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:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
supplier: Société SEPPIC
• name: SEPIWHITE MSH
batch number:
- on the Study plan and at receipt: LCAO4006
- on the analytical certificate: 0214400005
• description: white to yellowish powder container: one plastic flask
• date ofreceipt: 2 June 2004
storage conditions: at room temperature
• composition: see analytical certificate expiry date: 24 May 2005.
Confirmation of test item identity is the responsibility ofthe Sponsor.

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
Preliminary toxicity test: 9 male and 9 female mice were used,
Main cytogenetic test: 56 mice, 28 males and 28 females were used,
Blood sampling: 12 mice, 6 males and 6 females were used.
Strain: Swiss Ico: OF1 (IOPS Caw).
Reason for this choice: rodent species generaliy accepted by regulatory authorities for this type
of study.
Breeder: Charles River Laboratories France, l’Arbresle, France.
Age: on the day of treatment, the animais were approximately 6 weeks old.
Veterinary care at CIT: upon their arrivai at CIT, the animais 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 arrivaI, the animais were randomly allocated to the groups by sex.
Subsequently, each group was assigned to a different treatment group.
Identification: individuai tau marking upon treatment.

Environmental conditions
Upon their arrivai at CIT, the animais were housed in an animal room, with the following environmental conditions:
temperature: 22 ± 2°C, relative humidity: 30 to 70%, lightldark cycle: 12 h/12 h (07:00 - 19:00), ventilation: at ieast 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.
The animais were housed by groups in polycarbonate cages. Each cage contained autociaved sawdust (SICSA, Alfortviile, France).
Sawdust is anaiyzed by the supplier for composition and contaminant levels.

Food and water
Ail animais had free access to A04 C pelieted maintenance diet (SAFE, Viliemoisson-sur-Orge,
France).
Each batch of food is analysed by the supplier for composition and contaminant levels.
Drinking water filtered by a F0 Millipore membrane (0.22 micron) was provided ad libitum. Bacteriologicai and chemical analysis of water are performed regularly by extemal laboratories, These analyses inciude the detection of possible contaminants (pesticides, heavy metals and nitrosamines).

No contaminants were known to have been present in the diet, drinking water or bedding materiai at leveis which may be expected to interfere with or prejudice the outcome of the study.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
corn oil
Details on exposure:
Route for the vehicle and the test item: oral, since it is a possible route of exposure in Man, frequency: two treatments separated by 24 hours, volume: 10 mL/kg,
CPA: oral route, one treatment.
The quantity of each item administered to each animal was adjusted according to the most recently recorded body weight.
Frequency of treatment:
2 treatments, separated by 24 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:

Basis:
actual ingested
375
Remarks:
Doses / Concentrations:

Basis:
actual ingested
750
Remarks:
Doses / Concentrations:

Basis:
actual ingested
1500
No. of animals per sex per dose:
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 357 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 750 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1500 mg/kg; No. of animals: 8; Sacrifice time: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 357 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 750 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 1500 mg/kg; No. of animals: 8; Sacrifice times: 24 hours
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide

Examinations

Tissues and cell types examined:
Treatment of resuits
Ail the individual data are presented in tabular form. The number of MPEI2000 PE and the
PEINE ratio are given for each animal.
The means and the standard deviations of the frequency of MPEI1000 PE and the PEINE ratio
are given for each experimental group.
Details of tissue and slide preparation:
Blood samples for the determination of plasma levels ofthe test item were taken at the foliowing time:
• 1 and 4 hours following the second treatment.
Venous blood (approximately 1 mL) was taken into a tube containing lithium heparinate from the orbital sinus of the animais under light isoflurane anesthesia. After the blood sampling, the animals were killed by carbon dioxide asphyxiation and discarded without necropsy.
The biood was centrifuged (10 min at 4000 rpm, at +4°C) and the plasma was kept frozen in individual tubes at -20°C.
At the request of the Sponsor, no plasma analysis was performed and plasma samples which are kept stored at -20°C will be destroyed one year after the finalization of the study report.

Preparation ofthe bone marrow smears
At the time of sacrifice, ah the animais were killed by C02 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal caif serum. After centrifùgation, the supematant was removed and the ceils in the sediment were resuspended by shaking. A drop of this ceil suspension was placed and spread on a siide. The siides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group ofthe slide under evaluation (“blind” scoring).

Microscopic examination ofthe siides
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 estabiished by scoring a total of 1000 erythrocytes (PE + NE).
The analysis of the siides was performed at Microptic, cytogenetic services (2 Langiand Close Mumbies, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was Natalie Danford. Details conceming the method used, the format of the data, communication, quahity assurance and archiving are indicated in a PIDS (Principal Investigator Data Sheet).
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, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Loveli et al., 1989) (d), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the X2 value (Loveli et al., 1989) «».
When there was significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, l969). The student “t” test was used for the PEINE ratio comparison.
Probability values of p 0.05 was considered as significant.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No clinical signs and no mortaiity were observed in the animais of both sexes given
375 mg/kg/day.
At 750 mgfkg/day, no mortality was noted. Piloerection and loud breathing were observed from
24 hours foliowing the first treatment.
At 1500 mg/kg/day, 2/8 femaies were found dead 24 hours following the second treatment. In
surviving animais, piloerection, dyspnea, loud breathing and hypoactivity were noted.
The mean values of MPE as weli as the PEINE ratio in the groups treated with the test item, were equivalent to those of the vehicie group.
The mean values of MPE as well as the PEINE ratio for the vehicle and positive controis were consistent with our historical data.
Cyclophosphamide induced a highly significant increase (p <0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered vaiid.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under our experimental conditions, the test item SEPIWHITE MSH did flot induce any damage to the chromosomes or the mitotic apparatus of mice bone marrow ceils after two oral administrations, at a 24-hour interval, at the dose-levels of 375, 750 and 1500 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential of the test item SEPIWHITE MSH to induce structural or numerical damage in bone marrow celis of mice. The study was performed according to the international guidelines (OECD 474, Commission Directive No. B 12 and Japanese guidelines) and in compliance with the Princip les 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, one group of eight male and eight female Swiss Ico: OF1 (TOPS Caw) mice was given oral administrations of SEPIWHITE MSH at the dose-level of 1500 mgfkg/day, over a 2-day period. Two groups of five male and five female mice were given oral administrations of SEPT WHITE MSH at dose-levels of 375 and 750 mg/kg/day, under the same experimental conditions. One group of five males and five females received the vehicie (com oil) 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 mglkg. The animais of the treated and vehicle control groups were killed 24 hours after the last treatment and the animaIs 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 erytbrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 eiythrocytes (PE + NE).

Results

The top dose-level for the cytogenetic test was seiected according to the criteria specified in the international guidelines; since toxic effects were observed in the preliminary toxicity test, the choice of the top dose-level was based on the levei of toxicity, such that a higher dose-ievel was expected to induce lethality. Consequently, 1500 mglkg/day was selected as the top dose-level for the main test. The two other selected dose-levels were 375 and 750 mg/kg/day. The mean values ofMPE as well as the PEINE ratio in the groups treated with the test item, were equivalent to those of the vehicle group.

The mean values of MPE as welI as the PEINE ratio for the vehicle and positive controls were consistent with our historical data.

Cyclophosphamide induced a highly 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 SEPT WHITE MSH did flot induce any damage to the chromosomes or the mitotic apparatus of mice bone marrow celis after two oral administrations, at a 24-hour interval, at the dose-levels of 375, 750 and 1500 mg/kg/day.