New Approach Methodologies (NAMs) for ad hoc human health risk assessment of food and non-food products - Proceedings of a workshop.

RIVM convened a workshop on the use of New Approach Methodologies (NAMs) for the ad hoc human health risk assessment of food and non-food products. Central to the workshop were two case studies of marketed products with a potential health concern: the botanical Tabernanthe iboga which is used to facilitate mental or spiritual insight or to (illegally) treat drug addiction and is associated with cardiotoxicity, and dermal creams containing female sex hormones, intended for use by perimenopausal women to reduce menopause symptoms without medical supervision. The workshop participants recognized that data from NAM approaches added valuable information for the ad hoc risk assessment of these products, although the available approaches were inadequate to derive health-based guidance values. Recommendations were provided on how to further enhance and implement NAM approaches in regulatory risk assessment, specifying both scientific and technical aspects as well as stakeholder engagement aspects.

Prenatal exposure of the ovine fetus to androgens reduces the proportion of neurons in the ventromedial and arcuate nucleus that are activated by short-term exposure to estrogen.

In sheep, the steroid control of gonadotropin-releasing hormone (GNRH) release is sexually differentiated such that estrogen can trigger a GNRH surge and attendant reproductive behaviors in the female, but not the male. Furthermore, female lambs that have been exposed to testosterone during a critical window of in utero development are also unable to generate a GNRH surge. This study tests the hypothesis that exposure of the ovine fetus to androgens alters the development of key steroid-receptive neuronal inputs to the GNRH neurons. In adulthood, this results in reduced activation of specific neurons by estrogen in the male and testosterone-treated female. To make this determination, groups of ewes, rams, and testosterone-exposed ewes were treated with estrogen, and the activation of neurons in the mediobasal hypothalamus and brain stem determined by immunocytochemistry. A lower percentage of neurons in the ventrolateral aspect of the ventromedial nucleus (vlVMN) and the caudal arcuate nucleus (cARC), but not the brainstem, was activated by a 6-h exposure to estrogen in the androgenized and male animals. In the vlVMN, some of these neurons contain somatostatin; however, the phenotype of activated neurons in the cARC remains unknown. These data suggest that specific neural populations in these brain regions are involved in the estrogen feedback control of GNRH release in the sheep, and that the defeminization of the surge-generating system by in utero androgen exposure results, in part, from a failure of estrogen to activate key neural phenotypes.

Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1'-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells.

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1'-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can be metabolized to 1'-hydroxyestragole by cytochrome P450 enzymes. Basil extract addition to incubations of rat and human liver S9 homogenates with 1'-hydroxyestragole, the sulfotransferase cofactor PAPS, and 2'-deoxyguanosine resulted in a dose-dependent inhibition of N2-(trans-isoestragol-3'-yl)-2'-deoxyguanosine formation. Because the inhibition resembled the inhibition by the sulfotransferase inhibitor pentachlorophenol and since the inhibition was not observed in incubations with the direct electrophile 1'-acetoxyestragole it is concluded that the inhibition occurs at the level of the sulfotransferase mediated bioactivation step. Additional experiments in HepG2 cells revealed the same protective effect of basil extract in intact cells, demonstrating that the inhibitors are able to enter the cells. The results of this study suggest that bioactivation and subsequent adverse effects of 1'-hydroxyestragole might be lower in a matrix of other basil ingredients than what would be expected on the basis of experiments using 1'-hydroxyestragole as a single compound.

Consumption of plant food supplements in the Netherlands.

The use of food supplements containing herbs or other botanical ingredients (plant food supplements, PFS) is on the rise. In some cases, PFS can contain compounds that are toxic and may pose a health risk. To assess the potential health risks, information on the consumption of PFS is required, however, this was lacking for the Netherlands. In the current study, the consumption of PFS was investigated for several subgroups in the Dutch population, including children. Data from the Dutch National Food Consumption Surveys were used to get a first impression on the consumption of PFS. To obtain more detailed information, a specific PFS consumption survey was performed using online questionnaires. First, a screening survey was performed among a representative sample of 75 100 adults and children of the Dutch population, followed by a main survey among 739 selected PFS users in eight different age and gender subgroups. The prevalence of PFS users in the Dutch population was approximately 10% for men, 17% for women and 13% for children. A wide variety of PFS was used, with around 600 different PFS reported, containing 345 different botanicals. The most frequently used botanicals were echinacea (Echinacea purpurea), ginkgo (Ginkgo biloba), cranberry (Vaccinium macrocarpon), ginseng (Panax ginseng) and algae (such as species belonging to the genus Spirulina or Chlorella). Because PFS are widely used in the Dutch population, it is important to evaluate the potential risks associated with PFS consumption in the Netherlands, including potential herb-drug interactions. The data collected in this study are of great value to assess these risks.

Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products.

An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent metabolite resorufin. The system was tested with three P450 1A2 inhibitors, for which minimum detectable amounts (MDA) ranging from 0.7 to 9.5 ng were obtained. Analysis of a kava kava and a basil extract showed that the on-line system is applicable to complex mixtures, since in both extracts, peaks with P450 1A2 inhibiting activity were observed.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and biphenyls (PCBs) in home-produced eggs.

Home produced eggs from 62 addresses in the Netherlands were investigated for the levels of polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs) and biphenyls (PCBs), both dioxin-like (dl) and non-dioxin-like (ndl). Compared to commercial eggs, levels were relatively high with a median of 4.6 pg TEQ g(-1) fat for the sum of PCDD/Fs and dl-PCBs, and a highest level of 18.9 pg TEQ g(-1) fat. A number of samples showed clearly elevated ndl-PCB levels with a median of 13 ng g(-1) fat and a highest level of 80 ng g(-1) fat. There were no clear regional differences, even though part of the samples were derived from the rather industrial Rotterdam/Rijnmond area. Based on the congener patterns, former backyard burning of waste seems the most likely source for most eggs, with two exceptions where other sources contributed to the contamination. Similar is true for the ndl-PCBs. The study shows that average levels are about ten-fold higher than commercial eggs and may substantially contribute to the intake of PCDD/Fs and dl-PCBs by consumers. Intervention measures to reduce the intake of these contaminants by laying hens are advised.

Flavonoids and alkenylbenzenes: mechanisms of mutagenic action and carcinogenic risk.

The present review focuses on the mechanisms of mutagenic action and the carcinogenic risk of two categories of botanical ingredients, namely the flavonoids with quercetin as an important bioactive representative, and the alkenylbenzenes, namely safrole, methyleugenol and estragole. For quercetin a metabolic pathway for activation to DNA-reactive species may include enzymatic and/or chemical oxidation of quercetin to quercetin ortho-quinone, followed by isomerisation of the ortho-quinone to quinone methides. These quinone methides are suggested to be the active alkylating DNA-reactive intermediates. Recent results have demonstrated the formation of quercetin DNA adducts in exposed cells in vitro. The question that remains to be answered is why these genotoxic characteristics of quercetin are not reflected by carcinogenicity. This might in part be related to the transient nature of quercetin quinone methide adducts, and suggests that stability and/or repair of DNA adducts may need increased attention in in vitro genotoxicity studies. Thus, in vitro mutagenicity studies should put more emphasis on the transient nature of the DNA adducts responsible for the mutagenicity in vitro, since this transient nature of the formed DNA adducts may play an essential role in whether the genotoxicity observed in vitro will have any impact in vivo. For alkenylbenzenes the ultimate electrophilic and carcinogenic metabolites are the carbocations formed upon degradation of their 1'-sulfooxy derivatives, so bioactivation of the alkenylbenzenes to their ultimate carcinogens requires the involvement of cytochromes P450 and sulfotransferases. Identification of the cytochrome P450 isoenzymes involved in bioactivation of the alkenylbenzenes identifies the groups within the population possibly at increased risk, due to life style factors or genetic polymorphisms leading to rapid metaboliser phenotypes. Furthermore, toxicokinetics for conversion of the alkenylbenzenes to their carcinogenic metabolites and kinetics for repair of the DNA adducts formed provide other important aspects that have to be taken into account in the high to low dose risk extrapolation in the risk assessment on alkenylbenzenes. Altogether the present review stresses that species differences and mechanistic data have to be taken into account and that new mechanism- and toxicokinetic-based methods and models are required for cancer risk extrapolation from high dose experimental animal data to low dose carcinogenic risks for man.

Dioxins, PCBs and heavy metals in Chinese mitten crabs from Dutch rivers and lakes.

Chinese mitten crab is an invasive species in many European rivers and lakes. Data from the UK indicated high levels of dioxins and PCBs, in particular in the brown meat in the body. This was confirmed by studies in the Netherlands, showing average levels of dioxins and PCBs in the meat in the body of 43 pg TEQ g(-1) ww in crabs caught in the large rivers. Levels in crab of lakes in the Northern part of the Netherlands were on average 3.7-fold lower. Consumption of crabs from polluted areas results in a relatively high dose of dioxins and dl-PCBs and could significantly increase the intake above the TWI. However, in general consumption of these crabs is low, even in the Asian sub-population in the Netherlands. Cadmium and lead levels were higher in crabs from contaminated areas, but for mercury and arsenic there was no clear difference. Consumption of crabs would not result in significant risks for cadmium and mercury. For lead the daily intake could be raised above the BMDL01 for neurodevelopmental toxicity, but this would only occur on a limited number of days. For arsenic the exposure would exceed the lower end of the BMDL01 values for certain cancers, but again, the infrequent consumption by most consumers reduces this risk. Furthermore, speciation showed that most arsenic in crabs was probably not a toxic inorganic form, but likely to be in an organic form.

Evaluation of human interindividual variation in bioactivation of estragole using physiologically based biokinetic modeling.

The present study investigates interindividual variation in liver levels of the proximate carcinogenic metabolite of estragole, 1'-hydroxyestragole, due to variation in two key metabolic reactions involved in the formation and detoxification of this metabolite, namely 1'-hydroxylation of estragole and oxidation of 1'-hydroxyestragole. Formation of 1'-hydroxyestragole is predominantly catalyzed by P450 1A2, 2A6, and 2E1, and results of the present study support that oxidation of 1'-hydroxyestragole is catalyzed by 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2). In a first approach, the study defines physiologically based biokinetic (PBBK) models for 14 individual human subjects, revealing a 1.8-fold interindividual variation in the area under the liver concentration-time curve (AUC) for 1'-hydroxyestragole within this group of human subjects. Variation in oxidation of 1'-hydroxyestragole by 17beta-HSD2 was shown to result in larger effects than those caused by variation in P450 enzyme activity. In a second approach, a Monte Carlo simulation was performed to evaluate the extent of variation in liver levels of 1'-hydroxyestragole that could occur in the population as a whole. This analysis could be used to derive a chemical-specific adjustment factor (CSAF), which is defined as the 99th percentile divided by the 50th percentile of the predicted distribution of the AUC of 1'-hydroxyestragole in the liver. The CSAF was estimated to range between 1.6 and 4.0, depending on the level of variation that was taken into account for oxidation of 1'-hydroxyestragole. Comparison of the CSAF to the default uncertainty factor of 3.16 for human variability in biokinetics reveals that the default uncertainty factor adequately protects 99% of the population.

Human cytochrome p450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes.

Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1'-hydroxyestragole were identified and compared to the enzymes of importance for 1'-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that all enzymes tested, except P450 2C8, are intrinsically able to 1'-hydroxylate estragole. Experiments with Gentest microsomes, expressing P450 enzymes to roughly average liver levels, indicated that P450 1A2, 2A6, 2C19, 2D6, and 2E1 might contribute to estragole 1'-hydroxylation in the human liver. Especially P450 1A2 is an important enzyme based on the correlation between P450 1A2 activity and estragole 1'-hydroxylation in human liver microsomal samples and inhibition of estragole 1'-hydroxylation by the P450 1A2 inhibitor alpha-naphthoflavone. Kinetic studies revealed that, at physiologically relevant concentrations of estragole, P450 1A2 and 2A6 are the most important enzymes for bioactivation in the human liver showing enzyme efficiencies (kcat/Km) of, respectively, 59 and 341 min-1 mM-1. Only at relatively high estragole concentrations, P450 2C19, 2D6, and 2E1 might contribute to some extent. Comparison to results from similar studies for safrole and methyleugenol revealed that competitive interactions between estragole and methyleugenol 1'-hydroxylation and between estragole and safrole 1'-hydroxylation are to be expected because of the involvement of, respectively, P450 1A2 and P450 2A6 in the bioactivation of these compounds. Furthermore, poor metabolizer phenotypes in P450 2A6 might diminish the chances on bioactivation of estragole and safrole, whereas lifestyle factors increasing P450 1A2 activities such as cigarette smoking and consumption of charbroiled food might increase those chances for estragole and methyleugenol.

Human cytochrome p450 enzymes of importance for the bioactivation of methyleugenol to the proximate carcinogen 1'-hydroxymethyleugenol.

In vitro studies were performed to elucidate the human cytochrome P450 enzymes involved in the bioactivation of methyleugenol to its proximate carcinogen 1'-hydroxymethyleugenol. Incubations with Supersomes, expressing individual P450 enzymes to a high level, revealed that P450 1A2, 2A6, 2C9, 2C19, and 2D6 are intrinsically able to 1'-hydroxylate methyleugenol. An additional experiment with Gentest microsomes, expressing the same individual enzymes to roughly average liver levels, indicated that P450 1A2, 2C9, 2C19, and 2D6 contribute to methyleugenol 1'-hydroxylation in the human liver. A study, in which correlations between methyleugenol 1'-hydroxylation in human liver microsomes from 15 individuals and the conversion of enzyme specific substrates by the same microsomes were investigated, showed that P450 1A2 and P450 2C9 are important enzymes in the bioactivation of methyleugenol. This was confirmed in an inhibition study in which pooled human liver microsomes were incubated with methyleugenol in the presence and absence of enzyme specific inhibitors. Kinetic studies revealed that at physiologically relevant concentrations of methyleugenol P450 1A2 is the most important enzyme for bioactivation of methyleugenol in the human liver showing an enzyme efficiency (kcat/Km) that is approximately 30, 50, and > 50 times higher than the enzyme efficiencies of, respectively, P450 2C9, 2C19, and 2D6. Only when relatively higher methyleugenol concentrations are present P450 2C9 and P450 2C19 might contribute as well to the bioactivation of methyleugenol in the human liver. A 5-fold difference in activities was found between the 15 human liver microsomes of the correlation study (range, 0.89-4.30 nmol min(-1) nmol P450(-1)). Therefore, interindividual differences might cause variation in sensitivity toward methyleugenol. Especially lifestyle factors such as smoking (induces P450 1A) and the use of barbiturates (induces P450 2C) can increase the susceptibility for adverse effects of methyleugenol.

Human cytochrome p450 enzyme specificity for bioactivation of safrole to the proximate carcinogen 1'-hydroxysafrole.

In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1'-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1'-hydroxylation of safrole was characterized in a variety of in vitro test systems, including Supersomes, expressing individual human P450 enzymes to a high level, and microsomes derived from cell lines expressing individual human P450 enzymes to a lower, average human liver level. Additionally, a correlation study was performed, in which safrole was incubated with a series of 15 human liver microsomes, and the 1'-hydroxylation rates obtained were correlated with the activities of these microsomes toward specific substrates for nine different isoenzymes. To complete the study, a final experiment was performed in which pooled human liver microsomes were incubated with safrole in the presence and absence of coumarin, a selective P450 2A6 substrate. On the basis of the results of these experiments, important roles for P450 2C9*1, P450 2A6, P450 2D6*1, and P450 2E1 were elucidated. The possible consequences of these results for the effects of genetic polymorphisms and life style factors on the bioactivation of safrole are discussed. Polymorphisms in P450 2C9, P450 2A6, and P450 2D6, leading to poor metabolizer phenotypes, may reduce the relative risk on the harmful effects of safrole, whereas life style factors, such as the use of alcohol, an inducer of P450 2E1, and barbiturates, inducers of P450 2C9, and polymorphisms in P450 2D6 and P450 2A6, leading to ultraextensive metabolizer phenotypes, may increase the relative risk.