title: Miniaturization of cytotoxicity tests for concentration range-finding studies prior to conducting the pH 6.7 Syrian hamster embryo cell-transformation assay.
abstract: The Syrian hamster embryo (SHE) cell-transformation assay (SHE assay) is a promising alternative method to animal testing for the identification of potential carcinogens in vitro. Prior to conducting the SHE assay the appropriate concentration range for each test chemical must be established, with a maximum concentration causing approximately 50% cytotoxicity. Concentration range-finding is done in separate experiments, which are similar to the final SHE assay but with less replicates and more concentrations. Here we present an alternative for the cytotoxicity testing by miniaturization of the test procedure by use of 24-well plates and surpluses from feeder-cell preparations as target cells. In addition, we integrated the photometry-based neutral red (NR) assay. For validation of the assay, incubations with dimethyl sulf-oxide, p-phenylenediamine-2HCl, aniline, o-toluidine-HCl, 2,4-diaminotoluene, and 2-naphthylamine were carried out in the miniaturized approach and compared with the standard procedure in terms of calculating the relative plating efficiencies (RPEs). To directly compare both methods, concentrations that produced 50% cytotoxicity (IC50) were calculated. Excellent associations were observed between the number of colonies and NR uptake. For all test substances a concentration-dependent, concomitant decrease of NR uptake in the miniaturized approach and RPEs in the standard test was observed after a 7-day incubation. The results from both test setups showed a comparable order of magnitude and the IC50 values differed by a factor <2 (1.4-1.9), depending on the substance in question. Overall, the miniaturized approach should be considered an improved alternative for cytotoxicity testing in the SHE assay, as it saves valuable SHE cells and speeds-up the time, to obtain test results more rapidly.
authors.affiliation: Institut für Prävention und Arbeitsmedizin der Deutschen Gesetzlichen Unfallversicherung - Institut der Ruhr-Universität Bochum IPA, Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany. ploettner@ipa-dguv.de
authors.forename: Sabine
authors.initials: S
authors.lastname: Plöttner
authors.forename: Heiko U
authors.initials: HU
authors.lastname: Käfferlein
authors.forename: Thomas
authors.initials: T
authors.lastname: Brüning
mesh_terms: D015081:2-Naphthylamine / Q000633:toxicity; D000814:Aniline Compounds / Q000633:toxicity; D000818:Animals; D015197:Carcinogenicity Tests / Q000295:instrumentation / Q000379:methods*; D002273:Carcinogens / Q000633:toxicity; D018929:Cell Culture Techniques / Q000295:instrumentation; D002471:Cell Transformation, Neoplastic / Q000187:drug effects*; D002478:Cells, Cultured / Q000187:drug effects; D003114:Colony-Forming Units Assay; D004396:Coloring Agents; D006224:Cricetinae; D004121:Dimethyl Sulfoxide / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D005260:Female; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D008647:Mesocricetus / Q000196:embryology; D008904:Miniaturization; D009499:Neutral Red; D010655:Phenylenediamines / Q000633:toxicity; D010783:Photometry; D011247:Pregnancy; D015203:Reproducibility of Results; D014052:Toluidines / Q000633:toxicity
chemical_list: D000814:Aniline Compounds; D002273:Carcinogens; D004396:Coloring Agents; D010655:Phenylenediamines; D014052:Toluidines; D009499:Neutral Red; C023622:2-toluidine; D015081:2-Naphthylamine; C010914:2,4-diaminotoluene; C023650:aniline; C029728:4-phenylenediamine; D004121:Dimethyl Sulfoxide
keywords: Aromatic amines; Cytotoxicity; Neutral red uptake; Relative plating efficiency; SHE assay