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3Rs Topics

NICEATM supports efforts to replace, reduce, or refine the use of animals in research and testing. Methods that use fewer or no animals, or that refine animal use (reduce animal pain and distress), are referred to as alternative methods; replacement, reduction, and refinement are referred to as the "3Rs."

See below for information about topics relevant to NICEATM 3Rs activities.

Extrapolating From In Vitro Concentration to In Vivo Dose

January 2016

Current research in toxicology is focused on advancing cell- and biochemical-based assays that provide mechanistic information on chemical toxicity. Use of these in vitro assays is expected to facilitate prediction of human toxicity and reduce reliance on animal tests.

However, a key issue encountered with these assays is how to accurately relate chemical concentrations that induce in vitro responses to exposure concentrations that result in human or animal (“in vivo”) illness or injury. This relationship, established through in vitro to in vivo extrapolation (IVIVE), was the focus of a NICEATM webinar series. Scientists interested in the use of IVIVE for chemical screening and risk decision-making met in February 2016 at a workshop co-organized by NICEATM and the U.S. Environmental Protection Agency to develop best practices and identify areas for further research. A report from the workshop is in preparation.

Using pharmacokinetics to understand chemical fate

Estimating the tissue dose required to cause an illness or injury requires an understanding of what happens to the chemical as it passes from the point of exposure to the target organ. For example, an in vitro assay might show that a chemical is toxic to liver cells at a certain concentration. Relating that in vitro concentration to the amount of a chemical a person needs to swallow, inhale, or absorb to cause liver damage requires an understanding of the pharmacokinetics of the chemical.

Pharmacokinetics describes the fate of a chemical administered to a living organism, and includes (1) how much of the chemical is absorbed into the bloodstream, (2) how the chemical is distributed throughout the body, (3) the rate and extent to which a chemical is modified or broken down by the body, and (4) the rate and extent to which the chemical is excreted from the body. Each of these steps affects how much of the chemical from the original exposure reaches the target tissue, as well as the magnitude of its effects on that target tissue.

Models representing chemical pharmacokinetics can be built using mathematical equations for measured or estimated biological parameters, such as the fraction of chemical that will bind to blood protein, the rate of blood flow through the liver, the rate at which the chemical is removed from the blood by the liver and kidneys, and the size of the liver and kidneys.

Reversing pharmacokinetics for IVIVE analyses

Traditional pharmacokinetic models estimate the tissue concentration resulting from a known exposure dose. IVIVE analyses reverse that approach. Using similar equations and parameters, IVIVE analyses begin with a concentration known to cause a response in an in vitro assay to estimate the exposure dose required to reach that concentration in the blood.

One potential application of IVIVE is to prioritize screening study results for further testing. IVIVE analyses applied to a set of results from high throughput screening assays could estimate the exposure concentrations that would produce the blood concentrations equivalent to chemical concentrations producing assay responses. These estimates could be compared to what is known about actual human exposures to prioritize chemicals for more extensive testing.

NICEATM IVIVE studies on estrogen-active chemicals

A large amount of in vitro and in vivo data is available for estradiol, bisphenol A, and other chemicals known to interact with the estrogen receptor. NICEATM scientists used these data to develop a reverse pharmacokinetic model to derive exposure estimates from chemical concentrations known to cause effects in an in vitro assay. The model, described in Chang et al., 2014, also compared the exposure estimates to known human exposures. Subsequent analyses described in a poster presented by Chang et al. at the 2015 FutureTox III meeting refined the model by more closely examining the roles of various input parameters. Work is underway to expand the IVIVE analysis to include more in vitro assays and a wider range of environmental chemicals.

Reducing and Replacing Animal Use for Acute Toxicity Testing

December 2015

Acute systemic toxicity tests identify chemicals that could cause illness or death immediately or shortly after a single exposure. These tests assess whether a chemical could cause illness when swallowed (acute oral toxicity tests), absorbed through the skin (acute dermal toxicity tests), or inhaled (acute inhalation toxicity tests).

Acute systemic toxicity tests are traditionally conducted using rats. The results are expressed as an LD50 value, the dose expected to result in lethality in 50% of the animals tested. The LD50 value is used to assign substances such as pesticides or industrial chemicals to toxicity categories and determine what hazard phrases are required on product labels

Approaches such as the up-and-down procedure and use of cell culture assays to establish starting doses greatly reduce the numbers of animals needed for these tests. NICEATM scientists and colleagues continue to explore ways to further reduce and eventually replace animal use for acute toxicity testing.

Using oral toxicity data to estimate dermal hazards of pesticides

The Environmental Protection Agency requires acute systemic toxicity tests to classify pesticides in order to protect people and the environment during handling and use. NICEATM scientists are evaluating data from acute oral and dermal toxicity tests from over 200 pesticide active ingredients, including fungicides, herbicides, and insecticides, to determine if oral toxicity tests could be used to assign dermal hazard classifications and thereby eliminate the need for separate acute dermal toxicity tests.

Preliminary results presented in a poster at the 2015 Annual Meeting of the Society of Toxicology (Paris et al.) suggested that acute oral toxicity data on pesticide active ingredients may provide relevant information on dermal hazard and allow the number of animals used for dermal acute toxicity testing to be reduced. More recent analyses for pesticide formulations provide similar results.

Exploring approaches that could replace animal use

Using data from oral toxicity tests to predict dermal hazard classification could reduce animal use for acute systemic toxicity testing. NICEATM is exploring how alternative approaches such as computer models, high-throughput cell- and chemical-based tests, and tests using small model organisms such as microscopic worms and fish embryos would enable faster collection of acute toxicity data while further reducing reliance on animal use.

A NICEATM poster presented at the 2015 Society of Toxicology meeting (Polk et al.) described an approach to combining data from high-throughput tests and small model organism assays to predict outcomes of rat acute oral toxicity tests. Testing strategies based on human adverse outcome pathways (see article below) for acute toxicity might also be more relevant to human hazard than animal tests.

NICEATM and collaborators held a September 2015 workshop at which participants developed plans to advance alternative approaches that meet the needs of regulatory agencies while reducing the use of animal. Participants reviewed how acute systemic toxicity data is used for regulatory purposes, and then breakout groups crafted strategies for reducing or replacing rodent use within a three-year timeframe. Outcomes of the workshop will be summarized in a report to be submitted for publication in early 2016.

Adverse Outcome Pathways Support Better Understanding of Toxicity

January 2015

In our daily lives, we are exposed to many chemicals used in products such as personal care products and cleaning supplies. Even more chemicals are used in manufacturing and industrial processes. Ideally, we would have information about the health risks posed by these chemicals. But events such as the January 2014 Elk River chemical spill in West Virginia remind us that this is not the case. Little or no information was available about the safe exposure limits or health risks associated with the chemicals released in that spill.

Traditional methods used to gather this information involve treating animals and observing outcomes. However, the U.S. Environmental Protection Agency estimates that over 10,000 chemicals lack full toxicity data; hundreds of new chemicals are added to that total every year. Traditional testing methods are too expensive and labor-intensive to fully characterize the toxicity of these chemicals within a reasonable time frame.

There is increasing interest in approaches that gather toxicity data using high-throughput cell- and biochemical-based assays. Each of these assays is designed to assess a specific activity such as protein binding or receptor activation. Toxic effects are complex processes that cannot be predicted by any one of these assays. However, testing a chemical with a number of these assays and evaluating the combined data could provide the information needed to predict toxic effects. Key to integrating the data generated by this testing approach is the concept of the “adverse outcome pathway.”

Elements of an adverse outcome pathway

Adverse outcome pathways (AOPs) allow us to organize information about biological interactions and toxicity mechanisms into models that describe how chemical exposures might cause a toxic effect, or disease. AOPs are made up of specific elements.

  • A molecular initiating event is an interaction between the toxic chemical and the organism, such as binding of the chemical to a receptor or protein. This interaction begins the toxicity process.
  • Key events following the molecular initiating event characterize the progression of the toxicity. Early key events can include changes in protein production or molecular signaling that occur in individual cells; later key events can include altered tissue or organ function. The links between key events are described by key event relationships.
  • Adverse outcomes may occur at individual or population levels. An adverse outcome for an individual organism can include disease, impaired development, or impaired reproduction; population adverse outcomes can include changes in population structure or local extinction of a species.

Using AOPs for research and testing

Once an AOP is defined for an adverse outcome, researchers can identify specific cell- or biochemical-based assays that represent the molecular initiating events, key events, and key event relationships for that pathway. A set of assays covering many or all steps of the AOP can be used as a group to screen chemicals for those most or least likely to cause an identified adverse outcome in an efficient, cost-effective manner. This approach also supports animal welfare goals by minimizing or eliminating animal use.

AOPs clarify the events and mechanisms involved in toxicity, which can help with classification and prioritization of chemicals for further/future testing. For example, chemicals lacking the properties needed to cause a molecular initiating event would be unlikely to result in an adverse outcome, so testing of that chemical might not be necessary.

The process of defining AOPs can also help researchers and test method developers identify areas needing improved characterization. Knowledge gaps that prevent an AOP from being fully defined indicate the need for more basic research; key events that are not represented by any suitable assays suggest future areas for test method development.

AOP development is an international effort

The Organisation for Economic Co-operation and Development (OECD) supports international standardization of testing methods to assess chemical toxicity. In this role, the OECD is actively supporting AOP development. OECD maintains a wiki-based interface for developing descriptions of AOPs and issues formal descriptions of well-defined AOPs.

OECD scientists joined colleagues from U.S. government agencies, industry, and academia at a September 2014 workshop organized by NICEATM to discuss the current status of AOP development efforts and how AOPs could be applied to regulatory testing. The workshop featured demonstrations of the OECD wiki and of Effectopedia, a data collection and collaboration tool for developing AOPs. Presentations from the workshop and links to additional resources are available on the workshop webpage; a workshop report will be published in 2015.

Allergic Contact Dermatitis: What Is It, and Why Should I Care About It?

September 2014

If you've ever had a case of poison ivy, you know how unpleasant it is. It produces red, itching, weeping and blistered skin. The effects can last for weeks and it can have a major effect on your daily life.

Poison ivy is an example of allergic contact dermatitis (ACD). To get the rash, you have to be allergic to poison ivy, and your bare skin must come in contact with the plant or its sap. The resulting rash is the dermatitis of ACD.

Sensitizers can cause allergic reactions

An allergy is an immune reaction to a foreign substance. Such a foreign substance is called an allergen or, in the case of ACD, a sensitizer. Except for their allergenic properties, sensitizers are generally (though not always) relatively harmless substances. Why sensitizers result in ACD in some people but not others is not fully understood – we do know that allergies can be hereditary, so sensitivity may be part of your genetic makeup.

The first time you encounter a sensitizer, you might become sensitized. You'll have no visible symptoms, but the sensitizer triggers a complex physiological reaction. This will cause your cells to produce substances that damage your body the next time you are exposed to the sensitizer. The outward symptom of that damage is the ACD rash.

ACD is a common occupational illness

ACD is often considered an occupational illness because it can develop in a variety of workplace settings. Workers in professions as diverse as farming, construction, food handling, or hairdressing may be at risk. Even office workers might be exposed to sensitizers such as rubber, nickel, or glue.

According to the U.S. Bureau of Labor Statistics, occupational skin diseases, including contact dermatitis, account for 15% to 20% of occupational diseases and are the second most common type of occupational illness. ACD is estimated to constitute about 20% to 25% of all contact dermatitis cases. A severe rash can result in lost workdays, and if the sensitizer is not removed from the home or workplace, the rash can reoccur. Sometimes, a worker is forced to leave the job to avoid exposures. The estimated total annual cost of occupational skin diseases (including lost workdays and loss of productivity) may reach $1 billion annually.

ACD diagnosis and treatment

ACD is difficult to diagnose unless you can establish a direct connection between the sensitizer and the rash. You may be uncertain what you were exposed to or where the exposure occurred. In severe cases of ACD, the rash can persist for years, even if the patient avoids additional exposure to the sensitizer, and have a significant impact on the patient’s quality of life.

Your dermatologist can diagnose whether you have ACD with a patch test. This test involves placing small amounts of sensitizers (covered with patches) on your skin. The patches are left on for about 48 hours, long enough for an allergic reaction to occur. Then the skin beneath the patches is examined for signs of dermatitis. A positive reaction means that you have been sensitized to the applied substance or a closely related one.

Once the sensitizer has been identified, you can inspect your home and workplace for its presence and take steps to avoid exposure. Occasionally, all that is required is personal protective equipment such as gloves, long sleeves, or a mask, but sometimes you will need to completely avoid the environment where the exposure occurs. Allergy shots may provide a cure, but they are not always effective.

Testing and labeling can help prevent exposure to sensitizers

The best way to avoid developing ACD is to avoid becoming sensitized. This is easier said than done, however. Thousands of potential sensitizers are introduced into the environment every year. U.S. regulatory agencies require chemical and product testing to determine their potential to cause ACD. Substances identified as sensitizers must be labelled with a description of their potential hazard and the precautions necessary to minimize exposure. Proper labelling reduces the risk for inadvertent exposure.

Traditionally, substances were tested in guinea pigs to predict if they had the potential to be human sensitizers. Animals were exposed to the substance twice, several weeks apart, and observed after the second exposure to see if ACD developed. More recently, the mouse local lymph node assay has become the test of choice for determining the ACD hazard potential for most substances. Compared to the guinea pig tests, it requires fewer animals, takes less time to perform, and eliminates animal pain and distress.

Alternative tests have been developed that don’t use live animals at all. For these, sensitizing potential is assessed by:

  • Measuring specific responses in cells grown in culture
  • Measuring the reactivity of a chemical in a test tube, or
  • Examining and comparing chemical structures using computer models

While it is not yet possible to eliminate all animal testing for ACD, scientists are working to develop an integrated testing strategy that may replace animal testing in the future.

Sensitizers are everywhere

Among the top 10 identified sensitizers are nickel, the topical antibiotics neomycin and bacitracin, formaldehyde, and the chemicals Balsam of Peru and Quaterinium-15. Nickel is everywhere; it is found in coins and costume jewelry and has a myriad of other uses. Neomycin and bacitracin are common constituents of the nonprescription antibiotic ointments used to treat minor skin infections and abrasions. Formaldehyde is familiar to anyone who dissected a frog in high school as the strong-smelling preservative that made your eyes water; it’s used in home construction materials, personal care products, clothing, and furniture. Balsam of Peru is an ingredient in many fragrances and processed foods. Quaternium-15 is a preservative used in many cosmetics.

It is important to remember that exposure to a sensitizer does not mean that you will automatically develop ACD. Some people are not allergic to even very strong sensitizers like poison ivy. However, if you experience a recurring skin rash, especially on your hands or face, you may wish to visit your dermatologist to determine if ACD is the problem.