NTC Jan 26

8/2/2019 NTC Jan 26

1/8

PHAR 303 - January 26, 2012Lecture 6: Toxicity Testing in Vivo (Ch. 2)Professor Geraldine Delbes

- There are many toxicants surrounding us, and these amounts are increasing due toindustry

o What is the impact of these substances on both human health, and health of anyorganisms on the planet? environmental toxicology

- Toxicants can impact the health of future generations, not just our own- How do we determine the toxicity of a compound? Do you think that in vitro studies are

enough to determine that a compound is safe/healthy for exposure?

Risk Assessment and Risk

Management- Identification of hazards leads to

epidemiology, in vivo toxicology,

in vitro tests, and structure

activity analyseso E.g. phthalates in baby

toys high exposure dueto touching/biting/licking

o How do we go fromidentifying a compound,

to figuring out its effects

on theindividual/society/the

population?

- Characterization of risks:potency, exposure, susceptibility

Toxicity Tests- Are designed to characterize the toxic effects of a chemical

o Drug Development safety pharmacology + preclinical toxicology

Toxicity Testing Vs. Epidemiological Studies- Epidemiological studies establish an association between an etiological factor and the

observed effect

- Toxicological studies establish a direct cause-effect relationshipo This is done by animal studies to directly examine the effect of the toxicant on

animal health

- E.g. correlation between spread of Chernobyl radiation and thyroid cancer throughoutEurope

Agencies involved in Regulation of Toxic Chemicals- Screen compounds and make regulatory laws:

o Food and drugs, environment, water, air, industrial chemicals, pesticides, etc.- The industry themselves must test for the toxicity of the compounds that they are using;

can test in-house (in a research lab in the facilities) or through a contract lab

8/2/2019 NTC Jan 26

2/8


- Non-government institutes- Contract laboratories (CROs)test compounds for a company- Government Agencies (Environment Canada, Health Canada, Food & Drug Agency, etc.)- Academic Institutionsresearchers in universities

o The major difference between contract labs and government is the strict followingof GLP (good lab practice)

This doesnt necessarily occur in universities due to high variationbetween lab personnel

Various Purposes of Toxicity Testing- Mechanistic toxicology, Regulatory

Toxicology, Descriptive Toxicologyalllink into risk assessment

Ability to Conduct Health Hazard

Assessment- The sections that are well controlled are

pesticides, cosmetics, drugs and foods

- However, commercial chemicals are oftennot intensively studied

A Summary of Toxicity Tests1. Chemical and Physical properties of the compound2. Exposure and environmental fate: what will happen to the chemical after

exposure/presence in the environment

a. Degradation studiesb. Mobility and dissipation in soil/water/airc. Accumulation in plants/animals/etc

3. Standard animal toxicity tests

Animal Models- Used to predict the effects of human

exposure to a chemical- However, will animals react the exact

same way as a human? Are the

pharmacokinetics/dynamics the same?

- Is it inhumane to kill animals for research?The number animals killed for researchand education are almost insignificant in

proportion to animals killed for food, andanimals abandoned by their owners

Research, Teaching and Testing in

Canada

8/2/2019 NTC Jan 26

3/8


- The numbers of animals used for research have not changed very much through the years- Animals are mainly used in research, rather than testing or teaching

o E.g rodents, fish, birds, rabbits (but rodents are mostly used)o The rat is the favorite physiological model; most closely reflect human

physiological processeso There has been a large increase in the use of mice, perhaps due to the ease of

creating transgenic mice (in comparison to rats)

- Animals are usually used to test toxicity of the pharmaceutical industry, among othercompounds (e.g. in food, pesticides, etc)

o Check for toxic/negative effects (more subtle effects than death)o Acute toxicityglobal effect, versus organ-specific reaction and long-term

toxicity (e.g. tumor formation)

o Developmental/reproductive toxicity is also important- Very strict regulations for animal testing in labs (e.g. Canadian Council on Animal Care)

Animal Toxicity Tests - Assumptions1. The effects produced by a compound in laboratory animal are applicable to humans

o On the basis of dose per unit of body surface, toxic effects in humans are usuallyin the same dose range as those for experimental animals (not always true)

2. Exposure of experimental animals to toxic agents in high doses is a necessary and validmethod of discovering possible hazards in humans

o Based on quantal dose-response conceptthe number of animals in toxicologyexperiments is always small compared to the size of the human population at risk

Quantal dose Response- Dose-response curve for cumulative mortality testing for the LD50 (dose at which 50%

of the exposed animals will die)- How many animals should be tested so that youre sure that you can trust the results?

o According to statistics, about 200 per test groupbut this isnt feasible (cantkeep that many animals

in one spot)

o So we only use about 10animals; you initially

give 100% of the dose

that is found in nature

if that has no effect, thenyou can safely assume

that lower doses wontbe toxic, either

Regulatory Parameters- LOAEL (lowest observable

adverse effect level)statistically significant effect

8/2/2019 NTC Jan 26

4/8


- NOAEL (no observable adverse effect level) anything below the threshold of beingstatistically significant

- LOEL(lowest observable effect level) not harmful for health and life (non-adverseeffects, such as a subtle increase in heart rate)

- NOEL (no observable effect level)no- observed effect at all

Reference Dose- Based on the NOAEL (because you dont want any adverse effects)- Appropriate safety/uncertainty factors may be applied for risk assessment

o Population co-exposure to another potentially harmful compound test themixture!

o Genetic polymorphisms that might cause extreme sensitivity to a particular familyof compounds

o Significant portion of elderly or the very young in the population (changes intoxicokinetics in comparison to an adult population)

Benchmark Dose (BMD)- The benchmark dose is

based on modelling all of

the available dose

response data andgenerally estimates a risk

of 10% over background

(zero) for the response

of interest- Also you have to note that

the majority of the dose-response curve is due to

predictions which coverthe space between the

amounts of drug exposure

that was actually tested(rather than many

individual points, tested to

create a curve)

Risk Assessment: Exposure and Dose-Effect Data- What do you eat, drink, inhale used to calculate the exposure of an individual- Put this exposure level on the BMD graph and identify the chance of there being an

adverse effect

The Exposure-Response Paradigm- You may end up with a drug concentration in your tissue that is different from the dose

that you were actually exposed to

8/2/2019 NTC Jan 26

5/8


o Then compare this tissue dose to a biologically effective dose, and then examineresponses and potential pathology

Traditional Safety Tests

- Toxicokinetics measure ADME (absorption, distribution, metabolism, and excretion)- You have to question your animal model (e.g. there is a toxic metabolite produced in the

rat, which isnt produced in humans)

o You want to examine the location of the drug and its concentration in allorgans/tissues/fluids of the body (must find a way to obtain samples)

Dose Range- There must be extrapolation from what will affect a rat, to what will affect humans- Historically the LD50 was used; now we use the LD10 (lethal dose for 10% of the

population)

- Is always important to note the dose, time of exposure, and route of exposure

Duration and Frequency of Exposure- Acute Exposureless than 24 hours (can be either single or repeated)- Subchronic Exposurerepeated exposure between1-3 months- Chronic Exposurerepeated exposure form ore than 3 months

Acute Systemic Toxicity- Test by administering a drug to the animal, withdrawing the drug, and then observing for

effects after a 14-21 day period

o E.g. you can get the LD50 for ingestion of a certain compound- The LD50 can vary due to many factors:

o Species, age, weight, polymorphisms, gender, health, nutrition, route ofadministration, temperature, time of day, human error, etc.

o Age - newborn vs. adulto Route of exposureoral, dermal, subcutaneous

- You dont just have to measurethe LD50; you can also measure

motor function, respiration,

gastrointestinal effect, etc.

Acute Toxicity Tests

- Skin/eye irritation tests

topical toxicologyo Apply a drug and then

observe for a periodafterwards (redness,

corrosion, blistering,

etc.)

8/2/2019 NTC Jan 26

6/8


Long-Term Exposure

Experiments- Subchronic (12 months), Chronic

(45 months), Subacute (8 months)- Must consider comparison of

toxicity testing to reality (e.g. if

you are exposed to something for

a short time a day, youreprobably regularly exposed to it

in your lifestyle)

Long-term Toxicity- Determine:

o Therapeutic margins ineach species

o Dose-responserelationship of effects of a

drug when administeredrepeatedly

o Target organs for potential toxicityo Species and sex differenceso Whether the toxicity is reversible or not (e.g. if youre exposed during an accident

when you leave the site of exposure, will the effects go away?)

Histopathology- Histological examination of tissues and organs for changes and toxic effects- You can also sample body fluids and perform biochemical testsphysiological tests

o Your animal might look ok, but something will be impaired or affected

Chronic Exposure- Checking effect of a cumulative dose, carcinogenicity, reproductive and developmental

toxicity

Carcinogenicity- Studies measure the potential for a

compound to produce tumorsanimalsare exposed throughout their lives, and

resulting tumors are evaluated

o Highly expensive and time-consuming (not often conducted)

Developmental/Reproductive Toxicity- Measures the effects of chemical

exposure on sperm/eggs, fetaldevelopment and the ability to

reproduce

- Also observes delayed effects afterpubescence

8/2/2019 NTC Jan 26

7/8


- You want an organism with similar toxicokinetics as humans, a similarly designedplacenta and embryos that develop similarly to those of humans

o You want animals that are easy to breed, large litters and a short gestation period,and easy to maintain (inexpensive and well-behaved)

- For reproductive toxicity you can test the fertility of the male and female (testing howmany fetuses there are and how many losses there are)

o You can also let the fetuses be born, and then assess after birtho They can be sacrificed after the lactation period (identify compounds secreted in

the mothers milk)

o You can also test the second generation (effect of grandparents exposure)

Neurobehavioural Effects- Most are based on maze protocol put a rat in a maze and put food at the end; can

check memory and cognitive processes

o This can be checked during the development and also during the adult

Ecotoxicology- Effects on wildlife (fish, birds, earthworms)

o Measurement of the environmental effects of chemicals begins with alternativetargets

- E.g. estrogens present in the water feminization of male fish in the water

Testing Biologicals (vaccines, etc)- Expose an individual to a type of vaccine and test if there is an adverse effect (e.g. fever)

Types of toxicological studies- Acute toxicity irritation (skin/eye), skin sensitization- Embryotoxicity, teratogenicity, subacute/subchronic toxicity and reproductive toxicity- Chronic toxicity and carcinogenicity- Biokinetic and metabolic toxicity, and mutagenicity

Other possible model organisms- Bacteria (checking mutagenicity); insects, amphibians and lower vertebrates, also

transgenic mice- Are these relevant to human beings?- Ames testmutate the bacterial gene such that they will not survive in a medium lacking

a certain nutrient; if you apply the toxicant and the bacteria suddenly gain the ability to

survive in said media, it means that the compound is mutagenic- We know a lot about the

drosophila genome, so it can be

easier to test for mutations in the

drosophila genomeo Again, even if it does

affect the fly, how much

will it affect a humanbeing?

- Transgenic mice are often used totest carcinogenicityyou can

8/2/2019 NTC Jan 26

8/8


design a mouse that is more sensitive to carcinogenic agents

o It will then take less time and fewer amounts of the drug in order to observe animpact on tumor growth

Documents

NTC Jan 26