Anne-Marie van Zeller and Robert D. Combes
The relevance of the rodent bioassay for assessing human risk to carcinogens has long been questioned. This has prompted several regulatory authorities and the International Conference on Harmonisation (ICH) to discuss the need for studies in two rodent species. Currently, six alternative tests are being evaluated in an interlaboratory collaborative study being organised by the International Life Sciences Institute (ILSI). These tests include four transgenic carcinogenicity assays in mice (the c-Ha-ras, Tg.AC, p53+/– and XPA systems). These assays are discussed in this review, and it is concluded that, to date, the data suggest that none of these assays is appropriate for inclusion in a carcinogenicity testing strategy. It is suggested that more emphasis should be placed on developing replacement alternative assays which are capable of identifying and characterising carcinogens of human relevance, rather than focusing on tests which are likely to merely duplicate the results of the rodent chronic bioassay. In this respect, the outcome of studies using the Syrian Hamster Embryo cell transformation assay, also being evaluated as part of the ILSI programme, will be of great interest. Ultimately, it is expected that cell transformation systems based on human cells will provide useful data for predicting human hazard from carcinogen exposures, and efforts to develop such systems should be encouraged.
The Three Rs concept, which was developed by Russell & Burch in 1959, was implemented into the legal framework in the European Union (EU) for the protection of vertebrate animals used for experimental and other scientific purposes, when Directive 86/609/EEC was adopted in 1986. One focus of activity under this Directive is the use of animals and alternative methods in regulatory testing. To reduce or replace animal testing for regulatory purposes, non-animal tests must be independently validated to prove that they can provide information that is relevant and reliable for hazard prediction in relation to specific types of toxicity in vivo. At the end of the 1980s, no scientific concept existed for the formal validation of in vitro toxicity tests, so a small group of European and American scientists met to develop a set of principles for experimental validation, which were first adopted by ECVAM in Europe in 1995, and, after harmonisation with experts from the USA and Japan, accepted internationally by the OECD in 1996. ECVAM has directly funded a number of validation studies, and a major breakthrough in the year 2000 was the acceptance for regulatory purposes in the EU of cientifically validated in vitro toxicity tests for phototoxic potential and for skin corrosivity. These, and other examples which are discussed, confirm that the internationally harmonised ECVAM/ICCVAM/OECD validation concept is a practical and effective way of making possible the replacement of regulatory testing in animals.
The European Commission (EC) White Paper on a Strategy for a Future Chemicals Policy calls for the collection of adequate information about chemicals, in order to ensure their appropriate risk management. The White Paper proposes a stepwise and flexible approach to all chemicals produced in amounts above 1 tonne/year/manufacturer, including testing, if information cannot be provided by other means. The required information should be collected by the end of 2012. The EC services are currently preparing the drafts for the future chemicals legislation.
A Statistical Model to Allow the Phasing Out of the Animal Testing of Demineralised Bone Matrix Products
Samuel S. Murray, Elsa J. Brochmann, Judith O. Harker, Edward King, Ryan J. Lollis and Sameer A. Khaliq
Demineralised bone matrix (DBM) products are complex mixtures of proteins known to influence bone growth, turnover, and repair. They are used extensively in orthopaedic surgery, and are bioassayed in vivo prior to being used in clinical applications. Many factors contribute to the osteogenic potency of DBM, but the relative contributions of these factors, as well as the possibility of interactive effects, are not completely defined. The “gold standard” measure of the therapeutic value of DBM, the in vivo assay for ectopic bone formation, is costly, time-consuming, and involves the use of numerous animal subjects. We have measured the levels of five growth factors released by the collagenase digestion of DBM, and statistically related these levels with osteogenic potency as determined by a standard in vivo model, in order to determine which value or combination of values of growth factors best predict osteogenic activity. We conclude that the level of BMP-2 is the best single predictor of osteogenic potency, and that adding the values of other growth factors only minimally increases the predictive power of the BMP-2 measurement. A small, but significant, interactive effect between BMP-2 and BMP-7 was demonstrated. We present a statistical model based on growth factor (e.g. BMP-2) analysis that best predicts the in vivo assay score for DBM. This model allows the investigator to predict which lots of DBM are likely to exhibit in vivo bioactivity and which are not, thus reducing the need to conduct in vivo testing of insufficiently active lots of DBM. This model uses cut-point analysis to allow the user to assign an estimate of acceptable uncertainty with respect to the “gold standard” test. This procedure will significantly reduce the number of animal subjects used to test DBM products.