble in water and was soluble in Dimethyl sulphoxide up to 50 mg/ml. Insolubility was assessed as precipitation in the final mixture under the actual test conditions and was evident to the unaided eye. The test item was dissolved in DMSO at 50 mg/mL concentration and was checked for precipitation on agar. Precipitation was observed at 5 and 3.75 mg/plate concentrations, which was considered to interfere with the colony count. At 2.5 mg/plate concentration, slight precipitation was observed, which was considered non-interfering with colony count. Therefore 2.5 mg/plate was selected as the highest concentration for the pre-experiment. In the preliminary cytotoxicity test, bacterial cells of TA 98 and TA100 were exposed to test concentrations of 0.0 (NC, distilled water), 0.0 (VC, DMSO), 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 µg/plate in the presence and absence of S9 metabolic activation. Complete inhibition of the bacterial background lawn was observed at ≥1.25 µg/plate, while at 0.625 µg/plate, moderate inhibition of the background lawn was noted in TA98 and TA100 testers both in the presence and absence of S9 metabolic activation. Thus, the main test was performed with the flowing test item concentrations: 0.0 (VC), 0.0 (NC), 0.039, 0.078, 0.156, 0.313 and 0.625 µg/plate both in the presence and absence of S9 metabolic activation system using five Salmonella Typhimurium tester strains. For each strain and dose level, including negative, vehicle and positive controls, three plates (triplicate) were tested. The following positive control substances were used: sodium azide (TA 1535, TA100, without S9 mix), 4-Nitro-o-phenylenediamine (TA1537, TA98, without S9 mix), Methyl methanesulfonate (TA102 without S9), 2-Aminoanthracene (TA98, TA100, TA1535, TA1537 and TA102 wit S9 mix). Trial I was performed according to the plate incorporation method with five test concentrations along with the negative (Distilled water), vehicle (DMSO), and positive controls, and using three strains, i.e., TA1537, TA1535 and TA102.For TA98 and TA100 tester strains, the revertant colony counts were directly incorporated in Trial I from the pre-experiment up to the five concentrations (0.625 mg/plate to 0.039 mg/plate). Trial II was performed according to the preincubation method with all five tester strains along with the negative, vehicle, and positive controls. Results: No significant increase in the number of revertant colonies was observed following the treatment with the test substance up to the concentration of 0.625 µg/plate either in the presence or absence of S9 metabolic activation in all five tester strains in both trials (Trial I-II) when compared to the vehicle control. No trend of an increased number of revertant colonies with increased dosing of the test item was observed. Each strain-specific positive control showed a significant increase in the number of revertant colonies both in the presence and absence of metabolic activation, thus confirming the validity of the assay. Conclusion: The registered substance, i.e., 2-methoxynapthalene (CAS No. 93-04-9), did not induce gene mutation within the histidine operon by base-pair exchange or frameshift in Salmonella Typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) either in the presence or absence of S9 metabolic activation system.
In vitro mammalian chromosme aberration test:
The clastogenic potential of the registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), was assessed in human peripheral blood lymphocytes in the presence and absence of an exogenous metabolic activation system in an OECD 473 study. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction was derived from the Aroclor 1254-induced rats. The test concentrations were selected based on the solubility, precipitation and pH checks and a preliminary cytotoxicity test. The test substance was soluble in Dimethyl Sulfoxide (DMSO) up to 200 mg/ml to give the final treatment concentration of 2 mg/ml. No significant changes in pH were observed at 0 or 4 hours when compared with the vehicle control. Precipitation was observed at concentrations of 2.0, 1.0, 0.5, and 0.25 mg/ml. Slight precipitation was observed at 0.125 mg/m test concentration at the 0th hour, which was judged not to interfere with the conduct of the test. Hence, the concentration of 0.125 mg/ml was selected as the high dose for the cytotoxicity experiment.
In the cytotoxicity test, cells were exposed to test item concentrations of 0.0 (NC: distilled water), 0.0 (VC: DMSO), 0.125, 0.063 and 0.031 mg/ml in culture media in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control data. All the tested concentrations at the initial cytotoxicity experiment were cytotoxic both in the presence and absence of the S9 metabolic activation system. Next, the cytotoxicity was assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.015 mg/ml of culture media in a second cytotoxicity experiment (Experiment II). In cytotoxicity experiment (II), all tested concentrations were cytotoxic both in the presence and absence of the S9 metabolic activation system. Cytotoxicity was then assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml in culture media in the third cytotoxicity experiment (Experiment III). The test concentration of 0.0024 mg/ml produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and the presence of metabolic activation (+S9), respectively. The dose level that results in approximately 55 ± 5 % reduction in the mitotic index was selected as the highest test concentration in the main test. Hence, 0.0024 mg/ml was chosen as the highest test item concentration for the main study, both in the presence and absence of metabolic activation. In the CA test, proliferating peripheral blood cells (whole blood) were used for the test, and the treatment of cultures with the test item was conducted in two independent phases. In the Phase I experiment, cells were exposed to test item concentrations of 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml along with positive controls (EMS: 600 µg/ml without S9 mix and CPA: 30 600 µg/ml with S9 mix) for 4 hours both in the presence and absence of S9 metabolic activation system (1 v/v %) using duplicates. In the Phase II experiment, cells in duplicate cultures were treated with 0.0 (NC), 0.0 (VC), 0.0006, 0.0012 and 0.0024 mg/ml for 4 hours in the presence of the S9 metabolic activation system (2 v/v%) or 24 hours in the absence of S9 metabolic activation. Cells were harvested 24 hrs after treatments in both experiements. A minimum of 1000 cells was counted in different fields of slide per culture, and the number of metaphases was recorded for Mitotic Index (MI) calculation. Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides. Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, 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. Results: In the cytotoxicity test (Experiment III), the test concentration 0.0024 mg/ml produced a 58.59 % and 56.08 % decrease in the mitotic index compared to VC in the absence (-S9) and the presence of metabolic activation (+S9), respectively. The test concentration 0.0012 mg/ml produced 42.00 % and 44.16 % decreases in the mitotic index compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The test concentration 0.0006 mg/ml produced a 33.13 % and 31.67 % decrease in the mitotic index (cytotoxicity) compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively.
In Phase I experiment, the cultures were exposed to the test substance for 4 hours both in the presence (+S9) and absence (-S9) of the metabolic activation system (1%). The mean percentages of aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 ( at 0.0006 mg/ml), 0.667 (at 0.0012 mg/ml), 0.667 (at 0.0024 mg/ml) and 11.000 (PC: 600 µg/mL EMS) in the absence of metabolic activation. In the presence of metabolic activation (1 v/v%), the mean percentage of aberrant cells were 0.333 (NC), 0.333 (VC), 0.000 (at 0.0006 mg/ml), 0.333 (at 0.0012 mg/ml), 0.667 (at 0.0024 mg/ml) and 10.333 (PC: 30 µg/ml CPA). Treatment with positive control substances caused significant increases in the percent of aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were deemed biologically significant. A 55.74% and 52.16 % of reduction in the mitotic index, i.e., cytotoxicity, was observed at the tested concentration of 0.0024 mg/ml compared to vehicle control (VC) in the absence (-S9) and presence (+S9) of metabolic activation system, respectively. The observed mean mitotic index in the absence of metabolic activation were 9.87 (NC), 9.66 (VC), 6.87 (at 0.0006 mg/ml), 5.57 (at 0.0012 mg/ml), 4.28 (at 0.0024 mg/ml) and 8.37 (PC: 600 µg/mL EMS). In the presence of metabolic activation (1 v/v%), the mean mitotic index was 9.53 (NC), 8.66 (VC), 5.59 (at 0.0006 mg/ml), 4.92 (at 0.0012 mg/ml), 4.14 (at 0.0024 mg/ml) and 7.69 (PC: 30 µg/mL CPA). The Phase II experiment was performed to confirm the negative results obtained in Phase I in the presence and absence of S9 metabolic activation. In the absence of metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 (at 0.0006 mg/ml), 0.333 (at 0.0012 mg/ml), 0.333 (at 0.0024 mg/ml) and 10.667 (PC: 600 µg/mL EMS). In the presence of the metabolic activation, the mean percent aberrant cells were 0.667 (NC), 0.333 (VC), 0.000 (at 0.0006 mg/ml), 1.000 (at 0.0012 mg/ml ), 0.333 (at 0.0024 mg/ml) and 11.000 (PC: 30 µg/mL CPA). Treatment with positive control substances in the absence and presence of metabolic activation caused a significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increases were deemed biologically significant. A reduction of 54.67 and 41.22 in the mitotic index was observed at the tested concentration of 0.0024 mg/ml compared to the vehicle control (VC) in the absence (-S9) and presence (+S9) of the metabolic activation system, respectively. The observed mean mitotic index in the absence (-S9) of metabolic activation were 9.35 (NC), 9.12 (VC), 6.38 (at 0.0006 mg/ml), 5.82 (at 0.0012 mg/ml), 4.14 (at 0.0024 mg/ml) and 8.12 (PC: 600 µg/mL EMS). In the presence (+S9) of metabolic activation, the mean mitotic index was 8.71 (NC), 8.31 (VC), 7.12 (at 0.0006 mg/ml), 5.38 (at 0.0012 mg/ml), 4.89 (at 0.0024 mg/ml) and 7.62 (PC: 30 µg/mL CPA). No significant increase in the percent of aberrant cells was observed in the vehicle control groups (Dimethyl sulfoxide) in Phase I and Phase II when compared to the negative control (distilled water). The increased frequency of aberrant cells observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system suitability of the methods and conditions employed in the experiment. Conclusion: The registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), is non-clastogenic in human peripheral lymphocytes both in the presence (1% and 2%) and in the absence of metabolic activation.
In vitro gene mutation in mammalian cells:
An in vitro gene mutation study in mammalian cells, according to OECD TG 476 with the registered substance (CAS: 93-04-9) has been initiated. This information will be submitted at a later time point.
Justification for classification or non-classification
A combination of in vitro genotoxicity tests was conducted with the registered substance, i.e., 2-methoxynaphthalene (CAS: 93-04-9), to assess its genotoxic properties. The substance tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA1535, TA1537 and TA102 according to OECD TG 471 and GLP both in the presence and absence of cofactor-supplemented liver S9 microsomal fraction. It has also been tested non-clastogenic (negative) in the primary culture of human peripheral blood cells according to OECD TG 473 and GLP both in the presence and absence of cofactor-supplemented liver S9 microsomal fraction. An in vitro gene mutation study with the Substance according to OECD TG 476 and GLP is ongoing. Justification for germ cell mutagenicity will be provided after receiving the data from the OECD 476 study.
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