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.

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.

Neurochemical findings in a perinatal sudanophilic leukodystrophy rich in steryl ester.

Neurochemical and neuropathological studies have been made of a 10-day-old child who suffered from a sudanophilic leukodystrophy. The brain white matter contained abundant sudanophilic material. The patient's grey matter total cholesterol content was 30% higher than whole brain tissue derived from a comparable control. White matter cholesterol content was more than double the control value. Nearly 80% of the white matter cholesterol was esterified. Subcellular fractionation of the white matter resulted in a "floating fraction" rich in cholesteryl ester. The steryl ester fatty acid composition was not typical of control tissue or demyelinating tissue. Patient phospholipid fatty acid composition patterns differed from control, but white matter galactolipid fatty acid composition appeared normal. Cholesteryl ester hydrolase activity appeared normal. Myelin and myelin-like fractions, isolated from diseased and normal brain tissue, were of a primitive developing nature but appeared to be comparable. The findings indicate a neonatal sudanophilic leukodystrophy which doubtless began in prenatal life and which was rich in cholesteryl ester. The aetiology of the leukodystrophy is unknown.

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.

Safety evaluation of biological and biotechnology-derived medicines.

Evaluating the 'safety' of drugs produced by biotechnology resembles the assessment of conventional 'new chemical entities,' but with certain major differences. The 'quality' of the product requires careful control because of concern about the carry-over of DNA, immunogenic proteins, endotoxin and process chemicals. Equally the potency and purity of the product must also be considered, as well as its identity. The toxicity testing of rDNA-derived proteins, monoclonal antibodies and vaccines, although increasingly being swept under the umbrella of conventional studies, should be empirically devised according to the nature and physiological effects of the substance, taking account of the responsiveness of suitable species for non-clinical testing, the potential immunogenicity of heterologous proteins and any effect the drug may have on physiological mechanisms and the immune status of the test animals. Conventional types of single and multidose and reproduction toxicity experiments can then be adapted to detect and investigate any hazard of the novel drug. Kinetics, metabolism and drug interactions should be explored and the regulatory demand for genotoxicity data satisfied. If appropriate, immunological actions, including auto-immunity, can be sought. 'Safety-in-use' should then be predictable with some confidence, because of the extent of the toxicological investigations and because activities that cannot be examined will have been delineated, e.g. lack of a responsive species or of a suitable laboratory procedure.

Translocation of particulates across the gut wall--a quantitative approach.

The translocation of latex particles across the epithelium of the rat small intestine and their transfer to internal organs was determined and quantified. The rate of particle uptake was also established. Particle uptake from the gut lumen to internal tissues was rapid as they were detected in all tissues examined within 30 minutes of oral administration. The maximum number of particles per gram tissue was detected in tissues of the small intestine. Particle number in spleen, kidney, lung, liver and brain increased with time, and in mesenteric lymph node, and heart tissues they decreased with time. Particles were also detected in bone marrow samples. These findings suggest that this animal model is useful in the evaluation and quantification of particle uptake and the determination of the tissues to where they are transported.

Allergy to antimicrobial residues in food: assessment of the risk to man.

Meat and other dietary products from food animals and farmed fish in the West may contain residues of many antibiotics and antibacterial agents, or haptenised macromolecules, e.g. penicilloylated proteins. General surveys have shown a low incidence of detectable residues in most products (up to 0.5-2%). A notable proportion of the general population has true allergic sensitivity to these substances (up to 7-10% to "penicillin") due to prior medical treatment. However, cases of proven allergy to such substances in food are extremely rare, based on clinical and laboratory proof of an immunological reaction, whereas there are less well substantiated reports blaming antibiotics in up to 50% of cases of chronic urticaria. Consideration of nature of haptenisation by antimicrobial substances, of the inefficiency of the oral route for immunisation, and of the transient and unrepeatable nature of most examples of food-related reactions all suggest that allergy to antimicrobial residues in the diet is exceedingly rare.

Dominant lethal study of ribavirin in male rats.

Ribavirin, a new synthetic antiviral agent, was studied for dominant lethal effects in male CD rats. The drug was administered intraperitoneally at doses of 50, 100 and 200 mg/kg/day for 5 days. Males were mated weekly with 8 consecutive batches of female rats. Marginal increase in early foetal death detected in Assessment Weeks 3 and 8 in females mated with the low-dose and high-dose males were not dose-related and were most probably chance events caused by the particularly low vehicle control frequencies for these 2 weeks. Also, the slightly reduced pregnancy proportion among females mated with the high-dose treated males was to a substantial extent the effect of a single male rate which failed to fertilize any females. Ribavirin was, therefore, regarded as being devoid of any mutagenic potential demonstrable by a rat dominant lethal assay.

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.

Comparative subacute effects of dietary raw soya flour on the pancreas of three species, the marmoset, mouse and rat.

Subacute changes induced in the pancreas by raw soya flour have been studied in three species. The raw soya flour was fed to male Sprague-Dawley rats and male CD-1 mice for 2, 4 or 8 days at a level of 50% (w/w) in the diet, and to male and female marmosets for 2 or 7 days at 20% (w/w) in the diet. The stathmokinetic method was used to study mitotic activity in the pancreas, and mean acinar cell area was calculated using an image analysis system. The relative weight of the pancreas in the rat and mouse increased during dosing, but that of the marmoset did not. No proliferative or trophic effects were observed in marmoset pancreas, but in both the mouse and rat a transient increase in mitotic activity was followed by a steady increase in acinar cell size.

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.

The gastrointestinal absorption of neptunium, plutonium and americium in a primate (C. jacchus).

Mixtures of Np, Pu and Am were administered to primates (C. jacchus) by gastric intubation to measure their fractional gastrointestinal absorption (f1 values). The values obtained were about 2 x 10(-3) and 1 x 10(-3), respectively, for Np and Pu administered as the citrate, and 2 x 10(-3) and 6 x 10(-4), respectively, for Pu and Am in potato. The significance of these values in terms of absorption in humans is discussed.

Urinary bladder hyperplasia in the rat: non-specific pathogenetic considerations using a beta-lactam antibiotic.

Eight of the known chemical substances associated with neoplasia in man are known to target the urinary bladder urothelium. Preneoplastic changes have been identified following exposure to each of these chemicals, and they have also been seen to occur in many species of lab animals. The most important such change is preneoplastic hyperplasia. Adaptive hyperplasia is the first form of hyperplasia to appear. It can be seen both in untreated controls and dosed animals. The distinguishing features are that in treated groups it does not progress with dose or time, and the process is reversible. Reparative hyperplasia involves disruption of homeostasis. Its severity increases with dose and time. It is not seen in controls but it is still reversible during the recovery segment after exposure to a toxic substance. When reparative hyperplasia continues beyond a certain threshold of time and dose, it progresses to preneoplastic hyperplasia, which further progresses with continued stimulation to frank neoplasia. The synthetic beta-lactam penem antibiotic FCE 22891 and its metabolite FCE 22101 caused adaptive urothelial hyperplasia of the urinary bladder only in rats and in no other species. Based on the pharmacokinetic profile of FCE 22891 and FCE 22101, it can be deduced that the morphologic finding of adaptive urothelial hyperplasia is caused by reduction of intravesicular urine pH. This effect has no relevance to therapeutic use in humans. Further, it is important to distinguish adaptive and reparative hyperplasia in preclinical toxicity studies.

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.

Strength of meaning--strong words and a certain message?

The ability to convey specific meanings is important to scientists in their writing. However, difficulties arise when the ideas to be portrayed are judgmental. While extreme terms expressing strength of meaning are readily understood, more subtle ones appear not to be.

Effects of a cholecystokinin receptor antagonist on rat exocrine pancreatic response to raw soya flour.

1 Raw soya flour (RSF) in the diet induces pancreatic hypertrophy and hyperplasia in the rat, changes ascribed to production of a high circulating level of cholecystokinin (CCK) due to inhibition of trypsin in the duodenum. Prolonged ingestion results in pancreatic adenomas and carcinomas. 2 L-364, 718, a potent, highly specific CCK antagonist was used to investigate the short-term role of CCK. 3 In rats fed 50% RSF and L-364, 718 5 mg kg-1 p.o. twice daily for 4 d, there was inhibition of pancreatic hypertrophy and hyperplasia, which is further evidence that peripherally-acting CCK plays a major role in the generation of RSF-mediated changes in the pancreas.