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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 27 - June 19th, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Done under GLP and OECD Methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Corning Hazleton protocol 449PCO, edition 1, modified for Abbott Labs

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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)

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes

Details on mammalian cell type (if applicable):

Human venous blood

Metabolic activation:

with and without

Metabolic activation system:

Exogenous metabolic activation

Test concentrations with justification for top dose:

Target dose 2.5 mg/ml; dosing volume of 1 % (10 ul/ml). In DMSO.

Concentration range in the initial study: 2.95 ... 1500 µg/ml. (1500 ug/ml based on solubility) with and without metabolic activation in a 24 hr assay.

Reduction in mitotic index of 38% at 5.90 ug/ml; 58% at 11.8 ug/ml, 96% at 23.5 ug/ml and 100 % at 47.0 ug/ml dosed for 24 hours without metabolic activation as compared to vehicle controls.
Reduction in mitotic index of 15% at 23.5 ug/ml; 68% at 94 ug/ml, and 100 % at 188 ug/ml dosed for 24 hours with metabolic activation as compared to vehicle controls.

Chromosomal aberrations were analyzed from the cultures treated for 24 hrs, (without metabolic activation): 2.95, 5.90 and 11.8 µg/ml and harvested 24 and 48 hrs after treatment.
Chromosomal aberrations were analyzed from the cultures treated for 24 hrs, (with metabolic activation): 23.5, 47.0 and 94 µg/ml and harvested 24 and 48 hrs after treatment.
Higher concentration proved extremely toxic.

Concentrations in confirmatory test dosed for 24 and 48 hrs (without metabolic activation): 3- 18 µg/ml.
Concentrations in confirmatory test dosed for 24 and 48 hrs (with metabolic activation): 10-100 µg/ml.

Chromosomal aberrations were analyzed from the cultures treated with 3, 6, and 12 µg/ml from 24 hr without metabolic activation.
Chromosomal aberrations were analyzed from the cultures treated with 3, 6, 12 and 18 µg/ml from 48 hr without metabolic activation.

Chromosomal aberrations were analyzed from the cultures treated with 10-70 µg/ml from 24 hr with metabolic activation.
Chromosomal aberrations were analyzed from the cultures treated with 20-70 µg/ml from 48 hr with metabolic activation.
Higher concentration proved extremely toxic.

Vehicle / solvent:

vehicle : Dimethylsulfoxide

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

initial experiment:
Exposure period (with metabolic activation): 24 hrs
Exposure period (without metabolic activation): 24 hrs
harvested at 24 hrs after treatment

confirmatory experiment:
Exposure period (with metabolic activation): 24 and 48 hrs
Exposure period (without metabolic activation): 24 and 48 hrs
harvested at 24 hrs or 48 hrs after treatment

Duplicate cultures were treated and analyzed for each drug concentration condition.

at 2 hours before harvest each of the cultures were treated with colcemid solution ( 0.1µg/ml) to block cells at the metaphase stage of mitosis.

cultured medium-for each culture heparinsed whole blood was added to culture medium containing RPMI 1640 supplemented with 15% fetal bovine serum, 1% photohaemogglutinin, 1% penicillin streptomycin and 1% L-glutamine. Total culture volume: 10ml. The culture was incubated on a slope at 37°C in a humidified atmosphere at 5% CO2/95% air for 48 hours.

Evaluation criteria:

100 cells from each replicate culture at at least 3 concentrations and the negative and vehicle (solvent) control cultures were analyzed for the different types of chromosomal aberration. At least 25 cells were analyzed for chromosomal aberrations from the positive control cultures.
Mitotic index was assessed by analyzing the number of mitotic cells in 1,000 cells and the ratio was expressed as a ratio of the mitotic cells.

Statistics:

linear trend and Fisher's Exact test for multiple comparisons treated to vehicle controls at p<0.01 significance.

Results and discussion

Test results

Test results 1

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant increase in cells with chromosomal aberrations was observed at the concentrations analyzed with and without metabolic activation except for at a single toxic dose level from the extended harvest time (48 hour) with S9 activation.

vehicle and positive controls were acceptable and valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
ambiguous with metabolic activation at single toxic dose level at 48 hrs

No significant increase in cells with chromosomal aberrations was observed at the concentrations analyzed without metabolic activation and with metabolic activation at the 24 hr assay. A significant increase in cells with chromosomal aberrations were observed in cultures treated with 70.0 ug/ml from the 48 hrs assay, a single toxic dose level. No increase were observed in polyploid or endoreduplicated cells in the 48 hr with and without metabolic activation. Test substance was considered negative for inducing chromosomal aberrations without metabolic activation and results are equivocal in the presence of metabolic activation due to a positive result in a single toxic dose level from the extended assay at 48 hours.