A. Typical toxicology tests

Toxicity studies are conducted to determine what type of hazard a substance may pose to an organism. Some of the adverse effects toxicity tests are looking for include: lethality, organ damage, cellular effects, mutagenicity (damage to DNA), reproduction and developmental defects and cancer. Toxicity studies can be broadly divided into several categories: in vitro (conducted in isolated cells), in vivo (conducted in test animals), acute (short-term), and chronic (long term) (see Table below).

In vitro testing uses cells isolated from different organisms, including humans. The type of cell used depends on what effect the research is looking for. For example, if the researcher suspects a chemical will damage the liver, they will choose a cell type that comes from the liver. These cells are treated with a substance and the cells are observed for adverse changes (e.g. death, reduced metabolism, or changes in gene expression).

In vivo toxicity tests are categorized by how long the study is conducted and what potential effect the study is looking at. Acute toxicity studies are short-term studies designed to determine what dose is of immediate danger to an organism. Typically, a single dose is administered test animals to determine what dose is lethal to 50% of a test population (LD50 = lethal dose, 50%). Acute toxicity tests are used to establish the relative toxicity of a substance; the lower the LD50 (that is a smaller amount of the substance is needed to cause death), the more toxic a substance is.

Repeated- dose (sometimes also called sub-acute or chronic) studies evaluate potential adverse effects resulting from long-term exposure to a substance. The length of the repeated-dose study is determined by a number of factors, including the suspected potency of the chemical, the anticipated effect from chemical exposure, and the regulatory requirements. Typically, experimental animals are dosed from 28 days to up to 2 years; the test substance can be administered through the oral (gavage – force feeding via a tube or syringe- or in food), dermal (applied to the skin), inter-peritoneal (injected into the abdominal area), or inhalation (air) route. At the end of the study, the animals are killed and the entire animal is examined for adverse changes. Adverse changes could be organ structure changes or failure, tissue death, changes to the immune cells, pre-cancerous lesions, or tumors. Adverse changes to organs or tissues are reported and help inform toxicologist about potential risks to humans.

Special repeated-dose tests are used to determine if there are adverse effects to reproduction or to the development of the fetus. To test for adverse effects on the development of a fetus, pregnant rodents (called ‘dams’) are exposed to the substance throughout the pregnancy, usually by gavage. The dams and fetuses are killed near the end of gestation and the fetuses are examined for deformities. Reproductive toxicity is tested by exposing male and female rodents to the test substance before and during mating. Exposure continues until the resulting offspring reach maturity. The animals are sacrificed and all animals are examined for adverse effects with special attention to the reproductive systems and the health, growth, development, and function of the offspring.

As discussed in another section, in vivo tests have drawbacks. Some of the test procedures still commonly used today were developed early in the twentieth century. The OECD (Organisation for Economic Cooperation and Development) approves and modifies toxicity test protocols to reflect the state of the science and ethics. For example, the LD50 was introduced in the 1920s. In 2002, the OECD eliminated the traditional LD50 from its international testing guidelines;  the OECD guidelines now recommend alternative methods requiring fewer animals to estimate LD50 data. OECD recently approved a one generation, extended reproductive toxicity test protocol intended to replace the older two generation test. These initiatives, and others, are designed to reduce the number of animal used in toxicity testing.

In vitro tests have several benefits; they can be conducted relatively fast, a lot of data can be collected at once, are relatively inexpensive, and avoid using animals. While all of these benefits seem great, in vitro tests currently can’t answer all the questions researchers need to ask to provide safety. They are typically used to answer straightforward questions like whether or not a chemical can directly mutate DNA. There is a large effort underway by hundreds of scientists to improve the accuracy and reliability of these studies. It is the goal of the not-too-distant-future to use in vitro studies in most, if not all, toxicity tests to eliminate the need for animals.

B. Regulatory requirements for toxicological information

Toxicological information is used in a number of different ways, including: to set safe doses for drugs, to set exposure limits for pesticides, to develop protective measures for people who work with toxic substances, and for package labeling on consumer products. In recent decades, companies have also used toxicological information during drug and chemical development to avoid highly toxic substances.

For example, in the United States, toxicity information is used by the CPSC (Consumer Product Safety Commission), DOT (Department of transportation) and OSHA (Occupational Safety and Health Administration) for labeling purposes to protect consumers and workers, and is specifically required by the EPA (Environmental Protection Agency) for pesticides, new industrial chemicals and substances that are found in drinking water, and by the FDA (Food and Drug Administration) for drugs, biologics, medical devices and food additives. In addition, any person or entity can nominate a substance to the NTP (National Toxicology Program) at the National Institutes of Health for toxicity testing.

In Europe, toxicological testing is under the purview of both the European Commission (EC) and a group of independent agencies. The European Commission is led by one commissioner from each of the member countries (28 in 2013) and is responsible is responsible for drafting proposals for new European laws, which are then presented to the European Parliament and the Council for approval and enactment. The EC is divided into a number of Directorates General (DGs), each responsible for managing and/or providing technical support to different EU regulatory sectors. DGs with the greatest involvement in chemicals testing are: DG Enterprise (pharmaceuticals, biologics, cosmetics, and consumer products), DG Environment (biocides), DG Research (research policy and extramural funding), DG-SANCO (Health & Consumer Protection; agrochemicals, food additives, GMOs, nanomaterials, etc.) and the Joint Research Centre (research and validation of alternative methods).

Enforcement of DG laws and policies implemented by independent agencies, including the European Chemicals Agency (ECHA), the European Food Safety Authority (EFSA) and the European Medicines Agency (EMA). ECHA is in the process of implementing the world’s largest chemicals testing program, REACH (Registration, Evaluation, and of Chemicals), which is collecting a vast array of toxicological information for all industrial chemicals that are manufactured or imported into the EU in amounts of one metric ton or greater per year.

Type Test Description Subcategory Average no. Animals/ test OECD TG*
Acute Acute systemic toxicity – oral Animals are given different doses to identify the amount of a chemical that kills 50% of the animals by the oral route 7, 6 – 12, 4-15 rats 420, 423, 425
inhalation route 20 rats 403
dermal route 20 rats, rabbits or Guinea pigs 402
Skin irritation/corrosion Chemical is administered to the skin and animals are observed for reversible skin damage (irritation) or severe and irreversible skin damage (corrosion) in vivo 1-3 rabbits 404
in vitro 430, 431, 435, 439

Rat skin transcutaneous electrical resistance (TER) test method

Excised rat skin is exposed and measured for changes in electrical resistance. Ex vivo   430

Reconstructed human epidermis (RHE)

Reconstructed human skin is exposed and measured for damage over time. In vitro   431

Membrane Barrier

A synthetic membrane is treated and the time of a chemical to cross the barrier is measured. In vitro   435

Reconstructed human epidermis test (RHE)

Under conditions different than TG 431, reconstructed human tissue is exposed and measured for changes over time. In vitro   439
Eye irritation/corrosion Chemical is administered to the skin and animals are observed for reversible eye damage (irritation) or severe and irreversible skin damage (corrosion) in vivo 1-3 rabbits 405

Bovine corneal opacity permeability (BCOP) test

Chemical is administered to excised eye. Eye is measured for extent of damage over time ex vivo   437

Isolated chicken eye (ICE) test

ex vivo   438

Reconstructed Human Cornea-like Epithelium (RhCE)

Chemical is administered to reconstructed human cornea, which is measured for damage over time in vitro   492
Skin sensitization Animals are observed for allergic reaction after repeat skin application LLNA 16 mice 429
GPMT 32 Guinea pigs 406

Direct Peptide Reactivity Assay (DPRA)

Measures protein binding of the chemical in vitro   442C

KeratinoSensTM ARE-Nrf2 luciferase

Measures gene activation in keratinocyte skin cells in vitro   442D
Chronic Repeat dose (28 day) toxicity Animals are monitored for changes in behavior; animals are killed and examined for defects in appearance and histology of cells or organs caused by daily repeat exposure for 28 days, 90 days, or one year. oral route 40 rats 407
inhalation route 40 rats 412
dermal route 40 rats, rabbits or Guinea pigs 410
Subchronic (90 day) toxicity oral route 80 rats 408
inhalation route 80 rats 413
dermal route 411
non-rodent 32 dogs 409
Chronic (1-year) toxicity – rats 160 rats 452
  Non-rodent 32 dogs 452
Reproductive/developmental screen Adult animals are dosed then mated, and killed and examined for defects in reproductive tissues.   Number of successful pregnancies are counted 675 rats 421, 422
Prenatal developmental toxicity Pregnant mothers are dosed, usually orally and killed just before giving birth. Fetuses are examined for malformations. 80 rats or rabbits (pregnant females only) 414
Developmental toxicity – non-rodent Non-rodent 660 rabbits 414
Reproductive toxicity in 2 generations Adult animals are dosed daily and then mated. Pregnant females are dosed daily throughout pregnancy. Some animals are killed periodically to look for defects in reproductive tissues, number of successful pregnancies, and malformations of fetuses. Some of the treated babies are dosed daily until they are old enough to mate, then mated, and pregnant mothers again dosed daily throughout pregnancy. Again, some animals are killed periodically to look for defects in reproductive tissues, number of successful pregnancies, and malformations of fetuses. 2,600 rats 416
Extended 1 generation toxicity study Similar to the two generation test, adult animals are dosed daily and then mated. Pregnant females are dosed daily throughout pregnancy. Some animals are killed periodically to look for defects in reproductive tissues, number of successful pregnancies, and malformations of fetuses. Some of the treated babies are dosed daily until they are old enough to mate, then mated, and pregnant mothers again dosed daily until partially through pregnancy – when they are killed and examined for successful pregnancies and malformed fetuses. 1,600 – 2,600 rats 443
Cancer Gene mutation – in vitro (Ames) Bacterial cells are exposed and monitored for changes in specific gene sequences. 471
Gene mutation – in vitro (mammalian cells) Mammalian cells are   exposed and monitored for changes in specific gene sequences. 476
Mutagenicity –in vitro chromosomal aberration

micronucleus

Mammalian cells are exposed and observed for gross chromosomal malformations 473

487

Mutagenicity – mouse micronucleus Animals are dosed once or twice one day apart and samples taken 24, 36 and 48 hours later. Either peripheral blood is sampled, or animals are killed and bone marrow is sampled. 50 rodents 474
Mutagenicity – chromosomal aberration in vivo Animals are dosed once, and some are killed at 12 and 24 hours later. Bone marrow is sampled for gross chromosomal malformations. 50 rats or hamsters 475
Combined carcinogenicity/chronic toxicity – rodent Animals are dosed daily for two years and monitored for Animals are monitored for changes in behavior; some animals are killed and examined for defects in appearance and histology of cells or organs. After two years, remaining animals are killed and examined for signs of cancer in different organs. 400 rats 453
Carcinogenicity – rodent Animals are dosed daily for two years and monitored for Animals are monitored for changes in behavior; some animals are killed and examined for signs of cancer in different organs.   After two years, remaining animals are killed and examined for signs of cancer in different organs. 400 mice 451

*Test Guideline (TG) ID number from the Organization for Economic Cooperation and Development (OECD): these methods have been agreed by all member countries, which currently number 36 and include the US, EU member countries, Japan, South Korea, Mexico, Turkey, Canada, Australia, New Zealand and Switzerland.