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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
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- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
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- Solubility in organic solvents / fat solubility
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- 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
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- Endpoint summary
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- 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
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Carcinogenicity
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- Exposure related observations in humans
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- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
Materials and methods
Test guideline
- 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
Test material
- Reference substance name:
- Methyl 2-naphthyl ether
- EC Number:
- 202-213-6
- EC Name:
- Methyl 2-naphthyl ether
- Cas Number:
- 93-04-9
- Molecular formula:
- C11H10O
- IUPAC Name:
- 2-methoxynaphthalene
- Test material form:
- solid: flakes
Constituent 1
- 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
Method
Species / strain
- 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.
Controls
- 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.
Results and discussion
Test results
- 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)
Any other information on results incl. tables
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 |
Applicant's summary and conclusion
- 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.
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