Toxicologists -- come out and educate!

Toxicologists must be aware of the poor level of understanding among the non-scientific press and media, and presumably also within governments, of the quantitative aspects of environmental (including dietary) chemical hazards. The ignorance of the basic principles of dose response, and of the differences between hazard and risk and between a chance association and a causal relationship results in huge amounts of public money being misspent by focusing on the reduction or elimination of many trivial or even imaginary hazards. Toxicologists as a profession should give more effort to the promulgation of these concepts to journalists, the general public and, particularly, those responsible for risk management.

Rodent carcinogenicity tests need be no longer than 18 months: an analysis based on 210 chemicals in the IARC monographs.

The IARC Monographs (Vols 1-70) were studied to determine the time of onset of treatment-related tumorigenicity in long-term rodent studies for chemicals classified by IARC as having sufficient evidence of carcinogenicity in animals. The analysis excluded studies on metals and their salts, studies on particulates, studies by parenteral routes of administration that resulted in tumours only at the site of exposure, and studies that did not approximate to the current standard long-term rodent carcinogenicity bioassay, for instance transplacental or multigeneration studies, initiator-promoter studies, lung tumour assays in Strain A mice and studies in newborn animals. Data from a total of 210 chemicals revealed that, overall, evidence of treatment-related tumorigenicity was first apparent within 12 months for 66% of the chemicals and for only 7% were studies of longer than 18 months necessary. All IARC Group 1 chemicals were detected in animals within 18 months, and most within 12 months. Most of the tumour types that required more than 18 months for detection were of dubious relevance to human risk assessment. Termination of rodent carcinogenicity studies at 18 months or earlier would greatly reduce the complications that arise in interpreting the findings in aged animals which often have defective hepatic or renal function and would also markedly reduce the time required for histopathological examination of dozens of tissues taken from the approximately 500 animals routinely employed in these studies.

Evaluation of the toxicity of drug residues in food: the case for 'relay' toxicity studies.

The tissue residues of veterinary drugs given to food-producing animals are an ill-defined mosaic of parent drug and metabolites. Consumers are only ever exposed to this mixture in the presence of a vast excess of the excipient, food. Given the modifying effects of food on the absorption and disposition of co-administered xenobiotics, it follows that the toxicity of these residues can only be properly evaluated in the presence of a large excess of food. Adoption of a relay toxicity strategy addresses these two points. The proposed relay toxicity testing approach is as follows. The target species receives a recommended dosage regimen of the drug, but a dose level three- to five-fold higher than normal and the animals are then killed several days earlier than the projected withdrawal period. The tissues from these animals, containing the residue mixture at artificially high concentrations, are then administered to laboratory animals for conventional toxicological evaluation. In this approach the residues do not require individual identification and their potential toxicity is evaluated in the presence of the inescapable excipient, food. Determination of an 'exposure' level without observerable toxicity provides, in principle, the means of relating human safety to a No-Observed Effect Level in laboratory animals in the traditional manner.

Evaluation of the carcinogenic potential of pharmaceuticals. Opportunities arising from the International Conference on Harmonisation.

The evaluation of the carcinogenic potential of pharmaceuticals is currently undergoing dramatic changes. For the past 25 years the regulatory expectation for agents intended for long term use has been that lifespan studies (usually lasting 2 years) in 2 rodent species be conducted. These studies take at least 3 years to plan, execute and interpret, and use over 1200 animals. It is now recognised that the quality of the information obtained from these studies is unreliable for prediction of carcinogenic risk to humans. Over the past 4 years, the International Conference on Harmonisation (ICH) has recommended changes in approaches to assessing the carcinogenic potential of pharmaceuticals. In future, only one long term rodent study will be routinely required (usually in rats), provided this is complemented with a short or medium term test in one of the emerging new models for carcinogenicity, such as transgenic mice or newborn mice. However, the relevance of these new models to human cancer and their use in risk assessment is still largely unknown and this situation must be kept under review as knowledge accumulates. A long term study in a second rodent species is still an option. Dose selection has also been improved inasmuch as there are now several alternatives to the use of the maximum tolerated dose (MTD). In the past, the use of the MTD, when the normal homeostasis of the test animals is disturbed, has been considered one of the major problems with the rodent carcinogenicity bioassay. However, one of the alternative end-points to the use of the MTD, i.e. the comparison of plasma concentrations in rodents and humans, must be viewed with caution. While this may contribute to limiting the high dose level for agents of very low toxicity, the concept should not be interpreted as signifying that plasma concentrations provide a sound basis for comparing the carcinogenic activity of agents in different species. Recognition of the 4 properties (genotoxicity, immunosuppression, steroid hormonal activity and long term tissue damage), at least one of which is associated with each of the pharmaceuticals known to be carcinogenic to humans, should focus more attention on a search for these properties in patients. Absence of these properties at clinically relevant dose levels indicates that a pharmaceutical is highly unlikely to be carcinogenic to humans.

Veterinary drugs no longer need testing for carcinogenicity in rodent bioassays.

The putative carcinogenic risk to humans from ingestion of edible tissues containing traces of nongenotoxic veterinary drugs is so slight that the routine application of rodent cancer bioassays cannot be justified. This argument is based, first, on the pharmacological similarity of veterinary and human drugs: many of the latter that are carcinogenic to rodents have been deemed on mechanistic and/or potency grounds not to pose a cancer risk to humans. Second, the distribution of a veterinary drug through the target animal body before ingestion of a portion of edible tissue by humans days or weeks later means that the human dose from a residue is several orders of magnitude lower than the normal dose of human drugs. The dose of residue is also much lower than the exposure of humans to the most potent carcinogens.

Human carcinogenic risk assessment based on hormonal effects in a carcinogenicity study in rats with the antifungal agent, fluconazole.

Fluconazole is an orally active bis-triazole antifungal agent that acts by selective inhibition of lanosterol 14 alpha-demethylase, a key enzyme for maintenance of the fungal cell wall. It is not genotoxic. In a 2 year carcinogenicity study in Sprague-Dawley rats, fluconazole decreased mammary fibroadenomas in females and adrenal pheochromocytomas in males, and increased hepatic adenomas in males. The pattern of these changes is explicable in terms of a hormonal imbalance, corroborated in other studies with fluconazole in rats by changes in the weights of hormone-sensitive organs and circulating levels of 17 beta-estradiol. The decreases in mammary tumors are probably a consequence of aromatase inhibition by fluconazole at high dose levels. The tumor effects observed in this study are extremely unlikely to be of relevance to humans, since the hormone effects observed in this study do not occur in humans treated with therapeutic dose levels of fluconazole. This study illustrates the importance of seeking a mechanistic interpretation of rodent tumor findings, which may then be assessed for its relevance to the clinical use of a drug.

Diurnal exposure profile in rats from dietary administration of a chemical (doxazosin) with a short half-life: interplay of age and diurnal feeding pattern.

Doxazosin, an alpha-adrenergic blocking agent, has a plasma half-life in male rats of 1-2 h after i.v. administration. Plasma concentrations of doxazosin were measured in male rats receiving the drug mixed in the diet at dose levels from 5 to 40 mg kg-1. Samples taken at 4-h intervals during the light (0700-1900) and dark phases revealed peak concentrations at 0400 which were only about three times higher than the trough concentrations observed ca. 12 h later. The 24-h area under the curve (AUC) values increased disproportionately with dose and with age from 2 months up to 8 months of age; thereafter they were fairly stable to 24 months of age. This age-related effect may have been due to a reduction in clearance and/or a change in the feeding pattern of the rats. Young rats consumed ca. 84% and old rats only 45% of their daily feed during the nocturnal (active) phase. Given the known diurnal rhythms in absorption, protein binding and enzyme metabolising activity, such a change in feeding pattern with age may have wider toxicokinetic implications.

Interspecies comparisons in toxicology: the utility and futility of plasma concentrations of the test substance.

A classical dilemma in toxicology is how the dose administered relates to the dose delivered to the target site. Plasma concentrations of the test substance may be misleading since the concentration of any given substance in the plasma may not be representative of its concentration in tissues. Furthermore, a given tissue concentration of a xenobiotic can evoke responses which are highly species-dependent. While evaluating toxicity data within one species, plasma concentrations reflect the effects of route of administration, bioavailability, dose level, multiple dosing, age, gender, etc. However, when toxicity data is compared across species, the relevance of plasma concentrations depends on the nature of the toxicity. Reversible, pharmacodynamic effects often correlate with plasma concentrations, although there may be marked interspecies differences in dose-response relationships. Irreversible effects, if pharmacodynamic in origin, often correlate better with the intensity/duration of the pharmacodynamic response, rather than with plasma concentration. On the other hand, irreversible effects, if chemically mediated, may not correlate at all with plasma concentration, the lesions being caused by reactive metabolites of fleeting existence, which rarely survive long enough to leave their site of synthesis. They cannot be measured in the plasma nor predicted from plasma concentrations of the parent xenobiotic. The limitations of plasma concentrations in interpreting the toxicology of substances which are tissue-sequestered, which are subject to pharmacogenetic factors, or which show plasma concentrations that are not proportional to dose are also discussed. Mention is made of possible alternatives to plasma concentrations in assessing exposure in toxicology studies.

A rapidly reversible effect of a single dose of phenoxybenzamine on the fertility of male rats.

Male Sprague-Dawley rats received a single subcutaneous injection of phenoxybenzamine and each was allowed to mate with an untreated receptive female at 5, 29 or 53 h post-injection. At 5 h the number of males ejaculating was reduced and fertility virtually abolished; at 29 h all males ejaculated, but fertility was still low; at 53 h the performance of the males was normal.

The effect of luteinizing hormone on parturition in rats; imidazole antifungals may affect parturition via luteinizing hormone.

Studies were carried out to investigate the mechanism whereby the imidazole anti-fungal, tioconazole, affects parturition in rats. Administration s.c. of luteinizing hormone (LH) to pregnant rats on days 15-17 post-insemination (p.i.) or days 15-21 p.i. delayed the onset of parturition by a day and markedly reduced the ovarian production of 17 beta-estradiol, but not of progesterone. Administration of LH on days 18 and 19 p.i., which was already known to advance birth, reduced ovarian production of progesterone, but not of 17 beta-estradiol. The similarity of these results to those for tioconazole administered from days 15 p.i. or on days 18 and 19 p.i. suggests that tioconazole affects parturition in rats, at least in part, via LH.

A cytochemical study of the livers of rats treated with diethylnitrosamine/phenobarbital, with benzidine/phenobarbital, with phenobarbital, or with clofibrate.

Male Sprague-Dawley rats were treated with clofibrate (CLOF) in the diet for 2 years or with 4 i.p. injections of either diethylnitrosamine (DEN) or benzidine (BZ) followed by phenobarbital (PB) in the diet for 67 weeks, or just with PB for 41 weeks. Animals were killed at frequent intervals, some while still on treatment and others after 3 or 6 months withdrawal of treatment. The livers were subjected to cytochemical measurements of the parenchyma, foci, nodules and carcinomas. The parenchyma of the CLOF groups showed, in general, increases in glucose-6-phosphate dehydrogenase (G-6PD), alpha-glycerophosphate dehydrogenase (alpha-GPD), 5'-nucleotidase (5'-Nu), acid phosphatase (AP) and catalase and decreases in uricase and glutathione (GSH). CLOF induced a low incidence of GSH positive foci; nodules showed universally lower levels of catalase and GSH. In the DEN/PB and BZ/PB groups the parenchyma showed increases (even before PB treatment started) in G-6PD and in gamma-glutamyl transpeptidase (gamma-GT) and decreases in GSH. DEN raised and BZ lowered 5'-Nu. Neither initiator affected alpha-GPD. Both initiators caused a high incidence of foci positive for G-6PD and for gamma-GT; nodules induced by DEN/PB were mainly positive for gamma-GT and showed an erratic response to the other parameters. Carcinomas, found only after DEN/PB, were all positive for G-6PD and, with one exception, all were negative for alpha-GPD, 5'-Nu, AP and GSH. All changes regressed within 3 months of withdrawal of CLOF but not after withdrawal of PB from DEN-initiated animals. In conclusion G-6PD, alpha-GPD and 5'-Nu may be useful histocytochemical parameters for studying the precarcinogenic hepatic changes and nodules induced by peroxisome proliferators and by genotoxic hepatocarcinogens.

Effects of tioconazole on parturition and serum levels of 17 beta-oestradiol, progesterone, LH and PRL in the rat.

Tioconazole, an imidazole antifungal agent, was administered orally at 100 mg/kg/day to pregnant rats according to two regimens; in one, treatment started on day 15 post-insemination (p.i.) and in the other it started on day 18 p.i. The first regimen caused a delay in onset of parturition and a prolongation of labour. Serum progesterone was decreased from days 17 to 21 p.i., 17 beta-oestradiol decreased on day 21 p.i., LH increased on day 17 p.i., and the normal surge of prolactin on day 21 p.i. abolished. The parturition disorders disappeared when 17 beta-oestradiol (0.125 microgram/animal/day s.c.) was given with tioconazole from day 15 p.i. In the second regimen, tioconazole treatment advanced by about 24 hours the onset of parturition and the normal fall in serum progesterone and the surge in prolactin. Serum 17 beta-oestradiol was unaffected, but LH was raised on days 19 and 20 p.i. In animals receiving progesterone (2.5 mg/animal/day, s.c.) and tioconazole from day 18 p.i. parturition was no longer advanced. In conclusion, the parturition disorders observed in rats during tioconazole treatment are associated with a modification of progesterone and 17 beta-oestradiol serum levels. These findings have questionable relevance for the human situation as the roles of these steroid hormones in parturition in women are different from those in rats.

Hepatic foci of cellular and enzymatic alteration and nodules in rats treated with clofibrate or diethylnitrosamine followed by phenobarbital: their rate of onset and their reversibility.

The histologic appearance and cytochemical characteristics of foci of hepatic cellular alteration, hepatic nodules, and hepatocellular carcinomas occurring in male Sprague-Dawley rats treated with the hypolipidemic agent clofibrate (CAS: 637-07-0), with phenobarbital (CAS: 50-06-6), or with diethylnitrosamine [(DENA) CAS: 55-18-5] followed by phenobarbital were studied after treatment periods from 1 month to 2 years. Rats treated with clofibrate revealed foci of cellular alteration that were more often basophilic and occurred slightly sooner (wk 42) than those in untreated controls (wk 60). Of 36 rats that had received 68 or more weeks of continuous clofibrate, 19 had hepatic nodules. Of the 11 nodules examined cytochemically, none was gamma-glutamyltransferase (gamma-GT) positive and 2 were positive to glucose-6-phosphate dehydrogenase (G-6-PD) under oxygen. In rats withdrawn from clofibrate for 16-18 weeks after 68-95 weeks of clofibrate, 0 of 14 had nodules. In several of these rats zones of hepatic scarring were observed, suggesting the reversibility of the nodules. Phenobarbital alone had little effect on the incidence of foci of cellular alteration, although the number of gamma-GT-positive foci was increased. DENA followed by phenobarbital led to the early appearance of foci of cellular alteration (from wk 4), of nodules (from wk 13), and of hepatocellular carcinomas (from wk 26). gamma-GT activity was raised in most of these nodules and carcinomas, while G-6-PD activity was raised in only 3 of 9 nodules but in all 9 carcinomas examined. DENA-phenobarbital given for 13 or 26 weeks followed by withdrawal of phenobarbital for 28 and 26 weeks, respectively, produced an essentially similar pattern of lesions. In view of the growing recognition of the nonspecificity gamma-GT as a marker of carcinogen-initiated foci, the value of G-6-PD (under oxygen) as a marker merits further investigation.

Dazoxiben, a prototype inhibitor of thromboxane synthesis, has little toxicity in laboratory animals.

Dazoxiben, an orally active specific inhibitor of thromboxane synthetase, was administered by mouth daily to dogs and rats for 6 months. Dogs showed no evidence of toxicity up to 300 mg day-1 kg-1, the highest dose level used. Rats showed no evidence of toxicity after 100 mg day-1 kg-1, but at 300 mg day-1 kg-1 there were slight increases in plasma calcium and urea concentrations and a moderate incidence of focal nephrosis; males showed a slightly increased platelet count. Studies in rats and rabbits at dose levels up to 400 mg day-1 kg-1, by mouth, revealed no adverse effects on male or female fertility, embryogenesis, parturition or postnatal development. As dazoxiben is well absorbed after oral administration, the generally negative outcome to these toxicity studies suggests that selective inhibitors of thromboxane synthesis may be largely free of adverse effects which might impede their therapeutic or prophylactic use in clinical medicine.