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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
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
non GLP; Both doses by far exceeded the max. doses recommended by current OECD Guideline (474)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Both doses by far exceeded the max. doses recommended by current OECD Guideline (474)
Principles of method if other than guideline:
NA
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Purity batch
> 99.0 % 7.32% active oxygenBatch 247408973
99.27 % 9407226339; 247408999
99.6 % 0410105225413
> 97 % 4289070300; 0419101226056
99.1 % 11600 0010-108
99.3 % 247413988
99.4 % 7.35 % active oxygen Batch 247409800
98.9 % 0707518256
> 99.1 % 247412384
98.5 % 0707603153
99.0 % 1703465433
99.5 % 150726; 247413309
99.3 % 000055055

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Males: 27 - 35 g; Females: 24 - 29.9 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet (e.g. ad libitum): ad libitum, TEKLAD Certified Rodent Chow 7012C
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 23°C
- Humidity (%): 50 ± 20°C

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in corn oil at a concentration of 500 mg/mL, the maximum concentration tested.
Details on exposure:
NA
Duration of treatment / exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by oral gavage at a constant volume of 20 mL/kg bw. Mice were observed after dose administration for clinical signs of chemical effect.
Frequency of treatment:
24, 48, 72 hrs after dose administration
Post exposure period:
None
Doses / concentrations
Dose / conc.:
20 other: mL/kg bw
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP) will be administrated as the positive control at a dose of 60 mg/kg. CP will be administrated by the same route as the test article.

Examinations

Tissues and cell types examined:
The micronucleus study evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice.
Details of tissue and slide preparation:
At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
Evaluation criteria:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control (p ≤ 0.05, Kastenbaum-Bowman Tables).
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1979; Mackey and Mac Gregor, 1979)

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration (p>0.05, Kastenbaum-Bowman).

Any other information on results incl. tables

No remarks

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay described in this report, tert.-Butylperoxy- 2-ethylhexanoat did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

The test article, tert.-Butylperoxy- 2-ethylhexanoat, was tested in the mouse micronucleus assay. In the study, test and control articles were administered in a constant volume of 20 mL/kg bw by a single oral gavage.

Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in corn oil at a concentration of 500 mg/mL, the maximum concentration tested.

In the pilot assay (toxicity test), male mice were dosed with 1, 10, 100 or 1000 mg test article/kg bw and male and female mice were dosed with 5000 mg/kg bw. Mortality occurred an 1/5 males and 3/5 females at 5000 mg/kg. Clinical signs observed after dose administration included: lethargy in male mice at 1000 mg/kg and in male and female mice at 5000 mg/kg, prostration and crusty eyes in one male mouse and diarrhoea in one female mouse at 5000 mg/kg. Treatment-related mortality was observed to be less than 50 % in the pilot toxicity study. However, in females the mortality was estimated to be 60% at 5000 mg/kg. Therefore, the high dose in the micronucleus study for males was set at 5000 mg/kg. For females the high dose was set at 4000 mg/kg which was estimated to be approximately 80 % of 5000 mg/kg. Both doses by far exceeded the maximum doses recommended by current OECD guideline.

In the micronucleus assay, male mice were dosed with 1250, 2500 or 5000 mg/kg and female with 1000, 2000 or 4000 mg/kg bw of tert.-Butylperoxy- 2-ethylhexanoat. Mortality occurred in 1 /20 male mice at 5000 mg/kg and 2 /25 female mice at 4000 mg/kg. Clinical signs following dose administration included: lethargy in male and female mice at all dose levels, diarrhoea in male mice at 2500 and 5000 mg/kg and in female mice at 2000 and 4000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Moderate reductions (up to 36 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the vehicle control groups. A reduction of 36 % suggests bioavailability of the test article to the target organ, the bone marrow.

Under the conditions of the assay described in this report, tert.-Butylperoxy- 2-ethylhexanoat did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow even at pronounced systemically toxic doses and was concluded to be negative in the micronucleus test using male and female ICR mice.