James M. McKim

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing - t4 report

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

Absorption dynamics of organic chemical transport across trout gills as related to octanol-water partition coefficient

An in vivo fish preparation was used that allowed a direct measure of the transport rates of 14 different organic chemicals across the gills of rainbow trout (Salmo gairdneri). The chemicals, all C14 labeled, were selected from five classes, encompassing a range of octanol-water partition coefficient (log P) values, from 0.23 (ethyl formate) to 7.5 (mirex). The uptake efficiency (extraction efficiency) of each chemical was determined by monitoring the inspired and expired water of trout exposed to each chemical over an exposure period of 1 to 6 hr. The mean gill extraction efficiency for all chemicals tested varied from a low of 7% to a high of 60%, extracted in a single pall of the chemical across the gills. The extraction efficiency of chemicals with log P or 1 or less were low and showed no relationship to log P. These low extraction efficiencies seen at log P of 1 and below with molecular weights below 100 were indicative of aqueous pore transport. The mean extraction efficiency for chemicals with log P values of 1 to 3 seemed to vary directly with log P, to a maximum of slightly greater than 60%, suggesting that uptake was controlled by the lipid membrane. The mean extraction efficiency for chemicals with log P of 3 to 6 was independent of log P and remained at 60%, which suggested that gill uptake was controlled by aqueous diffusion rates rather than gill membrane permeability. The mean extraction efficiency with mirex (log P = 7.5) decreased to 20%. This behavior was consistent with previous physical models which predict that high log P and melting point (low water solubility) may substantially reduce the rate of accumulation in fish. These studies support the passive diffusion model for the uptake of organics at the gill-water interface.

A Physiologically Based Toxicokinetic Model for the Uptake and Disposition of Waterborne Organic Chemicals in Fish

A physiologically based toxicokinetic model was developed to predict the uptake and disposition of waterborne organic chemicals in fish. The model consists of a set of mass-balance differential equations which describe the time course of chemical concentration within each of five tissue compartments: liver, kidney, fat, and richly perfused and poorly perfused tissue. Model compartmentalization and blood perfusion relationships were designed to reflect the physiology of fishes. Chemical uptake and elimination at the gills were modeled as countercurrent exchange processes, limited by the chemical capacity of blood and water flows. The model was evaluated by exposing rainbow trout (Oncorhynchus mykiss) to pentachloroethane (PCE) in water in fish respirometer-metabolism chambers. Exposure to 1500, 150, or 15 micrograms PCE/liter for 48 hr resulted in corresponding changes in the magnitude of blood concentrations without any change in uptake kinetics. The extraction efficiency for the chemical from water decreased throughout each exposure, declining from 65 to 20% in 48 hr. Extraction efficiency was close to 0% in fish exposed to PCE to near steady state (264 hr), suggesting that very little PCE was eliminated by metabolism or other extrabranchial routes. Parameterized for trout with physiological information from the literature and chemical partitioning estimates obtained in vitro, the model accurately predicted the accumulation of PCE in blood and tissues, and its extraction from inspired water. These results demonstrate the potential utility of this model for use in aquatic toxicology and environmental risk assessment.

Changes in the blood of the brown bullhead (Ictalurus nebulosus (Lesueur)) following short and long term exposure to copper(II)

Abstract Blood from brown bullheads exposed to Cu(II) from 3.4 to 104 μg/l for 6, 30 and 600 days was analyzed to identify and evaluate changes in red blood cells, hematocrit, hemoglobin, plasma chloride, plasma total protein, plasma glucose, plasma glutamic oxaloacetic transaminase (PGOT, l -aspartate: 2-oxoglutarate aminotransferase) and plasma lactic dehydrogenase (PLDH, l -lactate: NAD oxido-reductase). After 6 days, glucose and hematocrit increased significantly above the controls; after 30 days, chloride and protein decreased, and hematocrit, hemoglobin and glucose increased; after 600 days, the transaminase decreased. The maximum concentration of Cu(II) having no detectable effect upon the brown bullhead was determined to be between 11 and 16 μg/l.

A MODEL FOR EXCHANGE OF ORGANIC CHEMICALS AT FISH GILLS : FLOW AND DIFFUSION LIMITATIONS

Abstract A mathematical model for the exchange of neutral organic chemicals at fish gills was formulated based on limitations imposed by flows of water and blood to the gills, diffusion barriers defined by gill morphology, and chemical binding relationships within water and blood. This model was parameterized independently of exchange measurements and validated against datasets on the relationship of chemical uptake rates for large rainbow trout and small guppies to chemical hydrophobicity. This model was found to closely predict the magnitude and trends of observed gill uptake rates in these datasets, predictions deviating from observed values by no more than a factor of two over a range of octanol:water partition coefficients from 2 to > 10 6 . Elimination rates for small guppies were also predicted. This analysis suggests that gill exchange can be understood and predicted on the basis of fundamental physiological and morphological variables.

A direct measure of the uptake efficiency of a xenobiotic chemical across the gills of brook trout (Salvelinus fontinalis) under normoxic and hypoxic conditions

1. An in vivo whole fish preparation was evaluated for measuring the uptake efficiency of chemicals directly from the water across the gills of transected brook trout. n n2. A continuous-flow system allowed a determination of the effects of environmental variables (i.e. dissolved oxygen) on the movement of chemicals across the blood-water interface at the gills. n n3. Endrin uptake efficiency at two water concentrations (0.046 and 0.072 μg endrin/1) was 81 ± 9% and 80 ± 8%, respectively, indicating no significant effect of endrin concentration on uptake efficiency. n n4. Total mean endrin taken up across the gills (dose) at 11–12°C was ca. 7.1 μg/kg per day at the low endrin concentration and 13.2 μg/kg per day at the high. n n5. Mean ventilation volume increased from 72 ± 21 ml/min at oxygen saturation to 310 ± 67 ml/min at 30% of saturation substantially increasing endrin exposure of the gills. n n6. Oxygen utilization (%) decreased from a mean of 60 ± 11% oxygen saturation to 44 ± 10% at 30% saturation, while endrin uptake efficiency dropped from a mean of 80 ± 11% at saturation to a low of 47 ± 11% at 30% of saturation. n n7. In spite of decreased oxygen utilization (%) and endrin uptake efficiency (%), the total uptake of endrin and oxygen increased as the oxygen content of the water was decreased, to 50% of saturation. n n8. A threshold for oxygen and endrin uptake occurred between 50 and 30% of water oxygen saturation beyond which brook trout could no longer increase uptake of oxygen or endrin despite a larger respiratory volume.

Physiologically based toxicokinetic modeling of three waterborne chloroethanes in rainbow trout (Oncorhynchus mykiss)

A physiologically based toxicokinetic model for fish was used to simulate the uptake and disposition of three waterborne chloroethanes in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 1,1,2,2-tetrachloroethane, pentachloroethane, and hexachloroethane in fish respirometer-metabolism chambers to assess the kinetics of chemical accumulation in arterial blood and chemical extraction efficiency from inspired water. Chemical residues in tissues were measured at the end of each experiment. Trout exposed to tetrachloroethane were close to steady-state in 48 hr. Fish exposed to pentachloroethane were near steady-state in 264 hr. Extraction efficiency data showed that systemic (extrabranchial) elimination of both chemicals was small. Hexachloroethane continued to accumulate in fish exposed for 600 hr. Parameterized with chemical partitioning data obtained in vitro, the model accurately simulated the uptake of all three chloroethanes in blood and tissues and their extraction from inspired water. These results provide support for the basic model structure and the accuracy of physiological input parameters.

Environmental Impacts on the Physiological Mechanisms Controlling Xenobiotic Transfer across Fish Gills

Fish physiologists have provided the basic information on gill morphology, gill function, and vascular dynamics with which to understand branchial flux of gases, water, and ions. In addition, pharmacologists and toxicologists, working in the area of drug action, have characterized the physicochemical attributes of xenobiotic chemicals that determine their rate of movement across biological membranes. Recently, aquatic toxicologists have applied this information to the question of what mechanisms control the movement of organic chemicals across fish gills and how exchange is affected by chemical properties. This research on gill transfer mechanisms was extended to consider environmental conditions that have been reported to influence chemical exchange (e.g., dissolved and/or suspended organic material, hydrogen ion concentration [pH], dissolved O₂, content, and water temperature). Mathematical models were developed that predict gill exchange as a function of basic processes such as water flow across the gills, blood flow through the gills, partitioning of the chemical between water and blood, and diffusion between blood and water across gill epithelia. Such mechanistic models can predict the effects of environmental conditions on exchange rates of xenobiotics. To fully develop a predictive capability for xenobiotic uptake and distribution by fish, it will be necessary to incorporate these gill models into emerging, physiologically based models for the entire animal.

Consensus Report on the Future of Animal-Free Systemic Toxicity Testing

Since March 2013, animal use for cosmetics testing for the European market has been banned. This requires a renewed view on risk assessment in this field. However, in other fields as well, traditional animal experimentation does not always satisfy requirements in safety testing, as the need for human-relevant information is ever increasing. A general strategy for animal-free test approaches was outlined by the US National Research Council`s vision document for Toxicity Testing in the 21st Century in 2007. It is now possible to provide a more defined roadmap on how to implement this vision for the four principal areas of systemic toxicity evaluation: repeat dose organ toxicity, carcinogenicity, reproductive toxicity and allergy induction (skin sensitization), as well as for the evaluation of toxicant metabolism (toxicokinetics) (Fig. 1). CAAT-Europe assembled experts from Europe, America and Asia to design a scientific roadmap for future risk assessment approaches and the outcome was then further discussed and refined in two consensus meetings with over 200 stakeholders. The key recommendations include: focusing on improving existing methods rather than favoring de novo design; combining hazard testing with toxicokinetics predictions; developing integrated test strategies; incorporating new high content endpoints to classical assays; evolving test validation procedures; promoting collaboration and data-sharing of different industrial sectors; integrating new disciplines, such as systems biology and high throughput screening; and involving regulators early on in the test development process. A focus on data quality, combined with increased attention to the scientific background of a test method, will be important drivers. Information from each test system should be mapped along adverse outcome pathways. Finally, quantitative information on all factors and key events will be fed into systems biology models that allow a probabilistic risk assessment with flexible adaptation to exposure scenarios and individual risk factors.

Physiological response of rainbow trout (Salmo gairdneri) to acute fenvalerate intoxication

Abstract The physiological responses of rainbow trout ( Salmo gairdneri ) to fenvalerate intoxication during aqueous exposure were examined to provide information about the pyrethroid mode of action in fish. Trout ( n = 4) were exposed to 412 ± 50 μg/liter fenvalerate and died in 10.9 ± 1.5 hr. Brain, liver, and carcass fenvalerate concentrations associated with mortality were 0.16 ± 0.05, 3.62 ± 0.57, and 0.25 ± 0.05 mg/kg, respectively. Visible signs of intoxication included elevated cough rate, tremors, and seizures. Histopathological examination of gill tissue showed damage consistent with irritation. An evaluation of respiratory-cardiovascular and blood chemistry responses indicated an elevated rate of metabolism associated with increasingly severe seizures. A cessation of ventilatory and cardiac activity, occurring with the seizures, was also observed. Finally, urine osmolality, Na + and K + concentrations, and Na + and K + excretion rates were elevated with intoxicated trout. The physiological responses of rainbow trout to fenvalerate intoxication suggest that besides effects on the nervous system, effects on respiratory surfaces and renal ion regulation may be associated with the mechanism of pyrethroid action in fish.