Communication of the three Rs within establishments: some problems and solutions. Report of a LASA Alternatives Section meeting. Laboratory Animal Science Association.

A meeting was organised by the Laboratory Animal Science Association (LASA) Alternatives Section to provide a forum in which ideas for strategies to raise awareness of the Three Rs could be discussed. One of the main objectives of the meeting was to identify effective strategies which could feasibly be implemented at establishments. In general, there are two main ways in which Three Rs information can be obtained and communicated. Individuals can be assigned to a role with specific responsibilities (for example, a named alternatives expert), or committees can be established, with a remit to find ways to reduce, refine and/or replace animal use (for example, an alternatives committee). The meeting involved invited speakers who are engaged in obtaining and communicating Three Rs information, and this report presents a summary of the advantages and limitations of various approaches that are being undertaken.

Prospects for reducing and refining the use of dogs in the regulatory toxicity testing of pharmaceuticals.

A workshop was held to critically discuss the need for a nonrodent species and the role of the dog in regulatory toxicity testing of pharmaceuticals; to discuss opportunities to reduce and refine the use of dogs in preclinical toxicology; and to identify a number of specific recommendations which could be feasibly achieved to move the process forward. To facilitate a preliminary evaluation of the contribution of dog studies to the risk assessment process, anonymised, unpublished data were provided from fully evaluated, repeat-dose toxicity studies in the rat and dog. Results of the International Life Sciences Institute (ILSI) Human Toxicity Project were also presented and discussed. Analysis of the data demonstrated that the dog can provide additional toxicity information, which, in some cases, was shown to be predictive for humans. Discussions indicated that there is potential for achieving a reduction in dog use and several possible approaches were identified. To further the progress of this initiative, there is a need to collate the results of pharmacology, toxicology, and clinical studies to address some of the proposed approaches. One of the outcomes of the workshop will be the establishment of a steering group to co-ordinate data collation for further analysis.

A Comparison of Three Cytotoxicity Endpoints in the Corneal HCE-T Model.

The prediction of ocular irritation potential from in vitro assays still presents a problem, despite a number of validation trials. A study with coded cosmetic formulations, for which historic in vivo data were available, has been conducted with a human corneal multi-layered model system. This corneal model, the HCE-T model, was developed by using HCE-T cells, a transfected human corneal epithelial cell line. The relative effectiveness of three endpoints that provide a measure of cytotoxicity in the HCE-T model was evaluated. Cell viability immediately after exposure to the test materials was determined by using the MTT and Alamar Blue™ (AB) assays, and, 24 hours later, by using the MTT, AB and lactate assays. Viability measurements with the MTT, AB and lactate assays gave similar dose-response curves at the 24-hour endpoint. One formulation (an anti-dandruff shampoo) caused a less severe drop in viability in assays conducted immediately after the exposure than at the 24-hour time-point. There was little deterioration in viability with the other test materials. The ranking of the test formulations on the basis of relative loss of viability and release of lactate resulted in the same order as for the Modified Maximum Average Draize Test Score. Comparison of the HCE-T model cytotoxicity assay results with historic in vitro data from two different cytotoxicity assays, conducted by using fibroblast monolayer cultures and the same materials, indicated that the multi-layered corneal model had a greater predictive ability. The results of a blind trial with the lactate assay in two laboratories indicated that the techniques required were transferable between laboratories. The lactate results were reproducible between laboratories, even when cultures derived from different passage human corneal cells were tested, provided that the passage number was below 20.

Differential cytotoxicity of Ifosfamide and its metabolites in renal epithelial cell cultures.

Ifosfamide (IF) chemotherapy is dependent on bioactivation by cytochrome P-450 and may be limited by concurrent nephrotoxicity. An in vitro approach utilizing the renal cell lines G401, MDCK Strains I and II, LLCPK1 and OK has been adopted to study nephrotoxic actions of IF and its known metabolites, 4-hydroperoxy-ifosfamide (4-OOH-IF, 4-OH-IF in solution), chloroacetaldehyde (CAA), isofosphoramide mustard (IF-mus) and acrolein using an MTT assay. IF is virtually non-toxic to the panel of renal cell lines used; prolonged incubation with ifosfamide led to a progressive reduction in cell MTT activity only in MDCKI (days 3, 4). 4-hydroxylation of IF was directly measured in MDCK microsomes; MDCKI microsomes gave an activity of 0.045nmol/min/mg, while in the MDCKII microsome preparation only 0.004nmol/min/mg was detected. The limited toxicity to IF observed in MDCKI may be explained in part by the ability of cell cytosol to activate IF by 4-hydroxylation. In contrast to IF, its metabolites applied extracellularly were toxic to the panel of renal cell lines used. This cytotoxicity varied within and between cell lines. In LLCPK1 cells after 48hr of exposure to IF metabolites, the toxicity as measured as a percentage of control MTT activity was: CAA (30 mum) 11+/-0.5%>4-OH-IF, (75 mum), 22.9+/-1.2%,>acrolein (75 mum), 42.0+/-2.9%>>IF (75 mum) 69.8+/-2.8%. The toxicity to IF-mus (exposure at 150 mum for 48hr) varied between cell lines, MTT activity measured as a percentage of controls gave: MDCKII 38.1+/-3.2>G401 41.6+/-1.7%>MDCKI 60.1+/-1.7% approximately LLCPK1 62.4+/-2.5>OK cells 92.0+/-5.4%. Whereas G401 and OK cells show marked sensitivity to exposure to acrolein (75 mum, 48hr, 1.7+/-0.2%, 14.2+/-0.6% of control MTT, respectively), MDCKII cells are relatively insensitive to acrolein toxicity (86.5+/-4.2% of control MTT). In contrast to the toxicity of acrolein, G401 cells are relatively insensitive to CAA (30 mum, 48hr exposure, 48.4+/-1.7% of control MTT), while MDCKII and OK cells are markedly sensitive (MTT 8.7+/-0.5, 3.3+/-0.4% of controls, respectively). Finally, the toxicity of 4-OH-IF (75 mum, 48hr exposure) varied considerably between cell lines, with MTT values as a percentage of control values of: G401 3.1+/-0.3,>LLCPK1, 22.9+/-1.2% approximately OK, 23.6+/-1.4,>MDCKI cells 43.6+/-0.8%.

Strategies for replacing animals in biomedical research.

In 1959, Russell and Burch published their recommendations for applying the Three Rs (reduction, refinement and replacement alternatives) to the use of animals in scientific experimentation. At this time, they could not have predicted the effect of this fundamental concept on academic and industrial animal experimentation and in biomedical education. Although more than 30 years have passed, their ideas are gaining respectability and wide acceptance. This article reviews the recent advances in the use of non-animal methods in biomedical research, and discusses the outlook for the further implementation of 'alternatives'.

Atrial natriuretic peptide in the eel, Anguilla anguilla L.: its cardiac distribution, receptors and actions on isolated branchial cells.

The presence of atrial natriuretic peptide (ANP) and the nature of its binding sites were studied in fresh-water (FW)- and seawater (SW)-adapted eels using a heterologous analogue, that of the rat (rANP). Rat ANP-like immunoreactivity was demonstrated in the cardiac atria and ventricles of both FW and SW eels, and electron-dense ANP-like granules were observed. The atria and ventricles of FW eels contained significantly more granules than those of SW animals and, in both types, the atria were more granular than the ventricles. Specific binding sites for rANP were demonstrated by displacement and uptake experiments using labelled rANP in dispersed eel branchial cell preparations, enriched in chloride cells. The concentration of rANP required to produce a 50% inhibition of binding in FW cells was significantly lower than that in SW cells. Scatchard analyses revealed the presence of two classes of binding site in SW eel branchial cells but only a single class of receptor in FW cells. The affinity of the FW receptor was not significantly different from that of the SW high affinity site. Rat ANP stimulated the production of cyclic GMP (cGMP) in a dose-dependent manner, and both basal and stimulated levels of cGMP were significantly greater in SW branchial cells. These studies suggest that ANP is involved in the adaptation of the euryhaline eel to differing environmental salinities; the levels of the peptide in the heart alter with changing salinity, and the nature of the receptors in the sodium chloride-transporting epithelium of the gill changes in response to the need either to eliminate or to absorb sodium chloride.