Toxicity Testing

Toxicity Testing

Introduction

• Over 100,000 chemicals are in regular use in the United States.
• Several thousand new chemicals are synthesized each year for personal and industrial use.
• There is no instrument that can directly assess toxicity.

Purposes of Toxicity Testing

• A quantitative effort to elucidate a dose–effect relationship.
• A qualitative determination of the toxicity of the agent relative to other known chemicals.
• Both purposes are accomplished using laboratory animals (in vivo) and in vitro methods.

Considerations for Toxicity Studies

• Toxicity studies are conducted for chemicals that have the potential for public exposure.
• The extent and complexity of toxicity testing depend on:
  • The specific type of chemical hazard.
  • How it is to be used.
  • The projected levels of human exposure, dose, or quantity.
  • The extent of its release into the environment.
  • The pathway(s) of exposure.
  • The pattern of the exposure.
  • The duration of the exposure.
• The importance of the dose or concentration and the hazardous nature of the chemical may vary considerably depending on the route of exposure.

Route-Specific Differences in Absorption

• A chemical may be poorly absorbed through the skin but well absorbed orally.
• Toxicants are often ranked for hazard in accordance with the route of exposure.
• A chemical may be relatively nontoxic by one route of exposure and highly toxic via another route of exposure.
• Toxicity testing should be conducted using the most likely routes for human exposure.

Methods for Obtaining Toxicity Information

• Use of laboratory animals (in vivo studies).
• Surrogate animal models such as cell culture systems (in vitro studies).
• Human data obtained from intentional or accidental exposures to chemical agents.
• Nonbiological models (computers, structure–activity relationships [SARs]).
• There are advantages and disadvantages for each of these types of studies.

In Vitro vs. In Vivo Studies

• In Vitro (Cells)
  • Advantages: Fast, easy, cheap, many potential readouts, good standardization.
  • Limitations: Simplified system, moderate predictivity, low translational value.
• In Vivo (Zebrafish)
  • Advantages: Fast, easy, cheap, ethical in vivo analysis, organism complexity.
  • Limitations: Moderate flexibility, moderate predictivity, moderate translational value.
• In Vivo (Rodents)
  • Advantages: Organism complexity, route of administration, proper predictivity.
  • Limitations: Expensive, time-consuming, stringent regulations, ethical constraints.
• In Vivo (Humans)
  • Advantages: Reliable and realistic, high translational value, dosing information.
  • Limitations: Expensive, time-consuming, stringent regulations, ethical constraints.

Animal Use Concerns

• Ethical concerns are 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.
• Responses vary between animal species because of anatomical, physiological, and biochemical differences.
• Limits to some extent our confidence as to human applicability.

Uncertainty Factors

• Extrapolation magnifies error, so standards have been developed using uncertainty factors (UF) and modifying factors.
• Lowest observable adverse effect limit (LOAEL).
• No observable adverse effect limit (NOAEL).

Basis for Toxicity Studies Using Laboratory Animals

• Understanding how a chemical may potentially produce an adverse response in humans.
• Demonstrating a range of exposure levels and gradation of toxicity from NOAEL to severe toxicity.
• Justification for public health risk assessments.
• Application of the results from animal testing can improve safety and help prevent injury.

Toxicity Test Objectives and Considerations

• Studies involving chemicals such as food additives, agricultural chemicals, pharmaceuticals, and veterinary drugs undergo more extensive toxicity testing than chemicals that have limited use.
• Toxicity testing using laboratory animals is often the only initial means by which human toxicity can be predicted.
• It is often the only acceptable means for safety testing that satisfies certain regulatory requirements.

Stipulations for Meaningful Toxicity Tests

• An appropriate biological model.
• An end-point that can be qualitatively and quantitatively assessed.
• A well-developed test protocol.

Appropriate Biological Model

• The model represents the system that is used for evaluation.
• This may involve the use of whole animals 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.

Measurement End-Point

• Measurement end-point is an appropriate parameter that can be used to predict toxicity.
• Toxicological end-points are the biological responses to chemical insult.
• Represent a measure of interaction between toxicant and living system.
• Can be as crude as lethality or as subtle as a nonclinically detectable change in cellular DNA.

Well-Developed Test Protocol

• Test protocol is the schedule that defines the conditions related to:
  • Dosing and time.
• Provides all experimental details.
• Includes statistical methodology.

Important Considerations in Toxicology

• One of the most important considerations in toxicology is the duration and frequency of exposure to a chemical.
• Also an important consideration in developing toxicity tests.
• There are basically four types of exposure durations:
  • Acute
  • Subacute
  • Subchronic
  • Chronic exposure

Types of Exposure Durations

• Acute
  • Generally refers to an exposure lasting less than 24 hours.
  • In most cases, it is a single or “continuous” exposure over a period of time within a 24-hour period.
  • 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.
• Subacute
  • Generally refers to repeated exposure to a chemical for a period of 1 month or less.
• Subchronic
  • Generally refers to repeated exposure for 1–3 months.
• Chronic
  • Generally refers to repeated exposure for more than 3 months.

Ecotoxicological Examples

• Acute
  • 48 hours
  • Assessing lethality
• Chronic
  • 7 days
  • Lethality and reproductive effects

Acute Systemic Toxicity Testing

• 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 LD_{50}.

LD_{50}

• Developed in the late 1920s as a measure of the toxicological potency of chemicals intended for human use such as insulin and digitalis.
• The use of the test was expanded to one that was generally recognized as an acceptable in vivo animal surrogate to rank chemical toxicity.
• 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.

Definition of LD_{50}

• 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.

Acute Lethality Example

• Hypothetical chemical “X” is used to determine acute lethality from oral doses in laboratory rats.
• Can be better approximated by drawing a horizontal line from the 50% lethality point on the curve and then a perpendicular line to the dose intercept.
• Because the dose–response curve is not a straight line, caution must be used in making an LD_{50} estimate.

Limitations of LD_{50}

• Poor indicator of human health effects because death is the least desirable measure of toxicity.
• No information on long-term effects.
• Doesn’t indicate mechanisms of toxicity.

Assessing Nonlethal Endpoints

• To assess nonlethal endpoints, toxicologists can expose organisms to much lower doses than the LD_{50}.
• Changes in appearance, behavior, appetite, thirst, growth, body chemistry can be observed.
• Subchronic tests also determine the NOAEL and LOAEL.
• Can reveal target organs, nature of tissue damage, differences in interspecific response.
• Assists in determining dosing needed for chronic testing.

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 ED_{50} would represent the lowest dose that is beneficial (efficacious) in 50% of the test population.
• For a chemical intended to produce some benefit to the body at a certain dose, there is a likelihood of some toxicity may also result from the same chemical at some dose.
• Any dose that results in a toxic end-point (nonlethal) can be abbreviated TD. Thus, a TD_{50} would represent the dose of a chemical toxic to 50% of the population.
• 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.

Margin of Safety (MOS)

• Perhaps a better designation to describe the safety of a drug is the margin of safety (MOS).
• Overcomes the problem of any significant differences in the response slopes between toxicity and efficacy curves.
• Represents the ratio of lethality at a very low level (e.g., 1%) compared with efficacy at the 99% level (MOS= LD{1}/ED{99}).
• 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.

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.

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.
• 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