tert-butyl peroxyisobutyrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in-vitro studies according to OECD guideline 471 and OECD guideline 476 were conducted. The results are described in the following sections:

 

Ames test

The test substance (in acetone as vehicle) was examined for its potential to induce reverse mutation in Salmonella typhimurium according to OECD No. 471 guideline and EC method B.14. The test substance induced significant and reproducible increases in the number of revertants, with and without S9 mix, in any of the five strains, except in the TA 1535 strain with S9 mix. Under the experimental conditions, the test substance showed mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.

 

HPRT test

The test item, TBPIB was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. TBPIB tested both without and with metabolic activation (S9 mix), induced increases in mutant frequency in this test in Chinese hamster ovary cells. TBPIB was mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

 

Both studies showed adverse effects. Because of that one further Comet assay in-vivo was conducted and is described in the section in-vivo. One in-vivo study according to OECD guideline 474 was conducted to fulfill the endpoint chromosome aberration.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in-vitro studies according to OECD guideline 471 and OECD guideline 476 were conducted. As both studies showed adverse effects one further in-vivo Comet assay was conducted. One in-vivo study according to OECD guideline 474 was conducted to fulfill the endpoint of chromosome aberration. The results are described in the following sections:

In-vivo Chromosome aberration test

In this study the test substance was examined for its potential to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice according to OECD guideline No. 474 and EU Method B.12. Under the experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 500,1000 or 2000 mg/kgbw/day.

 

In-vivo Comet assay

The test item was investigated by the means of the in vivo comet assay on isolated liver and stomach cells under alkaline conditions in the male WISTAR rats administered orally twice with 2000, 1000 and 500 mg/kg body weight/day, with one sampling time of about 3 to 4 hours after the second treatment. Under the experimental conditions, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or in stomach cells. The investigated test item is negative and did not show genotoxic activity in the examined tissues.

 

Conclusion: Both test system showed no genotoxic potential. Based on these results of the in-vivo genetic toxicity studies the substance is classified as not genetic toxic. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Two in-vitro studies according to OECD guideline 471 and OECD guideline 476 were conducted. As both studies showed adverse effects one further in-vivo Comet assay was conducted. One in-vivo study according to OECD guideline 474 was conducted to fulfill the endpoint of chromosome aberration. The results are described in the following sections:

 

In-vitro

Ames test

The test substance (in acetone as vehicle) was examined for its potential to induce reverse mutation in Salmonella typhimurium according to OECD No. 471 guideline and EC method B.14. Selection of concentrations was based on a preliminary toxicity test, thus, the selected treatment-levels were: without S9 mix: 30, 100, 300, 1000, 3000 ug/plate, -with S9 mix: 3, 10, 30, 100, 300 ug/plate except for the TA 102: 10, 30, 100, 300, 1000 ug/plate.

The test substance induced significant and reproducible increases in the number of revertants, with and without S9 mix, in any of the five strains, except in the TA 1535 strain with S9 mix. Under the experimental conditions, the test substance showed mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.

 

HPRT test

The test item, TBPIB was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in N,N-dimethylformamide (DMF) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix: 30, 45, 60, 75, 90 and 105 *µg/mL

Experiment 1, 5-hour treatment period with S9 mix: 10, 30, 50, 70, 90, 100, and 110* µg/mL

Experiment 2, 20-hour treatment period without S9 mix: 20, 40, 60, 70, 75, 80 and 85* µg/mL

Experiment 2, 5-hour treatment period with S9 mix: 10, 30, 50, 70, 90, 100, and 110* µg/mL

*:Theseconcentrations were very toxic and there was not enough cells start the phenotypic expression period after the treatment.

In Experiment 1, there was biologically and statistically significant increases in mutation frequency at concentrations of 90 and 105 *µg/mL without S9 mix. Five-hour treatment in the presence of S9 mix caused slight dose-related increases in mutant frequency. The increase was statistically significant at concentration of 100 *µg/mL.

In Experiment 2, the mutant frequency at concentrations of 40, 70 and 75 µg/mL showeda slight increasescompared to the concurrent control without S9 mix over a prolonged treatment period of 20 hours. In this experiment, a 5-hour treatment in the presence of S9 mix caused increases in mutantfrequency, which werestatistically significant at concentration of 70, 90 and 100 µg/mL, further indicating that the findings in Experiment 1 were out of normal biological variation.

TBPIB tested both without and with metabolic activation (S9 mix), induced increases in mutant frequency in this test in Chinese hamster ovary cells. TBPIB was mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

 

In-vivo

 

Micronucleus test

In this study the test substance was examined for its potential to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice according to OECD guideline No. 474 and EU Method B.12.

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice received two oral treatments of the test substance at dose-levels of 500,1000or 2000 mg/kg bw/day, at a 24-hour interval. One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test substance (cyclophosphamide) once by oral route at the dose-level of 50 mg/kgbw.

The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

For both males and females, the mean values of MPE in the groups treated with the test substance, were equivalent to those of the vehicle group. The PE/NE ratio was significantly (p < 0.05) lower when compared to that of the vehicle group, in the groups given 2000 mg/kg/day (for both males and females) or 1000 mg/kg/day (for females only), showing that the bone marrow cells were effectively exposed to the test substance. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.

Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under the experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 500,1000 or 2000 mg/kg bw/day.

 

Comet assay

The purpose of the comet assay (single cell gel electrophoresis assay) was to evaluate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organs, tissues as specified and requested by ECHA. Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 400, 200 and 100 mg/mL. The measured concentration values remained within the ±5% of nominal range at all concentration levels examined. The nominal concentration values 400, 200 and 100 mg/mL (and the corresponding dose levels: 2000, 1000 and 500 mg/kg body weight) were applied and referred throughout the study. Analysis of formulations (for checking of each concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility according to the validated analytical method (Study codes: 552.102.2994 and 552.102.3150). The test substance was administered orally by gavage; twice: once on the day 0 and 24 hours thereafter at the test item doses and negative controls. The positive control animals were treated by oral gavage once during the experiment on the day 1. The target Tissues were the stomach and the liver. The Sampling time was 3-4 hours after the second treatment (doses and vehicle control) and 3-4 hours after the treatment (positive control) the animals were euthanized and the cells of the target tissues were isolated. Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. Prior the scoring the DNA was stained with 50 µL of 20 µg/mL Ethidium bromide; The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 6.0 F (Andor Technology) was used. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection. The Numbers of treated Animals were 6 animals in the dose groups and negative control group; 4 animals in the positive control group. The Numbers of Analysed Animals, Cells: 5 animals in the dose groups and negative control group; 3 animals in the positive control group. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 450 per positive control).

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met. No mortality was observed during the treatments and expression period in the 1000 mg/kg body weight/day, 500 mg/kg body weight/day doses and in the controls (negative, positive). After the second treatment one animal died at the highest dose group of 2000 mg/kg body weight/day. Toxic symptoms or any clinical signs were not observed during the treatments in the controls (negative and positive) and in the 1000 and 500 mg/kg body weight/day doses. At the highest dose group of 2000 mg/kg body weight/day changed motility, reduced activity and incoordination was principally noticed after the first treatment. The signs discontinued, the animals were asymptomatic before the second treatment. The second treatment was performed as planned. Beside reduced activity and incoordination, salivation and piloerection were observed after the second treatment. At the tissue isolation after the opening of the stomach a characteristic strong chemical smell was noticed at the test item treatments and the smell intensity increased dose-dependently. Macroscopic change of gastric mucosa layer furthermore bedding material in the stomach was noticed at 1000 and 2000 mg/kg body weight/day. Hyperaemia in of the stomach and intestine was additionally observed at 2000 mg/kg body weight/day. The average body weights increased in negative and positive control and test item treatments at 1000 and 500 mg/kg body weights/day. The body weight increases remained in the same range. At 2000 mg/kg body weight/day slight body weight decrease was noticed. At the screening cytotoxicity measurements (using Trypan blue dye exclusion method) significant cytotoxicity was not noticed in any test item and control item treatments. In the stomach samples the number of ghost cells remained nearly in the same range at the test item doses and positive control; however the statistical evaluation established significant differences between the vehicle control and the 1000 and 2000 mg/kg body weight/day doses. At the liver samples the numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses and a statistically significant increase of ghost cells was noticed at the EMS treatments. The ghost cells are a possible indicator of cytotoxicity and/or apoptosis. According to the referred literature increased frequency of ghost cells may indicate cells with severe DNA damage (genotoxicity). To be conscious of the mutagenicity results and laboratory’s earlier experience, the relatively higher number of ghost cells in the stomach samples at the test item treated doses were considered to be a possible indicator of cytotoxicity. While ghost cells in the liver samples treated with EMS are rather considered to be a possible indicator of genotoxicity. DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, Olive Tail Moment (OTM) and tail length. The mean % tail DNA values of each dose remained in the vehicle control range at both, at the liver and stomach samples. The slightly different (higher or lower) values did not differ statistically significantly from that of the vehicle control up to the limit dose of 2000 mg/kg bw/day . Additionally the % tail DNA mean median values were calculated and investigated.In the liver and stomach samples the same tendencies were obtained as it was noticed at the mean values.Statistical significances were not obtained at all test item doses. The mean median value calculations were performed in the case of the % tail DNA parameter, only. The analysis of these values confirmed the results obtained at the mean value calculations. Additionally the olive tail moment (OTM) and tail length values of the vehicle control and each treatment were compared. All of the obtained statistical significances at the OTM values and tail length values of the liver and stomach samples were considered as not relevant for mutagenicity assessment since the significance is linked with lower (but acceptable) values than the corresponding vehicle control value. In the case of the tail length comparisons statistical significance was noticed in the liver samples at the dose of 500 mg/kg body weight/day. The statistical significance was considered as not relevant for mutagenicity assessment since the significance is linked to a lower value which is well within the historical control data range. The test item was investigated by the means of the in vivo comet assay on isolated liver and stomach cells under alkaline conditions in the male WISTAR rats administered orally twice with 2000, 1000 and 500 mg/kg body weight/day, with one sampling time of about 3 to 4 hours after the second treatment. Under the experimental conditions, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in liver or in stomach cells. The investigated test item is negative and did not show genotoxic activity in the examined tissues.          

 

Conclusion: The in-vivo studies showed no effects of genetic toxicity. Based on these results the substance was determined to be not genetic toxic.

Justification for classification or non-classification

Based on the available experimental data, the test substance is considered to be not genetic toxic according to Regulation (EC) No 1272/2008.