Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19/12/2011 to 19/01/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 474 and ICH S3A, ICH S2A and ICH S2B guidelines.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
other: ICH S3A (1995), ICH S2A (1996), ICH S2B (1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Jeffamine RFD-270
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Analytical purity: 100%
- Lot/batch No.: 880289
- Storage condition of test material: Ambient (15 to 30°C), protected from light

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: 6 weeks old
- Weight at study initiation: dose range finding study: male mice: 30.9 - 36.5 grams, female mice: 30.0 - 25.0 grams; definitive micronucleus study: male mice: 29.5 - 34.0 grams, female mice: 24.4 - 28.6 grams
- Assigned to test groups randomly: yes, 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 by sex, with up to five animals 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.
- Diet (e.g. ad libitum): Certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet); ad libitum
- Water (e.g. ad libitum): Tap water; Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 22.2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 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 test substance dose formulations were freshly prepared prior to administration. An appropriate amount of the test substance was weighed separately for each formulation, and ~ 80% of the required vehicle was added to the respective containers. Each formulation was vortexed briefly, and the remaining volume of the vehicle was added to achieve the required final volume. All formulations appeared clear formulations.
Duration of treatment / exposure:
Animals were treated once.
Frequency of treatment:
Animals were treated once, by oral gavage, using a dose volume of 20 mL/kg
Post exposure period:
Bone marrow was harvested at 24 or 48 hours after treatment and evaluated microscopically for the presence of micronucleated polychromatic erythrocytes (MPCEs), as well as for the proportion of PCEs (relative to total erythrocytes) as an indication of bone marrow cytotoxicity.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 125, 250, 500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5, with exception of the high dose group (500 mg/kg) where 15 mice/sex were tested (including 5 replacement mice/sex)
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:
bone marrow ; polychromatic erythrocytes were examined for the presence of micronuclei
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: 24 hours post dose: 5 animals per group, 48 hours post dose: 5 animals in the vehicle control group and 5 animals in the high dose group

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): sampling at 24 and 48 hours post dose

DETAILS OF SLIDE PREPARATION: The bone marrow was transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum, the cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was removed, leaving a small amount of serum with the remaining pellet.

The cells were re-suspended and a small drop of the suspension was spread onto a clean glass slide. Two slides were prepared from each mouse, labeled with the experiment and animal numbers, air dried, and fixed in methanol. One set of slides was stained with acridine orange and anallyzed microscopically.

METHOD OF ANALYSIS: Slides were initially scanned using a fluorescent microscope and medium magnification (400X; blue 440-490 nm excitation filter and 520 nm barrier filter) and an area acceptable quality was selected such that the cells were well spread and stained. Once selected, the following cell populations and cellular components were evaluated under high power magnification (1000X oil immersion).

- Polychromatic erythrocytes (PCEs):
PCEs are young erythrocytes in the early stage of erythropoiesis that stain orange-red; 2000 PCEs/animal (10000 PCEs/group) were scored for the presence of micronuclei.
- Normochromatic erychtrocytes (NCEs):
NCEs are mature erythrocytes, the final cell population formed during erythropoiesis, that stain light green; the number of NCEs/1000 total erythrocytes also was determined for each animal, to calculate the proportion of PCEs to total erythrocytes, as an index of bone marrow cytotoxicity. PCE proportions less than 20% of control values are considered excessively toxic.
- Micronuclei (M)
Micronuclei are round nuclear (chromosome) fragments, generally with a sharp contour and diameters of approximately 1/20 to 1/5 of an erythrocyte, that stain green. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs). The frequency of MNCEs observed while scoring 2000 PCEs was determined, but the results are not presented or used in analysis of genotoxic response since the primary target cells are the PCEs.
Evaluation criteria:
- A test substance is considered to be positive for the induction of micronuclei if it induces a significant increase in MPCE frequency (p<=0.05) at any dose level of sampling time
- A test substance is considered to be negative for the induction of micronuclei if no significant increase in MNPCE frequency is observed (p>0.05).
- If criteria for either a positive or negative clastogenic response were not met, the resuls would have been judged as equivocal.
Other criteria also may be used in reaching a conclusion about the study results(e.g. magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.).
Statistics:
Statisical significance, as compared to the concurrent vehicle control, was determined for each group using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately by sex and sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Complete mortality was observed on day 0 in all mice treated with Jeffamine RFD-270 at doses of 750, 1000 and 2000 mg/kg. All mice treated at a dose of 500 mg/kg exhibited piloerection after treatment and on day 1; males in this group also exhibited lethargy on day 0. All mice in the 100 and 250 mg/kg treatment groups appeared normal throughout the study, except for piloerection noted in the 100 mg/kg dose group on day 3.

RESULTS OF DEFINITIVE STUDY
Five males treated with Jeffamine RFD-270 at a dose of 500 mg/kg were found dead on day 0. Males from the replacement group were reallocated to ensure sufficient animals were available for each sampling time. Piloerection also was observed in some or all males at all dose levels on day 0 and 1, and in some or all females at a dose of 500 mg/kg on day 0 and 1. Lethargy also was observed in all males at a dose of 500 mg/kg on day 0.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results of this test indicate Jeffamine RFD-270 was negative in the mouse bone marrow erythrocyte micronucleus.