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:
2011-10-11 to 2011-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP study performed according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
Test material form:
liquid
Details on test material:
- State of aggregation : liquid
- Appearance : colourless to yellow brown
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1D403
- Purity: 100% (provided by sponsor)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15 to 30°C), in the dark without desiccant

OTHER SPECIFICS:
- Name of test material (as cited in study report): JEFFCAT DMDEE
- Substance type: light yellow liquid
- Physical state: liquid
- Other: assignment of code AD36HK; stability not determined
- Total amine content: 8.4 meq/g
- Water content: 0.04 wt%
- Viscosity: 29 cSt

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation: dose range finding study: male: 31.8 - 33.4 g, female: 24.3 - 25.4 g; definitive micronucleus study: male mice: 30.0 - 36.1 g, female mice: 24.5 - 30.3 g
- Assigned to test groups randomly: yes, under following basis: In each study, mice were assigned to the appropriate number of treatment groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight varation of the animals did not exceed ± 20% from their group mean. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during randomization process. The cage card contained the following information: the animal number, sex, study number, test substance identification, treatment group number, dose level and route of administration. Cage cards were color coded as a function of treatment group. Raw data records and specimens were also identified by the unique test animal number.
- Fasting period before study: no
- Housing: Animals were housed in an AAALAC-accredited facility with a controlled environment of
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days (dose-range finder) or 6 days (definitive study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: All dose formulations were administered at a dose volume of 20 mL/kg by a single oral administration using appropriate size disposable polypropylene syringes with gastric intubation tubes (needles).
The test substance dose formulations were prepared fresh for each phase of the study prior to dose administration. The formulation (100 mg/mL) for the DRF study and all formulations (25, 50 and 100 mg/mL) for definitive study were prepared as follows:
An appropriate amount of the test substance was weighed for each concentration separately.
An appropriate volume of the vehicle, ~80% of the final volume was added to the respective containers.
Each formulation was vortexed for 1 minute.
Remaining volume of the vehicle was added to achieve the final volume.
All formulations appeared as clear solutions.
Duration of treatment / exposure:
Single oral administration
Frequency of treatment:
Single oral administration
Post exposure period:
Dose range finding study: mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration on Study Day 0 and on Study Days 1 and 3. Following the last observation on Study Day 3, animals were euthanized by exposure to CO2 verified by toe pinch reflex and discarded without further examination.
Definitive micronucleus study: In-Life evaluations: mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity, which were recorded. Bone marrow collection and slide preparation: At the scheduled bone marrow collection time, five mice per sex per treatment were euthanized by CO2 asphyxiation verified by toe pinch reflex.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Based on the information provided by the Sponsor, the oral LD50 dose was > 2000 mg/kg. In order to confirm this information, in the dose-range finding study, 5 male and 5 female mice were orally exposed 2000 mg/kg, the highest regulatory recommended dose.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Based on the information provided by the Sponsor, the oral LD50 dose was > 2000 mg/kg. In order to confirm this information, in the dose-range finding study, 5 male and 5 female mice were orally exposed 2000 mg/kg, the highest regulatory recommended dose.
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Based on the information provided by the Sponsor, the oral LD50 dose was > 2000 mg/kg. In order to confirm this information, in the dose-range finding study, 5 male and 5 female mice were orally exposed 2000 mg/kg, the highest regulatory recommended dose.
No. of animals per sex per dose:
500 and 1000 mg/kg: 5
vehicle and 2000 mg/kg: 10
positive control: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Tissue: bone marrow of the femurs
Cell types examined:
- Polychromatic erythrocytes (PCEs): 2000 PCEs per animal were screened (scored) for the presence of micronuclei resulting in evaluation of a total of 10000 PCEs per each treatment group.
- Normochromatic erythrocytes (NCEs): The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) is determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs ratio).
- Micronuclei (M): Micronuclei are round, fluorescent green-stained nuclear (chromosome) fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs).
- The proportion of polychromatic erythrocytes to total erythrocytes were also determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal.
Details of tissue and slide preparation:
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 at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Slides were labelled with the experiment and animal numbers using a lead pencil. One set of slides was stained with a nucleic acid-specific stain, acridine orange, and was used in microscopic evaluation.
Evaluation criteria:
In this study, the test substance was jugded 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 was observed.
The test substance would have been considered to induce a positive response if the incidence of micronucleated polychromatic erythrocytes at one or more doses in statistically elevated relative to the vehicle control (p < or = 0.05, binomial distribution, Kastenbaum-Bowman tables).
However, the results of the statistical analysis would not be the only criteria in determination of the test substance positivity. A dose-dependent increase or biological relevance of the data would have been taken in consideration. In addition, if criteria for either a positive or negative genotox response were not met, the results would have been judged as equivocal.
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined.
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each mouse and treatment group (PCEs/ECs ratio). The proportion of polychromatic erythrocytes to total erythrocytes in test substance animals should not be less than 20% of the control value.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Piloerection and/or lethargy are among the clinical effects observed during the course of the study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the dose-range finding study, no mortality was observed. Piloerection and/or lethargy are among the clinical signs observed during the course of the study. No appreciable changes in the group mean body weights were seen.

RESULTS OF DEFINITIVE STUDY
In the definitive study, following results were generated:
- No mortality was observed in any of the treatment groups. All mice in the control substance (vehicle or positive) groups and all mice in the 500 and 1000 mg/kg treatment groups appeared normal during the study project. Piloerection was seen at 2000 mg/kg uring the course of the study.
- In the bone marrow, no appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test substance groups relative to the respective vehicle control groups were observed, suggesting that the substance did not inhibit erythropoiesis.
- No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, binomial distribution, Kastenbaum-Bowman Tables).
- CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p< or = 0.05, binomial distribution, Kastenbaum-Bowman Tables) in both male and female mice. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

Any other information on results incl. tables

Criteria for a valid test:

All criteria for a valid test were met because of the following:

- The incidence of micronucleated polychromatic erythrocytes in the vehicle control groups did not exceed the historical vehicle control range.

- The incidence of micronucleated polychromatic erythrocytes in the male and female positive control groups was significantly increased relative to the respective vehicle control groups (p < or = 0.05, binomial distribution, Kastenbaum-Bowman Tables).

However, the results of the statistical analysis would not be the only criteria in determination of the test substance positivity. A dose-dependent increase or biological relevance of the data would have been taken in consideration. In addition, if criteria for either a positive or negative genotoxic response were not met, the results would have been judged as equivocal.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study conduct, a single oral administration of the test substance at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. Therefore, the substance was concluded to be negative in the mouse micronucleus assay.
This test was performed to fulfil other regulatory requirements outside the EU.