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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
Study period:
2006-07-04 to 2007-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
: See below in section on "any other information on materials and methods".
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
: See below in section on "any other information on materials and methods".
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): JNJ-559728-AAA (T001250)
- Physical state: solid (powder)
- Appearance: White powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
00443418 RT001250G4A661
- Expiration date of the lot/batch:
Not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature, protected from moisture; Keep away from direct sunlight
- Stability under test conditions:
Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH; D-33178 Borchen
- Age at study initiation: The age of the animals at the start of acclimation was 8-10 weeks.
- Weight at study initiation: The mean weights of the animals at the start of treatment were 33.8 g (males) and 29.8 g (females).
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: Animals were housed singly in Makrolon Type I cages with wire mesh tops and provided with granulated soft wood bedding.
- Diet: Animals were provided with pelleted standard (Harlan Winkelmann GmbH) diet ad libitum.
- Water: Animals were provided with tap water ad libitum.
- Acclimation period: The animals were kept under quarantine in the animal house of the laboratory for a minimum of five days after their arrival, during which time the animals did not show any sings of illness or altered behavior.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-88
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: not indicated

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 30 % DMSO + 70 % PEG400 (polyethylene glycol 400)
- Justification for choice of solvent/vehicle: The vehicle was chosen based on its relative non-toxicity for the animals.
- Concentration of test material in vehicle: no data
- Amount of vehicle: 10 mL/kg bw
- Type and concentration of dispersant aid: not applicable
- Lot/batch no.: batch K32087831 (DMSO); S3783526 (PEG400)
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- On the day of the experiment, the test substance was formulated in the vehicle. All animals received a single standard volume (10 mL/kg bw) orally.

DIET PREPARATION
- Rate of preparation of diet: not applicable
- Mixing appropriate amounts with: not applicable
- Storage temperature of food: not applicable
Duration of treatment / exposure:
single gavage dose
Frequency of treatment:
single gavage dose
Post exposure period:
24 hours (all groups) and 48 hours (high dose group only)
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- Six animals per sex per dose were treated with 0, 500 and 1000 mg/kg bw and 12 animals per sex per dose were treated with 2000 mg/kg bw.
- Five animals per sex per dose were evaluated for micronuclei (the remaining 6th animal of each sex in the respective test group was usually evaluated in case an animal died in its test group spontaneously).
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide is one of the recommended positive controls of OECD Guideline 474.
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow of the mouse
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test substances. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. The toxicity test described in the results section determined that 2000 mg/kg bw (the maximum guideline recommended dose) was suitable for the highest dose level in the micronucleus assay.

TREATMENT AND SAMPLING TIMES:
- Twelve animals, six males and six females, were treated per dose group and sampling time, except for the highest dose group where 24 animals, twelve male and twelve females, were treated. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h; 24 h and 48 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 hours after treatment

DETAILS OF SLIDE PREPARATION:
- Animals were sacrificed at 24 and 48 hours using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minute and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald. Cover slips were mounted with Eukitt. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes were analyzed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

OTHER:
- The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1, 2-4, 6, 24 and 48 hours after treatment.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
A nonparametric Mann-Whitney test was used to assess statistical significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 1000 and 2000 mg/kg bw
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100-2000 mg/kg bw
- Solubility: no data
- Clinical signs of toxicity in test animals: Animals were examined for acute toxic symptoms at intervals of around 1, 2-4, 6, 24, 30 and 48 hours after administration of the test substance. The animals treated with 100 mg/kg bw did not express any toxic reaction. The animals treated with 1000 mg/kg bw had ruffled fur. The animals treated with 2000 mg/kg bw expressed the following toxic reactions: reduction of spontaneous activity, eyelid closure and ruffled fur. On the basis of these data 2000 mg/kg bw were estimated to be suitable.
- Evidence of cytotoxicity in tissue analyzed: not applicable
- Rationale for exposure: to determine dose levels to be used in the main mutagenicity study
- Harvest times: not applicable
- High dose with and without activation: 2000 mg/kg bw


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: not applicable
- Induction of micronuclei: Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group. The positive control showed a statistically significant increase of induced micronucleus frequency.
- Ratio of PCE/NCE: After treatment with the test substance the number of polychromatic erythrocytes was not substantially decreased as compared to the mean values of polychromatic erythrocytes of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow.
- Appropriateness of dose levels and route: The preliminary toxicity test determined the high dose used in the micronucleus assay to be appropriate.
- Statistical evaluation: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group. The positive control showed a statistically significant (p <0.0001) increase of induced micronucleus frequency.

Any other information on results incl. tables

Toxic Reactions and Plasma Concentrations:

The animals treated with 2000 mg/kg bw expressed toxic reactions as shown below:

Toxic Reaction

Hours post-treatment (male/female)

 

1 h

2-4 h

6 h

24 h

48 h*

Reduction of spontaneous activity

9/11

8/10

5/6

1/0

1/0

Abdominal position

4/6

1/1

1/0

0/0

0/0

Eyelid closure

6/4

4/3

3/1

0/0

0/0

Ruffled fur

0/0

3/4

6/6

4/3

1/0

Tremor

0/0

0/0

1/0

0/0

0/0

Death

0/0

1/0

0/1

0/0

0/0

Urine color

-

Dark yellow

Dark yellow

-

-

* Data only from 6 animals per sex

The animals treated with 1000 mg/kg bw expressed toxic reactions as shown below:

Toxic Reaction

Hours post-treatment (male/female)

 

1 h

2-4 h

6 h

24 h

Reduction of spontaneous activity

0/0

2/0

3/4

0/0

Ruffled fur

0/0

3/4

5/5

0/0

The animals treated with 500 mg/kg bw expressed toxic reactions as shown below:

Toxic Reaction

Hours post-treatment (male/female)

 

1 h

2-4 h

6 h

24 h

Reduction of spontaneous activity

0/0

0/0

2/2

0/0

Ruffled fur

0/0

0/0

2/2

0/0

 

The death of two animals at the high dose indicated the systemic distribution of the test substance and thus its bioavailability. The clear signs of systemic toxicity observed at the high dose corroborate this indication. The analysis of the plasma of animals treated with the high dose confirmed the bioavailability of the test substance. These animals had high levels of the test substance in their plasma at both sampling intervals (1 and 4 hours).

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
The test substance was evaluated for the potential to induce micronuclei in polychromatic erythrocytes in the bone marrow orally treated mice. Under the conditions of the study, the test substance did not induce micronuclei.