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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLp study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
• name: LCAO5 005
• batch number:
- Study plan and labeling: 173JG
- analytical certificate: 173 JG1 (the correspondence between both batch numbers was stated by the Study Monitor in an e-mail dated 02 February 2006)
• description:
- at receipt: slightly yellowish viscous liquid
- on the Study plan: none
- on the analytical certificate: hard orange wax
• container: one plastic flask
• date ofreceipt: 08 November 2005
• storage conditions: at room temperature
• purity: 99.3%
• retest date: 01 September 2006.

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
Preliminary toxicity test: 3 males and 3 females mice were used.
Main cytogenetic test: 56 mice, 28 males and 28 females were used.
Strain: Swiss Ico: OF1 (IOPS Caw).
Reason for this choice: rodent species generally accepted by regulatory authorities for this type ofstudy.
Breeder: Charles River Laboratories, l’Arbresle, France.
Age: on the day oftreatment, the animals were approximately 6 weeks old.
Weight: at the beginning oftreatment the mean body weight was 32.3 g for males (ranging from 29.7 to 33.6 g) and 26.3 g for females (ranging from 24.8 to 28.0 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 except for females from the positive control (CPA) treated group and supplementary females from the high dose group which had only 4 days of acclirnation.
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.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
water
Details on exposure:
For the vehicle and the test item:
• Route: oral, since it is a possible route ofexposure in Man
• Frequency: two treatments separated by 24 hours
• Volume: 10 mL/kg

For the positive control (CPA):
• Route: oral
• Frequency: one treatment
• Volume: 10 mL/kg
Frequency of treatment:
2, spaced by 24 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:

Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:

Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:

No. of animals per sex per dose:
Male: mg/kg; No. of animals: ; Sacrifice time: hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Control animals:
yes, historical
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 irnrnediately 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 C02 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 50 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:
All the individual data are presented in tabular form. The number of MPE/2000 PE and the
PEINE ratio are given for each animal.
The means and the standard deviations of the frequency of MPE/1000 PE and the PEINE ratio
are given for each experimental group.
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 ( 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 Dunn test (ifnecessary).
All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.4 17, SAS Institute mc), with a level of significance of 0.05 for all tests.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under our experimental conditions, the test item LCAO5 005 did not induce any damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mglkglday.
Executive summary:

The objective of this study was to evaluate the potential of the test item LCAO5 005 to induce structural or numerical damage in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practice Regulations.

Methods

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given oral administrations of LCAO5005 at dose-levels of 500, 1000 or 2000 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (water for injections) 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 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).

Results

The highest dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no toxic effects were observed, the highest dose-level was 2000 mg/kg/day. The two other selected dose-levels were 1000 and 500 mg/kg/day. For both males and females, the mean values of MPE as well as the PEINE ratio in the groups treated with the test item, were equivalent to those of the vehicle control group. The mean values of MPE as well as the PEINE 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.

Conclusion

Under our experimental conditions, the test item LCAO5 005 did flot induce any damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day.