Repeated dose toxicity testing using oral administration of a test substance in rodents for 28 and 90 days is used to evaluate chronic toxic effects, primarily effects on various organ systems, and to establish a no observed effect level (NOEL). Depending on the potential route of human exposure to the substance, similar testing using dermal and inhalation dosing may also be assessed. Doses are selected to be sublethal but still cause toxic effects. Long-term chronic toxicity studies with a minimum duration of 12 months are sometimes required.
Toxicity testing to detect acute, single exposure target organ toxicity is discussed under Toxicity Endpoints & Tests: Acute Systemic Toxicity.
The Animal Test(s)
Chronic toxicity testing consists of oral, dermal, and inhalation subacute repeated dose studies (28-day) and subchronic repeated dose studies (90-day) in rodents. Testing on both sexes is required. Some agencies may also require testing in a nonrodent species, typically dogs or nonhuman primates, and some agencies require longer testing periods (52 weeks). The most commonly performed studies are the 28-day and 90-day oral toxicity tests in rodents. The endpoints for repeat dose testing consist of an evaluation of clinical observations, blood analysis, whole body gross necropsy, and microscopic examination of all organs and tissues (histopathology). The target organs/systems evaluated may include liver, kidney, lung, neural (central nervous system), reproductive organs, the haematopoietic system, the immune system, and the endocrine system.
Regulatory Requirements & Test Guidelines
The Globally Harmonized System for Classification and Labeling of Chemicals (GHS) provides guidance for the classification of substances that cause "specific target organ/systemic toxicity arising from a repeated exposure" (Part 3, Chapter 3.9). The GHS classification is based on the weight of evidence from all data, including published studies, human incidents/epidemiology, and animal studies. Positive human data have precedence over animal data for classification decisions. In some cases, a chemical not specifically tested for target organ/systemic toxicity may be classified using data generated "from a validated structure activity relationship and expert judgment-based extrapolation from a structural analogue" (UNECE, 2004).
Six Organisation for Economic Cooperation and Development (OECD) Test Guidelines (TG) describe short-term repeat-dose toxicity testing:
- Repeated Dose 28-day Oral Toxicity Study in Rodents (TG407)
- Repeated Dose 90-Day Oral Toxicity Study in Rodents (TG 408)
- Repeated Dose Dermal Toxicity: 21/28-day Study (TG 410)
- Subchronic Dermal Toxicity: 90-day Study (TG 411)
- Repeated Dose Inhalation Toxicity: 28-day or 14-day Study (TG 412)
- Subchronic Inhalation Toxicity: 90-day Study (TG 413)
The OECD is updating TG 407, the repeat dose 28-day oral toxicity testing in rats, to validate a protocol suitable for detecting the endocrine disruption potential of test substances. A report on the validation of the updated TG 407 and a more recent summary report are available.
OECD TG 452, Chronic Toxicity Studies, describes a longer 12-month exposure for some types of studies such as pesticide testing. US Environmental Protection Agency (EPA) Health Effects Test Guidelines 870.4100 also specify chronic toxicity studies of 12 months.
In some cases, repeated-dose testing can be combined with other chronic toxicity testing and this may reduce the numbers of animals used. Examples include combinations with reproductive/developmental testing (OECD TG 422) and carcinogenicity testing TG 453, but in the latter, additional test groups and length of exposure are required, limiting the 3R gains of combining protocols.
Non-animal Alternative Methods
Expert working groups have noted that "inter-species differences limit the usefulness of animal studies for predicting long-term target-organ and target-system effects in humans" (ECVAM, 2002a) and that in vitro approaches often provide more relevant information for hazard assessment than the animal tests (Pfaller, et al., 2001). Therefore, efforts to replace animal chronic toxicity models have focused on the development of human cell-based models.
In 1998, a scientific working group assessed alternative methods for organ toxicity testing and made the following recommendations to promote the development and use of in vitro culture systems for organ toxicity testing (Spielmann, et al., 1998):
- The need for better access to (and techniques for preservation of) high quality human tissues for the use/development of in vitro cultures
- The availability of reference test substances for each organ system
- The availability of in vivo toxicity data, including human data
- Research to identify and validate biologically relevant toxicity endpoints for each organ system
The recommendations are still relevant today.
Potential non-animal alternatives for chronic toxicity testing include human and animal perfused organs; organ tissue slices; isolated, suspended cells; primary cultured cells; cultured cell lines; genetically engineered cell lines; reaggregating cell cultures; 3-dimensional cell cultures and co-cultures; and (quantitative) structure activity relationship ((Q)SAR) computational systems. The advantages and disadvantages of some of these non-animal systems are described by Spielmann, et al. (1998), ECVAM (2002a), Pfaller, et al. (2001), Prieto, et al. (2005), and Prieto, et al. (2006).
Prieto, et al. (2005) discussed the following in vitro and in silico methods as alternatives to repeated dose testing:
- Liver models: isolated human hepatocytes, liver slices, and perfused liver; perfused, collagen sandwich, and bioreactor test systems based on human liver cells
- Kidney models: renal epithelial primary cells and cell lines
- CNS models: neuronal primary cells and cell lines; reaggregating brain cell cultures; brain slices; astrocyte cell cultures; oligodendrocyte cell cultures; microglia cell cultures
- Pulmonary models: isolated, perfused rat and mouse lung; lung slices; human trachael/bronchial epithelial cell cultures; human alveolar cell model of Skinethic; primary rat pneumocyte type II cell cultures; and other cells models in development
- Haematopoietic models: long-term bone marrow cultures; long-term culture initiating cells; myeloid-lymphoid initiating cell assay; human cobblestone area-forming cells; blast colony-forming cell assay; high proliferative potential colony-forming cell assay; colony-forming unit-A method
- Novel long-term culture methods: hollow fiber bioreactors; perfusion culture models
The use of cultured cell models for chronic toxicity testing presents the challenge of maintaining and exposing the cells for the extended periods of time required to assess chronic toxic effects. Additional challenges for cell-based methods include the identification and use of cell culture conditions that retain the fundamental functions of the cell/tissue/organ type; identification and integration of all of the various biological parameters and tissue toxicities that occur in vivo with appropriate in vitro models; and extrapolation of the in vitro results to the in vivo system.
In vitro models are developed using cells or tissues from the organs that are the typical targets of toxicity. The liver is the primary site for the metabolism of many chemicals and drugs by the body and is also the primary site of potential toxic injury (hepatotoxicity). Like all organs, the liver is composed of various cell types; predominately hepatocytes. Liver cells contain phase 1 and phase 2 drug metabolizing enzymes, which convert many chemicals and drugs to other forms that the body can readily use or excrete (biotransformation). In some cases, the biotransformed chemical is the one that causes the toxicity (to the liver or to another organ). Li, et al. (2004; in press) developed a system to coculture hepatocytes with cells from other major organ systems for the concurrent evaluation of metabolism and toxicity. Species differences in the activities of the liver's drug metabolizing enzymes are one of the major contributors to the species differences observed in the toxicity of chemicals and drugs. Therefore, a human liver model for the prediction of human toxicity is particularly important. A major obstacle in using in vitro liver cells/models for chronic toxicity testing has been the limited time the cells maintain their normal functions in vitro (Pfaller, et al., 2001). Improvements in cryopreservation and culturing techniques for human hepatocytes that extend their time in culture and their predictive capabilities were recently reviewed (Li, 2007; in press).
The kidney is another organ susceptible to chemical-induced toxicity. Like the liver, perfused whole kidney and kidney slices have been used to test chemicals for short periods of time (Pfaller, et al., 2001). Kidney tubule cell cultures and cell lines have been developed, and some can be maintained for extended periods (Prieto, et al., 2006). Likewise, cells from other human organs such as lung and brain, as well as blood and immune cells, have been cultured for toxicity testing.
While good progress has been made in the development of predictive in vitro organ models, technological and scientific barriers remain. No matter how good individual in vitro organ models are, they will not replace animal testing for chronic toxicity until they are organized into a predictive integrated testing scheme. The biokinetics of a chemical in the human or animal body must be determined in order to develop appropriate in vitro methods and models. Combinations of in vitro and/or (Q)SAR models to determine the biokinetics (i.e., the Absorption/uptake, Distribution, Metabolism/biotransformation, and Excretion, or ADME) of a chemical must be evaluated to fully understand its potential chronic toxic effects (Blaauboer, et al., 2000; ECVAM, 2002b; Li, 2004).
Historical and current approaches to developing integrated testing schemes for the prediction of systemic toxicity are described in Toxicity Endpoints & Tests: Acute Systemic Toxicity. A similar but even more complex approach will be required to totally replace animals for the prediction of chronic toxicity testing.
Validation and Acceptance of Non-animal Alternative Methods
ICCVAM and ECVAM have not validated any non-animal methods for assessing chronic toxicity endpoints or repeated exposure target organ toxicity.