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EC number: 274-919-2 | CAS number: 70833-40-8
- 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
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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
- 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
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- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Based on the three negative in vitro genotoxicity tests (bacterial and mammalian gene mutation and chromosomal aberration studies), the test substance is shown to be non-mutagenic, non-clastogenic and non-aneugenic. The data from GLP studies are conclusive but not sufficient for classification.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011-12-19 until 2012-03-19
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- according to OECD 476 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine Kinase Locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: Clone 3.7.2C
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I (4 hours treatment):
without S9 mix: 20.3; 40.6; 81.3; 162.5; 243.8 (PS); 325.0 (PS) µg/mL
with S9 mix: 40.6; 81.3; 162.5; 325.0; 487.5 (PS); 650.0 (PS) µg/mL
Experiment II (24 hours treatment):
without S9 mix: 5.0; 10.0; 20.0; 40.0; 60.0; 80.0; 100.0 µg/mL
Experiment II (4 hours treatment):
without S9 mix: 10.0; 20.0; 40.0; 80.0; 160.0; 240.0; 320.0 µg/mL
PS = Phase Separation
Following the expression phase of 48 hours the cultures (printed in bold letters) at 20.3 µg/mL without metabolic activation in experiment I and at 5.0 µg/mL without metabolic activation and 10.0 µg/mL with metabolic activation in experiment II were not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at 650.0 µg/mL with metabolic activation in experiment I and 100.0 µg/mL without and 320.0 µg/mL with metabolic activation in experiment II were not continued due to exceedingly severe cytotoxic effects. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility properties - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days
SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Evaluation criteria:
- A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above
the corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. - Statistics:
- Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.54 measured in the solvent control versus pH 7.50 at 2600 µg/mL)
- Effects of osmolality: Not increased (382 in the solvent control versus 334 at 2600 µg/mL)
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation:
In the main experiments phase separation occurred at 243.8 and 325.0 µg/mL in experiment I without S9 mix, and at 487.5 and 650.0 µg/mL with S9 mix.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH and osmolarity were determined at the two highest concentrations of the test item and in the solvent control without metabolic activation. There was no relevant shift in either parameter.
1x107 cells (3x106 cells at the beginning of 24 h treatment) were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed twice by centrifugation (425 g, 10 min) and resuspended in "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3x105 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector..
According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiment.
The highest concentration should be 10 mM, but not higher than 5 mg/mL or 5 µL/mL, unless limited by the solubility or toxicity of the test item.
RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50% are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20%.
The pre-experiment was performed in the presence and absence of metabolic activation with a treatment time of 4 hours. Test item concentrations between 20.3 µg/mL and 2600 µg/mL equal to a molar concentration of approximately 10 mM were used. The dose calculation was not adjusted to purity.
Toxic effects leading to RSG values below 50% were observed at 162.5 µg/mL and above in the absence and at 325.0 µg/mL and above in the presence of metabolic activation (4 hours treatment). After 24 hours treatment relevant toxic effects as described above occurred at 81.3 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of the treatment period (4 hours) before the test item was removed. Phase separation was observed at 650 µg/mL with and without metabolic activation following 4 hours treatment.
Both, pH value and osmolarity were determined in the pre-experiment at the two highest concentrations of the test item and in the solvent control without metabolic activation. There was no relevant shift of both parameters.
The dose range of the main experiments was set according to the cytotoxicity data of the test item. In both main experiments the individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic or phase separating range more closely.
To overcome problems with possible deviations in toxicity and solubility the main experiments were started with more than four concentrations.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects indicated by a relative total growth of less than 50% were observed in at least one of the cultures of experiment I at
243.8 µg/mL and above without metabolic activation and at 325.0 µg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described were noted at 40.0 µg/mL and above without and 240 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation. - Remarks on result:
- other: strain/cell type: in vitro gene mutation assay with L5178Y cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the
cell line L5178Y in the absence and presence of metabolic activation. - Executive summary:
The study was performed to investigate the potential of O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate (CAS 70833-40-8) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation. Due to exceedingly severe cytotoxic effects at the three highest concentrations with and without metabolic activation, the second experiment was repeated using lower concentrations (experiment 2.2). The experimental part of this repeat experiment without metabolic activation was again repeated (experiment 2.3) based on a technical error. The results of experiment 2.2 with metabolic activation and experiment 2.3 without metabolic activation are reported in experiment II.
The main experiments were evaluated at the following concentrations with and without metabolic activation:
Experiment I
without S9 mix: 40.6; 81.3; 162.5; 243.8; and 325.0 µg/mL
with S9 mix: 40.6; 81.3; 162.5; 325.0; and 487.5 µg/mLExperiment II
without S9 mix: 10.0, 20.0; 40.0; 60.0; and 80.0 µg/mL
with S9 mix: 20.0; 40.0; 80.0; 160.0; and 240.0 µg/mLRelevant toxic effects indicated by a relative total growth of less than 50% were observed in at least one of the cultures of experiment I at 243.8 µg/mL and above without metabolic activation and at 325.0 µg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described were noted at 40.0 µg/mL and above without and 240 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. In the first culture of the first experiment with metabolic activation an isolated increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control occurred at 487.5 µg/mL. This isolated increase was judged as irrelevant since no comparable increase was noted in the parallel culture under identical conditions.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency using SYSTATâstatistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the first culture of experiment I with metabolic activation. However, this trend was based on the isolated increase of the mutation frequency discussed above and consequently, considered irrelevant.
MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations.
The cloning efficiency (viability) slightly exceeded the recommended range of up to 120% in the solvent control of the second culture of the first experiment without metabolic activation. This deviation was considered irrelevant since it was very minor (121% versus 120%) and the cloning efficiency of the parallel culture was well within the recommended range. The total suspension growth of the solvent control of the first culture of the second experiment with metabolic activation fell short of the lower limit of the recommended range (5.4 versus a lower limit of 8.0). Again, the total suspension growth of the parallel culture remained within the recommended range and the data were judged as valid.
Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: This is a well-performed study carried out according to the OECD 471 test guideline.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9-mix
- Test concentrations with justification for top dose:
- 100, 333, 1000, 3333, & 5000 ug/plate
- Vehicle / solvent:
- Dimethylsulfoxide
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene(2AA); 4-nitro-o-phenylene- diamine (4NPDI
- Details on test system and experimental conditions:
- Standard plate test
Top agar in top agar tubes is melted and heated to 45°C. The following
solutions are successively added to 3 ml of top agar: 0.1 ml of a fresh
bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of
a dilution of the test substance in dimethyl sulphoxide (DMSO), and in
the case of activation assays 0.5 ml of 59-mix. The ingredients are
mixed on a Vortex and the contents of the top agar tube are poured onto
a selective agar plate. After solidification of the top agar, the plates
are turned and incubated in the dark at 37°C for 48h. After this period
revertant colonies (histidine independent) are counted automatically
with an Artek model 880 colony counter or manually. - Evaluation criteria:
- All bacterial strains showed nega t ive responses over the ent ire dose range of the tes t substance, i.e. no statistically significant dose-related increase in the number of revertants (His+) colonies.
- Statistics:
- none
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535; TA1537; TA1538; TA98 and TA100) either with or without the addition of a metabolic system (Aroclor 1254-induced rat liver S9-mix). The test substance can, therefore, be considered as nonmutagenic in this test system.
- Executive summary:
The test substance was tested in the Ames Salmonella/microsome test up to a concentration of 5000 ug/plate. The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535; TA1537; TA1538; TA98 and TA100) either with or without the addition of a metabolic system (Aroclor 1254-induced rat liver S9-mix). The test substance can, therefore, be considered as nonmutagenic in this test system.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-01-12 to 2012-08-03
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: A GLP study performed according to OECD test guideline 487
- Qualifier:
- according to guideline
- Guideline:
- other: Conducted according to the OECD guideline 487
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9
- Test concentrations with justification for top dose:
- With metabolic activation:
Experiment I: 5.5, 9.7, 16.9, 29.6, 51.7, 90.5, 158.4, 277.2, 485.1, 849.0, 1485.7, 2600.0 µg/mL
Experiment II: 158.4, 277.2, 485.1, 849.0, 1485.7, 2600.0 µg/mL
Without metabolic activation:
Experiment I: 5.5, 9.7, 16.9, 29.6, 51.7, 90.5, 158.4, 277.2, 485.1, 849.0, 1485.7, 2600.0 µg/mL
Experiment II: 16.9, 29.6, 51.7, 90.5, 158.4, 277.2, 485.1, 849.0, 1485.7, 2600.0 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 487 - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: demecolcin
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Details on test system and experimental conditions:
- Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 20 hours without S9 mix. The chromosomes were prepared 40 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium
DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 20 hours (- S9 mix)
- Recovery after 4 hours treatment: 16 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours
CYTOKINESIS BLOCK (cytogenetic assays): Cytochalasin B 20 hours
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: about 1.5
NUMBER OF BINUCLEATED CELLS EVALUATED: 1000 per culture
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)
- Evaluation criteria:
- Evaluation of the slides will be performed using NIKON microscopes with 40 x objectives. The micronuclei will be counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture will be scored for cytogenetic damage on coded slides. The frequency of micronucleated cells will be reported as % micronucleated cells. To describe a cytotoxic effect the CBPI is determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
CBPI=(MONCx1)+(BINCx2)+(MUNCx3)/n
CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BINC Binucleate cells
MUNC Multinucleate cells
Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]
T Test item
C Solvent control - Statistics:
- Statistical significance can be confirmed by means of the Chi square test.
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The test item O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate (CAS 70833-40-8), dissolved in ethanol, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 2600.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
No precipitation of the test item in the culture medium was observed at the end of treatment. No relevant influence on osmolarity or pH value was observed. Phase separation was observed in Experiment I at 1485.7 µg/mL in the absence and presence of S9 mix. In Experiment II phase separation was observed at 485.1 µg/mL and above in the absence of S9 mix and at 158.4 µg/mL and above in the presence of S9 mix.
In the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration. However, in Experiment II in the absence of S9 mix a moderate cytotoxicity plateau was observed in a concentration range of 485.1 - 849.0 µg/mL not being evaluable due to a reduced cell number (Table 3 and 4).
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed (see Table 7, 8, 11 and 12). The micronucleus rates of the cells after treatment with the test item (0.15 - 0.85 % micronucleated cells) were within the range of the solvent control values (0.20 - 0.90 % micronucleated cells) and within the range of the laboratory historical control data (see ANNEX II).
Either Demecolcin (75.0 ng/mL), MMC (1.0 µg/mL) or CPA (10.0 or 12.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. After Demecolcin treatment an additional increase in mononucleate cells carrying micronuclei was observed.
In conclusion, it can be stated that under the experimental conditions reported, the test item O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate (CAS 70833-40-8) did not induce micronuclei in human lymphocytes in vitro when tested up to the highest required concentration. - Remarks on result:
- other: strain/cell type: human lymphocytes
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- In conclusion, in a study conducted according to the OECD guideline 487, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate (CAS 70833-40-8) is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
- Executive summary:
The test item O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate(CAS 70833-40-8), dissolved in ethanol, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:
Without S9-Mix
With S9-Mix
Exp. I
Exp. II
Exp. I and II
Exposure period
4 hrs
20 hrs
4 hrs
Recovery
16 hrs
-
16 hrs
Cytochalasin B exposure
20 hrs
20 hrs
20 hrs
Preparation interval
40 hrs
40 hrs
40 hrs
Total culture period
88 hrs
88 hrs
88 hrs
In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
The highest applied concentration in the this study (2600.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.
Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.
In the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration. However, in Experiment II in the absence of S9 mix a moderate cytotoxicity plateau was observed with a concentration range of 485.1 - 849.0 µg/mL not being evaluable due to a reduced cell number.
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item.
Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.
Referenceopen allclose all
Summary Table
relative | mutant | relative | mutant | |||||
conc. µg | S9 | total | colonies/ | total | colonies/ | |||
per mL | mix | growth | 106cells | threshold | growth | 106cells | threshold | |
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Experiment I / 4 h treatment | culture I | culture II | ||||||
Solv. control with ethanol | - | 100.0 | 83 | 209 | 100.0 | 92 | 218 | |
Pos. control with MMS | 19.5 | - | 52.5 | 311 | 209 | 43.8 | 332 | 218 |
Test item | 20.3 | - | culture was not continued# | culture was not continued# | ||||
Test item | 40.6 | - | 101.9 | 111 | 209 | 94.9 | 97 | 218 |
Test item | 81.3 | - | 122.6 | 99 | 209 | 98.2 | 88 | 218 |
Test item | 162.5 | - | 133.2 | 87 | 209 | 98.2 | 87 | 218 |
Test item | 243.8 (PS) | - | 33.7 | 142 | 209 | 77.6 | 89 | 218 |
Test item | 325.0 (PS) | - | 13.4 | 98 | 209 | 77.3 | 85 | 218 |
Solv. control with ethanol | + | 100.0 | 97 | 223 | 100.0 | 94 | 220 | |
Pos. control with CPA | 3.0 | + | 42.1 | 383 | 223 | 34.9 | 275 | 220 |
Pos. control with CPA | 4.5 | + | 36.9 | 517 | 223 | 31.8 | 262 | 220 |
Test item | 40.6 | + | 94.7 | 93 | 223 | 96.3 | 103 | 220 |
Test item | 81.3 | + | 114.7 | 83 | 223 | 67.5 | 121 | 220 |
Test item | 162.5 | + | 127.5 | 118 | 223 | 72.3 | 86 | 220 |
Test item | 325.0 | + | 43.2 | 199 | 223 | 35.8 | 117 | 220 |
Test item | 487.5 (PS) | + | 10.7 | 300 | 223 | 18.3 | 171 | 220 |
Test item | 650.0 (PS) | + | culture was not continued## | culture was not continued## | ||||
Experiment II / 24 h treatment | culture I | culture II | ||||||
Solv. control with ethanol | - | 100.0 | 124 | 250 | 100.0 | 82 | 208 | |
Pos. control with MMS | 13.0 | - | 13.1 | 522 | 250 | 23.5 | 596 | 208 |
Test item | 5.0 | - | culture was not continued# | culture was not continued# | ||||
Test item | 10.0 | - | 67.1 | 167 | 250 | 153.5 | 68 | 208 |
Test item | 20.0 | - | 66.5 | 137 | 250 | 123.7 | 85 | 208 |
Test item | 40.0 | - | 48.4 | 144 | 250 | 54.2 | 62 | 208 |
Test item | 60.0 | - | 16.5 | 142 | 250 | 51.8 | 73 | 208 |
Test item | 80.0 | - | 6.6 | 163 | 250 | 17.7 | 93 | 208 |
Test item | 100.0 | - | culture was not continued## | culture was not continued## | ||||
Experiment II / 4 h treatment | culture I | culture II | ||||||
Solv. control with ethanol | + | 100.0 | 135 | 261 | 100.0 | 104 | 230 | |
Pos. control with CPA | 3.0 | + | 121.1 | 435 | 261 | 110.8 | 280 | 230 |
Pos. control with CPA | 4.5 | + | 65.8 | 674 | 261 | 85.9 | 314 | 230 |
Test item | 10.0 | + | culture was not continued# | culture was not continued# | ||||
Test item | 20.0 | + | 134.0 | 182 | 261 | 111.8 | 173 | 230 |
Test item | 40.0 | + | 116.2 | 145 | 261 | 104.2 | 204 | 230 |
Test item | 80.0 | + | 193.0 | 137 | 261 | 117.9 | 193 | 230 |
Test item | 160.0 | + | 98.4 | 194 | 261 | 61.8 | 116 | 230 |
Test item | 240.0 | + | 65.5 | 132 | 261 | 13.5 | 142 | 230 |
Test item | 320.0 | + | culture was not continued## | culture was not continued## |
Threshold = number of mutant colonies per 106 cells of each solvent control plus 126
# culture was not continued since a minimum of only four analysable concentrations is required
## culture was not continued due to exceedingly severe cytotoxic effects
PS Phase Separation
Summary of
results of thein vitromicronucleustest
in human lymphocytes with
O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate (CAS 70833-40-8)
Exp. |
Preparation |
Test item |
Proliferation |
Cytostasis |
Micronucleated |
|
interval |
concentration |
index |
in %* |
cells |
|
|
in µg/mL |
CBPI |
|
in %** |
Exposure period 4 hrs without S9 mix |
|||||
I |
40 hrs |
Negative control |
2.00 |
|
0.35 |
|
|
Solvent control1 |
2.00 |
|
0.20 |
|
|
Positive control2 |
2.03 |
n.c. |
3.60S |
|
|
849.0 |
1.96 |
3.5 |
0.25 |
|
|
1485.7PS |
1.99 |
0.7 |
0.30 |
|
|
2600.0PS |
1.86 |
13.7 |
0.40 |
Exposure period 20 hrs without S9 mix |
|||||
II |
40 hrs |
Negative control |
1.79 |
|
0.25 / 0.35*** |
|
|
Solvent control1 |
1.70 |
|
0.35 |
|
|
Positive control3 |
1.51 |
35.9 |
2.25S/ 1.40S*** |
|
|
90.5 |
1.72 |
n.c. |
0.25 |
|
|
277.2 |
1.50 |
28.5 |
0.25 |
|
|
1485.7PS |
1.46 |
34.5 |
0.30 |
|
|
2600.0PS |
1.47 |
32.8 |
0.15 |
* For
the positive control groups, the relative values are related to the
negative controls;
for the test item treatment groups the values are related to the solvent
controls
** The number of micronucleated cells was determined in a sample of 2000 binucleated cells
*** The number of micronucleated cells was determined in a sample of 2000 mononucleated cells
PS Phase separation was observed at the end of treatment
S The number of micronucleated cells is statistically significantly higher than corresponding control values
n.c. Not calculated as the CBPI is equal or higher than the solvent control value
1 Ethanol 0.5
% (v/v)
2 MMC 1.0
µg/mL
3 Demecolcin 75.0 ng/mL
Table 2 (cont.): Summary of results of thein vitromicronucleus test in human lymphocytes with O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate (CAS 70833-40-8)
Exp. |
Preparation |
Test item |
Proliferation |
Cytostasis |
Micronucleated |
|
interval |
concentration |
index |
in %* |
cells |
|
|
in µg/mL |
CBPI |
|
in %** |
Exposure period 4 hrs with S9 mix |
|||||
I |
40 hrs |
Negative control |
2.19 |
|
1.40 |
|
|
Solvent control1 |
2.13 |
|
0.90 |
|
|
Positive control2 |
1.87 |
27.1 |
4.65S |
|
|
849.0 |
2.18 |
n.c. |
0.85 |
|
|
1485.7PS |
2.10 |
2.4 |
0.80 |
|
|
2600.0PS |
1.91 |
19.0 |
0.55 |
II |
40 hrs |
Negative control |
1.73 |
|
0.75 |
|
|
Solvent control1 |
1.75 |
|
0.85 |
|
|
Positive control3 |
1.45 |
38.7 |
2.80S |
|
|
849.0PS |
1.67 |
10.7 |
0.70 |
|
|
1485.7PS |
1.68 |
9.1 |
0.50 |
|
|
2600.0PS |
1.71 |
4.7 |
0.40 |
* For
the positive control groups, the relative values are related to the
negative controls;
for the test item treatment groups the values are related to the solvent
controls
** The number of micronucleated cells was determined in a sample of 2000 binucleated cells
PS Phase separation was observed at the end of treatment
S The number of micronucleated cells is statistically significantly higher than corresponding control values
n.c. Not calculated as the CBPI is equal or higher than the solvent control value
1 Ethanol 0.5
% (v/v)
2 CPA 10.0
µg/mL
3 CPA 12.5 µg/mL
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
Reference
Additional information
Additional information from genetic toxicity in vitro:
In vitro Mouse Lymphoma Assay: A GLP study (Wollny, 2012) was performed to investigate the potential of O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate (CAS 70833-40-8) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
In vitro bacterial reverse mutation assay: The test substance was tested in the Ames Salmonella/microsome test up to a concentration of 5000 ug/plate in a standard Ames test (bacterial reverse mutation assay) per OECD 471 test guideline (Debets, 1985). The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100) either with or without the addition of a metabolic system (Aroclor 1254-induced rat liver S9-mix). The test substance was, therefore, considered as nonmutagenic in this test system.
In a GLP study (Bohnenberger, 2012) “In vitro mammalian cell micronucleus test in humanlymphocytes with O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate”, the test item, dissolved in ethanol, was assessed for its potential to induce micronuclei in human lymphocytesin vitroin two independent experiments. In each experimental group two parallel cultures were analyzed. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. One-thousand binucleate cells per culture were scored for cytogenetic damage on coded slides. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens were used as positive controls and they induced statistically significant increases in cells with micronuclei. In conclusion, under the experimental conditions reported, the test item did not induce micronuclei and therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
Overall discussion of the endpoint summary: The test item was negative in all 3 in vitro genotoxicity tests, viz., in vitro bacterial reverse mutation assay, and two mammalian genotoxicty assays (in vitro mouse lymphoma assay, and in vitro micronucleau assay in humal lymphocytes) with or without metabolic activation. Thus the test item is non-mutagenic, non-clastogenic and non-aneugenic. In accordance with Endpoint Specific Guidance Chapter R.7A, Figure R.7.7 -1 "Flow chart of the mutagenicity testing strategy", no further testing (i.e., no in vivo testing) need be proposed in the event of a negative mouse lymphoma assay or hprt assay, regardless of whether or not the gene mutation test in bacteria is positive or negative. This, therefore, implies that when considering whether an in vivo gene mutation request is required for substances requiring Annex IX test proposals due to their volume bands, a negative mouse lymphoma assay is sufficient evidence to waive the need for an in vivo gene mutation test.
Justification for selection of genetic toxicity endpoint
In this key study conducted to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line, the test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells. The test item was considered to be non mutagenic in mammalian cells.
Justification for classification or non-classification
Based on the three negative in vitro genotoxicity tests (bacterial and mammalian gene mutation and chromosomal aberration studies), the test substance is shown to be non-mutagenic, non-clastogenic and non-aneugenic. The data are conclusive but not sufficient for classification.
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