Alternatives to Laboratory Animals

The lipophilicity, expressed as log P, has been estimated for 19 sesquiterpenoid unsaturated dialdehydes by the determination of the 1-octanol/water partition coefficient for 12 of the compounds, and the reversed-phase HPLC retention data for all the compounds. The log P values obtained varied between 0.5 and 3.02. The stability of 17 unsaturated dialdehydes in three common in vitro assay media at 37°C was investigated by HPLC analysis of samples dissolved in the media. All compounds investigated were essentially stable for up to 24 hours in a phosphate buffered saline, while several of them disappeared rapidly in media containing amino acids and fetal calf serum. This chemical instability must be considered when results from biological assays of unsaturated dialdehydes are compared.

Cells exposed to different types of stress usually exhibit morphological changes. These changes are often referred to as cell blebbing. The aim of this study was to compare a few different cytotoxins acting on freshly isolated rat hepatocytes through different molecular mechanisms. The toxins used were phalloidin and cytochalasin D (for cytoskeletal alterations), menadione (for oxidative stress), the Ca²⁺-ionophore A23187 (for intracellular Ca²⁺-elevation) and microcystin-LR (for protein phosphatase inhibition). Light and electron microscopy were used when comparing the morphological effects of the toxins. The morphological effects on microfilaments were studied by labelling F-actin with rhodamine-conjugated phalloidin. The results indicate that, although all the toxins caused cell blebbing, the blebs were morphologically markedly different and the redistribution of actin varied considerably, depending on the toxin used. The modification of the microfilamental architecture seemed to correlate with the morphological changes. Cell blebbing is inevitably a rather vague term for morphological alterations, which may look very different, depending on the subcellular cytotoxic mechanism involved.

The acute cytotoxicity of ten chemicals included in the MEIC (multicentre evaluation of in vitro cytotoxicity) list has already been estimated in various cell lines, e.g. primary rat hepatocytes, HeLa cells, human hepatoma cells, and 3T3 and mouse fibroma cells. In the present study, primary cultures of human lymphocytes, with or without an S9-mix (microsomes), were used to assay for in vitro toxicity. The cultured cells were initially treated with different concentrations of the respective drugs for 24 hours. Then cellular enzymatic activity was estimated using two assay systems, namely, measurement of cytosolic LDH activity and the mitochondrial (diaphorase) MTT test. Since the biological significance of the assay of LC50 values is dependent upon the slope of the dose-response curves, the results are expressed as LC20, LC50 and LC80 values in μM for the ten drugs.

A Spearman rank correlation analysis revealed a significant correlation between the results from the two assays in both cell culture systems (r=1.00 and r=0.99). The LC50 values found in the lymphocyte cultures, both with and without an S9-mix, correlated well with the results previously found using primary cultures of hepatocytes and HeLa cells.

Mixed glial cells in primary cultures were exposed to amitriptyline (AT) and diazepam (Dia). Morphological observations during 17–18 days of exposure, revealed that 10μM and 100μM were critical concentrations for AT and Dia, respectively. Exposure to concentrations above the critical concentrations led to culture death. Concentrations below 10μM AT and 100μM Dia stimulated the proliferation of glial cells. The microglia cells were stimulated to proliferate and individual cells to hypertrophy. On the other hand, in Dia-treated cultures, the development of microglia cells did not differ from that in control cultures. Both drugs stimulated the development of oligodendroglia cells (OG). With decreasing concentrations of the drugs, the number of OG increased and the enzymes 2'3’-cyclic nucleotide 3’-phosphodiesterase (CNP) and acid phosphatase (AcP), were elevated in inverse proportion to the concentration of drugs.

Water-soluble amphiphiles may, above a certain concentration, form colloidal aggregates (micelles or liquid crystals). If such aggregates are formed, there may be a competition between the micelles or the liquid crystals and the biological target for the monomelic form of the amphiphile. This may reduce the number of monomers available for the biological target, which in turn may be reflected in concentration-dependent processes. Therefore, caution should be taken when examining dose-response with soluble amphiphiles forming micelles at low concentrations.

The effects of acrylamide on CO₂ production from [¹⁴C]-labelled glucose, pyruvate and glutamine have been studied in a mouse neuroblastoma cell line C1300, clone N1E115. A rotation metabolism chamber, permitting closed incubation of monolayer, anchorage-dependent cell cultures under good physiological conditions, was developed for making the determinations. The cells were exposed to acrylamide (0.35mM) for 14 days. The total amunt of CO₂ produced from glucose and pyruvate was increased by exposure to acrylamide, whereas a slight inhibition was found in the production from glutamine. The production of lactate remained unchanged. Comparison of these results with other data obtained in our laboratory leads us to conclude that the method described is relevant for the determination of energy metabolic processes through the quantification of CO₂ production. Furthermore, we assume that acrylamide causes an increased demand for energy in the cells and that this demand is met by the cells through the increased oxidative phosphorylation of glucose.

The aim of the present study was to investigate the suitability of bovine spermatozoa cryopreserved in a “defined” medium as an in vitro model for the assessment of the cytotoxic potential of chemicals. The endpoints used for this purpose were motion activity and cellular ATP content. The evaluation of properties of cryopreserved sperm shortly after thawing and at the end of a one-hour incubation period, shows that the cryoprotective medium developed is able to provide suitable cellular material for cytotoxicity tests. Results from experiments employing substances with known modes of action are presented, and suggest that cryopreserved sperm can be used as successfully as native sperm in cytotoxicity tests.

An in vitro test protocol is reported, which, using primary cultured rat hepatocytes, allows for the screening of xenobiotic effects on biotransformation as well as on basal cellular functions. O-Deethylation of 7-ethoxycoumarin (7-EC) and subsequent conjugation of the metabolite 7-hydroxycoumarin (7-HC) with sulphate or glucuronic acid are determined, as representative parameters for the hepatic biotransformation. Cell viability is examined by measuring cellular ATP content and leakage of lactate dehydrogenase. With respect to immediate and delayed effects on biotransformation reactions, the standard test protocol includes exposure to xenobiotics for 1, 24 and 48 hours. Different response patterns could be demonstrated for the solvents dimethylformamide (DMF) and dimethylsulphoxide (DMSO), and the chlorinated phenols, pentachlorophenol (PCP) and hexachlorophene (HCP), which are known to uncouple mitochondrial respiration. Short-term incubation with the solvents resulted in decreased 7-EC-O-deethylation without signs of cytotoxicity. PCP and HCP inhibited 7-EC-O-deethylation and 7-HC-conjugation, affecting sulphate and glucuronide formation differently. 24-hour exposures to PCP and HCP resulted in decreased 7-ethoxycoumarin-O-deethylase activity, which correlated with diminished cell viability, while DMSO and DMF enhanced 7-EC-O-deethylation at sub-cytotoxic concentrations. After exposure for 48 hours to the solvents, enzyme induction was even more pronounced.

The spindle-inhibiting and aneuploidy-inducing potency of in vitro exposure of PHA-stimulated human lymphocytes to organotin compounds has been studied indirectly by quantitation of chromosomal contraction and directly by determination of the frequency of aneuploidy by chromosome counting. The effects of trimethyltin chloride (TMT), dimethyltin chloride (DMT), tributyltin chloride (TBT), dibutyltin chloride (DBT), triphenyltin chloride (TPhT), and diphenyltin chloride (DPhT) on chromosal contraction were studied at concentrations of 10^-3-10 ⁹M, by measurement of the average length of chromosome 1 from asynchronous cultures of human peripheral lymphocytes. TMT, TBT, TPhT and DPhT appear to be very strong inducers of chromosomal supercontraction, indicating that these compounds are possible spindle inhibitors, while DMT and DBT seem to be ineffective. TMT, TBT, TPhT and DPhT gave rise to a little more than 100% increase in the number of hyperdiploid cells, all the increases being statistically significant except for that induced by TBT, indicating that organotin compounds are capable of inducing aneuploidy, probably by affecting spindle function.

Primary astroglial cell cultures from neonatal rat cerebral cortex revealed morphological changes when incubated in HgCl₂ at various concentrations. After incubation for 30 minutes with 5 x 10^-6 HgCl₂, most cells changed their form from a flat to a more rounded shape and extended processes. After longer incubations (>60 minutes) or at higher concentrations (≥10^-5M), vacuoles appeared in many cells. [³H]-Thymidine incorporation into DNA was slightly increased after acute incubation in 10^-9M HgCl₂, while decreases were obtained after incubation in the μM range. The results are important due to the functions of astroglia during development and in the mature nervous system.

Tests, performed over a ten-year period, on 653 polymers intended for use in extracorporeal renal replacement therapy, were evaluated. The test battery used included animal tests, in vitro tests and chemical tests. Some tests were found to have a very low sensitivity. Thus, acute systemic toxicity testing in mice with sodium chloride, ethanol or paraffin oil as extractants was performed on a total of 806 occasions. Only two of these resulted in a “fail” decision. The final outcome of the tests for the majority of materials could be predicted by the results of the UV absorption, chloride, inhibition of cell growth, and tin tests. Of the materials that passed these four tests, less than 2% were not approved on the basis of the whole test battery. The experiments show that only a limited number of tests have to be performed when testing polymers intended for use in extracorporeal renal replacement therapy.

In recent years, conventional toxicity testing in animals has been reinforced by in vitro methods. As a result, toxicity testing in some sectors has become more effective and at the same time more ethical. This trend is probably only at its beginning, as many of the newly-developed methods have not yet won general acceptance as a basis for the large-scale replacement and reduction of animal experimentation. What limits the wider use of these methods is validation, i.e. the evaluation of their reliability and relevance. The present paper is a short review of the validation efforts made hitherto, including projects being planned and under discussion. Our own MEIC approach is compared with other strategies. Finally, our opinion on the effectiveness of one large consensus project relative to several different smaller validation programmes is expressed — we advocate the latter strategy, because it will save time and reduce costs.

In order to provide for greater assurance of the safety of cosmetic ingredients and products manufactured and/or marketed within the European Economic Community (EEC), without a significant increase in the need for animal testing, it is proposed that a Cosmetic Safety Review Committee (CSRC) be appointed to replace the Scientific Committee on Cosmetology (SCO, which currently advises the Commission of the European Communities (CEC). It is further proposed that a list of ingredients already in use be established, and that these ingredients then be ranked in order of need for safety review, on the basis of scale of use (tonnage), frequency of use (formulations), biological activity, etc. Proposals are also made for product safety assessment, and for the presentation of ingredient and product safety reviews to the CEC and the Member States of the EEC, via the CSRC.

The effects of chromate, vanadate, nickel, manganese, copper and barium, both separately and in combination with high pressure helium gas, have been studied in an epithelial-like cell line — Chinese hamster ovary (CHO) cells, using inhibition of cell growth as the cytotoxic endpoint.

High pressure (175 bar) by itself reduced the net growth of CHO cells to 71.7 ± 5.5% (mean ± SD) of normobaric control growth. The combined effects of high pressure and metal salts were additive for the lowest concentrations of chromate, copper, manganese and barium, more than additive for the lowest concentration of nickel, and less than additive for vanadate and the highest concentrations of chromate, copper, manganese and barium.

A ranking of the normobaric growth inhibition of the metal salts, estimated as the concentration giving 50% inhibition, was Cr>V>Mn>Ni>Cu>Ba. The growth inhibition of barium was less than 50% at a concentration of ImM BaCl₂. The combined exposure to high pressure and the metal salt did not change the toxicity ranking.

Details of the major pathways of metabolism have been elucidated, in large measure through work on experimental animals. It is unlikely that they differ qualitatively between mammalian species. The more important challenge facing researchers today is that of metabolic regulation, and of the integrated control of metabolic pathways in the whole organism. These aspects may well differ quantitatively or even qualitatively between species. In this review, methods for studying metabolism and metabolic regulation in humans are described. There are both scientific and practical advantages to performing studies of metabolic regulation in humans. The scientific advantages are clear from some fundamental differences in metabolic regulation between rats and humans, such as the importance of de novo lipogenesis to the deposition of body fat, and the metabolism of atherogenic triacylglycerol-rich lipoproteins. The practical advantages result mainly from the size of a human compared with that of most laboratory animals, enabling large blood samples to be obtained, and several measurement techniques to be applied at one time. Reasons for the persistence of animal experimentation as the norm, rather than the exception, among life science researchers are discussed.

We have evaluated a new in vitro opacitometer method as an alternative to the in vivo Draize test for ocular irritancy. Several concentrations of timolol maleate (timolol) with or without 0.005% benzalkonium chloride were applied to porcine isolated corneas which were either intact or with the epithelium, endothelium, or both epithelium and endothelium removed. Corneal opacities were measured using an opacitometer. In general, timolol with benzalkonium chloride caused a greater degree of opacity to develop in the cornea than did timolol alone. At the lower concentrations of timolol, the increased opacity probably represented additive effects of the two compounds. However, at the highest concentration of timolol (5 x 10 ²M), there was an enhanced opacification in the presence of benzalkonium chloride, which may have been due to an increase in penetration, particularly through the epithelium. Timolol caused a greater degree of opacity to develop in the isolated intact porcine corneas when the drug was applied to the endothelial surface, than when applied to the epithelial surface or to both the epithelial and endothelial surfaces. However, timolol with benzalkonium chloride caused a greater degree of opacity in the intact cornea, when the drug was applied to both surfaces than when it was applied only to the epithelial or the endothelial surface.

Computers are now used routinely as tools in pharmacology, particularly in the areas of teaching, data processing and collection, information retrieval and literature searching, and in molecular modelling and drug design. Their use in these areas has enhanced research activity and has extended and increased the availability of new teaching methods. Here, their impact on the use of animals in both teaching and research is discussed. It is concluded that computers may have some potential to reduce animal experimentation in the medium to long term, but their current use as alternatives to animals has made only a marginal impact on the total number of animals utilised for experimental purposes.