The refinement of uncertainty/safety factors in risk assessment by the incorporation of data on toxicokinetic variability in humans.

The derivation of safe levels of exposure in humans for compounds that are assumed to cause threshold toxicity has relied on the application of a 100-fold uncertainty factor to a measure for the threshold, such as the no observed adverse effect level (NOAEL) or the benchmark dose (BMD). This 100-fold safety factor consists of the product of two 10-fold factors allowing for human variability and interspecies differences. The International Programme on Chemical Safety has suggested the subdivision of these 10-fold factors to allow for variability in toxicokinetics and toxicodynamics. This subdivision allows the replacement of the default uncertainty factors with a chemical-specific adjustment factor (CSAF) when suitable data are available. This short review describes potential options to refine safety factors used in risk assessment, with particular emphasis on pathway-related uncertainty factors associated with variability in kinetics. These pathway-related factors were derived from a database that quantified interspecies differences and human variability in phase I metabolism, phase II metabolism, and renal excretion. This approach allows metabolism and pharmacokinetic data in healthy adults and subgroups of the population to be incorporated in the risk-assessment process and constitutes an intermediate approach between simple default factors and chemical-specific adjustment factors.     

Evolution of chemical-specific adjustment factors (CSAF) based on recent international experience; increasing utility and facilitating regulatory acceptance

The application of chemical-specific toxicokinetic or toxicodynamic data to address interspecies differences and human variability in the quantification of hazard has potential to reduce uncertainty and better characterize variability compared with the use of traditional default or categorically-based uncertainty factors. The present review summarizes the state-of-the-science since the introduction of the World Health Organization/International Programme on Chemical Safety (WHO/IPCS) guidance on chemical-specific adjustment factors (CSAF) in 2005 and the availability of recent applicable guidance including the WHO/IPCS guidance on physiologically-based pharmacokinetic (PBPK) modeling in 2010 as well as the U.S. EPA guidance on data-derived extrapolation factors in 2014. A summary of lessons learned from an analysis of more than 100 case studies from global regulators or published literature illustrates the utility and evolution of CSAF in regulatory decisions. Challenges in CSAF development related to the adequacy of, or confidence in, the supporting data, including verification or validation of PBPK models. The analysis also identified issues related to adequacy of CSAF documentation, such as inconsistent terminology and often limited and/or inconsistent reporting, of both supporting data and/or risk assessment context. Based on this analysis, recommendations for standardized terminology, documentation and relevant interdisciplinary research and engagement are included to facilitate the continuing evolution of CSAF development and guidance.     

Physiological-model-based derivation of the adult and child pharmacokinetic intraspecies uncertainty factors for volatile organic compounds

The intraspecies uncertainty factor (UF(HH)=10x) is used in the determination of the reference dose or reference concentration and accounts for the pharmacokinetic and pharmacodynamic heterogeneity within the human population. The Food Quality Protection Act of 1996 mandated the use of an additional uncertainty factor (UF(HC)=10x) to take into account potential pre- and postnatal toxicity and lack of completeness of the data with respect to exposure and toxicity to children. There is no conclusive experimental or theoretical justification to support or refute the magnitude of the UF(HH) and UF(HC) nor any conclusive evidence to suggest that a factor of 100 is needed to account for intrahuman variability. This study presents a new chemical-specific method for estimating the pharmacokinetic (PK) component of the interspecies uncertainty factor (UF(HH-PK) and UF(HC-PK)) for volatile organic compounds (VOCs). The approach utilizes validated physiological-based pharmacokinetic (PBPK) models and simplified physiological-model-based algebraic equations to translate ambient exposure concentration to tissue dose in adults and children the ratio of which is the UF(HH-PK) and UF(HC-PK). The results suggest that: (i) the UF(HH-PK) and UF(HC-PK) are chemical specific; (ii) for the chemicals used in this study there is no significant difference between UF(HH-PK) and UF(HC-PK); (iii) the magnitude of UF(HH-PK) and UF(HC-PK) varies between 0.033 and 2.85 with respect to tissue and blood concentrations; (iv) the body weight, the rate of ventilation, the fraction of cardiac output flowing to the liver, the blood : air partition coefficient, and the hepatic extraction ratio are the only parameters that play a critical role in the variability of tissue and blood doses within species; and (v) the magnitude of the UF(HH-PK) and UF(HC-PK) obtained with the simplified steady-state equations is essentially the same with that obtained with PBPK models. Overall, this study suggests that no adult-children differences in the parent chemical concentrations of the VOCs are likely to be observed during inhalation exposures. The physiological-model-based approaches used in the present study to estimate the UF(HH-PK) and UF(HC-PK) provide a scientific basis for their magnitude. They can replace the currently used empirical default approaches to provide chemical-specific UF(HH-PK) in future risk assessments.     

Metabolism,variability and risk assessment

For non-genotoxic carcinogens, “thresholded toxicants”, Acceptable/Tolerable Daily Intakes (ADI/TDI) represent a level of exposure “without appreciable health risk” when consumed everyday or weekly for a lifetime and are derived by applying an uncertainty factor of a 100-fold to a no-observed-adverse-effect-levels (NOAEL) or to a benchmark dose. This UF allows for interspecies differences and human variability and has been subdivided to take into account toxicokinetics and toxicodynamics with even values of 10^0.5 (3.16) for the human aspect. Ultimately, such refinements allow for chemical-specific adjustment factors and physiologically based models to replace such uncertainty factors.     

危险度评价最新进展

在过去的几十年里 ,人们开发了许多新的方法来改进化学品危险度评价中的剂量 反应关系的评定 :如采用定义较为完善的有效作用剂量来替代无可见有害作用水平(NOAEL) ,以及改进了的不确定 /安全系数的评定方法。本文将介绍目前国际上使用较为广泛的几种评定方法 :①采用剂量 反应关系数学模拟的方法来估计有效剂量以取代NOAEL作为安全剂量的出发起点值的剂量 反应关系的评定方法 ,包括 :“基准剂量法”和“分类回归法” ;②使用种系内和种系间毒代动力学和毒效动力学的资料来选择不确定系数的“基于数据的不确定系数法” ;③用于估计不确定系数的可信区间 ,以及计算安全剂量值的可信区间的“概率参考剂量法”。     

A scoping review of evaluations of and recommendations for default uncertainty factors in human health risk assessment

Uncertainty factors (UFs) are used to account for uncertainties and variability when setting exposure limits or guidance values. Starting from a proposal of a single UF of 100 to extrapolate from an animal NOAEL to a human acceptable exposure, the aspects of uncertainty and number of UFs have diversified and today there are several risk assessment guidelines that contain schemes of default UFs of varying complexity. In the present work, we scoped the scientific literature on default UFs to map developments regarding recommendations and evaluations of these. We identified 91 publications making recommendations for one or several UFs and 55 publications evaluating UFs without making explicit recommendations about numerical values; these were published between 1954 and 2021. The 2000s was the decade with the largest number of publications, interspecies differences and intraspecies variability being the most frequent topics. The academic sector has been the most active (76 out of 146 publications). Authors from the private sector more often presented UF recommendations, but differences between sectors regarding size of recommendations were not statistically significant. The empirical underpinning of the reviewed recommendations ranges from four to 462 chemicals, that is, relatively low numbers compared with the range of chemicals these default UFs are expected to cover. The recommended UFs have remained remarkably constant, with merely a slight decrease over time. Although chemical specific UFs are preferable, the widespread use of default UFs warrants further attention regarding their empirical and normative basis.     

Significance of Excursions of Intake above the Acceptable Daily Intake: Effect of Time and Dose in Developmental Toxicology

Three major factors to be considered in assessing the possible effects on developmental toxicology of excursions above the acceptable daily intake (ADI) are discussed. If maternal toxicity occurs at lower doses than developmental toxicity, then there may be adequate protection for the fetus if the mother is protected. In other cases, the first adverse developmental effects are usually small and reversible changes in fetal weight and it is unlikely that brief excursions, for a few days, above the ADI, would induce changes in final birth weight. The importance of excursions above the ADI on teratogenic responses would likely depend on the mechanism of teratogenesis and whether the agent acted primarily as a result of a high peak plasma level, a C_max effect, or depended more on the total body exposure, an area under the curve (AUC) effect. This type of information is usually not available from current safety study designs. Finally, the temporal equivalence factors must be taken into account. There are 10- to 100-fold differences in time span for pre- and postnatal development up to sexual maturity, in rodents compared with humans. This can be contrasted with the relatively small pharmacokinetic differences, perhaps of up to 2- or 3-fold in plasma levels of chemicals with the same administered dose in the two species. Thus, single episodes of high chemical exposure have greater opportunity to produce permanent effects on development in rodents compared with humans. The limited evidence from acute poisonings in pregnant women tends to support this hypothesis and it seems unlikely that occasional excursions by women above the ADI would result in developmental toxic effects.     

Food contamination by metals and pesticides in the European Union. Should we worry?

The estimation of the risk associated with dietary intakes of heavy metals and pesticide residues by the consumer is a vital and integral part of regulatory processes. The exposure of the consumer is compared directly to the acceptable daily intake (ADI) for pesticides and to the tolerable daily intake (TDI) for heavy metals. The exposure is obtained using the basic equation: Exposure (mg/kg b.w./day)=Consumption (mg/kg b.w./day) x Residue (mg/kg). The establishment of the ADI and the TDI is based on the results of toxicological studies that involve the determination of the lowest-no-observed-adverse-effect level/10 (SF1) x10 (SF2), where SF corresponds to 'Safety Factor'. SF1 and SF2 account for interspecies and intraspecies variability, respectively. In order to evaluate the risk for the consumer, that is associated to the presence of heavy metals and pesticides in food, a review of the level of contamination in European countries has been made. The exposure of European consumers to lead, cadmium, arsenic and mercury is superior to the TDI. For pesticides, the first step is to compare the detected amount of residues of a specific pesticide to the maximum residue level (MRL) authorized in foodstuffs. If the residue level in food exceeds the MRL, the theoretical maximum daily intakes and the ADI have to be taken into account in order to assess the risk for the consumer.     

Addressing human variability in risk assessment--the robustness of the intraspecies uncertainty factor

Addressing human variability and sensitive subpopulations is one of the challenges of risk assessment and is an important aspect of the Food Quality Protection Act, the law passed in 1996 that regulates food use pesticides in the United States. The intraspecies uncertainty factor is intended to address differences in susceptibility within the human population. This paper examines the history and scientific basis for the intraspecies uncertainty factor. Our best source of knowledge about human variability in the response to chemicals comes from clinical trials of pharmaceuticals. This large body of data allows both qualitative and quantitative characterization of variability in pharmacokinetic and pharmacodynamic parameters in the general population and in subgroups such as children. The preponderance of evidence in the areas of pharmacodynamics and pharmacokinetics supports the routine use of an intraspecies uncertainty factor in the range of 1-10 as being protective of greater than 99% of the human population. The intraspecies uncertainty factor is highly protective of various subpopulations, including infants and children.     

Humans appear no more sensitive than laboratory animals to the inhibition of red blood cell cholinesterase by dichlorvos

Inhibition of red blood cell (RBC) cholinesterase is a consistent and sensitive indicator of exposure to dichlorvos (DDVP). Absent human data, default 10-fold adjustment factors for potential interspecies and intraspecies sensitivity differences would be used in developing a reference dose from the no observed effect levels for this endpoint obtained in toxicological assessments of laboratory animals. However, many studies of the cholinesterase-inhibiting effects associated with DDVP exposure have been conducted in humans, including healthy male volunteers, other healthy subpopulations, and diverse clinical subpopulations. Indeed, ample human data exist to permit a data-based assessment of potential interspecies sensitivity differences in RBC cholinesterase inhibition associated with DDVP exposure. In aggregate, these data demonstrate that the DDVP doses producing inhibition in humans are virtually identical to those eliciting the same levels of inhibition in laboratory rats, mice, monkeys, and dogs. Thus, healthy humans appear to be no more sensitive than laboratory animals to DDVP's effects on RBC cholinesterase, and an interspecies uncertainty factor of 1 is appropriate and scientifically warranted for use in DDVP risk assessments.     

Pharmacokinetics, pharmacodynamics, and safety of inhaled cyclosporin A (ADI628) after single and repeated administration in healthy male and female subjects and asthmatic patients

Severe asthmatics treated with oral/inhaled corticosteroids are at risk of side effects (adrenal suppression). Oral cyclosporin A has been effective in asthma treatment, and nebulized cyclosporin A has been administered for approximately 6 months with no nephrotoxicity or hepatotoxicity, suggesting a wider therapeutic margin for an inhaled cyclosporin A for treatment of asthma. Single- and repeated-dose studies in healthy and asthmatic male and female subjects were conducted to determine the pharmacokinetics, pharmacodynamics, and safety of a new formulation of inhaled cyclosporin A (ADI628) metered-dose inhaler (MDI). ADI628 had roughly dose-linear increases in blood concentrations with moderate variability after single and multiple administration in healthy subjects. Steady-state ADI628 concentrations reflected an effective half-life of 7.0 to 12.5 hours. No overt gender-related differences were observed after single inhaled 10 mg ADI628 dose. However, asthmatics and females (20 mg dose group) had lower ADI628 concentrations as compared to healthy males, probably due to lower inspiratory flow rates and probably not due to disease- or gender-related differences in metabolism/elimination of ADI628. Renal excretion was a minor route of elimination for ADI628 with no dose- or gender-related differences. The blood ADI628 exposure in humans was 1/3- to 1/6-fold lower than the no-effect dose in dogs. Also, the blood ADI628 exposure after the highest inhaled dose was much lower than after the administration of the efficacious oral cyclosporin A dose (3 mg/kg) for treating asthma. The highest steady-state dose (10 mg bid) resulted in ADI628 concentrations that are not typically associated with systemic nephrotoxicity or immunosuppression. Furthermore, repeated inhaled doses of ADI628 were safe and generally well tolerated with no apparent systemic immunosuppressive activity in healthy and asthmatic subjects.     

Elucidation of Human Amphotericin B Pharmacokinetics: Identification of a New Potential Factor Affecting Interspecies Pharmacokinetic Scaling

Purpose. To elucidate the pharmacokinetics of amphotericin B in rats, mice and humans, and to perform interspecies scaling to humans using allometry. Methods. Plasma concentrations following intravenous bolus administration in rats, and mice were determined by HPLC. Human pharmacokinetic parameters elucidated from literature data were validated in a preliminary study involving a patient receiving daily infusion dose for 27 days. A critical literature review was conducted to identify appropriate pharmacokinetic parameter values in other species for interspecies scale-up. Interspecies allometric scale-up was performed across mice, rats, rabbits and dogs and the resulting predictions in humans were compared to observed values. Results. A triexponential decline in rat, mouse and human plasma concentrations were observed. No gender differences in rat pharmacokinetics were observed. In contrast to allometry, mouse CL was smaller (82 vs 116 ml/h/kg) and T_0.5 (33 vs 20 h) was longer compared to rat. In the preliminary human study, Cpeak and Cmin values remained relatively constant over the duration of therapy, and a CL, MRT, T_0.5, Vss and Vdarea of 26 ml/h/kg, 10 and 23 days, 6.2 and 20 L/kg, respectively, were estimated. The relative contributions of the terminal phase area in rat, mouse and human were 75%, 92% and 31%, respectively. Interspecies allometric scale-up predictions of human CL (41 ml/h/kg), CLu (467 ml/h/kg) and Vss (3.3 L/kg) were similar to reported values, whereas poor predictions of human Vuss (33 L/kg), Vdarea (4.1 L/kg) and T_0.5 (3 days) were obtained. Conclusions. Insignificant accumulation in humans inspite of the long terminal T_0.5 was rationalized to be due to the small terminal-phase area contribution. While human CL and Vss were sucessfully predicted in the interspecies scaling, poor predictions of human Vdarea and T_0.5 were obtained, which was attributed to disposition pattern differences between humans and other species, a potential new critical factor affecting interspecies scale-up.     

Derivation of an Oral Reference Dose (RfD) for the Nonphthalate Alternative Plasticizer 1,2-Cyclohexane Dicarboxylic Acid,Di-Isononyl Ester (DINCH)

1,2-Cyclohexanedicarboxylic acid, 1,2-diisononylester (DINCH), a polyvinyl chloride plasticizer, has food, beverage, and medical device applications that may result in general population exposure. Although no apparent toxicity information in humans was identified, there is a substantial data set in lab animals to serve as the basis of hazard identification for DINCH. Target tissues associated with repeated dietary DINCH exposure in lab animals included liver, kidney, and thyroid and mammary glands. In contrast to some phthalate ester plasticizers, DINCH did not show evidence of hepatic peroxisomal proliferation, testicular toxicity, or liver tumors in rats. Liver and thyroid effects associated with DINCH exposure were attributed to compensatory thyroid stimulation secondary to prolonged metabolic enzyme induction. The toxicological significance of mammary fibroadenomas in female rats is unclear, given that this common benign and spontaneously occurring tumor type is unique to rats. The weight of evidence suggests DINCH is not genotoxic and the proposed mode of action (MOA) for thyroid gland lesions was considered to have a threshold. No adverse reproductive effects were seen in a two-generation study. An oral reference dose (RfD) of 0.7 mg/kg-d was derived from a human equivalent BMDL₁₀ of 21 mg/kg-d for thyroid hypertrophy/hyperplasia seen in adult F₁ rats also exposed in utero. The total uncertainty factor of 30x was comprised of intraspecies (10×) and database (3×) factors. An interspecies extrapolation factor was not applied since rodents are more sensitive than humans with respect to the proposed indirect MOA for thyroid gland lesions.     

Toxicology and pharmacology of sodium ricinoleate.

Ricinoleic acid constitutes approximately 90% of the fatty acid content of castor oil. Castor oil is known for its purgative effects and can be used to induce labor. Both castor oil and ricinoleic acid are approved for use in food. The mechanistic basis for purgative actions likely includes the membrane-disruptive effects of detergent-like molecules, such as sodium ricinoleate (a 'soap'). These effects have been shown to be dose-related and to exhibit a threshold below which no laxative response was evident, in both animals and in humans. Castor oil was not toxic in subchronic feeding studies in rodents at doses ranging up to 10-20% of the diet. Sodium ricinoleate, as a surfactant, demonstrates predictable skin and mucus membrane irritant effects, and may induce a Type IV dermal sensitization response in those previously sensitized to it. However, food-grade castor oil and sodium ricinoleate are prepared in such a manner as to be free of the castor bean constituents that have been proven to be the source of reported Type I immediate hypersensitivity responses. Feeding studies with castor oil in rodents provide a basis for a no observable adverse effect level (NOAEL) estimate of 7,500 mg/kg/day and 5,000 mg/kg/day in mice and rats, respectively (). Applying an uncertainty factor of 100 to the lesser of these NOAELs, one can thus estimate an acceptable daily intake (ADI) in man to be 50 mg/kg, or 3,000 mg of castor oil per day in an average 60 kg person. As ricinoleic acid constitutes approximately 90% of castor oil, applying this calculation to the 3,000 mg/day estimated ADI in humans for castor oil (given the rapid hydrolysis of castor oil glyceride in the gastrointestinal tract), the acceptable daily intake of ricinoleic acid may be as high as 2,400 mg/person.     

An analysis of the need for an additional uncertainty factor for infants and children

Uncertainty factors have been used for over 40 years to establish safe intakes of threshold toxicants. Tenfold factors are used to allow for species differences and for human variability, with extra factors for database inadequacies. The proposal to introduce an additional 10-fold factor for pesticides when exposure of infants and children is anticipated implies either age-related differences between species or differences within humans which exceed those present in adults. Alternatively, the extra factor could be related to deficiencies of current testing methods or concerns over irreversibility in developing organ systems. Available data do not provide a scientific rationale for the extra factor due to inadequacy of inter- and intraspecies uncertainty factors. Justification for the factor therefore must relate to the adequacy and sensitivity of current methods or concern about irreversible effects in the developing organism.     

Assessment of molybdenum toxicity in humans.

In an attempt to define a tolerable daily intake (TDI) for molybdenum based on a toxicological risk analysis approach, a large literature survey was conducted. In man, absorption of molybdenum after oral intake is in the range of 28-77% and urinary excretion is 17-80% of the total dose. A low order of toxicity of molybdenum compounds has been observed in humans. However, with the available data, it is not possible to calculate any dose-response or dose-effect relationships. Because molybdenum toxicity is associated with copper intake or depleted copper stores in the body, humans who have an inadequate intake of dietary copper or some dysfunction in their copper metabolism that makes them copper-deficient could be at greater risk of molybdenum toxicity. In the absence of relevant human studies, animal studies were evaluated for the derivation of the TDI. Effects of Mo on reproduction and foetal development were found to be critical effects observed in rats and mice. A dose-response relationship was observed in a study by Fungwe et al., with a 'no observed adverse effect' level (NOAEL) and a 'lowest observed adverse effect' level (LOAEL) of 0.9 and 1.6 mg Mo kg(-1) day(-1), respectively. Applying uncertainty factors of 10 for intraspecies and 10 for interspecies differences to the NOAEL, a TDI of 0.009 mg Mo kg(-1) day(-1) was calculated. The TDI is given a medium confidence rating. This TDI is more than double the upper limit of adequate intake for adolescents and adults that was derived from the Mo content of the average diet in the USA.     

Magnitude and mechanistic determinants of the interspecies toxicokinetic uncertainty factor for organic chemicals

The interspecies uncertainty factor, UF(AH), of 10 was recently subdivided into two components to account separately for interspecies differences in toxicokinetics and toxicodynamics (UF(AH-TK)=3.16, UF(AH-TD)=3.16). Even though the UF(AH) in its composite or dissociated form is used during the health risk assessment of systemic toxicants, there is no convincing scientific basis to justify the use of the same UF for all chemicals. In this study, we use equations that describe the toxicokinetics of chemicals at steady-state to characterize the magnitude and mechanistic determinants of UF(AH-TK) for several volatile organic chemicals (VOCs). Further, algorithms have been developed to determine the magnitude of the components of UF(AH-TK), namely the UF(AH-TK-ABS) (accounting for interspecies differences in dose absorbed during identical inhalation exposure conditions), UF(AH-TK-MET) (referring to the factor by which the blood concentration of unchanged parent chemical differs from one species to another, due to metabolic clearance, when both species receive identical doses) and UF(AH-TK-DIS) (reflecting the magnitude of difference in chemical concentrations distributed in target tissues of two species when the arterial blood concentration in both species is identical). The results show that the body weight, the rate of alveolar ventilation, the fraction of cardiac output flowing to the liver, partition coefficients (blood:air and tissue:blood), and the hepatic extraction ratio are the only parameters that play a critical role in the extrapolation of tissue and blood concentrations across species. Further, the magnitude of the UF(AH-TK-ABS) (means+/-SD, 0.19+/-0.04), UF(AH-TK-MET) (means+/-SD, 0.24+/-0.05) and UF(AH-TK-DIS) (mean range: 1.76-0.93) obtained in this study for several VOCs compares well with that obtained previously using physiologically based toxicokinetic models.     

Uncertainty factors for chemical risk assessment: interspecies differences in glucuronidation

For the risk assessment of effects other than cancer, a safe daily intake in humans is generally derived from a surrogate threshold dose (e.g. NOAEL) in an animal species to which an uncertainty factor of 100 is usually applied. This 100-fold is to allow for possible interspecies (10-fold) and interindividual (10-fold) differences in response to a toxicant, and incorporates toxicodynamic and toxicokinetic aspects of variability. The current study determined the magnitude of the interspecies differences in the internal dose of compounds for which glucuronidation is the major pathway of metabolism in either humans or in the test species. The results showed that there are major interspecies differences in the nature of the biological processes which influence the internal dose, including the route of metabolism, the extent of presystemic metabolism and enterohepatic recirculation. The work presented does not support the refinement of the interspecies toxicokinetic default to species- and pathway-specific values, but demonstrates the necessity for risk assessments to be carried out using quantitative chemical-specific data which define the fundamental processes which will influence the internal dose of a chemical (toxicokinetics), or the interaction of toxicant with its target site (toxicodynamics).     

CYP2E1 expression in bone marrow and its intra- and interspecies variability: approaches for a more reliable extrapolation from one species to another in the risk assessment of chemicals

When characterizing the health risks for man by exposure to chemicals, species-specific differences have to be taken into consideration, otherwise extrapolation from animal data to the human situation would be inadequate. The site-specific toxicity of chemicals may be explained by the following alternatives: (1) reactive metabolites are generated in the liver and subsequently transported to the target tissue(s); (2) metabolism of the parent compound occurs in the target tissue, a pathway by which the enzymes necessary for activation must be expressed in the target tissue. Cytochrome P450 2E1 (CYP2E1) is an important phase-I enzyme activating several chemicals. In the study described in this paper, myeloid intra- and interspecies variability in the expression of CYP2E1 has been investigated in rats, rabbits and man, because the bone marrow represents an important target organ for toxic effects of several chemicals, e.g. benzene. CYP2E1 at the protein level was detected by Western blotting and enzyme activities were determined by CYP2E1-dependent hydroxylation of chlorzoxazone (CLX). In the bone marrow of Wistar rats, the CLX hydroxylase activities were within the same order of magnitude (range: 0.1–0.4 pmol/mg protein per min) as previously described for mice (range 0.2–0.8 pmol/mg protein per min), whereas the CYP2E1 activities in two strains of rabbits were significantly higher (range: 1.7–4.7 pmol/mg protein per min) than in the rodents (P < 0.05). In human CD34⁺ bone marrow stem cells, CYP2E1 could also be detected on the protein level by Western blotting. The data demonstrate a presence of CYP2E1 in the bone marrow of all species investigated, thus supporting the hypothesis of CYP2E1-dependent local metabolism of several chemicals as a factor possibly contributing to their myelotoxicity and haematotoxicity. The data show that intraspecies/intrastrain variability of CYP2E1 activity in rodents is small. However, CYP2E1 activity between rodents and a non-rodent species was quite different indicating considerable interspecies variability. Received: 5 August 1999 / Accepted: 5 November 1999     

Chemical-specific adjustment factors (inter-species toxicokinetics) to establish the ADI for steviol glycosides

The acceptable daily intake (ADI) of commercially available steviol glycosides is currently 0–4 mg/kg body weight (bw)/day, based on application of a 100-fold uncertainty factor to a no-observed-adverse-effect-level value from a chronic rat study. Within the 100-fold uncertainty factor is a 10-fold uncertainty factor to account for inter-species differences in toxicokinetics (4-fold) and toxicodynamics (2.5-fold). Single dose pharmacokinetics of stevioside were studied in rats (40 and 1000 mg/kg bw) and in male human subjects (40 mg/kg bw) to generate a chemical-specific, inter-species toxicokinetic adjustment factor. T_max values for steviol were at ∼8 and ∼20 h after administration in rats and humans, respectively. Peak concentrations of steviol were similar in rats and humans, while steviol glucuronide concentrations were significantly higher in humans. Glucuronidation in rats was not saturated over the dose range 40–1000 mg/kg bw. The AUC_0-last for steviol was approximately 2.8-fold greater in humans compared to rats. Chemical-specific adjustment factors for extrapolating toxicokinetics from rat to human of 1 and 2.8 were established based on C_max and AUC_0-last data respectively. Because these factors are lower than the default value of 4.0, a higher ADI for steviol glycosides of between 6 and 16 mg/kg bw/d is justified.