Len Levy

Sources of Variability in Biomarker Concentrations

Human biomonitoring has become a primary tool for chemical exposure characterization in a wide variety of contexts: population monitoring and characterization at a national level, assessment and description of cohort exposures, and individual exposure assessments in the context of epidemiological research into potential adverse health effects of chemical exposures. The accurate use of biomonitoring as an exposure characterization tool requires understanding of factors, apart from external exposure level, that influence variation in biomarker concentrations. This review provides an overview of factors that might influence inter- and intraindividual variation in biomarker concentrations apart from external exposure magnitude. These factors include characteristics of the specific chemical of interest, characteristics of the likely route(s) and frequency of exposure, and physiological characteristics of the biomonitoring matrix (typically, blood or urine). Intraindividual variation in biomarker concentrations may be markedly affected by the relationship between the elimination half-life and the intervals between exposure events, as well as by variation in characteristics of the biomonitored media such as blood lipid content or urinary flow rate. Variation across individuals may occur due to differences in time of sampling relative to exposure events, physiological differences influencing urinary flow or creatinine excretion rates or blood characteristics, and interindividual differences in metabolic rate or other factors influencing the absorption or excretion rate of a compound. Awareness of these factors can assist researchers in improving the design and interpretation of biomonitoring studies.

Response to the Reply on behalf of the ‘Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area’ (MAK Commission) by Andrea Hartwig Karlsruhe Institute of Technology (KIT)

Calculation error in the MAK Commission’s document on GBS [3] when using the rule of three in Pauluhn’s volumetric model (we emphasize that the comment did not dispute the arithmetical error lowering Model B’s GBS limit value erroneously from 2.0 mg/m to 0.5 mg/m). Use of an MPPD2 program version in [3] that is outdated and no longer available to enable to replicate the MAK Commission’s conclusions. Input values in [3] that cannot be reproduced from the references listed in [3] or are not state-of-the-art. Inconsistent use of varying input data by the MAK Commission in [3] although explicitly specified as guideline in the same document [3].

Potential for Effects of Land Contamination on Human Health. 1. The Case of Cadmium

A review of the epidemiological literature on the potential effects of land contamination shows that the largest body of contaminant-specific research relates to cadmium (Cd). First, a brief outline of the key issues related to the study of health impact of land contamination is presented. The recent literature is then reviewed for evidence of associations and possible causal relationships between exposure to Cd from land contamination and health impact. A large number of studies focusing on Cd arise because of the ready availability of biomarkers of exposure and effect and the demonstrated link between soil Cd and itai-itai disease (severe renal and bone disorders) via dietary exposure in Japan and China. Where dietary differences yield lower exposures, links have been established between Cd in soil and biomarkers of renal or bone dysfunctions, but not to health impacts per se. Potential effects of Cd exposure were also investigated for other health outcomes, including hypertension, cancer incidence, preterm delivery, and semen parameters. In contrast to renal and bone disorders, results are generally inconsistent and require further lines of evidence. Residence in locations with elevated concentrations of Cd in soil is a poor surrogate for exposure, and there are examples where residents in locations with elevated concentrations of Cd in soil did not appear to suffer serious health consequences.

Translational toxicology in setting occupational exposure limits for dusts and hazard classification – a critical evaluation of a recent approach to translate dust overload findings from rats to humans

BackgroundWe analyze the scientific basis and methodology used by the German MAK Commission in their recommendations for exposure limits and carcinogen classification of “granular biopersistent particles without known specific toxicity” (GBS). These recommendations are under review at the European Union level. We examine the scientific assumptions in an attempt to reproduce the results. MAK’s human equivalent concentrations (HECs) are based on a particle mass and on a volumetric model in which results from rat inhalation studies are translated to derive occupational exposure limits (OELs) and a carcinogen classification.MethodsWe followed the methods as proposed by the MAK Commission and Pauluhn 2011. We also examined key assumptions in the metrics, such as surface area of the human lung, deposition fractions of inhaled dusts, human clearance rates; and risk of lung cancer among workers, presumed to have some potential for lung overload, the physiological condition in rats associated with an increase in lung cancer risk.ResultsThe MAK recommendations on exposure limits for GBS have numerous incorrect assumptions that adversely affect the final results. The procedures to derive the respirable occupational exposure limit (OEL) could not be reproduced, a finding raising considerable scientific uncertainty about the reliability of the recommendations. Moreover, the scientific basis of using the rat model is confounded by the fact that rats and humans show different cellular responses to inhaled particles as demonstrated by bronchoalveolar lavage (BAL) studies in both species.ConclusionClassifying all GBS as carcinogenic to humans based on rat inhalation studies in which lung overload leads to chronic inflammation and cancer is inappropriate. Studies of workers, who have been exposed to relevant levels of dust, have not indicated an increase in lung cancer risk. Using the methods proposed by the MAK, we were unable to reproduce the OEL for GBS recommended by the Commission, but identified substantial errors in the models. Considerable shortcomings in the use of lung surface area, clearance rates, deposition fractions; as well as using the mass and volumetric metrics as opposed to the particle surface area metric limit the scientific reliability of the proposed GBS OEL and carcinogen classification.

Does carbon black disaggregate in lung fluid? A critical assessment.

Carbon black is an industrially produced particulate form of nearly pure elemental carbon. The basic building blocks of carbon black are (1) primary particles, minute pieces of matter with defined physical boundaries; (2) aggregates, collections of strongly bound or fused particles; and (3) agglomerates, collections of weakly bound aggregates. Industrial carbon black is produced within a closed reactor where the primary particles form aggregates, which become the indivisible entities of carbon black. These aggregates then form agglomerates, which are the typical form of carbon black in commerce. Carbon black is often used in in vitro and in vivo particle toxicology investigations as a reference nanoparticle. The toxicology studies often report the sizes of the primary particles but rarely the sizes of the aggregates or agglomerates. It appears in many cases that there is a limited understanding of the fact that carbon black typically does not exist as primary particles but instead exists as aggregates and agglomerates. Moreover, many toxicology studies manipulate carbon black particles in order to disperse them so that the form of carbon black used in these toxicology studies may be substantially different from the form that may be encountered in the workplace environment. Since the main exposure route for carbon black is inhalation, the question arose as to whether inhaled carbon black may deagglomerate or disaggregate to either smaller aggregates or primary particles when in contact with lung fluids. This question relates to the concern that there may be additional hazards of smaller particles, such as their ability to translocate to tissues and organs beyond the lung and the ability to pass through the blood-brain barrier. The purpose of this assessment is to review the existing literature for evidence as to whether carbon black deagglomerates or disaggregates into smaller aggregates or primary particles when in contact with lung fluid. On the basis of a review of the physical characteristics of commercial carbon black and various toxicology studies, it appears that commercially produced carbon black in contact with lung fluid is unlikely to deagglomerate or disaggregate into smaller aggregates or primary particles.

Inter- and intra-individual variation in urinary biomarker concentrations over a 6-day sampling period. Part 1: Metals

The aim of the current HBM-study is to further the understanding of the impact of inter- and intra-individual variability in HBM surveys as it may have implications for the design and interpretation of the study outcomes. As spot samples only provide a snapshot in time of the concentrations of chemicals in an individual, it remains unclear to what extent intra-individual variability plays a role in the overall variability of population-wide HBM surveys. The current paper describes the results of an intensive biomonitoring study, in which all individual urine samples of 8 individuals were collected over a 6-day sampling period (a total of 352 unique samples). By analyzing different metals (As, Cd, Mn, Ni) in each individual sample, inter- and intra-individual variability for these four metals could be determined, and the relationships between exposure, internal dose, and sampling protocol assessed. Although the range of biomarker values for different metals was well within the normal range reported in large-scale population surveys, large intra-individual differences over a 6-day period could also be observed. Typically, measured biomarker values span at least an order of magnitude within an individual, and more if specific exposure episodes could be identified. Fish consumption for example caused a twenty- to thirty-fold increase in urinary As-levels over a period of 2-6h. Intra-class correlation coefficients (ICC) were typically low for uncorrected biomarker values (between 0.104 and 0.460 for the 4 metals), but improved when corrected for creatinine or specific gravity (SG). The results show that even though urine is a preferred matrix for HBM studies, there are certain methodological issues that need to be taken into account in the interpretation of urinary biomarker data, related to the intrinsic variability of the urination process itself, the relationship between exposure events and biomarker quantification, and the timing of sampling. When setting up HBM-projects, this expected relationship between individual exposure episode and urinary biomarker concentration needs to be taken into account.

Framework for the development and application of environmental biological monitoring guidance values

Human biomonitoring (HBM) is widely recognised as a useful tool to aid assessment of exposure to chemical substances, but our ability to detect hazardous substances (or their metabolites and health effects) often exceeds our understanding of their biological relevance. There are only a few established frameworks for developing and using occupational and environmental biological guidance values (BGVs), mostly for data-rich substances that have been in use for some time. BGVs for new substances and those with unknown dose-response relationships are difficult to derive. An accepted framework based on current scientific knowledge and best practice is therefore urgently needed to help scientists, regulators, and stakeholders to design appropriate HBM studies, interpret HBM data (both for groups and individuals) understand the limitations and to take appropriate action when required. The development and application of such a tool is described here. We derived a conceptual framework that was refined by consultation with an advisory group and workshop. The resulting framework comprised four levels defined by increasing data, with increasing confidence for human health risk assessment. Available data were used for 12 chemicals with expert judgement to illustrate the utility of the framework.

Determination of ethylenethiourea in urine by liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry for monitoring background levels in the general population

This study reports a sensitive analytical method suitable for the quantitative analysis of ethylenethiourea (ETU) in human urine and its application to samples from the general population. Sample preparation involved the use of diatomaceous earth extraction columns to remove matrix interferences. Quantification was achieved by liquid chromatography-mass spectrometry using positive ion atmospheric pressure chemical ionisation. Within-day and between-day variability of 14% (n=10) and 11% (n=6), respectively, were obtained at 98 nmol/l (10 μg l(-1)). The assay was linear over the investigated range 2.5-245 nmol/l, with a limit of detection of 2.5 nmol/l. The method was applied to monitoring background levels of ETU in urine samples from the general population in the UK. Results obtained from 361 spot samples contained ETU levels ranging from less than the detection limit (54% of samples) to a maximum of 15.8 μmol/mol creatinine (14.3 μg/g creatinine). The 95th percentile was 5.7 μmol/mol creatinine (5.2 μg/g creatinine).

Relevance of the rat lung tumor response to particle overload for human risk assessment—Update and interpretation of new data since ILSI 2000

The relevance of particle-overload related lung tumors in rats for human risk assessment following chronic inhalation exposures to poorly soluble particulates (PSP) has been a controversial issue for more than three decades. In 1998, an ILSI (International Life Sciences) Working Group of health scientists was convened to address this issue of applicability of experimental study findings of lung neoplasms in rats for lifetime-exposed production workers to PSPs. A full consensus view was not reached by the Workshop participants, although it was generally acknowledged that the findings of lung tumors in rats following chronic inhalation, particle-overload PSP exposures occurred only in rats and no other tested species; and that there was an absence of lung cancers in PSP-exposed production workers. Since the publication of the ILSI Workshop report in 2000, there have been important new data published on the human relevance issue. A thorough and comprehensive review of the health effects literature on poorly soluble particles/lung overload was undertaken and published by an ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals) Task Force in 2013. One of the significant conclusions derived from that technical report was that the rat is unique amongst all species in developing lung tumors under chronic inhalation overload exposures to PSPs. Accordingly, the objective of this review is to provide important insights on the fundamental differences in pulmonary responses between experimentally-exposed rats, other experimental species and occupationally-exposed humans. Briefly, five central factors are described by the following issues. Focusing on these five interrelated/convergent factors clearly demonstrate an inappropriateness in concluding that the findings of lung tumors in rats exposed chronically to high concentrations of PSPs are accurate representations of the risks of lung cancer in PSP-exposed production workers. The most plausible conclusion that can be reached is that results from chronic particle-overload inhalation studies with PSPs in rats have no relevance for determining lung cancer risks in production workers exposed for a working lifetime to these poorly soluble particulate-types.

Potential for Effects of Land Contamination on Human Health. 2. The Case of Waste Disposal Sites

This review of the epidemiological literature shows that evidence for negative impacts of land contaminated by waste disposal on human health is limited. However, the potential for health impacts cannot be dismissed. The link between residence close to hazardous waste disposal sites and heightened levels of stress and anxiety is relatively well established. However, studies on self-reported outcomes generally suffer from interpretational problems, as subjective symptoms may be due to increased perception and recall. Several recent multiple-site studies support a plausible linkage between residence near waste disposal sites and reproductive effects (including congenital anomalies and low birth weight). There is some conflict in the literature investigating links between land contamination and cancers; the evidence for and against a link is equally balanced and is insufficient to make causal inferences. These are difficult to establish because of lack of data on individual exposures, and other socioeconomic and lifestyle factors that may confound a relationship with area of residence. There is no consistently occurring risk for any specific tumor across multiple studies on sites expected to contain similar contaminants. Further insights on health effects of land contamination are likely to be gained from studies that consider exposure pathways and biomarkers of exposure and effect, similar to those deployed with some success in investigating impacts of cadmium on human health.