Chapter 20: Toxicity Testing

There are two purposes of toxicity testing. –There is a quantitative effort to elucidate a dose–effect relationship –There is a qualitative determination of the toxicity of the agent relative to other known chemicals.

Toxicology testing, cont. Both purposes are accomplished using laboratory animals and in vitro methods. –There are ethical concerns associated with whole animal studies as the intent of toxicity testing is to produce harm to the animal and then extrapolate the results to humans. –Extrapolation magnifies error, so standards have been developed using uncertainty factors and modifying factors. –Application of the results from animal testing can improve safety and help prevent injury.

Toxicity Testing The toxic effects of chemicals are determined by: –The nature of the chemical hazard –The dose or quantity to which the individual is exposed –The pathway(s) of exposure –The pattern of the exposure –The duration of the exposure

Toxicology testing, cont. In toxicity testing the importance of the dose or concentration and the hazardous nature of the chemical may vary considerably depending on the route of exposure.

Toxicology testing, cont. A chemical may be poorly absorbed through the skin but well absorbed orally. –Because of such route-specific differences in absorption, toxicants are often ranked for hazard in accordance with the route of exposure. –For example, a chemical may be relatively nontoxic by one route of exposure and highly toxic via another route of exposure. –Accordingly, toxicity testing should be conducted using the most likely routes for human exposure.

Toxicity Information Toxicity information is obtained primarily by –Use of laboratory animals (in vivo studies) –Surrogate animal models such as cell culture systems [SARs] (in vitro studies) –Human data obtained from intentional or accidental exposures to chemical agents –Nonbiological models (computers, structure– activity relationships [SARs])

Toxicity Information, cont. A great deal of information is available on the toxicity of chemicals from whole animal studies, in vitro studies, and epidemiological studies. There are advantages and disadvantages for each of these types of studies.

Exposure Durations One of the most important considerations in toxicology is the duration and frequency of exposure to a chemical. This is also an important consideration in developing toxicity tests. There are basically four types of exposure durations: 1. Acute 2. Subacute 3. Subchronic 4. Chronic exposure

Exposure Duration: Acute 1. Acute: Generally refers to an exposure lasting less than 24 hours, and in most cases it is a single or “continuous” exposure over a period of time within a 24-hour period. For example, a single oral exposure to 10 ml of an organophosphate pesticide or the inhalation of toluene in the air that we are breathing at 150 ppm over a period of 3 hours would constitute examples of acute exposures.

Exposure Duration: Subacute 2. Subacute: Generally refers to repeated exposure to a chemical for a period of 1 month or less.

Exposure Duration: Subchronic 3. Subchronic: Generally refers to repeated exposure for 1–3 months.

Exposure Duration: Chronic 4. Chronic exposure: Generally refers to repeated exposure for more than 3 months.

Toxicity Studies Toxicity studies are conducted for chemicals that have the potential for public exposure; however, the extent of the toxicity testing and therefore the complexity of the study depend on several considerations: –The specific type of chemical hazard –How it is to be used –The projected levels of human exposure –The extent of its release into the environment

Toxicity Studies, cont. As you might anticipate, any study involving chemicals such as food additives, agricultural chemicals, pharmaceuticals, and veterinary drugs would undergo more extensive toxicity testing than chemicals that have limited use, perhaps in a specific industrial or research application.

Toxicity Studies, cont. Toxicity testing using laboratory animals is often the only initial means by which human toxicity can be predicted and is often the only acceptable means for safety testing that satisfies certain regulatory requirements.

Toxicity Testing To be meaningful, any test for toxicity must contain the following three stipulations: –An appropriate biological model –An end-point that can be qualitatively and quantitatively assessed –A well-developed test protocol

Meaningful Toxicity Testing An appropriate biological model The model represents the system that is used for evaluation. This may involve the use of whole animals (in vivo testing) or an appropriate in vitro test system. When in vitro models are used, one should be selected that best represents what is believed to be occurring in the whole animal.

Meaningful Toxicity Testing An end-point that can be qualitatively and quantitatively assessed The measurement end-point is an appropriate parameter that can be used to predict toxicity. Toxicological end-points are the biological responses to chemical insult. They represent a measure of interaction between toxicant and living system. The term toxicodynamics is sometimes used to refer to this dynamic interaction. This end-point can be as crude a measure as lethality or as subtle as a nonclinically detectable change in cellular DNA.

Meaningful Toxicity Testing A well-developed test protocol The test protocol is the schedule that defines the conditions related to dosing and time, and provides all experimental details, including statistical methodology.

Toxicity Studies Toxicity studies using laboratory animals thus provide a basis for: –Understanding how a chemical may potentially produce an adverse response in humans –Demonstrating a range of exposure levels and gradation of toxicity from no observable effects to severe toxicity –Justification for public health risk assessments

Toxicity Studies It must be recognized, however, that although the intent of such studies is to provide information that would be predictive of effects in humans, responses vary between animal species because of anatomical, physiological, and biochemical differences, and this limits to some extent our confidence as to human applicability.

Extrapolation of Animal Results to Humans

Equivalent Dose Levels in Several Species

Acute Local Toxicity Irritation and corrosion tests are examples of local tissue responses. –The chemical being tested is applied to the skin of the test animal and over a period of time, generally hours to a few days, the skin is examined for signs of inflammation. –When these types of tests are performed on the eyes, it is referred to as the Draize test.

Irritation and corrosion tests, cont. –The ocular toxicity of irritants is determined by the brief application of the substance to the eyes of several test animals, which are usually rabbits. –Examination of the eyes is conducted over a period of 3 days to assess for any injuries that may have been produced to the conjunctiva, cornea, or iris. –Substances have been demonstrated to produce a range of effects from no observable reaction or simple reversible irritation to severe irritation and corrosion.

Acute Local Toxicity, cont. Some chemicals have the potential to produce a direct irritating/inflammatory skin response while others may need to first be processed by immunological sensitization. –In the latter process, skin injury is not the direct effect of the chemical on the skin, but rather an indirect response from the release of mediators of inflammation upon reexposure in the sensitized individual. –Rabbits are generally used for these types of studies.

Acute Local Toxicity, cont. To determine whether the chemical produces a primary irritant response (contact dermatitis), the substance is applied to the skin and any changes are observed over the course of several days. To assess for an immunological response, guinea pigs are first treated with the chemical by its topical application to the skin for several hours (sensitization phase). –There should be no inflammatory changes to the skin over the course of a week or two. –The substance is then reapplied to the skin (skin challenge) and observations are made over a period of one to several days.

Acute Systemic Toxicity Toxicological prechronic tests typically use rodents of both sexes, over a period of either 24 hours (acute), 14 days (the subacute or 2-week study), or 90 days (the subchronic or 13-week study). –A simple end-point measure used for many years is the LD50. –This is a dose (generally orally administered) that is statistically derived from laboratory animals and represents the dose at which 50% of the test animals would be expected to die. –In the late 1920s the LD50 test was developed as a measure of the toxicological potency of chemicals intended for human use such as insulin and digitalis.

Acute Systemic Toxicity, cont. The use of the test was expanded to one that was generally recognized as an acceptable in vivo animal surrogate to rank chemical toxicity and became accepted for regulatory purposes as an important source of safety information for new chemicals, including drugs, household products, pesticides, industrial chemicals, cosmetics, and food additives.

LD 50 Curve

Efficacy, Toxicity, and Lethality For many chemicals that we intentionally use, some benefit is derived from their use. –For example, a prescribed medication is anticipated to produce a beneficial effect if properly taken. –The level of benefit (efficacy) can also be quantitatively measured; thus an ED50 would represent the lowest dose that is beneficial (efficacious) in 50% of the test population.

Efficacy, Toxicity, and Lethality, cont. It should be apparent that for a chemical intended to produce some benefit to the body at a certain dose, the likelihood of some toxicity may also result from the same chemical at some dose beyond therapeutic. –Any dose that results in a toxic end-point (nonlethal) can be abbreviated TD. –Thus, a TD50 would represent the dose of a chemical toxic to 50% of the population.

Efficacy, Toxicity, and Lethality, cont. For chemicals that produce a beneficial effect, e.g., a drug, a comparison of the doses that produce efficacy and those that produce toxicity can yield important information regarding its safety.

Common Abbreviations of Beneficial, Toxic, and Lethal Doses

Margin of Safety Perhaps a better designation to describe the safety of a drug is that of the margin of safety (MOS), which overcomes the problem of any significant differences in the response slopes between toxicity and efficacy curves. –The MOS represents the ratio of lethality at a very low level (e.g., 1%) compared with efficacy at the 99% level (MOS= LD1/ED99). –The higher the value, the safer the drug.

Human Studies Toxicity information from human studies may come from a number of sources: –Case reports from individuals that have been accidentally or intentionally poisoned –Reported adverse reactions to drugs –Clinical studies from various sized groups of individuals that have been intentionally exposed to an investigational chemical, such as a new pharmaceutical –Epidemiological studies that attempt to determine whether a causal relationship exists in a study population that has been exposed to a substance that may produce adverse health effects when compared with an unexposed population that has been matched for such factors as age, gender, race, and economic status.

Human Studies, cont. An example of such a study might be to determine whether a greater incidence of a specific disease (e.g., asthma) in a community is associated with the discharge of pollutants from a specific geographical area.

Human Studies Although epidemiological studies offer obvious advantages over laboratory studies, there are nonetheless a number of disadvantages: –The tests are often expensive to conduct. –Good quantification of exposures is frequently difficult. –Large numbers of individuals are acquired for meaningful statistical evaluation. –Exposure quantification in humans is frequently difficult because of simultaneous exposures to multiple chemical, physical, and biological agents. –Epidemiological studies generally require long periods of time before information is made available through the appropriate published resources.

Alternatives to Animal Testing Toxicologists as well as other scientists who use animals for research and testing purposes have been encouraged to explore the “3R’s” of animal alternatives: –Replace the animal with another appropriate test. –Reduce the total number of animals used. –Refine the study to reduce the distress of laboratory animals.

Alternatives to Animal Testing: in vitro limitations The replacement of laboratory animals with an appropriate in vitro test is often not a viable option. –Accepting an in vitro methodology as a suitable surrogate for an in vivo test requires its validation. –The in vitro methodology must be implementable by multiple laboratories, and consistent results must be produced, that is, the new methodology must be validated.

Alternatives to Animal Testing: in vitro limitations –Validation may be defined as a process by which the credibility of a new test is established for a specific purpose and its reliability and reproducibility have been verified by independent sources. –Although a large number of in vitro tests are available, most of them have not been validated and are unacceptable for regulatory purposes.

In Vitro Methodologies Mutagenicity and Chromosome Damage Tumor Promotion Cytotoxicity Eye Irritation Cardiac Muscle Toxicity Nephrotoxicity Hepatotoxicity Endocrine Toxicity Respiratory Toxicity Reproductive Toxicity Ecological Toxicity Tests

Websites Chemical Toxicity Database: National Toxicology Program: The Centers for Disease Control: The Department of Health and Human Services: The Environmental Protection Agency: The Food and Drug Administration: : The National Toxicology Program U.S. Department of Labor Occupational Safety & Health Administration: U.S. FDA Center for Food Safety and Applied Nutrition: