Session E5: Neurotoxicity and Developmental Toxicity: Development, Validation, and Implementation of Alternatives and New Methods

Chairs: Sidney Green (USA) and Hiroshi Ono (Japan)

E5: The Ecvam International Validation Study on Three In Vitro Embryotoxicity Tests H. Spielmann, E. Genschow, G. Scholz, A. Seiler, N. Brown, A. Piersma, M. Brady, N. Clemann, H. Huuskonen, F. Paillard, S. Bremer, and K. Becker. Center for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET), 12277 Berlin, Germany. zebet@bgvv.de.

From 1996-2000, ZEBET has coordinated the ECVAM pre-validation and validation study of three in vitro embryotoxicity tests: (a) a test employing embryonic stem cell lines (EST); (b) the micro-mass test (MM test); and (c) the post-implementation rat whole-embryo culture assay (WEC test). The main objectives of the study were to assess the performance of the three selected in vitro tests to discriminate among non-, weak-, and strong-embryotoxic compounds. Phase I of the study (1997) was designed as a pre-validation phase, and phase II (1998-2000) involved a formal validation trial under blind conditions, using 20 coded test chemicals. Applying the prediction model (PM) PM of the EST in the definitive phase of the formal validation study, in 78% of the experiments, chemicals were classified correctly. For the MM and the WEC test, the PM's provided 71% and 80% correct classifications, respectively, and more importantly, a predictivity of 100% was obtained with strong embryotoxic chemicals in each of the three in vitro embryotoxicity tests.

E5: Developmental Neurotoxicity Test Guidelines
Susan Makris. Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC 20460, USA. makris.susan@epa.gov.

A guideline for the conduct of a developmental neurotoxicity study that evaluates the effects of an agent on the developing nervous system was finalized by the U.S. EPA in 1991. The use of this study for the characterization of potential hazards to infants and children has increased substantially since the passage of the Food Quality Protection Act (1996). In the typical guideline study, maternal rats are treated through critical periods of gestation and lactation; therefore, offspring are exposed to the test substance in utero and via the milk. In addition to the assessment of growth and survival in pups, neurobehavioral testing of offspring includes a functional observational battery, motor activity testing, auditory startle measures, and cognitive testing. Both qualitative and quantitative neuropathological evaluations are conducted. The EPA continues to consider refinements to the developmental neurotoxicity testing paradigm based upon specific information about the test chemical. Additionally, the EPA encourages the reduction of the number of animals used for DNT testing and/or the more efficient utilization of test animals by incorporating neurobehavioral and neuropathological endpoints into other study designs.

E5: Genetically Altered Mouse Models: Short-Term Alternatives to Two-year Carcinogenicity Testing
John E. French. Laboratory of Molecular Toxicology, NIEHS/NIH, Research Triangle Park, North Carolina 27709, USA. french@niehs.nih.gov.

Genetically altered mouse models (GAMM) for human cancers have been critical for investigation of gene expression and function and characterization of the associated phenotype. To identify potential human carcinogens and mechanisms of carcinogen-gene interactions and tumorigenesis, we have used both the FVB/N-Tg.AC (mutated v-Ha-ras) and the B6.129 (N5) or FVB/N tumor suppressor gene knockout p53 mouse models. Many carcinogenicity studies have also been conducted in the CB6F₁-RasH2 (human ras) mouse. Together, more than ninety carcinogens, including 14 known (IARC Class 1) human carcinogens, have been tested in at least one of these three models. Using 26-week exposure protocols with fewer animals than required for the conventional two-year cancer bioassay, the summary results indicate the potential for genetically altered mouse models to identify and, possibly, classify chemicals of potential risk to humans in short-term carcinogenicity experiments. By identifying significant outcomes and responses to chemical exposure in appropriate short-term mechanism-based genetically altered rodent models, strategies for prevention and intervention may be developed and employed to the benefit of public health, while reducing the number of animals required and the level of morbidity observed.

E5: Cell Transformation Assay Using Balb/C 3T3 Cells or Bhas 42 Cells for the Efficient Detection of Tumor Promoters
Makoto Umeda. Hatano Research Institute, Food and Drug Safety Center, 729-5, Ochiai, Hadano City, Kanagawa Pref. 257-0025, Japan. GZH04502@nifty.ne.jp.

We modified the two-stage transformation assay using Balb/c 3T3 cells. The method uses a medium consisting of DMEM/F12 supplemented with 2% FBS and ITES. Validation studies of this method were conducted, and the method was proved efficient and relevant for the detection of not only initiators, but also tumor promoters (reported by Tsuchiya et al. and Nishiyama et al, in this meeting). The fact that this method can detect promoters is especially important because of the presence of a considerable number of non-genotoxic carcinogens. The method was registered as the Technical Report of Japanese Standard Association. In order to develop a more efficient method to detect promoters, further trials using Bhas 42 cells were conducted. Bhas 42 cells are the v-Ha-ras-transfected Balb/c 3T3 cells; therefore, treatment with initiators can be omitted. When DMEM/F12 supplemented with 5% FBS is used, transformed foci efficiently appeared by treatment with promoting chemicals (reported by Ohmori et al in this meeting). Validation study of this method is ongoing. The use of DMEM/F12 instead of MEM is essential for obtaining efficient transformation. (Partly supported by a Grant-in-Aid from Japan Chemical Industry Association.)

E5: The Alkaline Comet Assay: A Proposed Alternative Test Method for the Detection of Genotoxic Carcinogens
Raymond Tice. Integrated Laboratory Systems, PO Box 13501, RTP, NC 27709, USA. rtice@ils-inc.com.

The Alkaline (pH > 13) Comet Assay is a sensitive, cost-effective method for the detection of genotoxic substances with possible carcinogenic activity. Because of the small number of cells needed per sample, its ease of application, and its ability to detect multiple classes of DNA damage in individual cells, in vitro and in vivo Comet assay test methods are increasingly used to identify substances with genotoxic activity. Currently, its primary regulatory use is to provide mechanistic data on substances suspected of being carcinogenic. In vitro Comet assay test methods can be used to screen substances for genotoxic activity prior to conducting animal studies. When animal studies are required, the Comet assay can be used to collect data from multiple tissues, thereby refining and reducing animal use. Within the international scientific community, there is increasing interest in replacing the in vivo hepatocyte unscheduled DNA Synthesis (UDS) assay, a required regulatory test method, with this more sensitive and predictive test method. However, before the Comet assay can be accepted for this purpose, defined protocols must be established, and laboratory validation studies must be conducted following established international guidelines.