Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
Cultured peripheral human lymphocytes were used as test system.
Species / strain
Species / strain / cell type:
lymphocytes: human peripheral lymphocytes
Details on mammalian cell type (if applicable):
human peripheral lymphocytes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes S9
Test concentrations with justification for top dose:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. AHU377 was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
The highest tested concentration was determined by the solubility of AHU377 in the culture medium at the 3 h exposure time. At the 24 h exposure time, AHU377 was tested beyond the limit of solubility to obtain adequate toxicity data.
In the dose range finding test blood cultures were treated with 3, 10, 33, 100 and 333 µg
AHU377/ml culture medium with and without S9-mix.
Vehicle / solvent:
dimethyl sulfoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking male volunteers.
Blood samples

age 28, AGT = 14.5 h (Nov. 2003)
age 38, AGT = 17.0 h (Nov. 2003); without S9-mix age 25, AGT = 16.5 h (Feb. 2004); with S9-mix age 37, AGT = 14.2 h (Nov. 2003)

Blood samples were taken from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
Culture medium
Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L­ glutamine (2 mM) (Merck), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (lnvitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, the Netherlands).
Lymphocyte cultures
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Murex, Dartford, England) was added.
Rationale for test conditions:
The study procedures described in this report were based on the most recent OECD, EEC and ICH guidelines.
Evaluation criteria:
A test item was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test item was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Applicant's summary and conclusion

Conclusions:
AHU377 is not clastogenic in human lymphocytes under the experimental conditions described in this report.