The choice of methods to extrapolate toxicological data from animals to man largely depends on the mechanism underlying the toxicity of the substance under investigation. Traditionally, the extrapolation of toxicity data of substances which give positive results in chronic carcinogenicity studies as well as in genotoxicity studies is carried out by using methods based on the assumption that there is no threshold dose (see Section 18.2). Toxicity data of non-carcinogenic substances are extrapolated by using methods assuming a threshold value mechanism (see also Section 18.2). Although the latter method offers a rather clear-cut possibility to extrapolate toxicity data from one species to another, its application in everyday safety evaluation procedures is often more ambiguous. This will be explained for the food contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
TCDD is the dioxin with the highest toxicity. Dioxins are emitted into the environment by waste incineration and other combustion processes. They may enter the food chain. Chronic toxicity studies in experimental animals showed that TCDD is a liver carcinogen in the female but not in the male rat. Further studies revealed that TCDD was not capable of inducing genotoxic effects in in vitro-genotoxicity assays and that its carcinogenicity is probably associated with an altered function of female steroid hormones. These findings were used as a starting point for the evaluation of the toxicological risk from TCDD. In practice, however, different authorities took diverging scientific standpoints for the extrapolation of TCDD toxicity data to man. As a result, quite different estimates of the toxic potential of TCDD were reached. In the Netherlands, for example, an expert panel was of the opinion that the experimental data on the toxicity of TCDD provided sufficient evidence to classify this substance as a non-genotoxic carcinogen in experimental animals. The panel concluded that in the case of TCDD safe exposure levels, i.e., an ADI could be calculated in a valid way. For the calculation of the ADI, liver carcinogenicity (in rats) was chosen as the critical toxic effect. For this effect, a marginal-observed-adverse-effect level (MOAEL) of 1 ng/kg/day was established in a chronic experiment in rats. The MOAEL is the lowest found concentration of a substance which causes a marginal adverse effect. The MOAEL is between the NOAEL and the LOAEL. From this effect level a NOAEL was calculated by applying an extrapolation factor of 2.5. The panel considered this value for the MOAEL-NOAEL extrapolation factor adequate in view of the type of effect observed at the MOAEL. Application of inter- and intraspecies extrapolation factors of 10 then gave an ADI of 4 pg/kg/day. In contrast to the Dutch Health Authorities, the US Environmental Protection Agency (US EPA) concluded that the available information on the toxicity of
TCDD did not give conclusive evidence with regard to its carcinogenicity mechanism. The US EPA decided to consider TCDD as a genotoxic carcinogen and to base its safety evaluation on acceptable rather than safe exposure levels. To calculate the acceptable exposure level 1 extra liver tumor incidence per 10-6 after lifelong exposure to TCDD was taken as an acceptable risk level. The calculation of the exposure level was based on a quantitative dose-response relationship between the daily TCDD intake and liver tumor incidence; the relationship was assessed by using a multi-stage carcinogenesis model. This relationship was then used for the calculation of the risk specific dose (RSD, see Section 18.2). This calculation resulted in an acceptable exposure level of 6.4 fg/kg/day.
Whether or not TCDD is considered a genotoxic or a non-genotoxic carcinogen, the extrapolations mentioned above were based on the so-called external dose concept. This means that the toxic potential of a substance is proportional to the amount of the substance to which an organism is exposed. The exposure levels were expressed in terms of units of weight of the substance per kg body weight. The external dose concept has been used for the interspecies extrapolation of TCDD toxicity data up to 1991. In 1991, however, this concept was abolished. In that year a World Health Organization Expert Committee decided to use the actual concentration of TCDD in its target organ, i.e., the liver, rather than the ingested amount for the calculation of the safe human exposure levels to TCDD (internal dose concept). The reason to replace the external dose by the internal dose lies in the widely accepted view that the toxicity of a substance is best characterized by the following two factors: the disposition of the substance in the organism (toxicokinetics) and the mechanism underlying its toxicity. In order to assess the disposition of TCDD in mammals as a function of the dose, the Committee used a one-compartment model. Toxicokinetic analyses showed that, at equal exposure levels, TCDD concentrations are expected to be 10-fold higher in the human liver than in rat liver. On the basis of a NOAEL of 1 ng/kg/day for TCDD carcinogenicity in the rat, this analysis predicted a NOAEL of 100 pg/kg/day in man. By dividing this (estimated) NOAEL by a safety factor of 10 (for intraspecies variation) the ADI of TCDD was obtained, i.e., 10 pg/kg/day.
The kinetic extrapolation method used by the WHO Expert Committee is an example of classic toxicokinetic modeling. A limitation of this type of modeling is its inability to give a physiological interpretation of the various compartments forming part of the model. The classic toxicokinetic modeling does not allow organ-specific toxicokinetic and toxicodynamic processes to be taken into account in safety evaluation procedures. To obviate this limitation, alternative kinetic approaches have been developed in the last decade. These so-called physiologically based pharmacokinetic (PBPK) models describe the disposition (absorption, distribution, metabolism, and excretion) of substances in the organism on the basis of blood flows through the organs instead of distribution over compartments. Figure 18.3 gives a diagrammatic representation of a PBPK-model of TCDD disposition in the rat.
A system of five blood flows is shown: blood circulation, and four flows through the liver, fat tissue, slowly perfused organ system (SPO, mainly skin and muscle) and richly perfused organ system (RPO, mainly kidneys, lungs and spleen). After a physiological flow diagram as shown in Figure 18.3 has been defined, absorption and elimination of the substance concerned are included in the model. For TCDD, this refers to absorption, elimination by hepatic metabolism, and biliary excretion of the metabolites formed. The model also includes a toxicodynamic parameter, viz. the induction of hepatic P-450 mixed-function oxidase (MFO), a well-known effect of TCDD and structurally related chlorinated aromatic hydrocarbons. The mechanism underlying this induction has been found to consist of a sequence of events: uptake of TCDD by the liver, binding of TCDD to a cytosolic receptor protein (the aryl hydrocarbon or Ah receptor), and stimulation of the de novo P-450 MFO synthesis. The determination of the exposure level of the liver to TCDD
oral dose ka
Figure 18.3 Flow diagram for a PBPK model of TCDD disposition in the rat.
oral dose ka
Figure 18.3 Flow diagram for a PBPK model of TCDD disposition in the rat.
is based on this mechanism. The PBPK model has been used to analyze the disposition of TCDD in experimental animals (rat, mouse) and man. The results showed that the disposition of TCDD in these species could be described by one PBPK model, irrespective of the dose level (high to low dose extrapolation), the route of administration (route to route extrapolation) and the dosage schedule (acute, semi chronic or chronic exposure conditions). Further, these analyses showed that TCDD-induced de novo synthesis of P-450 MFO was the primary factor determining the disposition of TCDD (and thus its toxicological risk) in rodent liver but not in human liver. This underlines the importance of taking interspecies differences in toxicity into account in toxicological safety evaluation. In contrast to classical toxicokinetic modeling, PBPK models can predict the disposition and toxicity of substances in mammals on the basis of a common physiological approach of the organism. PBPK models enable the incorporation of detailed knowledge of toxicity mechanisms as well as variations in the physiological state (growth, pregnancy, sex, disease, age) into toxicological safety evaluation. These possibilities make PBPK models suitable for quantitative and physiologically valid interspecies extrapolation of toxicity data. In this connection, PBPK models are continuously the subject of extensive scientific research.
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