In vitro methods

In 1959, a book titled The Principles of Humane Experimental Technique was published, which introduced the concept of the 3Rs (Russell and Burch 1959). The 3Rs stand for reduction, refinement, and replacement. 

The authors, William Russell and Rex Burch (a zoologist and a microbiologist, respectively) recommended the reduction of the number of animals used in experiments to the minimum number required to obtain statistically relevant data, the refinement of procedures to minimize pain and distress in experimental animals and provide for their well-being based on their behavioral needs, and the replacement of whole, living animals with in vitro models like tissue and cell cultures when possible. 

The Johns Hopkins Center for Alternatives to Animal Testing (CAAT) maintains the alternatives to animal testing Web site (Altweb 2007), where the full text of Russell and Burch s book is freely available for download. 

The 3R concept lies behind efforts to improve ethical standards for the use of experimental animals throughout the scientific community, including toxicity testing. A number of in vitro methods for genetic toxicology testing have been established as guideline methods for many years, e.g., the bacterial reverse mutation test, more popularly known as the Ames test. 

Isolated cells, tissues, and organs may be grown in culture in a manner where their natural properties (in vivo) are maintained to some extent. Such in vitro biological systems have been used for many years in studies of mutagenic and genotoxic properties, and in studies of mechanisms. 

A variety of in vitro techniques may be employed to help elucidate mechanisms of chemical-induced nephrotoxicity. Toxic effects of chemicals may be evaluated in 

Several laboratories have developed methods to physically separate and characterize the metabolic capacity of glomeruli, proximal tubules, and distal tubules. These techniques can offer insight into biochemical changes associated with site-specific nephrotoxicity. Additionally, micropuncture and microperfusion experiments have been utilized to help identify specific loci of action of nephrotoxicants. 

While Franz-type diffusion cells are commonly used to assess in vitro penetration of compounds across the skin, they have also been used for the assessment of compound permeability across the buccal mucosa [19, 71, 104], In this system, buccal mucosa is sandwiched between two chambers, and compound solution is added to the donor chamber with compound-free buffer in the receptor chamber. The receptor chamber is then periodically sampled to assess the amount of compound that has permeated the tissue over time. 

Although more commonly used in permeation experiments for skin, there have been some research groups who use flow-through diffusion cells to assess 

The appropriate calculations for determining the flux and permeability coefficient across the buccal mucosa using this approach are detailed in the appendix containing the detailed method used in our laboratory for assessing buccal permeation. 

Issues Associated With In Vitro Permeability Assessment 

Because of the possible effects of active and carrier-mediated processes and metabolic biotransformation, the issue of tissue viability is important for in vitro buccal mucosal experiments. The barrier nature of the buccal mucosa resides in the upper layers of the epithelium, where unlike in the stratum corneum, the cells contain a variety of functional organelles [119, 122, 125, 150], and so tissue viability may be an important component of the barrier function of the tissue. Various methods have been employed to assess the viability of excised buccal mucosa, including measurement of biochemical markers, microscopic methods, and linearity of transport data [42], While biochemical methods, including measurement of adenosine 5 -triphosphate (ATP) levels and utilization of glucose, provide information on the metabolic activity of the tissue, this does not necessarily relate to the barrier function of the tissue. In excised rabbit buccal mucosa, levels of ATP were measured and found to decline by 40% in 6 h, and this correlated well with transmission electron microscopic evaluation of the tissue (intact superficial cells) [32], In addition, the permeability of a model peptide was unaltered up to 6 h postmortem, but at 8 h, a significant change in permeability was observed [32], These investigators therefore claimed that excised rabbit buccal mucosa could be used for diffusion studies for 6 h. 

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Whereas over 200 plant constituents are reported to have antiviral activity, as determined by in vitro methods, only 31 compounds have shown antiviral activity in vivo (19). Immunotherapeutic activity has not been determined. 

Van Paassen [57,67] reported a synergistic decrease of the skin and eye irritation level of sodium lauryl ether sulfate by combination with lauryl ether carboxylates. The investigations have been carried out using the Draize eye irritation test and human patch test (Tables 13 and 14). Furthermore, measurements by in vitro methods, the Zein test, and the red blood cell test show low to no irritancy [251-253]. 

In a broad evaluation also the sulfosuccinate disodium laureth sulfosuccinate (DLSS) was a part of a variety of surfactants tested for their dermatological mildness, and some different test methods were applied [16]. Products were compared applying in vitro methods (Zein test, hemolysis) and in vivo methods (Duhring-Chamber test, skin mildness by intracutaneous test on mice and topical application on hairless mice, mucous membrane irritation according to the Draize procedure on rabbit eyes). In the Duhring-Chamber test the DLSS elicited no reactions in the animal tests it ranged in the least irritant third of the 15 products tested. 

Adiotomre, Eastwood, M. A. Edwards, C. A. Brydon, W. G. (1990). Dietary fiber in vitro methods that anticipate nutrition and metabolic activity in humans. American Journal of Clinical Nutrition, Vol.52, No.l, Qanuary 1990), pp. 128-134, ISSN 0002-9165. 

Recently, there has been a growth of interest in the development of in vitro methods for measuring toxic effects of chemicals on the central nervous system. One approach has been to conduct electrophysiological measurements on slices of the hippocampus and other brain tissues (Noraberg 2004, Kohling et al. 2005). An example of this approach is the extracellular recording of evoked potentials from neocortical slices of rodents and humans (Kohling et al. 2005). This method, which employs a three-dimensional microelectrode array, can demonstrate a loss of evoked potential after treatment of brain tissue with the neurotoxin trimethyltin. Apart from the potential of in vitro methods such as this as biomarkers, there is considerable interest in the use of them as alternative methods in the risk assessment of chemicals, a point that will be returned to in Section 16.8. 

Screening Techniques for Detecting Toxicity. Simple toxicity screening techniques are necessary to identify toxic species and to monitor the efficacy of isolation and purification procedures used to purify toxins. Atterwill and Steele 108) have recently comprehensively reviewed in vitro methods for toxicology and so much of the following is in the nature of a general overview. 

Developmental effects of trichloroethylene exposure have been demonstrated with the FETAX (Frog Embryo Teratogenesis Assay Xenopus) bioassay, an in vitro method using whole frog embryos (Fort et al. 1991, 1993 Rayburn et al. 1991). Observed defects included gut miscoding, skeletal kinking, and heart malformations heart malformations have also been observed in rat developmental assays (Dawson et al. 1993). 

Schlesier, K. et ah. Assessment of antioxidant activity by using different in vitro methods. Free Radio. Res., 36, 177, 2002. 

In recent studies on perfused rats hearts (Veitch et al., 1992), it was found that differences in the sensitivity of complexes 1-lV to ischaemic damage were dependent upon the duration of ischaemia and the presence of oxygen. The demonstration that complex 1 is a major defective site dependent upon isolation of mitochondria from homogenates of the tissue by in vitro methods seemed important to us. We therefore decided to attempt to make noninvasive measurements of mitochondrial function soon after reperfusion in transplanted rabbit kidneys by surface fluorescence (for mitochondrial NADH levels) and near infra-red spectroscopy (NIRS) for the redox state of cytaas. 

In the framework of the phthalate controversy Wilkinson and Lamb [109] used various in vitro methods in which known amounts of soft PVC materials were shaken, stirred, impacted, or otherwise mechanically agitated in some type of simulated saliva under controlled conditions (T, f), and the saliva extracted into hexane for analysis. Shaking-flask (liquid-liquid) extraction was also used for the solvent extraction of nonionic surfactants of the general type R0(CH2CH20) H (where R... 

Biological evaluation of medical devices—Part 5 Tests for cytotoxicity in vitro methods. ISO 10993-5 1992(E). International Standards Organization, 1992. 

The growth of generic prescribing and generic substitution will clearly increase the role and responsibilities of pharmacists in drug product selection. This movement will also stimulate in many regions of the world further debate about how bioequivalence should be quantified and when in vitro methods may, in some... 

In the following chapter we will discuss recent experiments we have performed using transgenic mice (see Beermann et al., 1992b). This approach allowed us to address several questions that could not have been followed by using in vitro methods only. 

Alternative tests can be divided into two categories in vitro and in silico. In vitro methods refer to the fact that experiments are done in a tube, generally. In silico methods refer to the use of the computer to model a certain property of interest. Below, we will analyze these two categories, and which criteria can be used to choose a suitable methodology. 

Focusing on validation process of in vitro methods, it is possible to underline some differences between tools for research and ones for toxicological testing. A research model is validated when there are some specific evidences confirming that the information from the model is able to correctly describe the process in the intact animal. Tools for toxicity testing are often used to evaluate safety hypothesis so they can be used without requiring in vivo confirmation. They are validated using a subset of well-known materials and, once validated, systems will be applied to new unknown materials or mixtures in order to evaluate their toxicity and compare their potential with other chemicals. 

What we describe here is not necessarily the result of a formal validation process, but conversely we preferred to provide a list which is scientifically updated and may offer advanced, modem tools useful for stakeholders. However, for in vitro methods, the formal validation process should be followed. 

Qualitative Evaluation of Carcinogenic Potential of Some PFCs Using In Silico and In Vitro Methods... 

In recent years, the scientific community has focused on the need to develop alternative methods to animal experiments, including cell-based in vitro methods and in silico models, based on statistics and informatics. 

Sources The data are taken from Partilla et al.28 and Setola et al.29 Details concerning in vitro methods can be found in these papers. 

Hariparsad, N., Sane, R.S., Strom, S.C. and Desai, PB. (2006) In vitro methods inhuman drug biotransformation research implications for cancer chemotherapy. Toxicology In Vitro An International Journal Published in Association with BIBRA, 20 (2), 135-153. 

Y., Comparative studies on in vitro methods for evaluating in vivo function of MDR1 P-glycoprotein, Pharm. Res. 2001, 38, 1660-1668. 

There are several approaches to estimating absorption using in vitro methods, notably Caco-2 and MDCK cell-based methods or using methods that assess passive permeability, for example the parallel artificial membrane permeation assay (PAMPA) method. These are reviewed elsewhere in this book. The assays are very useful, and usually have an important role in the screening cascades for drug discovery projects. However, as discussed below, the cell-based assays are not without their drawbacks, and it is often appropriate to use ex vivo and/or in vivo absorption assays. 

Absorption and clearance are two of the fundamental parameters that determine oral bioavailability. There are many in vitro methods to assess the absorption and metabolic potential of a given molecule, and it can be argued that a combination of these data should produce a model capable of predicting oral bioavailability. Such a model, based on a graphical approach has recently been published [26]. 

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