Types of Animal Testing

Eye Irritancy

John H. Draize, Ph.D., a scientist at the United States Food and Drug Administration (FDA), developed the Draize eye test in 1944 to assess eye irritation caused by various chemicals. In the test, a substance is placed in one eye, with the other eye serving as a control. The rabbits are restrained, preventing them from responding naturally to the irritation, and their eyes are evaluated after one hour and then at 24-hour intervals for up to 14 days. Some continue to be evaluated up to three weeks later. The level of irritation to the eyes is scored numerically by observation of the three major tissues of the eye (cornea, conjunctiva, and iris). Rabbits suffer from redness, bleeding, ulcers, and even blindness, and are likely killed upon completion of the experiment.

The Draize eye test has been criticized for several reasons. The structure of the cornea of the eye of a rabbit differs significantly from that of a human. Rabbits also produce a smaller volume of tears than humans, allowing chemicals and other irritants placed in rabbit eyes to linger longer and cause more irritation. Not only does this make the Draize eye test unreliable, but it also adds to the immense suffering caused by this test. Finally, evaluated damage caused to the eye is highly subjective leading to a great deal of variation in results. While no non-animal alternative has yet been approved as a replacement for the Draize eye test, two alternatives have been created to allow for partial replacement of animal tests in a tiered testing scheme.

Acute Toxicity

Acute toxicity testing is used to determine the danger of exposure to a chemical by mouth, skin, or inhalation. For decades, acute toxicity testing meant poisoning large numbers of animals in Lethal Dose 50 (LD50) tests, which are conducted until at least one half of the test animals die. The LD50 test is conducted infrequently now as it is being replaced by several new, but still lethal, options. A different toxicity test is the fixed dose method, which does not use death as the endpoint for the experiment; signs of ailments or suffering will usually terminate the experiment. Other tests include the acute toxic class method and the up-and-down procedure, which typically involve the use of a smaller number of animals. However, during these tests, animals will often endure excruciating pain, convulsions, loss of motor function, and/or uncontrollable seizures. The animals are killed ay the end of the test so that a necropsy can be performed to determine internal damage. Rats and mice are the animals most often used in acute toxicity testing.

It is difficult to extrapolate information on human responses to chemicals based on these animal test methods because different species of animals have wide variations in their responses to chemicals. These differences include the varying degrees of sensitivity to the chemicals, as well as differences in metabolism and absorption.
Two in vitro test methods have been recommended to reduce the number of animals needed by estimating the starting doses for the fixed dose method and the up and down procedure, and are awaiting validation.

Repeated dose toxicity

Unlike acute toxicity that looks at the amount of substance required to create toxic effects in one dose, repeated dose toxicity is used to evaluate chronic toxic effects, primarily effects on various organ systems, and to establish a no-observed-effect-level (NOEL). Chronic toxicity testing consists of oral, dermal, and inhalation subacute repeated dose studies (28-day) and subchronic repeated dose studies (90-day) in rodents. Some agencies may also require these tests to be completed in a non-rodent species such as dogs or for longer periods of time. Animals are evaluated during the test period and then killed at the end to look for signs of organ or body system damage. Scaling up the results of repeated dose toxicity tests from small, short-lived animals to humans is difficult and there is great variation in how chemicals are absorbed and metabolized by different species. There are no validated non-animal alternatives for repeated dose toxicity.

Skin Corrosivity/Irritation

Skin corrosion tests assess the potential of a substance to cause irreversible damage to the skin. Skin irritation tests determine the level of damage caused to skin such as itching, swelling, and inflammation. Both tests are typically performed on rabbits. The skin irritation test is often referred to as the Draize skin test and involves placing a chemical on a shaved patch of skin and using another shaved patch as a control. Due to differences in the anatomy and structure of the skin of differing species, using animal data to determine skin irritation on humans is often inapplicable.
Alternatives have been validated to replace skin corrosivity tests is some cases; however, often the alternatives are part of a tiered testing strategy that requires at least some animal tests. The alternatives have not been validated for use as a replacement for skin irritation tests.

Skin Sensitization

The skin sensitization test is used to determine if a chemical causes an allergic reaction. In the past, skin sensitization testing was usually performed on guinea pigs who had substances applied on the surface or injected onto their shaved skin. In the Guinea Pig Maximization Test, a chemical adjuvant is injected with the test substance to the boost the immune reaction. In the Buehler test, no adjuvant is used but the test is less sensitive. In both of these skin sensitization tests, multiple doses are applied in order to create an allergic reaction. The guinea pig tests for skin sensitization are highly subjective as the substances are assessed based on the appearance of the skin. In addition, the method of applying these chemicals to the guinea pigs (i.e. injecting them or delivering with an adjuvant) is not consistent with the human use.

Most skin sensitization testing now occurs using the
Local Lymph Node Assay (LLNA). The procedure involves the application of test chemicals on the surface of the ears of mice. LLNA was the first test method to be validated under the ICCVAM process by a panel of peer reviewers, which concluded that the LLNA is a valid alternative to the guinea pig test methods because it reduces the number of animals required for testing and eliminates animal pain and distress. Compared to the traditional test, the LLNA can also be completed in a shorter timeframe and provides dose-response information. However, the mice are still killed after their use in these tests. Alternatives involving more refined versions of the LLNA are under consideration.

Pharmacokinetics/Toxicokinetics and Metabolism

Pharmacokinetic/toxicokinetic tests measure the rates of absorption, distribution, metabolism, and excretion of toxic substances. Some chemicals are inactivated by metabolism while others become more toxic as they are metabolized. Animals, usually rats or mice, are given single or multiple doses of test substances through force feeding, inhalation, intravenous injection, or through the skin and blood samples are taken to determine the rates of absorption, distribution, excretion, and metabolism. The animals are then killed and examined for the accumulation of the test chemicals in their organs. The differences between species and the amount and type of liver enzymes they possess makes it difficult to properly extrapolate human test data from these animal tests. Unfortunately, no non-animal alternative test method has been reviewed or approved for metabolism and pharmacokinetics/toxicokinetics.

Dermal Penetration

Dermal penetration or skin absorption tests analyze the movement of a chemical through the skin and into the bloodstream. In these tests, rats are most often used. After the test chemical is administered, the rats are killed and the amount of test substance absorbed is estimated. There are differences in the structure of the skin of rats and humans, which can lead to unreliable data for these tests. No non-animal tests have yet been approved for use in dermal penetration tests.


A mutagen is a physical or chemical agent that changes the genetic information of an organism and thus increases the frequency of mutations. As many mutations cause cancer, mutagens are typically also carcinogens. In the bone marrow cytogenetic test, chemicals are administered to the test animals, usually rats or mice, who are later killed. The animals’ bone marrow is then evaluated to examine the effects on the nucleus of the blood cells. In a similar procedure, the micronucleus test, a blood sample is obtained from the animals, usually mice, and analyzed for an increased presence of micronuclei, a sign of chromosomal damage. Because the chemical may have failed to reach the bone marrow, false negatives may occur. To mitigate this problem, large doses are often used but this leads to unnatural exposure to chemicals. A
non-animal alternative method that utilizes bacteria has been in use for years.


A carcinogen is a substance or mixture of substances that induces cancer or increases the incidences of cancer. Rats and mice are typically used for testing carcinogenicity. The test chemical is administered orally, placed on the skin, or inhaled in a two year duration. Animal health is monitored throughout the study but most information is obtained after the animals are killed and their tissues and organs are examined for evidence of cancer. Rodents are more prone to cancer than humans, making them poor models for studying carcinogenicity. Also, these tests results vary greatly from one species to another and one breed to another. While there are no approved replacements for animal carcinogenicity tests,
several non-animal alternatives have been created to allow for partial replacement of animal tests in a tiered testing scheme.

Reproductive & Developmental Toxicity

Reproductive toxicity includes the toxic effects of a substance on the reproductive ability of an organism, and the toxic effects on the development of its offspring. Developmental toxicity testing involves giving pregnant female animals, usually rats and rabbits, doses of chemicals administered orally. The animals are killed just prior to delivery and the fetuses are examined for any sign of toxic effects by the test substance. Rats and mice are typically used in reproductive toxicity tests. Both males and females are orally given test substances prior to mating and the female is also given a dose while pregnant. In a two-generation test, dosing with the test substance continues to the first generation offspring. The effects are determined by daily observation and necropsy. Reproductive and developmental toxicity tests use genetically similar animals that do not represent the genetic variability in humans. In addition, test animals have very different reproductive cycles and life spans, making it difficult to extrapolate valuable information for humans. However, the challenge in replacing animal tests for reproductive and developmental toxicity testing is the many possible organs and physiological processes that could be affected by a chemical. In vitro models for all the potential areas of toxicity need to be identified, developed, and organized into a testing scheme.


Neurotoxicity tests aim to find out if substances cause alterations to the nervous system. Neurotoxicity tests are often used to study the effects of pesticides and primarily involve the use of hens or rats. In the tests designed for using hens, the animals are given a single oral dose of a substance and observed for 21 days or they are given doses of the test substance orally on a daily basis for 28 days. The hens are observed during the test for weight changes, behavioral changes, etc. At the end of the tests, the remainder of the hens are killed and their bodies are evaluated for signs of neurotoxicity. In the rat neurotoxicity test, the animals are given daily doses of the test substance for 28 days, 90 days, or one year. The rats are observed for physical and behavioral changes during the test and are killed at the end of the test period and examined for signs of neurotoxicity. There are no regulatory accepted non-animal methods for neurotoxicity testing.


Ecotoxicity tests aim to determine the negative effects of chemicals entering the environment. Fish are often used to test for both acute and chronic toxic effects. The standard acute toxicity test is the fish 96-hour LC50 (lethal concentration 50%), which measures the concentration of a chemical that kills 50% of the fish in a 96 hour period. Chronic fish tests last from seven to more than 200 days and the fish are evaluated for growth, hatching and spawning success, and mortality. Both of these tests may also be conducted with crustaceans such as water fleas. No ICCVAM validated non-animal alternatives exist for aquatic toxicity, though some regulatory agencies will accept results from certain non-animal tests.


A pyrogen is a substance (often bacterial) that causes elevation to an animal’s body temperature. Pyrogenicity testing seeks to find any possible fever-causing contaminants in items such as vaccines and injectable drugs. The rabbit pyrogen test, in use since the 1940s, requires the injection of the material into the rabbits’ blood stream and then monitoring for temperature increases. The rabbits’ sensitivity to the test is greatly affected by the strain of the pyrogen, as well as differences in age and gender, which can lead to skewed data. Furthermore, the rabbit pyrogen test simply produces pass/fail results, but drugs injected under the skin or in the muscle in small doses require the formulation of maximum acceptable concentration, which is not obtained by this test. Another pyrogen test has replaced the rabbit test for many, but not all pyrogen testing needs. This test, the Limulus amoebocyte lysate (LAL) test, uses the amoebocytes from the blood of horseshoe crabs in order to demonstrate the immune system to response to pyrogens. Five alternative test methods to the rabbit pyrogen test are currently being evaluated.


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