A short description of the history of the 3Rs concept is given, which was developed as the scientific concept to refine, reduce and replace animal experiments by Russel and Burch more than 40 years ago. In addition, the legal framework in Europe for developing alternatives to animal experiments is given and the current status of in vitro systems in pharmacology and toxicology is described including an update on metabolising systems. The decrease in experimental animal numbers during the past decade in Europe is illustrated by the situation in Germany and the contribution of international harmonisation of test guidelines on reducing animal numbers in regulatory testing is described. A review of the development of the principles of experimental validation is given and the 3T3 NRU in vitro phototoxicity test is used as an example for a successful validation study, which led to the acceptance of the first in vitro toxicity test for regulatory purposes by the OECD. In addition, a promising QSAR approach to identify the skin irritation potential of chemicals is described, which was developed at the BfR and is proposed for regulatory acceptance after critical evaluation by the European Commission. Finally, the currently accepted alternative methods for standardisation and safety testing of drugs, biologicals and medical devices are summarised.
DNA double strand breaks are the most relevant damage related to the detrimental effects of DNA damaging agents. Previous studies have established various biochemical methods to measure DNA double strand breaks in mammalian cells, however, the application of these methods is limited to the experiments using higher doses of DNA damaging agents. Recently, it has been shown that DNA damaging checkpoint pathways are activated by phosphorylation of the checkpoint factors, such as ataxia-telangiectasia mutated (ATM), ATM-Rad3 related (ATR), CHK1/2, and p53 proteins. For example, ATM is activated through its autophosphorylation at serine 1981 (S1981) at the sites of DNA double strand breaks. Because the response is highly amplified, and when visualized with an antibody recognizing phosphorylated ATM at S1981, phosphorylated ATM appears as nuclear foci. Our group has examined the formation of phosphorylated ATM foci in exponentially growing normal human diploid cells exposed to ionizing radiation, and the induction of foci showed a linear dose-relationship with doses ranging from 10 mGy to 1 Gy. The number of phosphorylated ATM foci is well correlated with the estimated number of DNA double strand breaks, providing an unique and sensitive tool for the measure-ment of DNA double strand breaks at the physiological level in situ.
The objective of this study was to demonstrate that the chick embryo with hyperthyroidism induced by treatment with L-thyroxin (TRX) was a feasible model as an alternative to animal model and to examine the pharmacological effects of digoxin using this model. When 0.5 or 1.0µg/egg of TRX was injected into the eggs, the 16th day-chick embryos showed some characteristic changes including an increased heart rate (tachycardia), an increased relative heart weight (cardiac hypertrophy) and relative thyroid weight (thyroid hypertrophy), and an increased concentration of plasma T3 and T4. These findings suggest that TRX-treated chick embryos may be a model of the heart diseases associated with hyperthyroidism. When 50µg/egg of digoxin was injected into the TRX-treated eggs, negative chronic action reduced. This fact suggests that digoxin decreased the sensitivity to TRX in the heart of chick embryos with hyperthyroidism as well as in dog and man. In conclusion, the TRX-treated chick embryos may prove to be an alternative to animal experiments to predict unexpected effects of cardiovascular drugs.
The toxic interactions between miconazole and disopyramide were studied in chick embryos. Chick embryos have been widely used in pharmacologic and toxicologic experiments for evaluating drug action. Fertilized eggs of White Leghorns were incubated and investigated. Miconazole 1 mg/egg, 5 mg/egg, 10 mg/egg alone or disopyramide 0.3 mg/egg alone was injected into the air sac of each fertilized egg. Miconazole 1 mg/egg with disopyramide 0.3 mg/egg was injected into the air sac of each fertilized egg. Electrocardiograms (ECGs) were recorded after the drug injection, and heart rate was determined from ECG wave cycles. Changes in heart rate were expressed as mean-percent changes of the drug-treated groups to the matched control. After the administration of miconazole 1 mg/egg alone, the heart rate did not differ compared with that of the controls. However, the heart rate was significantly decreased with the administration of miconazole 5 mg/egg and 10 mg/egg. The heart rate was also significantly decreased by the administration of miconazole 1 mg/egg together with disopyramide 0.3 mg/egg. In addition, an arrhythmia was produced by miconazole and disopyramide. These findings in-dicate that the interaction between miconazole and disopyramide has a marked influence on the heart rate in chick embryos.
A hypoxanthine phosphoribosyltransferase (HPRT)-locus mutation assay system in mouse ES cells was established and several typical mutagens/carcinogens were examined. The results were as follows: 1) the frequency of spontaneous mutation was 1 per 106 cells, 2) direct-acting mutagens: N-methyl-N'- nitro-N-nitrosoguanidine (0.5-2µg/ml) and ethyl methane sulfonate (200-600µg/ml) respectively induced 30-150 and 6-70 mutants per 106 cells, 3) mutagens activated by ES cells: 4 nitroquino-line-1-oxide (0.5, 1µM) induced 20-50 mutants per 106 cells; mitomycinC, even at a highly toxic dose (0.25µg/ml), did not induce mutation, 4) ES cells seemed not to have metabolic activation system for polycyclic aromatic hydrocarbons (PAHs) but 7,12-dimethylbenz(a)anthracene(0.5-2µM) induced 20-80 mutants per 106 cells when added to ES cells on a feeder layer. Thus the frequencies of ES cell-mutation (spontaneous and induced) were approximately one tenth those of V79 cells, and 5) the degree of metabolic cooperation of the ES cells was almost the same as that reported for V79 cells. Thus feeder cells (unless 6TG-resistant feeder cells) should not be used and the cell number should be under 5 x 105 cells per 10 cm dish when 6TG selection is started.
To estimate the developmental toxicity of environmental chemicals on the formation of primordial germ cells (PGCs), Xenopus laevis embryos were exposed to caffeine at 200 mg/l during the migratory stage of presumptive PGCs. The PGC-containing region of the larvae at stage 46, which corresponds to genital ridge, was illuminated by cold light using a halogen lamp and photographed using a digital camera under a dissecting microscope. The length along the cephalocaudal axis and area of the PGC-containing region were measured using image-measuring software. The length and area of the PGC-containing region as well as its relative length compared to the 100-µn;m body length of caffeine-exposed larvae were significantly shorter and smaller, respectively, than those of the control. Though further studies concerning the effect of caffeine on PGC formation are needed, these findings suggest that the Xenopus embryo and larva system examined in this study is useful as a simple, rapid and low-cost initial method for the estimation of the developmental toxicity of environmental chemicals on PGC formation.
Several proposed modified local lymph node assays (LLNAs) use a non-RI method, but such methods tend to yield lower stimulation indices (SIs) than those obtained with the standard LLNA when used to assess moderate sensitizers. To address this issue, we propose a modified LLNA in which mice are exposed to the test material four times, instead of three times, and adenosine triphosphate (ATP) content is used as a measure of proliferation of lymph node cells (LNCs). The chemicals tested were 2.4-dinitro-chlorobenzene (DNCB), eugenol, and a-hexyl cinnamic aldehyde (HCA), which are classified as strong-to-moderate sensitizers in the LLNA. Methyl salicylate (MS) was used as an example of a non-sensitizer. Using an SI of 3 as a cut-off, as recommended in the standard LLNA, DNCB, eugenol and HCA were classified as positive, while MS was classified as negative using the ATP-based non-RI method. Furthermore, there were no marked differences in SI levels for 10% eugenol and 10% HCA (a moderate sensitizer) between the non-RI method and the standard LLNA. Thus, it is reasonable to con-clude that the non-RI method proposed in the present study could be a valid alternative to the standard LLNA.