Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-21 - 2010-11-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
other: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use (1994, 1996 and 1997)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Reference substance name:
Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
EC Number:
272-712-1
EC Name:
Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Cas Number:
68909-77-3
Molecular formula:
Not applicable (UVCB)
IUPAC Name:
Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Test material form:
liquid
Details on test material:
Name: Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Physical state: liquid
Appearance: brown liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C8
- Physical state: dark liquid
- Analytical purity: the test substance is a complex mixture, and the purity will be considered to be 100%
- Lot/batch No.: AD16UT
- Storage condition of test material: at ambient (15 to 30°C), protected from light

Test animals

Species:
mouse
Strain:
other: Hsd:ICR (CD-1)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation:
- Assigned to test groups randomly: yes, under following basis: Initially for the dose range finding study, animals are assigned to one group of 5 male and 5 female mice, as per study design. For the definitive micronucleus study animals are assigned to 7 groups of 5 males and 5 females each. The animals are assigned using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals will not exceed ± 20% of the mean weight. Following randomization, animals are identified by sequentially numbered ear tags assigned to each animal during randomization process. The cage card contains, at least, the animal number, sex, study number, test substance identification, treatment group number, dose level, and route of administration. Cage cards are color coded as a function of treatment group.
- Fasting period before study: no fasting
- Housing: Animals are housed in an AAALAC-accredited facility with a controlled environment. Mice of the same sex are housed up to five per rodent Micro-Barrier cage. Cages are placed on the racks equipped with an automatic watering system and MicroVENT full ventilation, HEPA filtered system. The purpose of this system is 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 indroducing the air back into the cage. If needed, alternative housing system will be implemented. Heat treated hardwood chips are used for bedding to absorb liquids.
- Diet (e.g. ad libitum): A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) is provided at libitum. The food is analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients.
- Water (e.g. ad libitum): Animals have free access to tap water, which meets U.S. EPA drinking water standards. Drinking water is monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens.
- Acclimation period: 5 days prior to dose administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 22°C
- Humidity (%): 50 ± 20% relative humidity
- Air changes (per hr): The animal rooms are supplied with at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: purified (deionized water)
- Justification for choice of solvent/vehicle: good workability/solubility of the test substance in the vehicle and compatibility of the vehicle with the test system.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For each study, the test substance formulations are be prepared prior to dose administration. Each concentration is prepared by mixing an appropriate amount of the test substance with the appropriate volume of the vehicle. The mixtures are vortexed, if needed homogenized and stirred in order to achieve workable or soluble formulations.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): not applicable (gavage)
- Storage temperature of food: no data
Duration of treatment / exposure:
The test substance dose formulations, the vehicle alone or the positive control (cyclophosphamide monohydrate) are given as a single administration.
Frequency of treatment:
The test substance dose formulations, the vehicle alone or the positive control (cyclophosphamide monohydrate) are given as a single administration. The volume of administration will be 20 mL/kg body weight.
Post exposure period:
In the definitive test, approximately 24 and 48 hours after dose administration, animals are euthanized by carbon dioxide inhalation followed by incision of the diaphragm. Animals in the positive control group are euthanized 24 hours after dose administration.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested gavage
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested gavage
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested gavage
No. of animals per sex per dose:
Dose range finding study: 30 animals/sex
Definitive Micronucleus Study: 35 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
- Route of administration: oral: gavage
- Doses / concentrations: animals will receive cyclophosphamide at a dose of 50 mg/kg body weight

Examinations

Tissues and cell types examined:
Immediately following euthanasia, the femurs are exposed, cut just above the knee and the bone marrow will be aspirated into a syringe containing fetal bovine serum.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In the definitive test, approximately 24 and 48 hours after dose administration, animals are euthanized by carbon dioxide inhalation followed by incision of the diaphragm. Animals in the positive control group are euthanized 24 hours after dose administration.

DETAILS OF SLIDE PREPARATION: The bone marrow cells are transferred to a labelled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells are pelleted by centrifugation and the supernatant is drawn off, leaving a small amount of fetal bovine serum with the remaining cell pellet. The cells are respudended and a small drop of the bone marrow suspension is spread onto a clean glass slide. Each slide will be identified by the experiment and animal number. At least two slides are prepared from each animal, air dried and fixed by dipping in methanol. One set of slides is stained with May-Gruenwald-Giemsa, permanently mounted and used for microscopic evaluation. The other set of slides (not stained) is kept as a backup set.

METHOD OF ANALYSIS: Using a light microscope and a medium magnification (400 X), an area of acceptable quality will be selected such that the cells are well spread and stained. The following cell populations and cell components are recorded using oil immersion (1000X):
-2000 polychromatic erythrocytes (PCEs) are scored per animal for the presence of micronuclei resulting in evaluation of 10000 PCEs per group.
- The number of micronucleated normochromatic erythrocytes (MNCEs) in the field of 2000 polychromatic erythrocytes are also enumerated, but are not used to evaluate the response of the test substance
- The proportion of polychromatic erythrocytes to total erythrocytes is also determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal.


Evaluation criteria:
The test substance will be considered to induce a positive response if the incidence of micronucleated polychromatic erythrocytes at one or more doses is statistically elevated relative to the vehicle control (p< or = 0.05, Kastenbaum-Bowman Tables).
The substance will be judged negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the concurrent negative (vehicle) groups is observed.
However, the results of statistical analysis may not be the only criteria in determination of the test substance potential to induce micronuclei. The following criteria may be taken into consideration: values that are statistically significant but do not exceed the range of historical negative or vechicle controls may be judged as not significant and relevant; a dose-dependent increase will also be taken in consideration in determination of the test substance positive response; if criteria for either a positive or negative genotoxic response are not met, the results are judged as equivocal or inconclusive.
Statistics:
Statistical analysis of data is performed using the Kastenbaum-Bowman tables which are based on the binomial distribution.

Results and discussion

Test results
Key result
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 500, 1000, 2000 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.

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