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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames + MLA, K1: The study was performed according to OECD guidelines and GLP. The substance is positive for mutagenicity with metabolic activation in vitro in an Ames test and negative in a Mouse Lymphoma Assay.

An in vitro cytogenicity, micronucleus assay (K1), is negative.

Link to relevant study records

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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:
17 March 2003 - 09 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to OECD guidelines and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: see below
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: see below
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Results of the dose rangefinding study were used to select doses tested in the mutagenicity assay. Doses tested with all tester strains in the presence and absence of S9 mix were 100, 333, 1000, 3330 and 5000 μg per plate.

Based on the toxicity observed in the presence of S9 mix in the first trial, the doses tested in the confirmatory assay were selected. Doses tested in the confirmatory assay were 10.0, 33.3, 100, 333, 500, 750, 1000, 2000 and 5000 μg per plate in the presence of S9 mix and 100, 333, 1000, 3330 and 5000 μg per plate in the absence of S9 mix.

Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 51 ± 4 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: Revertant colonies were counted by automated colony counter or by hand

DETERMINATION OF CYTOTOXICITY
Condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control and recorded along with the revertant counts for that dose.
Evaluation criteria:
Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows:

Tester Strains TA98, TA100, and WP2uvrA. For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
None
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
at 5000 µg/plate precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
at 5000 µg/plate precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+ S9 mix: from 750 µg/plate, -S9-mix: 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+ S9 mix: from 750 µg/plate, -S9-mix: 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+ S9 mix: from 750 µg/plate, -S9-mix: 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the initial mutagenicity assay, Trial 24908-B1, no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. In this trial, a 2.2-fold increase was observed with tester strain WP2uvrA in the presence of S9 mix. However, this increase was not clearly dose-responsive, and therefore did not meet the criteria for a positive evaluation. In order to clarify this increase, the test article was re-tested with tester strain WP2uvrA in the presence of S9 mix in Trial 24908-D1. All data generated in Trial 24908-B1 were acceptable with the exception of tester strains TA98 and TA1537 in the presence of S9 mix, where only two non-toxic doses were observed. For this reason, the test article was re-tested with these tester strains in Trial 24908-D1.

Based on the toxicity observed in the presence of S9 mix in Trial 24908-B1 (which was not observed in the dose rangefinding study), the doses tested in the confirmatory assay were selected. Doses tested in the confirmatory assay were 10.0, 33.3, 100, 333, 500, 750, 1000, 2000 and 5000 μg per plate in the presence of S9 mix and 100, 333, 1000, 3330 and 5000 μg per plate in the absence of S9 mix.

In the confirmatory mutagenicity assay, Trial 24908-C1, all data were acceptable and a 3.0-fold positive increase was observed in the mean number of revertants per plate with tester strain WP2uvrA in the presence of S9 mix. No positive increases were observed with any other tester strains in the presence or absence of S9 mix. In this trial, a 1.8-fold increase was observed with tester strain TA100 in the presence of S9 mix. However, this increase did not meet the 2-fold criteria to be considered a positive response. In order to clarify this increase, the test article was re-tested with tester strain TA100 in the presence of S9 mix in Trial 24908-D1.

In the repeat mutagenicity assay, Trial 24908-D1, all data were acceptable positive increases were observed in the mean number of revertants per plate with tester strains TA100 (3.0-fold) and WP2uvrA (2.3-fold)in the presence of S9 mix. In addition, increases in the mean number of revertants per plate were observed with tester strain TA1537 in the presence of S9 mix, however, these increases were not clearly dose-related. In addition, all observed values were within the acceptable vehicle control range for this strain. The observed increases appear to be the result of a lower than routinely observed TA1537 mean vehicle control value and were not
considered to be biologically relevant. No positive increases were observed with tester strain TA98 in the presence of S9 mix.

All criteria for a valid study were met.
Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation TA100 and WP2uvrA

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article, 1,1,3,3-Tetramethylbutyl peroxy-2-ethylhexanoate, did cause positive increases in the mean number of revertants per plate with tester strains TA100 and WP2uvrA in the presence of S9 mix. No positive increases were observed with any other tester strain either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).
Executive summary:

The objective of this study was to evaluate the test article, 1,1,3,3-Tetramethylbutyl peroxy-2 - ethylhexanoate, for its ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of

Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli tester strain WP2uvrA.

The doses tested in the mutagenicity assay were selected based on the results of a dose rangefinding study using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5000 μg per plate, one plate per dose, both in the presence and absence of S9 mix.

The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested in the mutagenicity assay with all tester strains in both the presence and absence of S9 mix were 100, 333, 1000, 3330 and 5000 μg per plate. An independent confirmatory experiment was conducted with all tester strains at doses of 10.0, 33.3, 100, 333, 500, 750, 1000, 2000 and 5000 μg per plate in the presence of S9 mix and at doses of 100, 333, 1000, 3330 and 5000 μg per plate in the absence of S9 mix. The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article, 1,1,3,3-Tetramethylbutyl peroxy-2-ethylhexanoate, did cause positive increases in the mean number of revertants per plate with tester strains TA100 and WP2uvrA in the presence of S9 mix. No positive increases were observed with any other tester strain either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 02 November 2017 Experimental completion date: 02 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Identification: 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3)
Product Name: Trigonox 421
Chemical Name: 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate
Batch Number: 1501442030
Physical state/Appearance: Clear colourless liquid
Purity: 93.3%
Expiry Date: 31 July 2018
Storage Conditions: Approximately -20 °C in the dark

Formulated concentrations were adjusted to allow for the stated water/impurity content of the test item.
Target gene:
not applicable
Species / strain / cell type:
other: whole blood
Details on mammalian cell type (if applicable):
CELLS USED
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: female, aged 21 years
Main Experiment: female, aged 33 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9 from Phenobarbital/B-Naphtha flavone male rats
Test concentrations with justification for top dose:
Preliminary toxicity test:
Due to formulation difficulties and the sensitivity of human lymphocytes to acetone, the maximum dose level that could be achieved was 1000 µg/mL.
The dose range of test item used was 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL.

Main test:
4-hour without S9: 4, 8, 16, 32, 40, 48, 64
4-hour with S9 (2%): 4, 8, 16, 32, 40, 48, 64
24-hour without S9: 4, 8, 16, 32, 40, 48, 64
Vehicle / solvent:
The test item was insoluble in aqueous media and dimethyl sulfoxide at 20 and 200 mg/mL, respectively, but was soluble in acetone at 200 mg/mL in solubility checks performed in house. Prior to each experiment, the test item was accurately weighed, formulated in acetone and serial dilutions prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/mL for 4-hour exposure
Positive control substance:
mitomycin C
Remarks:
Absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.075 µg/mL for 24-hour continuous exposure
Positive control substance:
other: Demecolcine
Remarks:
Absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/mL for 4-hour exposure
Positive control substance:
cyclophosphamide
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
Experimental Design and Study Conduct

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 µg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The Preliminary Toxicity Test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the exposures of the Main Experiment.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used in all three exposure groups was 0, 4, 8, 16, 32, 40, 48 and 64 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Assessments
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

Coding
The slides were coded before analysis using a computerized random number generator.

Cytokinesis Block Proliferation Index (CBPI)
A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:

% Cytostasis = 100 - 100{(CBPIT – 1) / (CBPIC – 1)}

Where:

CBPI = (No. mononucleate cells + (2 x No. binucleate cells) + (3 x No. multinucleate cells)) / Total number of cells

Key:
T = test chemical treatment culture
C = vehicle control culture

Scoring of Micronuclei
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
The criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.

In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
Statistics:
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.
Species / strain:
other: whole blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/mL. The maximum dose was the maximum achievable dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 62.5 µg/mL in all three exposure groups.
Hemolysis was observed following exposure to the test item at 31.25 µg/mL to 500 µg/mL in the 4-hour exposure group in the absence of metabolic activation (S9) and at and above 62.5 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. No hemolysis was observed in the 4-hour exposure group in the presence of S9.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 1000 µg/mL in the 4-hour exposure in the absence of S9. In the presence of S9, binucleate cells were present up to 62.5 µg/mL and then from 250 µg/mL to 1000 µg/mL. The absence of binucleate cells at 125 µg/mL in the presence of S9 coincides with the onset of precipitating dose levels. In the 24-hour continuous exposure group binucleate cells were present up to 62.5 µg/mL.
The CBPI data showed that the test item induced some evidence of toxicity in all of the exposure groups but mainly in the 24-hour group.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level with toxicity also being taken into account in the dose selection.

Micronucleus Test – Main Experiment
The qualitative assessment of the slides determined that there was a shift in toxicity compared to the Preliminary Toxicity Test. In the 4-hour exposure in the absence of S9 there were binucleate cells suitable for scoring up to 48 µg/mL. The dose level of 64 µg/mL had insufficient binucleate cells for scoring. In the presence of S9 there was no obvious toxicity demonstrated and there were binucleate cells present up to the maximum dose level of 64 µg/mL. The 24-hour exposure group demonstrated similar toxicity to that observed in the Preliminary Toxicity Test and had binucleate cells suitable for scoring up to 48 µg/mL.
A precipitate of the test item was observed in the parallel blood-free cultures at the beginning of exposure, at and above 48 µg/mL in all three exposure groups. Precipitate was observed at the end of exposure at 64 µg/mL in the blood-free cultures of the 24-hour continuous exposure group. This observation combined with the precipitate observations in the Preliminary Toxicity Test was taken to confirm that the lowest precipitating dose level was 64 µg/mL.
Hemolysis was observed following exposure to the test item at and above 32 µg/mL in the 4 hour exposure group in the absence of S9 only. No hemolysis was observed in the 4-hour in the presence of S9 or the 24-hour exposure groups.
The CBPI data for the short exposure groups and for the 24-hour exposure group confirm the qualitative observations in that a dose related inhibition of CBPI was observed in the absence of S9 only.
In the 4-hour exposure group in the absence of S9, 19% and 55% cytostasis was achieved at 16 and 32 µg/mL, respectively. The maximum dose level selected for analysis of binucleate cells was based on toxicity and was 32 µg/mL because this concentration achieved optimum toxicity as defined by the OECD test guideline 487 (55±5%).
In the 24-hour continuous exposure group in the absence of S9, 22%, 36% and 57% cytostasis was achieved at 16, 32 and 40 µg/mL, respectively. Therefore, the maximum dose level selected for analysis of binucleate cells was 40 µg/mL because this concentration achieved optimum toxicity as defined by the OECD test guideline 487 (55±5%).
In the presence of S9, no dose-related inhibition of CBPI was observed at any dose level of the test item tested. Therefore, the maximum dose level selected for analysis of binucleate cells was 64 g/mL which was considered to be the lowest precipitating dose level based on the precipitate observations of the Preliminary Toxicity Test and the Main Experiment.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in the absence of metabolic activation. In the 4-hour exposure in the presence of S9, there was a small but statistically significant increase in the number of micronuclei at 64 µg/mL, the maximum dose level scored. However, since this was compared against a low vehicle control value, there was no clear dose-related response and the mean percentage of micronuclei (1.40) only marginally exceeded the upper limit of the historical control data for a vehicle (1.20) it was considered to be of no toxicological significance.

The dose levels of the controls and the test item are given in the table below:

Exposure Group

Final concentration of test item1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3)(µg/mL)

4-hour without S9

0*, 4*, 8*, 16*, 32*, 40, 48, 64, , MMC0.2*

4-hour with S9 (2%)

0*, 4, 8, 16*, 32*, 40*, 48*, 64*, CP5*

24-hour without S9

0*, 4, 8*, 16*, 32*, 40*, 48, 64,DC0.075*


*             = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC= Mitomycin C

CP           = Cyclophosphamide

DC          = Demecolcine

Conclusions:
The test item, 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3), did not induce any toxicologically significant increases in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

Summary

Introduction

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to five dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by precipitate with toxicity also being taken into account in the dose selection. The dose levels selected for the Main Experiment were as follows:

Exposure Group

Final concentration of test item 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3) (µg/mL)

4-hour without S9

4, 8, 16, 32, 40, 48, 64

4-hour with S9 (2%)

4, 8, 16, 32, 40, 48, 64

24-hour without S9

4, 8, 16, 32, 40, 48, 64

Results

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item demonstrated toxicity in all three exposure conditions but did not induce any statistically significant increases in the frequency of cells with micronuclei in the absence of S9, using a dose range that included a dose level that induced cytostasis in the range of 55±5%. In the presence of S9 there was a statistically significant increase in the number of micronuclei at the maximum dose level scored, the lowest precipitating dose level, but this was considered to be of no toxicological significance since it was compared against a low vehicle control value, there was no clear dose-related response and the mean percentage of micronuclei (1.40) only marginally exceeded the upper limit of the historical control range for a vehicle of 1.20.

Conclusion

The test item,1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3)was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.


 

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 January 2012 - 20 February 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.
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
Remarks:
Date GLP of inspection: 19 - 21 July 2011 Date of signature on GLP form: 31 August 2011
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640

- Properly maintained:
yes

- Periodically checked for Mycoplasma contamination:
yes

- Periodically checked for karyotype stability:
no

- Periodically "cleansed" against high spontaneous background:
yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Vehicle and positive controls were used in parallel with the test item. Solvent (acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batch BCBC4573V at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0277203 at 2 µg/ml was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
Solvent (Acetone) treatment groups were used as the vehicle controls.

- Justification for choice of solvent/vehicle:
Formed a soultion suitable for dosing at the required concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (Acetone) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (Acetone) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
This study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al, 1980; Jotz and Mitchell, 1981).

The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances. The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).
Evaluation criteria:
Please see "Any other information on materials and methods incl. tables" section.
Statistics:
Please see "Any other information on materials and methods incl. tables" section.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test

The dose range of the test item used in the preliminary toxicity test was 5.31 to 1360 µg/ml. In all three of the exposure groups there was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item, with the greatest reductions observed in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. A greasy / oily precipitate of the test item was observed at and above 42.5 µg/ml in the 4-hour and 24 hour exposure groups in the absence of metabolic activation, and at and above 21.25 µg/ml in the 4-hour exposure group in the presence of metabolic activation. The greasy / oily precipitate increased in intensity in all three exposure groups with increase in dose concentration. The apparent “recovery” observed at 1360 µg/ml in the 4-hour exposure group in the absence of metabolic activation, and at and above 680 µg/ml in the 4-hour exposure group in the presence of metabolic activation, was considered to be due to the presence of precipitate effectively reducing exposure of the test item to the cells. However, with evidence of marked toxicity in all three of the exposure groups, the maximum dose level in the subsequent mutagenicity test was limited by test item-induced toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.
There was once again evidence of marked dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 3 and 6). There was also evidence of modest dose related reductions in viability (%V); therefore indicating that modest residual toxicity had occurred in both the absence and presence of metabolic activation. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at 35 µg/ml in the absence of metabolic activation, and at and above 127.5 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item induced statistically significant dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in both the absence and presence of metabolic activation (Tables 3 and 6). Statistically significant increases in mutant frequency were also observed at individual dose levels in both the absence and presence of metabolic activation. However, the increases were only observed at dose levels at or approaching the limit of acceptable toxicity, the GEF was not exceeded at any of the dose levels, and the mutant frequency values observed were within the acceptable range for vehicle controls in the absence of metabolic activation and only marginally exceeded in the presence of metabolic activation. It was considered that the increases were due to a cytotoxic mechanism and not a true genotoxic response. The responses observed were, therefore, considered to be spurious and of no toxicological significance. Greasy / oily precipitate of test item was observed at and above 30 µg/ml in the absence of metabolic activation, and at and above 21.25 µg/ml in the presence of metabolic activation.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 9 and 12). There was also once again evidence of a modest dose related reduction in viability (%V) in the presence of metabolic activation, therefore indicating that residual toxicity had occurred. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at 120 µg/ml in the presence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had a modest effect on the toxicity of the test item.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in the absence of metabolic activation (Tables 9). A statistically significant dose related (linear-trend) increase in the mutant frequency was observed in the presence of metabolic activation (Table12). Statistically significant increases in mutant frequency were also observed at two individual dose levels in the presence of metabolic activation. However, the increases were once again only observed at dose levels at or approaching the limit of acceptable toxicity, the GEF was not exceeded at any of the dose levels, and the mutant frequency values observed were within the acceptable range for vehicle controls. It was considered that the increases were once again due to a cytotoxic mechanism and not a true genotoxic response. The responses observed were, therefore, considered to be spurious and of no toxicological significance. Greasy / oily precipitate of test item was observed at and above 20 µg/ml in the presence of metabolic activation. The precipitate observations in the absence of metabolic activation varied from those seen previously with a greasy / oily precipitate observed at and above 2.5 µg/ml. The reason for this was not known. However, the expected level of toxicity was achieved, therefore, the study was considered unaffected.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.
Remarks on result:
other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Remarks:
Migrated from field 'Test system'.

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.
Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.
Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Thethod was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test, and in Experiment 1 was 2.5 to 35 µg/ml in the absence of metabolic activation, and 10.63 to 340 µg/ml in the presence of metabolic activation. In Experiment 2 the dose range was 0.31 to 20 µg/ml in the absence of metabolic activation and 5 to 120 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was observed at and above 2.5 µg/ml in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment using a dose range that induced optimum levels of test item-induced toxicity.

Conclusion. 

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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

Genetic toxicity in vivo

Description of key information

Since the in vitro micronucleus is negative further investigation of this endpoint in vivo is not required.

The substance is positive for mutagenicity with metabolic activation in vitro in an Ames test and negative in a Mouse Lymphoma Assay.

Kirkland et al. (Mutation Research 775-776 (2014) 69-80) conclude that “Thus, in the case of an Ames-positive chemical, negative results in 2 in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential.”This therefore implies for the mutagenicity endpoint a negative “In Vitro Mammalian Cell Gene Mutation Test” supported by in silico data is sufficient evidence to waive the need for in vivo gene mutation testing.”

 

QSAR models (see details in the waiver) predict a negative outcome for (Ames) mutagenicity.

 

In this situation the Ames is positive with metabolic activation, the MLA is negative and the in vivo micronucleus is negative. In silico methods indicate that the mutagenicity endpoint is negative. Based on the available information in vivo mutagenicity testing is not required.

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:

QSAR PREDICTIONS on the genotoxic potential of CAS 22288-43-3

Summary:

Five QSAR softwares were used to evaluate the potential genotoxicity of 1,1,3,3-tetramethylbutyl peroxy-2-ethylhexanoate (CAS # 22288-43-3).

 

In general, the predictions for Ames test are consistently negative. The OECD QSAR Toolbox suggests that a mechanistic basis for mutagenicity (mammalian and bacterial) is lacking.

QSAR predictions on carcinogenicity are also provided, as ultimately, this endpoint is of interest. This is a more complex endpoint the weight of the evidence suggest a negative outcome.

 

Kim Doornebosch

Toxicologist, AkzoNobel Specialty Chemicals

March 6, 2018

CAS

22288-43-3

EC name

1,1,3,3-tetramethylbutyl peroxy-2-ethylhexanoate

Molecular weight

272.4

SMILES

CCCCC(CC)C(=O)OOC(C)(C)CC(C)(C)C

Molecular formula

C16H32O3

Structure

QSAR profile

OECD Toolbox

 

Carcinogenicity (genotox & nongenotox) alerts by ISS

No alert found

DNA alerts by AMES by OASIS v. 1.4

No alert found

DNA alerts by CA and MNT by OASIS v. 1.1

No alert found

In vitro mutagenicity (Ames Test) alerts by ISS

No alert found

In vivo mutagenicity (micronucleus) alerts by ISS

H-acceptor-path3-H-acceptor.A potential Hydrogen bond acceptor.

Protein binding alerts for chromosomal aberration by OASIS v. 1.2

No alert found

QSAR profile

ACD/Tox Suite

 

Ames test

Probability of positive Ames test = 0.040

Reliability: Borderline (Reliability Index= 0.5)

Genotoxicity hazards

No hazardous fragments have been bound

TOPKAT

 

Weight of Evidence Rodent carcinogenicity

Non-carcinogen

Ames mutagenicity

Non-mutagen

DEREK NEXUS

 

Mutagenicity in vitro

Bacterium – inactive.Contains misclassified features, slightly lower confidence 

Carcinogenicitybacterium

Impossible. Alert: oxidising agent.

Carcinogenicitymammal

Plausible. Alert: oxidising agent.

VEGA

 

Mutagenicity (Ames test)CONSENSUSmodel (v. 1.0.2) [Based on 4 separate models]

Non-mutagenic 

Carcinogenicity model (ISS) (v. 1.0.2)

Carcinogen (low reliability)

Carcinogenicity model (IRFMN/Antares) (v. 1.0.0)

Non-Carcinogen (low reliability)

Carcinogenicity model (IRFMN/ISSCAN-CGX) (v. 1.0.0)

Non-Carcinogen (low reliability)

Carcinogenicity model (CAESAR) (v. 2.1.9)

Non-Carcinogen (low reliability)

 

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Available data shows a negative outcome for genotoxicity based on these results, the data is conclusive but not sufficient for classification.