2,5-dioxopyrrolidin-1-yl N-{[methyl[[2-(1-methylethyl)-4-thiazolyl]methyl]amino]carbonyl}-.sc.l.sc.-valinate

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:

May- September 1996

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:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EEC, Method B10.

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:

-

EC Number:

424-660-8

EC Name:

-

Cas Number:

224631-15-6

Molecular formula:

Hill formula: C18H26N4O5S CAS formula: C18H26N4O5S

IUPAC Name:

2,5-dioxopyrrolidin-1-yl (2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanoate

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes

Metabolic activation system:

Phenobarbital/Beta-Naphthoflavone induced rat-liver S9.

Test concentrations with justification for top dose:

Concentration range in the initial assay was 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000 and 5000 mM µg/ml.(with and metabolic activation).
Concentration range in the confirmatory assay (with and metabolic activation): 1, 2.5, 5, 10, 50, 100 and 150 mM µg/ml


cultured medium

for each culture heparinsed whole blood was added to culture medium containing a mutagen (photohaemogglutinin) and incubated at 37C in a humidified atmosphere at 5% CO2/95% air for 48 hours
dose levels for positive controls
without S9 mix- mitomycin C 0.125 ug/ml ( 3 hour of treatment or 2 ug/ml ( continuous treatment)
with S9, cyclophosphamide: 12.5 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

Initial assay:
Exposure period (with metabolic activation): 24 hr
Exposure period (without metabolic activation): 24 hr

Confirmatory assay:
Exposure period (with metabolic activation): 24 and 48 hrs
Exposure period (without metabolic activation): 24 and 48 hrs

Before harvest each cultured treated with colcemid solution ( 10 mg/ml) to block cells at the metaphase stage of mitosis.

Evaluation criteria:

Reproducible and statistically significant increase in frequency of cells with structural chromosome aberrations for at least one dose level and for 1 of 2 harvest times was considered as positive results.

Cytotoxicity evaluated based on mitotic index which is number cells with mitosis indicating mitotic inhibition. 1000 cells with mitosis were evaluated with no blind scoring.

analysis of 200 metaphases/dose-level with 44 to 46 chromosome were made with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analyst when at least 10% with structural chromosome aberrations were observed. Blind scoring was done.

Statistics:

each test and each harvest time- comparison of treatment verses vehicle control using Fischer Exact Test (2 tailed) and p<0.01.

mean aberrations per cells, number of cells with aberrations and any evidence for increasing amount of damage with increasing dose.

Results and discussion

Test results

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

Additional information on results:

Observations:
Analytical analysis completed:
intial assay : 90.4% and 100.5% ( acceptable)
confirmatory assay: 70.4% to 98.7% ( lower assay results in lower concentrations but did not impact the results)

vehicle and postive controls were acceptable and valid

Remarks on result:

other: other: initial assay

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
positive with metabolic activation

The test material did not induce a statistically significant dose-related increase in the frequency of cells with chromosome aberrations in cultured human lymphocytes in the presence and absence of metabolic activation.