Evaluation of the Inherent Toxicity Concept in Environmental Toxicology and Risk Assessment.

Intrinsic/inherent chemical properties are characteristic, irrespective of the number of molecules present. However, toxicity is an extensive/extrinsic biochemical property that depends on the number of molecules. Paracelsus, often considered the father of toxicology, noted that all things are poisonous. Because dose magnitude (i.e., number of molecules) determines the occurrence of poisonous effects, toxicity cannot be an intrinsic/inherent biochemical property. Thus, toxicology's task is to determine case-specific risks resulting in adverse effects produced by the interaction of toxic doses/exposures, toxic mechanisms, and case-specific influencing factors. Experimental testing results are known to vary within and between chemicals, test organisms, and experimental conditions and repetitions; however, hazard-based approaches treat toxicity as a fixed and constant property. A logical alternative is the standard-risk, case-specific risk model. In this approach, testing data are defined as standard risks where the nature, magnitude, and toxicity effect is standardized to the organism, chemical, and test conditions. Interpolation/extrapolation of standard risks to site-specific conditions (i.e., case-specific risks) is challenging, requiring understanding of the influences of the complex interactions within and between differing species, conditions, and toxicity-modifying factors. Therefore, Paracelsus's paradigm is perhaps better abbreviated as "dose-causality-response", because a key interpretive requirement is establishing toxicity causality by separating mode/mechanism of toxic action from modifying factor influences in overall toxicity responses. Unfortunately, the current knowledge base is inadequate. Moving to a standard-risk-specific-risk paradigm would highlight the importance of improving the toxicity causality knowledge base. Thereby, a rationale would be provided for enhancing the design and interpretation of toxicity testing that is necessary for achieving advances in routine translation of standard-risk to specific-risk estimates-the raison d'être of regulatory risk decision making. Environ Toxicol Chem 2020;39:2351-2360. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The regulatory challenge of chemicals in the environment: Toxicity testing, risk assessment, and decision-making models.

Environmental assessment for chemicals relies on models of fate, exposure, toxicity, risk, and impacts. Together, these models should provide scientific support for regulatory risk management decision-making, assuming that progress through the data-information-knowledge-wisdom (DIKW) hierarchy is both appropriate and sufficient. Improving existing regulatory processes necessitates continuing enhancement of interpretation and evaluation of key data for use in decision-making schemes, including ecotoxicity testing data, physical-chemical properties, and environmental fate processes. Yet, as environmental objectives also increase in scope and sophistication to encompass a safe chemical economy, testing, risk assessment, and decision-making are subject to additional complexity due to the ongoing interaction between science and policy models. Problems associated with existing design and implementation choices in science and policy have both limited needed development beyond chemo-centric environmental risk assessment modeling and constrained needed improvements in environmental decision-making. Without a thorough understanding of either the scientific foundations or the disparate evaluation processes for validation, quality, and relevance, this results in complex technical and philosophical problems that increase costs and decrease productivity. Both over- and under-management of chemicals are consequences of failure to validate key model assumptions, unjustified standardized views on data selection, and inordinate reification (i.e., abstract concepts are wrongly treated as facts).

Data quality and relevance in ecotoxicity: The undocumented influences of model assumptions and modifying factors on aquatic toxicity dose metrics.

A model-based approach using hypothetical organic chemicals examines how aquatic toxicity test results are influenced by toxicity modifying factors such as hydrophobicity, exposure duration, body size, lipid content, mode of toxic action (via Critical Body Residue differences), and metabolic degradation. Differences of up to one to three orders of magnitude were identified for modeled LC50s. Dominance of CBR by low log Kow chemicals can cause further influences. Such differences cause significant changes in the relationship between exposure- and organism-based doses and create substantial difficulties for both interpretation of test results and extrapolation to other laboratory or field exposure conditions. The resulting variability is not readily evident in toxicity testing as insufficient data are collected to validate fundamental assumptions. Consequently, results obtained with standard aquatic toxicity test protocols do not yield consistent, comparable measures of relative toxicity and are inappropriate for quantitative toxicology and risk applications. The substantial uncertainties in testing results created by such undocumented variability must also be given serious consideration in data quality and relevance assessments. Necessary improvements in aquatic toxicity testing methodology should include explicit estimation of toxicokinetics and toxicodynamics and routine validation of toxicological model assumptions.

Are we in the dark ages of environmental toxicology?

Environmental toxicity is judged to be in a "dark ages" period due to longstanding limitations in the implementation of the simple conceptual model that is the basis of current aquatic toxicity testing protocols. Fortunately, the environmental regulatory revolution of the last half-century is not substantially compromised as development of past regulatory guidance was designed to deal with limited amounts of relatively poor quality toxicity data. However, as regulatory objectives have substantially increased in breadth and depth, aquatic toxicity data derived with old testing methods are no longer adequate. In the near-term explicit model description and routine assumption validation should be mandatory. Updated testing methods could provide some improvements in toxicological data quality. A thorough reevaluation of toxicity testing objectives and methods resulting in substantially revised standard testing methods, plus a comprehensive scheme for classification of modes/mechanisms of toxic action, should be the long-term objective.

Evaluation of critical body residue data for acute narcosis in aquatic organisms.

The Environmental Residue Effects Database was evaluated to identify critical body residues of organic chemicals causing acute baseline neutral narcosis in aquatic organisms. Over 15 000 records for >400 chemicals were evaluated. Mean molar critical body residues in the final data set of 161 records for 29 chemicals were within published ranges but varied within and among chemicals and species (~3 orders of magnitude), and lipid normalization did not consistently decrease variability. All 29 chemicals can act as baseline neutral narcotics, but chemicals and/or their metabolites may also act by nonnarcotic modes of action. Specifically, nonnarcotic toxicity of polycyclic aromatic hydrocarbons and/or their biotransformation derivatives may be a significant source of variability. Complete testing of the narcosis-critical body residue hypothesis was confounded by data gaps for key toxicity modifying factors such as metabolite formation/toxicity, lipid content/composition, other modes of toxic action, and lack of steady-state status. Such problems impede determination of the precise, accurate toxicity estimates necessary for sound toxicological comparisons. Thus, neither the data nor the chemicals in the final data set should be considered definitive. Changes to testing designs and methods are necessary to improve data collection and critical body residue interpretation for hazard and risk assessment. Each of the toxicity metrics discussed-wet weight and lipid weight critical body residues, volume fraction in organism lipid, and chemical activity-has advantages, but all are subject to the same toxicity modifying factors.

Model validation in aquatic toxicity testing: implications for regulatory practice.

Toxicity test validity is contingent on whether models and assumptions are appropriate and sufficient. A quality control evaluation of the acute toxicity testing protocol using the US. EPA fathead minnow database focused around three key assumptions that ensure results represent valid toxicological metrics: 1, it must be possible to estimate steady-state LC50s; 2, LC50s should occur at equivalent exposure durations; 3, all substantive toxicity modifying factors should be adequately controlled. About 8% of the tests failed the first assumption and are invalid and unusable. Examination of remaining data indicated variance from unquantified effects of toxicity modifying factors remained in LC50s, thereby failing assumption three. Such flaws in toxicity data generated via recommended LC50 testing protocols means resultant data do not represent consistent, comparable measures of relative toxicity. Current regulations employing LC50 testing data are acceptable due to the use of semiquantitative, policy-driven development guidance that considers such data uncertainty. Quantitative applications such as QSARs, mixture toxicity, and regulatory chemical grouping can be compromised. These validation failures justify a formal quality control review of the LC50 toxicity testing protocol. Interim improvements in the design, execution, interpretation, and regulatory applications of LC50 and related protocols using exposure-based dose surrogates are warranted.

The human relevant potency threshold: reducing uncertainty by human calibration of cumulative risk assessments.

The 2008 National Research Council report "Phthalates and Cumulative Risk Assessment: Tasks Ahead," rejected the underlying premises of TEQ-like approaches - e.g., chemicals are true congeners; are metabolized and detoxified similarly; produce the same biological effects by the same mode of action; exhibit parallel dose response curves - instead asserting that cumulative risk assessment should apply dose addition (DA) to all chemicals that produce "common adverse outcomes" (CAOS). Published mixtures data and a human health risk assessment for phthalates and anti-androgens were evaluated to determine how firmly the DA-CAOS concept is supported and with what level of statistical certainty the results may be extrapolated to lower doses in humans. Underlying assumptions of the DA-CAOS concept were tested for accuracy and consistency against data for two human pharmaceuticals and its logical predictions were compared to human clinical and epidemiological experience. Those analyses revealed that DA-CAOS is scientifically untenable. Therefore, an alternative approach was developed - the Human-Relevant Potency-Threshold (HRPT) - that appears to fit the data better and avoids the contradictions inherent in the DA-CAOS concept. The proposed approach recommends application of independent action for phthalates and other chemicals with potential anti-androgenic properties at current human exposure levels.

Advancing environmental toxicology through chemical dosimetry: external exposures versus tissue residues.

The tissue residue dose concept has been used, although in a limited manner, in environmental toxicology for more than 100 y. This review outlines the history of this approach and the technical background for organic chemicals and metals. Although the toxicity of both can be explained in tissue residue terms, the relationship between external exposure concentration, body and/or tissues dose surrogates, and the effective internal dose at the sites of toxic action tends to be more complex for metals. Various issues and current limitations related to research and regulatory applications are also examined. It is clear that the tissue residue approach (TRA) should be an integral component in future efforts to enhance the generation, understanding, and utility of toxicity testing data, both in the laboratory and in the field. To accomplish these goals, several key areas need to be addressed: 1) development of a risk-based interpretive framework linking toxicology and ecology at multiple levels of biological organization and incorporating organism-based dose metrics; 2) a broadly applicable, generally accepted classification scheme for modes/mechanisms of toxic action with explicit consideration of residue information to improve both single chemical and mixture toxicity data interpretation and regulatory risk assessment; 3) toxicity testing protocols updated to ensure collection of adequate residue information, along with toxicokinetics and toxicodynamics information, based on explicitly defined toxicological models accompanied by toxicological model validation; 4) continued development of residue-effect databases is needed ensure their ongoing utility; and 5) regulatory guidance incorporating residue-based testing and interpretation approaches, essential in various jurisdictions.

Improving the quality of risk assessments in Canada using a principle-based approach.

In 2000, the Government of Canada issued the "Framework for Science and Technology Advice" (FSTA). This official government policy directive mandates that decision-making to protect human health and the environment should be carried out in a timely, predictable, and efficient manner. It also leaves no doubt that quality assurance and quality control (QA/QC) are required for both technical data and the decision-making process itself. Since current publicly available Canadian risk assessment guidance does not directly address quality management for the decision-making process, an evaluative scheme for CEPA risk assessments was created. For each of the six FSTA principles (early issue identification, inclusiveness, sound science and science advice, uncertainty and risk, openness and transparency, review) specific expectations were developed for performance measurement purposes. This scheme addresses the FSTA requirement for a policy compliance checklist. It could serve as the basis for quality assurance audits during risk assessment production and quality control audits in subsequent review of the risk assessment process. Among other things, it could also improve the integration of science advice, policy objectives and general risk communication by enhancing decision-making openness and transparency.

Review of the toxicity of chemical mixtures containing at least one organochlorine.

An analysis of current research on mixture toxicity was conducted by critically reviewing published journal articles. The scope was limited to complex mixtures (more than two components) where at least one component was a chlorinated organic chemical. Although the basics of dose-response are widely accepted for mixtures, a number of technical issues, including dose metrics and the unquantified influence of toxicity modifying factors, confound data interpretation and restrict the ability to establish reliable determinations of the presence, nature, and extent of additivity. Lack of knowledge about dose level influences and species-specific variations contribute further interpretational limitations. Within this context, available data indicates that most tested mixtures are near or below simple dose/concentration additivity. Exceptions (both positive and negative) tend to occur when tested mixtures have only a few components or where sensitive whole organism or sub-organismal changes are used as the response metric. Available information does not routinely identify the presence of chlorine as a marker either of a particular type of toxicity or consistently greater potency. The most profound difficulty is the problem of clearly defining when and why similarity and dissimilarity of toxic action is expected for a particular mixture. This impediment largely results from the lack of a generally accepted, technical classification for mode/mechanism of toxic action coupled with the lack of a generally accepted classification scheme for mode/mechanism of toxicity interactions.

Review of the toxicity of chemical mixtures: Theory, policy, and regulatory practice.

An analysis of current mixture theory, policy, and practice was conducted by examining standard reference texts, regulatory guidance documents, and journal articles. Although this literature contains useful theoretical concepts, clear definitions of most terminology, and well developed protocols for study design and statistical analysis, no general theoretical basis for the mechanisms and interactions of mixture toxicity could be discerned. There is also a poor understanding of the relationship between exposure-based and internal received dose metrics. This confounds data interpretation and limits reliable determinations of the nature and extent of additivity. The absence of any generally accepted classification scheme for either modes/mechanisms of toxic action or of mechanisms of toxicity interactions is problematic as it produces a cycle in which research and policy are interdependent and mutually limiting. Current regulatory guidance depends heavily on determination of toxicological similarity concluded from the presence of a few prominent constituents, assumed from a common toxicological effect, or presumed from an alleged similar toxic mode/mechanism. Additivity, or the lack of it, is largely based on extrapolation of existing knowledge for single chemicals in this context. Thus, regulatory risk assessment protocols lack authoritative theoretical underpinnings, creating substantial uncertainty. Development of comprehensive classification schemes for modes/mechanisms of toxic action and mechanisms of interaction is needed to ensure a sound theoretical foundation for mixture-related regulatory activity and provide a firm basis for iterative hypothesis development and experimental testing.

On the validity of classifying chemicals for persistence, bioaccumulation, toxicity, and potential for long-range transport.

It is argued that chemical substances can be meaningfully ranked or classified according to their persistence (P), bioaccumulation (B), toxicity (T), and potential for long-range transport (LRT) only if these attributes can be shown to be intensive, as distinct from extensive, properties of the substance, i.e., they are independent of quantity of substance. It is shown that P, B, and LRT can be considered intensive or quasi-intensive properties, but toxicity is more problematic. To obtain an intensive metric of toxicity requires selection of one of several possible extensive quantities that define exposure or dose. Ranking of a group of chemicals by toxicity is shown to be very dependent on which quantity is selected. It is suggested that toxicity metrics, such as lethal concentration to 50% of the population (LC50), lethal dose to 50% of the population (LD50), and threshold limit value (TLV) suffer the severe disadvantage of being dependent on the efficiency of delivery of the substance to the site(s) of toxic action in the organism. The use of measured or calculated internal dose is a preferable measure of toxicity since it reduces ambiguities inherent in the other metrics. Also, the primary concern is not the quasi-intensive property of toxicity; rather, it is the risk of toxic effects, an extensive quantity. To adequately assess the risk of toxic effects, both the toxic hazard and the degree of exposure must be characterized. Since exposure cannot be estimated without knowledge of the emission rate of chemicals to the environment, a compelling case can be made that screening to identify priority P, B, T, and LRT substances should be expanded to include quantity released to the environment as an additional factor.

Residue-based interpretation of toxicity and bioconcentration QSARs from aquatic bioassays: polar narcotic organics.

Bioconcentration and toxicity estimation for a group of substituted phenols often categorized as "polar narcotics" can be confounded by pH-dependent ionization. Two methods of correction for ionization were applied to toxicity data obtained by U.S. EPA-Duluth for fathead minnows exposed to 30 different phenols in 37 bioassays. Toxicity QSARs with corrected data were substantially different from those obtained with raw toxicity data. When ionization-corrected toxicity data were used in the critical body residue (CBR) estimation process previously successful with neutral narcotic organics, several categories of CBR, apparently related to different modes of toxic action, resulted. Published data on lethal CBR for substituted phenols were in general agreement, although such information is limited. Elimination half-life rate constants, estimated from nonlinear curve fitting to time-toxicity information, were relatively constant for the Duluth bioassay data, averaging 0.3 days. Half-life information for small aquatic organisms, both from toxicity- and bioconcentration-based tests in the literature, was in a similar range. Much of the relatively high variability encountered experimental data for substituted phenols may in large part be due to differences in metabolic degradation between chemicals and species.

Interpreting aquatic toxicity QSARs: the significance of toxicant body residues at the pharmacologic endpoint.

Aquatic QSAR investigations employ bioassay data where a biological response is associated with a toxicant concentration in the exposure water. Although convenient this ignores the fundamental principle that the response is more closely related to an amount present in the organism. This problem has been examined using acute toxicity and bioconcentration QSARs employing log Kow as the molecular descriptor. Combining these QSAR relationships enables estimates of the amount of toxicant in organisms to be made. For acute narcosis (50% mortality) the toxicant level in the hydrophobic (lipid) phase of an exposed organism is, as a first approximation, constant at approximately 50 mmol l-1 of lipid. For the acute toxicity of a variety of hydrophobic narcotic organics examined by US EPA-Duluth in tests with fathead minnows, this corresponds to a whole-body residue of about 4 mmol kg-1. Although the level in the hydrophobic phase remains constant, for hydrophilic chemicals (log Kow less than 1.5) the contribution of the hydrophilic (water) phase of the organism dominates and total body residues should be similar to the respective threshold LC50s. This has important implications in simple pharmacokinetic modelling and in interpreting residue levels in organisms collected in environmental monitoring studies.

The acute and chronic toxicity of ten chlorinated organic compounds to the American flagfish (Jordanella floridae).

The acute toxicity (96-hr median lethal concentrations (LC50s) of ten chlorinated isomers of benzene, phenol, ethane, and ethylene to the American flagfish (Jordanella floridae) were determined in both static and flow-through systems. Chronic toxicity to embryo-larval fish was also estimated from hatching success and post-hatch survival as well as fry growth rates and survival. Maximum acceptable toxicant concentrations (MATC) were estimated where possible. In general, for both acute and chronic toxicity tests, the order of increasing relative toxicity based on the water-borne exposure concentrations was: chloroethanes, chloroethylenes, chlorobenzenes, and chlorophenols. Within groups, more highly chlorinated isomers were usually more toxic. The presence of suspended or colloidal 1,2,4,5-tetrachlorobenzene was observed in acute toxicity testing and affected toxicity estimates.

Carbonic anhydrase (acetazolamide-sensitive esterase) activity in the blood, gill and kidney of the thermally acclimated rainbow trout, Salmo gairdneri.

1. Gill, kidney and blood levels of acetazolamide-sensitive esterase (carbonic anhydrase) activity were estimated at acclimation temperature and at a common temperature (25 degrees C) in rainbow trout acclimated to 2, 10 and 18 degrees C. Plasma levels of sodium, potassium and chloride were also examined for possible acclimatory variations. 2. Plasma sodium and chloride levels, and the sodium:chloride ratio were unaffected by thermal acclimation; potassium concentrations were significantly elevated at 18 degrees C. 3. Significant, but modest changes in renal and branchial carbonic anhydrase activity were observed under physiologically realistic incubation temperature conditions. Blood carbonic anhydrase activity was sharply elevated at higher acclimation temperatures. 4. The data are discussed in relation to the hypothesis that carbonic anhydrase in this relatively stenothermal freshwater salmonid, through its intimate association with the coupled HCO-3/CL- and H+ +NH+4/Na+ exchange systems may provide for relatively thermostable basal rates of sodium and chloride uptake from the medium and recovery from urine. The renal, and more notably the branchial (Na+/K+)-stimulated ATPase systems, and erythrocytic carbonic anhydrase may then serve primarily as high-temperature amplifiers of sodium and chloride recruitment respectively.