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EC number: 807-534-0 | CAS number: 1228284-89-6
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2013-10-29 to 2014-06-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study was conducted under GLP and according to OECD 473 without deviations. The method is well documented; the concurrent negative and positive control data, signs of precipitation, changes in ploidy and definition of aberrations were reported.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- 1-(2,4,6-trichlorophenyl)acetone O-methyloxime
- EC Number:
- 807-534-0
- Cas Number:
- 1228284-89-6
- Molecular formula:
- C10H10Cl3NO
- IUPAC Name:
- 1-(2,4,6-trichlorophenyl)acetone O-methyloxime
- Test material form:
- not specified
- Details on test material:
- - Storage condition of test material: at room temperature, light protected, moisture protected
Constituent 1
Method
- Target gene:
- Not applicable
Species / strain
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- - Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a female donor (37 years old) for Experiment I and from a male donor (20 years old) for Experiment II. Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to Harlan CCR to initiate cell cultures within 24 h after blood collection. If necessary, the blood was stored before use at 4 °C.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Mammalian Microsomal Fraction S9 Mix: Phenobarbital/beta-naphthoflavone induced rat liver S9. Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
- Test concentrations with justification for top dose:
- The highest concentration used in the pre-test was chosen with regard to the current OECD guideline 473 for in vitro mammalian cytogenetic tests requesting for the top concentration to induce clear cytotoxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation. In the case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible.
With regard to the molecular weight of the test substance, 2666.0 µg/mL of the test item (approx. 10 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test substance concentrations between 17.3 and 2666.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test substance was observed at the end of treatment at 30.3 µg/mL and above in the absence of S9 mix and at 53.0 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, clear toxic effects were observed after 4 hours treatment with 92.8 µg/mL and above in the absence of S9 mix and with 284.3 µg/mL and above in the presence of S9 mix. Therefore, 200.0 µg/mL (without S9 mix) and 400.0 µg/mL (with S9 mix) were chosen as top concentrations in Experiment II. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Concurrent solvent controls (culture medium with 0.5 % DMSO) were performed.
Name: DMSO
Supplier: Fisher Chemicals
Purity: 99.9 %
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubilisation properties and its relative non-toxicity to the cell cultures.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
On the day of the experiment (immediately before treatment), the test substance was dissolved in DMSO. The final concentration of DMSO in the culture medium was 0.5 % (v/v). The culture medium was replaced with serum-free medium containing the test substance. All cultures were incubated at 37 °C in a humidified atmosphere with 5.5 % CO2.
DURATION
- Preincubation period: about 48 h after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks (Nunc GmbH & Co. KG, 65203 Wiesbaden, Germany).
- Exposure duration:
-- Exposure time 4 hours: The culture medium was replaced with serum-free medium containing the test substance. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 h the cells were spun down by gentle centrifugation for 5 minutes (approx. 900 x g). The supernatant with the dissolved test substance was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once as described. The "saline G" solution was composed as follows (per litre):
NaCl: 8000 mg
KCl: 400 mg
glucose x H2O: 1100 mg
Na2HPO4x 2H20: 192 mg
KH2PO4: 150 mg
pH was adjusted to 7.2
After washing the cells were re-suspended in complete culture medium and cultured until preparation.
-- Exposure time 22 hours (without S9 mix): The culture medium was replaced with complete medium (with 10 % FBS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
- Fixation time (start of exposure up to fixation or harvest of cells): The cultures were treated with the metaphase-arresting substance Colcemid (final concentration: 0.2 µg/mL) approximately three hours before the requested harvest time. The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g) the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concentration: 0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 well-spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II without S9 mix, where only 50 metaphases were evaluated.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis)
OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. - Evaluation criteria:
- A test substance is classified as non-clastogenic if:
- the number of induced structural chromosomal aberrations in all evaluated concentration groups is in the range of the laboratory historical control data.
- no statistically significant increase of the rate of structural chromosomal aberrations is observed in comparison to the respective solvent control.
A test substance is classified as clastogenic if:
- the number of induced structural chromosomal aberrations is not in the range of the historical laboratory control data
and
- either a concentration-related or a statistically significant increase in the number of cells carrying structural chromosomal aberrations is observed.
A test substance not meeting the criteria for classification as non-mutagenic or mutagenic may be considered equivocal in this assay and may be subject to further investigation. - Statistics:
- Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test substance are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Results and discussion
Test results
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: test item dissolved in DMSO
- Precipitation: Precipitation of the test substance in the culture medium was observed at the end of treatment in Experiment I at 30.3 µg/mL and above in the absence of S9 mix and at 53.0 µg/mL and above in the presence of S9 mix and in Experiment II at 37.3 µg/mL and above in the absence of S9 mix and at 100.0 µg/mL and above in the presence of S9 mix.
- Other confounding effects: no
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test substance and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 22 h after start of the exposure.
COMPARISON WITH HISTORICAL CONTROL DATA: range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I in the absence of S9 mix and in Experiment II in the absence and presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration (35.3, 49.9 and 36.9 % of control, respectively). In Experiment I in the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, the mitotic index was markedly reduced to 56.1 % at the highest evaluable concentration. - Remarks on result:
- other: strain/cell type:
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table 1 Summary of Results of the chromosomal aberration study without S9 mix
Exp. | Preparation interval | Test item concentration | Mitotic indices | Aberrant cells | ||
( µg/mL) | % of control | incl. gaps* | excl. gaps* | carrying exchanges | ||
Exposure period 4 hrs without S9 mix | ||||||
I | 22 hrs | Solvent control1 | 100 | 1.5 | 1.5 | 0 |
Positive control2 | 57.4 | 12 | 11.5S | 3 | ||
17.3 | 93.2 | 3 | 3 | 0.5 | ||
30.3P | 92.6 | 1 | 1 | 0 | ||
53.0P | 86.3 | 2 | 2 | 0 | ||
92.8P## | 35.3 | 3.5 | 3.5 | 0 | ||
Exposure period 22 hrs without S9 mix | ||||||
II | 22 hrs | Solvent control1 | 100 | 1.5 | 1.5 | 0 |
Positive control3# | 44.5 | 38 | 38.0S | 11 | ||
21.3 | 86.1 | 1.5 | 1.5 | 0 | ||
37.3P | 93.2 | 0.5 | 0.5 | 0 | ||
65.3P | 49.9 | 0.5 | 0.5 | 0 |
*: Including cells carrying exchanges
#: Evaluation of 50 metaphases per culture
##: Evaluation of 200 metaphases per culture
P: Precipitation occurred at the end of treatment
S: Aberration frequency statistically significant higher than corresponding control values
1: DMSO0.5 % (v/v)
2: EMS770.0 µg/mL
3: EMS550.0 µg/mL
Table 2 Summary of Results of the chromosomal aberration study with S9 mix
Exp. | Preparation interval | Test item concentration | Mitotic indices | Aberrant cells | ||
( µg/mL) | % of control | incl. gaps* | excl. gaps* | carrying exchanges | ||
Exposure period 4 hrs with S9 mix | ||||||
I | 22 hrs | Solvent control1 | 100 | 0.5 | 0.5 | 0 |
Positive control2 | 49.8 | 16 | 15.5S | 1 | ||
30.3## | 103.8 | 3.3 | 3.0S | 0 | ||
53.0P## | 102.9 | 2 | 1.8 | 0 | ||
92.8P## | 109.2 | 3.5 | 3.5S | 0 | ||
162.4P## | 56.1 | 4.5 | 3.8S | 0.3 | ||
Exposure period 22 hrs with S9 mix | ||||||
II | 22 hrs | Solvent control1 | 100 | 0.5 | 0.5 | 0 |
Positive control2 | 31.6 | 16.5 | 16.5S | 4.5 | ||
50 | 96.9 | 0 | 0 | 0 | ||
100.0P | 59.1 | 0.5 | 0.5 | 0 | ||
125.0P | 36.9 | 2.5 | 2.5 | 0 |
*: Including cells carrying exchanges
##: Evaluation of 200 metaphases per culture
P: Precipitation occurred at the end of treatment
S: Aberration frequency statistically significant higher than corresponding control values
1: DMSO0.5 % (v/v)
2: CPA7.5 µg/mL
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
In a valid, conclusive and reliable study according to OECD 473 (1997), EPA OPPTS 870.5375 (1998) and EC 440/2008 B. 10 (2008), the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating or the highest evaluable concentrations. - Executive summary:
This in vitro assay was performed to assess the potential of the test item to induce structural chromosomal aberrations in cultured human lymphocytes in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II without S9 mix, where only 50 metaphases were evaluated. The highest applied concentration in this study (2666.0 µg/mL of the test substance, approx. 10 mM) was chosen with regard to the molecular weight of the test substance and with respect to the current OECD Guideline 473. Concentration selection for the cytogenetic experiments was performed considering the toxicity data and test substance precipitation and in accordance with OECD Guideline 473.
In Experiment I in the absence of S9 mix and in Experiment II in the absence and presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In Experiment I in the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, the mitotic index was markedly reduced to 56.1 % at the highest evaluable concentration. Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. In Experiment I in the presence of S9 mix, statistically significant increases were observed after treatment with 30.3, 92.8 and 162.4 µg/mL (3.0, 3.5 and 3.8 % aberrant cells, excluding gaps). The highest value slightly exceeded the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps). However, no concentration-dependency could be observed and the findings were not confirmed in Experiment II. No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic and/or precipitating concentrations.
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