Registration Dossier

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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
-Purity: 87.9%.
- Received by BioReliance on August 07, 1998
- The lot number: 1-10307-120
- Expiration date of July 06, 1999
- Upon receipt, the test article was described as a pale yellow liquid and was stored at -5°C to°- 30°C, protected from exposure to light.

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
- ICR mice were obtained from Harlan Sprague Dawley, Inc., Frederick, MD. At the initiation of the study, the mice were 6 to 8 weeks old. Animal body weights recorded at randomization were within the following ranges:
- Pilot study: Males, 27.4 - 31.7 grams Females, 26.4 - 27.6 grams
- Toxicity study: Males, 25.8 - 30.2 grams Females, 23.5 - 26.3 grams
- Micronucleus assay: Males, 27.0 - 31.7 grams Females, 22.7 - 27.5 grams
- 74±6°F, 50±20% relative humidity, and a 12 hour light/dark cycle
- Food and water: ad libitum

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil. C max 200 mg/l.
Details on exposure:
Single intraperitoneal injection at a constant volume of 10 mL/kg body weight. The test article was soluble in corn oil at 200 mg/mL, the maximum concentration tested
Post exposure period:
3 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 113, 225 or 450 mg test article/kg body weight.
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes Cyclophosphamide 50 mg/kg

Examinations

Details of tissue and slide preparation:
Slide Preparation:
At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by C02 asphyxiation.
Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal 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:
- Scoring for micronuclei: 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1 /5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
- The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group.
- Quantification of the proliferation state of the bone marrow (indicator of bone marrow toxicity): by determination of the proportion of polychromatic erythrocytes to total erythrocytes
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Pilot study: 3-hydroxy-1,1-dimethylbutylperoxyneodecanoate was administered by single intraperitoneal injection to male mice at 1, 10, 100, or 1000 mg/kg and to male and female mice at 2000 mg/kg which was administered in a total volume of 10 mL test article-vehicle mixture/kg body weight. Mortality occurred within three days of dose administration as follows: 5/5 males and 5/5 females at 2000 mg/kg and 2/2 males at 1000 mg/kg. Clinical signs, which were noted after dose administration, included: lethargy in all male mice at 100 and 1000 mg/kg, piloerection in all male mice at 1000 mg/kg, prostration in ail mice at 1000 and 2000 mg/kg and irregular breathing in all mice at 2000 mg/kg. All other animals appeared normal throughout the observation period.

Toxicity study: 3-hydroxy-1,1-dimethylbutylperoxyneodecanoate was administered by a single intraperitoneal injection to male and female mice at 200, 400, 600, or 800 mg test article/kg body weight which was administered in a total volume of 10 mL test article-vehicle mixture/kg body weight. Mortality occurred within three days of dose administration as follows: 1 /5 males and 2/5 females at 600 mg/kg and 3/5 males and 3/5 females at 800 mg/kg. Clinical signs, which were noted on the days following dose administration, included: lethargy and piloerection in ail male and female mice at all dose levels and hunched position in ail mice at 600 and 800 mg/kg. The LD5013 was calculated by probit analysis to be approximately 759 mg/kg for male and female mice. The high dose for the micronucleus test was set at 450 mg/kg which was estimated to be approximately 60% of the LD50.

Any other information on results incl. tables

Clinical Signs Following Dose Administration of 3-hydroxy-1,1-dimethylbutylperoxyneodecanoate
Pilot Toxicity Study

Treatment

Clinical Observation

Number of Animals

with Clinical

Number of Animals

Died/Total Number

of Animals Dosed

Signs/Total

Number of Animals

Dosed

Males

Females

Males

Females

Test Article,

1 mg/kg

Normal

2/2

N/A

0/2

N/A

Test Article,

10 mg/kg

Normal

2/2

N/A

0/2

N/A

Test Article,

100 mg/kg

Lethargy

2/2

N/A

0/2

N/A

Test Article,

1000 mg/kg

Lethargy

Piloerection

Prostration

2/2

2/2

2/2

N/A

2/2

N/A

Test Article,

2000 mg/kg

Prostration

Irregular breathing                                         _

5/5

5/5

5/5

5/5

5/5

5/5

Pilot Toxicity Study Using 3-hydroxy-1,l-dimethylbutylperoxyneodecanoate In ICR Mice:
Body Weight and Mortality Data

Group Mean Body Weights (gms)

% Changer

Treatment

Sex

Pretreatment

Day 1

Day 3

Day 1

Day 3

Mortality`

TA, 1 mg/kg

M

30.3

30.1

30.9

-0.7%

2.0%

0 /

2

± 3.3

± 3.3

± 3.5

10 mg/kg

M

30.8

30.5

31.6

-1.0%

2.6%

0 /

2

±     1.0

±1.3

± 1.6

100 mg/kg  .

M

31.5

31.9

33.0

1.3%

4.8%

0 /

2

± 2.3

± 3.2

± 2.8

1000 mg/kg

M

30.5

28.4

3ND

-6.9%

3ND

2 /

2

±     1.5

±1.6

2000 mg/kg

M

31.2

3ND

3ND

3ND

3ND

5 /

5

±     1.4

F

27.4

3ND

3ND

3ND

3ND

5 /

5

±     1.0

TA =3-hydroxy-1,1-dimethytbutylperoxyneodecanoate

1%Change =(Post-treatment weiqht - Pretreatment weiqht) x 100Pretreatment weight

2Reported as number of animais dead 3± days after doseadministration/totalnumber tested.3No datadueto mortality.

Clinical Signs Following DoseAdministration of 3-hydroxy-1,l-dimethylbutylperoxyneodecanoate
Toxicity Study

Treatment

Clinical Observation

Number of Animais

with Clinical

Number of Animais

Died/Total Number

of Animais Dosed

Signs/Total

Number of Animais

Dosed

Males

Females

Males

Females

Test Article,

Lethargy

5/5

5/5

0/5

0/5

200 mg/kg

Piloerection

5/5

5/5

Test Article,

Lethargy

5/5

5/5

0/5

0/5

400 mg/kg

Piloerection

5/5

5/5

Test Article,

Lethargy

5/5

5/5

1/5

2/5

600 mg/kg

Piloerection

5/5

5/5

Hunched position

5/5

5/5

Test Article,

Lethargy

5/5

5/5

3/5

3/5

800 mg/kg

Piloerection

5/5

5/5

Hunched position

5/5

5/5

Clinical Signs Following Dose Administration of 3-hydroxy-1,1-dimethylbutylperoxyneodecanoate
Micronucleus Assay

Treatment

Clinical Observation

Number of Animais

with Clinical

Number of Animais

Died/Total Number

of Animais Dosed

Signs/Total

Number of Animais

Dosed

Males

Females

Males

Females

Corn Oil,

10 mL/kg

Normal

10/10

10/10

0/10

0/10

Test Article,

113 mg/kg

Normal

Lethargy

5/5

3/5

2/5

0/5

0/5

Test Article,

225 mg/kg

Lethargy

Piloerection

5/5

5/5

5/5

5/5

0/5

0/5

Test Article,

450 mg/kg

Lethargy

Piloerection

15/15

15/15

15/15

15/15

0/15

0/15

CP,

50 mg/kg

Normal

5/5

_

5/5

0/5

0/5

Summary of bone marrow micronucleus study using the substance:

treatment sex time (hours) number of mice PCD.Total Erythrocytes (mean +/-sd) Changes from control Micronucleated polychromatic number per 1000 PCDs (mean +/-sd) Erytrhocytes number per PCEs scored /10000
Corn oil 10ml/kg M 24 5 0.47 (0.1) / 0.2 (0.27) 2
  F 24 5 0.46 (0.09) / 0.3 (0.27) 3
Test substance 113 mg/kg M 24 5 0.41 (0.06) -13 0.4 (0.42) 4
  F 24 5 0.35 (0.07) -24 0.4 (0.42) 4
Test substance 225 mg/kg M 24 5 0.31 (0.08) -34 0.5 (0.00) 5
  F 24 5 0.42 (0.06) -9 0.4 (0.22) 4
Test substance 450 mg/kg M 24 5 0.28 (0.05) -40 0.2 (0.27) 2
  F 24 5 0.35 (0.08) -24 0.5 (0.35) 5
CP 50 mg/kg M 24 5 0.29 (0.05) -38 29.3 (10.84) 293
  F 24 5 0.31 (0.06) -33 29.4 (9.85) 244
Corn oil 10ml/kg M 24 5 0.45 (0.09) / 0.3 (0.27) 3
  F 24 5 0.48 (0.11) / 0.2 (0.27) 2
Test substance 450 mg/kg M 48 5 0.42 (0.05) -7 0.2 (0.27) 2
  F 48 5 0.36 (0.06) -25 0.3 (0.27) 3

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
3-hydroxy-l, 1- dimethylbutylperoxyneodecanoate was concluded to be negative in the in vivo mouse micronucleus assay.
Executive summary:

The purpose of this study was to evaluate the clastogenic potential of the test article as measured by its ability to induce micronucleated polychromatic erythrocytes in mouse bone marrow (OECD 474 test, GLP study)

The test article, 3-hydroxy-1,1-dimethylbutylperoxyneodecanoate, was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay followed by a toxicity study. The second phase, 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. In both phases of the study, test and control articles were administered in a constant volume of 10 mL/kg body weight by a single intraperitoneal injection.

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 200 mg/mL, the maximum concentration tested. Dosing preparations were delivered to the test system as solutions.

In the pilot assay, male mice were dosed with 1, 10, 100, or 1000 mg test article/kg body weight and male and female mice were dosed with 2000 mg test article/kg body weight. Mortality was observed in 2/2 male mice at 1000 mg/kg and in 5/5 male and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included lethargy in male mice at 100 mg/kg and 1000 mg/kg, piloerection in male mice at 1000 mg/kg, prostration in all mice at 1000 and 2000 mg/kg and irregular breathing in male and female mice at 2000 mg/kg.

In the toxicity assay, male and female mice were dosed with 200, 400, 600, or 800

mg test article/kg body weight. Mortality was observed in 1/5 male mice and 2/5 female mice at 600 mg/kg and in 3/5 male mice and 3/5 female mice at 800 mg/kg. Clinical signs following dose administration included lethargy and piloerection in ail male and female mice at ail dose levels and hunched position in ail male and female mice at 600 and 800 mg/kg. The high dose for the micronucleus test was set at 450 mg/kg which was estimated to be approximately 60% of the LD50.

In the micronucleus assay, male and female mice were dosed with 113, 225 or 450 mg test article/kg body weight. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs following dose administration included: lethargy in 2/5 female mice at 113 mg/kg and lethargy and piloerection in ail male and female mice at 225 and 450 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. A moderate reductions (up to 40%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test article to the bone marrow target.

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 or 48 hours after dose administration (p>0.05, Kastenbaum-Bowman). The results of the assay indicate that under the conditions described in this report, 3-hydroxy-l,ldimethylbutylperoxyneodecanoate did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. 3-hydroxy- l ,1 dimethylbutylperoxyneodecanoate was concluded to be negative in the mouse micronucleus assay.