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REACH

Di-tert-pentyl peroxide

EC number: 234-042-8 | CAS number: 10508-09-5

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this endpoint, OECD 471, 473 and 487 tests are available on di-tert-amyl peroxide and an OECD 476 assay on the analogue substance di-tert-butyl peroxide.

Gene mutation assays

Di-tert-amyl peroxide (purity 84.8 %) was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays. The procedures used complied with OECD TG no. 471 (issued 1983) (May, 1992). The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of test item from 25 to 2500 µg per plate, selected following a preliminary toxicity test in strain TA 98, and included solvent (ethanol) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strains, either in the presence or absence of S-9 mix. Only on one occasion of testing in strain TA 100 was any evidence of toxicity towards the bacterial strains obtained (observed as a slight reduction in revertant colony numbers), although the test item had clearly shown marked toxicity (reduction in revertant colony numbers and thinning of the background lawn) at 2500 µg per plate in the preliminary toxicity tests. Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

It was concluded that di-tert-amyl peroxide was devoid of mutagenic activity under the conditions of the test.

The potential of the analogue substance, di-tert-butyl peroxide (CAS No. 110-05-4), to induce mutations at the mouse lymphoma thymidine kinase locus was investigated using the cell line L5178Y (Wollny, 2010). This study was conducted according to the OECD TG no. 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 4 h with and 24 h without metabolic activation. The maximum tested concentration was equal to about 10 mM. Both main experiments were evaluated at 92.5; 185; 370; 740; and 1480 µg/mL with and without S9 mix.

The range of the solvent controls was from 117 up to 197 mutant colonies per 10e6 cells; the range of the groups treated with the test item was from 76 up to 279 mutant colonies per 10e6 cells. The highest solvent control values (182, 194, and 197 colonies per 10e6 cells) exceeded the recommended range of 50 – 170 x 10e6 cells. The data are judged as acceptable however, since the range of up to 200 cultures per 10e6 cells recommended by the IWGT in 2003 was covered. MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.

No relevant toxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment with the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. The threshold of 126 plus each solvent control count was not exceeded in any of the experimental parts. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT 11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

It was concluded that di-tert-butyl peroxide was devoid of mutagenic activity under the conditions of the test.

Chromosomal aberration assays

Cultures treated with di-tert amyl peroxide in the absence and presence of S-9 (Experiment 1 and 2) resulted in frequencies of cells with structural aberrations, which were similar to those seen in concurrent negative controls. All cultures receiving the test article had numbers of cells with structural aberrations that were within historical negative (normal) control ranges.

It was concluded that di-tert-butyl peroxide was devoid of clastogenic activity under the conditions of the test.

The clastogenic and aneugenic activity of di-tert-amyl peroxide was assessed by means of the in vitro micronucleus test (OECD TG no. 487) in TK6 lymphoblastoid human cells treated in presence and in absence of metabolic activation, either with a short-term or with a continuous treatment according to OECD guideline (OECD 487, 2014) (Simar, 2018). The top concentrations were chosen in accordance with the cytotoxicity of the test item, i.e. 0.5 to 1.25 mM depending on the treatment program. The acceptance criteria for the assay were considered as fulfilled. No clastogenic and aneugenic activity was revealed in absence of metabolic activation, with a short-term or a continuous treatment, or with metabolic activation.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, K1a

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

activating system derived from rat liver (S-9 mix)

Test concentrations with justification for top dose:

25-2500 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: Sodium azide: without S9 for TA 1535 and TA 100 / 2-aminoanthracène for TA1535 with and without S9 / 9-Aminoacridine forTA1537 with S9 / 2-Nitrofluorenefor TA 98 without S9 / Benzo-a-pyrene for TA1537, 98 and 100, with and without S9

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Young male CD rats, ca. 200 g bodyweight, were obtained from Charles River Breeding Laboratories (U.K.), Margate, Kent. Aroclor 1254(500 mg/kg bodyweight in corn oil) was administered as a single intraperitoneal injection to induce microsomal enzyme activity.

Four days after treatment, the animals were fasted overnight and then killed by cervical dislocation. The livers were removed, washed in cold 0.15M KCl, then homogenised with more of the same medium (approximately 3 ml per g wet liver)in an homogeniser. Homogenates were centrifuged at 9000 g for 10 minutes and supernatants collected and stored at -80°c until required for preparation of the S-9 mix. Supernatant is used within 3 months of preparation.

Conclusions:

Di-tert-amyl peroxide was devoid of mutagenic activity under the conditions of the test.

Executive summary:

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of test item from 25 to 2500 µg per plate, selected following a preliminary toxicity test in strain TA 98, and included solvent (ethanol) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strains, either in the presence or absence of S-9 mix.

Only on one occasion of testing in strain TA 100 was any evidence of toxicity towards the bacterial strains obtained (observed as a slight reduction in revertant colony numbers), although the test item had clearly shown marked toxicity (reduction in revertant colony numbers and thinning of the background lawn) at 2500 µg per plate in the preliminary toxicity tests.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

It was concluded that di-tert-amyl peroxide was devoid of mutagenic activity under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a: GLP, OECD 473 Guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 with 20% fetal calf serum, 2mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin + phytohemagglutinin for stimulation
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

: Rat S9 mix. Liver S9 homogenate was prepared from rats that have been induced with Arochlor 1254.

Test concentrations with justification for top dose:

• 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2236 µg/mL, for the first experiment with and without S9 mix.
• 78.1, 156.3, 312.5, 625, 1250 and 2236 µg/mL, for the second experiment with and without S9 mix.

Vehicle / solvent:

- Vehicle: ethanol

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

mitomycin C (-S9) and cyclophosphamide (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: 48H
- Exposure duration:
- 3H (+/-S9) for the 1st experiment.
- Until harvest: 20-44H (-S9), 3H (+S9) for the 2nd experiment
- Fixation time (start of exposure up to fixation or harvest of cells): 20H (1st experiment - 2nd experiment with S9) ; 44H in the second experiment without S9.

SPINDLE INHIBITOR: 10 µg/mL of colcemid solution (1.5H prior harvest)
STAIN: Giemsa (time)

NUMBER OF REPLICATIONS: 2 experiments with duplicates for each experiment

NUMBER OF CELLS EVALUATED: 100 metaphases per culture (200 per dose-level). Only 50 metaphases/culture were analyzed when at
least 10% cells with structural chromosome aberration were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A cell having one or more of the above-mentioned structural chromosome aberration was recorded as a single cell with structural chromosome aberration. Therefore the total frequency of cells with structural chromosome aberration was not necessarily equivalent to the total number of aberrations
Reproducible and statistically increase in the number of cells with structural chromosome aberration for at least one dose level and one of the two harvest times is considered as positive.
Reference to historical data and consideration to biological relevance may also be taken into account.

Statistics:

Frequency of cells with structural chromosome aberrations compared to that of the vehicle control. Test of “Chi-2’ is used when necessary with p=0.05 as the lowest significant level.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Without S9, no significant increase in the frequency of cells with structural chromosomal aberration was noted after 3-, 20- as well as 44-hour treatments.
With S9, no significant increase in the frequency of cells with structural chromosomal aberration was noted in both experiments and at both harvest times.
The frequency of cells with structural chromosome aberration of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.

Conclusions:

Di-tert-amyl peroxide was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three female donors in two independent experiments both in the absence and presence of metabolic activation (S9 mix), according to the OECD n° 473 Guideline in compliance with the Principles of Good Laboratory Practice.
Di-tert-amyl peroxide was tested in two independent experiments, both with and without a metabolic activation system. In the first experiment lymphocytes cultures were exposed for 3 hours to solvent vehicle or di-tert-amyl peroxide either with or without activation at 0, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2236 µg/mL.
The dose-levels for the additional experiment were selected from the results of cytotoxicity of the first experiment as follows: , 78.1, 156.3, 312.5, 625, 1250 and 2236 µg/mL. The cultured cells were exposed for 3 hours with S9 mix and 20 hours without S9 mix.
All cells were harvested 20 hours after treatment in the first experiment and 20 and 44 hours in the second one.
Appropriate negative control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges.
Cultures treated with di-tert amyl peroxide in the absence and presence of S-9 (Experiment 1 and 2) resulted in frequencies of cells with structural aberrations, which were similar to those seen in concurrent negative controls. All cultures receiving the test article had numbers of cells with structural aberrations that were within historical negative (normal) control ranges.
Under these experimental conditions, di-tert-amyl peroxide did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in any experiment or at any harvest time.

Executive summary:

Di-tert-amyl peroxide was tested in two independent experiments, both with and without a metabolic activation system. In the first experiment lymphocytes cultures were exposed for 3 hours to solvent vehicle or di-tert-amyl peroxide either with or without activation at 0, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2236 µg/mL.

The dose-levels for the additional experiment were selected from the results of cytotoxicity of the first experiment as follows: , 78.1, 156.3, 312.5, 625, 1250 and 2236 µg/mL. The cultured cells were exposed for 3 hours with S9 mix and 20 hours without S9 mix.

All cells were harvested 20 hours after treatment in the first experiment and 20 and 44 hours in the second one.

Appropriate negative control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges.

Under these experimental conditions, di-tert-amyl peroxide did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in any experiment or at any harvest time.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

September 2014

Deviations:

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (American Type Culture Collection – Rockville, MD 20852 - USA).
- Cell cycle length, doubling time or proliferation index: 16-18 hour doubling time.
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable: RPMI 0 medium containing sodium bicarbonate, non-essential aminoacids, penicillin, streptomycin and HCl.
- Modal number of chromosomes: stable caryotype (47, XY, 13+)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Cytokinesis block (if used):

Metabolic activation:

with and without

Metabolic activation system:

Hepatic S9 fraction from male rat OFA Sprague Dawley induced by Aroclor 1254

Test concentrations with justification for top dose:

0.156, 0.313, 0.625, 1.25, 2.5, 5, and 10 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: griseofulvin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 200 000 cells/mL

DURATION
- Exposure duration: 3h +/- S9 and 27h without S9

STAIN (for cytogenetic assays): Giemsa reagent in mineral water (2%).

NUMBER OF REPLICATIONS: none

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For each culture, the cells were collected by centrifugation for 6 minutes at 1000 rpm and washed twice with PBS. Thereafter, the cells were subjected to hypotonic shock for 4 min using RPMI 1640 containing 1% of pluronic acid and sterile water: RPMI 1640 medium – sterile water (1:1). A step of pre-fixation was realized by adding 0.5 mL of a Carnoy-ethanol mix: ethanol-acetic acid (3:1). After centrifugation, as much as possible of the supernatant was eliminated and the cells were fixed for at least one night at +5±3°C using 2 mL of a Carnoy-ethanol mix.
After centrifugation, the supernatant was eliminated and replaced by 0.5 mL of Carnoy-ethanol mix. The cells were gently resuspended, spread on slides (5-6 drops per slide, 4 slides per culture), exposed to air for at least one night at room temperature A total of 4 slides per culture was prepared, i.e. 2 slides for the standard micronucleus test and 2 provision slides for the FISH test.

NUMBER OF CELLS EVALUATED: The incidence of micronucleated cells out of 1000 mononucleated cells per culture was counted (2000 mononucleated cells/concentration).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The frequency of the number of incidence of micronucleated cells is assessed in mononucleated cells.Micronuclei are identified according to the criteria of Fenech et al. (2000, 2003).
Micronuclei are morphologically identical, but smaller, than nuclei. They also have the following characteristics:
- The diameter of micronuclei usually varies between 1/16th and 1/3rd of the mean diameter of the main nuclei;
- Micronuclei are non-refractile and they can therefore be readily distinguished from artefact such as staining particles;
- Micronuclei are not linked or connected to the main nuclei;
- Micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary;
Micronuclei usually have the same staining intensity as the main nuclei but occasionally staining may be more intense.

DETERMINATION OF CYTOTOXICITY
- Method: :population doubling
- Any supplementary information relevant to cytotoxicity: a preliminary assay which was performed using the protocol used for the main assay. Three treatment schedules were performed as described in the relative paragraph, except that a single culture was performed instead of 2 and no positive control was
included. The cytotoxicity assay was carried out with a wide range of concentrations generally according to a half progression.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase (X²) compared with the concurrent negative control and,
- the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test and,
- any of the results are outside the distribution of the historical negative control data (95% control limits).
When all of these criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control and,
- there is no concentration-related increase when evaluated with an appropriate trend test and
- all results are inside the distribution of the historical negative control data (95% control limits).
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

Statistical analysis of the results obtained in the cells treated at each concentration level was performed using the ƒÓ2 test in comparison with those in control groups. ANOVA trend test was also applied, using the statistical software Stat viewR, version 5.

Species / strain:

mouse lymphoma L5178Y cells

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
Compound Concentration
in mM pH Osmolality Osmolality variation (mOsmol/kg)
(mOsmol/kg) Compared to Solvent Control
Solvent Control
(culture medium +
1% ethanol) 0 7.66 457 -
Di-tert-amyl peroxide 10 7.66 426 -31
5 7.67 440 -17
2.5 766 441 -16

RANGE-FINDING/SCREENING STUDIES:
The recapitulative results are depicted in Table 1.

Individual results of preliminary cytotoxicity assays for the choice of the concentrations in the main genotoxicity assays are shown in Tables 3 to 5.
Results of cytotoxicity assessed during the definitive genotoxicity assays for the choice of the concentrations to be actually analyzed for genotoxicity are presented in Tables 6 to 8.

In the preliminary toxicity assay using a 3-hour treatment without metabolic activation followed by a recovery period (Table 3), a very important toxicity was noted at the 4 highest concentrations ranging from 10 to 1.25 mM. A strong toxicity was still noted at 0.625 mM with a cytostasis of 77 % (i.e. RPD of 23%), and a slight toxicity was noted at the immediately lower concentration of 0.313 mM with a cytostasis of 5.5% for a corresponding RPD of 94.5%.
The concentration of 2 mM was hence retained as the top concentration to be tested in the corresponding genotoxicity assay. A large and narrowed range of concentrations was tested.
In the corresponding genotoxicity assay (Table 6), a very important toxicity was noted at the 2 highest concentrations of 2 and 1.25 mM. A strong toxicity was still noted at 1 mM with a cytostasis of 81.1 % (i.e. RPD of 18.9%), and a slight toxicity was noted at the immediately lower concentration of 0.625 mM with a cytostasis of 18.7% for a corresponding RPD of 81.3%. This concentration was thus retained for genotoxicity assessment.

In the preliminary toxicity assay using a 3-hour treatment with metabolic activation followed by a recovery period (Table 4), a very important toxicity was noted at the 4 highest concentrations ranging from 10 to 1.25 mM. The concentration of 0.625 mM induced a slight toxicity with a cytostasis of 30%, i.e. 70% RPD.
The concentration of 2 mM was hence retained as the top concentration to be tested in the corresponding genotoxicity assay. A large and narrowed range of concentrations was tested.
In the corresponding genotoxicity assay (Table 7), a very important toxicity was noted at the highest concentration of 2 mM. A moderate toxicity was noted at 1.25 mM with a cytostasis of 41.2% (i.e. RPD of 58.8%). This concentration was thus retained for genotoxicity assessment.

In the preliminary toxicity assays using a 27-hour treatment without metabolic activation (Table 5), a very important toxicity was noted at the 4 highest concentrations ranging from 10 to 1.25 mM. A strong toxicity was still noted at 0.625 mM with a cytostasis of 77.7 % (i.e. RPDs of 22.3%), and a slight toxicity was noted at the immediately lower concentration of 0.313 mM with a cytostasis of 12.5% for a corresponding RPD of 87.5%.
The concentration of 2 mM was hence retained as the top concentration to be tested in the corresponding genotoxicity assay. A large and narrowed range of concentrations was tested.
In the corresponding genotoxicity assay (Table 8), a very important toxicity was noted at the 4 highest concentrations ranging from 2 to 0.625 mM. A strong but acceptable toxicity was noted at 0.5 mM with a cytostasis of 57.6% (i.e. RPD of 42.4%). This concentration was thus retained for genotoxicity assessment. Two lower concentration were also analysed.

NUMBER OF CELLS WITH MICRONUCLEI
In the short-term treatment without metabolic activation followed by a 24-hour recovery period (assay S9- 3h/+ 24h), the test item Di-tert-amyl peroxide induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed ranging from 0.313 to 0.625 mM. Indeed, 3.5 micronucleated mononucleated cells were observed per 1000 cells, vs. 3.5 in the solvent control (Table 9).
The test item Di-tert-amyl peroxide was thus not genotoxic under this condition.

In the short-term treatment with metabolic activation followed by a 24-hour recovery period (assay S9+ 3h/+ 24h), the test item Di-tert-amyl peroxide induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed ranging from 0.625 to 1.25 mM. Indeed, 4 to 6.5 micronucleated mononucleated cells were observed per 1000 cells, vs. 5 in the solvent control (Table 10).
The test item Di-tert-amyl peroxide was thus not genotoxic under this condition.

In the continuous treatment without metabolic activation without recovery period (assays S9- 27h/+0h), the test item Di-tert-amyl peroxide induced neither statistically nor biologically significant increase in the number of micronucleated cells at all the concentrations analyzed ranging from 0.25 to 0.5 mM. Indeed, 2 or 3.5 micronucleated mononucleated cells were observed per 1000 cells, vs. 2 in the solvent control (Table 11).
The test item Di-tert-amyl peroxide was genotoxic under this condition.

HISTORICAL CONTROL DATA
- Positive historical control data: see attached document
- Negative (solvent/vehicle) historical control data: see attached document

Conclusions:

The genotoxic activity of the test item Di-tert-amyl peroxide was assessed by means of the in vitro micronucleus test in TK6 lymphoblastoid human cells treated in presence and in absence of metabolic activation, either with a short-term or with a continuous treatment according to OECD guideline (OECD 487, 2014). The top concentrations were chosen in accordance with the cytotoxicity of the test item, i.e. 0.5 to 1.25 mM depending on the treatment program. The acceptance criteria for the assay were considered as fulfilled. The study is valid. Under these experimental conditions, no genotoxic activity was revealed in absence of metabolic activation, with a short-term or a continuous treatment, or with metabolic activation.

Executive summary:

The investigation of a genotoxic activity of Di-tert-amyl peroxide has been carried out in compliance with the OECD Guideline No. 487 (September 2014), using the in vitro mammalian cell micronucleus test on TK6 lymphoblastoid human cells. After a preliminary cytotoxicity test, Di-tert-amyl peroxide diluted in ethanol, was tested at the concentrations of 2, 1.25, 1, 0.625, 0.5, 0.313, 0.25 mM with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. The treatment conditions in duplicates were as follows: 3 h treatment + 27 h recovery with and whitout S9 mix and 24 h treatment + 0 h recovery without S9 mix. Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. After the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). When the slide analysis was undertaken, micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

In the preliminary toxicity assays performed at the concentrations of 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM using a 3-hour treatment without and with metabolic activation followed by a recovery period and a 27-hour treatment without metabolic activation, a very important toxicity was noted at the 4 highest concentrations ranging from 10 to 1.25 mM. A strong toxicity was still noted at 0.625 mM in both treatment schedules without metabolic activation with cytostasis of 77 and 77.7% in the short and long-term treatment, respectively (i.e. RPDs of 23 and 22.3%), and a slight toxicity was noted at the immediately lower concentration of 0.313 mM with percents cytostasis of 5.5 and 12.5% for corresponding RPDs of 94.5 and 87.5%. With metabolic activation, the concentration of 0.625 mM induced a slight toxicity with a cytostasis of 30%,i.e.70% RPD. The concentration of 2 mM was hence retained as the top concentration to be tested in the main genotoxicity assays. A large and narrowed range of concentrations was tested.

	Genotoxic activity study using in vitro micronucleus test on TK6 cells
	Conc. in mM	Relative Population Doubling (%)	Cytostasis (%)	Mean number of micronucleated cells per 1000 mononucleated cells	p	ANOVA
S9- 3h/+24h
Negative control	0	100	0	3.5	-	-
Mitomycin C	0.5 µg/mL	51.1	48.9	178.5	<0.001	-
Di-tert-amyl peroxide	*0.625*	81.3	18.7	3.5	N.S.	*
	*0.5*	87.5	12.5	3.5	N.S.
	*0.313*	106.9	-6.9	3.5	N.S.

*No calculation could be performed as the same number of micronucleates were noted in all treated and control groups

S9+ 3h/+24h
Negative control	0	100	0	5	-	-
Cyclophosphamide	10 µg/mL	23.6	76.4	262	<0.001	-
Di-tert-amyl peroxide	*1.25*	58.8	41.2	4.5	N.S.	N.S.
	1	66.0	34.0	4	N.S.
	*0.625*	99.6	0.4	6.5	N.S.

S9- 27h/+0h
Negative control	0	100	0	2	-	-
Mitomycin C	0.1 µg/mL	58.6	41.4	199.5	<0.001	-
Griseofulvin	10 µg/mL	57.4	42.6	33	<0.001	-
Di-tert-amyl peroxide	*0.5*	42.4	57.6	3.5	N.S.	N.S.
	*0.313*	106.6	-6.6	3.5	N.S.
	*0.250*	103.8	-3.8	2	N.S.

Chi 2 test for number of micronucleates

N.S.= not statistically significant at the threshold of p+ 0.05

Di-tert-amyl peroxide induced no statistically or biologically significant increases in the number micronucleated TK6 cells in the short-term treatment with and without metabolic activation or in the long-term treatment without S9-mix. Di-tert-amyl peroxide was not genotoxic under these experimental conditions.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-conducted GLP study performed according to established guideline OECD 476 with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

Principles of method if other than guideline:

first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I
without S9 mix 46.3; 92.5; 185; 370; 740; 1480 µg/mL
with S9 mix 46.3; 92.5; 185; 370; 740; 1480 µg/mL
Experiment II
without S9 mix 46.3; 92.5; 185; 370; 740; 1480 µg/mL
with S9 mix 46.3; 92.5; 185; 370; 740; 1480 µg/mL
Following the expression phase of 48 hours the cultures at 46.3 µg/mL in experiment I and II were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the corresponding solvent control or negative control, respectively. A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in negative and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration. Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Water solubility: --
- Precipitation: not observed
- Other confounding effects:none

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 11.6 µg/mL and 1480 µg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the purity (99.0 %) and the molecular weight (146 g/mol) of the test item.
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 and 24 hours of treatment.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation was observed by the unaided eye up to the maximum concentration.
Therefore, the maximum concentration of the main experiment was again 1480 µg/mL or approximately 10 mM. The lower concentrations were spaced by a factor of 2. To overcome problems with possible deviations in toxicity or solubility both main experiments

COMPARISON WITH HISTORICAL CONTROL DATA: complies

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10⁶cells	threshold	growth	10⁶cells	threshold
Experiment I / 4 h treatment			culture I			culture II
Solv. control with THF		-	100.0	194	320	100.0	117	243
Pos. control with MMS	19.5	-	27.1	417	320	85.1	291	243
Test item	46.3	-	culture was not continued^#			culture was not continued^#
Test item	92.5	-	98.4	159	320	180.4	195	243
Test item	185.0	-	101.8	141	320	125.7	177	243
Test item	370.0	-	114.4	123	320	148.8	125	243
Test item	740.0	-	79.6	165	320	159.2	128	243
Test item	1480.0	-	69.9	157	320	210.6	161	243
Experiment I / 4 h treatment			culture I			culture II
Solv. control with THF		+	100.0	194	320	100.0	139	265
Pos. control with CPA	3.0	+	32.2	321	320	47.2	237	265
Pos. control with CPA	4.5	+	50.7	388	320	41.4	313	265
Test item	46.3	+	culture was not continued^#			culture was not continued^#
Test item	92.5	+	136.0	170	320	104.7	122	265
Test item	185.0	+	123.2	179	320	104.1	141	265
Test item	370.0	+	132.7	184	320	114.4	184	265
Test item	740.0	+	94.1	212	320	131.5	109	265
Test item	1480.0	+	126.8	172	320	71.5	226	265
Experiment II / 24 h treatment			culture I			culture II
Solv. control with THF		-	100.0	182	308	100.0	197	323
Pos. control with MMS	13.0	-	25.8	529	308	37.1	564	323
Test item	46.3	-	culture was not continued^#			culture was not continued^#
Test item	92.5	-	92.5	145	308	150.6	98	323
Test item	185.0	-	51.1	279	308	120.9	79	323
Test item	370.0	-	84.5	176	308	181.9	76	323
Test item	740.0	-	73.6	210	308	151.3	107	323
Test item	1480.0	-	69.7	241	308	177.8	95	323
Experiment II / 4 h treatment			culture I			culture II
Solv. control with THF		+	100.0	151	277	100.0	160	286
Pos. control with CPA	3.0	+	51.0	240	277	56.7	262	286
Pos. control with CPA	4.5	+	34.6	302	277	47.9	379	286
Test item	46.3	+	culture was not continued^#			culture was not continued^#
Test item	92.5	+	90.0	163	277	124.8	154	286
Test item	185.0	+	96.1	143	277	84.4	224	286
Test item	370.0	+	90.6	214	277	76.5	206	286
Test item	740.0	+	111.8	152	277	128.4	111	286
Test item	1480.0	+	66.6	273	277	105.8	216	286

Threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

# culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The potential of di-tert-butyl peroxide to induce mutations at the mouse lymphoma thymidine kinase locus was investigated using the cell line L5178Y. This study was conducted according to the OECD TG no. 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 4 h with and 24 h without metabolic activation. The maximum tested concentration was equal to about 10 mM. Both main experiments were evaluated at 92.5; 185; 370; 740; and 1480 µg/mL with and without S9 mix.

The range of the solvent controls was from 117 up to 197 mutant colonies per 10e6 cells; the range of the groups treated with the test item was from 76 up to 279 mutant colonies per 10e6cells.The highest solvent control values (182, 194, and 197 colonies per 10e6 cells) exceeded the recommended range of 50 – 170 x 10e6 cells. The data are judged as acceptable however, since the range of up to 200 cultures per 10e6 cells recommended by the IWGT in 2003 was covered. MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.

It was concluded that di-tert-butyl peroxide was devoid of mutagenic activity under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

For this endpoint, an OECD 474 test is available on di-tert-amyl peroxide and OECD 474 and 483 assays are available on the analogue substance di-tert-butyl peroxide.

Study on di-tert-amyl peroxide

Di-tert-amyl peroxide was tested in an in vivo micronucleus test for its ability to induce structural or numerical damage in bone marrow cells of mice (Haddouk, 2006). The study was performed according to the international guidelines (OECD 474). Three groups of 5 male and 5 female mice were given intraperitoneal administrations of di-tert-amyl peroxide at dose-levels of 0, 500, 1000 and 2000 mg/kg/day in corn oil, over a 2-day period. One group of five males and five females received the positive control test item (Cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals were killed 24 hours after the last. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE +NE). For both males and females, the mean values of MPE in the groups treated with the di-tert-amyl peroxide, were higher than the concurrent vehicle control groups and out of the vehicle control historical range: 1.5-3.7 MPE/1000 PE for males versus 0.2-1.7 MPE/1000 PE for historical mean vehicle control value and 1.2-3.3 MPE/1000 PE for females versus 0.0-1.4 MPE/1000 PE for historical mean vehicle control value. Statistical significance was observed only in groups of females treated at 1000 and 2000 mg/kg/day. However, in view of the dose-related increase observed in both males and females and the frequencies of MPE which were almost all out of the vehicle control historical range, the effect observed was considered as biologically significant in both males and females. Under these experimental conditions, di-tert-amyl peroxide induced an increase damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

Study on the analogue substance di-tert-butyl peroxide (CAS No. 110-05-4)

Micronucleus assays in bone marrow

Di-tert-butyl peroxide was tested in an in vivo micronucleus test for its ability to induce structural or numerical damage in bone marrow cells of mice (Sire, 2005). The study was performed according to the international guidelines (OECD 474). Three groups of five male and five female Swiss mice were given intraperitoneal administrations of di-tert-butyl peroxide at dose-levels of 500, 1000 and 2000 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

No clinical signs and no mortality were observed in the animals of both sexes given 500 mg/kg/day. At the dose-levels of 1000 and 2000 mg/kg/day, no mortality was noted. Piloerection was observed in the animals from 24 hours following the first treatment. Statistically significant and dose-related increases in the frequency of MPE were observed in male and female mice of the test item treated groups. Cyclophosphamide induced a highly significant increase (p < 0.01) in the frequency of MPE, indicating the sensitivity of the test system under these experimental conditions. The study was therefore considered valid.

In conclusion, di-tert-butyl peroxide induced damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

Di-tert-butyl peroxide was tested in an in vivo micronucleus test for its ability to induce structural or numerical damage in bone marrow cells of mice (Gudi and Putman, 1996). The study was performed according to the international guidelines (OECD 474). In the absence of mortality in a pilot assay, the high dose for the micronucleus test was set at 5000 mg/kg. Test and control articles were administered in a constant volume of 20 ml/kg body weight by a single oral gavage. In the initial micronucleus assay, male and female mice were dosed with 1250, 2500 or 5000 mg/kg body weight of di-t-butyl peroxide. No mortality occurred in male or female mice. Clinical signs observed in males and females after dose administration included diarrhea at all test article dose levels and lethargy at 2500 and 5000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Reductions up to 15% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the vehicle control groups. Statistically significant increases in micronucleated polychromatic erythrocytes relative to the respective vehicle control group were observed in female mice at 1250 and 5000 mg/kg at the 24 hour time point only (p<0.05, Kastenbaum-Bowman Tables). To confirm the results, the micronucleus study was repeated with a 24 hour harvest only. For the repeat micronucleus assay, male and female mice were dosed with 1250, 2500 or 5000 mg/kg body weight of di-t-butyl peroxide. No mortality occurred in either sex. Clinical signs observed in males and females after dose administration included diarrhea at 2500 and 5000 mg/kg. Bone marrow cells, collected 24 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. No apparent reductions in the ratio of polychromatic erythrocytes to total erythrocytes were observed in the test article-treated groups relative to the vehicle control groups. Statistically significant increases in micronucleated polychromatic erythrocytes relative to the respective vehicle control group were observed in male mice at 5000 mg/kg and in female mice at 2500 and 5000 mg/kg (p<0.05, Kastenbaum-Bowman Tables).

The results of the initial assay indicated a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes as 1250 mg/kg and 5000 mg/kg at the 24 hour sacrifice time in female mice with no evidence of a dose response. In the repeat assay, although a statistically significant increase was observed, at 2500 and 5000 mg/kg in female mice and at 5000 mg/kg in male mice, the number of micronucleated polychromatic erythrocytes induced in both assays were within the historical solvent control range (0-8 per animal) and within the criteria for the determination of a valid test for negative control (5/1000) with the exception of 1/5 female mice at 1250 and 5000 mg/kg in the initial assay and 1/5 male mice at 500 mg/kg in the repeat assay (9/1000). Based on these findings of the study, di-t-butyl peroxide was concluded to be weakly positive in the mouse micronucleus assay.

Di-tert-butyl peroxide was tested in an in vivo micronucleus test for its ability to induce structural or numerical damage in bone marrow cells of mice (Jonker and Reus, 2014). The study was performed according to the international guidelines (OECD 474). This micronucleus test was part of a sub-chronic (13-week) inhalation toxicity study in which Wistar Hannover rats were exposed nose-only to target concentrations of 0 (control, clean air), 100, 300 and 1000 mg/m3 of the test material 6 hours/day, 5 days/week for 13 consecutive weeks (resulting in 65 exposure days in total). At scheduled necropsy at the end of the 13-week study period, bone marrow cells of one of the femurs of five male rats per group (negative control, low, mid and high concentration) were collected, processed into smears and examined microscopically. The study included a positive control group of five male rats treated with the mutagen Mitomycin C (single intraperitoneal injection; 1.5 mg/kg body weight) and sacrificed 24 hours after administration of the mutagen.

The target concentrations were accurately achieved as demonstrated by the results of total carbon analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation of the daily mean concentration) were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and high concentration level respectively. Di-tert-butyl peroxide did not adversely affect the general health, appearance or body weight development of the animals. Microscopic examination of bone marrow smears of male rats revealed no signs of toxicity to the bone marrow and no evidence of chromosomal damage and/or damage to the mitotic apparatus of bone marrow erythrocytes. There was no reason to assume that the negative bone marrow response was due to lack of systemic exposure because treatment-related systemic effects (including increases in liver and kidney weight) occurred in male rats of the high-concentration group. Positive controls (five male rats treated with the mutagen Mitomycin C) showed the expected bone marrow response (cytotoxicity and increased number of micronucleated polychromatic erythrocytes).

Under the conditions of this study exposure to di-tert-butyl peroxide did not induce chromosomal damage or damage to the mitotic apparatus of bone marrow erythrocytes of male rats.

Chromosome aberration assay in germ cell

Di-tert-butyl peroxide was tested in the mammalian spermatogonial chromosome aberration test using male ICR mice (Gudi, R. and Krsmanovic, 2005). The study was performed according to the international guidelines (OECD 473). The chromosome aberration assay was designed to evaluate the potential of the test article to induce chromosome aberrations in spermatogonial cells. The chromosome aberration assay consisted of five groups, each containing 5 male ICR mice. Animals in these groups were intraperitoneally exposed to the controls (negative or positive) or to di-t-butyl peroxide at a dose of 200, 1000 or 2000 mg/kg/day. The test article was formulated in corn oil. Corn oil was used as the negative control (vehicle) and Mitomycin C (MMC), at a dose of 4 mg/kg, as the positive control article. The test and negative control article were administered on two consecutive days, separated by approximately 24 hours. Each administration was conducted at a dose volume of 20 mL/kg body weight. Animals were observed following each dose administration and during the course of the study. Colchicine was given 4-5 hours prior to euthanasia to arrest cells in metaphase. Twenty-four hours after the last dose, animals were euthanized and testes were removed from animal body cavity. Spermatogonial cells were isolated from the tubules and smeared onto the microscope slide. The smears were stained with Giemsa stain. One hundred metaphase cells per each animal were scored for structural chromosome aberrations. A statistically significant difference between the test article treated groups relative to the concurrent negative (vehicle) control was determined using Fisher’s exact test for level of significance of p< 0.05. Mitotic index (MI) was calculated for each animal as the ratio of spermatogonial cells in mitosis per 1000 cells observed. No mortality or clinical signs were observed in any of the mice during the course of the study. No statistically significant increase in the percentage of aberrant cells and no dose-related decrease of the mitotic index were observed in the test article-treated groups relative to the vehicle control (p > 0.05 Fisher’s exact test). The results of the study indicate that under the conditions described in this report, di-t-butyl peroxide, when intraperitoneally administered on two consecutive days (at doses up to 2000 mg/kg/day), did not induce a significant increase in the percentage of spermatogonial cells with structural chromosome aberrations. Therefore, di-t-butyl peroxide was concluded to be negative in the spermatogonial chromosome aberration test.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, Klimisch 1a

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Breeder: Charles River Laboratories, l'Arbresle, France.
- Age: on the day of treatment, the animals were approximately 6 weeks old.
- Weight: at the beginning of treatment the mean body weight was 31.8 g for males (ranging from 30.3 to 33.2 g) and 22.8 g for females (ranging from 19.1 to 25.1 g).
- Veterinary care at CIT: upon their arrival at CIT, the animals were given a complete examination to ensure that they were in good clinical conditions.
- Acclimation: at least 5 days before the day of treatment.
- Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex.
- Subsequently, each group was assigned to a different treatment group.
- Identification: individual tail marking upon treatment.

Upon their arrival at CIT, the animals were housed in an animal room, with the following
environmental conditions:
• temperature: 22 ± 2°C,
• relative humidity: 30 to 70%,
• light/dark cycle: 12 h/12 h (07:00 - 19:00),
• ventilation: at least 12 cycles/hour of filtered non-recycled fresh air.
The temperature and relative humidity were under continuous control and recording. The
housing conditions (temperature, relative humidity and ventilation) and corresponding
instrumentation and equipment were verified and calibrated at regular intervals.

Food and water: were provided ad libitum

Route of administration:

intraperitoneal

Vehicle:

corn oil

Duration of treatment / exposure:

2 days

Frequency of treatment:

Frequency: two treatments separated by 24 hours, volume: 10 mL/kg

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5 males and 5 females at 500 mg/kg
5 males and 5 females at 1000 mg/kg
8 males and 8 females at 2000 mg/kg (but since no mortality occurred, only five animals of each sex were subjected to bone marrow analysis. The
supplementary animals were humanely killed and bone marrow smears were not prepared)
3 additional males and 3 additional females at 2000 mg/kg for determination of plasma level of the test item

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control was Cyclophosphamide (CPA, Endoxan, Baxter, Maurepas, France), batch No. 4A121 dissolved in distilled water at a concentration of 5 mg/mL. The preparation was stored at -20°C and thawed immediately before use.

Details of tissue and slide preparation:

Preparation of the bone marrow smears
At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

Microscopic examination of the slides
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
The analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was Natalie Danford. Details concerning the method used, the format of the data, communication, quality assurance and archiving are indicated in the corresponding PIDS (Principal Investigator Data Sheet) reported in appendix 3.

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data (appendix 4), or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

Normality and homogeneity of variances were tested using a Kolmogorov Smirnov test and a
Bartlett test.
If normality and homogeneity of variances were demonstrated, the statistical comparison was
performed using a Student "t" test (two groups) or a one-way analysis of variance
(= three groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Withney test
(two groups) or a Kruskall Wallis test (= three groups) was performed followed by a Dunnett test
(if necessary).
All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for all tests.

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

For both males and females, the mean values of MPE in the groups treated with the test item DI-TERT-AMYL PEROXIDE, were higher than the concurrent vehicle control groups and out of the vehicle control historical range: 1.5-3.7 MPE/1000 PE for males versus 0.2-1.7 MPE/1000 PE for historical mean vehicle control value and 1.2-3.3 MPE/1000 PE for females versus 0.0-1.4 MPE/1000 PE for historical mean vehicle control value.

Statistical significance was observed only in groups of females treated at 1000 and 2000 mg/kg/day. However, in view of the dose-related increase observed in both males and females and the frequencies of MPE which were all (except for the low dose groups) out of the vehicle control historical range, the effect observed was considered as biologically significant in both males and females.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Clinical signs:

Piloerection was noted in all animals treated at 500, 1000 or 2000 mg/kg/day. In one male from the high dose group (supplementary animal) half-closed eyes were also observed at 24 hours following the second treatment.

One female from the 1000 mg/kg/day dose group was found dead (cannibalised animal) 24 hours following the second treatment.

Results:

			Results of the cytogenetic test: data summary
			MPE/1000PE		PE/NE ratio
	Group	Doses (mg/kg/day)	mean	sd	mean	sd	time of sacrifice after the last administration
Males	Vehicle	_	1,4	0,7	0,4	0,1	24 h
	DTA	500	1,5	1,1	0,5	0,1
	DTA	1000	2,8	1,2	0,4	0
	DTA	2000	3,7	1,8	0,4	0,1
	cyclophosphamide	50	27,6*	2,2	0,8**	0,1
Females	Vehicle	_	0,8	0,6	0,5	0,1
	DTA	500	1,2	1	0,6	0,2
	DTA	1000	2,4*	1,1	0,5	0,3
	DTA	2000	3,3**	0,8	0,4	0,1
	cyclophosphamide	50	21,2**	6,8	0,6	0,3

Five animals per group (except for females treated at 1000 mg/kg/day: 4 animals) MPE: Micronucleated Polychromatic Erythrocytes

PE: Polychromatic Erythrocytes NE: Normochromatic Erythrocytes sd: standard deviation

Vehicle and test item:

Number of administrations: two administrations separated by a 24-hour interval

Route: intraperitoneal route

Vehicle: corn oil

Cyclophosphamide:

Number of administrations: one

Route: oral

Vehicle: water

Statistical significance:

* p<0,05

** p<0,01

Conclusions:

Di-tert-amyl peroxide induced an increase damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential of di-tert-amyl peroxide, to induce structural or numerical damage in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474) and in compliance with the Principles of Good Laboratory Practice Regulations.

Three groups of 5 male and five female mice were given intraperitoneal route administrations of di-tert-amyl peroxide at dose-levels of 0, 500, 1000 and 2000 mg/kg/day in corn oil, over a 2-day period.

One group of five males and five females received the positive control test item (Cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals were killed 24 hours after the last. Bone marrow smears were then prepared.

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

For both males and females, the mean values of MPE in the groups treated with the di-tert-amyl peroxide, were higher than the concurrent vehicle control groups and out of the vehicle control historical range: 1.5-3.7 MPE/1000 PE for males versus 0.2-1.7 MPE/1000 PE for historical mean vehicle control value and 1.2-3.3 MPE/1000 PE for females versus 0.0-1.4 MPE/1000 PE for historical mean vehicle control value.

Statistical significance was observed only in groups of females treated at 1000 and

2000 mg/kg/day. However, in view of the dose-related increase observed in both males and females and the frequencies of MPE which were almost all out of the vehicle control historical range, the effect observed was considered as biologically significant in both males and females.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under these experimental conditions, di-tert-amyl induced an increase damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

72 hours (2x24 for treatment plus 24hr post-treatment examination)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented study conducted according to modern standards of internationally accepted protocol and Good Laboratory Practice. Test Material analytically confirmed for purity; however, analyses to determine the uniformity or concentration of the test article dose formulations or their stability were not included in the final report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Swiss Ico: OF1 (IOPS Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Preliminary toxicity test: 3 male and 3 female mice were used.
Main cytogenetic test: 56 mice, 28 males and 28 females were used.
Blood sampling: 6 mice, 3 males and 3 females were used.
Strain: Swiss Ico: OF1 (IOPS Caw).
Reason for this choice: rodent species generally accepted by regulatory authorities for this type of study.
Breeder: Charles River Laboratories, l'Arbresle, France.
Age: on the day of treatment, the animals were approximately 6 weeks old.
Veterinary care: upon their arrival, the animals were given a complete examination to ensure that they were in good clinical condition.
Acclimation: at least 5 days before the day of treatment.
Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex. Subsequently, each group was assigned to a different treatment group.
Identification: individual tail marking upon treatment.

Environmental conditions
Upon their arrival, the animals were housed in an animal room, with the following environmental conditions:
• temperature: 22 ± 2°C,
• relative humidity: 30 to 70%,
• light/dark cycle: 12 h/12 h (07:00 - 19:00),
• ventilation: at least 12 cycles/hour of filtered non-recycled fresh air.
The temperature and relative humidity were under continuous control and recording. The housing conditions (temperature, relative humidity and ventilation) and corresponding instrumentation and equipment were verified and calibrated at regular intervals. The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analyzed by the supplier for composition and contaminant levels.

Food and water
All animals had free access to A04 C pelleted maintenance diet (SAFE, Villemoisson-sur-Orge, France). Each batch of food is analysed by the supplier for composition and contaminant levels. Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analysis of water are performed regularly by external laboratories, These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines). No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to interfere with or prejudice the outcome of the study.

Route of administration:

intraperitoneal

Vehicle:

The vehicle was corn oil, batch No. 122K0131 (Sigma, Saint-Quentin-Fallavier, France).

Details on exposure:

Route for the vehicle and the test item: intraperitoneal (at the request of the Sponsor)
• Frequency: two treatments separated by 24 hours
• Volume: 10 mL/kg
• Positive Control (cyclophosphamide): oral route, one treatment.
The quantity of each item administered to each animal was adjusted according to the most recently recorded body weight.

Duration of treatment / exposure:

2 administrations

Frequency of treatment:

two injections, 24-hours apart

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5M/5F per dose at 0, 500, 1000 mg/kg bw
8M/8F per dose at 2000 mg/kg bw

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control was cyclophosphamide (CPA), batch No. OD203A (Laboratoire Asta Médica, Mérignac, France) dissolved in distilled water at a concentration of 5 mg/mL. The preparation was stored at -20°C and thawed immediately before use. CPA: oral route, one treatment.

Tissues and cell types examined:

femur bone marrow

Details of tissue and slide preparation:

At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

Microscopic examination of the slides
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). The analysis of the slides was performed in compliance with GLP at a separate site. Details concerning the method used for slide examination, the format of the data, communication, quality assurance and archiving are indicated in a PIDS (Principal Investigator Data Sheet) appended to the final report.

Evaluation criteria:

Treatment of results
The final report includes all the individual data presented in tabular form. The number of MPE/2000 PE and the PE/NE ratio are given for each animal.
The means and the standard deviations of the frequency of MPE/1000 PE and the PE/NE ratio are given for each experimental group.

Evaluation criteria
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

Statistical analysis
When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Lovell et al., 1989), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the ¿2 value (Lovell et al., 1989). When there was significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969). The student "t" test was used for the PE/NE ratio comparison. Probability values of p = 0.05 was considered as significant.

Sex:

male/female

Genotoxicity:

positive

Remarks:

at all levels

Toxicity:

yes

Remarks:

at mid and high dose, piloerection noted for 24 hours following first treatment

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Under the described experimental conditions, the test item DITERTIOBUTYL PEROXIDE (batch No. 11648 995404-109, purity: 98.9%) induced damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

Group	Dose (mg/kg/day)	MPE/1000 PE		PE/NE ratio		sacrifice time
		mean	SD	mean	SD
males	0 (vehicle)	0.5	1.1	0.3	0.1	24 h
	500	1.9	0.9	0.5	0.1
	1000	2.9*	1.5	0.4	0.2
	2000	5.4**	3.2	0.4	0.1
CPA	50	26.3**	8.1	0.5	0.1
females	0 (vehicle)	1.4	1.6	0.6	0.1	24 hours
	500	4.2*	1.3	0.8	0.3
	1000	4.9	3.3	0.9	0.2
	2000	6.0*	2.7	0.9	0.2
CPA	50	18.0**	7.5	0.8	0.2

Conclusions:

Interpretation of results (migrated information): positive
Under the described experimental conditions, the test item DITERTIOBUTYL PEROXIDE (batch No. 11648 995404-109, purity: 98.9%) induced damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

Executive summary:

Three groups of five male and five female Swiss mice were given intraperitoneal administrations of DITERTIOBUTYL PEROXIDE at dose-levels of 500, 1000 and 2000 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

No clinical signs and no mortality were observed in the animals of both sexes given 500 mg/kg/day. At the dose-levels of 1000 and 2000 mg/kg/day, no mortality was noted. Piloerection was observed in the animals from 24 hours following the first treatment. Statistically significant and dose-related increases in the frequency of MPE were observed in male and female mice of the test item treated groups.Cyclophosphamide induced a highly significant increase (p < 0.01) in the frequency of MPE, indicating the sensitivity of the test system under these experimental conditions. The study was therefore considered valid.

Under these experimental conditions, the test item DITERTIOBUTYL PEROXIDE (batch No. 11648 995404-109, purity: 98.9%) induced damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well-conducted GLP study with no major deviations. Test material identified only by trivial chemical name and batch number; neither trade name, chemical name, CAS number nor purity were provided.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

ICR mice were obtained from Harlan Sprague Dawley, Inc., Frederick, MD. At the initiation of the study, the mice were 6 to 8 weeks old. Animal body weights were recorded within the following ranges:

Pilot study
Males, 30.6 - 33.9 grams at randomization
Females, 25.0 - 28.4 grams at randomization

Micronucleus assay:
Males, 27.0 - 35.0 grams at randomization
Females, 24.0 - 29.9 grams at randomization

Repeat Micronucleus Assay:
Males, 31.5 - 37.0 grams at randomization
Females, 24.3 - 29.2 grams at randomization

Mice were obtained from a source monitored for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas and appropriate murine viruses and were quarantined for no less than 5 days after receipt. Mice were housed in an AAALAC-accredited facility with a controlled environment of 74 +/- 6 °F, 50 +/- 20% relative humidity, and a 12-hour light-dark cycle. 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. Heat treated hardwood chips were used for bedding. Mice had free access to certified laboatory rodent chow which had been analyzed for environmental contaminants and to tap water. No contaminants that could be expected to alter the outcome of the study are known to be present in the diet or water.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test article-vehicle mixture, the vehicle alone, or the positive control was administered by oral gavage at a constant volume of 20 ml/kg body weight. All mice in the experimental and control groups were weighed individually prior to dose administration and the dose volume was based on individual body weights. Mice were observed after dose administration for clinical signs of ehemical effect.

For the pilot study. di-t-butyl peroxide was administered by oral gavage to male mice at 1, 10. 100, or 1000 mg test article/kg and to male and female mice at 5000 mg/kg which was administered in a total volume of 20 ml test article-vehicle mixture/kg body weight. No mortality occurred at any test article dose levels. Clinical signs observed after dose administration included diarrhea in males and females at 5000 mg/kg. All other animals appeared normal throughout the observation period. In the absence of mortality in the pilot study, a toxicity study was not required. The highest dose level for the micronucleus study was set at 5000 mg/kg. For the repeat micronucleus assay, male and female mice were dosed with 1250, 2500 or 5000 mg/kg body weight of di-t-butyl peroxide.

Duration of treatment / exposure:

One treatment.

Frequency of treatment:

Once.

Post exposure period:

Three days.

Remarks:

Doses / Concentrations:
1250, 2500 and 5000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

See Table below.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, 60 mg/kg

Tissues and cell types examined:

Bone marrow cells from the femur.

Details of tissue and slide preparation:

At scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediate 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 a capped 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. Slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

Evaluation criteria:

The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group. In order to quantify the test article effect on erythropoiesis, as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to toal 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 elevetaed relative to the vehicle control (p

Statistics:

Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970; Mackey and MacGregor, 1979). All analyses were performed separately for each sex and sampling time.

Sex:

male/female

Genotoxicity:

other: weakly positive

Toxicity:

yes

Remarks:

diarrhea observed in males at 1250, 2500 and 5000 mg/kg and females at 5000 mg/kg; lethargy in females at 2500 and 5000 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the pilot assay, male mice were dosed with 1, 10, 100 or 1000 mg test article/kg body weight and male and female mice were dosed with 5000 mg/kg. No mortality occurred at any test article dose levels. Clinical signs observed after dose administration included diarrhea in males and females at 5000 mg/kg. In the absence of mortality in the pilot assay, the high dose for the micronucleus test was set at 5000 mg/kg.

In the initial micronucleus assay, male and female mice were dosed with 1250, 2500 or 5000 mg/kg body weight of di-t-butyl peroxide. No mortality occurred in male or female mice in the micronucleus study. Clinical signs observed in males and females after dose administration included diarrhea at all test article dose levels and lethargy at 2500 and 5000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Reductions up to 15% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the vehicle control groups. Statistically significant increases in micronucleated polychromatic erythrocytes relative to the respective vehicle control group were observed in female mice at 1250 and 5000 mg/kg at the 24 hour time point only (p < 0.05, Kastenbaum-Bowman Tables). To confirm the results, the micronucleus study was repeated with a 24 hour harvest only.

For the repeat micronucleus assay, male and female mice were dosed with 1250, 2500 or 5000 mg/kg body weight of di-t-butyl peroxide. No mortality occurred in either sex. Clinical signs observed in males and females after dose administration included diarrhea at 2500 and 5000 mg/kg. Bone marrow cells, collected 24 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. No apparent reductions in the ratio of polychromatic erythrocytes to total erythrocytes were observed in the test article-treated groups relative to the vehicle control groups. Statistically significant increases in micronucleated polychromatic erythrocytes relative to the respective vehicle control group were observed in male mice at 5000 mg/kg and in female mice at 2500 and 5000 mg/kg (p<0.05. Kastenbaum-Bowman Tables).

Summary of bone marrow micronucleus study using di-t-butyl peroxide

Treatment	Sex	Time (hr)	Number of mice	PCE/total erythrocytes (mean+SD)	Change from control (%)	Micronucleated polychromatic erythrocytes
						number per 1000 PCEs (mean +/- SD)	Number per PCEs scored
Corn Oil 20 ml/kg	M	24	5	0.54+0.07	…	1.4+1.14	7/5000
	F	24	5	0.55+0.03	…	0.6+0.35	3/5000

di-t-butyl peroxide
1250 mg/kg	M	24	5	0.55+0.06	2	1.6+1.34	8/5000
	F	24	5	0.56+0.08	2	3.6+3.71	18/5000

2500 rng/kg	M	24	5	0.51+0.13	-6	2.6+1.52	13/5000
	F	24	5	0.59+0.04	7	1.8+2.05	9/5000

5000 mg/kg	M	24	5	0.52+0.12	-4	3.6+2.07	18/5000
	F	24	5	0.55+0.06	0	3.4+3.36	17/5000

CP,60 mg/kg	M	24	5	0.47+0.10	-13	32.8+8.58	164/5000
	F	24	5	0.52+0.07	-5	29.6+7.37	148/5000

Treatment	Sex	Time (hr)	Number of mice	PCE/total erythrocytes (mean+SD)	Change from control (%)	Micronucleated polychromatic erythrocytes
						number per 1000 PCEs (mean +/- SD)	Number per PCEs scored
Corn Oil 20 ml/kg	M	48	5	0.55+0.04	…	1.4+2.61	7/5000
	F	48	5	0.51+0.06	…	1.4+0.89	7/5000

di-t-butyl peroxide
1250 mg/kg	M	48	5	0.55+0.08	0	1.2+1.30	6/5000
	F	48	5	0.57+0.04	12	0.8+0.45	4/5000

2500 rng/kg	M	48	5	0.59+0.05	7	1.4+0.55	7/5000
	F	48	5	0.58+0.04	14	1.2+0.84	6/5000

5000 mg/kg	M	48	5	0.47+0.11	-15	2.6+2.30	13/5000
	F	48	5	0.45+0.17	-12	2.8+2.17	14/5000

Treatment	Sex	Time (hr)	Number of mice	PCE/total erythrocytes (mean+SD)	Change from control (%)	Micronucleated polychromatic erythrocytes
						number per 1000 PCEs (mean +/- SD)	Number per PCEs scored
Corn Oil 20 ml/kg	M	72	5	0.53+0.11	…	1.0+1.41	5/5000
	F	72	5	0.59+0.05	…	0.8+0.45	4/5000

di-t-butyl peroxide
1250 mg/kg	M	72	5	0.52+0.08	-2	0.2+0.45	1/5000
	F	72	5	0.57+0.06	-3	0.4+0.55	2/5000

2500 rng/kg	M	72	5	0.55+0.13	4	1.2+1.35	6/5000
	F	72	5	0.59+0.04	0	0.8+0.84	4/5000

5000 mg/kg	M	72	5	0.51+0.09	-4	1.4+1.14	7/5000
	F	72	5	0.56+0.06	-5	1.0+0.17	5/5000

Conclusions:

Interpretation of results (migrated information): other: weakly positive
Based on these findings of the study, the results of the assay indicated that under the conditions described in this report, di-t-butyl peroxide was concluded to be weakly positive in the mouse micronucleus assay.

Executive summary:

The test article, Di-t-butyl peroxide, was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay. The second phase, the micronucleus study, evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control articles were administered in a constant volume of 20 ml/kg body weight by a single oral gavage.

In the initial micronucleus assay, male and female mice were dosed with 1250, 2500 or 5000 mg/kg body weight of di-t-butyl peroxide. No mortality occurred in male or female mice in the micronucleus study. Clinical signs observed in males and females after dose administration included diarrhea at all test article dose levels and lethargy at 2500 and 5000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Reductions up to 15% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the vehicle control groups. Statistically significant increases in micronucleated polychromatic erythrocytes relative to the respective vehicle control group were observed in female mice at 1250 and 5000 mg/kg at the 24 hour time point only (p<0.05, Kastenbaum-Bowman Tables). To confirm the results, the micronucleus study was repeated with a 24 hour harvest only.

For the repeat micronucleus assay, male and female mice were dosedwith1250, 2500 or 5000 mg/kg body weight of di-t-butyl peroxide. No mortality occurred in either sex. Clinical signs observed in males and females after dose administration included diarrhea at 2500 and 5000 mg/kg. Bone marrow cells, collected 24 hours after treatment, were examined microscopically formicronucleatedpolychromatic erythrocytes. No apparent reductions in the ratio of polychromatic erythrocytes to total erythrocytes were observedin the test article-treated groups relative to the vehicle control groups. Statistically significant increases in micronucleated polychromatic erythrocytes relative to the respective vehicle control group were observed in male mice at 5000 mg/kg and in female mice at 2500 and 5000 mg/kg (p<0.05.Kastenbaum-BowmanTables).

The results of the initial assay indicated a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes as 1250 mg/kg and 5000 mg/kg at the 24 hour sacrifice time in female mice with no evidence of a dose response. In the repeat assay, although a statistically significant increase was observed, at 2500 and 5000 mg/kg in female mice and at 5000 mg/kg in male mice, the number of micronucleated polychromatic erythrocytes induced in both assays were within the historical solvent control range (0-8 per animal) and within the criteria for the determination of a valid test for negative control (5/1000) with the exception of 1/5 female mice at 1250 and 5000 mg/kg in the initial assay and 1/5 male mice at 500 mg/kg in the repeat assay (9/1000). Based on these findings of the study, the results of the assay indicated that under the conditions described in this report, di-t-butyl peroxide was concluded to be weakly positive in the mouse micronucleus assay.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2012 - March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study conducted according to modern standards of internationally accepted protocol and Good Laboratory Practice. Test Material analytically confirmed for purity

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

Principles of method if other than guideline:

Animals were exposed for 90 days via inhalation.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

The study was conducted with albino rats. Young adult, male and female Wistar Hannover outbred rats (RccHan®:WIST) were obtained from a colony maintained under specific pathogenfree (SPF) conditions at Harlan Laboratories, The Netherlands (for the in vivo MN test, only male rats were used). On the day of randomization (shortly before the first exposure day), the age of the rats was about 7-8 weeks, and the initial body weight variation did not exceed ± 20% of the mean weight for each sex. Mean body weights at the start of treatment were 264 and 176 grams for male and female animals, respectively.

Upon arrival, the rats were taken to a quarantine room and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. Two days after arrival, the
results of serological tests were passed on by telephone and indicated an acceptable microbiological status. Subsequently, the animals were released for experimental use and moved to their definitive room. The duration of the acclimatization period to the conditions in the experimental room
prior the first exposure was 10 days (males) or 11 days (females). Shortly before initiation of exposure (study days -4 and -5 for males and females, respectively), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals were kept in reserve to serve as sentinels (four/sex). These animals were discarded at the end of the in-life phase of the study.

From their arrival, the rats were housed under conventional conditions in one room separated by sex. No other test system was housed in the same room during the study. Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark. The room was ventilated with about 10 air changes per hour. The temperature and relative humidity in the room were 22 ± 2°C and 45-65%, respectively, with a few exceptions.

The animals were housed in groups of five, separated by sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment (Lillico, Betchworth, England). During exposure, the animals were kept individually in the exposure unit. Immediately after each exposure, the animals were returned to their home cages. After treatment with the mutagen Mitomycin C, the five animals of the positive control group were kept in smaller Makrolon® cages with filter tops (one or two animals per cage; bedding: wood shavings; enrichment: strips of paper) until sacrifice the next day.

Feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period before scheduled sacrifice. The animals received a proprietary cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of RM3 diet is analysed by the supplier for nutrients and contaminants. The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The feed in the feeders was replaced with fresh portions once weekly and filled as needed. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the test facility for a limited number of variables.

Route of administration:

inhalation: vapour

Vehicle:

Air

Details on exposure:

The animals were exposed to the test atmosphere in a nose-only inhalation chamber (Institute’s design) consisting of a cylindrical PVC column with a volume of about 75 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column and was exhausted at the top. Each column included three rodent tube levels of 20 ports each. The animals were placed at the top level. Empty ports were used for measurement of temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column. Only the nose of the rats protruded into the interior of the column. The remaining ports were closed. Male and female rats were placed in alternating order. Animals were rotated weekly with respect to the position in the column. From 25 February 2013, larger sized animal holders were used for the male rats because these animals had reached a size for which the standard holders were too small.
The animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing
a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose. This way, dilution of test atmosphere at the animals’ noses was avoided.
The units were illuminated externally by normal laboratory fluorescent tube lighting. The total airflow through the unit was at least 1 litre/min per animal. The air entering the unit was maintained between 22 ± 3°C and the relative humidity between 30% and 70%.

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a peristaltic pump (Gilson France SA, Villiers le Bel, France), was evaporated in a glass evaporator. The temperature of the evaporator was controlled at 22.5°C (exceptions: 22.7 or 22.8°C on a few occasions) using a temperature controlled flow of circulating water. The vapour was transported in a stream of humidified compressed air, the flow of which was controlled by a mass flow controller (Bronkhorst, Hi Tec, Ruurlo, The Netherlands). All three test atmospheres (target concentrations 0.1, 0.3 and 1 g/m3) were obtained by diluting a pre-mixture containing about 3 g/m3 of the test material in humidified compressed air. First, mass flow controlled streams of the pre-mixture were supplemented with a mass flow controlled stream of humidified compressed air via an eductor to obtain the low- and mid-concentration. Next, the remaining stream of the pre-mixture was diluted with a mass flow controlled stream of humidified compressed air to obtain the high-concentration. The generated test atmospheres were directed to the bottom inlets of the exposure units. The exposure unit for the control animals was supplied with a measured stream of humidified compressed air only. The animals were placed in the exposure unit after stabilization of the test atmosphere.

The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak EuroFID total hydrocarbon analyser; Sick Instruments Benelux, Hedel, the Netherlands). The response of the analyser was recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line.
Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using PET sample bags with known volumes of clean dry air and known amounts (by weighing) of test material. For each target concentration three calibration concentrations were prepared, at least in duplicate, and analysed (approximately 80, 100 and 120% of the target concentration). The calibrations were checked weekly by measuring the concentration in a sample bag with a theoretical concentration close to the target concentration. If the measured concentration deviated more than 5% from the theoretical concentration and this was confirmed with a second sample bag, the TCA was recalibrated.

The nominal concentration was determined, for each exposure day, by dividing the total amount of test material used (by weight) by the total volume of air passed through the exposure unit. The nominal concentration was calculated for the low-, mid- and high-concentration as well as for the pre-mixture which was diluted to obtain the test atmospheres. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration).

The chamber airflow of the test atmospheres was recorded about hourly using a Rotameter (group 1) or a mass flow controller (groups 2-4). The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). For group 4 of the sub-chronic study the temperature and relative humidity were additionally measured about hourly by means of a RH/T device (TESTO 635-1, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany).

The overall mean actual concentrations (+/- standard deviation) of the test material in the test atmospheres as measured by total carbon analysis were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and high-concentration, respectively. These actual concentrations were very close to the target concentrations (100, 300 and 1000 mg/m3).

The overall mean nominal concentrations, calculated from the daily consumption of test material, the airflow and the duration of test atmosphere generation, were 96, 290 and 1036 mg/m3 for the low-, mid- and high-concentration, respectively. The corresponding generation efficiencies were close to the expected 100%, namely 105, 103 and 96%, respectively.

The overall mean (± standard deviation) chamber airflows were 27.8 (± 0.0), 26.2 (± 0.2), 27.6 (± 0.3) and 24.3 (± 0.2) L/min for exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high), respectively.

The air temperature in the exposure chambers during exposure was within the target range of 19 – 25°C. The overall mean temperature was about 23°C for each chamber. The relative humidity during exposure was within the target range of 30-70%. The overall mean relative humidity was 46, 40, 39 and 44% in exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high), respectively.

Duration of treatment / exposure:

90 days (65 exposure days)

Frequency of treatment:

6 h/day, 5 days/week

Post exposure period:

Not applicable

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/m3
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0, 101, 299, and 993 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5 males/concentration
5 males positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C was administered by a single intraperitoneal injection at a dose level of 1.5 mg/kg body weight as a solution in physiological saline (dose volume 10 ml/kg body weight; concentration 0.15 mg/ml).

Tissues and cell types examined:

At sacrifice, bone marrow cells of one of the femurs (left femur) were collected from five male animals per group.

Details of tissue and slide preparation:

The bone marrow cells were immediately collected into foetal calf serum and processed into glass drawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald-Giemsa solution. The other fixed unstained smear was kept in reserve and discarded after completion of analysis.

The bone marrow smears of the males of groups 1-5 were examined microscopically. The slides were randomly coded by a person not involved in the
scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear.
The following criteria were used for the scoring of cells:
¿ A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
¿ A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
¿ A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain. The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. The incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

The test was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and the negative controls were within the historical range.
A test material was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a dose-related positive response or a clear increase of micronucleated cells in a single dose group.
A test material was considered to be negative in the micronucleus test if it did not produce a positive response at any of the dose levels analysed.
The test material or its metabolites were considered to be cytotoxic to the bone marrow via the general circulation, if the test material statistically significantly reduced the mean number of PE.
Both statistical significance and biological relevance were considered together in the evaluation.

Statistics:

Statistical tests were performed using GraphPad Prism®, Version 5.03, Copyright © 1992-2010 GraphPad Software, Inc., CA, USA.. In all tests a significance level of 5% was used (a = 0.05). Data on PE and MPE (PE/200 E and MPE/2000 PE) were analysed by one-way analysis of variance [Anova]. Prior to Anova, it was checked if the Anova assumptions were met (i.e. variances equal). In case assumptions were not met non-parametric testing was performed using the Mann-Whitney test (positive control compared with negative control) or Kruskal-Wallis analysis of variance (test substance groups compared with negative control). Two Anova models were applied for both PE/200 E and MPE/2000 PE. In the first Anova model it was tested if the positive control differed from the negative control (t-test). In the second Anova model (including Dunnett’s test as post-hoc test) it was tested if the test material (different doses) differed from the negative control.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MPE results (see Table 5 below)
The mean number of MPE/2000 PE in the negative control (group 1) was within the historical range. The mean number of MPE/2000 PE in the positive control group treated with mitomycin C (group 5) was within the historical positive control range and statistically significantly increased (p value: 0.0097) compared to the concurrent negative control (group 1). This indicates that the positive control substance mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells under the conditions of this study. These results, together with the normal MPE/PE ratio in the negative control group, demonstrate the validity of the test system.
The mean numbers of MPE/2000 PE in the groups exposed to the test material (groups 2-4) did not differ statistically significantly from the mean MPE/2000 PE in the negative control group (group 1). This indicates that treatment with the test material under the conditions of this study did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells.

PE results (see Table 5 below)
Compared with the negative control group (group 1), positive controls treated with mitomycin C showed a statistically significant decrease (p value: 0.0001) in the number of PE/200 E, indicating that mitomycin C was cytotoxic to the bone marrow. The mean numbers of PE/200 E in the groups exposed to the test material did not differ statistically significantly from the mean PE/200 E in the negative control (group 1). This indicates that treatment with the test material under the conditions of this study did not result in cytotoxicity to the bone marrow.

Clinical signs and mortality

All animals survived until scheduled sacrifice. No treatment-related clinical signs were observed in animals exposed to the test material or treated with the positive control substance. The few signs observed were incidental findings unrelated to treatment.

Body weight

Mean body weights of animals exposed to the test material showed no biologically or statistically significant differences from controls.

Organ weights

The organ weight results for male animals showed the following statistically significant differences between animals exposed to the test material and controls:

- Higher relative liver weight at the high-concentration (relative difference from control 9%).

- Higher relative kidney weight at the high-concentration (relative difference from control 11%).

Conclusions:

Interpretation of results (migrated information): negative
The results of this micronucleus test incorporated in a sub-chronic (13-week) toxicity study did not provide any indication of chromosomal damage or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats exposed via inhalation to di-tert-butyl peroxide CAS# 110-05-4 for 6 hours/day, 5 days/week (total of 65 exposure days) at concentrations of 101 (± 3), 299 (± 3) or 993 (± 10) mg/m3 (mean actual concentrations ± standard deviation, determined by total carbon analysis). As treatment-related systemic effects were observed in male rats of the high-concentration group (increased weights of the liver and kidneys), there is no reason to assume that the negative bone marrow response was due to lack of systemic exposure.

Executive summary:

The purpose of this mammalian in vivo micronucleus test was to examine the potential of di-tert-butyl peroxide CAS# 110-05-4 to cause damage to the chromosomes and/or the mitotic apparatus of erythroblasts (micronuclei). This micronucleus test was part of a sub-chronic (13-week) inhalation toxicity study in which Wistar Hannover rats were exposed nose-only to target concentrations of 0 (control, clean air), 100, 300 and 1000 mg/m3 of the test material 6 hours/day, 5 days/week for 13 consecutive weeks (resulting in 65 exposure days in total).

The micronucleus test was conducted in accordance with the OECD Guideline for the Testing of Chemicals 474. Mammalian Erythrocyte Micronucleus Test, adopted 21st July 1997. At scheduled necropsy at the end of the 13-week study period, bone marrow cells of one of the femurs of five male rats per group (negative control, low, mid and high concentration) were collected, processed into smears and examined microscopically. The study included a positive control group of five male rats treated with the mutagen Mitomycin C (single intraperitoneal injection; 1.5 mg/kg body

weight) and sacrificed 24 hours after administration of the mutagen.

Di-tert-butyl peroxide CAS# 110-05-4 did not adversely affect the general health, appearance or body weight development of the animals. Microscopic examination of bone marrow smears of male rats revealed no signs of toxicity to the bone marrow and no evidence of chromosomal damage and/or

damage to the mitotic apparatus of bone marrow erythrocytes. There was no reason to assume that the negative bone marrow response was due to lack of systemic exposure because treatment-related systemic effects (including increases in liver and kidney weight) occurred in male rats of the high-concentration group. Positive controls (five male rats treated with the mutagen Mitomycin C) showed the expected bone marrow response (cytotoxicity and increased number of micronucleated polychromatic erythrocytes).

Under the conditions of this study exposure to di-tert-butyl peroxide CAS# 110-05-4 did not induce chromosomal damage or damage to the mitotic apparatus of bone marrow erythrocytes of male rats.

Reference 5

Endpoint:: in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 25 days (total); ~48 hrs in vivo exposure
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: see 'Remark'
Remarks:: Well documented study conducted according to modern standards of internationally accepted protocol and Good Laboratory Practice. Test Material analytically confirmed for purity, even though "Analyses to determine the uniformity or concentration of the test article dose formulations or their stability were not performed by the testing facility or the Sponsor."
Qualifier:: according to guideline
Guideline:: OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)
Deviations:: yes
Remarks:: "Analyses to determine the uniformity or concentration of the test article dose formulations or their stability were not performed by the testing facility or the Sponsor."
GLP compliance:: yes (incl. QA statement)
Type of assay:: chromosome aberration assay
Species:: mouse
Strain:: ICR
Sex:: male
Details on test animals or test system and environmental conditions:: Animal Receipt and Quarantine
Mice were obtained from a source monitored for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were quarantined (acclimatized) for no less than 5 days after receipt. At BioReliance, mice were observed each day for signs of illness, unusual food and water consumption, and other conditions of poor health. The animals were judged to be healthy prior to utilization in the assay.

Animal Welfare Provisions and Animal Care
The number of animals, animal procedures and experimental design used for this study have been reviewed and were approved by the BioReliance Institutional Animal Care and Use Committee. All procedures involving animals performed at BioReliance follow the specifications recommended in The Guide for the Care and Use of Laboratow Animals (National Academy Press, Washington, D.C., 1996). The mice were housed in an AAALAC-accredited facility with a controlled environment of 72 * 3°F temperature, 50 * 20% relative humidity, and a 12 hour lightldark cycle. Mice of the same sex were housed up to five per each rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of this system was to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air Jiom each cage and re-filter the air (HEPA) prior to introducing the air back into the room. Heat-treated hardwood clnps were usedfor bedding. Animals had f?ee access to tap water and a certified laboratory rodent chow, (Harlan 2018C Certified Global Rodent Diet) which has been analyzed for environmental contaminants.
Route of administration:: intraperitoneal
Vehicle:: corn oil
Details on exposure:: Spermatogonial Chromosome Aberration Assay
The spermatogonial chromosome aberration assay was conducted using established procedures (Adler, I.D., 1984, Adler et al. 1994). Animals were assigned to five groups of five male mice each. An additional group of five males was designated as a replacement group to be used in the event of mortality at the high dose. Each animal was given a sequential number and identified by an ear tag. The study outline was as follows:

Number of Animals Used For
Spermatogonial Cell Collection 24
Number of Hours After the Last Dose
Treatment (20 mL/kg/treatment) Animals Dose Administration
Vehicle Control: Corn Oil 5 5
Test Article: LUPEROX DI
Low dose (200 mg/kg/day) 5 5
Mid dose (1000 mg/kg/day) 5 5
High dose (2000 mg/kg/day) 10* 5
Positive Control: MMC (50 mg/kg) 5 5

*Including 5 replacement animals per sex to ensure the availability of five animals for chromosome aberration analysis

Dose Preparation and Administration
The test article-vehicle mixture and the vehicle alone were intraperitoneally administered on two consecutive days, separated by approximately 24 hours at a volume of 20 mlkgttreatment. The positive control article was administered by a single intraperitoneal injection at 20 ml/kg. All mice in the ex erimental groups were weighed immediately prior to dose administration and the dose volume was based on individual body weights. Following dose administration and during the course of the study, animals were observed for mortality and clinical signs of toxicity.
Duration of treatment / exposure:: see above "details of exposure"
Frequency of treatment:: see above "details of exposure"
Post exposure period:: see above "details of exposure"
Remarks:: Doses / Concentrations:
200, 1000 and 2000 mg/kg bw
Basis:
No. of animals per sex per dose:: 5 males
[at the 2000 mg/kg dose, 10 males were dosed to ensure that at least 5 would be available for harvesting cells]
Control animals:: yes, concurrent vehicle
Positive control(s):: Mitomycin, 50 mg/kg; single dose
Tissues and cell types examined:: spermatogonial metaphase cells
Details of tissue and slide preparation:: Approximately four to five hours before the sacrifice time, animals were exposed to colchicine, at a dose of 4 mgkg. Colchicine, used to arrest dividing cells at metaphase, was administered to the animals by a single intraperitoneal injection at a volume of 10 mLkg. 24 hours after the last dose admin stration, animals were euthanized by C02 asphyxiation. Immediately following sacrifice, the testes were excised and placed in a small dish. The tunica albuginea was removed and the tubules were minced. The minced tubules were suspended in 3-4 rnL phosphate buffered saline (PBS) and transferredinto 15 mL. centrifuge tubes. The fragments were repeatedly pipetted to remove meiotic cells. The tubes were allowed to stand until all the fragments were sedimented. The supernatant was discarded and the fragments were washed two more times with PBS. The sedirnented fragments were resuspended with 0.5-1 mL of 0.1% trypsin, transferred to 15 mL tubes and incubated for 15 minutes in a 37'C shaker incubator. After incubation, 1-2 mL of the same trypsin was added to the tubes and the tubes were allowed to stand at room temperature for 5-10 minutes. The supernatant, containing liberated spermatogonia, was collected in 15 mL centrifuge tubes and placed on ice. The remaining fragments were treated with 0.1% trypsin, as described above, 2-3 times for 5 minutes each and the supernatants were pooled. Sufficient fetal bovine serum was added to the supernatants to yield 10% final concentration and the tubes were centrifuged at 1000-1200 rpm for 5 minutes. The cell pellets were treated with 5 mL of 0.06 M KC1 (hypotonic) for 15 minutes at room temperature. After hypotonic treatment, a few drops of fresh fixative (methano1:acetic acid, 3:1, vlv) was added to the tubes and centrifuged at 1000-1200 rpm for 5 minutes. The supernatant was aspirated and the pellets resuspended in about 5 mL of fixative. The cells were fixed for 10 minutes, centrifuged and the supernatant was aspirated and discarded. The pellets were resuspended in about 5 mL fresh fixative and stored overnight or longer at about 2-8'C until the slides were prepared.

Slide Preparation
At the time of slide preparation, cells were suspended by centrifugation, the supernatant was removed and a few drops of the re-suspended cells were placed onto a wet slide. The slides were air-dried, stained with Giemsa and permanently mounted. At least two slides were prepared from each animal. The slides were identified by the experiment and animal number.
Evaluation criteria:: The mitotic index and the total number and types of aberrations found in each animal are presented. Gaps are presented in the data but not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell. The percentage of damaged cells in the total population of cells scored is calculated for each treatment group. The severity of damage withm the cells is reported as the average number of aberrations per cell for each treatment dose. The conclusion of the study was based on sound scientific judgment; however as a guide to interpretation of the data, the test article was considered to induce a positive response if the number of aberrant cells is significantly increased in a dose responsivemanner relative to the negative control (vehicle). A significant increase at the high dose only with no dose-response was considered suspect. A significant increase at one dose other than the high dose with no dose-response was considered equivocal. The test article was judged to be negative if no statistically significant increase in percent of aberrant cells was observed relative to the vehicle control group at any sampling time. The percentage of cells in the vehicle control group demonstrating aberrations of any type, other than gaps, must not exceed 5%. The percentage of aberrant cells in thepositive control group must be statistically increased above that in the negative (vehicle) control group (p<0.05, Fisher's exact test)..
Statistics:: The Fisher's exact test was used for pairwise comparisons of the percentage of aberrant cells between each treatment and negative (vehicle) control group. If a statistically significant difference was observed, the Cochran-Annitage trend test would have been performed to test for evidence of a dose response.
Sex:: male
Genotoxicity:: negative
Toxicity:: no effects
Vehicle controls validity:: valid
Negative controls validity:: valid
Positive controls validity:: valid
Additional information on results:: In this assay, male mice were exposed either to the control articles (negative or positive) or LUPEROX DI at a dose of 200, 1000 or 2000 mgkglday. No mortality or clinical signs were observed in any of the mice during the course of the study (Table 8.0-1). Spermatogonial cells chromosome aberration summary data are presented in Table 8.0-2 and individual data in Table 8.0-3. Based on analysis, the percentage of aberrant cells was not statistically increased in the test article-treated groups relative to the vehicle control group (p > 0.05 Fisher's exact test). The mitotic index was reduced, up to 43.5 %, relative to the vehicle control, only in the 200 mgkg-treated group. No significant effect on the mitotic index was observed in animals at 1000 and 2000 mg/kg. In this study, all criteria for a valid test were met as specified in the protocol. CP induced a significant increase in the number of aberrant cells (p< 0.05, Fisher's exact test) and the mitotic index was reduced to 69.4%. No aberrant cells were observed in the negative control group.
Conclusions:: Interpretation of results (migrated information): negative
The results of the study indicate that under the conditions described in this report, Di-t-butyl peroxide (LUPEROX DI), when intrapentoneally administered on two consecutive days (at doses up to 2000 mgkgtday), did not induce a significant increase in the percentage of spermatogonial cells with structural chromosome aberrations. Therefore, Di-t-butyl peroxide (LUPEROX DI) was concluded to be negative in the spermatogonial chromosome aberration
test.
Executive summary:: The test article, Di-t-butyl peroxide (LUPEROX DI), hereafter referred to as LUPEROX DI, was tested in the mammalian spermatogonial chromosome aberration test using male ICR mice. The chromosome aberration assay was designed to evaluate the potential of the test article to induce chromosome aberrations in spermatogonial cells. The chromosome aberration assay consisted of five groups, each containing 5 male ICR mice. Animals in these groups were intraperitoneally exposed to the controls (negative or positive) or to LUPEROX DI at a dose of 200, 1000 or 2000 mgikgday. The test article was formulated in corn oil as per the Sponsor's request. Corn oil was used as the negative control (vehicle) and Mitomycin C (MMC), at a dose of 4 mgkg, as the positive control article. The test and negative control article were administered on two consecutive days, separated by approximately 24 hours. Each administration was conducted at a dose volume of 20 mLkg body weight. Animals were observed following each dose administration and during the course of the study. Colchicine was given 4-5 hours prior to euthanizia to arrest cells in metaphase. Twenty-four hours after the last dose, animals were euthanized and testes were removed from animal body cavity. Spennatogonial cells were isolated from the tubules and smeared onto the microscope slide. The smears were stained with Giemsa stain. One hundred metaphase cells per each animal were scored for structural chromosome aberrations. A statistically significant difference between the test article treated groups relative to the concurrent negative (vehicle) control was determined using Fisher's exact test for level of significance of pS 0.05. Mitotic index (MI) was calculated for each animal as the ratio of spermatogonial cells in mitosis per 1000 cells observed. No mortality or clinical signs were observed in any of the mice during the course of the study. No statistically significant increase in the percentage of aberrant cells and no dose-related decrease of the mitotic index were observed in the test article-treated groups relative to the vehicle control (p > 0.05 Fisher's exact test). The results of the study indicate that under the conditions described in this report, Di-t-butyl peroxide (LUPEROX DI), when intraperitoneally administered on two consecutive days (at doses up to 2000 mg/kg/day), did not induce a significant increase in the percentage of spermatogonial cells with structural chromosome aberrations. Therefore, Di-t-butyl peroxide (LUPEROX DI) was concluded to be negative in the spermatogonial chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

According to the ECHA final decision of July 7th, 2017, an in vitro mammalian cell micronucleus test with fluorescence in situ hybridisation (FISH) or immunochemical labelling of kinetochores (CREST) (OECD 487/EU B.49) was requested to discriminate between clastogenic and aneugenic effects in the OECD 474 study ( Haddouk, 2006). ECHA deems that this assay will clarify the mechanism of genotoxicicity of the registered substance. The results of this assay will be used to orientate the further experimental strategy including the possibility to request new data on germ cells or on carcinogenicity. Accordingly, the genotoxic activity of di-tert-amyl peroxide was assessed by means of the in vitro micronucleus test in TK6 lymphoblastoid human cells treated in presence and in absence of metabolic activation (Simar, 2018). Under these experimental conditions, no genotoxic activity was revealed, confirming the results of a previous in vitro cytogenetics assay (Sire, 2005) in human lymphocyte cultures. Di-tert-amyl peroxide did not appear to be clastogenic and aneugenic in the conditions of these in vitro assays whereas it induced an increase of the frequencies of the micronucleated polychromatic erythrocytes in the bone marrow of mice.

Both di-tert-amyl peroxide and di-tert-butyl peroxide induced a positive response in the micronucleus assay after i.p. and/or oral administrations to mice, whereas di-tert-butyl peroxide was negative in rats after an inhalation exposure. The negative in vitro mammalian cell micronucleus test (OECD Guideline 487) on L5178Y cells performed with di-tert-amyl peroxide allows to exclude an aneugenic mechanism. Therefore, the mechanism of the genotoxic action could be related to the production of reactive oxygen species either in situ in the bone marrow and/or after hepatic metabolism. Overwhelming of antioxidant and DNA repair mechanisms in the cell by a massive reactive oxygen species (ROS) production after an oral or i.p. bolus administrations of high dose levels (at the opposite of a diffuse inhalation exposure at lower dose levels) may result in oxidative stress and oxidative damage to the cell. This resulting oxidative stress can damage critical cellular macromolecules like DNA and/or modulate gene expression pathways.

As hepatocytes are the primary cell type involved in metabolism of xenobiotics, the ability ofdi-tert-amyl peroxide and di-tert-butyl peroxideto elicit production of ROS in plated male rat hepatocytes (McMahon, 2019). Both di-tert-amyl peroxide and di-tert-butyl peroxide caused a statistically significant, dose-dependent, elevation in ROS production indicating a possible genotoxic activity via this mode of action in the conditions of a bolus administration.

Additional information

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), di-tert amyl peroxide is classified as mutagenic category 2 (H341). Based on the positive result from an in vivo somatic cell micronucleus assay without evidence that an analogue substance can be clastogenic to germ cells.

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Registration Dossier

Registration Dossier

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Di-tert-pentyl peroxide

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Summary Table

Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

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Endpoint conclusion

Mode of Action Analysis / Human Relevance Framework

Additional information

Justification for classification or non-classification

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