Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Test substance has provided negative gene tox results in two separate reverse mutation assays using bacterial cells with and without S9 activiation. Negative in both Chinese Hamster Lung fibroblast and Human Lymphocyte chromosomal aberation assays. No in vivo assays available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-72 hrs
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study according to OECD 471 and OECD & French Government GLP. Study report includes, QA & GLP statement and C of A for test article. Study report does not include analytical verification of test substance concentration or stability in study media.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
The five strains (Ames et aI., 1975; Maron and Ames, 1983): TA 1535, TA 1537, TA 98, TA 100 and TA 102, are supplied by B.N. Ames' Laboratory (University of California, Berkeley, U.S.A.). They are stored in a cryoprotective medium containing 1 ml nutrient broth and 0.09 ml dimethylsulfoxide in a liquid nitrogen container.

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine. In addition, to increase their sensitivity to mutagenic substances, additional mutations have been added:
-the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to
large molecules that do not penetrate the normal bacteria cell wall,
-the uvr B mutation is a deletion of a gene code for the DNA excision repair system, which renders the bacteria unable to use this repair
mechanism to remove the damaged DNA,
-the addition of the pKM 10 1 ampicillin resistant plasmidic R -factor in the strains T A 98, TAI00 and TAl 02 enhances their detection
sensitivity to some mutagens.
-in addition, the pAQl tetracycline resistant plasmidic factor has been added to the TAI02 strain.

Genotype of the bacterial strains
Strains Histidine mutation Additional mutations
TA 1535 His G46 rfa uvrB
TA 100 His G46 rfa uvrB Factor R
TA 102 His G 428 (pAQl) rfa Factor R
TA 1537 His C 3076 rfa uvrB
TA 98 His D 3052 rfa uvrB Factor R

The T A 1535, TAl 00 and TAl 02 strains are reverted by base-pair substitution mutagens and the T A 1537 and T A 98 strains by frameshift
mutagens. In addition, the TAl 02 strain detects oxidative mutagens.
Species / strain / cell type:
other: TA1535, TA100, TA102, TA1537, TA98
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500, 5000 ug/plate
Vehicle / solvent:
The vehicle was acetone, batch No. 074BR (Prolabo, 75011 Paris, France).
Untreated negative controls:
yes
Remarks:
acetone
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
other: Five known mutagens, dissolved in dimethylsulfoxide (except for Mitomycin C in distilled water), were used to check the sensitivity of the test system: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C, 2-anthramine
Details on test system and experimental conditions:
Rationale for dose selection
The top dose was selected according to the following criteria specified in international regulations:
• for non-toxic, freely soluble test substances, the top dose is 5000 ug/plate,
• for non-toxic, poorly soluble test substances, the top dose is the lowest precipitating dose.
• for toxic test substances, irrespective of solubility, the top dose is based on the level of toxicity: clearing of the bacterial lawn and/or reduction in the number of revertants when compared to the controls. However, precipitation should not interfere with the scoring of the test.

Treatment
The day before treatment, cultures were inoculated from frozen permanents: a crystal was sampled under sterile conditions and put into approximately 6 ml of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37°C for about 14 hours.

The tests were performed according to:
-direct plate incorporation method (both tests without S9 mix, first test with S9 mix): 0.05 ml of the test substance solution, 0.5 ml of S9 mix when required and 0.1 ml of the strain were mixed with 2 ml of overlay agar containing traces of the relevant amino-acid and biotin and maintained at 45°C.After rapid homogenization, the mixture was spread out on a Petri plate containing minimum medium.
-preincubation method (second test with S9 mix): 0.05 ml of the test substance solution, 0.5 ml of S9 mix and 0.1 ml of the strain were incubated for 60 minutes at 37°C prior adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Artek counter, model 880, O.S.L, 75015 Paris, France).

Preliminary toxicity test
To assess the toxicity of the test substance to the bacteria, six doses (one plate/dose) were tested in the T A 98, TAl 00 and TAl 02 strains, with or without S9 mix.The sterility of the test substance was checked during this test and was found to be satisfactory.

Mutagenicity tests
In two independent tests, five doses of the test substance (three plates/dose) were tested on each strain, with or without S9 mix. A third test was performed with S9 mix.
During each test, the following controls were made using triplicate plates:
-vehicle control: strain treated with the vehicle,
-positive control: strain treated with the known mutagens previously mentioned.
The sterility of the S9 mix was checked during each test (before the beginning and at the end of the experiment) and was found to be satisfactory.
Evaluation criteria:
Treatment of results
During each test, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtainedin the presence of the vehicle), are presented in a table in the attached study report.
Acceptance criteria:
This study was considered valid because the following criteria were fully met:
• the number of revertants of the controls was within the range of our historical data,
• the number of revertants of the positive controls was higher than that of the controls and
within the range of our historical data.
Evaluation criteria:
Biological relevance of the results was considered first. In addition, the following criteria may be used as an aid for determining a positive response:
• a dose-related increase in the number of revertants,
and/or
• a reproducible increase in the number of revertants (i.e. a doubling in at least one strain when compared to that of the controls) for at least one of the doses.
Species / strain:
other: TA1535, TA100, TA102, TA1537, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the reported experimental conditions, the test substance LUPEROX 231 (1, I-DI-(tertBUTYLPEROXY)- 3,3,5-TRIMETHYLCYCLOHEXANE) did not show mutagenic activity in the reverse mutation assay on Salmonella typhimurium.
Executive summary:

RESULTS

MUTAGENICITY TESTS: The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria and within the range of our historical data. The top dose was selected according to the criteria specified in the international regulations. Since the test substance was non-toxic, freely soluble, the top dose was 5000 ug/plate. The selected range dose was: 312.5,625, 1250,2500 and 5000 ug/plate. The test substance did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the five strains. Since an increase in the number of revertants was obtained at 312.5 and 625 ug/plate for the TA 1537 strain in the first test with S9 mix using the direct incorporation method, but not in the second test using the preincubation method, a third test with S9 mix using the direct incorporation method, was performed using the following doses: 125, 250, 500, 1000 and 2000 ug/plate. No increase was recorded.

CONCLUSION: Under the reported experimental conditions, the test substance LUPEROX 231 (1, I-DI-(tertBUTYLPEROXY)- 3,3,5-TRIMETHYLCYCLOHEXANE) did not show mutagenic activity in the reverse mutation assay on Salmonella typhimurium.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
up to 24 hour cell treatment
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study which appears to conform to modern standards of study conduct. However, there is no separate QA or GLP statement. the study report does not purport that the study conforms to either a national/international protocol nor national/international GLP standards. Though the study report includes a statement of chemical purity (97.9%), including some identification of impurities, there is no separate C of A. as well, the study report asserts that a separate assessment of compound stability was conducted, showing that compound purity was 97.8% following the conduct of the study, however, there is no separate report of this incorporated into this study report. neither is there an assessment of chemical substance stablity in the study medium. The testing facility address is not provided. The study report is not dated, though the compound receipt date is given as October 16, 1998 which seems to comport with the study number 98-113.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
No study protocol is referenced in the report. However, the study report appears consistent with an approach essentially equivalent to OECD 473.
Deviations:
not specified
Remarks:
No study protocol is referenced in the report. However, the study report appears consistent with an approach essentially equivalent to OECD 473.
Principles of method if other than guideline:
An in vitro chromosomal study was carried out with a Chinese hamster lung fibroblast cell line (CHL/IU).
GLP compliance:
not specified
Remarks:
while there is no separate GLP certification, the study report reflects a standard consistent with international GLP criteria
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung fibroblast cell line (CHL/IU)
Details on mammalian cell type (if applicable):
Test cell line
Chinese hamster lung fibroblast cells (CHL/IU) donated on January 13, 1985 by the National Institute of Health Sciences, Division of Genetics and Mutagenesis (previously National Health Institute, Division of Mutagenicity) were used. The cells were stored frozen with liquid nitrogen after adding 10% DMSO to the cell suspension, it was thawed in the culture medium, and subcultures up to 7 generations were used after thawing.

Culture medium
An Eagle-MEM powdered culture medium (Gibco Laboratories, Lot Nos. 1015566, 1019178) was prepared with conventional procedures, and 10% inactivated (heat treatment at 56°C for 30 min) bovine serum (Gibco Laboratories, Lot Nos. 1013320, 1015812, 1019034) was added.

Culture conditions
The test cells were cultured in a CO2 incubator (Napco Co.) with 5% CO2 and 95% air at 37°C under humidified conditions.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix The S9 mix used was a frozen product of the drug metabolizing enzyme fraction (S9) of rat liver homogenate with the cofactor added, which was purchased from Kikkoman Corp. (Lot No. CAM-394, manufactured on December 4, 1998.
Test concentrations with justification for top dose:
the doses for the chromosomal aberration test in the brief treatment method were set at 93.75, 187.5, 375, 750, 1500 and 3000 µg/mL in the absence of S9 mix and 375, 750, 1500 and 3000 µg/mL in the presence of S9 mix. Furthermore, the doses in the continuous 24 h treatment method were set at 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL.
Vehicle / solvent:
acetone (99.5%)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: The positive control substance used was 1-methyl-3-nitro-1-nitrosoguanidine (MNNG, Aldrich Chemical Company, Lot No. 00613PN, purity of 97%) for the continuous treatment method, and it was 3,4-benzo[a]pyrene (B[a]P, Sigma Chemical Company, Lot No. 57F-343
Details on test system and experimental conditions:
Preparation of study substance test solution
For study substance test solutions, the test substance was dissolved in acetone to prepare a test solution at the maximum dose (stock solution). Then a portion of the stock solution was successively diluted with acetone to prepare test solutions of the desired concentrations. The amount of those study substance test solutions to be added was set at 0.5 vol% of the amount of culture media in each Petri dish.

Cell treatment
In the brief treatment method, 5 mL of the culture medium containing 4 x 103 cells/mL was added to a 6 cm diameter plastic Petri dish (Becton-Dickinson Co.), 3 days after starting the culture in the absence of S9 mix, the culture medium was removed, except for 3 mL in each Petri dish, and 0.015 mL of acetone (negative control) or study substance test solution was added to the dish. Furthermore, in the presence of S9 mix, the culture medium was removed, except for 2.5 mL in each Petri dish, 0.5 mL of S9 mix was added, and 0.015 mL of acetone or study substance test solution was added to the dish. After 6 h of cultivation, the culture medium was removed, and a fresh batch of 5 mL culture medium was added to continue culturing for 18 h. On the other hand, in the continuous treatment method, the cells were cultured with the same procedures as those in the brief treatment method, 3 days after starting the culture, 0.025 mL of acetone or study substance test solution was added, and culturing was continued for 24 or 48 h. After completing culturing, the culture medium was removed, the cell surface was washed twice with saline, and then an aqueous solution of 10% formaldehyde was added for fixation for about 10 min. After fixation, it was washed with water and stained with an aqueous solution of 0.1% w/v crystal violet for about 10 min. After washing with water, it was dried at room temperature by allowing it to stand overnight.
Incidentally, if the dose was 100 µg/mL or higher, the oil droplet or oil film-like precipitation of the study substance was observed when the test solutions were added to the culture medium, and the precipitates formed remained until culturing ended.

Cell proliferation rate measurement
The cell density of the fixed and stained cells in the above section 8-2) was determined by measuring the stain’s contrast with a monolayer culture cell densitometer (Monocellator II, Olympus Corp.), and the cell proliferation rate was determined for each dose group with the cell proliferation in the negative control group set at 100%.
As shown in the following table, slightly over 50% cell proliferation inhibition was observed at 500 µg/mL in the absence of S9 mix in the brief treatment method, but no cell proliferation inhibition over 50% was observed at other doses. Furthermore, in the presence of S9 mix, no cell proliferation inhibition over 50% was observed at any of doses, and the 50% cell proliferation inhibition dose was judged to be over 3000 µg/mL. In the continuous treatment method, the 50 µg/mL and over doses exhibited over 50% cell proliferation inhibition in the 24 h treatment, and the 50% cell proliferation inhibition dose was judged to be in the range of 25-50 µg/mL. In the 48 h treatment, all doses exhibited over 50% cell proliferation inhibition, and approximately 50% cell proliferation inhibition was observed at 125 µg/mL.
Chromosomal aberration test
Doses of test and positive control substances
The doses of the study substance were determined from the results of the cell proliferation inhibition experiments, with 3000 µg/mL, corresponding to 10 mM as the maximum dose, 6 doses of 93.75, 187.5, 375, 750, 1500 and 3000 µg/mL (common ratio of 2) in the absence of S9 mix and 4 doses of 375, 750, 1500 and 3000 µg/mL (common ratio of 2) in the presence of S9 mix. In the continuous 24 h treatment method, the doses were set at 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL so that doses with less and more than the 50% cell proliferation inhibition dose were included, and data for more than 3 doses was obtainable. The respective doses used for the positive control substances were 2.5 µg/mL MNNG and 10 µg/mL B[a]P.

Preparation of study and positive control substance test solutions
For study substance test solutions, the test substance was dissolved in acetone to prepare a test solution at the maximum dose (stock solution). Then a portion of the stock solution was successively diluted with acetone to prepare test solutions at the desired concentrations. The positive control substance test solutions prepared were 0.5 mg/mL MNNG and 2.0 mg/mL B[a]P.

Cell treatment
To a 6 cm round plastic Petri dish (Becton-Dickinson Co.), 5 mL of the culture medium containing 4 x 103 cells/mL was added, cultured for 3 days, and then treated using the following procedures. Four Petri dish cultures were used per dose, two dishes were for preparation of chromosome specimens, and the remaining 2 dishes were for cell proliferation rate measurement. However, no cell proliferation rate measurement was carried out in the positive control groups, and only two Petri dishes were used to prepare chromosome specimens.
In the absence of S9 mix, the culture medium was removed, except for 3 mL in each Petri dish, and 0.015 mL of acetone, study substance test solution, or MNNG test solution was added to the dish. Furthermore, in the presence of S9 mix, the culture medium was removed, except for 2.5 mL in each Petri dish, 0.5 mL of S9 mix was added, and 0.015 mL of acetone, study substance test solution or B[a]P test solution was added to the dish. In both the absence and presence of S9 mix, the culture medium was removed after culturing for 6 h, and 5 mL of a fresh batch of the culture medium were added to continue culturing for 18 h.
In the continuous treatment method, 0.025 mL of acetone, study substance test solution or MNNG test solution was added to the Petri dish, and cultured for 24 h.
Incidentally, if the dose was 93.75 µg/mL or higher, the oil droplet or oil film-like precipitation of the study substance was observed when the test solutions were added to the culture medium, and the precipitates formed remained until culturing ended.

Chromosomal sample preparation and cell proliferation measurement
At 2 h before specimen preparation, colcemid (Gibco Laboratories, Lot No. 1010169) was added to each Petri dish culture so that the final concentration was 0.2 µg/mL. After completing culturing, the culture medium was removed, the cells were treated with 2 mL of an aqueous solution of 0.2% trypsin to peel them off the Petri dish, placed in a centrifugation tube containing 5 mL of the culture medium, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, the cell sediment was suspended in 4 mL of an aqueous hypotonic solution of 75 mM potassium chloride for 37°C for 15 min. After hypotonic treatment, 1 mL of a cold mixture (v/v) of methanol and acetic acid (3:1) prepared immediately before use was added for fixation. The supernatant was discarded after centrifugation at 1000 rpm for 5 min, and the cell sediment was suspended and fixed in 4 mL of the freshly prepared fixative. After repeating the procedure 3 times, the cells were suspended at a suitable density in a small amount of fixative, one drop each was dropped at 2 sites on a slide glass and dried at room temperature by allowing them to stand overnight. After drying, the cells were stained for 15 min with a 1.4 vol% Giemsa solution diluted with Sørensen’s buffer (pH 6.8, manufactured by Iatron Inc., Lot No. 1478). After washing with water, the stained cells were dried at room temperature to obtain a chromosome specimen. Three specimens were prepared per Petri dish.
For cell proliferation rate measurement, the culture medium was removed after culture, the cell surface was washed twice with saline, and an aqueous solution of 10% formaldehyde was added to carry out fixation for about 10 min. After fixation, the cells were washed with water, stained for about 10 min with an aqueous solution of 0.1 w/v violet, washed with water and dried. A monolayer culture cell densitometer (Monocellator II, Olympus Corp.) was used, and the cell proliferation rate was determined for each dose group with the cell proliferation in the negative control (solvent) group set at 100%.

Chromosome observation
The uncovered chromosomes were observed with a 60X objective lens and a total magnification of 600X. All specimens were coded, and the blinded observation method was used. For clearly distinguishable metaphase images of 25 ± 2 chromosomes at the respective doses, 100 units per dish, that is, a total of 200 units from 2 dishes, per dose of the chromosomes were observed.

Chromosomal aberration classification and tally
In the case of structural abnormalities, the chromosomal aberrations observed were chromatid breaks and exchanges, chromosome breaks and exchanges, and others (fragmentation, etc.) In the case of numerical aberrations, only polyploid cells (polyploids) were recorded.
Gaps (chromatid and chromosome) were recorded as an abnormality, but they were not included in the structural abnormalities. The gaps are achromatic sites narrower than the chromatid width.
For tallying the chromosomal aberrations, any cell having at least one of the abnormalities classified above was recorded as an abnormal cell and tallied by the kind of abnormality. The total number of structural aberrations was the number of abnormal cells found in the 200 cells observed.


Evaluation criteria:
For test result assessment, the frequency of structural aberrations and the appearance of polyploid cells was tested for a significant difference (the significance level used was 5% or 1% divided by the number of groups treated considering the multiplicity) between the negative control and the respective dose groups with Fisher’s direct probability method if the multiple sample Chi Squared test carried out showed a significant difference (significance level less than 5%). If the frequency of the appearance of chromosomal aberration cells in the study substance groups significantly increased at 2 doses or more compared with the negative control group, and dose-dependency or reproducibility was simultaneously observed, a positive result was assessed.
Statistics:
Chi Squared
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The frequency of the appearance of cells having structural chromosomal aberrations was low, 1.5% in the negative control group. Furthermore, the results in the study substance groups were also low, in the range of 0-1.0%. The frequency of the appearance of structural chromosomal aberrations with MNNG in the positive control group was 98.0%, confirming significant chromosomal aberration induction.

           No polyploids exhibiting numerical aberrations were observed in the negative and positive control groups. In the study substance groups, they were observed at a low frequency in the range of 0-0.5%.

 

Chromosomal aberration study (brief treatment: in the presence of S9 mix)

           The results obtained are shown in Table 2. The frequency of the appearance of cells having structural chromosomal aberrations was low, at 1.0% in the negative control group. Furthermore, the results in the study substance groups were also low, in the range of 0-0.5%. The frequency of the appearance of structural chromosomal aberration with B[a]P in the positive control group was 46.5%, confirming significant chromosomal aberration induction.

           No polyploids exhibiting numerical aberrations were observed in the negative and positive control groups. Furthermore, in the study substance groups, they were observed at a low frequency of 0.5% at 750 µg/mL.

 

Chromosomal aberration study (continuous treatment: 24 h treatment)

           The results obtained are shown in Table 3. Because there was no apparent increase in chromosomal aberrations by the brief treatment method, continuous 24 h treatment was carried out. As a result, no cells having structural chromosomal aberrations were observed in the negative control group. In the study substance groups, they were observed at low frequencies in the range of 0-1.0%, but the results were not statistically significantly different from the negative control group. The frequency of the appearance of structural chromosomal aberration with MNNG in the positive control group was 97.5%, confirming significant chromosomal aberration induction.

           The frequency of the appearance of polyploids was low, at 1.0% in the negative control group, and the results were also low, in the range of 0-1.0% in the study substance groups. No polyploidy was observed in the negative control group.

no chromosomal aberration induction was observed by the brief treatment method in the presence or absence of S9 mix, and by the continuous 24 h treatment method.

           Therefore, the chromosomal aberration induction ability of 1,1-bis(tert-butylperoxy)- 3,3,5-trimethylcyclohexane in CHL/IU cells under the experimental conditions employed was assessed as negative. The results of the present study also provided an apparent negative assessment by the biological assessment standards [4], that if the frequency of the appearance of CHL/IU cells having chromosomal aberrations was less than 5%, the assessment was negative.

           Incidentally, no suspicious finding of any significant delay in the cell cycle was observed, and thus no confirmation tests such as continuous 48 h treatment, etc., were carried out.

Conclusions:
No apparent increase in the cells having chromosomal aberrations was observed in the absence or presence of S9 mix in the brief treatment method. Furthermore, no apparent increase of chromosomal aberrations was observed in the continuous 24 h treatment method. From the results obtained, the chromosomal aberration inducing ability of 1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane in CHL/IU cells was determined to be negative under the test conditions employed in this study.
Executive summary:

The doses for the chromosomal aberration test in the brief treatment method were set at 93.75, 187.5, 375, 750, 1500 and 3000 µg/mL in the absence of S9 mix and 375, 750, 1500 and 3000 µg/mL in the presence of S9 mix. Furthermore, the doses in the continuous 24 h treatment method were set at 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL. No apparent increase in the cells having chromosomal aberrations was observed in the absence or presence of S9 mix in the brief treatment method. Furthermore, no apparent increase of chromosomal aberrations was observed in the continuous 24 h treatment method.

From the results obtained, the chromosomal aberration inducing ability of 1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane in CHL/IU cells was determined to be negative under the test conditions employed in this study

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: according to OECD 476
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 without metabolic activation, 4 hours treatment with 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:
without S9 mix: 46.9, 93.8, 187.5, 375.0 (P), 750.0 (P), 1500.0 (P) µg/mL
with S9 mix 46.9, 93.8, 187.5, 375.0 (P), 750.0 (P), 1500.0 (P) µg/mL
Experiment II
without S9 mix: 12.5, 25.0, 50.0, 100.0, 150.0, 200.0 µg/mL
with S9 mix: 150.0, 200.0, 250.0, 300.0, 350.0, 375.0 (P)

p = precipitation

Following the expression phase of 48 hours the cultures at 46.9 µg/mL in experiment I with metabolic activation, at 12.5 µg/mL without and at 150 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 1500 µg/mL in experiment I without metabolic activation were not continued due to exceedingly severe cytotoxic effects.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)
- 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
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Water solubility: immiscible
- Precipitation: Precipitation of the test item visible to the naked eye was noted in both experiments at 375 µg/mL and above with and without metabolic activation.
- 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 23.4 µg/mL and 3000 µg/mL were used. The highest concentration in the pre-experiment was about 10 mM based on the purity (99.16 %) and the molecular weight (302 g/mol) of the test item.
Relevant toxic effect (relative suspension growth, RSG below 50% of the solvent control) occurred at 750 µg/mL and above following 4h treatment without metabolic activation. After 24h treatment cytotoxic effects were noted at 187.5 µg/mL and above without metabolic activation. Following 4h treatment with metabolic activation the RSG fluctuated around 50% from 23.4 to 750 µg/mL. At higher concentrations the RSG showed a distinct cytotoxic effect.
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 750 µg/mL and above in all parts of the pre-experiment.
Therefore, the maximum concentration of the main experiments was adjusted according to the results of the pre-experiment. To overcome problems with possible deviations in toxicity or solubility both main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: complies


ADDITIONAL INFORMATION ON CYTOTOXICITY: none
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 THF - 100.0 128 254 100.0 159 285
Pos. control with MMS  19.5 -  27.0 369 254  32.4 297 285
Test item  46.9 -  86.6 123 254 109.4 189 285
Test item  93.8 - 101.3 146 254  85.3 147 285
Test item  187.5 -  48.9 166 254  25.1 220 285
Test item 375.0 (p) -  35.8 227 254  9.0 321 285
Test item 750.0 (p) -  23.9 213 254  14.8 142 285
Test item 1500.0 (p) - culture was not continued# culture was not continued#
       
Solv. control with THF + 100.0 141 267 100.0 182 308
Pos. control with CPA   3.0 +  65.0 230 267  54.3 206 308
Pos. control with CPA   4.5  +   33.1 363 267  10.6 1257 308
Test item  46.9  +  culture was not continued## culture was not continued##
Test item  93.8  +   71.6 158 267  52.9 249 308
Test item  187.5  +   71.5 147 267  43.9 382 308
Test item 375.0 (p)  +   39.6 169 267  31.6 351 308
Test item 750.0 (p)  +   34.2 208 267  17.1 435 308
Test item 1500.0 (p)  +   16.3 314 267  22.5 425 308
Experiment II / 24 h treatment   culture I culture II
Solv. control with THF - 100.0 162 288 100.0 180 306
Pos. control with MMS  13.0 -  17.9 725 288  22.1 562 306
Test item  12.5 - culture was not continued# culture was not continued#
Test item  25.0 -  84.7 174 288  69.3 310 306
Test item  50.0 -  65.4 237 288  90.8 172 306
Test item  100.0 -  85.6 181 288  63.0 215 306
Test item  150.0 -  37.0 231 288  38.3 219 306
Test item  200.0 -  6.2 244 288  14.1 193 306
Experiment II / 4 h treatment   culture I culture II
Solv. control with THF + 100.0 155 281 100.0 139 265
Pos. control with CPA   3.0 +  51.8 226 281  71.0 157 265
Pos. control with CPA   4.5 +  58.5 298 281  18.3 410 265
Test item  150.0 + culture was not continued# culture was not continued#
Test item  200.0 + 125.1  91 281  92.2 183 265
Test item  250.0 + 156.7 121 281  86.4 171 265
Test item  300.0 +  58.0 155 281  37.9 167 265
Test item  350.0 +  39.5 148 281  38.0 209 265
Test item 375.0 (p) +  55.2 170 281  46.1 243 265

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

#    culture was not continued due to strong toxic effects

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

p    precipitation or phase separation observed by the naked eye

 

Conclusions:
In conclusion it can be stated that 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 3,3,5-trimethylcyclohexylidene diperoxide to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

This study was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations:

Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998,
adopted July 21, 1997, Guideline No. 476 “In vitro Mammalian Cell Gene Mutation Test”.

Commission Regulation (EC) No. 440/2008, B17: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:          46.9; 93.8; 187.5; 375; and 750 µg/mL
with S9 mix:    93.8; 187.5; 375; 750; and 1500 µg/mL

Experiment II:

without S9 mix:          25; 50; 100; 150; and 200 µg/mL
with S9 mix:    200; 250; 300; 350; and 375 µg/mL

Precipitation of the test item visible to the naked eye was noted in both experiments at 375 µg/mL and above with and without metabolic activation.

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed in experiment I at 187.5 µg/mL and above without and at 375 µg/mL and above with metabolic activation. In experiment II cytotoxic effects as described above occurred at 150 µg/mL and above without and at 350 µg/mL and above with metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments up to the first precipitating concentration of 375 µg/mL. The threshold of 126 plus each solvent control count was exceeded in the second culture of the first experiment without metabolic activation at 375 µg/mL. In the presence of metabolic activation the threshold was exceeded at 1500 µg/mL in the first culture of the first experiment and at 187.5 µg/mL and above in the second culture of the first experiment. In the second experiment the threshold was solely exceeded at the lowest analysed concentration of 25.0 µg/mL in culture II without metabolic activation. The increased values of the mutation frequency were judged as biologically irrelevant since, with two irreproducible exceptions they all occurred at precipitating concentrations. Precipitation of a test item is a known source of artefacts in the mouse lymphoma assay since L5178Y cells grow as suspension. At the end of treatment the test item is removed by centrifugation. However, a precipitate can not completely be removed and arbitrary amounts of test item are carried over to the following steps of the assay leading to non-defined treatment intervals.

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 determined in the first culture of experiment I with and without metabolic activation and in the second culture of the second experiment with metabolic activation. Those trends however were not considered relevant since they were not reproduced in the parallel cultures. Furthermore, the mutation frequency did not exceed the threshold in the second experiment with metabolic activation. The trend observed in the first culture of the first experiment with metabolic activation is based on increased mutation frequency values generated at precipitating concentrations.

In this study the range of the solvent controls was from 128 up to 182 mutant colonies per 106 cells; the range of the groups treated with the test item was from 91 up to 435 mutant colonies per 106 cells. The solvent controls of the experimental part of the first experiment with metabolic activation (culture II) and of the second experiment without metabolic action (culture II) slightly exceeded the recommended 50 – 170 x 106 control range as stated under paragraph 8.12, acceptability of the assay of this report. The data are considered acceptable however, since the solvent control of the parallel culture (141 and 162 colonies per 106 cells, respectively) were fully acceptable. Furthermore, the IWGT recommendations published in 2003 (11) recommended an acceptable range from 50 to 200 colonies per 106 cells.

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.

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

Genetic toxicity in vivo

Description of key information

waived as all in vitro studies are negative

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Additional information

Test substance has provided negative gene tox results in two separate reverse mutation assays using bacterial cells with and without S9 activiation. Negative in both Chinese Hamster Lung fibroblast and Human Lymphocyte chromosomal aberation assays. No in vivo assays available.

Justification for selection of genetic toxicity endpoint

All in vitro studies were reliable and negative.  

Short description of key information:

Test substance has provided negative gene tox results in two separate reverse mutation assays using bacterial cells with and without S9 activiation. Negative in both Chinese Hamster Lung fibroblast and Human Lymphocyte chromosomal aberation assays. No in vivo assays available.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Test substance has provided negative gene tox results in two separate reverse mutation assays using bacterial cells with and without S9 activiation. Negative in both Chinese Hamster Lung fibroblast and Human Lymphocyte chromosomal aberation assays. No in vivo assays available.