principles of toxicology........

1. Principles of Toxicology

2. The Basics
Toxicology – study of exposure to and adverse
effects of chemicals in living organisms
Risk Assessment – the integration of quantitative
measures of exposure and effect in order to
estimate the potential for risk
Effect – Absorbed chemicals interact with a
molecular target and cause a (generally
adverse) change
Exposure – Chemicals come in contact with, and
are absorbed into organisms

3. Areas of Toxicological Study
Descriptive Toxicology
• Toxicity testing for safety and regulatory needs
• Appropriate tests based on chemical type, use and anticipated exposure
• Observational data generate hypotheses for mechanistic studies
Mechanistic Toxicology
• Study of physiological, biochemical and molecular mechanisms by which toxic effects occur
• Alternative chemicals for drugs and therapeutics
• Chemical scalpels in biochemistry and physiology
• Differential toxicity mechanisms for age, sex, genotype and species variability
Regulatory Toxicology
• Decides if chemical poses sufficiently low risk to allow marketing
• Uses data from descriptive and mechanistic studies
• Specific enforcement responsibilities
- Food and Drug Administration
- Environmental Protection Agency
- Department of Labor - OSHA
- Consumer Product Safety Commission
- Department of Transportation

4. Other Areas of Toxicology
• Forensic Toxicology (Medical Examiners office)
Medical-legal aspects of poisonings.
Identification and quantification of poisons.
Establish relationship between tissue residual level and
probable cause of death.
• Clinical Toxicology (hospital setting)
Deal with emergencies such as overdoses, poisonings,
attempted suicides.
Compound identification and quantification.
Sign and symptom management.
Emergency care--home poisoning.
Environmental Toxicology
• Effects of compounds on water, wildlife etc.
• Movement of chemicals in the environment i.e., soil, air, water.
• Residual life of chemicals in the environment

5. • “What is there that
is not poison?
• All things are poison
and nothing is
without poison.
• Solely the dose
determines that a
thing is not a
poison.”
Paracelsus

6. Classification of Toxic Agents
Target organ; Use ; Source ; (Toxin vs Toxicant) ; Physical state ;
Chemical structure ; Reactivity ; Poisoning potential ;
Biochemical mechanism
Exposure
For toxic effects to occur, a chemical must reach a particular target site at
a sufficient concentration for a sufficient duration.
This is dependent on the disposition of the chemical.
Disposition: Absorption
Distribution
Metabolism
Excretion

7. Major Routes of Chemical Entry
• Ingestion – via the gastro-intestinal [GI] tract
• Inhalation – via the lungs
• Dermal – via the skin
• Other Parenteral (non-intestinal) routes:
Intravenous (IV) – direct injection into a vein
Intraperitoneal (IP) – injection into the peritoneal
cavity
Subcutaneous (SC) – injection between the skin and
the muscle
Intramuscular (IM) – injection directly into the
muscle
Intradermal – injection into the dermis
• Occupational Exposures – Inhalation, Dermal
• Accidental / Suicide Exposures – Ingestion

8. Duration and Frequency of Exposure
• Acute – Less than 24 hours – generally a single dose
Oral intubation/gavage, dermal, IP, SC, IV
Inhalation usually 4 hr
• Repeated Exposures – usually dietary
• Subacute – Repeated exposure for 1 month or less
• Subchronic – Repeated exposure for 1 to 3 months
• Chronic – Repeated exposure for greater than 3
months

9. Acute and Chronic Exposures
Can Lead to Different Outcomes
• Benzene
Acute exposure – CNS narcosis
Chronic exposure – bone marrow damage and leukemia
• Anticoagulant pesticides (1st generation)
Acute exposure – no effect
Chronic exposure – loss of blood clotting ability
• Cigarette Smoke
Acute exposure – Nervous system stimulation (nicotine)
Chronic exposure – Cancer of mouth, pharynx, larynx,
lung, esophagus, pancreas and bladder; emphysema

10. Frequency of Exposure
• Degradation and elimination
of a chemical can decrease
the magnitude and duration
of a chemical exposure.
• The half life is the time it
takes to remove half the
chemical from the
bloodstream.
• Chemical A – very long half
life ~ 1 yr
• Chemical B – half life of ~ 1
day
• Chemical C – short half life
~6 hr

11. With repeated exposures,
toxicity occurs when the
chemical reaches a critical
concentration at a target
receptor.
The time to toxicity depends on
- the frequency of
administration,
- the dose level, and
- the time to recovery
before subsequent doses.
Time to toxicity
Chemical A – 2 days
Chemical B – 4 days
Chemical C – never

12. Chronic effects occur, therefore:
• If the chemical accumulates to toxic levels in
the biological system
(i.e., absorption > metabolism and excretion)
• If the chemical causes irreversible effects at
exposure levels even though they decline
below toxic levels after each dose or
• If insufficient time is allowed for recovery
from toxic damage within the exposure
frequency interval

13. Effects of Chemical Exposure
• Allergic Reactions occur in hypersensitive individuals or
after sensitization in allergic or sensitized persons.
• Often requires binding of chemical (hapten) to endogenous
protein in order to be recognized by the immune system.
• Reaction ranges from skin irritation to anaphylactic shock
• Idiosyncratic Reactions occur in individuals who have
genetic polymorphisms that lead to structural changes in
biomolecules, making them very sensitive or insensitive to a
chemical. A broad range of effects can be found with many
drugs and chemicals with sufficient exposure.

14. Chemical Effects (cont.)
• Immediate vs Delayed Toxicity – Most chemicals exert their
effects soon after exposure. Others may be delayed for days
(delayed neuropathic OPs) to years (cancer)
• Reversible vs Irreversible Effects – Depends on tissue’s
ability to regenerate itself at a variety of levels: molecular,
cellular and tissue. Liver vs CNS
• Local vs Systemic Toxicity – Corrosives and irritants act
locally, little goes systemic. Acids (GI) Cl2 (lung).
• Systemic effects in sensitive (not always highest
concentration) tissues. CNS, circulatory and blood, visceral
organs. Pb DDT

15. Chemical Interactions
• Additive – combined effect is the same as the sum
of effects when given alone Ex. OPs
• Synergistic - combined effects are much greater than the sum
of effects when given alone
Ex. CCl4 and EtOH Ex. Pyrethroids and piperonyl butoxide
• Potentiation – exposure to a chemical with no toxicity
increases the toxicity of another compound
Ex. CCl4 and isopropanol
• Antagonism – co-administration of two chemicals
interferes with the toxicity of both or one of them
Ex. Antidotal therapies

16. Antagonism
• Functional – Chemicals counterbalance each other by
exerting opposite effects on a physiological function.
Ex. Convulsions treated with benzodiazepines
• Chemical (or inactivation) – Chemical reaction between
two compounds leads to less of the toxic compound.
Ex. Chelators and metals Ex. Antivenins
• Dispositional – Disposition of toxic chemical is changed
so that concentration and/or duration is diminished.
Ex. Ipecac, charcoal, pH alteration, metabolism induction or
inhibition
• Receptor – Chemicals compete for the same receptor,
decreasing effective binding of toxic compound.
Ex: Naloxone and morphine Ex: Tamoxifen and estradiol Ex:
OPs Atropine and AChR

17. Tolerance
• A state of decreased responsiveness due to a prior
exposure to the same or a structurally similar
chemical in an individual
• Dispositional Tolerance – A decreased amount of
chemical reaches the site where the effect is
produced
Ex. CCl4 – metabolism inhibition Ex. Cd and
metallothionein
• Receptor Tolerance – Same amount of chemical
reaches the site, but target receptor response
decreased
Ex. OPs and muscarinic AChR Ex. Nicotine in
cigarettes Ex. Morphine and Opioid receptors

18. Resistance
• A change in the susceptibility to a chemical at the
population level.
• A selective process (evolution) by which sensitive
individuals do not survive and only those with a
genetic trait that accommodates the chemical
survive.
• Subsequent selection through numerous
generations fixes the trait, stabilizing the resistant
population

19. Dose-Response Relationships
• Fundamental relationship that brings
together the dose of a chemical and the
resulting effect.
• Variability in the expression of dose and the
measurement of effect
• Evaluated at two different levels:
Individual, or “graded”, dose-response
and
Population, or “quantal”, dose-response

20. • Graded Dose-Response –
the Individual Level
• A dose-related increase in
the severity of a response
at the individual level
Can be documented for a wide
variety of endpoints
• Leading to families of
curves, each with their
individual dose-response
relationships.
• For a particular dose, a
profile of anticipated
responses might be found.
Ex. 7 mg/kg chlorpyrifos
ChE – 80% inhibited, CaE –
25% inhibited

21. At the Population Level – The Quantal Dose-Response
Based on a quantal or “all-or-none” response rather than a
gradation of potential responses
Consider the LD50. “The statistically derived single dose of
a substance that can be expected to cause death in 50% of
the animals tested”
Groups of animals are given a gradation of doses and the
number of mortalities and survivors is tallied.
Ex.
Dose # dosed Deaths/survivors % mortality
1 10 0 / 10 0
2 10 1 / 9 10
3 10 4 / 6 40
4 10 7 / 3 70
5 10 9 / 1 90
6 10 10 / 0 100

22. • If the # of mortalities at each dose
is
reduced by the # in each
previous dose, we get a mortality
frequency
distribution
• Quantal dose-responses usually
exhibit a normal or gaussian
distribution.
• Biological variation causes the
differences in sensitivity.
• Animals at the low end:
Hypersusceptable
• Animals at the high end: Resistant

23. Summary: Probit transformation adjusts
quantal data (i.e., mortality) to an
assumed normal population
distribution, resulting in a straight
line.
Differences in slope reflect the biological
variability,between individuals in the
test species, in the dispositional and
receptor
characteristics associated with a
chemical’s toxicity in an animal’s body.
The slope of the LD50 curves can
vary between compounds, with
shallow and steep slopes.
Shallow Slope (A) – Greater variability
between individuals
Steep Slope (B) – Less variability between
individuals

24. Dose-Response Curves for
Essential Compounds

25. Hormesis – Non-nutritional
toxicants impart beneficial
or stimulatory effects at low
doses, but adverse effects at
higher doses.
Ex.Chronic alcohol
consumption leads to liver
cirrhosis & cancer and
cancer of the esophagus. (A)
Moderate consumption
decreases
coronary heart disease and
stroke (B)
Taken together they result in
“U-shaped” dose-response
curves (C)

26. For therapeutic chemicals, it is important to
know how an effective dose (ED)
compares with a toxic dose (TD) and a
lethal dose (LD).
The Therapeutic Index is a ratio of the dose
that is lethal to the dose that is
therapeutic
TI = LD50 / ED50 = 300 / 20 = 15
The Margin of Safety is more protective:
Margin of Safety = LD1 / ED99 = 55 / 95 = 0.58
“Safe” as larger numbers indicate values
farther apart.
Does not consider the slopes of the effective
and lethal dose curves, however
Therapeutic Index and Margin of Safety
Values > 1 indicate no overlap.
< 1 indicate adverse effects with efficacy

27. • Potency: Chemical A is more
potent than Chemical B
based on relative position
along the dosage axis. The
lower the dose to lead to an
effect, the more potent the
compound. C is also more
potent than D.
• Maximal Efficacy: Chemicals
A and B have similar
maximal potencies. The
maximal efficacy of
Chemical C is less than that
of Chemical D.

28. • Animal Toxicity Animal
Toxicity Tests
• Acute Lethality
• Skin and Eye Irritation
• Sensitization
• Subacute
• Subchronic
• Chronic
• Developmental and
• Reproductive Toxicity

29. PRECLINICAL TOXICOLOGY
Acute Toxicity
SubAcute Toxicity
Subchronic
Chronic Exposure
Drug Disposition/Pharmacokinetics (ADME)
In Vitro Permeation Studies
In Vivo Absorption Studies
LOCAL TOLERANCE STUDIES
Irritation And Sensitization
Immunotoxicity
Reproductive Toxicity
Genotoxicity/Mutagenicity
Teratogenicity
Carcinogenicity

30. Acute Toxicity
Objectives
• To determine the Median Lethal Dose (LD50) after a single dose
administered through one or more routes, one of which is the intended route
of administration in humans.
• To determine Maximum Tolerated Dose (MTD) and No Observable Effect
Level (NOEL).
• To identify potential target organs for toxicity.
• To help select doses for repeated-dose toxicity tests.
Duration
A few days to 2 weeks after a single doseTest System/Animal System
2 species required. Mice, rats, sometimes rabbits or dogs.
Dose Administration
• Oral (by gavage or with food); Subcutaneous; Intraperitoneal; Intradermal;
Inhalation;
• Intranasal; Topical (epicutaneous); Intravenous
Parameters
• Mortality
• Clinical pathology
• Gross necropsy
• Weight change
• Signs of toxicity

31. Subacute Toxicity (Repeated Dose)
• Objectives
• To determine toxicity after repeated administration of the test
material.
• To help establish doses for subchronic studies.
Duration
14 days
Test System/Animal System
2 species required. Mice, rats, rabbits, guinea pigs, dogs.
Dose Administration
3 to 4 doses given by the same routes as previous toxicity tests.
Parameters
• Mortality
• Signs of toxicity
• Pathology and histopathology
• Weight change
• Clinical pathology

32. Subchronic And Chronic Exposure
• Objectives
• To establish a “no observable effect level" (NOEL)
• To characterize dose-response relationships following repeated doses
• To identify and characterize specific organs affected after repeated administration
• To predict a reasonable and appropriate dose for chronic exposure studies
(maximum tolerated dose or MTD)
Duration
Commonly 90 days, but varies from 2 weeks to 6 months or up to 10% of species’
lifespan.
Test System/Animal System
2 species required. Rodents, dogs.
Dose Administration
At least 3 doses given by the same routes as previous toxicity tests; the lowest
producing no apparent toxicity and the highest producing toxicity but less than or
equal to 10% mortality.
Parameters
• Mortality
• Weight change
• Signs of toxicity
• Clinical pathology
• Pathology and histopathology

33. Chronic Exposure
Objectives
• To evaluate the cumulative toxicity of chemicals.
• To assess carcinogenic potential.
Duration
Rodents - 6 to 24 months; non-rodents - 12 months or longer or up
to 10% of species’ lifespan. Length depends on intended period of
human exposure.
Test System/Animal System
2 species required. Rodents, dogs.
Dose Administration
As in subacute/ subchronic toxicity studies.
Parameters
• Mortality
• Pathology and histopathology
• Weight change
• Clinical pathology of all animals (mortalities and survivors)

34. Drug Disposition/Pharmacokinetics (ADME)
Objective
Evaluate the bioavailability, tissue
distribution, active metabolite formation,
and elimination of test materials.
Test System/Animal System
Rat, Dog, Swine.
Parameters
• AUC (Area Under Curve)
• Cmax (max concentration in blood)
• Tmax (Time Cmax is reached)

35. In Vitro Permeation Studies
Objective
To study the effect of test materials on skin metabolism, or the effect
of skin metabolism on xenobiotics
Test System/Animal System
Excised skin, mucosa, or other biological membranes cultured on
specialized diffusion cells.
Parameters
• Evaporation rate
• Quantitation of deposition in different skin layers
• Degree of percutaneous penetration
In Vivo Absorption Studies
Objective
To study percutaneous absorption of drugs or environmental
contaminants.
Test System/Animal System
Small rodents (guinea pigs), swine.
Parameters
Absorption rate (quantitation in blood, urine, feces, vaginal fluids,
and tissue samples)

36. LOCAL TOLERANCE STUDIES
Irritation And Sensitization
Objectives
• To determine the potential of a test material to provoke ocular irritation,
dermal irritation, or sensitization.
Duration
Irritation - one hour to three weeks after a single topical or corneal
administration. Sensitization - intradermal or topical induction doses
followed by topical challenges with a non-irritating dose (6 - 8 weeks total).
Test System/Animal System
Rodents, rabbits
Dose Administration
• Single patch administration
• Multiple doses over 2—4 weeks
• Topical (epicutaneous), intradermal, or corneal
Parameters
• Degree of pruritis, erythema, edema, papules, and vesicles
• Corneal irritation, swelling, or injury
• Microscopic integrity of corneal endothelium
• Other features of the eye (conjuctive, cornea, iris, lens, anterior portion of
vitreous humor)

37. Immunotoxicity
Objective
To determine the potential of a test material to induce immune suppression or
immune enhancement.
Duration
Subacute (14 days) or sub¬chronic (90 days) exposure
Test System/Animal System
Rodents
Dose Administration
Repeated doses administered as in subacute/subchronic toxicity studies
Parameters
Level I
• Hematology
• Histopathology or lymphoid organs
• Quantity of T- and B-cells (cellularity of lymphoid organs)
• Blastogenesis (mitogen responsiveness; mixed lymphocyte reaction)
• Quantitation and funciton of natural killer cells
• Macrophage function
• Cytokine production
Level II
• Kinetics of antibody production to T-dependent antigens
• Quantity of IgM/IgG-producing (plaque-forming) cells
• Delayed hypersensitivity responses to known sensitizers
• Immune response to infectious agents (e.g., Listeria, Streptococcus)
• Immune response to transplantable tumors

38. Reproductive Toxicity
Objectives
• To determine potential adverse effects of a test material on mammalian gametogenesis, fetal
organogenesis, and neonatal development.
• To determine potential adverse effects on delivery, lactation, neonatal survival and vitality.
Test System/Animal System
Rodents, rabbits.
Parameters
• Preconception
• Mating behavior
• Preimplantation and fertilization rates
• Integrity and quantity of sperm and egg cells
• Post Conception
• Maternal weight gain
• Time from conception to delivery
• Litter size
• Number of corpora lutea and implanted fetuses
• Fetal mortality and viability
• Placental weight
• Pup weight and crown-rump length
• Postnatal
• Problems at parturition
• Maternal-newborn relationship; maternal ability to rear young
• Postnatal growth; time of occurrence of developmental landmarks
• 21-day survival of young
• Functional parameters after 21 days

39. Teratogenicity test
Objective
The objective of this test is to administer the test substance to pregnant
animals during the period that the internal organs of the fetus are forming,
and determine what damage to the birth of the fetus is caused by the test
substance, in particular the teratogenicity of the test substance.
Test Animals
Animal type
• Use rodents, such as rats or mice, and non-rodents such as rabbits.
• When selecting the animal type, species and breed, take into consideration
knowledge concerning reproduction, such as knowledge about fertility, the
frequency of occurrence of naturally-occurring abnormalities, sensitivity of the
animal to substances known to have reproductive or birth toxicity, etc.
• Also, it is desirable to select animals with a low frequency of occurrence of
naturally-occurring abnormalities.
Number of animals
With rats and mice, use 20 or more animals for each dosage group
With rabbits, use 12 or more animals.

40. Test substance
Method of administration
As a rule, the test substance is administered forcibly and orally.
Dosage
 In order to determine the dosage-response relationship and estimate the maximum dosage
with no effect, establish dosage test groups with, as a rule, at least three stages.
 Make the maximum dosage the amount that causes the mother animals to show slight
indications of toxicity, such as declining food intake or suppression of increase in weight, but
does not cause a death rate of over 10%.
 In the case that the mother animal shows no indications of toxicity even from the maximum
amount of the test substance that can be administered (make the limit 1000mg/kg), make that
amount the maximum dosage.
 Make the minimum dosage the amount for which toxic impact on the birth of the fetus is not
shown. Set up a control group separately to administer the solvent only.
Administration period
 Administer the test substance every day during the period that the internal organs of the fetus
are forming.
 Normally, when the date of verification of copulation is defined to be day 0 of pregnancy, the
administration period is from the 6th day to the 18th day of pregnancy for mice,
 4th
day to 15th
day for hamsters
 from the 6th day to the 20th day of pregnancy for rats,
 and from the 6th day to the 29h day of pregnancy for rabbits.

41. Mating Procedures
• In-house mating of the animals is
recommended. A sufficient number of males
should be mated to ensure a large gene pool.
• Each male may be mated to either one or two
females.
• The following morning, each female should be
examined for the presence of sperm in the
vaginal lavage or the presence of a sperm plug.
• The presence of sperm in the vaginal lavage or
the presence of a vaginal plug is considered day
zero of gestation.

42. Maternal Toxicity and its Significance
• End points which may serve as indicators
of maternal toxicity include mortality, body
weight, body weight gain, organ weights,
feed and water consumption, clinical signs
of toxicity, and gross or microscopic
lesions.
• The calculation of a corrected mean
maternal weight gain (difference between
initial and terminal maternal body weight
less the gravid uterus weight) may also be
used as an index of maternal toxicity.

43. Clinical Observation
• Throughout the study, each animal should
be observed at least twice daily. The first
observation should be a thorough clinical
examination.
• Relevant behavioral changes and all signs
of toxicity, morbidity, or mortality should be
recorded.

44. • Fetuses should be evaluated for skeletal and soft-tissue anomalies. For
rodents, approximately one-half of the rodent fetuses should be preserved in
Bouin's solution and sectioned by the Wilson serial section technique to
evaluate alterations of the soft tissues.
• The remaining fetuses should be prepared and stained for skeletal
anomalies (Alizarin red stain for bone and optional Alcian blue stain for
cartilage). The assignment to soft-tissue or skeletal examination should be
done randomly or alternately. The alternation procedure is sometimes not
followed when an abnormality is found which would be better observed by a
different technique.
• For example, a specimen with an obvious skeletal defect would be prepared
for skeletal examination.
• Alternatively, all rodent fetuses may be freshly dissected to discover soft-
tissue abnormalities, then fixed and examined for skeletal anomalies.
• Each rabbit fetus should be examined for both soft-tissue and skeletal
malformations and variations.
• The bodies should be evaluated for soft-tissue anomalies by fresh
dissection, followed by fixation and an examination for skeletal anomalies.
• Internal head structures should be evaluated in at least one-half of the fetal
heads of rabbit fetuses.
• This evaluation should include at least the eyes, brain, nasal passages, and
tongue.

45. End Points Measured
For litters with live fetuses, mean male and female body weights and
the incidence per litter of all divergences from normal fetal
development (skeletal and visceral analysis) should also be
reported.
Analysis of Data
Reporting the Results of Developmental Toxicity Studies
Reports of all studies should contain the information required by the
Good Laboratory Practice Regulations, including a copy of the study
protocol and all amendments, absolute values for all parameters,
complete data (individual pups) and tables of data summarized and
analyzed by litter.