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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EEC, Method B10.
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

Constituent 1
Chemical structure
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:
(S)-2,5-dioxopyrrolidin-1-yl 2-(3-((2-isopropylthiazol-4-yl)methyl)-3-methylureido)-3-methylbutanoate

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
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
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.