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:
27 August 1996 to 03 March 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

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

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories- Weight at study initiation: males 29-38g and females 20-29g for the preliminary test, males 26-34g and females 20-26g for the main test.- Assigned to test groups randomly: Yes- Fasting period before study: No- Housing: Stainless steel wire mesh caging- Diet (e.g. ad libitum): PMI Certified Rodent Diet ad libitum- Water (e.g. ad libitum): Tap water ad libitum- Acclimation period: 5 to 12 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18-26°C- Humidity (%): >=25%- Photoperiod (hrs dark / hrs light): 12 hour light-12 hour darkIN-LIFE DATES: From: 27 August 1996 To: 03 March 1997

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: OS#114461 was diluted in corn oil.
Duration of treatment / exposure:
24, 48 and 72 hours
Frequency of treatment:
Single dose
Post exposure period:
24, 48, 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:500, 750, 1500, 1750, 2250, 3000, 5000 mg/kgBasis:other: actual injected
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide- Justification for choice of positive control(s): It is a known clastogen- Route of administration: Intraperitoneal- Doses / concentrations: 60 mg/kg in sterile water for injection

Examinations

Tissues and cell types examined:
Bone marrow polychromatic and normochromatic cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Maximum tolerated dose level, and two lower dose levels TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24, 48 and 72 hoursDETAILS OF SLIDE PREPARATION: Bone marrow cell suspension spread onto slide, fixed and stained before coding.METHOD OF ANALYSIS: 1000 erythrocytes (PCE and NCE) were scored for the PCE/NCE ratio. 2000 PCEs were scored for the presence of micronuclei.OTHER:
Evaluation criteria:
A test article is considered to have produced a positive response if it induces a statistically significant increase in the frequency of MPCEs at one or more test article concentration for a single sacrifice time or sex (p<0.05), and the increase in MPCE frequency is dose-dependent (p<0.05). If the test article induces either a statistically significant or dose-dependent increase in MPCE frequency it is considered to have produced an equivocal response. A test article producing neither type of increase is considered to be negative (non-clastogenic) in this assay.The above criteria are used as a guide in evaluating test results. however, the Study Director may take other factors (e.g., historical control values) into consideration in evaluating the test results, since biological and statistical significance should be considered inevaluation of the asay results.
Statistics:
The frequency of MPCEs i each group was compared to its respective negative control group using a one-tailed Student's t-test. In addition, the Cochran-Armitage test was used to determine the statistical significance of any possible dose trends. Statistical significance was evaluated at the p<0.05 probability level. PCE/NCE ratios were analysed as described above. All comparisons were made separately for each sacrifice time.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY- Dose range: 500, 750, 1500, 1750, 2250, 3000, 5000 mg/kg- Solubility: Dissolved in corn oil- Clinical signs of toxicity in test animals: Yes, including premature mortalityRESULTS OF DEFINITIVE STUDY- Induction of micronuclei (for Micronucleus assay):- Ratio of PCE/NCE (for Micronucleus assay): Changed ratio of PCE/NCE in higher dose groups- Statistical evaluation: Performed

Applicant's summary and conclusion

Conclusions:
The test material is considered to be negative in the In Vivo Micronucleus Test in the Mouse Bone Marrow Erythropoetic Cells, under the conditions, and according to the criteria, of the test protocol.
Executive summary:

Test Guidance

OECD Guideline No 474

Method

This study was designed to evaluate the potential of the test material to induce micronuclei in the newly formed polychromatic erythrocytes (PCEs) in mouse bone marrow.

A preliminary toxicity screen was conducted to select the appropriate doses for the definitive micronucleus test (MNT). Eight groups of mice (two/sex/dose level) were administered a single intraperitoneal (ip) injection of the test or negative control articles at a dose volume of 10 mL/kg. The test material was evaluated at doses of 500, 750, 1500, 1750, 2250, 3000 and 5000 mg/kg. Mice were observed for mortality and pharmacotoxic signs immediately (0 -2 hours), and at 24, 48 and 72 hours, after dosing. Except for one female treated at a dose of 5000 mg/kg, all females treated at dose =>1750 mg/kg, and all males treated at doses =>3000 mg/kg, died within 48 hours after treatment. All surviving mice were sacrificed at the last observation time by cervical dislocation. Bone marrow was harvested from these animals and scored for the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) per 1000 total erythrocytes, as an index of toxicity

Results

The test material did not produce any statistically significant depressions in PCE/NCE ratios at any dose level, as compared to the concurrent negative control. In the definitive MNT, nine groups of mice (five/sex/dose) were administered a single ip injection of the test material at doses of 250, 1250 and 2500 mg/kg (males), or at dose of 150, 750 and 1500 mg/kg (females), for sacrifice at 24, 48 or 72 hours after injection. The negative control, corn oil, was administered concurrently to three groups of mice (five/sex) for sacrifice at 24, 48 or 72 hours, while the positive control, cyclophosphamide (CP; 60 mg/kg), was administered concurrently to a single group of mice (five/sex) for sacrifice 24 hours after treatment. All test and control articles were administered in a volume of 10 mL/kg. Mice were observed for mortality and pharmacotoxic signs immediately (0 -2 hours), and approximately 24, 48 or 72 hours after dosing. Bone marrow slides were prepared, stained, coded and scored for the number of micronucleated PCEs (MPCEs) in 2000 PCEs/mouse, as well as for the number of micronucleated normochromatic erythrocytes (MNCEs) present in the optical fields containing 2000 PCEs. The PCE/NCE ratio per 1000 erythrocytes also was determined for each mouse as an index of toxicity. MPCE frequencies for all negative control groups were within acceptable ranges, and the CP positive control induced statistically significant increase in MPCE frequency (combined- and by-sex; p<0.01). Analysis of the by- and combined-sex indicated that the test material did not induce any statistically significant or dose-dependent increases in MPCE frequencies, at any harvest time evaluated, as compared to the compared to the concurrent negative controls. In addition, all observed MPCE frequencies in the animals treated with the test material were within historical ranges. Analysis of the PCE/NCE ratios revealed that the test material induced statistically significant depressions in female mice treated at 750 and 1500 mg/kg and harvested at 24 hours (p<0.05), as well as in male mice treated at 2500 mg/kg and harvested at 48 hours (p<0.05). A statistically significant depression in PCE/NCE ratios was also observed in the high dose groups, at the 48 -hour harvest time, when combining sexes (p<0.05; females treated at 1500 mg/kg, males treated at 2500 mg/kg).

Conclusion

The test substance is considered to be negative in the In Vivo Micronucleus Test in Mouse Bone Marrow Erythropoetic Cells, under the conditions, and according to the criteria, of the test protocol.