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 2011-11-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 474 without deviations.

Data source

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

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:
Details described in study specific records
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffcat DPA
- Substance type: Light yellow liquid
- Physical state: Liquid
- Analytical purity: 100%
- Composition of test material, percentage of components: 0.24 wt % water, total amine: 8.8 meq/g
- Lot/batch No.: 0H519
- Stability under test conditions: no data
- Storage condition of test material: The substance is stored at ambient (15 to 30°C) in the dark without desiccant.
- Other: synonym: 2-propanol, 1, 1'-((3-(dimethylamino)propyl)imino)bis- ; CAS# 63469-23-8

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: 7 weeks old
- Weight at study initiation: range:
dose range finding study: males: 31.1 - 34.0 grams, females: 26.3 - 28.1 grams
definitive micronucleus study: males: 29.9 - 34.0 grams, females: 23.9 - 29.9 grams
- Assigned to test groups randomly: yes, 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 variation of animals did not exceed ± 20% from their group mean.
- Fasting period before study: no
- Housing: Animals were housed in an AAALAC-accredited facility with a controlled environment
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Purified water
Details on exposure:
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 oral route of administration has been routinely used, and is widely-accepted for use in the mammalian bone marrow erythrocyte micronucleus assay.
Duration of treatment / exposure:
One single dose
Frequency of treatment:
Once
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: mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity, which were recorded. Bone marrow was collected 24 hours post-dose (all groups) and 48 hours post-dose (vehicle control and high dose group).
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Dose range finding study: 5 (2000 mg/kg)
Definitive study: 10 (vehicle control), 5 (500 and 1000 mg/kg), 15 (2000 mg/kg), 5 (positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide monohydrate
- Route of administration: oral: gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
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 labeled centrifuge tube containing aprpoximately 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. Two slides were prepared from each mouse. The slides were air dried and fixed in methanol. One set of slides was stained with a nucleid acid-specific stain, acridine orange, and was used in microscopic evaluation.
Details of tissue and slide preparation:
To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Using a fluorescent microscope and medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. using oil immersion (1000 X), the following cell populations and cellular components were evaluated and enumerated:
- polychromatic erythrocytes (PCEs; 2000 PCEs per animal were scored)
- normochromatic erythrocytes (NCEs; number of NCEs and micronucleated NCEs determined by scoring 1000 total erythrocytes per animal)
- micronuclei
The proportion of polychromatic erythrocytes to total erythrocytes were also determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined.
Statistics:
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. The test substance was judged negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groupos relative to the concurrent negative (vehicle) groups is obersved. The test substance would have been 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< = 0.05, binomial distribution, Kastenbaum-Bowman Tables).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
apart from piloerection in all animals.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
No mortality was observed. Piloerection was seen at a dose of 2000 mg/kg, in all males following dose administration and on Study Day 1, and in 3/5 females on Study Day 1. No appreciable changes in the group mean body weights were seen.

RESULTS OF DEFINITIVE STUDY
No mortality was observed in any of the treatment groups. All mice in the control substance (vehicle or positive) groups and all female mice in the 500 and 1000 mg/kg treatment groups appeared normal during the study period. Piloerection was seen in all male mice at 500, 1000 and 2000 mg/kg. This clinical sign persisted in the male mice at 2000 mg/kg at 24 hours post dose.
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 test 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≤ 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.

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 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test substance was concluded to be negative in the micronucleus test using male or female ICR mice.