Currently available in vitro methods used in the regulatory toxicology

About two decades ago in vitro mutagenicity tests were adopted as the first in vitro methods in regulatory toxicology. For reasons of animal welfare and better science, many validation studies of various in vitro methods were performed between 1985 and 1995 for their potential to replace the Draize eye irritation test. Albeit the fact that four in vitro methods (HET-CAM, BCOP, IRE, and ECE) have gained regulatory tolerance in Europe for the classification of severe eye irritants, the lessons learned mainly from these validation studies led to the definition of internationally harmonised OECD validation principles and acceptance criteria (Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Tests Methods, 1996. OECD Publication Office, Paris, France). Application of these principles to prevalidation and validation studies, and additional special studies performed in Europe with the support of ECVAM, ended in the scientific validation of new in vitro methods for the prediction of skin corrosivity and phototoxicity. They were accepted for regulatory use in Europe and adopted on 8 June 2000 as test methods B. 40 and B. 41 of Annex V of Directive 67/548/EEC. In quite a different approach, European industry has submitted in-house validation data to support a Draft OECD Test Guideline for in vitro dermal absorption testing, which-after peer review and a long lasting international discussion-will now be adopted by the OECD. The increasing importance of regulatory measures derived from quantitative cytotoxicity tests, and some examples of regulatory accepted in vitro methods, where the specific purpose is restricted to a specific regulation (e.g. testing of medicinal products, or medical devices), are also addressed.     

Opportunities to minimise animal use in pharmaceutical regulatory general toxicology: a cross-company review

Toxicity studies in animals are carried out to identify the intrinsic hazard of a substance to support risk assessment for humans. In order to identify opportunities to minimise animal use in regulatory toxicology studies, a review of current study designs was carried out. Pharmaceutical companies and contract research organisations in the UK shared data and experience of standard toxicology studies (ranging from one to nine months duration) in rodents and non-rodents; and carcinogenicity studies in the rat and mouse. The data show that variation in study designs was primarily due to (i) the number of animals used in the main study groups, (ii) the use of animals in toxicokinetic (TK) satellite groups, and (iii) the use of animals in off-treatment recovery groups. The information has been used to propose a series of experimental designs where small adjustments could reduce animal use in practice, while maintaining the scientific objectives.     

Hazard classification of chemicals inducing haemolytic anaemia: An EU regulatory perspective

Haemolytic anaemia is often induced following prolonged exposure to chemical substances. Currently, under EU Council Directive 67/548/EEC, substances which induce such effects are classified as dangerous and assigned the risk phrase R48 'Danger of serious damage to health by prolonged exposure.' Whilst the general classification criteria for this endpoint are outlined in Annex VI of this Directive, they do not provide specific information to assess haemolytic anaemia. This review produced by the EU Working Group on Haemolytic Anaemia provides a toxicological assessment of haemolytic anaemia and proposes criteria that can be used in the assessment for classification of substances which induce such effects. An overview of the primary and secondary effects of haemolytic anaemia which can occur in rodent repeated dose toxicity studies is given. A detailed analysis of the toxicological significance of such effects is then performed and correlated with the general classification criteria used for this endpoint. This review intends to give guidance when carrying out an assessment for classification for this endpoint and to allow for better transparency in the decision-making process on when to classify based on the presence of haemolytic anaemia in repeated dose toxicity studies. The extended classification criteria for haemolytic anaemia outlined in this review were accepted by the EU Commission Working Group on the Classification and Labelling of Dangerous Substances in September 2004.     

Genetic toxicology in the pharmaceutical industry

The main objective of the pharmaceutical industry is health protection. Drugs not intended for use in life-threatening diseases should be free from toxic effects, but every natural or man-made chemical has potential toxicity depending on the exposure dose. The pharmaceutical industry considers genetic toxicology as a part of overall safety evaluation. No single genetic toxicity test is satisfactory; a battery of test is necessary to cover the whole spectrum of genetic events. Numerous tier approaches have been proposed for mutagenicity testing but from the toxicological viewpoint a phylogenic testing model seems more appropriate than a sequential step model. Evaluation of the mutagenic potential of drugs should rely on in vivo animal models, although for screening purposes and to promote understanding of the mutagenic action, in vitro tests using different systems can be used. Rejection solely on the basis of in vitro tests can lead to the unnecessary loss of a valuable drug. More inexpensive and relatively short tests on non-mammalian and mammalian cells are needed for studying structure-mutagenic activity relationships. For extrapolating to man and in the framework of clinical studies, it might be worthwhile to focus more time on developing inexpensive and simple tests using models directly relevant to man (e.g. human body fluids).     

The use and interpretation of in vitro data in regulatory toxicology: cosmetics,toiletries and household products

There is currently a drive to eliminate animal testing for cosmetics, toiletries and household products; indeed, the European Union Cosmetics Directive aims to prohibit the use of experimental animals for the testing of finished cosmetic products after 2002. At present, national prohibitions are in place in the UK, Germany, Austria and the Netherlands, for the testing of finished cosmetic products and cosmetic ingredients. In the USA animal testing for certain types of finished products is mandatory. Against this background, the currently available regulatory in vitro tests comprise methods for eye irritation, skin corrosivity, genotoxicity, dermal penetration and photoirritation. The draft updates to the Organisation for Economic Co-operation and Development guidelines for eye and skin irritation advocate the use of in vitro or ex vivo methods prior to the commencement of animal studies. At present, testing for these endpoints cannot be completed in vitro, but potentially corrosive substances and products can be classified without the need for animal studies. Regulatory genotoxicity testing can be completed using only in vitro methods, provided that a clear negative outcome is obtained for each test. Data from dermal penetration studies may be used to refine risk assessments. Current developments in areas such as skin sensitisation and skin irritation promise that in the reasonably near future such information may be generated without the use of animals.     

A perspective on the toxicology of marine toxins

Although there has been much progress with regard to marine toxins from dinoflagellates, much remains to be done. Because these compounds are a seafood consumer risk, the demands cover from legislative to scientific aspects. Legislation is required for all new toxins that appear in the coasts. On the other hand, it is important to understand the toxicity of the different analogues, in terms of both the relative toxicity to reference compounds and the mechanism of toxicity itself, both acute and long-term. For this, a uniform approach to do toxic studies is necessary, especially acute toxicity. The need for pure standards in sufficient supply and the understanding of the mode of action of some of the compounds (such as yessotoxin or azaspiracids) will help the development of another important field, the use of marine toxins as drug leads, and the chemistry around them.     

The limits of regulatory toxicology

The Acceptable Daily Intake (ADI) has been used by regulatory and public health organizations (e.g., the U.S. Food and Drug and Administration, and the World Health Organization) for chemicals for more than 50 years. The ADI concept was also initially employed at the U.S. Environmental Protection Agency at its inception in 1971, although with the adoption of newer terminology, it later became known as the Reference Dose (RfD). It is clear from the literature that both were first devised as instruments of regulatory policy. In the intervening years, it has become common to use language that implies that these standards are statements of scientific fact. Similarly, some of the discretionary or default values that are used to derive regulatory standards are represented as scientific assumptions when in fact they also represent regulatory policy. This confusion impedes both the best use of the available science and informed public participation in policy making. In addition, the misconception of the ADI or the RfD as statements of scientific fact may impede the consideration of alternative means to reduce exposure to chemicals that may be harmful, including regulatory measures that do not involve prescribing a regulatory concentration limit.     

Lipid excipients and delivery systems for pharmaceutical development: a regulatory perspective

The use of lipid-based dosage forms for enhancement of drug absorption or delivery has drawn considerable interest from pharmaceutical scientists. The unique characteristics of these dosage forms, however, present significant challenges to pharmaceutical industry and regulatory agencies in many ways. For example, safety assessment is necessary when the use of a new lipid excipient is considered. An important question for lipid formulation is whether the drug remains in solubilised form along the gastrointestinal (GI) tract after it is administered. Certain lipid excipients and surfactants have been reported to change intestinal permeability or interfere with enzyme/transporter activity, thereby affecting drug bioavailability. The potential influence of biopharmaceutical and/or pathophysiological factors on the drug or lipid excipient(s) needs to be explored. For a complex lipid-based dosage form, the conventional in vitro dissolution methods may not be appropriate for predicting in vivo performance in view of the convoluted GI processing of the lipid vehicle and formulation Of paramount importance is to identify any gaps in the scientific understanding of lipid-based dosage forms so that regulatory issues can be addressed. More mechanistic studies should be encouraged to facilitate a better understanding of the pharmaceutical characteristics of lipid formulations and complex interactions between lipid excipient, drug and physiological environment. This review discusses some regulatory considerations in the use of lipid excipients and delivery systems for pharmaceutical development. Implications in the regulatory determination of pharmaceutical equivalence, bioequivalence and therapeutic equivalence are also illustrated.     

A proposal for the use of biomarkers for the assessment of chronic pollution and in regulatory toxicology

Despite a wealth of information on biomarkers, they are not routinely used for regulatory purposes, even though the potential benefits of biomarkers to rationalise complex exposure–response relationships are clear. Biomarkers can be inappropriately applied or misinterpreted, because the fundamental assumptions in exposure–response relations have not been considered. Factors causing temporal and spatial variability in biomarker responses are reviewed. These include numerous geochemical and biotic variables. The variation can be minimised by appropriate study site selection, experimental replication, multivariate epidemiological approaches, normalised controls, and temporal calibration of responses; so that the regulatory use of biomarkers for biomonitoring and tracking pollution events, including chronic or multiple exposures to complex mixtures is possible. We propose and define the characteristics of biomarkers of chronic exposure or effect, which must measure changes in pollution/effect against long-term changes in other general stresses (disease, nutrition, environmental quality), relate to cumulative injury, and remain responsive over months or years. Neuroendocrine, immunological, and histological biomarkers are suggested for chronic pollution. We propose a regulatory framework for biomarkers based on a weight of evidence approach that can integrate biomarkers in risk assessment and long-term monitoring programmes.     

In vitro toxicology methods: impact on regulation from technical and scientific advancements

The impressive advancement of technologies in biomedical research, and particularly in the area of in vitro experimental models, has opened up new possibilities related to co-cultures, micromass or stem cell cultures. Engineered cells to study specific targets and/or mechanisms are also available. Moreover, a very subtle approach in the study of toxicological effects is represented by the very recent genomics and proteomics techniques. New mechanistically based methods could be established from all these approaches, which, once validated, could enter the regulatory procedure. So far, in toxicology, only a few in vitro tests are accepted for regulatory purposes, such as those related to corrosion, phototoxicity and absorption. Many others are in the pre-validation or validation phase. An area where in vitro tests play a key role is the genetic toxicology. In this context, the most recent testing strategies and test methods will be presented, with particular attention to the recently updated guidelines for food additives by the EU Scientific Committee on Food. An improvement in the implementation of validated methods could arise from a better coordination on the matter at national and international levels, the harmonisation of different legislations, and a strict control of the national rules in order to make them up-to-date with respect to validated methods.     

International pharmaceutical social risk regulation: An ethical perspective

Pharmaceutical production and distribution constitute big business. For the companies the rewards can be substantial. Rates of return on drug company investments tend to be higher than many other manufacturing enterprises. But reward is only one side of the story. There is also the issue of social risk, the focus of this article. Social risk for pharmaceutical production is especially pronounced. An ineffective or, worse, dangerous drug, can have dire consequences for the population at large. For this reason, there is elaborate government regulation and oversight of drug safety and risk. These systems, especially in the US and Europe, will be the main focus of this paper. The two systems will be described, and then compared and contrasted in terms of their framing of social risk and actions governments take to limit it. Systems elsewhere, especially in the developing world, are increasing in relative importance and these will be briefly discussed as well. Ethical issues that have arisen in these various systems will be surfaced and analysed. The paper will close with some conclusions and suggestions for further research.     

In silico toxicology for the pharmaceutical sciences

The applied use of in silico technologies (a.k.a. computational toxicology, in silico toxicology, computer-assisted tox, e-tox, i-drug discovery, predictive ADME, etc.) for predicting preclinical toxicological endpoints, clinical adverse effects, and metabolism of pharmaceutical substances has become of high interest to the scientific community and the public. The increased accessibility of these technologies for scientists and recent regulations permitting their use for chemical risk assessment supports this notion. The scientific community is interested in the appropriate use of such technologies as a tool to enhance product development and safety of pharmaceuticals and other xenobiotics, while ensuring the reliability and accuracy of in silico approaches for the toxicological and pharmacological sciences. For pharmaceutical substances, this means active and impurity chemicals in the drug product may be screened using specialized software and databases designed to cover these substances through a chemical structure-based screening process and algorithm specific to a given software program. A major goal for use of these software programs is to enable industry scientists not only to enhance the discovery process but also to ensure the judicious use of in silico tools to support risk assessments of drug-induced toxicities and in safety evaluations. However, a great amount of applied research is still needed, and there are many limitations with these approaches which are described in this review. Currently, there is a wide range of endpoints available from predictive quantitative structure-activity relationship models driven by many different computational software programs and data sources, and this is only expected to grow. For example, there are models based on non-proprietary and/or proprietary information specific to assessing potential rodent carcinogenicity, in silico screens for ICH genetic toxicity assays, reproductive and developmental toxicity, theoretical prediction of human drug metabolism, mechanisms of action for pharmaceuticals, and newer models for predicting human adverse effects. How accurate are these approaches is both a statistical issue and challenge in toxicology. In this review, fundamental concepts and the current capabilities and limitations of this technology will be critically addressed.     

药物毒理学技术方法研究进展

药物是否安全和有效是药物研发成功与否的决定性因素。就药物研发的整个流程而言,毒性是终止药物研发的重要原因之一。药物毒理学研究贯穿于新药发现阶段、临床前安全性评价和上市后跟踪监督的全过程,因此药物的毒理学研究至关重要。简要综述了药物毒理学研究过程中所涉及的技术方法以及研究新动向,并主要介绍了组学技术的发展。     

Methods of in vitro toxicology.

In vitro methods are common and widely used for screening and ranking chemicals, and have also been taken into account sporadically for risk assessment purposes in the case of food additives. However, the range of food-associated compounds amenable to in vitro toxicology is considered much broader, comprising not only natural ingredients, including those from food preparation, but also compounds formed endogenously after exposure, permissible/authorised chemicals including additives, residues, supplements, chemicals from processing and packaging and contaminants. A major promise of in vitro systems is to obtain mechanism-derived information that is considered pivotal for adequate risk assessment. This paper critically reviews the entire process of risk assessment by in vitro toxicology, encompassing ongoing and future developments, with major emphasis on cytotoxicity, cellular responses, toxicokinetics, modelling, metabolism, cancer-related endpoints, developmental toxicity, prediction of allergenicity, and finally, development and application of biomarkers. It describes in depth the use of in vitro methods in strategies for characterising and predicting hazards to the human. Major weaknesses and strengths of these assay systems are addressed, together with some key issues concerning major research priorities to improve hazard identification and characterisation of food-associated chemicals.