Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
to be determined
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: Room temperature
Specific details on test material used for the study:
- Name of test material (as cited in study report): Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)-
- Analytical purity: The test substance was a complex mixture, and the purity was considered to be 100%.
- Storage condition of test material: ambient (15-30 degrees C), protected from light

Test animals

Species:
mouse
Strain:
other: Hsd:ICR (CD-1)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD or other alternate Harlan location
- Age at study initiation: ~6-8 weeks
- Weight at study initiation: 25.5-37.0 grams (males), 19.5-31.0 grams (females)
- Assigned to test groups randomly: yes; Animals were assigned to groups using a randomization procedure based on equalization of group mean body weights (MiniTab based program). At the time of randomization, the weight of variation of animals did not exceed +/- 20% of the mean weight.
- Fasting period before study:
- Housing: Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of the system was to supply uninterrupted positive air to each individual rodent Micro-barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the cage. If needed, alternated housing system was implemented.
- Diet (e.g. ad libitum): ad libitum (Harlan 2018C Certified Global Rodent Diet)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/- degrees F
- Humidity (%): 50 +/- 20 %
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: As per the Sponsor information, purified water was used as the vehicle based on good workability/solubility of the test substance in the vehicle and compatibility of the vehicle with the test system.
- Concentration of test material in vehicle:
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:The test substance was prepared prior to dose administration. Each concentration was prepared by mixing an appropriate amount of the test substance with the appropriate volume of the vehicle. The mixtures were vortexed, if needed, homogenized and stirred in order to achieve workable or soluble formulations.
The samples of the dosing formulations were not collected, and analysis of accuracy of preparation, homogeneity or stability of the formulations was not performed. However, by following the standard operating procedures of the laboratory it was to believe that the formulations were within commonly acceptable range of +/-20 % of target.
Animals received 20ml/kg via oral gavage.


Duration of treatment / exposure:
Animals received a single oral gavage administration.
Frequency of treatment:
Animals were treated once.
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals/sex/dose/sacrifice time point
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): CP is a known clastogen and is commonly used as the positive control in genetic toxicology assays.
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
femoral bone marrow (erythrocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A dose range finding study was used to determine the doses to be used in the definitive study. In the dose range-finding study, a highest guideline recommended dose of 2000 mg/kg was administered to mice five male and five female mice. If mortality occurred at this dose level, additional dose levels were tested. Mice were observed after dose administration and each day thereafter for 3 days for clinical signs of toxicity. Body weights were recorded prior to dose administration (Study Day 0) and on Study Days 1 and 3 (prior to euthanasia). Following observation period, all surviving animals were euthanized by carbon dioxide inhalation followed by incision of the diaphragm, and discarded without further examination. If 2000 mg/kg produced no treatment related morality, the high dose for the definitive micronucleus test would be 2000 mg/kg. Otherwise, the high dose would be the maximum tolerated dose. To additional doses, one half and one-fourth of the high dose, would be tested.


DETAILS OF SLIDE PREPARATION:
In the definitive study, approximately 24 and 48 hours after dose administration, animals were euthanized by carbon dioxide inhalation followed by incision of the diaphragm. Animals in the positive control group were euthanized 24 hours after treatment. Immediately following euthanasia, 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 labelled centrifuge tube containing approximately 1 mL fetal bovine serum.
The bone marrow cells were pelleted by centrifugation and the supernatant was drawn off, leaving a small amount of fetal bovine serum with the remaining cell pellet. The cells were resuspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. Each slide as identified by the experiment and animals number. At least two slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of slides was stained with May-Gruenwald-Giemsa, permanently mounted and use d for microscopic evaluation. The other set of slides (not stained) was kept as a backup set.


METHOD OF ANALYSIS:
Slides were coded using a random number table by an individual not involved with the scoring process. Using a light microscope and a medium magnification (400X), an area of acceptable quality was selected such that the cells were well spread and stained. The following cell populations and cell components were recorded using oil immersion (1000X):
1. 2000 polychromatic erythrocytes were scored per animal for the presence of micronuclei resulting in evaluation of 10000 PCEs per group.
2. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes were also enumerated, but were not used to evaluate the response of the test substance.
3. The proportion of polychromatic erythrocytes to total erythrocytes were determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal.
4. In the event of high reduction and suppression in PCEs/ECs ration, it may not have been possible to evaluate 2000 PCEs per animal for the presence of micronuclei.



Evaluation criteria:
See below.
Statistics:
Statistical analysis of data was performed using the Kastenbaum-Bowman tables which are based on the binomial distribution.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In an in vivo micronucleus test on mouse bone marrow erythrocytes, performed according to OECD 474, the animals were exposed orally (via gavage) with 125, 250 and 500 mg/kg. This did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay. No toxicity was observed. Vehicle and positive controls were valid.