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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October - Novemmber 4, 1996
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in reference to OECD Method in accordance with GLP. Study material is well characterized

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
A mixture of: bis(1S,2S,4S)-(1-benzyl-4-tert-butoxycarboxamido-2-hydroxy-5-phenyl)pentylammonium succinate; isopropyl alcohol
EC Number:
EC Name:
A mixture of: bis(1S,2S,4S)-(1-benzyl-4-tert-butoxycarboxamido-2-hydroxy-5-phenyl)pentylammonium succinate; isopropyl alcohol
Cas Number:
Molecular formula:
Empirical formula: C25H35N2O5
butanedioic acid; propan-2-ol; bis(tert-butyl N-[(4R)-5-amino-4-hydroxy-1,6-diphenylhexan-2-yl]carbamate)

Test animals

Details on test animals or test system and environmental conditions:

- Age at study initiation: 9 weeks
- Weight at study initiation: males- 19-36 grams; 22-33 grams females
- Assigned to test groups randomly: [no/yes, under following basis: ] random
- Fasting period before study: 30 minutes
- Housing: hanging metal rack cages with feeders and automatic waterers, ventilated rack system
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): free assess tap water
- Acclimation period:one week

- Temperature (°C): 69-72 hrs
- Humidity (%): recorded
- Photoperiod (hrs dark / hrs light):12/24

Administration / exposure

Route of administration:
oral: gavage
0.2 % HPMC
Details on exposure:
Doses of test material: 156, 313, 625 mg/kg; 20 ml/kg dosage volume based on body weight on day of treatment
dose of positive control (cyclophosphamide): 50 mg/kg
Duration of treatment / exposure:
The mice were dosed once daily for 2 consecutive days and one group of mice from each dose level (half and all positive controls) were killed 24 hours following treatment and a second group (other half of treatment group and vehicle) were killed after 48 hours.
Frequency of treatment:
3 dose groups, each of 10 mice ( male and female), were dosed once daily for 2 days via the oral gavage route with test material (total 20 mice treated per dose group); one positive control dose group - 5 males and females each and one vehicle only group of 10 animlas males and females, each.
Post exposure period:
Initial body weight was recorded for each mouse on day of treatment. All animals were observed for signs of overt toxicity and death twice daily
Doses / concentrationsopen allclose all
Doses / Concentrations:
625 mg/kg
nominal conc.
prepared daily on day of treatment
Doses / Concentrations:
313 mg/kg
nominal conc.
prepared daily on day of treatment
Doses / Concentrations:
156 mg/kg
nominal conc.
No. of animals per sex per dose:
total 20 animals per dose group- 10 females and 10 males
total 20 vehicle only with 10 males and 10 females
Positive control(s):
A positve control group of 10 animals were dosed once at 50 mg/kg using cyclophosphamide and terminated 24 hours.

vehicle control- total 20 animals with 10 per sex ( vehicle: 0.2 % hydroxypropyl methylcellulose)


Tissues and cell types examined:
Bone marrow from the femurs of each animal used for smears. Cell types examined for the presence of micronuclei in Polychromatic (PCE) and normochromatic (NCE) erythrocytes.
Details of tissue and slide preparation:
Immediately following termination, bone marrow smears were prepared from sections of the femurs from each animal and stained with May-Grinwald/Giemsa.Stained bone marrow smears were coded and examined. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE- blue stained immature cells) per animal was scored. An additional 500 slides were read per each male mouse at high dose group at 24 and 48 hr post treatment to compensate for 2 male mice that died prior to bone marow collection.

Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining.

The frequency of polychromatic to normochromatic erythrocytes was calculated. Also the percent micronucleated PCEs were determined.
Evaluation criteria:
The criteria to determine a positive result is a dose-related statistically significant increase in micronucleated polychromatic erythrocytes occurring or if no dose related increase than at least a statistically significant increase in micronucleated polychromatic erythrocytes a for at least one dose level

A test substance that did not induce a dose-related or a statistically significant increase in micronucleated polychromatic erythrocytes in at least one dose group would be considered negative.
The data was analysed using Fischer's Exact Test

Results and discussion

Test results
no effects
Vehicle controls validity:
Positive controls validity:
Additional information on results:
clinical signs: all decreased activity and squinting shortly after dosing, also mid dose group experienced dyspnea and the high dose group dyspnea and ataxia with recovery except for squinting in high dose group mice.
All but 2 mice survived until termination. There were 2 mortalities in the male high dose group around 24 hrs after treatment.

Any other information on results incl. tables

The analytical assay reported that the concentrations ranged from 104.5 to 108.3% of intended and are within the 10% criteria.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test up to 625 mg/kg/day, a dose at which clinical signs and limited mortalities were seen. The test material was considered to be non-genotoxic under the conditions of the test.