A mixture of: bis(1S,2S,4S)-(1-benzyl-4-tert-butoxycarboxamido-2-hydroxy-5-phenyl)pentylammonium succinate; isopropyl alcohol

Genetic toxicity: in vivo

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:

October - Novemmber 4, 1996

Reliability:

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

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

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:

414-810-0

EC Name:

A mixture of: bis(1S,2S,4S)-(1-benzyl-4-tert-butoxycarboxamido-2-hydroxy-5-phenyl)pentylammonium succinate; isopropyl alcohol

Cas Number:

144163-85-9

Molecular formula:

Empirical formula: C25H35N2O5

IUPAC Name:

butanedioic acid; propan-2-ol; bis(tert-butyl N-[(4R)-5-amino-4-hydroxy-1,6-diphenylhexan-2-yl]carbamate)

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- 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

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
625 mg/kg
Basis:
nominal conc.
prepared daily on day of treatment

Doses / concentrations 2

Remarks:

Doses / Concentrations:
313 mg/kg
Basis:
nominal conc.
prepared daily on day of treatment

Doses / concentrations 3

Remarks:

Doses / Concentrations:
156 mg/kg
Basis:
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)

Examinations

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.

Statistics:

The data was analysed using Fischer's Exact Test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

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

Conclusions:

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.