Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test

The test item (75 % in solvent) was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA according to OECD guideline no.471. Based on the results of this study it is concluded that tert-butylperoxyneodecanoate is mutagenic in the Salmonella typhiurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

HPRT

The test item, TBPND (100 %) was tested in a Mammalian Gene Mutation Test in CHO-K1 cells.

TBPND tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. TBPND was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

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:
1999-10-19 to 1999-10-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (based on 75 % test substance), equivalent to 2.25, 7.5, 24.75, 75, 249.75, 750, 2497.5 and 3750 µg/plate of pure substance.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation for all strains
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535: without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537: Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycine
Remarks:
TA98, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA100, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
no
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
E.coli WP2 uvr A: Without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:

- Dose range finding test:
Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assay was the recommened 5 mg/plate.

- Mutation assay:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain.
Top agar in top agar tubes was molten and heated to 45 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10xe9 cells/mL) of one of the tester strains, 0.1 mL of dilution of the test substance in ethanol and either 0.5 mL S9 mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer ( in case of non-activation assays). The ingredients were mixed on a Votex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 °C for 48 hours. Inadvertently, the plates of the tester strains TA1535, TA1537 (in absence of S9-mix) and TA98 were incubated for 72 hours. Since, the values of the negative and positive controls were within our historical control data range, this has no further effect on the integrity of the results. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan idependent for Escherichia coli) were counted.

Evaluation criteria:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent value, with or without metabolic activation.
b) The negative response should be resproducible in at least one independently repeated experiment.

A test substance is considered positiv (mutagenic) in the test if:
- It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.

The proceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
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 examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the test item concentration of 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 examined
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 examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test item was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (75 % substance in solvent) in the absence and presence of S9-mix.
Precipitate: The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 3330 and 5000 µg/plate. At the end of the incubation no precipitation on the plates was visible anymore.
Toxicity: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
No reduction of the bacterial background lawn and no decraese in the number of revertant colonies were observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-spefic positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

MUTATION ASSAY
Precipitate: The test item preciptated in the top agar at the concentrations of 1000 µg/plate and upwards. Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 3330 and 5000 µg/plate. At the end of the incubation no precipitation on the plates was visible anymore.
Toxicity: In the absence of S9-mix, the bacterial background lawn was not reduced at all concnetrations tested and no decrease in the number of revertants was observed.
In the presence of S9-mix in tester strain TA1535, a moderate reduction of the bacterial background lawn was observed at the test substance concentrations of 3330 and 5000 µg/plate. In tester stratins TA98, a slight reduction of te bacterial background lawn was observed at the test substance concentration of 1000 µg/plate. an exterme reduction of the bacterial background lawn and an increase in the size of the microcolonies was observed at test substance concentrations of 3330 and 5000 µg/plate. In tester strain TA 1537, TA 100 and WP2uvrA no toxicity observed.
Mutagenicity:
In the absence of S9-mix in tester strain TA1535, the test item induced an up to 2.7-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA1537, the test item induced an up to 6.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.2-fold, dose related, increase in the number of revertant colonies compared to the solvent control.
In the tester strains TA100 and WP2uvrA, the test item did not induce a dose-related increase in number of revertant colonies.
In the presence of S9-mix in tester strain TA1537, the test item induced an up to 6.0-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA100, the test item induced an up to 5.3-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain WP2uvrA, the test item induced an up to 4.6-fold, dose related, increase in the number of revertant colonies compared to the solvent control.
In the tester strains TA1535, the test item did not induce a dose-related increase in number of revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Based on the results of this study it is concluded that tert-butyl peroxyneodecanoate is mutagenic in the Salmonella typhiurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Tert-butyl peroxyneodecanoate (75 % in solvent) was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA according to OECD guideline no.471.

The test item was tested up to concentrations of 5000 µg/plate corresponding to 3750 µg/plate of pure substance in the absence and presence of S9 -mix. The test item did not precipitate on the plates at this dose-level. The test item showed toxicity in the tester strains TA98 and TA1535 in the presence of S9 -mix. In the other tester strains, no toxicity was observed.

In the absence of S9-mix in tester strain TA1535, the test item induced an up to 2.7-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA1537, the test item induced an up to 6.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.2-fold, dose related, increase in the number of revertant colonies compared to the solvent control.

In the tester strains TA100 and WP2uvrA, the test item did not induce a dose-related increase in number of revertant colonies.

In the presence of S9-mix in tester strain TA1537, the test item induced an up to 6.0-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA100, the test item induced an up to 5.3-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain WP2uvrA, the test item induced an up to 4.6-fold, dose related, increase in the number of revertant colonies compared to the solvent control.

In the tester strains TA1535, the test item did not induce a dose-related increase in number of revertant colonies.

Based on the results of this study it is concluded that tert-butyl peroxyneodecanoate is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-16 to 2011-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
, adopted 1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
, 2008-05-31
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
, August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Mammalian cell gene mutation assay
Target gene:
The objective of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of phenobarbital and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
Experiment 1, 5-hour treatment without S9 Mix:
15, 20, 25, 30, 35, 40* µg/ml

Experiment 1, 5-hour treatment with S9 Mix:
10, 20, 30, 40, 50, 60*, 70* µg/ml

Experiment 2, 5-hour treatment without S9 Mix:
4, 15, 20, 25, 30, 35, 40* µg/ml

Experiment 2, 5-hour treatment with S9 Mix:
10, 20, 30 ,40, 50, 60*, 70* µg/ml

*These concentrations were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: suitable solvent
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
yes
Positive controls:
no
Positive control substance:
no
Remarks:
Solvent control was run concurrently with treatment groups.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 5 and 20 hours
- Expression time (cells in growth medium): 1, 3, and 6 days
- Selection time: 5 days

SELECTION AGENT: Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 500000

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
- The assay is valid.
- The mutant frequency at one or more doses is significantly greater than that of the relevant control.
- Increase of the mutant frequency is reproducible.
- There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.
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:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: None observed


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 (6 concentrations) or in presence (6 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 (6 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.
In addition, pH and osmolality were considered for dose level selection. Results of the Pre-test on cell toxicity were used for dose selection of the test item used in the Main Mutation Assays. In Experiment 1 and in Experiment 2 six (without S9 mix) and seven (with S9 mix) were selected for the treatment.

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.

Remarks on result:
other: strain/cell type: Sub-line (KI) of Chinese hamster ovary cell line CHO
Remarks:
Migrated from field 'Test system'.
Conclusions:
TBPND tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. TBPND was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.
Executive summary:

The test item, TBPND (100 %) was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO 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:

15, 20, 25, 30, 35 and 401 *g/mL

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

10, 20, 30, 40, 50, 601 and 701 *g/mL

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

15, 20, 25, 30, 35 and 401 *g/mL *g/mL

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

10, 20, 30, 40, 50, 601 and 70 1 *g/mL

 *:These concentrations were very toxic and there were not enough cells to 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 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 1, 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.

 

TBPND tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. TBPND was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Tert-butyl peroxyneodecanoate (75 % in solvent) was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erthrocytes in bone marrow. It is concluded that tert-butyl peroxyneodecanoate is not mutagenic in the micronucleus test under the experimental conditions.

A structural analogue substance (CAS 109 -13 -7) was tested in a Comet assay in vivo according to OECD 489 and found to be negative under the experimental conditions.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to Read-across justification attached to IUCLID chapter 13.
Reason / purpose:
read-across source
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: At the chosen concentration level mortality was not observed. Any clinical sign, any suffering of animals were not observed one hour after the treatment, and reduced activity, incoordination and laxity were observed after two and four hours after the first treatment. Any clinical signs or suffering of animals were not observed before the second treatment and reduced activity, incoordination laxity and, at one animal, salivation were observed after two and three hours after the second treatment. The symptoms decreased and eliminated about 7 hours after the second treatment and due to the noticed intensity and elimination of the observations; 2000 mg/kg body weight/day was chosen as the highest dose level in the present study.

RESULTS OF DEFINITIVE STUDY:
- Appropriateness of dose levels and route: The highest dose (2000 mg/kg bw/day) is selected according to the criteria required by the OECD 489 guideline. Exposure via oral route was chosen as this route was considered to be the most relevant exposure route.

Results of Analytical Measurements

Based on the OECD 489 guideline three doses: 2000, 1000 and 500 mg/kg body weight/day were selected in this study. For these doses the following corresponding treatment solutions were prepared: 400, 200 and 100 mg/mL (the treatment volume was 5 mL/kg body weight). The treatment solutions were prepared freshly before each treatment and corresponding analytical samples were taken and immediately measured or stored in refrigerator until analysis.Based on the results of the available validation study and partial validation study the test item proved to be adequately stable in Sunflower oil formulations at 1, 10 and 500 mg/mL concentration levels at least for 4 hours at room temperature and for 4 days in refrigerator (5 ± 3°C), (Study Nos.: 552.102.2994 and 552.102.3150). The homogeneity of the samples was good, the test item in Sunflower oil formulations was considered to be homogeneous. The measured concentration values remained within the ±5% of nominal range at all concentrations. The nominal concentration values of 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.

Cytotoxicity, Ghost Cells

In this study a first indication of possible cytotoxicity was estimated by Trypan blue dye exclusion technique. This screening technique as an indicator provided preliminary information from the effectiveness and success of the single cell preparation. The cell concentrations of the isolated cell suspensions were in the 105 106 order of magnitude. The viability values of both the liver and the stomach cell suspensions remained in the same, vehicle control range at all test item treatment doses and positive controls. The screened average viability values varied between 81-85 % at the liver cell preparations and 79-83 % at the stomach cell preparations. The decrease of viability was not more than 30 % compared to the concurrent control in any case. In addition, each slide was examined for presence of ghost cells (possible indicator of cytotoxicity and/or apoptosis). Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum. However, the increased frequency of ghost cells may indicate that cells with severe DNA damage continue to be alive, possibly due to protective functions such as DNA repair. The interpretation of ghost cell data should always be done in the context of additional cytotoxicity assessments. According to the testing laboratory’s experience the number of ghost cells is in average about 3-8/slide in the liver preparations and 3-10/slide at the stomach preparations, higher numbers were observed at the 1,2-Dimethylhydrazine dihydrochloride positive control treatments in earlier studies. In the present study, in the stomach samples the number of ghost cells remained nearly in the same range (a clear dose-dependent change was not noticed) at the test item doses and positive control; however its statistical evaluation established significant differences between the vehicle control and the 1000 and 2000 mg/kg body weight/day doses. In these cases, the higher frequency of ghost cells was not accompanied with increased DNA migration. The relatively higher number of ghost cells in stomach samples was predominantly associated with toxic effects attributable to the test item, which was observed during macroscopic inspection of the tissue prior to isolation of cells and a generally decreased cell number in the cell isolations. According to the laboratory expertise at the cell isolations the average cell numbers are in 105-106/mL order of magnitude. In the present study most of the preparations were in 105/mL order of magnitude. At the evaluation of ghost cells a relatively poorer cell suspension quality was also found as it was described as possible explanation for higher frequency of ghost cells by the expert group in the JaCVAM validation trial.At the liver samples the numbers of ghost cells did not differ statistically significantly from that of the vehicle control at the examined doses but a statistically significant increase of ghost cells was noticed at the EMS treatments. At the EMS treatments all parameters that are relevant for the mutagenicity assessment (% tail DNA, tail length OTM values) were within the acceptable ranges of the testing laboratory and were in accordance with the referred literature. The relatively higher number of ghost cells at the EMS treatments is usually not observed in the testing laboratory. The referred JaCVAM validation trial report does not mention the known positive control EMS as ghost cell increasing chemical (such as e.g.: 1,2-Dimethylhydrazine dihydrochloride in the liver) either. In the present study the higher frequency of ghost cells at the EMS treatments was considered to be attributable to the severe DNA damage (genotoxicity) of the reference compound in the absence of any macroscopic tissue changes or a generally lower cell number in the cell isolates.

% Tail DNA, Olive Tail Moment and Tail Length Comparisons

The test item TBPIB-75-AL was investigated in three dose levels 2000, 1000 and 500 mg/kg body weight/day. The treatments were performed in two consecutive days (x2). Two target organs (tissues) of each animal were investigated: liver and stomach. 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 controls). DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity. The mean % tail DNA values of each dose remained in the vehicle control range at both, at the liver and stomach samples. Samples allowing the conclusion that the substance did not cause any effect. 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 body weight/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 the 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. Statistical significances at the OTM values of the liver and stomach samples were noticed at the 500 and 2000 mg/kg body weight/day doses. The statistical significances were considered as not relevant for mutagenicity assessment since the significance is linked to 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.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2002-12-03 to 2003-01-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Directive 2000/32/EC
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian vivo Micronucleus Test
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: male: 32.4 g; female: 26.3 g
- Fasting period before study: no
- Housing: Group of five animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material. Paper bedding was provided as nest material.
- Diet: Free access to standard pelleted laboratory animal diet.
- Water: Free access to tap-water.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 2°C
- Humidity: 30-70 %
- Air changes: 15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light and 12 hours dark per day

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
The mice received a single intraperitoneal injection of a tolerated (high), an intermediate and a low dose of the test item.
Duration of treatment / exposure:
Single treatment
Frequency of treatment:
Single treatment
Post exposure period:
The animals were sacrificed by cervical dislocation 24 or 48 hours after dosing.
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
250 mg/kg bw (total dose)
No. of animals per sex per dose:
5 mice
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide dissolved in physiological saline
- Route of administration: single intraperitoneal injectin
- Doses / concentrations: 50 mg salt/kg bw
Tissues and cell types examined:
Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a dose range finding study 14 animals were dosed intraperitoneally with 2000, 1500 and 1000 mg/kg bw.

TREATMENT AND SAMPLING TIMES:
The mice received a single intraperitoneal injection of a tolerated (high), an intermediate and a low dose of the test item. The animals were sacrificed by cervical dislocation 24 or 48 hours after dosing.

DETAILS OF SLIDE PREPARATION:
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with serum by aspiration with the remaining serum. Two slides were prepared per animal.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. At frist the slids were screened at a magnification of 100 x for regions of suitable technical quality. Slides were scored at magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:
Equivocal results should be clasified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups seperately.
A test subsatnce is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups seperately.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
Statistics:
Wilcoxon Rank Sum Test, two-sided test at p< 0.05
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
In a dose range finding study 14 animals were dosed intraperitoneally with 2000, 1500 and 1000 mg/kg bw. Based on the results of this dose range finding study dose levels of 1000, 500 and 250 mg/kg bw were selected as appropriate doses for the Micronucleus Test.

RESULTS OF DEFINITIVE STUDY
- Mortality and systemic toxic signs:
The animals of the groups treated with 500 and 250 mg test item/kg bw and the animals of the negative and positive control groups showed no abnormalities.
During the first hour after dosing seven (3 male and 4 female) animals of the groups treated with 1000 mg/kg bw were lethargic. All other animals showed no reaction to treatment. Within 18 hours after dosing all animals of the groups treated with 1000 mg/kg bw were lethargic and had a hunched posture, one animal also had a rough coat. Within 42 hours after dosing four (2 male and 2 female) animals had a rough coat and a hunched posture, all animals recovered from the treatment.
-Micronucleated polychromatic erythrocytes:
The mean number of micronucleated polychromatic erythrocytes scored in the test item treated groups were compared with the correponding solvent control group.
No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item.
- Ratio polychromatic to normochromatic erythrocytes:
The animals of the groups, which were, treated with the test item and the negative control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphaminde showed a decrease in the ratio of polychromatic to normochromatic erythrocytes.
Conclusions:
It is concluded that tert-butyl peroxyneodecanoate is not mutagenic in the micronucleus test under the experimental conditions.
Executive summary:

Tert-butyl peroxyneodecanoate (75 % in solvent) was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erthrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection.

Two groups were dosed with 1000 mg/kg bw, one group was dosed with 500 mg/kg bw and one group was dosed with 250 mg/kg bw. After dosing the animals of the dose level of 1000 mg/kg bw showed the following toxic signs: lethary, rough coat and a hunched posture. The animals of the dose levels of 500 and 250 mg/kg bw showed no abnormalities after dosing.

A vehicle treated group served as negative control, a group treated with a single intraperitoneal injection of cyclophosphamide at 50 mg/kg bw served as positive control.

Bone marrow of the groups treated with the test item was sampled 24 or 48 hours after dosing. Bone marrow from the negtive control group was harvested at 24 hours after dosing only and bone marrow from the positive control group harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a staistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item.

The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic erythrocytes compared to the vehicle controls.

Based on the clinical signs observed at the highest dose (lethargy, hunched posture and rough coat) it is assumed that the substance is taken up in the body and is systhemically available.

It is therefore concluded that this test is valid and that tert-butyl peroxyneodecanoate is not mutagenic in the micronucleus test under the experimental conditions.

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

Additional information

Ames test

Tert-butyl peroxyneodecanoate (75 % in solvent) was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA according to OECD guideline no.471.

The test item was tested up to concentrations of 5000 µg/plate (equivalent to 3750 µg/plate of pure substance) in the absence and presence of S9 -mix. The test item did not precipitate on the plates at this dose-level. The test item showed toxicity in the tester strains TA98 and TA1535 in the presence of S9 -mix. In the other tester strains, no toxicity was observed.

In the absence of S9-mix in tester strain TA1535, the test item induced an up to 2.7-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA1537, the test item induced an up to 6.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.2-fold, dose related, increase in the number of revertant colonies compared to the solvent control.

In the tester strains TA100 and WP2uvrA, the test item did not induce a dose-related increase in number of revertant colonies.

In the presence of S9-mix in tester strain TA1537, the test item induced an up to 6.0-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, the test item induced an up to 2.4-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA100, the test item induced an up to 5.3-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain WP2uvrA, the test item induced an up to 4.6-fold, dose related, increase in the number of revertant colonies compared to the solvent control.

In the tester strains TA1535, the test item did not induce a dose-related increase in number of revertant colonies.

Based on the results of this study it is concluded that tert-butyl peroxyneodecanoate is mutagenic in the Salmonella typhiurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

HPRT test

The test item, TBPND (100 %) was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO 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:

15, 20, 25, 30, 35 and 401 *g/mL

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

10, 20, 30, 40, 50, 601 and 701 *g/mL

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

15, 20, 25, 30, 35 and 401 *g/mL *g/mL

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

10, 20, 30, 40, 50, 601 and 70 1 *g/mL

 *:These concentrations were very toxic and there were not enough cells to 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 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 1, 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.

 

TBPND tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. TBPND was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

In vivo micronucleus test

Tert-butyl peroxyneodecanoate (75 % in solvent) was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erthrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection.

Two groups were dosed with 1000 mg/kg bw, one group was dosed with 500 mg/kg bw and one group was dosed with 250 mg/kg bw. After dosing the animals of the dose level of 1000 mg/kg bw showed the following toxic signs: lethary, rough coat and a hunched posture. The animals of the dose levels of 500 and 250 mg/kg bw showed no abnormalities after dosing.

A vehicle treated group served as negative control, a group treated with a single intraperitoneal injection of cyclophosphamide at 50 mg/kg bw served as positive control.

Bone marrow of the groups treated with the test item was sampled 24 or 48 hours after dosing. Bone marrow from the negtive control group was harvested at 24 hours after dosing only and bone marrow from the positive control group harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a staistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item.

The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic erythrocytes compared to the vehicle controls.

Based on the clinical signs observed at the highest dose (lethargy, hunched posture and rough coat) it is assumed that the substance is taken up in the body and is systhemically available.

It is therefore concluded that this test is valid and that tert-butyl peroxyneodecanoate is not mutagenic in the micronucleus test under the experimental conditions.

Comet assay with read-across substance CAS 109 -13 -7

A comet assay (single cell gel electrophoresis assay) was conducted with a structural analogue substance (CAS 109 -13 -7) to evaluate the mutagenic potential of the analogue substance by measuring its ability to induce DNA damage in the target organs, tissues as specified and requested by ECHA (for read-across justification please refer to IUCLID Chaper 13).

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 (See: Validity of the Study). 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:

TBPND revealed a positive result in the bacterial reverse mutation assay in vitro (Ames test). However, gene mutation was not observed with mammalian cells in vitro (HPRT assay). A structural analogue substance (CAS 109 -13 -7), being positive in both in vitro tests for mutagenicity (Ames and HPRT), was, however, negative for mutagenicity when tested in a comet assay in vivo. This is in line with other peroxy-compounds for which sometimes mutagenic effects in vitro are observed but no effects in vivo were seen. This finding is probably based on rapid transformation/detoxification processes in the living organism. These processes, especially metabolism of substances, can only be partially addressed in an in vitro system. Therefore, the test item, TBPND, is considered to be not mutatgenic in vivo and positive results of an in vitro assay with bacterial cells are considered not relevant in this case.

In addition, based on the negative outcome of the micronucleus assay in vivo the test item is not considered to induce cytogenicity.

Taken together, the test item is judged to be not genotoxic.

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 on genotoxicity the test item is not classified genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.