Bisisobutyryl peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro mutation studies were conducted with the registered substance in phlegmatizer. In a bacterial reverse mutation assay, there was an increase in the number of revertants, in the absence of metabolic activation, in one tester strain. In an in vitro mouse lymphoma assay, the registered substance in phlegmatizer, did not induce an increase in mutation frequency with or without metabolic activation.

In an in vitro micronucleus assay, the registered substance in phlegmatizer induced micronuclei frequency in binucleated cells only at a prolonged exposure time without metabolic activation.

The registered substance is thermally and hydrolytically unstable. The peroxide will completely decompose within half an hour at ambient temperature with a significant amount decomposing within 10 minutesprimarily to isobutyric acid which is not considered mutagenic or genotoxic. Propene may also be a major breakdown product. However, due to its volatility, this could not be verified. Other decomposition products may include, to a lesser degree, isopropanol and acetone which are not considered mutagenic or genotoxic. Under the conditions of this assay, the parent compound would quickly decompose. The positive in vitro results are likely due a thermal decomposition product.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-Dec-2011 to 08-Jun-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without S9-mix, 24 exposure; 24 hr fixation: 1, 3, 10, 33 and 100 µg/mL
Combined dose range finding test/First cytogenetic test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 10, 33 and 100 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 30, 60 and 75 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 Migrated to IUCLID6: MMC-C 0.25 µg/mL for a 3 hours exposure period and 0.15 µg/mL for a 24 hours exposure period

Positive control substance:

other: colchicine: 0.1 µg/mL

Remarks:

without S9

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: 15 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics:

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

only after the prolonged treatment period

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only after the prolonged treatment period

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 75 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was only observed observed at dose levels of 50 µg/ml and above in the absence of S9 for the continuous treatment of 24 hr

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the highest precipitating tested dose after the 3 h exposure time. Toxicity was reached at the dose levels selected for scoring for the continuous treatment of 24 h.

Conclusions:

Interpretation of results: positive

Bisisobutyryl peroxide solution induces the formation of micronuclei in human lymphocytes in the absence of S9 metabolic activation at a prolonged exposure period. Since Bisisobutyryl peroxide solution induces the micronuclei frequency in binucleated cells only, it is considered to be an aneugenic or clastogenic compound, under the conditions of this assay.

Executive summary:

The possible clastogenicity and aneugenicity of Bisisobutyryl peroxide solution was tested in two independent experiments. The study procedures described in this report were based on the most recent OECD guideline 487.

In the first cytogenetic assay, Bisisobutyryl peroxide solution was tested up to 100 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Bisisobutyryl peroxide solution precipitated in the culture medium at this dose level. In the second cytogenetic assay, Bisisobutyryl peroxide solution was tested up to 75 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level and Bisisobutyryl peroxide solution precipitated in the culture medium at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the first cytogenetic assay, Bisisobutyryl peroxide solution did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

In the second cytogenetic assay with a 24 hours continuous exposure time, Bisisobutyryl peroxide solution induced a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. These results indicate that Bisisobutyryl peroxide solution is positive in the in vitro micronucleus study and, can be considered an aneugenic or clastogenic compound, under the conditions of this assay.

The registered substance is thermally and hydrolytically unstable. The peroxide will completely decompose within half an hour at ambient temperature with a significant amount decomposing within 10 minutes primarily to isobutyric acid which is not considered mutagenic or genotoxic. Propene may also be a major breakdown product. However, due to its volatility, this could not be verified. Other decomposition products may include, to a lesser degree, isopropanol and acetone which are not considered mutagenic or genotoxic. Under the conditions of this assay, the parent compound would quickly decompose. The positive results are likely due a thermal decomposition product.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-Nov-2011 to 02-May-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/ml
Additional dose range finding test:
Without S9-mix, 3 hours treatment: 3, 10, 20, 30, 40, 50, 65, 80 and 100 μg/ml exposure medium.
With S9-mix, 3 hours treatment: 3, 10, 30, 40, 601, 70, 100, 125 and 150 μg/ml exposure medium.

Experiment 1:
Without S9-mix, 3 hours treatment: 1, 10, 15, 20, 25, 30, 35 and 40 µg/mL
With S9-mix, 3 hours treatment: 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 1, 3, 10, 20, 30, 35, 40 and 42.5 µg/mL
With S9-mix, 3 hours treatment: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: cyclophosphamide 7.5 µg/mL

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 42.5 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 50 µg/mL and above in the absence of S9, 3 hours treatment; no toxicity was observed up to the highest tested dose level of 150 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 33 µg/mL and above in the absence of S9, 24 hours treatment.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 92 and 81% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

Bisisobutyryl peroxide solution is not mutagenic in the mouse lymphoma L5178Y test system.

Executive summary:

Evaluation of the mutagenic activity of Bisisobutyryl peroxide solution in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat).

The study procedures were based on the most recent OECD, EC and ICH guidelines.

In the first experiment, Bisisobutyryl peroxide solution was tested up to concentrations of 40 and 100 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. Bisisobutyryl peroxide solution was tested up to a cytotoxic level of 92% in the absence of S9-mix. No toxicity was observed at the precipitating dose level of 100 μg/ml in the presence of S9-mix. In the second experiment, Bisisobutyryl peroxide solution was tested up to concentrations of 42.5 and 100 μg/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Bisisobutyryl peroxide solution was tested up to a cytotoxic level of 81% in the absence of S9-mix. No toxicity was observed at the precipitating dose level of 100 μg/ml in the presence of S9-mix.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

 

Mutation frequencies in cultures treated with positive control chemicals were increased by 7.6- and 8.7-fold forin the absence of S9-mix, and by 9.0- and 5.9-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, Bisisobutyryl peroxide solution did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, Bisisobutyryl peroxide solution did not induce a biologically relevant significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performe according to OECD guideline and GLP. Deviation from the current guideline: With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. An independent repeat of the study was performed only with TA 100, the negative results from the other strains were not confirmed by and independent repeat and a justification was not provided.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

- With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have.
- An independent repeat of the study was performed only with TA 100, the negative results from the other strains were not confirmed by and independent repeat and a justification was not provided.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see below

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First study
0, 0.025, 0.07, 0.22, 0.67, and 2.0 µl/pl mutagenicity test in the absence of the S-9 mix
0.0025, 0.007, 0.022, 0.067 and 0.2 µl/pl mutagenicity test in the presence of the S-9 mix

Second study:
0, 0.025, 0.074, 0.22, 0.67, and 2.0 µl/pl mutagenicity test in the absence of the S-9 mix
0, 0.5, 1.0, 2.0, 3.0, 4.0 and 10.0 µl/pl mutagenicity test in the absence of the S-9 mix repeat
0.0025, 0.0074, 0.022, 0.067 and 0.2 µl/pl mutagenicity test in the presence of the S-9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: suggested by the sponsor

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 days

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate plates

NUMBER OF CELLS EVALUATED: the his+ revertants were counted by hand.

DETERMINATION OF CYTOTOXICITY
- Method: The background lawn of bacterial growth was examined microscopically.

OTHER: S-9 and S-9 mix were checked for sterility. Each culture was examined for the number of spontaneous revertants, histidine requirement and sensitivity to ampicillin, crystal violet and UV radiation.

Evaluation criteria:

A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose-response.

Statistics:

None

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tox. test: No growth at 10µ/pl -S9 mix, No growth at 1.0µg/pl and slightly less dense background lawn at 0.1 µg/pl. Test 1: No cytotox. observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tox test: No growth at 10µ/pl -S9 mix, No growth at 1.0µg/pl +S9 mix and slightly less dense background lawn at 0.1 µg/pl +S9 mix/-S9 mix. Test 1: slightly less dense background lawn at 2.0 µg/pl -S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tox test: No growth at 10µ/pl -S9 mix, less dense background lawn of bacterial growth at 1.0 µg/pl. No growth at 1.0µg/pl +S9 mix and slightly less dense background lawn at 0.1 µg/pl +S9 mix. Test 1: less dense background lawn at 2.0 µg/pl -S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tox test: No growth at 10µ/pl -S9 mix. No growth at 1.0µg/pl +S9. Test 1: slightly less dense background lawn at 2.0 µg/pl -S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tox test: No growth at 1.0µg/pl +S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tox test: No growth at 10.0µg/pl -S9. Test 1: slightly less dense background lawn at 2.0 µg/pl -S9 mix. Test 2: less dense background lawn at 3.0 µg/pl and no growth above that -S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Test 1: From the results obtained in the experiment it appeared that incubation of the test substance with the bacteria did not increase the number of his+ revertants with s. typhimurium TA 1535, TA 1537, TA 1538, and TA 98, either in the absence or in the presence of the S-9 mix.
However, It appeared that the test substance induced, at the higher dose-levels, a clear increase in the number of revertants with strain TA 100, only in the absence of the S-9 mix. This increase was also observed with the toxicity test. The observed increase was not clearly dose-related, but because of the toxicity of the test substance, higher dose levels were not tested.
At 2.0 µI/plate, in the absence of the S-9 mix, the test substance was slightly toxic for TA 1537, TA 1538, TA 98 and TA 100, as was seen by the (slightly) less dense background lawn of bacterial growth.
The positive controls used in the present assay gave the expected strong increase in the number of his+ revertants, both in the absence and in the presence of the 5-9 mix.

Test 2: It appeared that also in the repeat experiment the test substance induced, at the highest dose level (2 µl per plate), a clear increase in the number of revertants with strain TA 100, only in the absence of the S-9 mix.
The observed increase was not dose-related, but because of the toxicity of the test substance, higher dose levels were not tested.
However, in the repeat experiment the background lawn of bacterial growth was normal. In view of the observed result it was decided to repeat the Ames test with Salmonella typhimurium mutant TA 100, in the absence of the S-9 mix and with other dose levels.
The results show that the test substance was toxic at a concentration of 3 µl per plate and above, as was seen from the reduced number of his+ revertants, the observation of pin points and the less dense background lawn of bacterial growth.
Again at the highest dose level (2 µl per plate), a clear increase in the number of revertants with strain TA 100 (in the absence of the S-9 mix) was observed.
Also at the dose level of 1.0 µl per plate a small increase in the number of revertants was observed.

See tables for details.

Conclusions:

Interpretation of results: positive without metabolic activation TA100

At the highest dose level that could be tested with diisobutyrylperoxide there was an increase in the number of revertants (in test 1 and test 2 + repeat) with Salmonella typhimurium TA 100, only in the absence of the S-9 mix.
The observed increase was not dose-related, but because of the toxicity of the test substance, higher dose levels resulted in absence of bacterial growth. In the test 2 repeat also at the dose level of 1.0 µl per plate a small increase in the number of revertants was observed.

Executive summary:

Test 1: "diisobutyrylperoxide" was examined for mutagenic activity in the Ames test using the histidine requiring Salmonella typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (5-9 mix), in compliance with OECD guideline 471 ("Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay"). At the request of the sponsor the test was carried out only once.

"diisobutyrylperoxide" was tested at five different concentrations ranging from 0.025 to 2.0 µ/l per plate (in the absence of the S-9 mix)

and from 0.0025 to 0.2 µ/l per plate (in the presence of the S-9 mix). Negative and positive controls were run simultaneously with the test substance. Due to the toxicity of the test substance, higher levels could not be tested.

It was concluded that "diisobutyrylperoxide" did not show mutagenic activity in the strains TA 1535, TA 1537, TA 1538 and TA 98 of Salmonella typhimurium, either in the absence or in the presence of the S-9 mix. It appeared that at the highest dose level that could be tested there was an increase in the number of revertants with Salmonella typhimurium TA 100, only in the absence of the S-9 mix.

Test 2: An independent repeat of the study was performed only with TA 100. "diisobutyrylperoxide" was tested at five different concentrations ranging from 0.025 to 2.0 µ/l per plate (in the absence of the S-9 mix) and from 0.0025 to 0.2 µ/l per plate (in the presence of the S-9 mix). Negative and positive controls were run simultaneously with the test substance. The test was repeated in the absence of S-9 mix at higher concentrations ranging from 0.5 -10.0 µg/plate. It appeared that also in the repeat experiment the test substance induced, at the highest dose level (2 µl per plate), a clear increase in the number of revertants with strain TA 100, only in the absence of the S-9 mix. The observed increase was not dose-related, but because of the toxicity of the test substance, higher dose levels were not tested. However, in the repeat experiment the background lawn of bacterial growth was normal. In view of the observed result it was decided to repeat the Ames test with Salmonella typhimurium mutant TA 100, in the absence of the S-9 mix and with other dose levels. The results show that the test substance was toxic at a concentration of 3 µl per plate and above, as was seen from the reduced number of his+ revertants, the observation of pin points and the less dense background lawn of bacterial growth.

Again at the highest dose level (2 µl per plate), a clear increase in the number of revertants with strain TA 100 (in the absence of the S-9 mix) was observed. Also at the dose level of 1.0 µl per plate a small increase in the number of revertants was observed. At the highest dose level that could be tested with diisobutyrylperoxide there was an increase in the number of revertants with Salmonella typhimurium TA 100, only in the absence of the S-9 mix.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

To further investigate the positive results from the in vitro micronucleus assay a comet asay was performed as part of an OECD 408, 90 -day oral gavage study. The results of this study were as follows: no effect were observed in the jejunum or the glandular stomach of male rats. In the liver of male rats at all dose levels, small increases in the mean and median percentage tail intensities were noted in relative to vehicle controls achieving statistical significance in most instances at 100 and 300 mg/kg bw/day (p<0.001-p<0.01). There was no strict dose-relationship but most individual values exceeded the historical vehicle control data ranges. The overall results were considered not to fully meet the requirements of the OECD 489 guideline for a positive response and therefore the outcome of this study is considered as equivocal.

Since the outcome of the first study was equivocal a second comet assay was commissioned. This assay was performed according to OECD 489 and the outcome of this assay is negative. A range-finding test was performed to identify the MTD which is higher than the upper dose in the 90-day study. The glandular stomach and the liver were the tissues selected for the assay. The liver was the equivocal result in the first test. The glandular stomach is the site of first contact.  ECHA made a reference in their final decision that testing in germ cells may be required in the event the comet assay is positive. As a backup up in this study slides were made of the testes. These would only be analysed in case the other tissues are positive.

In the final assay males rats were dosed by gavage in archais oil at 500, 1000 and 2000 mg/kg bw. A control group of 5 male was dosed with the vehicle alone (Arachis oil BP) and another group was dosed with the positive control (MNU). Animals are dosed twice at 0 and 24 hours. The comet assessment in male rats treated with the test item up to a dose level of 2000 mg/kg bw/day showed there are no increases in % tail intensity in either the liver or glandular stomach over the vehicle controls and the values were within the current historical range for a vehicle. Since these tissues are negative the testes slides have not been scored. The positive control worked well and demonstrated significant increases in the % tail intensity in both the liver and glandular stomach. The overall results from comet assay assessment were considered to fully meet the requirements of the OECD 489 guideline and the results were clearly negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 January 2016 and 02 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

A comet assay was performed as part of "3437-84-1, Repeated dose toxicity: oral, 90-day, ENVIGO, 2016, RS".

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Identification : Bisisobutyryl peroxide (CAS# 3437-84-1)
Physical State/Appearance : Clear colorless liquid
Purity : Diisobutyryl peroxide (38.91%)
Batch Number : BYK004856-171
Date Received : 25 November 2015
Storage Conditions : Approximately -20 °C
Expiry Date : 30 April 2016

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male

Details on test animals or test system and environmental conditions:

Animal information:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for at least nine days during which time their health status was assessed. A total of one hundred and twenty-five animals (sixty-five males and sixty females) we re accepted into the study. At the start of treatment the males weighed 181 to 227g, the females weighed 153 to 204g, and were approximately six to eight weeks old.

Animal Care and Husbandry:
Groups 1 to 4 animals were housed in groups of three or four by sex whilst Group 5 animals were housed in groups of two or three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limit ed, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Annex 6 of the study report; expiry date for the batch number 070715MA was extended to 07 July 2016. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were considered not to contain any c
ontaminant at a level that might have affected the purpose or integrity of the study. The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similaritybetween the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Arachis oil. The stability and homogeneity of the test item formulations were determined as part of Envigo Study Number 41502236 where formulations were shown to be stable for at least four
hours when kept on crushed ice. Formulations were therefore prepared daily and kept on crushed ice before and during use.
Samples were taken from test item formulation on various occasions during the study and were analyzed for concentration of Bisisobutyryl peroxide (CAS# 3437-84-1) at Envigo Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained are given in A
nnex 2 of the study report. The results indicated that the test item formulations were inside the acceptance criteria of ±20% on most occasions; see deviations from Study Plan.

Reference Item Preparation and Analysis
For the purpose of this study, N-Nitroso-N-methylurea was prepared at the appropriate concentration as a solution in distilled water.
Fresh formulations were prepared and used within two hours of being prepared; it is assumed this formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of N-Nitroso-Nmethylurea formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Duration of treatment / exposure:

90 days
The first five males from each non-recovery dose group (Groups 1 to 4) were dosed for ninety-one consecutive days whilst the remaining males and females from these dose groups were dosed for ninety consecutive days.
An additional group of five males (Group 5), used as positive control for comet assay assessment, was administered with 25 mg/kg bw/day N-Nitroso-N-methylurea by oral gavage on Days 90 and 91.

Frequency of treatment:

Daily

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Low dose level treatment group. Concentration: 2 mg/mL

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose level treatment group. Concentration: 20 mg/mL

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

High dose level treatment group. Concentration: 400 mg/mL.

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

High dose level treatment group.
Dose level increased to 300 mg/kg bw/day from Day 32 (males) and Day 31 (females)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

Positive control

No. of animals per sex per dose:

5 males

Control animals:

yes

Positive control(s):

N-Nitroso-N-methylurea

Tissues and cell types examined:

liver, glandular stomach and jejunum

Details of tissue and slide preparation:

Tissue sample requirements:

Following removal and weighing (where applicable), tissue sampes taken for the comet assay were placed in a small volume of the appropriate ice-cold buffer and quickly transferrred to the Department of Cell and Molecular Sciences where they were processed for the comet assay.
The tissue samples were processed under subdued lighting and over ice to provide single cell suspaensions, providing sufficient cells for scoring for the comet assay as follows:

Glandular Stomach - A small section of the glandular stomsch was immersed in stomach buffer (Hanks balanced salt solution supplemneted with EDTA and EGTA) and incubated for approximately 15 munites on ice. The mucosla layer of the glandular stomach was removed by gentle scraping and then a single cell suspension was obtained by scraping the remaining tissue into a small volume of stomach buffer.

Jenunum - approximately a 2 cm piece of Jejunum was processed. This was immersed briefly in liver buffer and then scraped gently to remove any contants, incubated in liver buffer (approximately 10mL), on ice for 5 to 10 minutes. A single cell suspension was obtained by gentle scraping into approximately 1 mL of fresh liver buffer.

Liver - A small piece of liver (approximately 1cm3) was washed in liver buffer (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a sibgle cell suspension.

Slide preparation:

Adequate numbers of slides were pre-coated with 0.5% normal meltingpoint agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay. Approximately 30 microliter of the cell suspensionw as added to 270 microliter of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 microliter of this agarose/cell suspension mix was placed onto a precoted slide. two gells were placed on each slide, and 4 gels were proepared for each tissue. Two of the gells were scoreed for comets ( A and B replicates) and tow ( C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4C in the dark for approximately 20 minutes to allow it to solidify.

Once te LMP agarose had set, the coverslips were removed and the slides gently lowered into the freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.

Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, with was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slodes were then left for 20 minutes to allow the DNA to unwind, after which they were subject to eletrophoresis at approximately 0.7 V/cm (calculated between the ectrodes), 300 mA for 20 minutes. The buffer batch was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of the electrophoresis to ensure the electrophiresis solution was maintained at low temperature (2-10C).

At the end of the electrophoresis period, the bacth was switched of, the slides genltly removed and placed onto a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% menthanol for 5 minutes and allowed to air dry, after which they were stored prior to staining and scoring.

For the glandular stomach and jejunum, two of the four processed slide gels were scored and the remaining two slides were stored as backup slides. For the liver slides, four of the processed slide gells were scored.

The processed comet slides were coded to allow 'blind' scoring suing a computer generated code and stained just prior to analysis for comets. To each dry slide gel, 75 microliter of propidium iodide (20 microg/l) was placed on top of the slide and overlaid with aclean coverslip. After a short period to allow hydration and staining of the DNA, the slide was placed onto the stage of a fluorescence mcroscope and scored for comets using a CCD camera attached to a PC-based image analysis program (comet IV version 4.3.1).

Two slide gels for each tissue for each animal were scored with a maximum of 100 cells per sIide gel giving accumulative total of 200 cells per tissue per animal. A further 200 cells per tissue per animal were scored from the backup slides for the vehicle and the test item dose levels of the liver tissue only. The slide score data for each tissue was processed using the excel macro program provided in comet IV version 4.3.1. Comparison between the vehicel control group response and that of the test item dose groups was made. the primary end-point was percentage DNA in the tail (percentage tail intencity), although other endpoints such as tail moment and length may also be utilized.

Each slide was also assessed for the incedence of 'hedgehog' cells to give and indication of the cell integrity.

Evaluation criteria:

The test item is considered to be clearly negative if:
- none of the test concentrations exhibits a significant increase compared with the concurrent negative control
- there is no evidence of a dose response relationship
- the results are within the laboratory historical vehicle control range
- there is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved

The test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase compared to the concurrent negative control
- the response is considered to be dose-related
- the results are substantially outside the laboratory historical behicle control range.

Statistics:

Where appropriate, statistical anaylsis was performed on the mean % tail intensity and media % tail intensity data using a students t-test on the transformed data using a v(x+1) transformation. Indivudual slide score values for the % tail intensity or median % tail intensity were used. Statistical analysis was also performed on the liver weights using Students t-test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: glandular stomach and jenunum

Key result

Sex:

male

Genotoxicity:

ambiguous

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: liver

See attached file

Conclusions:

The comet assessment in male rats treated with the test item up to a dose level of 300 mg/kg bw/day resulted in possible treatment-related increases in the mean and median tail intensities in the liver. Although these observations did not fully meet the requirements of the OECD 489 guideline for a positive response, in the absence of any evidence to show that the effect was due to anything other than DNA damage, a relationship to treatment with the test item cannot be ruled out and the it is concluded the results are equivocal.

Executive summary:

The test item was administered by oral gavage to three groups of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, at dose levels of 10, 100 or 200/300 mg/kg bw/day. Animals from the high dose group were dosed at 200 mg/kg bw/day up to Day 30 (females) or Day 31 (males) with the dose level increased to 300 mg/kg bw/day thereafter; as these animals were dosed at 300 mg/kg bw/day for approximately 2/3 of the dosing period, the dose level for this group will generally be referred to as 300 mg/kg bw/day throughout this study report. A control group of ten males and ten females was dosed with the vehicle alone (Arachis oil BP). The first five males from each non-recovery dose group (Groups 1 to 4) were dosed for ninetyone consecutive days whilst the remaining males and females from these dose groups were dosed for ninety consecutive days. An additional group of five males (Group 5), used as positive control for comet assay assessment, was administered with 25 mg/kg bw/day NNitroso-N-methylurea by oral gavage on Days 90 and 91. Animals treated on Days 90 and 91 were dosed approximately twenty-seven and three hours (respectively) before euthanasia and selected tissues from these animals were used for comet assay assessment.

Treatment with the test item up to a dose level of 300 mg/kg bw/day was considered not to induce any treatment-related increases in the mean or median percentage tail intensities in the jejunum or the glandular stomach from the male rat and as such was considered to be nongenotoxic to these tissues. At all dose levels, increases in the mean and median percentage tail intensities in the liver were observed achieving statistical significance in most instances at 100 and 300 mg/kg bw/day. Although the overall results from comet assay assessment were considered not to fully meet the requirements of the OECD 489 guideline for a positive response, in the absence of any evidence to prove that the effect was due to anything other than DNA damage, a relationship to treatment with the test item cannot be ruled out.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 10 October 2017 Experimental completion date: 05 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Identification: Bisisobutyryl peroxide (CAS# 3437-84-1)
Lot/Batch Number: 1705447077
Physical state/Appearance: Clear colourless liquid
Expiry Date: 16 July 2018
Purity:
Tert-Butyl Hydroperoxide (0.566%)
Diisobutyryl peroxide (38.91%)
Isododecane (60.5%)
No adjustment for purity was made.
Storage Conditions: Stored at approximately -20 °C in the dark

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Envigo (UK). At the start of the main test the males weighed 186.0 g to 219.1 g, and were approximately eight to ten weeks old. Details of the individual animal weights, group means and standard deviations for the animals used in the main test are presented in Table 1. After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.
The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study. The diet and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The animals were provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). These enrichment items were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil.

Details on exposure:

Determination by analysis of the concentration, homogeneity and stability of the test item preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.

Positive Control Preparation
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant Batch no.3012436).

Duration of treatment / exposure:

28 hours

Frequency of treatment:

dosed twice with a 24-hour interval

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Groups each of five male rats per dose

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

N-Nitroso-N-methylurea (MNU). MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.

Tissues and cell types examined:

Liver
Glandular Stomach
Testes

Details of tissue and slide preparation:

Comet Test
Groups each of five male rats were dosed twice with a 24 hour interval via the oral route with the test item at 2000, 1000 or 500 mg/kg. The groups of rats from each dose level were killed by humane euthanasia (carbon monoxide asphyxiation) approximately 4 hours following the second administration, 28 hours after the start of the test. In addition, two further groups of rats were included in the study; one group (five male rats) was dosed twice with a 24-hour interval via the oral route with the vehicle alone (Arachis oil) and a second group (three male rats) was dosed twice orally with 24-hour interval with N-Nitroso-N-methylurea (MNU). MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test. The vehicle and positive controls were killed approximately 4 hours following the second administration, 28 hours after the start of the test.

Tissue Sample Requirements
Humane euthanasia was performed on the animals at the end of the exposure period using a method that did not affect the integrity of the required tissues (carbon monoxide asphyxiation). Samples of liver, glandular stomach, and testes were obtained from each animal for comet processing.
Sub-samples of the liver, glandular stomach and testes were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathology investigations. Assessment of cytotoxicity by histopathology may be conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.
The tissue samples were processed under subdued lighting and over ice to provide single cell suspensions, providing sufficient cells for scoring for the comet assay as follows:
Liver - A small piece of liver (approximately 1 cm3) was washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered through gauze to provide a single cell suspension.

Glandular Stomach– The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides.

Testes – These were dissected from each animal. One was retained for possible histopathology. The second one was placed in liver buffer and chopped with scissors to provide a single cell suspension which was filtered through gauze before use for the comet slides.


Slide Preparation
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
Approximately 30 µL of the cell suspension was added to 270 µL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period, the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.
Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.


Scoring
Comet slides from the liver and glandular stomach were scored. Comet slides from the testes were prepared but were not scored at the request of the Sponsor.
The slides were coded prior to scoring to allow “blind” scoring. The slides are stained just prior to analysis for comets. To each dry slide, 75 L of propidium iodide (20 g/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, i.e. Comet IV version 4.3.1.
Two slide gels for each tissue per animal were scored with a maximum of 75 cells per slide gel giving an accumulative total of 150 cells per tissue per animal. Care was taken to guarantee that a cell is not scored twice. The slide score data from each experiment was processed using the Excel macro program provided in Comet IV. Comparisons between the vehicle control group response and that of the test item dose groups were made. The primary end-points are percentage tail DNA (%Tail intensity) and median percentage tail intensity.
Each slide was assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

Evaluation criteria:

Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly Negative if:
• None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
• There is no evidence of a dose-related response
• The results are within the laboratory historical vehicle control range
• There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved
The test item is then considered unable to induce DNA strand breakage in the tissues studied in the test system.
Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly Positive if:
• At least one of the test doses exhibits a statistically significant increase compared to the concurrent negative control.
• The response is considered to be dose related
• The results are substantially outside the laboratory historical vehicle control range.
The test item can be considered to induce DNA strand breakage in a particular tissue if all three conditions are met.
There is no requirement for verification of a clearly positive or negative response.
Although most experiments will be expected to give clear negative or positive results in rare cases the data set will preclude making a definite judgment. This may require the scoring of additional slides to increase the number of cells and, therefore, add more power to the data. If this does not resolve the issue then the result will be given as equivocal or questionable, and may require the histopathological assessment of the tissues to see if cell toxicity may be the causative agent rather than any genotoxic mechanism.

Statistics:

When a less than clear response is observed a comparison will be made between the vehicle control groups and each corresponding treatment dose group, on the percentage tail intensity data using individual slide scored values.

Additional information on results:

Range-Finding Toxicity Test
In animals dosed with test item there were no premature deaths and no clinical signs observed.
Bone marrow slides were prepared for quantitative assessment from the final range-finding experiment at 2000 mg/kg.
The quantitative assessment revealed that moderate bone marrow toxicity had occurred at 2000 mg/kg in one of the animals. This was considered to give an indication of systematic absorption of the test item in the absence of clinical signs.
Based on the above data the maximum recommended dose (MRD) of the test item, 2000 mg/kg, was selected for use in the main test, with 1000 and 500 mg/kg as the lower dose levels.

Comet Assay
Mortality Data and Clinical Observations
One premature death was observed in the 2000 mg/kg (MRD) dose group and one in the 1000 mg/kg group prior to the second dose being administered. In the 2000 mg/kg dose group one animal (11) exhibited hunched posture and one animal (13) exhibited hunched posture, lethargy and increased salivation one hour after the first dose was administered. Hunched posture was observed in three animals in the 2000 mg/kg dose group prior to termination. No clinical signs were seen in the animals of the 1000 mg/kg or the 500 mg/kg dose groups.
It is considered that the premature deaths were not due to true test item toxicity and resulted in each group not meeting the minimum animal number of five per group as stated in the test guideline. However, the Comet assay results for both tissues were well within the historical control range and did not indicate any genotoxic activity and, therefore, it was considered there was no impact on the integrity or purpose of the study.

Evaluation of Comet Assay Slides
The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control item (MNU) produced a marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach, comparable with the laboratory historical control range for these tissues. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

There were no marked increases in percentage tail intensity for any of the test item dose levels in the glandular stomach or liver tissues which exceeded the current historical control range for a vehicle, confirming the test item did not induce DNA damage in the liver or glandular stomach.

There was no marked increase in hedgehog frequency for any of the test item dose levels in either of the tissues investigated. High numbers of hedgehogs observed in the glandular stomach are characteristic of gastro-intestinal tract where there is a high turnover of cells.

Range-Finding Toxicity Test

The mortality data are summarized as follows:

Dose Level (mg/kg)	Sex	Number of Animals Treated	Route	Deaths on Day		Total Deaths
				0	1
450	Male	2	oral	0	0	0/2
600	Male	2	oral	0	0	0/2
1000	Male	2	oral	0	0	0/2
2000	Male	2	oral	0	0	0/2
2000	Male	2	oral	0	0	0/2

Bone marrow slides were prepared for quantitative assessment from the final range-finding experiment at 2000 mg/kg. The slides were scored per 1000 cells and the data is presented below.  

Animal Code	Dose level (mg/kg)	Number of polychromatic erythrocytes	Number of normochromatic erythrocytes	PCE/NCE ratio
4-0	2000	536	464	1.16
4-1		404	596	0.68

 

Summary of Results for Percentage Tail Intensity, Median Percentage Tail Intensity and Percentage Hedgehogs for each Tissue

Liver

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Median % Tail Intensity per Animal
Vehicle (Arachis oil)	0	0.29	0
500 mg/kg (MRD/4)	0	0.16	0
1000 mg/kg (MRD/2)	0	0.17	0
2000 mg/kg (MRD)	0	0.16	0
Positive control (N-Nitroso-N-Methylurea)	2.07	21.10	20.95

 

Glandular Stomach

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Median % Tail Intensity per Animal
Vehicle (Arachis oil)	7.65	5.48	3.61
500 mg/kg (MRD/4)	7.32	4.06	2.09
1000 mg/kg (MRD/2)	6.65	4.39	2.53
2000 mg/kg (MRD)	8.90	4.15	1.60
Positive control (N-Nitroso-N-Methylurea)	10.22	30.41	28.82

Conclusions:

The test item, Bisisobutyryl peroxide (CAS# 3437-84-1) did not induce any increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.

Executive summary:

 Introduction

The Comet Assay has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC 1999, as described by Ticeet al., 2000. The method is designed to be compatible with the procedures indicated in the OECD 489 Guideline (2016).

The primary target tissues of the comet assay were liver and glandular stomach. Comet slides were also prepared from the testes but these were not scored.

 

Methods

A range-finding test was performed to find suitable dose levels of the test item. The Comet assay main test was conducted at the maximum recommended dose (MRD), 2000 mg/kg with 1000 and 500 mg/kg as the lower dose levels. Groups of rats (five per group) were dosed a 0 hours and 24-hours and were killed at 28 hours (4 hours after the second dose administration). 

In addition, two further groups of rats were included in the study; one group (five rats) were dosed via the oral route with the vehicle alone (arachis oil) at 0 and 24 hours, and a second group (three rats) were dosed orally with N-Nitroso-N-methylurea (NMU) at 0 and 24 hours to act as the positive control.

 

The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 28 hours after the start of the test. The glandular stomach, liver, and testes were processed for comet slides. Samples of liver, testes and glandular stomach were preserved in formalin for possible histopathology in the event of a positive response. 

 

The slides prepared from the liver and glandular stomach were scored for the presence of Comets.

 

Results

The presence of clinical signs indicated that systemic absorption had occurred.

One premature death was observed in the 2000 mg/kg (MRD) dose group and one in the 1000 mg/kg group prior to the second dose being administered. The following clinical signs were observed in some animals dosed with test item at 2000 mg/kg: hunched posture, lethargy and increased salivation.It is considered that the premature deaths were not due to true test item toxicity and resulted in each group not meeting the minimum animal number of five per group as stated in the test guideline. However, the Comet assay results for both tissues were well within the historical control range and did not indicate any genotoxic activity and, therefore, it was considered there was no impact on the integrity or purpose of the study.

 

The positive control group induced a marked increase in percentage tail intensity and median percentage tail intensity in the liver and glandular stomach indicating that the test method itself was operating as expected. The vehicle control groups all had percentage tail intensity and median percentage tail intensity values within the expected range.

 

There was no statistically significant marked increase in percentage tail intensity or median percentage tail intensity for any of the test item dose groups in the liver or glandular stomach when compared to the vehicle control, confirming that the test item did not induce DNA damage in the tissue investigated under the conditions of the test.

 

Conclusion

The test item,Bisisobutyryl peroxide (CAS# 3437-84-1) did not induce any increases in the percentage tail intensity or median percentage tail intensity values in the liver or glandular stomach when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The mutagenicity study in bacterial was repeated several times in order to confirm the positive in one tester strain. The registered substance is not expected to be mutagenic based on the negative MLA assay.

Although, positive in the in vitro micronucleus assay, the registered substance would have undergone thermal decomposition under the conditions of this assay. The outcome of a comet assay (in combination with a 90 -day study) was equivocal. A second comet assay was commissioned under standard conditions and the outcome of this assay is negative.

Based on the available information the substance is not-classified for this endpoint.