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:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP complaint, guideline study, available as unpublished DRAFT report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 97.6%
Batch/Lot Number: 2030337

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: approximately 7 to 9 weeks old
- Weight at study initiation:
Dose Range Finding Study: Males: 29.5 – 32.6 g; Females: 29.4 – 32.9 g
Definitive Micronucleus Study: Males: 30.3 – 34.8 g
Toxicokinetic Portion of the Study: Males: 29.1 – 33.6 g
- Assigned to test groups randomly: yes, based on equalization of group mean body weights.
- 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 this 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.
- Diet (e.g. ad libitum): A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients.
- Water (e.g. ad libitum): Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards.
- Acclimation period: no less than 5 days after receipt

ENVIRONMENTAL CONDITIONS
Animals were housed in an AAALAC accredited facility with a controlled environment of 50 ± 20% relative humidity and 72 ± 3°F temperature with a 12 hour light/dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil was used as the test article vehicle in preparing the formulations and as a vehicle control in the definitive study. Corn oil (CAS number: 8001 30 7, Lot No.: 3783J, expiration date: 26 February 2011) was obtained from MP Biomedicals.
The vehicle control was characterized as per the Certificate of Analysis on file with the testing facility.
Details on exposure:
All dose formulations were administered at a dose volume of 20 mL/kg by a single oral gavage (per os, PO). Oral gavage was performed using appropriate size disposable polypropylene syringes with gastric intubation tubes (needles).
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
Dose Range Finding Study: Mice were observed immediately after dose administration, ~ 1 hour post-dose, ~ 4 hours post-dose, and daily thereafter for 3 days for clinical signs of toxicity
Micronucleus Test: Mice were observed immediately after dose administration, ~1 hour, 4 hours post-dose, and daily thereafter for clinical signs of toxicity.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
437.5 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
875 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1250 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Dose range-finding study: 3 male and 3 female mice per dose group
Micronucleus test: 5 male mice per dose group per sacrifice time
Control animals:
yes, concurrent vehicle
Positive control(s):
5 male mice; 50 mg/kg bw Cyclophosphamide monohydrate
(CP, CAS No.: 6055-19-2, lot no.: 036K1225, expiration date: 31 March 2009) was obtained from Sigma-Aldrich Company and was dissolved in sterile water for injection (B.Braun, CAS No.: 7732 18 5, lot no.: J8H010, expiration date: 31 March 2009) at a concentration of 2.5 mg/mL for use as the positive control article.
The accuracy of preparation and stability of the positive control dosing formulation was demonstrated by acceptable results that met the criteria for a valid test.

Examinations

Tissues and cell types examined:
At the time of euthanasia, femoral bone marrow was collected and bone marrow smears (slides) were prepared and stained with Acridine orange stain (a nucleic acid specific stain). Bone marrow cells [polychromatic erythrocytes (PCEs); 2000 PCEs/animal] were examined microscopically for the presence of micronuclei (micronucleated PCEs; MPCEs). A statistical analysis of data was performed using one way analysis of variance (ANOVA, significant level of p ≤0.050) and Dunnett’s t-test (post hoc). In addition, the ratio of polychromatic erythrocytes to total of 1000 erythrocytes (PCEs/ECs ratio) was determined as an indicator of bone marrow exposure to the test article and subsequently as a measure of test article cytotoxicity.
Details of tissue and slide preparation:
In the micronucleus test, at the scheduled bone marrow collection time, five mice per treatment were euthanized by CO2 asphyxiation and the incision of the diaphragm. 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 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 resuspended in the remaining serum 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 fixed in methanol, and one set of slides was stained with Acridine orange (a nucleic acid specific stain) and used in microscopic evaluation.
Evaluation criteria:
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 (400X; blue excitation filter in the range of 440-490 nm and barrier filter combination at 520 nm), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (1000X), the following cell populations and cellular components were evaluated and enumerated:
• Polychromatic erythrocytes (PCEs); PCEs stain orange-red. PCEs are young erythrocytes (early stage of erythropoiesis) and are the target cells for evaluation of the test article clastogenicity. Two-thousand PCEs per each animal were screened (scored) for the presence of micronuclei resulting in evaluation of a total of 10,000 PCEs per each treatment group.
• Normochromatic erythrocytes (NCEs); NCEs appeared light green in color. NCEs are mature erythrocytes (red blood cells) and are the final cell population formed during erythropoiesis. 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). The incidence of MNCEs per 2000 PCEs was enumerated for each animal, but the results were not presented in this report or used in analysis of the test article clastogenic response since the primary target cells are the PCEs.
• 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 was also recorded per 1000 erythrocytes per each animal (PCEs/ECs ratio).
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using one way analysis of variance (ANOVA, p≤ 0.050) and Dunnett’s t-test (post hoc) for group mean comparison and regression analysis (p ≤ 0.001) for detecting any trend using square-root transformed data (square root of MPCEs/2000 PCEs +1).
All analyses were performed separately for each sampling time.
Any significantly increased response in any of the test article groups would have been indicated with the corresponding “p” value where a “p” value ≤ 0.05 was considered significant.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
• No significant differences in the toxicity between male and female mice were observed during the conduct of DRF study. Therefore only male mice were used in the micronucleus test.
• Based on the mortality, clinical signs and reductions in body weights observed during DRF study, the maximum tolerated dose for male mice was estimated to be a dose of 1250 mg/kg Delta Damascone.
• Delta Damascone formulations were accurately prepared indicating that the intended doses of 1250, 875, and 437.5 mg/kg in the micronucleus test were achieved.
• Measurable concentrations of Delta Damascone were detected in the plasma samples of animals dosed with the test article indicating systemic exposure during the 24 hr post-dose period.
• A single oral administration of Delta Damascone at doses up to and including 1250 mg/kg (estimated maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male ICR mice.
Based on the all of the above, Delta Damascone was concluded to be negative in the In vivo micronucleus assay.

Applicant's summary and conclusion