Evaluating the evidence for non-monotonic dose-response relationships: A systematic literature review and (re-)analysis of in vivo toxicity data in the area of food safety.

This study aims to evaluate the evidence for the existence of non-monotonic dose-responses (NMDRs) of substances in the area of food safety. This review was performed following the systematic review methodology with the aim to identify in vivo studies published between January 2002 and February 2015 containing evidence for potential NMDRs. Inclusion and reliability criteria were defined and used to select relevant and reliable studies. A set of six checkpoints was developed to establish the likelihood that the data retrieved contained evidence for NMDR. In this review, 49 in vivo studies were identified as relevant and reliable, of which 42 were used for dose-response analysis. These studies contained 179 in vivo dose-response datasets with at least five dose groups (and a control group) as fewer doses cannot provide evidence for NMDR. These datasets were extracted and analyzed using the PROAST software package. The resulting dose-response relationships were evaluated for possible evidence of NMDRs by applying the six checkpoints. In total, 10 out of the 179 in vivo datasets fulfilled all six checkpoints. While these datasets could be considered as providing evidence for NMDR, replicated studies would still be needed to check if the results can be reproduced to rule out that the non-monotonicity was caused by incidental anomalies in that specific study. This approach, combining a systematic review with a set of checkpoints, is new and appears useful for future evaluations of the dose response datasets regarding evidence of non-monotonicity.

Does EU legislation allow the use of the Benchmark Dose (BMD) approach for risk assessment?

Hazard characterisation is largely based on an approach of (statistically) comparing dose groups with the controls in order to derive points of departure such as no-observed-adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs). This approach suggests the absence of any relevant effect at the NOAEL. The NOAEL approach has been debated for decades. A recent Scientific Opinion by the European Food Safety Authority (EFSA) concluded that the Benchmark Dose (BMD) approach should be preferred over the NOAEL approach for deriving human (health-based) limit or guidance values. Nonetheless, the BMD approach is used infrequently within European regulatory frameworks. The reason for this may lie in legislation or guidelines requiring the use of the NOAEL approach. In this context, various EU regulatory frameworks were examined on such demands. Interestingly, no single legislation was identified containing statutory requirements in conflict with the use of the BMD approach.

Gaussian Quadrature is an efficient method for the back-transformation in estimating the usual intake distribution when assessing dietary exposure.

In dietary exposure assessment, statistical methods exist for estimating the usual intake distribution from daily intake data. These methods transform the dietary intake data to normal observations, eliminate the within-person variance, and then back-transform the data to the original scale. We propose Gaussian Quadrature (GQ), a numerical integration method, as an efficient way of back-transformation. We compare GQ with six published methods. One method uses a log-transformation, while the other methods, including GQ, use a Box-Cox transformation. This study shows that, for various parameter choices, the methods with a Box-Cox transformation estimate the theoretical usual intake distributions quite well, although one method, a Taylor approximation, is less accurate. Two applications--on folate intake and fruit consumption--confirmed these results. In one extreme case, some methods, including GQ, could not be applied for low percentiles. We solved this problem by modifying GQ. One method is based on the assumption that the daily intakes are log-normally distributed. Even if this condition is not fulfilled, the log-transformation performs well as long as the within-individual variance is small compared to the mean. We conclude that the modified GQ is an efficient, fast and accurate method for estimating the usual intake distribution.

Carcinogenic activity of benzo[a]pyrene in a 2 year oral study in Wistar rats.

Because of the relatively high human oral exposure to polycyclic aromatic hydrocarbons (PAHs) compared to the inhalation exposure, the known carcinogenicity of this type of compounds and the limited data from oral studies available with polycyclic aromatic hydrocarbons, an oral carcinogenicity study was performed using benzo[a]pyrene (B[a]P) as a PAH representative. Wistar rats, 52 animals per sex and group were exposed daily (5 days a week) to 0, 3, 10 or 30 mg B[a]P/kg bw/day by gavage for 104 weeks and were subject to gross- and histopathology. The main tumours observed were hepatocellular carcinomas and forestomach tumours. Other tumours induced in this study were tumours of the auditory canal, skin and appendages, oral cavity, small intestine, kidney, and soft tissue sarcomas. For hepatocellular carcinomas and forestomach tumours, the BMDL10 were 3 and 1 mg/kg bw/day, respectively. The incidence of altered hepatic foci was increased in the 3mg/kg bw/day group. The increase in liver tumours is considered the most relevant effect for human risk assessment in terms of pathogenesis and sensitivity, and is proposed as the basis for human cancer risk assessment for oral PAH exposure.

Approaches to the risk assessment of genotoxic carcinogens in food: a critical appraisal.

The present paper examines the particular difficulties presented by low levels of food-borne DNA-reactive genotoxic carcinogens, some of which may be difficult to eliminate completely from the diet, and proposes a structured approach for the evaluation of such compounds. While the ALARA approach is widely applicable to all substances in food that are both carcinogenic and genotoxic, it does not take carcinogenic potency into account and, therefore, does not permit prioritisation based on potential risk or concern. In the absence of carcinogenicity dose-response data, an assessment based on comparison with an appropriate threshold of toxicological concern may be possible. When carcinogenicity data from animal bioassays are available, a useful analysis is achieved by the calculation of margins of exposure (MOEs), which can be used to compare animal potency data with human exposure scenarios. Two reference points on the dose-response relationship that can be used for MOE calculation were examined; the T25 value, which is derived from linear extrapolation, and the BMDL10, which is derived from mathematical modelling of the dose-response data. The above approaches were applied to selected food-borne genotoxic carcinogens. The proposed approach is applicable to all substances in food that are DNA-reactive genotoxic carcinogens and enables the formulation of appropriate semi-quantitative advice to risk managers.

Human variability in polymorphic CYP2D6 metabolism: is the kinetic default uncertainty factor adequate?

Human variability in the kinetics of CYP2D6 substrates has been quantified using a database of compounds metabolised extensively (>60%) by this polymorphic enzyme. Published pharmacokinetic studies (after oral and intravenous dosing) in non-phenotyped healthy adults, and phenotyped extensive (EMs), intermediate or slow-extensive (SEMs) and poor metabolisers (PMs) have been analysed using data for parameters that relate primarily to chronic exposure (metabolic and total clearances, area under the plasma concentration time-curve) and primarily to acute exposure (peak concentration). Similar analyses were performed with the available data for subgroups of the population (age, ethnicity and disease). Interindividual differences in kinetics for markers of oral exposure were large for non-phenotyped individuals and for EMs (coefficients of variation were 67-71% for clearances and 54-63% for C(max)), whereas the intravenous data indicated a lower variability (34-38%). Comparisons between EMs, SEMs and PMs revealed an increase in oral internal dose for SEMs and PMs (ratio compared to EMs=3 and 9-12, respectively) associated with lower variability than that for non-phenotyped individuals (coefficients of variation were 32-38% and 30% for SEMs and PMs, respectively). In relation to the uncertainty factors used for risk assessment, most subgroups would not be covered by the kinetic default of 3.16. CYP2D6-related factors necessary to cover 95-99% of each subpopulation ranged from 2.7 to 4.1 in non-phenotyped healthy adults and EMs to 15-18 in PMs and 22-45 in children. An exponential relationship (R(2)=0.8) was found between the extent of CYP2D6 metabolism and the uncertainty factors. The extent of CYP2D6 involvement in the metabolism of a substrate is critical in the estimation of the CYP2D6-related factor. The 3.16 kinetic default factor would cover PMs for substrates for which CYP2D6 was responsible for up to 25% of the metabolism in EMs.

Mathematical modelling and quantitative methods.

The present review reports on the mathematical methods and statistical techniques presently available for hazard characterisation. The state of the art of mathematical modelling and quantitative methods used currently for regulatory decision-making in Europe and additional potential methods for risk assessment of chemicals in food and diet are described. Existing practices of JECFA, FDA, EPA, etc., are examined for their similarities and differences. A framework is established for the development of new and improved quantitative methodologies. Areas for refinement, improvement and increase of efficiency of each method are identified in a gap analysis. Based on this critical evaluation, needs for future research are defined. It is concluded from our work that mathematical modelling of the dose-response relationship would improve the risk assessment process. An adequate characterisation of the dose-response relationship by mathematical modelling clearly requires the use of a sufficient number of dose groups to achieve a range of different response levels. This need not necessarily lead to an increase in the total number of animals in the study if an appropriate design is used. Chemical-specific data relating to the mode or mechanism of action and/or the toxicokinetics of the chemical should be used for dose-response characterisation whenever possible. It is concluded that a single method of hazard characterisation would not be suitable for all kinds of risk assessments, and that a range of different approaches is necessary so that the method used is the most appropriate for the data available and for the risk characterisation issue. Future refinements to dose-response characterisation should incorporate more clearly the extent of uncertainty and variability in the resulting output.

Ranking of allergenic potency of rubber chemicals in a modified local lymph node assay.

A modified local lymph node assay (LLNA) with ex vivo tritium thymidine (3H-TdR) labeling of the proliferating lymph node cells was used for determination of the allergenic potency of chemicals used in the production of rubber for latex medical gloves. Fifteen chemicals known to induce contact hypersensitivity reactions in man, including various thiuram, carbamate, and benzothiazole compounds, and one amine were tested. The EC3 (effective concentration inducing a 3-fold increase in proliferation of lymph node cells [Stimulation Index, SI = 3]) was calculated with nonlinear regression analysis, including a bootstrap method for determination of the 5-95% confidence interval of the EC3 value. This procedure identified 14 out of the 15 chemicals tested as sensitizers, while for one chemical, ZDBC, no EC3 could be calculated due to low responses and a lack of a dose-response relationship in the data obtained. The ranking order of the chemicals with increasing EC3 values (and thus decreasing allergenic potency) was found to be in the following order: ZDEC < TMTD < TETD < ZPC < ZDMC < MBTS < PTD < TMTM < MBT < MBI < PTT < ZMBT < TBTD < DEA < ZDBC. Our results indicate that the chemicals of choice for use in the production of natural rubber latex products would be for the thiuram compounds, TBTD; for the carbamates, ZDBC; and for the benzothiazoles, ZMBT. However, one has to be aware that besides potency, the total amount of residual chemical present in the final product is also important for allergy induction.

Comparison of dose-responses of contact allergens using the guinea pig maximization test and the local lymph node assay.

The guinea pig maximization test (GPMT) has been used as a method for the prediction of skin sensitizing potential for over 30 years. Besides hazard identification, risk assessment of sensitizing chemicals requires the assessment of potency. For the determination of potency based on lowest effective dose levels, dose-response studies are required. In the standard GPMT a single concentration is used for intracutaneous and topical induction and the assay provides a qualitative assessment of allergenicity. This paper presents data derived from quantitative evaluation of the sensitizing potency of chemicals in the GPMT, based on multiple concentrations. We performed the GPMT in accordance with the original procedure of Magnusson and Kligman; and included in this procedure a range of intradermal and topical concentrations for induction. Three allergens with different sensitizing potencies, diethylamine (DEA), tetramethyl thiuram disulfide (TMTD) and zinc dimethyl dithiocarbamate (ZDMC) were tested. The data obtained with this test procedure were compared to data we previously obtained using the local lymph node assay (LLNA). Both the GPMT and the LLNA showed dose response relationships for the three chemicals tested. For the chemicals tested, both tests differed in the relative potencies based on benchmark concentrations. While both tests ranked DEA as the least potent allergen, the GPMT ranked ZDMC more potent than TMTD, the reverse being found in the LLNA. The nature of the data provided in the LLNA makes it likely that benchmarks as defined with this test are more reliable than that defined in the GPMT. However, further validation with human data is necessary.

The benchmark approach applied to a 28-day toxicity study with Rhodorsil Silane in rats. the impact of increasing the number of dose groups.

The OECD study design, aimed at obtaining a no-observed-adverse-effect level (NOAEL), may be suboptimal for deriving a benchmark dose. Therefore the present subacute (28-day) study was carried out to evaluate a multiple dose study design and to compare the results with the common OECD design. Seven groups of 10 female rats each were intragastrically administered corn oil without (controls) or with 50, 150, 300, 450, 600 or 750 mg Rhodorsil Silane/kg body weight/day, once daily (7 days/week) for 4 weeks. From the complete dataset, two subsets were selected, one representing a study design with seven dose groups of five animals (7 x 5 design), the other representing a study design with four dose groups of 10 animals (4 x 10 design). Under the conditions of the present study, the NOAEL for Rhodorsil Silane 198 was assessed at 50 mg/kg body weight/day, based on the data of the 4 x 10 design. The benchmark approach resulted in a benchmark dose of 19 mg/kg body weight/day, based on the data of the 7 x 5 design. Comparison of the results demonstrated that the multiple dose (7 x 5) design led to a more reliable result than the OECD (4 x 10) design, despite the smaller total number of animals. The dose-response analysis showed that at "the NOAEL" the effect on relative spleen weight was larger than 10%, illustrating that at the NOAEL, adverse effects may occur.

Developmental toxicity of butyl benzyl phthalate in the rat using a multiple dose study design.

The developmental toxicity of butyl benzyl phthalate (BBP) was investigated in the rat using ten dose groups between 270 and 2100 mg/kg/day. Exposure was by daily gavage from gestation day 5 through 16 or gestation day 5 through 20. Dose-response data were analyzed using the benchmark approach by fitting dose-response models to the various endpoints. BBP induced increased liver and kidney weights in dams, accompanied by liver enzyme increases in maternal serum. Extramedullary hematopoiesis, which was already substantial in control pregnant animals, was increased after BBP treatment. Fetotoxicity included increased resorptions, reduced fetal weights, increased incidence of skeletal anomalies, and reduced fetal testis weights in the presence of an increased incidence of retarded testicular descent. As embryotoxicity was found at lower dosages compared to observed maternal toxicity, BBP appeared to be a specifically embryotoxic compound. The extended exposure protocol (gestation day 5 through 20) appeared more sensitive for measuring fetotoxic effects. We recommend the use of more doses in toxicity tests, together with the benchmark approach as an appropriate and more accurate method for analyzing dose-response data compared to the NOAEL approach.

A quantitative method for assessing the sensitizing potency of low molecular weight chemicals using a local lymph node assay: employment of a regression method that includes determination of the uncertainty margins.

Risk assessment of sensitizing chemicals requires, besides hazard identification, the assessment of potency. To examine the sensitizing capacity of low molecular weight chemicals, a murine local lymph node assay (LLNA) was used. The sensitizing capacity of known allergens was quantified by dose-response modeling. At a stimulatory index (SI) of 3, the corresponding estimated concentration was calculated (EC(3)), together with a confidence interval to take account of the quality of the particular data set. We tested ten allergens (ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), 4-ethoxymethylene 2-phenyl oxazol-5-one (oxazolone), phthalic anhydride (PA), toluene diisocyanate (TDI), trimellitic anhydride (TMA), tetramethylthiuramdisulfide (TMTD) and zincdimethyldithiocarbamate (ZDMC)). Oxazolone showed the strongest sensitizing potency followed in this order by DNCB, TDI, TMA, PA, TMTD, ZDMC, MBT, benzocaine and DEA. The approach performed in this study is a way to accurately assess the potency of sensitizing chemicals and thus a possibility for classification.

Balancing the risks and benefits of drinking water disinfection: disability adjusted life-years on the scale.

To evaluate the applicability of disability adjusted life-years (DALYs) as a measure to compare positive and negative health effects of drinking water disinfection, we conducted a case study involving a hypothetical drinking water supply from surface water. This drinking water supply is typical in The Netherlands. We compared the reduction of the risk of infection with Cryptosporidium parvum by ozonation of water to the concomitant increase in risk of renal cell cancer arising from the production of bromate. We applied clinical, epidemiologic, and toxicologic data on morbidity and mortality to calculate the net health benefit in DALYs. We estimated the median risk of infection with C. parvum as 10(-3)/person-year. Ozonation reduces the median risk in the baseline approximately 7-fold, but bromate is produced in a concentration above current guideline levels. However, the health benefits of preventing gastroenteritis in the general population and premature death in patients with acquired immunodeficiency syndrome outweigh health losses by premature death from renal cell cancer by a factor of > 10. The net benefit is approximately 1 DALY/million person-years. The application of DALYs in principle allows us to more explicitly compare the public health risks and benefits of different management options. In practice, the application of DALYs may be hampered by the substantial degree of uncertainty, as is typical for risk assessment.

Estimation of dioxin and furan elimination rates with a pharmacokinetic model.

Quantitative description of the pharmacokinetics of dioxins and furans in humans can be of great help for the assessment of health risks posed by these compounds. To that the elimination rates of sixteen 2,3,7,8-chlorinated dibenzodioxins and dibenzofurans are estimated from both a longitudinal and a cross-sectional data set using the model of Van der Molen et al. [Van der Molen G.W., Kooijman S.A.L.M., and Slob W. A generic toxicokinetic model for persistent lipophilic compounds in humans: an application to TCDD. Fundam Appl Toxicol 1996: 31: 83-94]. In this model the elimination rate is given by the (constant) specific elimination rate multiplied with the ratio between the lipid weight of the liver and total body lipid weight. Body composition, body weight and intake are assumed to depend on age. The elimination rate is, therefore, not constant. For 49-year-old males, the elimination rate estimates range between 0.03 per year for 1,2,3,6,7,8-hexaCDF to 1.0 per year for octaCDF. The elimination rates of the most toxic congeners, 2,3,7,8-tetraCDD, 1,2,3,7,8-pentaCDD, and 2,3,4,7,8-pentaCDF, were estimated at 0.09, 0.06, and 0.07, respectively, based on the cross-sectional data, and 0.11, 0.09, and 0.09 based on the longitudinal data. The elimination rates of dioxins decrease with age between 0.0011 per year for 1,2,3,6,7,8-hexaCDD and 0.0035 per year for 1,2,3,4,6,7,8-heptaCDD. For furans the average decrease is 0.0033 per year. The elimination rates were estimated both from a longitudinal and a cross-sectional data set, and agreed quite well with each other, after taking account of historical changes in average intake levels.

Increased frequencies of diploid sperm detected by multicolour FISH after treatment of rats with carbendazim without micronucleus induction in peripheral blood erythrocytes.

The purpose of the present study was to determine the effect of a single oral dose of carbendazim (CARB) on the frequencies of numerical chromosome aberrations in sperm and on micronuclei in peripheral blood erythrocytes of rats. Dual colour FISH on epididymal sperm of rats treated 31 days before sacrifice (0, 50, 150, 450 and 800 mg/kg body wt CARB in corn oil), corresponding to exposure during late pachytene, revealed a clear induction of diploid sperm. Induction of aneuploid sperm was not observed. Although the absolute frequencies of diploidy were low, ranging from 0.03% in the control group to 0.22% in the highest dose group, the observed dose-response relationship was highly significant. In sperm of rats killed 50 days after treatment with CARB (corresponding to exposure of spermatogonial stem cells) the effect was no longer apparent. In a second experiment, in addition to more dose groups in the low dose range, the peripheral blood micronucleus assay was incorporated. Results of triple colour FISH on epididymal sperm of rats treated with CARB (0-800 mg/kg body wt) again showed induction of diploid, but not of aneuploid sperm. Induction was less prominent than in the first experiment, but the dose-response relationship for diploidy was again significant. In blood samples drawn from the tail vein 48 h after treatment with CARB induction of micronuclei in peripheral blood erythrocytes was not observed, whereas the micronucleus frequency was significantly increased after a single i. p. dose of mitomycin C (3 mg/kg body wt). In conclusion, the present results show that CARB induces diploidy in sperm, without an accompanying induction of micronuclei in erythrocytes. This finding suggests that in rats the peripheral blood micronucleus assay is a less sensitive indicator for the genotoxic potential of CARB than the epididymal sperm aneuploidy/diploidy assay.

Assessment factors for human health risk assessment: a discussion paper.

The general goal of this discussion paper is to contribute toward the further harmonization of human health risk assessment. It first discusses the development of a formal, harmonized set of assessment factors. The status quo with regard to assessment factors is reviewed, that is, the type of factors to be identified, the range of values assigned, as well as the presence or absence of a scientific basis for these values. Options are presented for a set of default values and probabilistic distributions for assessment factors based on the state of the art. Methods of combining default values or probabilistic distributions of assessment factors are also described. Second, the effect parameter, the no-observed-adverse-effect level (NOAEL), is discussed. This NOAEL as selected from the toxicological database may be a poor substitute for the unknown, true no-adverse-effect level (NAEL). New developments are presented with respect to the estimation of the NAEL. The already widely discussed Benchmark Dose concept can be extended to obtain an uncertainty distribution of the Critical Effect Dose (CED). This CED distribution can be combined with estimated uncertainty distributions for assessment factors. In this way the full distribution of the Human Limit Value will be derived and not only a point estimate, whereas information on dose-response relations is taken into account. Finally, a strategy is proposed for implementation of the new developments into human health risk assessments.

The estimation of elimination rates of persistent compounds: a re-analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin levels in Vietnam veterans.

The elimination rate of TCDD was re-estimated from measurements of internal concentrations in Vietnam veterans with a model that can account for age dependent body composition, and both age and (calendar) time dependent background intake. Estimates of the specific elimination rate, which is independent of body composition, did not differ much between fits with different simplifications of this model for this particular data set. However, the assumption that the associated half-life is constant over the human lifespan is improper. The overall elimination rate and half-life are only constant when background intake and body composition are approximately constant. For example, our model predicts that half-life ranges between 5.5 in young adults and 11 years in elderly men. The change of half-life with age should be accounted for in toxicokinetic calculations.

Comparative immunotoxicology of ultraviolet B exposure I. Effects of in vitro and in situ ultraviolet B exposure on the functional activity and morphology of Langerhans cells in the skin of different species.

Ultraviolet (UV) B-induced morphological and functional changes in the skin of mice, rats and humans were investigated. Changes in the morphological structure of Langerhans cells (LC), the major antigen-presenting cells in the skin, using confocal laser scanning microscopy, were found in mouse and rat skin after in situ exposure to high doses of UVB radiation (FS40) (3-9 kJ/m2). Similar UVB doses failed to induce alterations in the morphological structure of human LC. Alterations in the function of epidermal cells (especially LC) were studied, using the mixed skin lymphocyte response (MSLR). In vitro UVB exposure of epidermal cells (EC), derived from the skin of the different species, revealed that low doses of UVB radiation impaired the stimulatory capacity of these cells dose-dependently; mouse epidermal cells were most UVB-susceptible, while human cells were least UVB susceptible. For suppression of the stimulatory capacity of EC after in situ UVB exposure of skin tissue, higher doses of UVB radiation than the in vitro UVB exposure were needed in all species tested. Also in this in situ set-up mouse epidermal cells were most UVB-susceptible, and human epidermal cells were least UVB-susceptible. The magnitude of differences in susceptibility for UVB-induced changes in the stimulatory capacity of EC after in situ and after in vitro exposure experiments was similar. Firstly, it may be concluded that UVB impairs the functional activity of LC at a lower dose than that which alters the morphology of these cells. Secondly, it is clear that epidermal cells, especially LC, from the skin of rodents are more susceptible to UVB than epidermal cells derived from human skin. It is important to account for these differences in susceptibility when data on the effects of UVB radiation on the immune system in rodents are extrapolated to humans.

Evaluation of the uncertainty factor for subchronic-to-chronic extrapolation: statistical analysis of toxicity data.

For the extrapolation of a subchronic no-observed-adverse-effect level (NOAEL) to a chronic NOAEL an uncertainty factor of 10 is routinely applied. We evaluated this uncertainty factor by statistically analyzing a database comprising 149 oral NOAEL(subchronic)/NOAEL(chronic) ratios. Since any database forms only a limited sample of all existent chemicals, we believe that estimation errors should be taken into account. Therefore, in addition to the 95th percentile (P95) of the ratio distribution, we calculated the 95% confidence interval (CI) of this percentile. The geometric mean (GM), geometric standard deviation (GSD), P95, and CI were 1.7, 5.6, 29, and 20-46, respectively. These data do not support a lowering of the uncertainty factor as has been suggested by others. Furthermore, we analyzed the LOAEL/NOAEL ratio for subacute, subchronic, and chronic toxicity studies. However, the size of the LOAEL/NOAEL ratio only depends on the spacing between doses of the studies reviewed. Therefore, though the results may be considered supportive for an uncertainty factor of 10, we believe that there is no justification for the use of such a factor. Instead, we recommend the use of dose-response modeling which would make LOAEL-to-NOAEL extrapolation redundant.

Immunotoxicology: extrapolation from animal to man--estimation of the immunotoxicologic risk associated with TBTO exposure.

Bis(tri-n-butyltin)oxide (TBTO) has been shown to be immunotoxic in rodents, resulting in decreased resistance to infections. The no-effect level assessed by estimating effects on host resistance in rats has been found to lie between 0.5 and 5.0 mg TBTO/kg food (0.025 and 0.25 mg/kg body weight). For risk assessment such animal data need to be extrapolated to the human situation. In risk assessment procedures uncertainty factors are used to account for interspecies variation (extrapolation from animal to man) and for variation within the human species. For both factors a value of 10 is often used, based on international guidelines. Hence, exposures below 0.00025 mg/kg body weight should not pose a risk for the human population. In the present study we have taken an alternative approach. We have produced dose-response curves for the effect of TBTO exposure on resistance to Trichinella spiralis. To extrapolate this curve to the human situation, we produced additional dose response data concerning in vitro effects of TBTO exposure on the mitogen responsiveness of both rat lymphoid cells and human blood cells. Using regression analyses of these dose-response data, we calculated a factor that accounts for interspecies variation (IEV) and a factor that accounts for intraspecies variation (IAV) within the human samples. Using these factors, we estimated the dose that decreases resistance in man to an infection. We choose 10% increase of the infectious load as a reference point which in our view is of biological significance. Based on these considerations, we estimated the dose that may affect resistance in adult humans at 0.04 mg/kg body weight. Pre- and postnatal exposure will probably result in effects at lower concentrations, due to the vulnerability of the developing immune system.