Analgesic use of inhaled methoxyflurane: Evaluation of its potential nephrotoxicity.

Methoxyflurane is a volatile, halogenated analgesic, self-administered in a controlled low dose from the Penthrox(®) inhaler for short-term pain relief. It was formerly used in significantly higher doses to produce anaesthesia, when it caused a specific type of dose-related renal tubular damage. The pathogenesis of the renal damage and clinical use of methoxyflurane are discussed here with evidence that a low but effective analgesic dose is not associated with the risk of renal adverse effects. The maximum dose employed to produce analgesia is limited to methoxyflurane 6 mL/day and 15 mL/week, producing a minimum alveolar concentration (MAC) of 0.59 MAC-hours. Renal damage is due to the metabolism of methoxyflurane and release of fluoride ions. Exposure of humans to methoxyflurane ≤2.0 MAC-hours, resulting in serum fluoride ≤40 µmol/L, has not been associated with renal tubular toxicity. The safety margin of analgesic use of methoxyflurane in the Penthrox ((®)) inhaler is at least 2.7- to 8-fold, based on methoxyflurane MAC-hours or serum fluoride level, with clinical experience suggesting it is higher. It is concluded from clinical experience in emergency medicine, surgical procedures and various experimental and laboratory investigations that the analgesic use of methoxyflurane in subanaesthetic doses in the Penthrox inhaler does not carry a risk of nephrotoxicity.

A review of mammalian carcinogenicity study design and potential effects of alternate test procedures on the safety evaluation of food ingredients.

Extensive experience in conducting long term cancer bioassays has been gained over the past 50 years of animal testing on drugs, pesticides, industrial chemicals, food additives and consumer products. Testing protocols for the conduct of carcinogenicity studies in rodents have been developed in Guidelines promulgated by regulatory agencies, including the US EPA (Environmental Protection Agency), the US FDA (Food and Drug Administration), the OECD (Organization for Economic Co-operation and Development) for the EU member states and the MAFF (Ministries of Agriculture, Forestries and Fisheries) and MHW (Ministry of Health and Welfare) in Japan. The basis of critical elements of the study design that lead to an accepted identification of the carcinogenic hazard of substances in food and beverages is the focus of this review. The approaches used by entities well-known for carcinogenicity testing and/or guideline development are discussed. Particular focus is placed on comparison of testing programs used by the US National Toxicology Program (NTP) and advocated in OECD guidelines to the testing programs of the European Ramazzini Foundation (ERF), an organization with numerous published carcinogenicity studies. This focus allows for a good comparison of differences in approaches to carcinogenicity testing and allows for a critical consideration of elements important to appropriate carcinogenicity study designs and practices. OECD protocols serve as good standard models for carcinogenicity testing protocol design. Additionally, the detailed design of any protocol should include attention to the rationale for inclusion of particular elements, including the impact of those elements on study interpretations. Appropriate interpretation of study results is dependent on rigorous evaluation of the study design and conduct, including differences from standard practices. Important considerations are differences in the strain of animal used, diet and housing practices, rigorousness of test procedures, dose selection, histopathology procedures, application of historical control data, statistical evaluations and whether statistical extrapolations are supported by, or are beyond the limits of, the data generated. Without due consideration, there can be result conflicting data interpretations and uncertainty about the relevance of a study's results to human risk. This paper discusses the critical elements of rodent (rat) carcinogenicity studies, particularly with respect to the study of food ingredients. It also highlights study practices and procedures that can detract from the appropriate evaluation of human relevance of results, indicating the importance of adherence to international consensus protocols, such as those detailed by OECD.

What killed Socrates? Toxicological considerations and questions.

The death of Socrates in 399 BCE, as reported by Plato in the Phaedo, is usually attributed to poisoning with common hemlock. His progressive centripetal paralysis is characteristic of that poison. Socrates is said to have had a prominent loss of sensation extending centrally from his legs, which is not a feature of hemlock poisoning, and he seems not to have had the unpleasant taste or common gastrointestinal effects of that poison. It is suggested that Plato gave a modified account of the death of Socrates for political and other reasons by describing a more "noble" death.

Risk assessment of triclosan [Irgasan] in human breast milk.

Triclosan is an established bacteriostatic compound widely used in topical and dental preparations. Its pharmacokinetics and toxicology have been extensively studied in humans and animals. It is known to be absorbed from the gastrointestinal tract and across the skin. A recent report noted its occurrence in human breast milk and this has now been further investigated. Sixty two unselected samples of human milk from Breast Milk Banks in California and Texas have been analysed for triclosan; the concentration ranged from 0 to 2100 microg/kg lipid. A risk assessment of triclosan in human milk has been made, based on a conservative calculation of exposure of neonates and experimental toxicity test results. The broad set of reproduction toxicity tests of triclosan includes a 2-generation study in the rat, in which there was considerable exposure of dams and pups to triclosan throughout fetal development and up to sexual maturity in the F2 generation, and a further study in which pups of dosed dams were followed to weaning. They established an oral NOAEL for pups of 50 mg/kg/d. The maximum exposure of babies via breast milk calculated using very conservative additive assumptions is approximately 7.4 microg/kg/d. The 'Margin of Exposure' between the NOAEL and that calculated in breast fed babies is approximately 6760-fold. It is concluded that there is no evidence to indicate that the presence of a miniscule amount of triclosan in breast milk presents a risk to babies.

Albendazole, mebendazole and praziquantel. Review of non-clinical toxicity and pharmacokinetics.

The pharmacokinetics and toxicity of albendazole, mebendazole and praziquantel are extensively reviewed, drawing on original published work and reviews in the open scientific literature and on assessments by international agencies and official regulatory bodies in Europe and the USA. Information about human and veterinary medical uses and adverse reactions is evaluated. The totality of the non-clinical information available about these long-established drugs may not comply with current official guidelines for new medicines but reasons are given why the "deficiencies" are only apparent and the data gaps can be replaced by other results, largely obtained from the target species and the many years of clinical experience of safe use of these drugs in humans and animals.

Development immunotoxicology and risk assessment. Conclusions and future directions: a personal view from the session chair.

Developmental immunotoxicity is new both as an area of scientific study and as a potential source of concern in the protection of the public health. It is a combination of three nascent sciences and one older established area of study--immunology, 'Development', toxicological science and the practical application of experimental findings to indicate risks to man and means to control them. This hardworking and very successful meeting, organized by the Immunotoxicology Technical Committee (ITC) of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) with input from the U.S. Environmental Protection Agency (EPA), provided an appropriate setting in which scientists from industrial, academic and regulatory backgrounds were able to debate how the development of the immune system might be affected by toxicity, and ways in which transient and permanent harmful effects might occur. There was considerable interest in how the basic processes and structures of immunity and development might be affected by model substances, which could afford examples to demonstrate the value of laboratory methods to detect developmental immunotoxicity. There were vigorous discussions about testing strategies, about techniques, about how to 'validate' laboratory methods when there was relatively little consistent information from man to identify suitable positive control substances and cardinal disorders, and about how best to employ the sparse information to support the effective prediction of risk.

Carcinogenicity testing of IL-10: principles and practicalities.

IL-10 is a cytokine with actions at many levels of the immune system. In the course of development of recombinant human IL-10 (rhuIL-10) as a potential treatment for a number of chronic diseases of man, the question 'What about its carcinogenicity testing?' was repeatedly asked, based on scientific evaluation by toxicologists, beliefs about regulatory requirements, and questions considered likely to be raised by physicians, patients, and lawyers. The feasibility of various approaches to the carcinogenicity testing of rhuIL-10 is critically discussed here as a contribution to rational consideration of the general need for and value of such testing, and its particular feasibility for a recombinant human protein with profound effects on the immune system. The physiological functions of IL-10 in man and rodents are reviewed in detail, as there are notable differences between species in its normal activities, followed by detailed evaluation of the potential procedures and practical problems of its carcinogenicity testing as a heterologous, immunogenic protein in rodents. The value of information that might be obtained from transgenic mice is also evaluated, and so are the results of studies exploring its actions on human tumour cell biopsies and rodent and human cell lines. It is concluded that despite the probable popular and regulatory expectations that carcinogenicity test results would be provided, all the physiological and pathological information reveals no indication that rhuIL-10 would pose a carcinogenic risk to humans on prolonged administration, and that it would not be feasible to undertake such experimentation. It is argued that in this, as in other instances, professional and popular expectations have run beyond practical feasibility or theoretical justification. Cautious and critical evaluation should be made every time shorter or longer term toxicity studies of any candidate drug are planned or even considered, especially if it is a recombinant protein, to decide on objective grounds whether the studies are really necessary and whether they can be done in a way that will give meaningful results that will help in risk assessment.

Mechanisms of chromium toxicity, carcinogenicity and allergenicity: review of the literature from 1985 to 2000.

Laboratory and clinical reports about the pathogenesis of the carcinogenicity and allergenicity of chromium compounds published between 1985 and 2000 have been reviewed as a basis for consideration of the pathogenetic mechanisms involved. There is good evidence from the clinic and the laboratory that Cr[VI] is the ion responsible for most of the toxic actions, although much of the underlying molecular damage may be due to its intracellular reduction to the even more highly reactive and short-lived chemical species Cr[III] and Cr[V]. Exposure to Cr[VI] can result in various point mutations in DNA and to chromosomal damage, as well as to oxidative changes in proteins and to adduct formation. The relative importance of these effects of chromium ions and of the free oxidising radicals they may generate in the body in causing tumours and allergic sensitisation remain to be demonstrated. Biochemical studies of the DNA-damaging effects and of the pathogenesis of the allergic reactions to chromium ions have not kept up with advances in understanding of the molecular basis of the effects of other carcinogens and allergens.

Pharmacokinetic and toxicology assessment of INTERCEPT (S-59 and UVA treated) platelets.

The pathogen inactivation process developed by Cerus and Baxter Healthcare Corporations uses the psoralen, S-59 (amotosalen) in an ex vivo photochemical treatment (PCT) process to inactivate viruses, bacteria, protozoans, and leukocytes in platelet concentrates and plasma. Studies were performed by intravenous infusion of S-59 PCT formulations +/- compound adsorption device (CAD) treatment and with non-UVA illuminated S-59, using doses that were multiples of potential clinical exposures. The studies comprised full pharmacokinetic, single- and repeated-dose (up to 13 weeks duration) toxicity, safety pharmacology (CNS, renal, and cardiovascular), reproductive toxicity, genotoxicity, carcinogenicity testing in the p53(+/-) mouse, vein irritation, and phototoxicity. No specific target organ toxicity (clinical or histopathological), reproductive toxicity, or carcinogenicity was observed. S-59 and/or PCT formulations demonstrated CNS, ECG, and phototoxicity only at supraclinical doses. Based on the extremely large safety margins (>30,000-fold expected clinical exposures), the CNS and ECG observations are not considered to have any toxicological relevance. Additionally, after a complete assessment, mutagenicity and phototoxicity results are not considered relevant for the proposed use of INTERCEPT platelets. Thus, the results of an extensive series of in vitro and in vivo studies have not demonstrated any toxicologically relevant effects of platelet concentrates prepared by the INTERCEPT system.

Future problems requiring scientific consideration.

Water is essential for life, a precept that should control all other considerations. Water and its toxicological safety represent a paradigm for much of what is happening in other areas of society in evolving perceptions of what is 'toxicity', what is 'risk' and what we wish to or are prepared to do about such 'risks' at what cost to ourselves and others. Water is an universal solvent and may contain a wide diversity of substances arising from sources and supply systems and modified by treatment and storage. The problems that are already present, and which are closely linked to others just beginning to be recognisable, arise from the growing sensitivity of analytical techniques showing exposure to novel or ever smaller amounts of known substances in water, how to recognize and evaluate their conventional and possibly new (i.e. previously unrealized) toxic actions, how to measure exposure to water as drunk and as in foods and drinks, in what way can safe exposure levels be set, and is it feasible to people to demonstrate that 'safety' has been achieved? Behind those lie the very real problems of deciding what is 'safety', what level of notional safety do we demand in our assessments, and how do we, the people, come to realise and accept the costs that we shall have to pay in demanding any given level of 'safety'? The latter includes deciding what is an appropriate level of safety for the community as a whole and any specially susceptible groups within it. These questions are deceptively simple to pose and tortuously difficult even to attempt to answer. But they are not all the future problems. Behind them lie the socio-political uncertainties of risk and its place in society-what real or perceived risks do we accept and why, and how in a democratic society are we, the people, to be informed about risks, how should we decide what to accept or reject, and how are we to balance our beliefs and wishes against the costs of prevention or acceptance of diseases that unclean or contaminated water can bring.

Safety evaluation of human living skin equivalents.

Human living skin equivalents (LSEs) offer an alternative to the use of split-thickness autografts for the treatment of hard-to-heal wounds. LSEs consist of 4 active components: a well-differentiated stratum corneum derived from epidermal keratinocytes, dermal fibroblasts, and an extracellular collagen matrix. Neonatal foreskins are used as the source of keratinocytes and dermal fibroblasts for the manufacture of LSEs. Following isolation and expansion in vitro, the cells are cultured on a 3-dimensional scaffold to give an upper epidermal layer and supporting dermal layer. The resulting product has the appearance and handling characteristics of human skin. Safety evaluation of LSEs begins with insuring that foreskins are obtained only from healthy infants whose mothers are negative for a panel of adventitious agents. Keratinocyte and fibroblast cell banks are characterized using morphologic, biochemical, and histologic criteria; checked for the absence of contaminating cell types such as melanocytes, macrophages, lymphocytes, and Langerhans cells; subjected to rigorous microbiological testing (with any production materials of biological origin); and evaluated for in vivo tumorigenicity. The consistency of certain key morphologic and functional characteristics are regularly assessed. Because an LSE represents an allogeneic graft, preclinical safety studies include in vitro and in vivo determinations of its potential immunogenicity. Immunocompromised (SCID) mice reconstituted with human leukocytes or engrafted with human fetal hematolymphoid organs have been useful animal models for assessing possible immunologic responses to LSEs. Additional preclinical studies are being conducted to show that LSEs are noncytotoxic and lack allergenic, sensitizing, or irritation potential.

The risks of risk assessment in foods.

This report is the outcome of a workshop organized by the International Life Sciences Institute-European Branch (ILSI Europe), on the "Risks of Risk Assessment in Foods" held on 18 February 1998 in Brussels, Belgium. The meeting discussed Risk Assessment as the principal means by which the European Union evaluates the potential harm arising from the use of existing and new products. The experiences of the parties involved have often shown that the concepts underlying risk assessment are complex and not always fully understood. There is an urgent need to familiarize industry, policy makers and the scientific community with developments in the basic principles and terminology of risk assessment. Therefore, the workshop aimed to review key areas in risk assessment and to provide an open forum for learning and discussion between all interested parties.

Transepithelial transport of large particles in rat: a new model for the quantitative study of particle uptake.

The transport of large particles across the intestinal mucosa and the mechanisms of transfer of the particles into the body are still little understood. Fluorescent polystyrene latex particles (2 microm diam.) were administered orally to young male Sprague-Dawley rats in doses of 2.33 x 10(3), 2.33 x 10(6) and 2.33 x 10(9) particles. After 60 min, Peyer's patches and Peyer's patch-free tissues were collected from the small intestine and colon. A novel technique was used to exclude non-translocated particles adherent to the mucosal surface; the intestinal epithelium was stripped from the intestine by immersion in Hanks' balanced salt solution containing 1.5 mM EDTA. Particles in solubilized samples of intact and epithelium-stripped Peyer's patches and Peyer's patch-free intestinal tissue and colon were quantified by fluorescence microscopy. The location of particles within the intact and epithelium-stripped gut samples was revealed by confocal microscopy. Particles were shown to have been taken up along the entire length of the small and large intestines via both Peyer's patches and the normal intestinal epithelium. The number of particles detected in the distal region was greater than in the proximal part of the small intestine, although the difference was not statistically significant. This study has revealed that large numbers of non-translocated particles adhered to the mucosal surface resulting in a high background count. The assay system was considerably improved by the epithelium-stripping technique. The process of transepithelial uptake is a potentially important route of uptake of toxic, immunologically active and radioactive substances. These particles are much larger than the conventionally accepted upper limit for absorbed materials.

Intestinal uptake of particulate material by dexamethasone-treated rats: use of a novel technique to avoid intestinal mucosal contamination.

The aim of this study was to investigate the effect of immune suppression on the uptake of particles across the wall of the intestine and the dissemination of the particles to systemic organs. Normal and dexamethasone-immunosuppressed rats were dosed orally with 0.5 mL distilled water or fluorescent polystyrene latex particle suspension containing 2.33 x 10(9) 2-microm diameter particles. One hour after particle dosing, the animals were killed by CO2 asphyxiation. The intestinal tissues and systemic organs were sampled for particle quantitation. To avoid contamination by particles adherent to intestinal mucosa the epithelium of intestinal tissue samples was removed before quantification. The number of fluorescent particles in tissues was determined by fluorescence microscopy of digests of selected samples. The uptake of particulate material across the intestinal wall was significantly (P < 0.05) increased in rats treated with dexamethasone but the number of particles transferred to systemic organs did not differ from values found for control animals. The results suggest that although dexamethasone increased intestinal permeability the apparatus or mechanisms involved in particle transport to distal sites were not affected during immune suppression.

Neurotoxicity and artemisinin compounds do the observations in animals justify limitation of clinical use?

High parenteral doses of certain artemisinin derivatives can produce a limited and unique, selective brain stem neuronopathy in laboratory animals. There is necrosis of a small number of nerve cells in certain brain stem nuclei and more extensive chromatolysis of neurons in the same nuclei a few days after intramuscular or intravenous injection of dihydroqinghaosu, artemether and arteether in doses exceeding about 6mg/kg/d intramuscular or intravenous for about 3-5 days (in an oily solvent) in the dog, or after a single parenteral dose exceeding about 100 mg/kg. The limited information available about the monkey suggests that it is only affected after doses several times higher. The probable order of sensitivity of species is dog > rat > monkey, but this is based on only few results. No lesions have been reported after various intramuscular and oral dosages of artesunate and artelinate. The limited reports of clinical observations have not suggested any specific pattern of abnormalities. The lesion is unique in its distribution, in the small number of neurons that become necrotic and the occurrence in nearby cells of chromatolysis. The latter is almost certainly reversible because more prolonged or higher dose studies have not shown more extensive neuronal damage. As the pathogenesis of this toxic response is not known, evaluation of the risk to man must be based on conventional assessment of active doses in animals versus those employed in the treatment of cerebral malaria. It is argued that there is no reason to anticipate a particular risk of conventional regimes employing up to artemether 3-6mg/kg/d intramuscular or other regimes involving artesunate per rectum for a few days.

Effect of solid and liquid diet on uptake of large particulates across intestinal epithelium in rats.

The effect of diet composition on the uptake of particulates across the gastrointestinal epithelium has been examined in fasted male weanling Sprague-Dawley rats by estimating the systemic uptake of orally administered 2-microns latex polystyrene microspheres. Using a tissue solubilization assay, particle transfer in animals maintained on a fluid diet was determined. A larger number of particles was transferred from the gut lumen to the internal organs, including the mesenteric lymph node, spleen, bone marrow, liver, kidney, and heart of animals fed solid pelleted diet than those maintained on a fluid-diet 4 hr after oral administration of particles. The increase in particle number in rats fed the solid diet was only statistically significant (P < 0.05) for brain tissue in the analysis for trend. However, the number of particles retained in the proximal region of the gut at the end of this period was greater in animals fed the fluid diet. This work demonstrates that diet composition is important in gastrointestinal transepithelial translocation of microspheres.