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Toxicological information

Genetic toxicity: in vivo

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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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline is not mentioned. The study was conducted in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Heddle, 1973; Hayashi et al., 1994; Mavournin et al., 1990
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Y-4036
- Physical state: Clear, colourless liquid
- Analytical purity: 99.3%
- Lot/batch No.: 17036-86
- Storage condition of test material: room temperature; protected from exposure to light

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: Pilot assay: Males, 26.4 - 29.4 grams and Females, 25.8 - 28.0 grams; Micronucleus assay: Males, 26.9 - 32.0 grams and Females, 24.3 - 27.6 grams

- Assigned to test groups randomly: yes; according to a computer-generated program which is based on distribution according to body weight
- Housing: In an AAALAC-accredited facility. Mice of the same sex were housed up to fiver per cage in polycarbonate cages which were maintained on stainless steel racks.
- Diet (e.g. ad libitum): Mice had free access to certified laboratory rodent chow.
- Water (e.g. ad libitum): Mice had free access to tap water.
- Acclimation period: mice were quarantined for no less than 5 days after receipt.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 74 ± 6 °F
- Humidity (%): 50 ±20%

- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: PEG 400
- Justification for choice of solvent/vehicle: Request by the Sponsor and compatibility of the vehicle with the test system animals.
- Concentration of test material in vehicle: The test article was soluble in PEG 400 at 400 mg/l, the maximum concentration tested. Dosing concentrations were delivered to the test system as solutions.
- Lot/batch no. (if required): 01904LQ
- Expiration date: 2001-01-09
Details on exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by intraperitoneal injection at a constant volume of 5 ml/kg body weight.
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
24 hours and 48 hours after dose administration, animals were sacrificed by carbon dioxide asphyxiation. The positive control group was sacrificed 24 hours after dose administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate CP, CAS number 6055-19-2
- Route of administration: IP injection, same route as the test article
- Doses / concentrations: CP was dissolved in sterile distilled water at a concentration of 10 mg/ml. Dose: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Pilot toxicity study. Male mice were dosed with 1, 10, 100 or 1000 mg test article/kg bw (2 males each) and 5 male and 5 female mice were dosed with 2000 mg/kg; no vehicle group was included. Due to less than 50% mortality at 2000 mg/kg, the high dose for the micronucleus test was set at 2000 mg/kg.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Immediately following sacrifice times (24h or 48h after treatment) for the vehicle control and the highest test dose and 24 hours for the 500 and 1000 mg/kg doses and the positive control.

DETAILS OF SLIDE PREPARATION: 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 capped 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 by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:
The test article was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one ore more doses were statistically elevated relative to the vehicle control (<=0.05, Kastenbaum-Bowman Tables) at any sampling time.
Statistics:
Statistical significance determined using Kastenbaum-Bowman Tables

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Pilot Assay:
All animals tested with a total volume of 20 ml test article-vehicle/kg bw at all dose levels died immediately due to solvent toxicity. The pilot toxicity study was repeated at 5 ml/kg dosing volume. Mortality occurred within two days of dose administration as follows: 1/5 males and 1/5 females at 2000 mg/kg. Significant loss of body weight was observed (>10%) in animals treated with 100, 1000 and 2000 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at the higher dose levels, bunt not in the 1 mg/kg dose group. Piloerection was observed in male mice at 1000 mg/kg and piloerection, crusty eyes, diarrhea and hunched position in male and female mice at 2000 mg/kg. Due to less than 50% mortality at 2000 mg/kg, the high dose for the micronucleus test was set at 2000 mg/kg.


Micronucleus Assay:
Obersvations:
Mortality was observed in 2/15 male and 4/15 female mice receiving 2000 mg/kg. Clinical signs following dose administration included piloerection in male and female mice at all test article dose levels, and irregular breathing, crusty eyes, tremors and hunched position in male and female mice at 2000 mg/kg. Lethargy was observed in all male and female mice administered with the test article and in all animals in the vehicle group. Therefore, lethargy as a clinical sign was not attributed to the test article, but is likely due to the vehicle.

Significantly low excreta was observed with male and female mice dosed 1000 and 2000 mg/kg.

Reductions (up to 20%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability to the test article to the bone marrow target.

Any other information on results incl. tables

 Treatment  Sex  Time (hour)  Number of Mice  PCE/Total Erythrocytes (Mean +/- SD)  Change From Control %  Micronucleated Polychromatic Number per 1000 PCEs ( Mean +/- SD) Erythrocytes number per PCEs Scored* 
PEG 400 (neat) 5 ml/kg      M  24  5  0.49 ± 0.06  - 0.3 ± 0.27  3 / 10000 
 F   24  5  0.56 ± 0.03  - 0.7 ± 0.76   7 / 10000 
    Y-4036 500 mg/kg   M   24  5  0.53 ± 0.02  8 0.8 ± 0.91   8 / 10000 
  F   24  5  0.56 ± 0.04  0  0.9 ± 0.96  9 / 10000 
 Y-4036 1000 mg/kg       M   24  5  0.56 ± 0.04  14  0.5 ± 0.00  5 / 10000 
  F   24  5  0.52 ± 0.08  -7  0.3 ± 0.45  3 / 10000 
     Y-4036 2000 mg/kg  M    24  5  0.52 ± 0.09  6  0.3 ± 0.45  3 / 10000 
  F   24  5  0.45 ± 0.10  -20 0.2 ± 0.45   2 / 10000 
     CP 50 mg/kg   M   24  5  0.51 ± 0.07  4 25.6 ± 7.24    *256 / 10000
  F   24  5  0.49 ± 0.06  -13 20.0 ± 5.91    *200 / 10000
     PEG 400 (neat) 5 ml/kg       M  48  5  0.48 ± 0.04  - 0.4 ± 0.22    4 / 10000
  F  48  5  0.47 ± 0.07  -  0.2 ± 0.27   2 / 10000
        Y-4036 2000 mg/kg   M  48  5  0.39 ± 0.03  -19  0.3 ± 0.45   3 / 10000
  F  48  5  0.41 ± 0.10  -13  0.1 ± 0.22  1 / 10000 

*p ≤ 0.05 (Kastenbaum-Bowman Tables)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results of the assay indicate that under the conditions described in this report, Y-4036 did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

The test article Y-4036, was tested in the mouse micronucleus assay. The micronucleus study evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. A pilot study was conducted to set the dose levels for the micronucleus assay. Test and control articles were administered in a constant volume of 5 ml/kg body weight by a single intraperitoneal injection. PEG 400 used as the vehicle at the request of the Sponsor. The dose levels of the micronucleus assay were 500, 1000, or 2000 mg/kg body weight.

Mortality was observed in 2/15 male and 4/15 female mice receiving 2000 mg/kg. Clinical signs following dose administration included piloerection in male and female mice at all test article dose levels, and irregular breathing, crusty eyes, tremors and hunched position in male and female mice at 2000 mg/kg. Lethargy was observed in all male and female mice administered with the test article and in all animals in the vehicle control group. Therefore, lethargy as a clinical sign was not attributed to the test article, but is likely due to the vehicle. Significantly low excreta was observed with male and female mice dosed 1000 and 2000 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Reductions (up to 20%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability to the test article to the bone marrow target.

No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control group was observed in male or female mice at 24 or 48 hours after dose administration (p>0.05, Kastenbaum-Bowman).