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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
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:
2011
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
Details on test material:

- Name of test material (as cited in study report): di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide
- Molecular weight (if other than submission substance): 290 g/mol
- Substance type: Industrial chemical
- Physical state: Liquid, colourless
- Analytical purity: 96.2% (dose calculation adjusted to purity)
- Composition of test material, percentage of components: --
- Purity test date: 2010-03-26
- Lot/batch No.: 1002516115
- Expiration date of the lot/batch: 2012-03-01
- Storage condition of test material: At room temperature (15-25 °C)
- Other:
On the day of the experiment (immediately before treatment), the test item was formulated in ethanol (purity 99.8 %). 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 : 1.5, 2.9, 5.9, 11.8, 23.5, 47.0, 70.5, 94.0 µg/mL
with S9 mix: 2.9, 5.9, 11.8, 23.5, 47.0, 94.0 (only culture II analysed), 141.0, 188.0 µg/mL

Experiment II (24 hours treatment)
without S9: 2.9, 5.9, 11.8, 23.5, 47.0, 70.5, 94.0 µg/mL

Experiment II (4 hours treatment)
with S9 mix: 2.9, 5.9, 11.8, 23.5, 47.0, 70.5, 94.0 µg/mL
Following the expression phase of 48 hours the cultures at the maximum concentration of 70.5 and 94.0 µg/mL in experiment I without metabolic activation and at 94.0 (in culture I only), 141.0, and 188.0 µg/mL with metabolic activation were not continued due to exceedingly severe cytotoxic effects. In experiment II the cultures at 70.5 and 94.0 µg/mL with and without metabolic activation were not continued for the same reason. The cultures at 1.5 µg/mL in experiment I without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.
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(R) 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 (solvent control: pH 7.26; test item at 1500 µg/mL: pH 7.28 )
- Effects of osmolality: (solvent control: osmolarity 379; test item at 1500 µg/mL: osmolarity 354)
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation:
Pre-experiment:
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye at 187.5 µg/mL and above in the parts following 4 hours treatment. After 24 hours treatment precipitation occurred at the maximum concentration of 1500 µg/mL.

Main experiments: No precipitation occurred

- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 11.7 µg/mL and 1500 µg/mL were used. Relevant toxic effect (relative suspension growth, RSG below 50% of the solvent control) occurred at 11.7 µg/mL and above following 4h treatment without metabolic activation. Following 4h treatment with metabolic activation the RSG was reduced <50% at 23.4 µg/mL. After 24h treatment cytotoxic effects were noted at 46.9 µg/mL and above without metabolic activation.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed.
Precipitation was observed by the unaided eye at 187.5 µg/mL and above in the parts following 4 hours treatment. After 24 hours treatment precipitation occurred at the maximum concentration of 1500 µg/mL.
Therefore, the maximum concentration of the main experiments was adjusted according to the toxicity data of the pre-experiment. To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations. The individual concentrations were spaced by a factor of 2. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.


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 the first experiment at 23.5 µg/mL and above with and without metabolic activation. In the second experiment cytotoxic effects as described were noted at 47.0 µg/mL with and without 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  68 194 100.0 112 238
Pos. control with MMS  19.5 -  47.4 304 194  46.5 415 238
Test item   1.5 - culture was not continued# culture was not continued#
Test item   2.9 - 115.3  72 194 152.3  73 238
Test item   5.9 - 125.1  67 194  94.2 119 238
Test item  11.8 - 125.6  64 194 149.4  47 238
Test item  23.5 -  55.3  56 194  34.6  97 238
Test item  47.0 -  10.8 123 194  10.6 102 238
Test item  70.5 - culture was not continued## culture was not continued##
Test item  94.0 - culture was not continued## culture was not continued##
       
Solv. control with ethanol + 100.0  72 198 100.0 117 243
Pos. control with CPA   3.0 +  51.3 146 198  53.2 294 243
Pos. control with CPA   4.5  +   21.0 302 198  21.5 779 243
Test item   2.9  +   90.2  46 198 culture was not continued#
Test item   5.9  +   62.1  93 198 147.1 156 243
Test item  11.8  +   61.0  62 198 333.0 103 243
Test item  23.5  +   47.2 140 198  77.3  87 243
Test item  47.0  +   9.3 150 198  60.3  83 243
Test item  94.0  +  culture was not continued##  47.8 102 243
Test item  141.0  +  culture was not continued## culture was not continued##
Test item  188.0  +  culture was not continued## culture was not continued##
Experiment II / 24 h treatment   culture I culture II
Solv. control with ethanol - 100.0  74 200 100.0 107 233
Pos. control with MMS  13.0 -  34.2 333 200  47.1 353 233
Test item   2.9 - 115.0  60 200  86.8 129 233
Test item   5.9 -  78.3  96 200 105.4  87 233
Test item  11.8 -  99.2  84 200 137.3  97 233
Test item  23.5 - 104.5  85 200  91.0 122 233
Test item  47.0 -  4.3 115 200  4.5  91 233
Test item  70.5 - culture was not continued## culture was not continued##
Test item  94.0 - culture was not continued## culture was not continued##
Experiment II / 4 h treatment   culture I culture II
Solv. control with ethanol + 100.0  48 174 100.0 117 243
Pos. control with CPA   3.0 +  45.4 126 174  47.8 228 243
Pos. control with CPA   4.5 +  23.9 248 174  36.1 336 243
Test item   2.9 + 106.7  65 174 105.5 107 243
Test item   5.9 +  91.8  68 174  93.3 122 243
Test item  11.8 +  72.4  80 174  94.6 120 243
Test item  23.5 +  73.2  52 174  78.4 146 243
Test item  47.0 +  8.7  61 174  7.6 170 243
Test item  70.5 + culture was not continued## culture was not continued##
Test item  94.0 + culture was not continued## culture was not continued##

threshold = number of mutant colonies per 106cells of each solvent control plus 126

#    culture was not continued since a minimum of only 4 analysable concentrations is required

##   culture was not continued due to exceedingly severe cytotoxic effects

 

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 ofdi-tert-butyl 1,1,4,4 -tetramethyltetramethylene diperoxide (CAS 78 -63 -7) 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 h. The first main experiment was terminated prior to the generation of any data on mutagenicity since the cytotoxic range was not reached even at the highest concentrations. The dose range was adjusted and the experiment repeated under identical general experimental conditions. The data of the repeat experiment are reported as experiment I.

The second experiment was performed with a treatment period of 4 h with and 24 h without metabolic activation. The second experiment with metabolic activation was terminated prior to the generation of any data on mutagenicity since exceedingly severe cytotoxicity occurred already at low concentrations. Again, the dose range was adjusted and the experiment repeated with metabolic activation. The data of the repeat experiment are reported as experiment II with metabolic activation.

Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 23.5 µg/mL and above with and without metabolic activation. In the second experiment cytotoxic effects as described were noted at 47.0 µg/mL with and without 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. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control in any of the experimental parts.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of the first experiment and the second culture of the second experiment with metabolic activation. However, both trends were judged as biologically irrelevant since the mutation frequency remained within the historical range of solvent controls.

In this study the range of the solvent controls was from 48 up to 117 mutant colonies per 106cells; the range of the groups treated with the test item was from 47 up to 170 mutant colonies per 106cells. In experiment II, culture I without metabolic activation the solvent control fell just short of the recommended 50 – 170 per 106control range as stated under paragraph 8.12,acceptability of the assay of this report. However, the deviation was rather minor (2 colonies per 106cells) and the spontaneous rate of the parallel culture and the mean value of both cultures was fully acceptable (48 and 117 colonies, equal to a mean of 82.5).

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 positive control of the second experiment, culture I with metabolic activation (at 4.5 µg/mL) and culture II without metabolic activation just failed to reach the lower limit of induced small colonies of 150 per 106cells. The data are acceptable however since the positive controls showed a substantial increase of induced total colonies compared to the corresponding solvent controls of 248 versus 48 colonies with and 353 versus 107 colonies without metabolic activation. Furthermore the positive controls of the parallel cultures exceeded the lower limit of 150 induced small colonies with and without metabolic activation.