Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this endpoint, three in vitro tests are available. There is no in vivo test.

Ames test:

The potential of the test item (tert-butyl cumyl peroxide at a 90 -98 % purity) to induce reverse mutation in Salmonella typhimurium(strains: TA 1535, TA 1537, TA 1538, TA 98 and TA 100) was evaluated in an Ames test (Willems, 1977). At 1evels of 500 and/or 1000 µg/ plate the test substance had a clearly toxic effect on the strains TA 1538 and TA 100. At 1000 µg/plate, the test substance had no toxic effects on strains TA 1535, TA 1537 and TA98. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.  Under these experimental conditions, tert-butyl cumyl peroxide did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

 

In vitro chromosomal aberration test:

Tert-butyl cumyl peroxide was tested in an in vitro cytogenetic assay using duplicate human lymphocyte cultures prepared from the pooled blood of two human donors in two independent experiments both in the absence and presence of metabolic activation (S9 mix), according to the OECD n° 473 guideline and in compliance with the GLP (CIT, 2004).

Following the 3- and 20-hour treatments without S9, a slight to moderate decrease in mitotic index was noted at dose-levels = 5 mM (33-62% decrease).Following the 44-hour treatment without S9 , a slight decrease in mitotic index was noted at 10 mM (40% decrease). The dose-levels selected for metaphase analysis were as follows: 2.5, 5 and 10 mM, for the 3-hour and the 20-hour treatments, the latter inducing 54 and 62% decrease in mitotic index in the 3-hour and the 20-hour treatments, respectively, 10 mM, for the 44-hour treatment, this dose-level being the highest achievable dose-level.

No significant increase in the frequency of cells with structural chromosomal aberration was noted after 3-, 20- as well as 44-hour treatments.

At the 20-hour harvest time with S9, a slight decrease in mitotic index was noted at 10 mM (28% decrease). Atthe 20-hour harvest time with S9, a slight to marked decrease in mitotic index was noted at dose-levels = 2.5 mM (30-67% decrease). At the 44-hour harvest time with S9 , a moderate decrease in mitotic index was noted at dose-levels = 2.5 mM (42-63% decrease). The dose-levels selected for metaphase analysis were as follows: 2.5, 5 and 10 mM, for the 20-hour harvest time in the first experiment, the latter being the highest achievable dose-level; 5, 7.5 and 10 mM, for the 20-hour harvest time in the second experiment, the latter inducing 67% decrease in mitotic index; 10 mM, for the 44-hour harvest time, this dose-level inducing 60% decrease in mitotic index.

Significant increases in the frequency of cells with structural chromosomal aberration were noted at 5 and 7.5 mM in the second experiment at the 20-hour harvest time (3 versus 0% for the vehicle control, p < 0.05). These increases were neither dose-related, nor reproducible (not observed in the first experiment). In addition the frequency of aberrant cells observed (3%) was clearly within the vehicle control historical range (0-4%). Therefore, these very slight increases were considered as non-relevant. No significant increase in the frequency of cells with structural chromosomal aberration was observed at the 44-hour harvest time.

In conclusion under these experimental conditions, tert-butyl cumyl peroxide did not induce chromosome aberrations in cultured human lymphocytes.

 

In vitro assay for gene mutations in mammalian cells

Tert-butyl cumyl peroxide was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method (, 2012). The study was designed to comply with the experimental methods indicated in OECD Guideline for the testing of chemicals No. 476 and was GLP compliant.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours) at46.2, 35.5, 32.6, 27.3, 21.0 and 16.2 µg/ml without S9, and 101, 78.0, 69.0, 60.0, 52.2, 46.2 and 35.5 µg/ml with S9. The second experiment with S9, 3-hour treatment, was performed with the following concentrations: 78.0, 60.0, 46.2, 35.5, 27.3, 21.0 and 16.2 µg/ml. Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a longer treatment time (24 hours) with the following concentrations: 65.0, 56.5, 49.1, 42.7, 37.2, 32.3 and 28.1 µg/ml.

In the first experiment, without S9, severe toxicity reducing relative total growth (%RTG) to 1% was noted at the highest dose level (46.2 µg/mL). The next lower dose level selected (35.5 µg/mL) yielded slight toxicity reducing RTG to 74% of the concurrent negative control value. No toxicity was noted over the remaining dose levels tested. In the presence of S9 metabolic activation, no cells survived to treatment at the three higher dose levels and marked toxicity reducing RTG to 14% of the concurrent negative control value was observed at 60.0 µg/mL. Mild toxicity, reducing RTG in a range of 34-43%, was noted over the remaining dose levels tested.In the second experiment, without S9 and using a long treatment time, the highest dose level selected (65.0 µg/mL) yielded severe toxicity reducing RTG to 6% of the concurrent negative control value. The next lower dose level of 56.5 µg/mL yielded mild toxicity reducing RTG to 32% while slight toxicity was noted at 49.1 and 42.7 µg/mL reducing RTG to 74% and 79%, respectively.

In the presence of S9 metabolism, no cells survived at the highest dose level selected of 78.0 µg/mL. Marked toxicity reducing RTG to 17% was seen at the next lower dose level (60.0 µg/mL), while no relevant toxicity was noted over the remaining dose levels tested.

No increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the substance at any concentration level. Therefore the test item was concluded not to induce mutation in the Mouse Lymphoma Assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: test in accordance with recognised standards, with acceptable restricition: not GLP, and no data on potential cytotoxicity at highest concentrations than 1000 µg:plate for 3 of the tested strains. In addition, no positive controls included in this test
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test was performed according to the method of Ames et al. (1975).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1598, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
: Rat S9 mix. Liver S9 homogenate was prepared from rats that have been induced with Arochlor 1254
Test concentrations with justification for top dose:
0, 4, 20, 100, 500 and 1000 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
other: not in this test. The reversion properties of each strain are regularly checked
Positive control substance:
other: Methylmethanesulfonate, 4-aminobiphenyl, 9-aminoacridine, N-mehtyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:


METHOD OF APPLICATION:

The procedures used in this mutagenicity assay are described in detail by Ames et al. (1975). Briefly the procedure was as follows:
to 2.5 ml molten soft agar were added 0.1 ml of a fully grown culture of one of the tester strains, 0.1 ml of an appropriate dilution/suspension of the test compound and the liver microsome system if indicated.
The ingredients were thoroughly mixed and immediately poured onto minimal glucose agar plates. After the top agar had been allowed to harden, the plates were incubated at 370c for three days. Then the colonies (revertants which are histidine­ independent) were counted, and the background lawn of bacterial growth examined.
Based on the results of preliminary toxicity tests, the test materials were examined at levels up to 1000 µg/plate. All determinations were carried out in triplicate and appropriate controls were included in each assay.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1538, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes, at the highest concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Number of revertants per plate (mean of 3 plates)
  TA 1535 TA 1537 TA 1538 TA 98 TA 100
Conc. [unit] - MA +MA - MA + MA - MA + MA - MA + MA - MA + MA
0  25 17  12  15   11 14   16  20 61   54
 4  30 13  20  12   13 8 17  22  47   62
 20  24 23  11  24   17  14  20 21   55 54 
 100  19  14 13   15  10  10  14  26 45  69 
 500  23  17  13 14 * PP   17 24  47  54 
1000   13 13   11  12 11 * PP  19   35  PP PP 

*: background lawn of bacterial growth less dense than in control plates

PP: background lawn of bacterial growth sparse or absent, plates crowded with small his- colonies

Conclusions:
tert-butyl cumyl peroxide did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The potential of the test item (tert-butyl cumyl peroxide at a 90 -98 % purity) to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 1538, TA 98 and TA 100) was evaluated in an Ames test.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Incorporation of 4 µg up to non-inhibitory amounts of the test material per plate (i.e. 20-1000 µg) did not induce an increase in the number of his+ revert plates in any of the tester strains, either in the presence or in the absence of the S-9 mix.

 

At 1evels of 500 and/or 1000 µg/ plate the test substance had a clearly toxic effect on the strains TA 1538 and TA 100. At 1000 µg/plate, the test substance had no toxic effects on strains TA 1535, TA 1537 and TA98.

Under our experimental conditions, tert-butyl cumyl peroxide did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January to 19 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline; coherence between data, results and conclusions
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
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 IU/ml + Penicillin G 50.000 units/ml
F 68 Pluronic

Minimal medium B

RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 units/ml + Penicillin G 50.000 units/ml

Complete medium (5%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium (10%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium A (20%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium B (20%)

Minimal medium B
Horse serum (heat-inactivated)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix:
46.2, 35.5, 32.6, 27.3, 21.0 and 16.2 ug/mL for the first experiment (3-hour treatment)
65.0, 56.5, 49.1, 42.7, 37.2, 32.3 and 28.1 ug/mL for the second experiment (24-hour treatment)

Experiments with S9 mix:
101, 78.0, 69.0, 60.0, 52.2, 46.2 and 35.5 ug/mL for the first experiment (3-hour treatment)
78.0, 60.0, 46.2, 35.5, 27.3, 21.0 and 16.2 ug/mL for the second experiment (3-hour treatment)

Vehicle / solvent:
Vehicle used: dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: fluctuation method in medium

DURATION
- Exposure duration: 3 hours in the absence and presence of S9 metabolis
24 hour hours in the absence of S9 metabolism
- Expression time (cells in growth medium): Two days after treatment (48 hours)
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF CELLS EVALUATED: 1.6 cells/well plated in each of 96-well plates (two)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

DETERMINATION OF MUTATION
- Method: Induced mutant frequency (IMF)
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:

(i) The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell
method (126 x 10-6) at one or more doses.

(ii) There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive
responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological
significance. Any increase in mutant frequency should lie outside the historical control range to have biological
relevance.

At low survival levels the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder
effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and
not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is
generally recommended that such data are treated with caution or excluded from consideration.
Statistics:
Dunnett test. Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).
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 specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No precipitate was noted upon addition of the test item to the cultures in all treatment series and by the end of treatment incubation period.
The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.

RANGE-FINDING/SCREENING STUDIES (Cytotoxicity test):
On the basisi of solubility results, a citotoxicity assay was performed in the absence and presence of S9 metabolic activation, at a maximum dose level of 2080 µg/mL and at a wide range of lower dose levels: 1040, 520, 260, 130, 65.0, 32.5, 16.3 and 8.13 µg/mL.

In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived to treatment at the six higher dose levels tested. At the next lower dose level (32.5 µg/mL) slight toxicity was observed reducing relative survival (RS) to 77% of the concurrent negative control value, while no toxicity was noted over the remaining two levels. Using the 24 hour treatment time, no cells survived to treatment at the five higher dose levels. Test item treatment at 65.0 µg/mL yielded moderate toxicity reducing RS to 15%. No relevant toxicity was observed over the remaining dose levels tested.
Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived to treatment at the five higher dose levels (2080, 1040, 520, 260 and 130 µg/mL). Mild toxicity was seen at 65.0 µg/mL (40% RS), while no toxicity was observed over the remaining dose levels tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
Solvent and positive control treatments were included in the mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

ADDITIONAL INFORMATION ON MUTAGENICITY:
Based on the cytotoxicity results obtained in the preliminary assay, two independent assays for mutation to trifluorothymidine resistance were performed using the dose levels described in the following table:

First experiment:
46.2, 35.5, 32.6, 27.3, 21.0 and 16.2 ug/mL (3-hour treatment time in the absence of S9 mix)
101, 78.0, 69.0, 60.0, 52.2, 46.2 and 35.5 ug/mL (3-hour treatment time in the presence of S9 mix)

Second experiment:
65.0, 56.5, 49.1, 42.7, 37.2, 32.3 and 28.1 ug/mL (24-hour treatment time in the absence of S9 mix)
78.0, 60.0, 46.2, 35.5, 27.3, 21.0 and 16.2 ug/mL (3-hour treatment time in the presence of S9 mix)


No increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the test item at any concentration level.
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.

The dose levels for the positive controls were:

-Without S9 mix:methylmethanesulphonate (MMS) 10.0 ug/mL (3 -hour treatment time); 5.00 ug/mL (24 -hour treatment time)

-With S9 mix:benzo(a)pyrene (B(a)P) 2.00 ug/mL

A cell suspension (1 x 106cells/ml) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.

The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS). Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a longer treatment time (24 hours).

After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2 x 105cells/ml.The cultures were incubated at 37°C in a 5% CO2atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2 x 105cells/ml.

Plating for 5-trifluorothymidine resistance

After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0µg/ml) and an estimated 2 x 103cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

Plating for viability

After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Results

A main assay for mutation to trifluorothymidine resistance was performed using the dose levels described in the following table:

 Experiment no

 S9

Treatment time (h)

 Dose levels (ug/mL)

 1

 -

 3

 46.2, 35.5, 32.6, 27.3, 21.0 and 16.2

 1

 +

 3

 101, 78.0, 69.0, 60.0, 52.2, 46.2 and 35.5

 2

 -

 24

 65.0, 56.5, 49.1, 42.7, 37.2, 32.3 and 28.1

 2

 +

 3

78.0, 60.0, 46.2, 35.5, 27.3, 21.0 and 16.2

 

 

 

 

 

Solvent and positive control cultures were included in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range (50-200 x 10-6viable cells). The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). The cloning efficiencies at Day 2 in the negative control cultures and the control growth factor over 2 days fell within the range.The study was accepted as valid.

Survival after treatment

In the first experiment, in the absence of S9 metabolic activation, severe toxicity reducing relative total growth (%RTG) to 1% was noted at the highest dose level (46.2 µg/mL). The next lower dose level selected (35.5 µg/mL) yielded slight toxicity reducing RTG to 74% of the concurrent negative control value. No toxicity was noted over the remaining dose levels tested.

In the presence of S9 metabolic activation, no cells survived to treatment at the three higher dose levels and marked toxicity reducing RTG to 14% of the concurrent negative control value was observed at 60.0 µg/mL. Mild toxicity, reducing RTG in a range of 34-43%, was noted over the remaining dose levels tested.

In the second experiment, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (65.0 µg/mL) yielded severe toxicity reducing RTG to 6% of the concurrent negative control value. The next lower dose level of 56.5 µg/mL yielded mild toxicity reducing RTG to 32% while slight toxicity was noted at 49.1 and 42.7 µg/mL reducing RTG to 74% and 79%, respectively.

In the presence of S9 metabolism, no cells survived at the highest dose level selected of 78.0 µg/mL. Marked toxicity reducing RTG to 17% was seen at the next lower dose level (60.0 µg/mL), while no relevant toxicity was noted over the remaining dose levels tested.

 

At low survival levels the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicityper secan lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration. Accordingly, we have excluded from the statistical analyses, mutation data obtained in the absence of S9 metabolism at 46.2 µg/mL and 65.0 µg/mL in the first and second experiment, respectively.

Mutation results

No increases in mutant frequency were observed in the absence or presence of S9 metabolic activation,following treatment withthe test itemat any concentration level.

Conclusions:
It is concluded that TERT-BUTYL alpha, alpha-DIMETHYLBENZYL PEROXIDE does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

The test item,TERT-BUTYL alpha, alpha-DIMETHYLBENZYL PEROXIDE, was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

The study was designed to comply with the experimental methods indicated in OECD Guideline for the testing of chemicals No. 476 and was GLP compliant.

The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours) at46.2, 35.5, 32.6, 27.3, 21.0 and 16.2 µg/ml without S9, and 101, 78.0, 69.0, 60.0, 52.2, 46.2 and 35.5 µg/ml with S9. The second experiment with S9, 3-hour treatment, was performed with the following concentrations:78.0, 60.0, 46.2, 35.5, 27.3, 21.0 and 16.2 µg/ml.Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a longer treatment time (24 hours) with the following concentrations:65.0, 56.5, 49.1, 42.7, 37.2, 32.3 and 28.1 µg/ml.

In the first experiment, in the absence of S9 metabolic activation, severe toxicity reducing relative total growth (%RTG) to 1% was noted at the highest dose level (46.2 µg/mL). The next lower dose level selected (35.5 µg/mL) yielded slight toxicity reducing RTG to 74% of the concurrent negative control value. No toxicity was noted over the remaining dose levels tested.

In the presence of S9 metabolic activation, no cells survived to treatment at the three higher dose levels and marked toxicity reducing RTG to 14% of the concurrent negative control value was observed at 60.0 µg/mL. Mild toxicity, reducing RTG in a range of 34-43%, was noted over the remaining dose levels tested.

In the second experiment, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (65.0 µg/mL) yielded severe toxicity reducing RTG to 6% of the concurrent negative control value. The next lower dose level of 56.5 µg/mL yielded mild toxicity reducing RTG to 32% while slight toxicity was noted at 49.1 and 42.7 µg/mL reducing RTG to 74% and 79%, respectively.

In the presence of S9 metabolism, no cells survived at the highest dose level selected of 78.0 µg/mL. Marked toxicity reducing RTG to 17% was seen at the next lower dose level (60.0 µg/mL), while no relevant toxicity was noted over the remaining dose levels tested.

No increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment witht he test item at any concentration level.

It is concluded thatTERT-BUTYL alpha, alpha-DIMETHYLBENZYL PEROXIDEdoes not induce mutation at the TK locus of L5178Y mouse lymphoma cellsin vitroin the absence or presence of S9 metabolic activation, under the reported experimental conditions.

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:
2003-2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable (not a gene mutation assay)
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin
(100 µg/mL) and phytohemagglutinin (PHA: a mitogen to stimulate lymphocyte division)
- Human lymphocytes were prepared from whole blood samples obtained from two healthy donors
(one male and one female for each experiment) and collected into heparinized sterile tubes.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
With a treatment volume of 27.5 µL/5.5 mL culture medium, the treatment-levels were as follows:
• 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5 and 10 mM, for the first experiment, both with and without S9 mix,
• 0.63, 1.25, 2.5, 5, 7.5 and 10 mM, for the second experiment, without S9 mix,
• 1.25, 2.5, 3.75, 5, 7.5 and 10 mM, for the second experiment, with S9 mix.
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice: test item was soluble in DMSO at 413,66 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION:
see Executive summary

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes.
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account.
Statistics:
For each test and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the 2 test, in which p = 0.05 was used as the lowest level of significance.
Species / strain:
lymphocytes: human
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:
Interpretation of results (migrated information):
negative

The test item tert-butyl cumyl peroxide was not clastogenic to mammalian cells in the presence and in the absence of metabolic activation.
Executive summary:

Tert-butyl cumyl peroxiode was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of two human donors in two independent experiments both in the absence and presence of metabolic activation (S9 mix), according to the OECD n° 473 Guideline and EC 92/69/EEC B.10 guidelines in compliance with the Principles of Good Laboratory Practice.

The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254.

The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account.

In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:

⋅ without S9 mix, cells were exposed continuously to the test or control items until harvest,

⋅ with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

Experiments without S9 mix:

Following the 3- and 20-hour treatments, a slight to moderate decrease in mitotic index was noted at dose-levels ≥ 5 mM (33-62% decrease).

Following the 44-hour treatment, a slight decrease in mitotic index was noted at 10 mM (40% decrease). The dose-levels selected for metaphase analysis were as follows: 2.5, 5 and 10 mM, for the 3-hour and the 20-hour treatments, the latter inducing 54 and 62% decrease in mitotic index in the 3-hour and the 20-hour treatments, respectively, 10 mM, for the 44-hour treatment, this dose-level being the highest achievable dose-level.

No significant increase in the frequency of cells with structural chromosomal aberration was noted after 3-, 20- as well as 44-hour treatments.

Experiments with S9 mix:

At the 20-hour harvest time in the first experiment, a slight decrease in mitotic index was noted at 10 mM (28% decrease). At

the 20-hour harvest time in the second experiment, a slight to marked decrease in mitotic index was noted at dose-levels ≥ 2.5 mM (30-67% decrease). At the 44-hour harvest time, a moderate decrease in mitotic index was noted at dose-levels ≥ 2.5 mM (42-63% decrease).

The dose-levels selected for metaphase analysis were as follows: 2.5, 5 and 10 mM, for the 20-hour harvest time in the first experiment, the latter being the highest achievable dose-level; 5, 7.5 and 10 mM, for the 20-hour harvest time in the second experiment, the latter inducing 67% decrease in mitotic index; 10 mM, for the 44-hour harvest time, this dose-level inducing 60% decrease in mitotic index.

Significant increases in the frequency of cells with structural chromosomal aberration were noted at 5 and 7.5 mM in the second experiment at the 20-hour harvest time (3 versus 0% for the vehicle control, p < 0.05). These increases were neither dose-related, nor reproducible (not observed in the first experiment). In addition the frequency of aberrant cells observed (3%) was clearly within the vehicle control historical range (0-4%). Therefore, these very slight increases were considered as non-relevant.

No significant increase in the frequency of cells with structural chromosomal aberration was observed at the 44-hour harvest time.

In conclusion under these experimental conditions, the test item t-BUTYL-CUMYL PEROXYDE did not induce chromosome aberrations in cultured human lymphocytes.

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

Genetic toxicity in vivo

Description of key information

No in vivo testing is required based on all in vitro results

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), Tert-butyl-a,a-dimethylbenzyl peroxide (TBCP) is not classified for germ cells mutagenicity.