Di-tert-pentyl peroxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, Klimisch 1a

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-pentyl peroxide

EC Number:

234-042-8

EC Name:

Di-tert-pentyl peroxide

Cas Number:

10508-09-5

Molecular formula:

C10H22O2

IUPAC Name:

2-methyl-2-[(2-methylbutan-2-yl)peroxy]butane

Test material form:

other: liquid

Details on test material:

• name: Study plan: DI-TERT-AMYL PEROXIDE
• CAS number: 10508-09-5
• batch number: 11650 920511-025


• description: at receipt: colorless to slightly yellowish liquid
• date of receipt: 26 January 2006
• storage conditions: at room temperature and protected from humidity
• peroxid content : 98.3%
• expiry date: July 2006

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 31.8 g for males (ranging from 30.3 to 33.2 g) and 22.8 g for females (ranging from 19.1 to 25.1 g).
- Veterinary care at CIT: upon their arrival at CIT, the animals were given a complete examination to ensure that they were in good clinical conditions.
- Acclimation: at least 5 days before the day of treatment.
- Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex.
- Subsequently, each group was assigned to a different treatment group.
- Identification: individual tail marking upon treatment.

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

Food and water: were provided ad libitum

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

corn oil

Duration of treatment / exposure:

2 days

Frequency of treatment:

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

Post exposure period:

24 hours

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Positive control(s):

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

Examinations

Details of tissue and slide preparation:

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

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

Evaluation criteria:

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

Statistics:

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

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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

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

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

Any other information on results incl. tables

Clinical signs:

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

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

Results:

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

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

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

Vehicle and test item:

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

Route: intraperitoneal route

Vehicle: corn oil

Cyclophosphamide:

Number of administrations: one

Route: oral

Vehicle: water

Statistical significance:

* p<0,05

** p<0,01

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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

 

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

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

 

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

 

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

 

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

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

 

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

 

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