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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Identity: O-(2-ethylhexyl) O,O,-tertpentyl peroxycarbonate
CAS No.: 70833-40-8
Batch-No.: 1012442557
Molecular Weight: 260.37 g/mol
Purity: 96.5% (dose calculation not adjusted to purity)
Physical State: Liquid, colorless
Storage: At approx. -20 °C
Solubility in Water: Unknown
Stability in Solvent: Not indicated by the sponsor
Expiration Date: January 01, 2013
On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol (purity 99.9 %). The final concentration of ethanol in the culture medium was 0.5 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cells.

Method

Target gene:
Thymidine Kinase Locus
Species / strain
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
Controlsopen allclose all
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)

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.