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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2011-02-16 to 2011-09-29
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997
GLP compliance:
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Identity: CD08467, also known as RN001351
- Appearance: white to almost white powder
- Purity (LC): 98.4%

- Source and lot/batch No. of test material: Batch No.: 10.01639 (CS10.038A.1001)
- Expiration date of the lot/batch: 2011-05-02

- Storage condition of test material: 2-8 °C in the dark

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan UK Ltd., Oxon UK or Charles River (UK) Ltd, Margate, UK
- Age at study initiation: 6-10 weeks old
- Assigned to test groups randomly: Yes
- Fasting period before study: No fasting
- Housing: Animals housed in groups of up to six
- Diet (e.g. ad libitum): ad libitum; SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham)
- Water (e.g. ad libitum): ad libitum; mains water
- Acclimation period: at least 5 days

- Temperature (°C): 20 - 24 °C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
- Vehicle(s)/solvent(s) used: PEG400/EtOH/NaCl 0.9%
- Justification for choice of solvent/vehicle: Previous trials confirmed that the maximum solubility of CD08467 in the vehicle, PEG400/EtOH/NaCl 0.9% (70/10/20 (w/w/w)), was 2 mg/mL.
- Concentration of test material in vehicle: PEG400/EtOH/NaCl 0.9% (70/10/20 (w/w/w))

Details on exposure:
All treatments were given via intraperitoneal injection in order to maximise exposure of the target organ to the test article. Animals were not fasted prior to dose administration.
Duration of treatment / exposure:
The test article was given as two administrations, 24 hours apart and animals were sampled 24 hours after the final administration, thus enabling examination of cells exposed to the test article over a period of 24 to 48 hours prior to sampling.
Frequency of treatment:
Two administrations, 24 hours apart
Post exposure period:
All animals were observed daily for signs of ill health or overt toxicity. An individual record was maintained of the clinical condition of all Range-Finder and Micronucleus animals dosed in the study.
In the Range-Finder Experiment, post-dosing observations were performed immediately after
each dose administration, at least four times in the 4 hours following each administration and prior to the second dose. Observations were also recorded at least once on each of the non-dose administration days.
The Range-Finder animals were killed and discarded without necropsy or sampling by an overdose of sodium pentobarbitone, given via intraperitoneal injection and subsequently ensured by cervical dislocation.
In the Micronucleus Experiment post-dosing observations were performed immediately after each dose administration, at least twice in the 4 hours following each dose and prior to the second administration. Observations were also recorded at least once on the day of bone marrow sampling.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
2.5 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control used: Cyclophosphamide (CPA) prepared in saline
- Route of administration: intraperitoneal
- Dose of CPA administered (mg/kg): 20
- Concentration of CPA solution (mg/mL): 4
- Dose volume (mL/kg): 5


Tissues and cell types examined:
Tissue: Bone marrow
Cell type examined: Erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on Range-Finder studies

DETAILS OF SLIDE PREPARATION: One femur from each animal was exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. An additional 4 mL of serum was added to each bone marrow sample prior to adding to prepared cellulose filtration columns.
Once filtered, the bone marrow cells were centrifuged at 200g for 5 minutes at room temperature and the majority of the supernatant removed. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. A second centrifugation step at 200g for approximately five minutes followed with the serum aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette. From each tube one drop of suspension was placed on the end of each of three slides, labelled appropriately with details such as study number, assay type, sampling time, sex, date of preparation and animal number. A smear was made from the drop by drawing the end of a
clean slide along the labelled slide. Slides were allowed to air-dry and then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. One slide from each set was taken and stained on the same day as slide preparation. Any remaining slides were kept in reserve at < -10 °C. Slides were stained for 5 minutes in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then allowed to dry and stored in the dark at room temperature prior to analysis. Following analysis a reserve slide from vehicle animal 4 was stained for analysis. Prior to staining (as above), the slide was fixed again for 10 minutes in absolute methanol and rinsed several times in distilled water.

METHOD OF ANALYSIS: fluorescence microscopy
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
- A statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MN PCE) occurred at one or more dose levels
- The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
- The group mean MN PCE value at such a point exceeds the 95% calculated confidence interval for the mean historical vehicle control data
- A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation. The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p < 0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
- Dose range: Based on the findings and the maximum test article solubility, 10 mg/kg/day was considered a suitable estimate of the maximum practicable dose. Two lower doses of 5 and 2.5 mg/kg/day were also selected for testing.
- Clinical signs of toxicity in test animals: On both days of dosing ataxia and aggressive behaviour were noted immediately after administration. On Day 1 female animals also displayed vocalization immediately after dosing. Decreased activity was seen in all animals up to 1hour post dose Day 1. The vehicle control was assessed for clinical toxicity at dose volumes of 10mL/kg and 5mL/kg. At 10mL/kg, clinical signs, including ataxia, decreased activity, lethargy, piloerection and hunched posture, persisted throughout post dose observations on Day 1. On Day 2 ataxia onlywas observed. With the 5mL/kg dose volume clinical signs were seen on Day 1 only, 2 & 4 hours post dose. These signs were limited to males and included decreased activity, hunched posture and aggressive behaviour. Due to the nature and severity of clinical signs observed in animals dosed with the vehicle control at 10mL/kg, the dose volume was limited to 5mL/kg for Range-Finder testing in this study.
As no substantial difference in toxicity was observed between males and females in the
Range-Finder Experiment, male animals only were used in the Micronucleus Experiment. - High dose with and without activation: 10 mg/kg/day

- Clinical signs: No clinical signs of toxicity were observed in the vehicle or positive control (CPA) animals. In test article treated animals (2.5, 5 and 10 mg/kg/day) clinical signs were limited to ataxia, which was noted in all animals 0.5 hour post the first administration. Following the first administration reductions in group mean bodyweight (ranging between 7-16 g) were noted (Day 1-2) in the vehicle and test article groups. There was no significant weight change following the second administration.
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): Groups of rats treated with CD08467 exhibited %PCE values that were acceptable compared to the vehicle control (Appendix 3 and Appendix 5) and which were within acceptable ranges. There was no evidence of any test article-induced toxicity to the bone marrow (as would usually be indicated by a notable decrease in %PCE values compared to the vehicle control group or dose dependent decrease).
- Statistical evaluation: For all dose groups, the group mean frequencies of MN PCE were comparable with, and not statistically (chi-square) different from those seen in concurrent vehicle control group. Individual frequencies of MN PCE for all treated animals were consistent with historical vehicle control distribution data and similar to frequencies observed in the concurrent controls.

For individual results see Table 1 in box "Any other information on results incl. tables".

Applicant's summary and conclusion

Under the reported experimental conditions, CD08467 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 10 mg/kg/day (the maximum practicable dose for this study).
Executive summary:

In an in vivo micronucleus study conducted according to OECD guideline 474, 6 -10 weeks old, male Han Wistar rats (6/dose) were exposed intraperitoneally to the test item CD08467 (purity: 98.4%) at a dose volume of 5 mL/kg at concentrations of 2.5, 5 and 10 mg/kg/day. The doses were given twice at an interval of 24 hours. The organisms were checked for clinical signs of toxicity and the bone marrow was sampled. Rats treated with CD08467 at all doses exhibited MN PCE frequencies that were similar to the values for the vehicle control group and which also fell within the laboratory's historical distribution data. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article. The positive control chemical (CPA) induced a statistically significant (p < 0.001) increase in the frequency of micronucleated PCE, exhibiting a group mean of 30.17 MN/2000 PCE compared to the concurrent vehicle control which displayed a group mean of 3.00 MN/2000 PCE. It is concluded that CD08467 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 10 mg/kg/day (the maximum practicable dose for this study). Based on these results, the test item is considered to be non-mutagenic according to the results of the in vivo micronucleus test reported.