AltTox.org

Non-animal Methods for Toxicity Testing

Pharmacokinetics & Metabolism

Last Updated: December 6, 2007

Print |
Email

Related Forum Topics

Pharmacokinetics/toxicokinetics is "defined as the study of the rates of absorption, distribution, metabolism, and excretion [ADME] of toxic substances or substances under toxicological study" (OECD, n.d.). Pharmacokinetics/toxicokinetics testing involves describing "the bioavailability of a substance and its kinetic and metabolic fate within the body" (Coecke, et al., 2005). Pharmacokinetics is also the term used to describe the assessment of ADME in the context of drug preclinical testing.

Metabolism has been "defined as all aspects of the fate of a substance in an organism ..." (OECD, n.d.); however, metabolism generally refers to the biotransformation of a substance (via an enzymatic or nonenzymatic process) within the body to other molecular species (usually called the metabolites). For ingested substances, metabolism primarily takes place in the liver, although many organs and tissues have metabolic capability. Two types of enzymes are involved in metabolism: phase 1 (cytochrome P450 enzyme family) and phase 2 enzymes.

An understanding of the metabolism of a substance in the body is critical to understanding its toxicity. For example, biotransformation sometimes results in a molecular species being generated that is more toxic than the original substance. The lack of metabolism of a substance can result in its bioaccumulation in the body. Understanding a substance's metabolism can also facilitate identification of possible target organs and the route of clearance (ECVAM, 2002).

Pharmacokinetic/toxicokinetic data may be used to: 1) assist in the interpretation of other toxicological data, 2) select doses for other toxicological studies, and/or 3) extrapolate data from animals to the human (OECD, n.d.).


The Animal Test(s)

Toxicokinetics studies are described by the Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 417. The route of administration, number of doses, and animal species may vary with the purpose of the study. "The substance and/or metabolites are determined in body fluids, tissues and/or excreta."


Non-animal Alternative Methods

Due to significant species differences in the types and activities of the enzymes involved in metabolism, this is one area where human-based models are critical for protecting human health. Many types of in vitro methods and test systems have been developed to assess metabolism. The advantage is that models using human cells provide species-specific results. A major disadvantage has been the rapid loss of metabolic enzyme activity by cultured cells and tissues. Specific assays and approaches for the non-animal assessment of metabolism have not been validated, but they are widely used by industry given the need for human data.

Recent European Centre for the Validation of Alternative Methods (ECVAM) publications have reviewed in vitro/in silico approaches, including tiered testing strategies, for assessing metabolism (ECVAM, 2002; Coecke, et al., 2005). These methods are briefly summarized here.

  • Microsomes: These are a cellular organelle containing many of the metabolic enzymes and are used in biochemical assays to identify the molecular products of metabolism
  • Suspended cells: This includes liver cells used immediately after isolation in a suspended state (floating in culture media) rather than being grown in culture; the advantage is better retention of metabolic activity, however, most attached cells do not behave "normally" when removed from their normal matrix, extracellular fluids, and interaction with other cell types; additionally, cells sustain some degree of cell membrane "injury" during the isolation process
  • Cells cultured as monolayers: The types of cells include primary liver cells, liver cell lines, and genetically engineered cell lines; human gene polymorphisms will not be detected using cells from only one source
  • Human hepatocyte sandwich cultures: The three dimensional (3D) cultures retain some of the in vivo features lost during monolayer cell culture
  • Precision-cut liver slices: Tissue sections retain the liver's 3D architecture, the cell-to-cell interactions, and the contributions of the different liver cell types
  • Computer-based systems: Three types of in silico systems are available: 1) structure-activity modeling [SAR and (Q)SAR]; 2) rule-based expert systems (METEOR, MetabolExpert, COMPACT, and META); and 3) pharmacophore or protein modeling

The proposed approach to in vitro methods is to start with the simple cell monolayer or microsome assays and progress to the more complex in vitro models. This provides an early identification for "the most important metabolic pathways, including metabolism-mediated toxic effects, metabolic stability, and enzyme inhibition (ECVAM, 2002). Additional tests are then needed to assess enzyme induction and polymorphism effects.

Non-animal alternatives for assessing pharmacokinetics/toxicokinetics consist of mathematical models that describe rates of ADME. These models are commonly referred to as biokinetic models when studying chemical ADME. The most useful are the physiologically based biokinetic (PBBK) models, which can integrate physiochemical and in vitro data (ECVAM, 2002). Validated models are needed that can be used for broader groups of chemicals.

ECVAM proposed a new testing scheme that takes into account the preliminary assessment of whether a substance is likely to result in a systemic exposure (Coecke, et al., 2005). This involves barrier-function assays to determine whether a compound can be absorbed in the intestine, penetrate the skin, or breach the lung epithelial barrier. In vitro models exist for the study of all of these barriers, but they have not been formally validated. Other assessments proposed are plasma protein binding (PPB)--the binding of a chemical to proteins in the blood--which can effect the action, distribution, and elimination of a chemical from the body, and blood-tissue partitioning--used to estimate a substance's blood and tissue levels. Both of these can be assessed by analytical methods, but the in vitro partitioning estimates do not account for active transport processes.

In vivo human testing of extremely low doses of a labeled compound (microdosing) has been used as part of human drug clinical testing to evaluate drug ADME (Coecke, et al., 2005). This approach has not been widely accepted for testing chemicals.


Validation and Acceptance of Non-animal Alternative Methods

The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), ECVAM and the OECD have not yet reviewed or endorsed as scientifically valid any non-animal methods for metabolism and pharmacokinetics/toxicokinetics. In vitro dermal absorption methods are defined by an OECD Test Guideline (TG 428) and are accepted by some regulatory authorities.