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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Bisisobutyryl peroxide
EC Number:
222-340-0
EC Name:
Bisisobutyryl peroxide
Cas Number:
3437-84-1
Molecular formula:
C8H14O4
IUPAC Name:
2-methylpropanoyl 2-methylpropaneperoxoate
Details on test material:
- Name of test material (as cited in study report): Bisisobutyryl peroxide solution
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Storage condition of test material: In freezer (≤ -25°C) in the dark


Method

Species / strain
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

Controlsopen allclose all
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:

Results and discussion

Test resultsopen allclose all
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.

Applicant's summary and conclusion

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.