In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

In a prevalidation study, a Standard Operating Procedure (SOP) for human and mouse in vitro tests was developed, for evaluating the potential haematotoxicity of xenobiotics in terms of their direct, adverse effects on the myeloid colony-forming unit (CFU-GM). Based on the adjustment of the mouse-derived maximum tolerated dose (MTD), a prediction model was set up to calculate the human MTD, and an international blind trial was designed to apply this model to the clinical neutropenia of 23 drugs including 17 antineoplastics. The model correctly predicted the human MTD for 20 drugs out of the 23 (87%). This high percentage of predictivity, and the reproducibility of the SOP testing, confirmed the scientific validation of this model, and suggested promising applications for developing and validating other in vitro methods for use in haematotoxicology.

A prevalidation study was established to develop two independent in vitro tests to quantify the potential haematotoxicity of xenobiotics from their direct adverse effects on clonal colony formation by a neutrophil progenitor called CFU-CM. In an initial phase of the study (Protocol Refinement), we assessed a standard operating procedure (SOP) for two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to both human (bone-marrow and umbilical cord blood) and mouse (bone-marrow) CFU-GM. In a second phase (Protocol Transfer), the SOP was transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. A further phase (Protocol Performance), was dedicated to the generation of a prediction model by testing a panel of six anticancer drugs (adriamycin, flavopiridol, morpholino-doxorubicin. pyrazoloacridine, taxol and topotecan). Our results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 90% of all tests attempted. The 90% inhibitory concentration values (IC90) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we propose that the formal Phase II validation study could be pursued by using the Test A SOPs for 14 additional xenobiotics that represent the spectrum of haematotoxic potential.

Das von der ECVAM “Task Force Prevalidation” 1994 entwickelte Konzept der Prävalidierung soll verhindern, daß neue toxikologische in vitro-Tests in Validierungsstudien unter blinden Bedingungen geprüft werden, bevor ihre Reproduzierbarkeit und Relevanz ausreichend geprüft wurde. Bei dem ECVAM-Prävalidierungskonzept unterscheidet man drei Phasen: Phase I: Überarbeitung und Standardisierung der Methode in einem erfahrenen Labor (Protocol Refinement), Phase II: Transfer der Methode in ein zweites Labor (Protocol Transfer) und Phase III: Ermittlung der Leistungsfähigkeiten und Grenzen der Methode unter blinden Bedingungen und unter Hinzunahme eines weiteren Labors (Protocol Performance). Die vorliegende Studie wurde im Rahmen des ECVAM-Projektes “Evaluation of the Prevalidation Process” durchgeführt, in dem von 1996-1998 die Praktikabilität des ECVAMPrävalidierungskonzeptes exemplarisch an fünf in vitro-Tests überprüft wurde.

To date, no standardized international guideline for the testing of chemicals for phototoxic potential has been accepted for regulatory purposes. In 1991, the European Commission (EC), represented initially by the Directorate General XI and later by ECVAM (the European Centre for the Validation of Alternative Methods) and COLIPA (the European Cosmetic, Toiletry and Perfumery Association), agreed to establish a joint EU/COLIPA programme on the development and validation of in vitro phototoxicity tests. The first phase (phase I, 1992–93) was designed as a prevalidation study, to identify in vitro test procedures and test protocols for a formal validation trial under blind conditions. In the second phase (phase II, 1994–95), the formal validation study, the most promising in vitro phototoxicity tests were validated with 30 carefully selected test chemicals in 11 laboratories in a blind trial. The 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT) was performed as a core test in nine laboratories, since it provided the best results in phase I of the study. The purpose of phase II was to confirm the reliability and relevance of the in vitro tests for predicting phototoxic effects and for identifying phototoxic chemicals. In phase II the phototoxic potential of test chemicals in the 3T3 NRU PT test was either assessed by determining the phototoxicity factor (PIF) by using a cut-off value of 5 as in phase I of the study, or by determining the mean photo effect (MPE) by using a cut-off value of 0.1, as recently proposed by Holzhütter (1997). Results obtained with both approaches in the 3T3 NRU PT test in phase II were reproducible in the nine laboratories, and the correlation between in vitro and in vivo data was very high. Therefore, ECVAM and COLIPA conclude from this formal validation trial under blind conditions that the 3T3 NRU PT test is a scientifically validated in vitro test which is ready to be considered for regulatory purposes for assessing the phototoxic potential of chemicals. A draft OECD Guideline for “In Vitro Phototoxicity Testing”, incorporating the standard protocol of the 3T3 NRU PT test, will be submitted to the OECD test guidelines programme in due course.