Robert A. Budinsky

Critical analysis of literature on low-dose synergy for use in screening chemical mixtures for risk assessment

There is increasing interest in the use of tiered approaches in risk assessment of mixtures or co-exposures to chemicals for prioritization. One possible screening-level risk assessment approach is the threshold of toxicological concern (TTC). To date, default assumptions of dose or response additivity have been used to characterize the toxicity of chemical mixtures. Before a screening-level approach could be used, it is essential to know whether synergistic interactions can occur at low, environmentally relevant exposure levels. Studies demonstrating synergism in mammalian test systems were identified from the literature, with emphasis on studies performed at doses close to the points of departure (PODs) for individual chemicals. This search identified 90 studies on mixtures. Few included quantitative estimates of low-dose synergy; calculations of the magnitude of interaction were included in only 11 papers. Quantitative methodology varied across studies in terms of the null hypothesis, response measured, POD used to test for synergy, and consideration of the slope of the dose-response curve. It was concluded that consistent approaches should be applied for quantification of synergy, including that synergy be defined in terms of departure from dose additivity; uniform procedures be developed for assessing synergy at low exposures; and the method for determining the POD for calculating synergy be standardized. After evaluation of the six studies that provided useful quantitative estimates of synergy, the magnitude of synergy at low doses did not exceed the levels predicted by additive models by more than a factor of 4.

Cross-species Comparisons of Transcriptomic Alterations in Human and Rat Primary Hepatocytes Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

A toxicogenomics approach was used to qualitatively and quantitatively compare the gene expression changes in human and rat primary hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes from five individual rats and five individual humans were exposed for 24 h to 11 concentrations of TCDD ranging from 0.00001 to 100nM and a vehicle control. Gene expression changes were analyzed using whole-genome microarrays containing 13,002 orthologs. Significant changes in expression of individual orthologs at any concentration (fold change [FC] ± 1.5 and false discovery rate < 0.05) were higher in the rat (1547) compared with human hepatocytes (475). Only 158 differentially expressed orthologs were common between rats and humans. Enrichment analysis was performed on the differentially expressed orthologs in each species with 49 and 34 enriched human and rat pathways, respectively. Only 12 enriched pathways were shared between the two species. The results demonstrate significant cross-species differences in expression at both the gene and pathway level. Benchmark dose analysis of gene expression changes showed an average 18-fold cross-species difference in potency among differentially expressed orthologs with the rat more sensitive than the human. Similar cross-species differences in potency were observed for signaling pathways. Using the maximum FC in gene expression as a measure of efficacy, the human hepatocytes showed on average a 20% lower efficacy among the individual orthologs showing differential expression. The results provide evidence for divergent cross-species gene expression changes in response to TCDD and are consistent with epidemiological and clinical evidence showing humans to be less sensitive to TCDD-induced hepatotoxicity.

Mode of action and dose-response framework analysis for receptor-mediated toxicity: The aryl hydrocarbon receptor as a case study

Abstract Dioxins and dioxin-like compounds are tumor promoters that cause liver cancer in rats and mice. The aryl hydrocarbon receptor (AHR) has been implicated as a key component in this tumor promotion response. Despite extensive knowledge of the toxicology of dioxins, no mode of action (MOA) hypothesis for their tumorigenicity has been formally documented using the Human Relevance MOA framework developed by the International Programme on Chemical Safety (IPCS). To address this information gap, an expert panel was convened as part of a workshop on receptor-mediated liver tumorigenicity. Liver tumors induced by ligands of the AHR were assessed using data for dioxins and related chemicals as a case study. The panel proposed a MOA beginning with sustained AHR activation, eventually leading to liver tumors via a number of other processes, including increased cell proliferation of previously initiated altered hepatic foci, inhibition of intrafocal apoptosis and proliferation of oval cells. These processes have been identified and grouped as three key events within the hepatocarcinogenic MOA: (1) sustained AHR activation, (2) alterations in cellular growth and homeostasis and (3) pre-neoplastic tissue changes. These key events were identified through application of the Bradford-Hill considerations in terms of both their necessity for the apical event/adverse outcome and their human relevance. The panel identified data supporting the identification and dose–response behavior of key events, alteration of the dose–response by numerous modulating factors and data gaps that potentially impact the MOA. The current effort of applying the systematic frameworks for identifying key events and assessing human relevance to the AHR activation in the tumorigenicity of dioxins and related chemicals is novel at this time. The results should help direct future regulatory efforts and research activities aimed at better understanding the potential human cancer risks associated with dioxin exposure.

Proposing a scientific confidence framework to help support the application of adverse outcome pathways for regulatory purposes.

An adverse outcome pathway (AOP) describes the causal linkage between initial molecular events and an adverse outcome at individual or population levels. Whilst there has been considerable momentum in AOP development, far less attention has been paid to how AOPs might be practically applied for different regulatory purposes. This paper proposes a scientific confidence framework (SCF) for evaluating and applying a given AOP for different regulatory purposes ranging from prioritizing chemicals for further evaluation, to hazard prediction, and ultimately, risk assessment. The framework is illustrated using three different AOPs for several typical regulatory applications. The AOPs chosen are ones that have been recently developed and/or published, namely those for estrogenic effects, skin sensitisation, and rodent liver tumor promotion. The examples confirm how critical the data-richness of an AOP is for driving its practical application. In terms of performing risk assessment, human dosimetry methods are necessary to inform meaningful comparisons with human exposures; dosimetry is applied to effect levels based on non-testing approaches and in vitro data. Such a comparison is presented in the form of an exposure:activity ratio (EAR) to interpret biological activity in the context of exposure and to provide a basis for product stewardship and regulatory decision making.

The human AHR: identification of single nucleotide polymorphisms from six ethnic populations

Background The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like chemicals are mediated through binding-dependent activation of the cytosolic aryl hydrocarbon receptor (AHR). The human AHR is a low-affinity receptor relative to most rodents, but some reports suggest that there may be individuals with polymorphic high-affinity receptors, thereby possibly increasing the sensitivity to dioxins in such people. Methods Although no polymorphisms have been reported in the ligand binding region of the AHR in the over 100 reported sequences, we sequenced 108 additional human AHR genes in an effort to further identify single single nucleotide polymorphisms (SNPs) within the open reading frames of the AHR locus. The DNA was sequenced from six ethnic populations that included Japanese, Chinese, European/Caucasian, African–American, South East Asian, and Hispanic. Results Six exonic SNPs were identified; four had been described as previously reported and two seem to be novel. Four of the SNPs identified lead to amino acid changes in the AHR protein and two of the SNPs lead to synonymous substitutions. An additional four SNPs have been reported elsewhere that were not identified in the current analysis. With these new sequences, more than 200 human AHR gene sequences have been analyzed for SNPs. Conclusion The results indicate a very limited presence of polymorphisms in the core ligand binding region of the human AHR. Other regions, such as the transactivation domain, seem to be slightly more polymorphic in the human population and the impact on functionality should be further examined.

Mortality rates among workers exposed to dioxins in the manufacture of pentachlorophenol.

Objective: We sought to determine if workers exposed to dioxins in pentachlorophenol (PCP) manufacturing were at increased risk of death from specific causes. Methods: We examined death rates among 773 workers exposed to chlorinated dioxins during PCP manufacturing from 1937 to 1980 using serum dioxin evaluations to estimate exposures to five dioxins. Results: Deaths from all causes combined, all cancers combined, lung cancer, diabetes, and ischemic heart disease were near expected levels. There were eight deaths from non-Hodgkin lymphoma (standardized mortality ratios = 2.4, 95% CI = 1.0 to 4.8). We observed no trend of increasing risk for any cause of death with increasing dioxin exposure. However, the highest rates of non-Hodgkin lymphoma were found in the highest exposure group (standardized mortality ratios = 4.5, 95% CI = 1.2 to 11.5). Conclusions: Other than possibly an increased risk of non-Hodgkin lymphoma, we find no other cause of death related to the mixture of the dioxin contaminants found in PCP.

AH receptor agonist activity in human blood measured with a cell-based bioassay: Evidence for naturally occurring AH receptor ligands in vivo

In the present study, an aryl hydrocarbon receptor (AHR)-driven reporter gene bioassay was used to measure the activity, measured as an induction equivalent (IEQ) as compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or IEQ concentration in human blood samples from 10 volunteers under different dietary regimens. Blood concentrations of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs), as determined by analytical chemistry (HR-GC/MS), and expressed as toxic equivalents (TEQs) with the use of TCDD equivalency factors (TEFs), were within a range that has been reported in the general US population, ranging from 0.022 to 0.119 ppt (whole blood basis). However, the human blood IEQ measured directly via bioassay ranged from 13.4 to 218 ppt (whole blood basis). These order of magnitude greater IEQs compared to the TEQs for dioxins, furans, and certain PCBs suggests that human blood contains a relatively high level of AHR agonists able to activate the CYP1A1 dioxin response element (DRE)-linked reporter gene bioassay and that this AHR activity is not accounted for by PCDDs/Fs and dioxin-like PCBs based on standard HR-GC/MS and TEF analysis. When study participants switched from a “baseline” to a high-vegetable diet, increases in bioassay IEQ were observed that were statistically significant (P<0.05). In addition, IEQ activity was elevated above levels observed following dietary intervention in two subjects given indole-3-carbinol (I3C) supplements. We conclude that a substantial portion of the IEQ activity occurred as a result of the increased intake of natural AHR agonists (NAHRAs) present in many fruits, vegetables. and herbs. Our findings also suggest that dietary NAHRAs constitute a substantial daily dietary intake of AHR-active compounds, and these NAHRAs could influence AHR status in humans and play a role in a basal level of AHR activation.

Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species.

The challenge of using read-across within the EU REACH regulatory framework; how much uncertainty is too much? Dipropylene glycol methyl ether acetate, an exemplary case study

The use of read-across of data within a group of structurally similar substances potentially allows one to characterise the hazards of a substance without resorting to additional animal studies. However the use of read-across is not without challenges, particularly when used to address the needs of a regulatory programme such as the EU REACH regulation. This paper presents a case study where a previously accepted read-across approach was used to address several data gaps in a REACH registration dossier but was subsequently rejected in part by the European Chemicals Agency (ECHA), resulting in the requirement to perform a developmental toxicity study in rodents. Using this case study, this paper illustrates some of the practical challenges faced when making use of read-across, particularly with respect to addressing the uncertainty associated with the use of read-across; showcasing the scientific justification and highlighting some of the potential implications/opportunities for future cases.

Estimates of Cancer Potency of 2,3,7,8-Tetrachlorodibenzo(p)dioxin Using Linear and Nonlinear Dose-Response Modeling and Toxicokinetics

Linear and nonlinear toxicity criteria were derived for 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD) using the recent National Toxicology Program rat cancer bioassay. Dose-response relationships were assessed for combined liver tumors based on lifetime average liver concentrations (LALCs) estimated with a toxicokinetic model. Rat LALC estimates at the 1% point of departure (POD) were obtained with benchmark dose (BMD) modeling to yield the BMD(01) in terms of LALC. The same toxicokinetic model was used to back-extrapolate the human-equivalent external dose (HED). A linear cancer slope factor (CSF) with a value of 1 x 10(-4) per pg/kg/day was calculated as the ratio between the benchmark response rate and the HED at the lower confidence limit of the benchmark dose (BMDL)(01). A nonlinear reference dose (RfD) with a value of 100 pg/kg/day was developed from the BMD(01) value by applying uncertainty factors to rat internal and human external doses. The RfD was 100 times higher than the 10(-4) risk-specific dose (RSD) based on the linear CSF. For comparison, BMD(01) and BMDL(01) values were developed for key events in the tumor promotion mode of action (MOA) of TCDD. This MOA involves dysregulation of the normal function of the aryl hydrocarbon receptor and its associated biological processes and results in pathologies that drive tumor promotion and progression. The BMD(01) values for key events were consistent with the timing of the key events within the MOA and provided support for the choices of the 1% tumor rate as a POD and dichotomous Hill model for representing receptor-mediated carcinogenicity. Because a threshold toxicity criterion most accurately reflects the MOA, the RfD for TCDD with a value of 100 pg/kg/day is considered appropriate for regulatory purposes, consistent with a 2006 NRC panels recommendation to develop a threshold-based cancer potency factor for TCDD and with the methodology in U.S. Environmental Protection Agencys Cancer Guidelines.