Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

In vivo

 

Micronucleus Test

Tert-butyl peroxypivalate was tested in mouse micronucleus essay according OECD guideline no. 474 and EU method B.12. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay followed by a toxicity study and a supplemental toxicity study. The second phase, the micronucleus study, evaluated the potential of the test item to increase the incidene of micronucleated polychromatic erythocytes in bone marrow of male and female mice. In both phases of the study, test and control items were administered in a constant volume of 20 mL/kg body weight by single intraperitoneal injection.

In the pilot assay, male mice were dosed with 1, 10, 100, or 1000 mg test item/kg body weight and male and female mice were dosed with 5000 mg/kg. Mortality was observed in 1/2 male mice at 1000 mg/kg and 5/5 male mice and 5/5 female mice at 5000 mg/kg. Clinical signs following dose administration included lethary in male mice at 100, 1000 mg/kg.

In the toxicity assay, male and female mice were dosed with 400, 800, 1200, or 2000 mg test item/kg body weight. Mortality was observed in 5/5 male mice and 5/5 female mice at 1200 and 2000 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 400 and 800 mg/kg and prostration in male and female mice at 1200 and 2000 mg/kg. Due to the lack of a test item dose level with an intermediate level of toxicity, a supplemental toxicity assay was performed.

In the supplemental toxicity assay, male and female mice were dosed with 900, or 1100 mg test item/kg body weight. mortality was observed in 2/5 male mice and 1/5 female mice at 900 mg/kg and 5/5 male mice and 5/5 female mice at 1100 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 900 and 1100 mg/kg and prostration in male mice at 900 mg/kg and in mice at 1100 mg/kg. The high dose for the micronucleus test was set at 760 mg/kg which was estimated to be approximately 80% of the LD50/3.

In the micronucleus assay, male and female mice were dosed with 190, 380 or 760 mg/kg body weight of tert-butyl peroxypivalate. Mortality was observed in 3/20 male and 1/20 female mice receiving 760 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at all test item dose levels. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examinedmicroscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 28 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some ofte test item-treated groups relative to the respective vehicle controls.

No significant increase in micronucleated polychromatic erythrocytes in test item-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration.

Tert-butyl peroxypivalate did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.

 

In vitro

 

Ames test

Tert-butyl peroxypivalate was tested in the bacterial reverse mutation assay using S. typhimurium tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 and E.coli tester strains WP2 uvrA (pKM101) and WP2 (pKM101) in the presence and absence of Aroclor-induced rat liver S9 according to OECD guideline no.471 and EU method B.13/14. The assay was performed in two phases, using the plate incorporation method. The first phase, the dose range-finding study, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay, was used to evaluate the mutagenic potential of the test item.

Ethanol was selected as solvent of choice based on solubility of the test item and compatility with the target cells.

In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a stock concentration of 100 mg/mL and a 50 µg plating aliquot. Precipitate was observed at >= 3333 µg per plate and toxicity was generally observed from 667 to 5000 µg per plate. Based on the findings of the toxicity assay, the maximum doses plated in the mutagenicity assay were 1000 µg per plate for Salmonella in the presence of S9 activation and 5000 µg per plate for the remaining tester strain/activation conditions.

In the mutagenicity assay positiv responses were observed. Precipitate was generally observed at >= 3333 µg per plate and toxicity was generally observed at >= 1000 µg per plate in the presence of S9 activation.

Tert-butyl peroxypivalate did cause positive responses with tester strains TA 98, TA 100, TA 1537, TA 102, WP2 uvra (pKM101) and WP2 (pKM101) in the presence of acroclor-induced rat liver S9 and with tester strains TA 100, TA 1537, TA 102 and WP2 (pKM101) in the absence of S9.

 

In a supporting study, TBPPI was tested in an Ames test with Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays according to OECD guideline no. 471. No increase in reversion to prototrophy were obtained with any of the four bacterial strains at the tert-butyl peroxypivalate levels tested, either in the presence or absence of S9 -mix. Inhibition of growth, observed as thinning of the background lawn of non-revertant cells and reduction in revertant colony numbers, occurred in all strains following exposure to tert-butyl peroxypivalate at 2500 µg per plate. The test item, tert-butyl peroxypivalate, was devoid of mutagenic activity under the conditions of the test.

HPRT test

The test item, TBPPI 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:

 

60, 75, 80, 85, 90, 95 and 100*µg/mL

Experiment 1, 5-hour treatment period with S9 mix:

30, 45, 60, 75, 80, 85 and 90*µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

20, 40, 60, 65, 70, 75 and 80 µg/mL

Experiment 2, 5-hour treatment period with S9 mix:

30, 45, 60, 75, 80, 85 and 90* µg/mL

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

 

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistical differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment with in the presence of S9 mix did not cause significant increases in mutant frequency, further indicating that the findings in Experiment 1 were within the normal biological variation.

 As in Experiment 2 no statistical differences between treatment and solvent control groups and no doseresponse relationships were noted.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

 

TBPPI tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. TBPPI was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells. Conclusion: Tert-butyl peroxypivalate did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test. In addition, TBPPI was not mutagenic in gene mutation tests on bacteria and mammalian cells in the absence and presence of metabolic activation. Despite of a positive result obtained in a further Ames Test in the absence and presence of metabolic activation, TBPPI is considered to be not mutagenic in vivo, since negative results were obtained in the in vivo micronucleus assay and the in vitro HPRT test on mammalian cells.

Justification for selection of genetic toxicity endpoint
In vivo GLP and guideline study

Short description of key information:
Tert-butyl peroxypivalate did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice. In addition, TBPPI was not mutagenic in gene mutation tests on bacteria and mammalian cells in the absence and presence of metabolic activation. However, positive results were obtained in a further Ames Test in the absence and presence of metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on Regulation (EC) No 1272/2008 (CLP), tert- butyl peroxypivalate is not classified for genetic toxicity.