of a Neurotoxic Pyrethroid - ACS Publications - American Chemical

Chapter 15

Downloaded by COLUMBIA UNIV on July 26, 2012 | http://pubs.acs.org Publication Date (Web): July 25, 2012 | doi: 10.1021/bk-2012-1099.ch015

Extrapolating Dose in Vitro to Dose in Vivo of a Neurotoxic Pyrethroid Pesticide Using Empirical Approaches and a PBPK Model Michael F. Hughes,*,1 Melissa P. L. Chan,2,5 James M. Starr,3 Timothy J. Shafer,1 Edward J. Scollon,4 and Michael J. DeVito2 1U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, MD B105-03, Research Triangle Park, North Carolina 27711 2National Institute of Environmental Health Sciences, National Toxicology Program, P.O. Box 12233, Research Triangle Park, North Carolina 27709 3U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, MD D205-05, Research Triangle Park, North Carolina 27711 4U.S. Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention, Office of Pesticide Programs, 1200 Pennsylvania Avenue NW, MD 7509P, Washington, DC 20460 5Current Address: Southern Illinois University-Edwardsville, Environmental Sciences Program, Box 1099, Edwardsville, Illinois 62026 *E-mail: [email protected]

Pyrethroids are a class of neurotoxic synthetic insecticides. Exposure to pyrethroids can be widespread because of their use in agriculture, medicine, and in residential homes and schools. Our studies are focused on generating in vitro and in vivo data for the development of physiologically-based pharmacokinetic models (PBPK) for pyrethroids. Using deltamethrin as a model compound, in vitro metabolic and in vivo tissue time-course data in the rat were determined and used in development of a PBPK model. This model adequately simulated the in vivo blood time course data after intravenous and oral administration of deltamethrin. The model was then used to predict the in vivo administered dose of deltamethrin that would result in blood concentrations equal to in vitro media concentrations of

Not subject to U.S. Copyright. Published 2011 by American Chemical Society In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

deltamethrin that reduce hippocampal cell firing. The in vitro media concentrations that resulted in a 10 and 50% decrease in cell firing were 10 times lower than the predicted in vivo concentrations. This suggests that the in vitro system may not account for the kinetic or dynamic differences between the two systems or that neuronal cells are more responsive to deltamethrin in vitro than in vivo.


Introduction Pyrethroid insecticides are a class of synthetic pesticides structurally based on the botanical pyrethrins. Pyrethrins are contained in the extracts of flowers of chrysanthemum plants (Chrysanthemum cineraraefolium). Pyrethrins have insecticidal properties, but are light sensitive and thus have limited agricultural use. Chemists have synthesized many different pyrethroids, using the structure of the pyrethrins as a base model. The persistence and insecticidal activity of most pyrethroids are greater than that of the pyrethrins. A recent survey of United States residences detected at least one pyrethroid in 89% of the households surveyed (1). The many uses of pyrethroids include agricultural, residential, medical and veterinary applications. Thus, humans may potentially be exposed to multiple pyrethroids by oral (dietary), inhalation or dermal routes. There is a need to adequately assess the potential exposure, disposition (metabolism and pharmacokinetics) and toxicity of the pyrethroids. Pyrethroids consist of an acid and an alcohol moiety as well as chiral centers (Figure 1). Pyrethroid with 2-3 chiral centers results in the formation of 2-4 enantiomer pairs (cis and trans isomers). These enantiomer pairs have different toxic potencies and are metabolized at different rates (2). Chiral HPLC columns are required to separate the isomers making up all the enantiomer pairs. Achiral HPLC columns separate the enantiomer pairs, but not the individual isomers making up the pairs. Pyrethroids are frequently classified into two types, Type I and Type II. Structurally, the alcohol moiety of Type I pyrethroids is either primary or secondary, whereas Type II pyrethroids are only secondary alcohols and have a cyano group on the alpha carbon of the alcohol moiety. Biologically, Type I pyrethroids elicit tremors in rodents (T syndrome). Examples of Type I pyrethroids include permethrin and allethrin. Type II pyrethroids elicit in rodents choreoathetosis (abnormal body movement) and salivation (CS syndrome). Examples of Type II pyrethroids include deltamethrin (Figure 1) and cypermethrin. Some pyrethroids show a mixed type response and are classified as Type I/II. An example includes fenpropathrin. The mechanism of action of the pyrethroids is thought to be alteration of the opening and closing of voltage-sensitive sodium channels in nerve membranes, and perhaps other channels as well (2). Pyrethroids are metabolized by oxidation and ester hydrolysis (3). The oxidation is catalyzed by cytochrome P450s (4, 5) and hydrolysis by carboxylesterases (4–6). The oxidized and hydrolyzed products of the pyrethroids 230 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


can be further metabolized (hydrolyzed and oxidized, respectively), conjugated and excreted in urine (3). Studies suggest that the oxidative and hydrolytic metabolism of the pyrethroids is a detoxication mechanism for their neurotoxic effect (7, 8). Direct injection of pyrethroids into the brains of mice results in the classic neurotoxic syndromes (T or CS) (7). Rats do not develop tremors following parenteral administration of the hydrolyzed products of pyrethroids (8).

Figure 1. The structure of deltamethrin. The dashed and bold wedges show the sites of chirality of deltamethrin. The dashed wedges are oriented behind the plane of the paper and the bold wedges are oriented to the front of the paper. The objective of our studies was to develop physiologically-based pharmacokinetic (PBPK) models for the pyrethroid insecticides in the rat, which upon extrapolation will inform risk assessors with estimates of the disposition of these compounds in humans following exposure. In addition, with in vitro and in vivo effects data, these models can be used for in vitro to in vivo extrapolation to estimate daily intakes resulting in steady state blood concentrations that are equivalent to in vitro media concentrations at which a chemical elicits 50% of its maximum response (e.g., inhibition of neuronal action potential). Deltamethrin (CAS No. 52918-63-5) was chosen for the initial investigation because it is marketed as one specific isomer (cis-deltamethrin; (αS)-cyano-3-phenoxybenzyl(1R,3S)-3-(2,2-dibromovinyl)-2,2-dimethyl-cyclopropanecarboxylate). Thus, the metabolism and toxicity of only one deltamethrin isomer needs to be considered in this modeling study.

Data Needs for PBPK Model Development PBPK models depend on quality and relevant data to be predictive of the disposition of a chemical. Quality data decreases the uncertainty and variability of the predictions of the disposition of the chemical of interest. A listing of data needs for PBPK model development is shown in Table 1. 231 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Table 1. Informational and data needs for PBPK models Physiological data of species (e.g., cardiac output; organ volumes) Physicochemical parameters (e.g., molecular weight, Log P, partition coefficient) Biochemistry and clearance data (e.g., protein binding; hepatic and renal clearance) In vivo tissue time course data In vivo dose response data


Physiological Information Rodent and human physiological information needed for development of PBPK models are available in the literature (9). Examples of the type of information needed include cardiac output, tissue volume, and the blood volume fraction of an organ.

Physicochemical Parameters Physicochemical parameters of the pyrethroids such as molecular weight, log octanol:water partition coefficient (Log P), water solubility and other information can be found in the literature or material safety data sheets. Some of the important parameters for deltamethrin include a molecular weight of 505.2 and a Log P of 6.5 (calculated) (10). Some data, such as tissue partition coefficients, can be estimated computationally using the approach of Poulin and Thiel (11). Tissue to blood partition coefficients of deltamethrin and other important parameters were calculated computationally (12).

Biochemical and Clearance Data A potentially important piece of data for PBPK model development is the extent of binding of a chemical to plasma proteins. This is a potential storage depot of chemicals. As the binding is a reversible process, displacement of a chemical by another agent can elevate the free plasma concentration of the chemical, which may result in a toxic reaction. However, as the extent of binding of deltamethrin to plasma proteins is not known, this parameter was not included in the PBPK modeling effort. Clearance takes into account how the body processes a chemical absorbed systemically. Two major types of clearance are metabolic and renal. Pyrethroids are extensively metabolized and the products are not considered neurotoxic. For the present study, the major focus was on metabolic clearance. The liver was considered to be the main metabolizing organ, thus, the in vitro hepatic clearance of deltamethrin was determined (6). Renal clearance of deltamethrin was not considered an important parameter in the PBPK model development for this insecticide. The in vitro hepatic clearance of a chemical can be determined by one of two methods. The first method involves the determination of the Michaelis-Menten 232 In Parameters for Pesticide QSAR and PBPK/PD Models for Human Risk Assessment; Knaak, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


parameters Km and Vmax of an enzymatic (in this case, metabolic) reaction. The chemical of interest is incubated with microsomes, cytosol or a cellular system such as hepatocytes, and metabolites are quantified over time. Using this method, clearance can be determined by the following formula (13):

With the pyrethroids, using this approach can be problematic because there are multiple metabolites formed by the oxidative and hydrolytic metabolic pathways. A relevant metabolite must be selected and quantified in in vitro assays. However, standards for many of the metabolites are not readily available. Also, Vmax must be measured accurately to precisely determine Km (substrate concentration at ½Vmax). The second method (parent depletion) involves monitoring the loss of parent in a subcellular or cellular incubation over time. When using the parent depletion approach, Km is first estimated in a pilot study (6). This is because the concentration of substrate in the incubations must be

of a Neurotoxic Pyrethroid - ACS Publications - American Chemical

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