Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was mutagenic in the key Bacterial Reverse Mutation Assay according to OECD 471 but no mutagenicity was observed in a second gene mutation test in bacteria which was conducted under similar conditions as described in OECD 471. In the key In Vitro Cell Gene Mutation Test according to OECD 476, no mutagenic potential of the test item was observed in the absence and presence of metabolic activation.


As in vitro mutagenicity testing showed ambiguous results, the in vivo mutagenic potential has to be addressed.

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
Study period:
1995-10-17 to 1995-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
May 1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
E. coli, other: WP2 (pKM101)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
- 100, 333, 450, 1000, 5000 µg/plate (for all strains without S9-mix)
- 10, 33, 100, 333, 450, 1000 µg/plate (for strains TA98, TA100, TA1535, T1537)
- 33, 100, 333, 450, 1000, 5000 µg/plate (for strains WP2 uvrA(pKM101) and WP2)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene for strains TA98, TA100, TA1535, TA1537
Remarks:
with S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sterigmatocystin for WP2 (pKM101) and TA102
Remarks:
with S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) Test item dilutions were prepared immediately before use. One-half (0.5) mililiter of S9 or Sham mix, 100 µL of tester strain and 50 µL of the vehicle or test item were added to 2.0 mL of molten selective top agar at 45 +/-2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 or 100 µL aliquot of appropriate positve control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 +/-2°C. Plates that were not counted immediately following the incubation period were stored at 4+/-2°C until colony counting could be conducted.

DURATION
- Exposure duration: 48-72 hours

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: 1 in triplicates

DETERMINATION OF CYTOTOXICITY
- Method: cell count

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.
For the test item to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of a least one tester strain with a minimum of two increasing concentrations of the test item. Data sets for strains TA 1535 and TA 1537 were judged positive if the increase in mean revertants at the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA 98, TA 100, TA 102, WP2 uvrA (pKM101) and WP2 (pKM101) were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli, other: WP2 uvrA (pKM101) and WP2 (pKM101)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli, other: WP2 (pKM 101)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: 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.
Conclusions:
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.
Executive summary:

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-13 to 2011-10-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase enzyme locus
Species / strain / cell type:
other: Sub-line (KI) of Chinese hamster ovary cell line CHO
Details on mammalian cell type (if applicable):
- Type and identity of media: F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
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
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: N,N-dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: suitable solvent
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 5 and 20 h
- Expression time (cells in growth medium):
During the phenotypic expression period the cultures were subcultured. Aliquots of 5x105 cells were taken on days 1, 3, and 6, and evaluated on
day 8
- Selection time (if incubation with a selection agent):
At the end of the expression period the cultures from each of the dose levels were aliquotted at 2x10^5 cells per 100-mm dish (five dishes) in selection medium.
- Fixation time:
After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

SELECTION AGENT (mutation assays):
EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL containing 10 µM/mL of 6-thioguanine (6 TG)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
- The mutant frequency in the negative (solvent) control cultures is within the range (min-max) of historical laboratory control data.
- The positive control chemicals induce a statistically significant and biologically relevant increase in mutant frequency.
- The cloning efficiency of the negative controls is between the range of 60% to 140% on Day 0 and 70% to 130% on Day 7.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) and the test item or positive control item treated groups.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test.
Key result
Species / strain:
other: Sub-line (KI) of Chinese hamster ovary cell line CHO
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: yes
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. A dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay, 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12 medium. Cells were seeded into 90 mm petri dishes (tissue culture quality: TC sterile) at 106 cells each and incubated in culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence (9-9 concentrations) of S9 mix (50 µL/mL) and incubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12 medium for 19 hours. Additional groups of cells were treated for 20 hours without metabolic activation (10 concentrations). 24 hours after the beginning of treatment, the cultures were washed with Ham's F12 medium covered with trypsin-EDTA solution and counted and the cell concentration was adjusted to 40 cells/mL with Ham's F12 medium. For each dose, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37°C in a humidified atmosphere of 5 % CO2 in air for 5-7 days for colony growing. Colonies were then fixed with methanol, stained with Giemsa and the colonies were counted. Survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was examined visually at beginning and end of the treatments.

COMPARISON WITH HISTORICAL CONTROL DATA:
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. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The concentrations covered a range from no toxicity to little or maximum toxicity in the highest doses in accordance with EU Method B.17 and OECD Guideline 476.
Conclusions:
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.
Executive summary:

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 thephenotypic expression periodafter 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 dose‑response 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 thisin vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In an in vivo Mammalian Erythrocyte Micronucleus Test according to OECD 474, the test item 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.


To further exclude the mutagenic potential of the test item, an in vivo alkaline Comet assay was conducted according to OECD guideline 489 in male rats. No increase in DNA strand breaks was observed in stomach, duodenum and liver cells and thus, the test item was considered non-mutagenic and non-clastogenic.


The test was shown to be neither clastogenic nor mutagenic in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-09-03 to 2021-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Council Regulation (EC) No 2017/735, Annex Part B, B.62: In vivo Mammalian Alkaline Comet Assay
Version / remarks:
2017-02-14
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Remarks:
HAN:WIST
Details on species / strain selection:
Rats are routinely tested in this test and the chosen Wistar rat strain was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at the test laboratory.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90, Hungary
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 254-286 g, the weight variation in animals involved at the start of the study did not exceed ± 20 %.
- Assigned to test groups randomly: All animals were sorted according to body weight by computer and grouped according to weight ranges.
- Fasting period before study: Animals were not fasted before treatment.
- Housing: 3 animals/cage (treatment+vehicle groups), 2 animals/ cage (positive control) in Type III polypropylene/polycarbonate cages with certified laboratory wood bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3-23.8
- Humidity (%): 43-65
- Air changes (per hr): More than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From September 8th to September 09th
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Sunflower oil
- Justification for choice of vehicle: Based on the information about previously performed studies with the test item and based on a 14-Day Oral Gavage Dose Range Finding Study (see section 7.5.1).
- Concentration of test material in vehicle: 300, 150 and 75 mg/mL
- Amount of vehicle: 5 mL (test groups and negative control) and 10 mL (positive control)
- Batch No: 8005514002
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The treatment solutions were prepared freshly before each treatment.
Duration of treatment / exposure:
27-28 hours (The test item was administered by oral gavage twice: Once on the day 0 and 24 hours thereafter. The negative control animals were treated concurrently with the vehicle, only. The positive control animals were treated by oral gavage once during the experiment on the day 1. Samples were taken 3-4 hours after the second treatment.)
Frequency of treatment:
Treatment and vehicle control groups were treated twice (once on day 0 and 24 hours thereafter) and the positive control group was treated once on day 1.
Post exposure period:
Samples were taken 3-4 hours after the last treatment.
Dose / conc.:
375 mg/kg bw/day (nominal)
Remarks:
The measured concentration values were slightly higher (9-15 % higher) than the nominal values
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
The measured concentration values were slightly higher (9-15 % higher) than the nominal values
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
The measured concentration values were slightly higher (9-15 % higher) than the nominal values
No. of animals per sex per dose:
6 male animals per test item dose or vehicle control groups and 4 male animals in the positive control group. Only samples of 5 animals per dose and vehicle group and samples of 3 animals of the positive control group were analyzed.
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethylmethanesulphonate (EMS)
- Route of administration: Oral by gavage
- Doses / concentrations: 200 mg/kg bw
Tissues and cell types examined:
Stomach, duodenum and liver tissues
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
The sampling time is a critical variable because it is determined by the period needed for the test chemicals to reach maximum concentration in the target tissue and for DNA strand breaks to be induced but before those breaks are removed, repaired or lead to cell death. A suitable compromise for the measurement of genotoxicity is to sample at 2-6 hour after the last treatment. In this particular test and based on the experience of the laboratory over the last years, the sampling was performed 3-4 hours after the second treatment.

DETAILS OF SLIDE PREPARATION:
Conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. Thereafter the underside of the slides was wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and were allowed to dry. Before use, a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA and covered with a glass coverslip. The slides were placed on a tray until the agarose hardened (~ 5 minutes). After the cell isolations, each cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose (LMPA). Thereafter, a cell suspension (100 or 65 μL) containing ~104 – 105 cells was added on the microscope slide after gentle slide off the coverslip. The microscope slides were covered with a new coverslip. After the LMPA cell mixture hardened, an additional 70 μL of N MA was dropped on the microscope slide after a gentle slide off the (second) coverslip and a new coverslip was laid on the slide. After the repeated NMA layer hardened the coverslip was removed. After the top layer of agarose solidified and the last glass coverslip was removed, the slides were immersed in chilled lysing solution overnight at 2-8 °C in the dark. After the incubation period, the slides were rinsed to remove residual detergents and salts prior to the alkali unwinding step. This rinsing procedure was performed in electrophoresis buffer. The slides were removed from the lysing solution and randomly placed on a horizontal gel electrophoresis unit. The slides were left in the electrophoresis tank for 30 min for the DNA to unwind. Thereafter, the electrophoresis was conducted for 30 min by applying a constant voltage of 0.7 V/cm and an electric current of about 300 mA (actual values: 270-302 mA). After electrophoresis, the slides were removed from the electrophoresis unit, were covered with neutralization solution, allowed to stand covered for about 5 minutes, thereafter blotted and covered again with neutralization solution. This procedure was repeated twice. Subsequently, the slides were exposed for additional 5 minutes to absolute ethanol in order to preserve all of the slides. The slides were air dried and then stored at room temperature until they were scored for comets. Just prior the scoring the DNA, the slides were stained using 2 μg/mL Ethidium bromide.

METHOD OF ANALYSIS
The slides were examined with an appropriate magnification (200x) using fluorescent microscope (Olympus BH-2) equipped with an appropriate excitation filter (TRITC) and with an Alpha DCM 510B CMOS camera. For image analysis, the Andor Kinetic Imaging Komet 6.0 (Andor Technology) was used. For each tissue sample, fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment and per vehicle control and 450 per positive control). DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. 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.
Evaluation criteria:
The test chemical is clearly negative if:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
• there is no concentration-related increase when evaluated with an appropriate trend test;
• all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administrations;
• direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

The test chemical is clearly positive if:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
• the increase is dose-related when evaluated with an appropriate trend test;
• any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administrations;

There is no requirement for verification of clearly negative or positive response.
Statistics:
The statistical significance of % tail DNA values, tail length, OTM values and number of ghost cells was calculated using the appropriate statistical method, using SPSS PC+ software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the intergroup comparisons were performed using Mann-Whitney U-test.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Body weight reduction, strongly reduced activity, piloerection, increased respiration rate, general lethargy, strong diarrhoea, narrow palpebral fissure, incoordination, prone position, cyanosis (2000 mg/kg bw); reduced activity, increased respiration frequency, incoordination, general lethargy, abdominal distension, piloerection (1800 mg/kg bw) (1800 mg/kg bw); General lethargy, piloerection, at one animal reduced activity, increased respiration frequency (1500 mg/kg bw)

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: According to OECD guideline 489, a dose where some signs of toxicity but no severe pain or suffering occurs shoud be selected as highest dose. In the highest test dose used in this assay, animals showed some behavioural changes and clinical signs (piloerection, decreased activity, increased respiration rate, diarrheoa, unstable motion) one hour after the second treatment and symptoms were intensified just before sacrifice. At this time point, also animals in the 750 mg/kg bw group showed piloerection, decreased activity and increased respiration (2 animals). These symptoms were in line with the requirements of the OECD guideline and thus, dose selection was considered appropriate. The oral route was considered to be the most relevant exposure route and the glandular stomach, the duodenum and the liver were selected as the tissues of interest as most exposed organs by this exposure route.
- Statistical evaluation: The statistical evaluation of data (% tail DNA, tail length and OTM) of stomach and duodenum samples did not show statistically significant differences from that of the vehicle control in any case. In the case of liver samples, no statistically significant differences were obtained between the mean median % tail DNA value of the vehicle control and each dose. The % tail DNA values showed a slightly increasing tendency with the increasing doses, but the linear trend analysis did not show significance, consequently no clear dose related increase in % tail DNA values was obtained. The effect ratio (ratio indicated an increase of means of % tail DNA of each dose over the vehicle control value) values were in the range of 1.02-1.20, showing a slight change that was no significant from a mutagenicity point of view.
Conclusions:
The test item was investigated by the means of the in vivo comet assay on isolated stomach, duodenum and liver cells under alkaline conditions in the male WISTAR rats. The test item was administered twice via oral gavage at the dose levels 1500, 750 and 375 mg/kg body weight/day on two consecutive days. Sampling was performed about 3 to 4 hours after the second treatment. Under the experimental conditions presented in this report, the test item TBPPI-75- AL did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum or liver cells. Concurrent controls confirmed the sensitivity and validity of the test. The investigated test item TBPPI-75-AL did not show genotoxic activity in the examined tissues in this Comet Assay.
Executive summary:

In a GLP compliant In Vivo Alkaline Comet Assay on the Rat Stomach, Duodenum and Liver according to OECD guideline 489, the clastogenic potential of the test item was investigated. Male Wistar rats were treated on two consecutive days with the test item at concentrations of 375, 750 and 1500 mg/kg bw/day by oral gavage. Sunflower oil was used as negative control and ethyl methanesulfonate (EMS) was used as positive control. Formulations were prepared freshly before each treatment. The test item was formulated in the vehicle in nominal concentrations of 300, 150 and 75 mg/mL. The test item in sunflower oil formulations was considered to be homogeneous. The measured concentration values were slightly higher (9-15 % higher), than the nominal values in both analytical occasions at all concentrations. The slightly higher concentration levels of treatments solutions (and in consequence doses) did not cause significantly different observations, clinical signs, symptoms at the investigated animals, than already noticed in the preliminary tests. The slightly higher measured concentrations were considered acceptable and the nominal concentration values of 300, 150 and 75 mg/mL (corresponding dose levels: 1500, 750 and 375 mg/kg body weight/day) were applied throughout the study. The animals of the test item dose groups and the negative control animals were treated by oral gavage twice, once on the day 0 and once 24 hours thereafter. The positive control animals were treated by oral gavage once during the experiment on day 1. 5 mL/kg body weight at the vehicle control and test item doses, and 10 mL/kg body weight at the positive control. 3-4 hours after the second treatment (test item treatments 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 using a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. Comet Assay steps were the following: Embedding the cells; Lysis (pH=10); Unwinding (pH>13; for 30 min.); Electrophoresis (pH>13; for 30 min. at 25V and about 300 mA);Neutralisation (pH=7.5; 3 times for 5 min.) and Preservation (abs. ethanol for 5 min. and air dried) of slides. Prior to scoring, the DNA was stained with 2 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. 6 animals in the test item treated groups and negative control group, respectively; 4 animals in the positive control group. 5 animals in the test item treated groups and negative control group, respectively; 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 and per vehicle control; 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 any dose group up to the limit dose of 1500 mg/kg body weight/day and in the controls. During the treatment period, after the second treatment, unequivocal signs of test item toxicity, obvious behavioural changes and clinical signs that appeared in dose-related manner were noticed. Piloerection, decreased or strongly decreased activity, increased respiration rate were the most characteristic signs that were most obvious at the highest dose of 1500 mg/kg body weight/day. At the time of tissue isolation, normal appearance and anatomy of stomach, duodenum and liver was noticed at the vehicle control animals and at three animals of the positive control. Unequivocal signs of toxicity and local test item effects (e.g.: tympanites in the stomach or gastrointestinal tract, characteristic chemical smell at the stomach opening, characteristic stomach content: bedding material or significant volumes of liquid (assumed: water) were noticed at all test item treated groups. The intensity, degree of these toxic effects, observations showed a dose dependent tendency. Furthermore, at the highest dose of 1500 mg/kg body weight/day, a mosaic pattern was noticed on the liver in three animals. The average body weights slightly increased (by 0.19-1.32 %) in the negative control and in the treated dose groups of 375 and 750 mg/kg body weight/day when comparing the weight values measured on day 0 and just before the sacrifice, which is in the range of the expected increase for such exposure times. At the dose group of 1500 mg/kg body weight/day, on average 4.66 % weight reduction was noticed, also indicating a test item toxic effect (see above). At the positive control, on average 1.95 % weight reduction was noticed. In the cytotoxicity screening measurements (using Trypan blue dye exclusion method), no cytotoxicity was noticed in any test item and control item treatments in any target tissue. In the stomach, the percentage of ghost cells differed statistically significant from that of the of the vehicle control at the dose level of 750 mg/kg body weight/day. The percentage of ghost cells was ~12 % in the vehicle control group and ~18 % at 750 mg/kg body weight/day. The percentage of ghost cells at 750 mg/kg body weight/day was slightly above the laboratory’s historical control data range for stomach preparations. However, dose dependent increase was not established in the percentage of ghost cells at the stomach samples, (i. e. at 1500 mg/kg body weight/day the ghost cell percentage was 16 %, within the historical control data range and not statistically significant and the linear trend analysis did not show significance). The slightly higher ghost cell frequency was not accompanied unequivocally with increased DNA migration. Therefore, the slightly higher percentage of ghost cells was considered acceptable and being within the biological variability range of the test. At the examined test item treated groups, the number of ghost cells in the duodenum samples remained nearly in the same range and did not differ statistically significantly from that of the vehicle control. In the liver, the percentage of ghost cells (the mean value was 2 % at the vehicle control and 4 % at 1500 mg/kg body weight/day) differed statistically significant from that of the vehicle control at the highest dose level of 1500 mg/kg body weight/day. The ghost cell percentages for liver preparations (means and individual values) remained well within the laboratory’s historical control data range in all doses; however, the percentage of ghost cells showed a dose related increase (also confirmed by linear trend analysis). The slightly higher frequency of ghost cells was not accompanied with unequivocally increased DNA migration: the linear trend analysis of changes of % tail DNA, tail length and OTM values did not show significance, consequently no clear dose related increase was obtained at these parameters. Therefore, the relatively higher number of ghost cells in liver samples especially at the highest dose level was predominantly associated with toxic effects attributable to the test item, which was observed during macroscopic inspection (see above) of the tissue prior to cell isolation which also could have influence on the prepared tissue (cell suspension) quality. A statistically significant increase of ghost cells was noticed in all tissues after EMS treatment. The ghost cells are a possible indicator of cytotoxicity and/or apoptosis. According to the literature increased frequency of ghost cells may also indicate cells with severe DNA damage (genotoxicity). Based on the mutagenicity results and the laboratory’s earlier experience, the relatively higher number of ghost cells at the positive control, EMS treatment are considered being a possible indicator of genotoxicity. The mean median % tail DNA values of each dose remained in the vehicle control range at the examined tissues, and the slightly different (higher or lower) values did not differ statistically significantly from that of the vehicle control up to the highest dose of 1500 mg/kg body weight/day. The mean median % tail DNA values of the vehicle control and test item doses in the stomach, duodenum and liver samples fell within the corresponding historical control data ranges within the 95 % confidence intervals, C-charts. Additionally, the tail length values and Olive Tail Moment (OTM) of the vehicle control and each treatment were compared. The tail length values of the stomach, duodenum and liver samples did not differ statistically significantly from that of the vehicle control in whole examined dose range. The Olive Tail Moment values in the stomach, duodenum and liver of the test item treated groups did not differ statistically significant from that of the vehicle control. Additionally, all of the tail length and OTM values (vehicle control and dose groups) remained well within the established historical control data ranges, within the 95 % confidence intervals, C-charts. In summary, under the experimental conditions presented in this report, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum or liver cells and is thus not considered clastogenic.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
June 1989
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Spargue Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Pilot study: 28.2 - 30.6 g at randomization (males), 26.0 - 28.3 g at randomization (females); Toxicity study: 28.4 - 34.6 g at randomization (males), 25.5 - 29.4 g at randomization (females); Supplemental toxicity study: 29.0 - 35.3 g at randomization (males), 25.8 - 30.7 g at randomization (females); Micronucleus assay: 28.0 - 34.4 g at randomization (males), 26.0 - 29.3 g at randomization (females)
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material.
- Diet: free access to rodent chow
- Water: free access to tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 °C
- Humidity: 50 +/- 20 %
- Photoperiod: 12 hour light/dark cycle

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Test and control articles were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
once
Post exposure period:
none
Dose / conc.:
190 mg/kg bw/day (actual dose received)
Dose / conc.:
380 mg/kg bw/day (actual dose received)
Dose / conc.:
760 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
- Vehicle control and the low test dose: 15 animals
- High test dose: 20 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide
- Doses / concentrations: 60 mg/kg
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: 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.

TREATMENT AND SAMPLING TIMES: Test and control articles were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examinedmicroscopically for micronucleated polychromatic erythrocytes.

DETAILS OF SLIDE PREPARATION: At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to acapped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Scoring for micronuclei with microscope.
Evaluation criteria:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5 %) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p<=0.05, Kastenbaum-Bowman Tables).
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed seperately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the relative to the vehicle control (p<=0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or uncofirmed positive and a repeat assay recommended. The test item was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1, 10, 100, 1000, 5000 mg test item/kg (total volume of 20 mL test ite/vehicle miture/kg bw
- Solubility: test item is insoluble in water; test item was soluble in corn oil at a maximum concentration of 500 mg/mL
- Clinical signs of toxicity in test animals: Mortality occurred within three days of dose administration as follows: 5/5 males and 5/5 females at 5000 mg/kg and 1/2 males at 1000 mg/kg. Clinical signs, which were noted within four hours or later after dose administration, included lethargy in male mice at 100 and 1000 mg/kg. All other animals appeared normal throughout the observation period.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The number of micronucleated polchromatic erythrocytes per 1000 polychromatic erythrocytes in test item-treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of the dose level or bone marrow collection time.
- Ratio of PCE/NCE: Reductions of up to 28 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in test item-treated mice relative to their respective vehicle controls.
Conclusions:
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.
Executive summary:

Tert-butyl peroxypivalate was tested in mouse micronucleus assay 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 oft the 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.

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

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.


 


Comet Assay


In a GLP compliant In Vivo Alkaline Comet Assay on the Rat Stomach, Duodenum and Liver according to OECD guideline 489, the clastogenic potential of the test item was investigated. Male Wistar rats were treated on two consecutive days with the test item at concentrations of 375, 750 and 1500 mg/kg bw/day by oral gavage. Sunflower oil was used as negative control and ethyl methanesulfonate (EMS) was used as positive control. Formulations were prepared freshly before each treatment. The test item wasformulated in the vehicle in nominal concentrations of 300, 150 and 75 mg/mL. The test item TBPPI-75-AL in sunflower oil formulations was considered to be homogeneous. The measured concentration values were slightly higher (9-15 % higher), than the nominal values in both analytical occasions at all concentrations. The slightly higher concentration levels of treatments solutions (and in consequence doses) did not cause significantly different observations, clinical signs, symptoms at the investigated animals, than already noticed in the preliminary tests. The slightly higher measured concentrations were considered acceptable and the nominal concentration values of 300, 150 and 75 mg/mL (corresponding dose levels: 1500, 750 and 375 mg/kg body weight/day) were applied throughout the study. Analysis of formulations (for checking of each concentration and homogeneity) was performed by a HPLC method with UV detection in the Analytical Laboratory of the Test Facility according to the validated analytical method. The animals of the test item dose groups and the negative control animals were treated by oral gavage twice, once on the day 0 and once 24 hours thereafter. The positive control animals were treated by oral gavage once during the experiment on the day 1. 5 mL/kg body weight at the vehicle control and test item doses, and 10 mL/kg body weight at the positive control. 3-4 hours after the second treatment (test item treatments 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 using a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. Comet Assay steps were the following: Embedding the cells; Lysis (pH=10); Unwinding (pH>13; for 30 min.); Electrophoresis (pH>13; for 30 min. at 25V and about 300 mA);Neutralisation (pH=7.5; 3 times for 5 min.) and Preservation (abs. ethanol for 5 min. and air dried) of slides. Prior to scoring, the DNA was stained with 2 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. 6 animals in the test item treated groups and negative control group, respectively; 4 animals in the positive control group. 5 animals in the test item treated groups and negative control group, respectively; 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 and per vehicle control; 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 (See: Validity of the Study). No mortality was observed during the treatments and expression period in any dose group up to the limit dose of 1500 mg/kg body weight/day and in the controls. During the treatment period, after the second treatment, unequivocal signs of test item toxicity, obvious behavioural changes and clinical signs that appeared in dose-related manner were noticed. Piloerection, decreased or strongly decreased activity, increased respiration rate were the most characteristic signs that were most obvious at the highest dose of 1500 mg/kg body weight/day. At the time of tissue isolation, normal appearance and anatomy of stomach, duodenum and liver was noticed at the vehicle control animals and at three animals of the positive control. Unequivocal signs of toxicity and local test item effects (e.g.: tympanites in the stomach or gastrointestinal tract, characteristic chemical smell at the stomach opening, characteristic stomach content: bedding material or significant volumes of liquid (assumed: water) were noticed at all test item treated groups. The intensity, degree of these toxic effects, observations showed a dose dependent tendency. Furthermore, at the highest dose of 1500 mg/kg body weight/day, a mosaic pattern was noticed on the liver in three animals. The average body weights slightly increased (by 0.19-1.32 %) in the negative control and in the treated dose groups of 375 and 750 mg/kg body weight/day when comparing the weight values measured on day 0 and just before the sacrifice, which is in the range of the expected increase for such exposure times. At the dose group of 1500 mg/kg body weight/day, on average 4.66 % weight reduction was noticed, also indicating a test item toxic effect (see above). At the positive control, on average 1.95 % weight reduction was noticed. In the cytotoxicity screening measurements (using Trypan blue dye exclusion method), no cytotoxicity was noticed in any test item and control item treatments in any target tissue. In the stomach, the percentage of ghost cells differed statistically significant from that of the of the vehicle control at the dose level of 750 mg/kg body weight/day. The percentage of ghost cells was ~12 % in the vehicle control group and ~18 % at 750 mg/kg body weight/day. The percentage of ghost cells at 750 mg/kg body weight/day was slightly above the laboratory’s historical control data range for stomach preparations. However, dose dependent increase was not established in the percentage of ghost cells at the stomach samples, (i. e. at 1500 mg/kg body weight/day the ghost cell percentage was 16 %, within the historical control data range and not statistically significant and the linear trend analysis did not show significance). The slightly higher ghost cell frequency was not accompanied unequivocally with increased DNA migration. Therefore, the slightly higher percentage of ghost cells was considered acceptable and being within the biological variability range of the test. At the examined test item treated groups, the number of ghost cells in the duodenum samples remained nearly in the same range and did not differ statistically significantly from that of the vehicle control. In the liver, the percentage of ghost cells (the mean value was 2 % at the vehicle control and 4 % at 1500 mg/kg body weight/day) differed statistically significant from that of the vehicle control at the highest dose level of 1500 mg/kg body weight/day. The ghost cell percentages for liver preparations (means and individual values) remained well within the laboratory’s historical control data range in all doses; however, the percentage of ghost cells showed a dose related increase (also confirmed by linear trend analysis). The slightly higher frequency of ghost cells was not accompanied with unequivocally increased DNA migration: the linear trend analysis of changes of % tail DNA, tail length and OTM values did not show significance, consequently no clear dose related increase was obtained at these parameters. Therefore, the relatively higher number of ghost cells in liver samples especially at the highest dose level was predominantly associated with toxic effects attributable to the test item, which was observed during macroscopic inspection (see above) of the tissue prior to cell isolation which also could have influence on the prepared tissue (cell suspension) quality. A statistically significant increase of ghost cells was noticed in all tissues after EMS treatment. The ghost cells are a possible indicator of cytotoxicity and/or apoptosis. According to the literature increased frequency of ghost cells may also indicate cells with severe DNA damage (genotoxicity). Based on the mutagenicity results and the laboratory’s earlier experience, the relatively higher number of ghost cells at the positive control, EMS treatment are considered being a possible indicator of genotoxicity. The mean median % tail DNA values of each dose remained in the vehicle control range at the examined tissues, and the slightly different (higher or lower) values did not differ statistically significantly from that of the vehicle control up to the highest dose of 1500 mg/kg body weight/day. The mean median % tail DNA values of the vehicle control and test item doses in the stomach, duodenum and liver samples fell within the corresponding historical control data ranges within the 95 % confidence intervals, C-charts. Additionally, the tail length values and Olive Tail Moment (OTM) of the vehicle control and each treatment were compared. The tail length values of the stomach, duodenum and liver samples did not differ statistically significantly from that of the vehicle control in whole examined dose range. The Olive Tail Moment values in the stomach, duodenum and liver of the test item treated groups did not differ statistically significant from that of the vehicle control. Additionally, all of the tail length and OTM values (vehicle control and dose groups) remained well within the established historical control data ranges, within the 95 % confidence intervals, C-charts. In summary, under the experimental conditions presented in this report, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum or liver cells and is thus not considered clastogenic.


 


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:


The test item did not induce gene mutations in a in vitro Mammalian Cell Gene Mutation assay. Results from two different Ames test were inconclusive (one positive and one negative test result). To rule out a mutagenic potential, an in vivo Mammalian Alkaline Comet assay was performed and showed no increase in DNA strand breaks and thus no mutagenic potential of the test item. It was thus concluded that the test item is non-mutagenic. As no significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow was observed in an in vivo Mammalian Erythrocyte Micronucleus Test, it was concluded that the test item furthermore does not possess a clastogenic potential.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification as genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.