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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Apparently well conducted GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Cyclohexanone, peroxide
EC Number:
235-527-7
EC Name:
Cyclohexanone, peroxide
Cas Number:
12262-58-7
Molecular formula:
UVCB (equilibrium mixture of a number of cyclohexyl based organic peroxides, hydrogen peroxide, cyclohexanone and water)
IUPAC Name:
Peroxycyclohexanone
Test material form:
other: Liquid
Details on test material:
Identification: Cyclohexanone peroxide (CAS No.012262-58-7)
Batch: 1405426797
Purity: 33.9% Cyclohexanone peroxide (mixture of cyclohexyl peroxy components)
Expiry / Retest Date: 18 February 2016
Storage Conditions: Ambient 10 oC not to exceed 25 oC.

Test animals

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 32 g for males (ranging from
29 to 34 g) and 24 g for females (ranging from 22 to 27 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.

Environmental conditions
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.
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 SsniffR/M-H pelleted maintenance diet (SSNIFF Spezialdiäten
GmbH, Soest, Germany).
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.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil
Details on exposure:
For the main test, the test item was dissolved in the vehicle in order to achieve the
concentrations of 1.875, 3.75, 7.5 and 15 mg/mL and then homogenized using a magnetic
stirrer. Using a treatment volume of 10 mL/kg, the target dose-levels were 18.75, 37.5, 75 and
150 mg/kg/day.
The preparations were made immediately before use.
Duration of treatment / exposure:
two treatments
Frequency of treatment:
two treatments separated by 24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
18.75, 37.5, 75 and 150 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide

Examinations

Tissues and cell types examined:
Bone marrow
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.
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 3), or other considerations of biological relevance was also taken into
account in the evaluation of data obtained.
Statistics:
Normality and homogeneity of variances will be tested using a Kolmogorov Smirnov test and a
Bartlett test.
If normality and homogeneity of variances were demonstrated, the statistical comparisons was
performed using a Student t-test (two groups) or a one-way analysis of variance (≥ 3 groups)
followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test
(two groups) or a Kruskall Wallis test (≥ 3 groups) was performed followed by a Dunn 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.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
In order to select the top dose-level for the cytogenetic study, 50, 75, 100, 150, 200, 300 and
2000 mg/kg were administered to three males and three females.
At 2000 mg/kg/day, the three females treated and one male out of three died following the
first treatment. The two surviving males were sacrificed before the second administration of the
test item.
At 300 mg/kg/day, two females out of three and two males out of three died following the
first treatment. The two surviving animals (one male and one female) were sacrificed before the
second administration of the test item.
At 200 mg/kg/day (administered to females only), two females out of three died following the
second treatment.
At 150 mg/kg/day (administered to females only), no mortality was induced. Piloerection and in
addition hypoactivity for one female, were observed.
At 100 mg/kg/day, one male out of three died following the second treatment and piloerection
was noted in the two surviving males. No clinical signs and no mortality were noted in the
three females treated.
At 75 mg/kg/day (administered to males only), piloerection was noted in the treated animals and
no mortality was induced. No clinical signs and no mortality were noted in the three females.
At 50 mg/kg/day, no mortality was induced. Piloerection and sometimes half-closed eyes were
noted in males. No clinical signs and no mortality were noted in the three females treated.
The top dose-level for the cytogenetic test was selected according to the criteria specified in the
international guidelines; since toxic effects were observed, the choice of the top dose-level was
based on the level of toxicity, such that a higher dose-level was expected to induce lethality.
Consequently, 75 mg/kg/day for males or 150 mg/kg/day for females were selected as the top
dose-level for the main test. The two other selected dose-levels were 18.75 and 37.5 mg/kg/day,
for males, and 37.5 and 75 mg/kg/day, for females.

Except for piloerection regularly observed in males (solvent control and the three dose-levels
treated groups) and sometimes noted in females (highest dose group only), no clinical signs and
no mortality were observed in treated animals.
For either males or females, the mean values of MPE as well as the PE/NE ratio in the groups
treated with the test item, were equivalent to those of the vehicle control or solvent groups.
The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were
consistent with our 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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under experimental conditions, the test item (35% of cyclohexanone peroxide in a solvent melange) did
not induce 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 18.75, 37.5 and
75 mg/kg/day and 37.5, 75 and 150 mg/kg/day for males and females respectively.
Executive summary:

The objective of this study was to evaluate the potential of the test item (35% of cyclohexanone peroxide in a solvent melange) to induce structural or numerical damage in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474, Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practice Regulations.

In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given intraperitoneal administrations of CYCLOHEXANONE PEROXIDE at dose-levels of 18.75, 37.5 and 75 mg/kg/day, for males, and of 37.5, 75 and 150 mg/kg/day for females, over a 2-day period. One group of five males and five females received the solvent control (Mixture diacetonealcohol/Diisobutylphtalate/Cyclohexanone) at 65% of the highest dose-level of the test item (i.e. 48.75 mg/kg/day for males and 97.5 mg/kg/day for females), under the same experimental conditions. 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, solvent 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).

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since toxic effects were observed, the choice of the top dose-level was based on the level of toxicity, such that a higher dose-level was expected to induce lethality. Consequently, 75 mg/kg/day for males or 150 mg/kg/day for females were selected as the top dose-level for the main test. The two other selected dose-levels were 18.75 and 37.5 mg/kg/day, for males, and 37.5 and 75 mg/kg/day, for females. Except for piloerection regularly observed in males (solvent control and the three dose-levels treated groups) and sometimes noted in females (highest dose group only), no clinical signs and no mortality were observed in treated animals. For either males or females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were equivalent to those of the vehicle control or solvent groups.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our 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.