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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.

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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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
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. certificate)
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'.
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

 

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/mL

Experiment 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/mL

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.

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
other: Conducted according to the OECD guideline 487
Deviations:
no
GLP compliance:
yes (incl. certificate)
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'.

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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

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.