[1,1'-Biphenyl]-4-pentanoic acid, .gamma.-[(3-carboxy-1-oxopropyl)amino]-.alpha.-methyl-, 4-ethyl ester, calcium salt (2:1), (.alpha.R,.gamma.S)-

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

calcium bis(3-{[(2S,4R)-1-{[1,1'-biphenyl]-4-yl}-5-ethoxy-4-methyl-5-oxopentan-2-yl]carbamoyl}propanoate)

EC Number:

935-847-3

Cas Number:

1369773-39-6

Molecular formula:

C48H56CaN2O10

IUPAC Name:

calcium bis(3-{[(2S,4R)-1-{[1,1'-biphenyl]-4-yl}-5-ethoxy-4-methyl-5-oxopentan-2-yl]carbamoyl}propanoate)

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.