Toxicity Testing

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• Chapter 20
• Toxicity Testing

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Toxicity Testing

• There are two purposes of toxicity testing.
• There is a quantitative effort to elucidate a
  doseeffect relationship
• There is a qualitative determination of the
  toxicity of the agent relative to other known
  chemicals.

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Toxicology testing, cont.

• Both purposes are accomplished using laboratory
  animals and in vitro methods.
• There are ethical concerns 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.
• Extrapolation magnifies error, so standards have
  been developed using uncertainty factors and
  modifying factors.
• Application of the results from animal testing
  can improve safety and help prevent injury.

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Toxicity Testing

• The toxic effects of chemicals are determined by
  
• The nature of the chemical hazard
• The dose or quantity to which the individual is
  exposed
• The pathway(s) of exposure
• The pattern of the exposure
• The duration of the exposure

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Toxicology testing, cont.

• In toxicity testing the importance of the dose or
  concentration and the hazardous nature of the
  chemical may vary considerably depending on the
  route of exposure.

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Toxicology testing, cont.

• A chemical may be poorly absorbed through the
  skin but well absorbed orally.
• Because of such route-specific differences in
  absorption, toxicants are often ranked for
  hazard in accordance with the route of exposure.
• For example, a chemical may be relatively
  nontoxic by one route of exposure and highly
  toxic via another route of exposure.
• Accordingly, toxicity testing should be conducted
  using the most likely routes for human exposure.

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Toxicity Information

• Toxicity information is obtained primarily by
• Use of laboratory animals (in vivo studies)
• Surrogate animal models such as cell culture
  systems SARs (in vitro studies)
• Human data obtained from intentional or
  accidental exposures to chemical agents
• Nonbiological models (computers,
  structureactivity relationships SARs)

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Toxicity Information, cont.

• A great deal of information is available on the
  toxicity of chemicals from whole animal studies,
  in vitro studies, and epidemiological studies.
  There are advantages and disadvantages for each
  of these types of studies.

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Exposure Durations

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

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Exposure Duration Acute

• 1. Acute Generally refers to an exposure lasting
  less than 24 hours, and in most cases it is a
  single or continuous exposure over a period of
  time within a 24-hour period. For 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 would constitute
  examples of acute exposures.

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Exposure Duration Subacute

• 2. Subacute Generally refers to repeated
  exposure to a chemical for a period of 1 month or
  less.

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Exposure Duration Subchronic

• 3. Subchronic Generally refers to repeated
  exposure for 13 months.

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Exposure Duration Chronic

• 4. Chronic exposure Generally refers to repeated
  exposure for more than 3 months.

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Toxicity Studies

• Toxicity studies are conducted for chemicals that
  have the potential for public exposure however,
  the extent of the toxicity testing and therefore
  the complexity of the study depend on several
  considerations
• The specific type of chemical hazard
• How it is to be used
• The projected levels of human exposure
• The extent of its release into the environment

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Toxicity Studies, cont.

• As you might anticipate, any study involving
  chemicals such as food additives, agricultural
  chemicals, pharmaceuticals, and veterinary drugs
  would undergo more extensive toxicity testing
  than chemicals that have limited use, perhaps in
  a specific industrial or research application.

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Toxicity Studies, cont.

• Toxicity testing using laboratory animals is
  often the only initial means by which human
  toxicity can be predicted and is often the only
  acceptable means for safety testing that
  satisfies certain regulatory requirements.

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Toxicity Testing

• To be meaningful, any test for toxicity must
  contain the following three stipulations
• An appropriate biological model
• An end-point that can be qualitatively and
  quantitatively assessed
• A well-developed test protocol

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Meaningful Toxicity Testing

• An appropriate biological model
• The model represents the system that is used for
  evaluation.
• This may involve the use of whole animals (in
  vivo testing) 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.

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Meaningful Toxicity Testing

• An end-point that can be qualitatively and
  quantitatively assessed
• The measurement end-point is an appropriate
  parameter that can be used to predict toxicity.
• Toxicological end-points are the biological
  responses to chemical insult.
• They represent a measure of interaction between
  toxicant and living system.
• The term toxicodynamics is sometimes used to
  refer to this dynamic interaction.
• This end-point can be as crude a measure as
  lethality or as subtle as a nonclinically
  detectable change in cellular DNA.

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Meaningful Toxicity Testing

• A well-developed test protocol
• The test protocol is the schedule that defines
  the conditions related to dosing and time, and
  provides all experimental details, including
  statistical methodology.

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Toxicity Studies

• Toxicity studies using laboratory animals thus
  provide a basis for
• Understanding how a chemical may potentially
  produce an adverse response in humans
• Demonstrating a range of exposure levels and
  gradation of toxicity from no observable effects
  to severe toxicity
• Justification for public health risk assessments

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Toxicity Studies

• It must be recognized, however, that although the
  intent of such studies is to provide information
  that would be predictive of effects in humans,
  responses vary between animal species because of
  anatomical, physiological, and biochemical
  differences, and this limits to some extent our
  confidence as to human applicability.

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Extrapolation of Animal Results to Humans
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Equivalent Dose Levels in Several Species
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Acute Local Toxicity

• Irritation and corrosion tests are examples of
  local tissue responses.
• The chemical being tested is applied to the skin
  of the test animal and over a period of time,
  generally hours to a few days, the skin is
  examined for signs of inflammation.
• When these types of tests are performed on the
  eyes, it is referred to as the Draize test.

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Irritation and corrosion tests, cont.

• The ocular toxicity of irritants is determined by
  the brief application of the substance to the
  eyes of several test animals, which are usually
  rabbits.
• Examination of the eyes is conducted over a
  period of 3 days to assess for any injuries that
  may have been produced to the conjunctiva,
  cornea, or iris.
• Substances have been demonstrated to produce a
  range of effects from no observable reaction or
  simple reversible irritation to severe irritation
  and corrosion.

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Acute Local Toxicity, cont.

• Some chemicals have the potential to produce a
  direct irritating/inflammatory skin response
  while others may need to first be processed by
  immunological sensitization.
• In the latter process, skin injury is not the
  direct effect of the chemical on the skin, but
  rather an indirect response from the release of
  mediators of inflammation upon reexposure in the
  sensitized individual.
• Rabbits are generally used for these types of
  studies.

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Acute Local Toxicity, cont.

• To determine whether the chemical produces a
  primary irritant response (contact dermatitis),
  the substance is applied to the skin and any
  changes are observed over the course of several
  days.
• To assess for an immunological response, guinea
  pigs are first treated with the chemical by its
  topical application to the skin for several hours
  (sensitization phase).
• There should be no inflammatory changes to the
  skin over the course of a week or two.
• The substance is then reapplied to the skin (skin
  challenge) and observations are made over a
  period of one to several days.

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Acute Systemic Toxicity

• 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 LD50.
• This is a 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.
• In the late 1920s the LD50 test was developed as
  a measure of the toxicological potency of
  chemicals intended for human use such as insulin
  and digitalis.

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Acute Systemic Toxicity, cont.

• The use of the test was expanded to one that was
  generally recognized as an acceptable in vivo
  animal surrogate to rank chemical toxicity and
  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.

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LD50 Curve
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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 ED50 would
  represent the lowest dose that is beneficial
  (efficacious) in 50 of the test population.

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Efficacy, Toxicity, and Lethality, cont.

• It should be apparent that for a chemical
  intended to produce some benefit to the body at a
  certain dose, the likelihood of some toxicity may
  also result from the same chemical at some dose
  beyond therapeutic.
• Any dose that results in a toxic end-point
  (nonlethal) can be abbreviated TD.
• Thus, a TD50 would represent the dose of a
  chemical toxic to 50 of the population.

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Efficacy, Toxicity, and Lethality, cont.

• 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.

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Common Abbreviations of Beneficial, Toxic, and
Lethal Doses
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Margin of Safety

• Perhaps a better designation to describe the
  safety of a drug is that of the margin of safety
  (MOS), which overcomes the problem of any
  significant differences in the response slopes
  between toxicity and efficacy curves.
• The MOS represents the ratio of lethality at a
  very low level (e.g., 1) compared with efficacy
  at the 99 level (MOS LD1/ED99).
• The higher the value, the safer the drug.

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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 when
  compared with an unexposed population that has
  been matched for such factors as age, gender,
  race, and economic status.

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Human Studies, cont.

• An example of such a study might be to determine
  whether a greater incidence of a specific disease
  (e.g., asthma) in a community is associated with
  the discharge of pollutants from a specific
  geographical area.

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Human Studies

• Although epidemiological studies offer obvious
  advantages over laboratory studies, there are
  nonetheless a number of disadvantages
• The tests are often expensive to conduct.
• Good quantification of exposures is frequently
  difficult.
• Large numbers of individuals are acquired for
  meaningful statistical evaluation.
• Exposure quantification in humans is frequently
  difficult because of simultaneous exposures to
  multiple chemical, physical, and biological
  agents.
• Epidemiological studies generally require long
  periods of time before information is made
  available through the appropriate published
  resources.

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Alternatives to Animal Testing

• Toxicologists as well as other scientists who use
  animals for research and testing purposes have
  been encouraged to explore the 3Rs 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.

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Alternatives to Animal Testing 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, that is, the new methodology
  must be validated.

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Alternatives to Animal Testing in vitro
limitations

• Validation may be defined as a process by which
  the credibility of a new test is established for
  a specific purpose and its reliability and
  reproducibility have been verified by independent
  sources.
• Although a large number of in vitro tests are
  available, most of them have not been validated
  and are unacceptable for regulatory purposes.

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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

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Websites

• Chemical Toxicity Database
• http//wwwdb.mhlw.go.jp/ginc/html/db1.html
• National Toxicology Program
• http//ntp-server.niehs.nih.gov/
• The Centers for Disease Control
• http//www.cdc.gov/
• The Department of Health and Human Services
• http//www.hhs.gov/
• The Environmental Protection Agency
• http//epa.gov/
• The Food and Drug Administration
• http//www.fda.gov/
• The National Toxicology Program
• http//ntp.niehs.nih.gov/
• U.S. Department of Labor Occupational Safety
  Health Administration
• http//osha.gov/
• U.S. FDA Center for Food Safety and Applied
  Nutrition
• http//www.cfsan.fda.gov/