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