Clinical Toxicology

Phar 3132
 Chapter 1

Origin & Scope of Toxicol-

ogy
Outline

•Introduction to Toxicology

•History of Toxicology

•Different Areas of Toxicology

 I. Introduction to Toxicology

Toxicology: Derived from Greek word, toxikon

and logos •
the study of the adverse effects of chemi-
cal, physical, or biological agents on living
organisms and the ecosystem, including
the prevention and amelioration of such
adverse effects."
·The science of poisons that studies toxic sub-
stances with respect to their:
Sources , Properties , Mechanism of toxicity
Toxic effects, Detection , Clinical manifesta-
3

tions
Introduction to Toxicology…..

· Chemical Sources: like drug, corrosives

· Plant sources: hashish, cocaine
· Animal sources: animal venoms( scorpion,
spider, Snake
· A toxicologist is trained to examine & com-
municate the nature of those effects on hu-
man, animal, & environmental health
Toxicological research examines the cellu-
lar, biochemical, & molecular MOT as well
as functional effects such as:
• Neurobehavioral & immunological, & as-
4
sesses the probability of their occur-
 Cont……

· Toxicologists they are involved in the

recognition, identification, and quantification
of hazards resulting from occupational expo-
sure to chemicals and the public health as-
pects of chemicals in air, water, other parts of
the environment, foods, and drugs.
Molecular toxicologist
Clinical toxicologist 5
 Toxicological terms and definitions

· Toxin- a poison of natural (biological) origin, specifies

the source (living cells/organisms).
Bacterial toxins: Botulinum toxin (from Clostrid-
ium botulinum), Tetanus toxin.
Plant toxins (Phytotoxins): Ricin (from castor
beans), Aconitine (from Monkshood).
Animal toxins (Zootoxins): The chemical com-
ponent of venom (e.g., from snakes, spiders, scor-
pions).
Fungal toxins (Mycotoxins): Aflatoxins.

 6
 Cont……
· Poisons :any substance -chemical, physical, or bio-
logical—that, when administered, inhaled, absorbed,
or ingested in sufficient quantity, produces disease
conditions, tissue injury or interrupts natural life pro-
cesses when in contact with or absorbed into the
body.
Is broader category focuses on the effect (causing
harm/toxicosis).
Toxicosis describes the disease state that results
from exposure to poison or poisoning and intoxica-
7
8
 Cont……
· Toxicity-any toxic (adverse) effect that a chemical or
physical agent might produce within a living organ-
ism 
· Toxicant : poisonous substance produced by hu-
man activity (e.g., industrial chemicals, pollution).
• chemical that can injure or kill humans, ani-
mals, or plants; (poison)
Systemic toxin: Affects the entire body/many or-
[Link] site specific
Organ toxin: Affects only specific tissues or organ
· Hazard - is the likelihood that injury will occur in a
given situation or setting: the conditions of use and 9
exposure are primary considerations
 Cont…….
· Risk - is defined as the expected frequency of
the occurrence of an undesirable effect aris-
ing from exposure to a chemical or physical
agent
RISK= HAZARD + EXPOSURE
· The toxicity depends on a variety of factors:
dose, duration and route of exposure, shape
and structure of the chemical itself, and indi-
vidual human factors.
· toxicity is a relative term applied in compar-
ing one chemical with another.
· A highly toxic substance causes damage to an organ-
10
 II. History of Toxicology

A. Antiquity
Toxicology dates back to the earliest humans, who used
animal venom & plant extracts for hunting, warfare, & as-
sassination
The Ebers papyrus (circa 1500 B.C.) describes many rec-
ognized poisons- hemlock, aconite, opium, lead, copper,
antimony, digitalis, belladonna alkaloids
Theophrastus (370–286 B.C.), a student of Aristotle, wrote
De Historia Plantarum
Socrates (470-399 B.C.) received poison as part of state
execution

11
Antiquity…

· Demosthenes (385– 322 B.C.) and Cleopatra’s

(69–30 B.C.)
Voluntary took poison
· King Mithridates (134-63 B.C.) of Pontus
Poisoned many criminals in his experiments to
identify antidotes
· Epidemic Poisonings in Rome lasted from 4th -1st
century B.C
· Dioscorides (Greek physician) 60 A.D.
 Described many poisons, devised categorization system
(plant, animal, mineral)

12
 B. Middle Ages

· Moses Ben Maimon (Maimonides 1135–1204 A.D.)

Wrote poisoning and their Antidotes
· Renaissance Italians brought poisoning to a zenith
Catherine de Medici
Catherine Deshayes ( “La Voisine” )

13
C. Age of Enlightenment

· Paracelsus (1493-1541)
Led the change from the science of Hippocrates and
Galen to that of the 17th century giants
Main Contributions by Paracelsus:
• Described the concept that “the dose makes the poi-
son”
• Toxicity is related to chemical structure
• Described the main clinical manifestations of two
common poisoning: arsenic & mercury

14
D. Modern Toxicology

· Exponential growth parallels the World War II

with greatly increased production of drugs, pesti-
cides, organic chemicals

· During this period, the use of “patent” medicines

was prevalent, and there were several incidents
of poisonings from these medicaments

15
Categories of Toxic effects

Toxic effects may be grouped into:

 Local and systemic effect
Reversible and irreversible effect
Immediate and delayed effect
Morphological effect
Functional effect , Biochemical effect

16
 Principal aspect of Toxicology
The principal aspects of Toxicology:
[Link]: The study of the mechanisms of
action by which toxicants exert their adverse effects
on living organisms (e.g., receptor binding, enzyme in-
hibition, DNA damage)..
[Link]: The study of the absorption, distri-
bution, biotransformation (metabolism), and ex-
cretion of toxicants (ADME).
[Link]: deals with the measurement and
quantitative assessment of the toxicity of sub-
stances..

17
 Cont…..
· Toxicometrics is fundamental to determining safety
standards and establishing appropriate exposure lim-
its for drugs, industrial chemicals, and environmental
agents.
 It provides the numerical data for risk assess-
ment.
1. Measurement of Poisonous Amounts
· It quantifying how much of a substance is required to
produce a specific adverse effect.
Key parameters measured include:
· LD-50,TD50, ED50 18
· LC50(Lethal Concentration 50): The concentra-
2. Dose-Response Relationship
·Toxicometrics is centered on the analysis of the dose-
response curve. This curve plots the magnitude of
the response (effect) against the dose (or concentra-
tion) of the substance.
·Toxicometrics helps to determine the :
No Observed Adverse Effect Level (NOAEL)
or
Lowest Observed Adverse Effect Level
(LOAEL), which are critical points used to set safe
exposure limits for humans.
·Slope: The steepness of the dose-response curve
provides a measure of the margin of safety or haz-
ard; a steeper slope means a small increase in dose
results in a large increase in response. 19
 3. Risk Assessment and Regulatory Toxicol-
ogy
· The quantitative data generated by Toxicometrics
forms the scientific basis for:
Calculating the Therapeutic Index (TI):

A high TI indicates greater safety.

· Setting Regulatory Standards: Agencies like the
FDA, EPA, and OSHA use toxicometric parameters
to establish Maximum Allowable Concentrations
(MACs) or Permitted Exposure Limits (PELs) for
chemicals in food, water, and the workplace.
· Predictive Toxicology: Developing mathematical
models (often integrating Toxicokinetics and Toxico-
dynamics) to predict the toxicity of new, unstudied20
chemicals.
III. Different Areas of Toxicology

· Toxicology is broadly divided into different

classes Depending on: 
A. Research methodology 
B. Socio-medical 
C. Organ/specific effects
I. Based on research methodology
· The professional activities of toxicologists
fall into 3 main categories:
Descriptive, mechanistic, & regulatory
21
 I. Based on research method-
· ology
Descriptive, mechanistic, & regulatory
· Although each has distinctive characteristics,
each contributes to the other, & all are vitally
important to chemical risk assessment
A. Mechanistic Toxicology:
 Concerned with identifying & understanding the cellu-
lar, biochemical, & molecular MZMs by which chemi-
cals exert toxic effects on living organisms
 In risk assessment, mechanistic data may be very
useful:
i. In demonstrating that an adverse outcome ob-
served in lab animals is directly relevant to hu-
mans
22
ii. This is important for rational treatment – Facili-
 Different Areas of Toxicology …

· Mechanistic Toxicology…
E.g., the relative toxic potential of
organophosphate insecticides in mammalians &
insects can be accurately predicted on the basis of
an understanding of:
• Common mechanisms (inhibition of AchE )&
• Differences in biotransformation for these insec-
ticides among the different species
· Malathione has low mammalian toxicity B/c mam-
mals have carboxylesterases, that readily hydrolyze
the carboxyester link, detoxifying the compound.
While Insects, by contrast, do not readily hydrolyze
this ester, and the result is its selective insecticidal
23
action
 Different Areas of Toxicology …
· Mechanistic Toxicology…

ii. In identifying adverse responses in experimental

animals that may not be relevant to humans
• E.g. , the propensity of the widely used artificial
sweetener saccharin to cause bladder cancer
in rats may not be relevant to humans at
normal dietary intake rates
• B/c bladder cancer is induced only under condi-
tions where saccharin is at such a high conc in
the urine that it forms a crystalline precipitate24
Different Areas of Toxicology …
B. Descriptive Toxicology:
Concerned directly with toxicity testing, which pro-
vides information for safety evaluation & regulatory re-
quirements
The appropriate toxicity tests in cell culture systems or
experimental animals are designed to yield info to
evaluate risks posed to humans & the environment
from exposure to specific chemicals

25
 Different Areas of Toxicology …

C. Regulatory Toxicology:

Decide, on the basis of data provided by

descriptive & mechanistic toxicologists,
whether a drug or other chemical poses a
sufficiently low risk to be marketed for a
stated purpose or subsequent human or
environmental exposure resulting from its
use. 26

·
II. Based on specific socio-medical issues

· Occupational toxicology – It deals with

chemical found in the workplace
E.g. – Industrial workers may be exposed to
these agents during the synthesis, manu-
facturing or packaging of substances
· Agricultural workers may be exposed to
harmful amounts of pesticides during the
27
application in the field
Different Areas of Toxicology …

· In addition to the above categories, there are

other specialized areas of toxicology such as:
Forensic toxicology
Clinical toxicology
Environmental toxicology

28
Different Areas of Toxicology …

a) Forensic toxicology:
A hybrid of analytic chemistry & fundamental
toxicological principles
Concerned primarily with the medico-legal
aspects of the harmful effects of chemicals on
humans & animals
Primarily to aid in establishing the cause of
death & determining its circumstances in a
postmortem investigation

29
Different Areas of Toxicology …

b) Environmental Toxicology
Focuses on the impacts of chemical pollu-
tants in the environment on biological organ-
isms
Although toxicologists concerned with the ef-
fects of environmental pollutants on human
health fit into this definition, it is most com-
monly associated with studies on the impacts
of chemicals on nonhuman organisms such
as fish, birds, terrestrial animals, and plants

30
Different Areas of Toxicology …

· Clinical toxicology:
Designates an area of professional em-
phasis in the realm of medical science
that is concerned with disease caused by
or uniquely associated with toxic sub-
stances
Efforts are directed at treating pts poi-
soned with drugs or other chemicals & at 31
III. Based on the organ/system effect

· Cardiovascular toxicology
· Renal toxicology
· Central nervous system toxicology –
· Gastrointestinal toxicology
· Respiratory toxicology

32
Fig. Graphical representation of the interconnections b/n different
areas of toxicology
33
34
 Scope and Importance of Toxicology

· Toxicology deals with the toxicity studies of

chemicals used:
[Link] medicine for diagnostic, preventive and
therapeutic purposes.
[Link] food industry as direct and indirect addi-
tives.
[Link] agriculture as pesticides, growth regula-
tors, artificial pollinators, and animal food ad-
ditives.
[Link] chemical industry as solvent, components,
and intermediates of plastics and many other 35
· Toxicology may be helpful in the development of:
i. Suitable and safer food additives,
ii. Suitable and safer pesticides,
[Link] drugs against any specific disease.
2. The data on acute toxicity tests Provides an idea
of toxic, sub-lethal & lethal dose of a specific toxi-
cant for specific animal

36
Cont……

3. helpful in the antidotal therapy.

4. helpful in the monitoring of environmental
pollution.
5. helpful in monitoring of risk assessment.
6. An understanding of the mechanism of
toxic action also contributes to the basic
knowledge of pharmacology, physiology,
biochemistry and cytology.
7. Analytical toxicology provides suitable
procedures to evaluate the presence or ab-
sence of different types of substances and 37
Principles of toxicology
Learning objectives

· By the end of this chapter you should be able to:

Classify toxic agents in a varity of ways
Describe the spectrum of undesired effects
Differentiate b/n quantal & graded dose re-
sponse relationships
Explain about variations in toxic responses

39
Chapter Outline

· Spectrum of toxic dose

· Classification of toxic agents
· Characteristics of exposure
· Spectrum of undesired effects
· Dose-response relationships
· Variation in toxic responses

40
 I. Spectrum of toxic dose
· It refers to the range of doses of a chemical (xeno-
biotic) that elicit different biological responses, rang-
ing from beneficial effects at low doses to lethal ef-
fects at high doses. This spectrum is typically visual-
ized and analyzed using the Dose-Response
Curve.
· Paracelsus (1493–1541), phrased this well when he
noted:
“What is there that is not poison? All things are
poison & nothing [is] without poison. solely the
dose determines that a thing is not a poison” 41


 Spectrum of toxic dose…
· Among chemicals there is a wide spectrum of
doses needed to produce deleterious effects, seri-
ous injury, or death
Some chemicals produce death in µg doses & are
commonly thought of as being extremely poi-
sonous
Other chemicals may be relatively harmless
after doses in excess of several grams
· It should be noted, however, that measures of acute lethal-
ity such as LD50 may not accurately reflect the full spec-
trum of toxicity, or hazard, associated with exposure to a
chemical
• E.g., some chemicals with low acute toxicity may
have carcinogenic or teratogenic effects at doses
that produce no evidence of acute toxicity 42
Table: Approx acute oral LD50 of some representative
chemical agents

43
44
45
II. Classification of toxic agents

· Toxic agents are classified in a variety of ways,

depending on the interests & needs of the clas-
sifier
· Eg, toxic agents are discussed in terms of their:
Target organs: liver, kidney, hematopoietic
system, etc
Use: pesticide, solvent, food additive, etc
Effects: cancer, mutation, liver injury, etc
Source: Toxin and Toxicant

46
Classification of toxic agents…

· Toxin: refers to toxic substances that are pro-

duced by biological systems such as plants, ani-
mals, fungi or bacteria
E.g. Zeralanone, produced by a mold

· Toxicant: used in speaking of toxic substances

that are produced by or are a by-product of an-
thropogenic (human-made) activities
E.g. Dioxin [2,3,7,8-tetrachlorodibenzop- dioxin
(TCDD)], produced during the combustion of
certain chlorinated organic chemicals
47
Classification of toxic agents…

· Some toxicants can be produced by both

E.g. polyaromatic HCs are produced by the
combustion of organic matter
• Natural processes, e.g., forest fires &
• Anthropogenic activities, e.g., combustion of
coal for energy production; cigarette smoking
· Generally, such toxic substances are referred to as
toxicants, rather than toxins
B/c, although they are naturally produced, they
are not produce by biological systems

48
Classification of toxic agents…

· Other means of classifications:

Physical state: gas, dust, liquid
Chemical stability or reactivity: explosive,
flammable, oxidizer
General chemical structure: aromatic amine,
halogenated HC, etc.
 Poisoning potential: extremely toxic, very toxic,
slightly toxic, etc
Biochemical MOA: e.g., alkylating agent,
sulfhydryl inhibitor, methemoglobin producer

49
III. Spectrum of undesired effects

· Spectrum of undesired effects of chemicals is broad

· Some effects are deleterious & others are not

· E.g. In therapeutics each drug produces a number

of effects, but usually only one effect is associated
with the primary objective of the therapy
All the other effects are referred to as undesir-
able or side effects of that drug for that therapeu-
tic indication

50
Spectrum of undesired effects

· However, some of these side effects may be de-

sired for another therapeutic indication
E.g. the “1st -gen” antihistamine diphenhydramine
• It is effective in reducing histamine responses
associated with allergies, but it readily enters
the brain & causes mild CNS sedation
• However, it is widely used as an ‘OTC”
sleep remedy, taking advantage of the
CNS-depressant effects

51
Spectrum of undesired effects…

· Some side effects of drugs are never desirable &

are always deleterious to the well-being of hu-
mans
These are referred to as the adverse, deleteri-
ous, or toxic effects of the drug

52
Spectrum of undesired effects…

a) Allergic reactions:
Chemical allergy: an immunologically mediated
adverse rxn to a chemical resulting from pre-
vious sensitization to that chemical or to a
structurally similar one

Once sensitization has occurred, allergic rxns

may result from exposure to relatively very low
doses of chemicals
Table: Classification of immunologic reactions

SJS/TEN: Stevens-Johnson syndrome/ Toxic epidermal necrolysis

AGEP: Acute generalized exanthematous pustulosis
DRESS syndrome: Drug Rxn (or Rash) with Eosinophilia and Systemic Symptoms

53
Spectrum of undesired effects…

b) Idiosyncratic rxn:
Refers to a genetically determined abnormal re-
activity to a chemical
c) Immediate vs delayed toxicity:
Immediate toxic effects
• Can be defined as those that occur/ develop
rapidly after a single administration of a sub-
stance
Delayed toxic effects:
• Those that occur after the lapse of some time

54
Spectrum of undesired effects…

d) Reversible vs irreversible toxic effects:

If a chemical produces pathological injury to a tis-
sue, the ability of that tissue to regenerate
largely determines whether the effect is reversible
or irreversible
• For a tissue such as liver, which has a high ability
to regenerate, most injuries are reversible
• Whereas injury to the CNS is largely irreversible
Carcinogenic & teratogenic effects of chemicals,
once they occur, are usually considered irre-
versible toxic effects
55
Spectrum of undesired effects…

e) Local Vs systemic toxicity:

Local effects:
• Those that occur at the site of first contact b/n
the biological system & the toxic agent
• Produced by the ingestion of caustic substances
or the inhalation of irritant materials
• E.g. chlorine gas reacts with lung tissue at the
site of contact, causing damage & swelling of
the tissue, with possibly fatal consequences,
even though very little of the chemical is ab-
sorbed into the bloodstream
56
Spectrum of undesired effects…

· Local Vs systemic toxicity…

Systemic effects:
• Require absorption & distribution of a toxicant
from its entry point to a distant site, at which delete-
rious effects are produced
Most substances except highly reactive agents pro-
duce systemic effects
For some agents, both effects can be demonstrated
• E.g. Tetraethyl Pb produces effects on skin at the site of
absorption & then is transported systemically to produce
its typical effects on the CNS & other organs

57
Spectrum of undesired effects…

· Local Vs systemic toxicity…

Most chemicals that produce systemic toxicity do
not cause a similar degree of toxicity in all organs;
instead, they usually elicit their major toxicity in
only one or two organs
• Target organs of toxicity of a particular chemical
The target organ of toxicity is often not the site of
the highest conc of the chemical
• E.g., Pb is concentrated in bone, but its toxicity is due to
its effects in soft tissues
• DDT is concentrated in adipose tissue but produces no
known toxic effects in that tissue
58
Spectrum of undesired effects…

f) Interaction of chemicals:
Chemical interactions are known to occur by a
number of MZMs, such as:
• Alterations in absorption, protein binding, & the
biotransformation & excretion of one or both of
the interacting toxicants

In addition to these modes of interaction, the re-

sponse of the organism to combinations of toxi-
cants may be ↑ed or ↓ed b/c of toxicologic re-
sponses at the site of action
59
Spectrum of undesired effects…

· Interaction of chemicals…
a) Additive effect:
• Occurs when the combined effect of 2 chemicals
is equal to the sum of the effects of each agent
given alone (E.g. 2 + 3 = 5)
• The effect most commonly observed when 2
chemicals are given together is an additive effect
• E.g. when 2 organophosphate insecticides are
given together, the cholinesterase inhibition is
usually additive

60
Spectrum of undesired effects…

· Interaction of chemicals…

b) Synergistic effect:
• Occurs the combined effects of 2 chemicals
are much greater to the sum of the effects of
each agent given alone (e.g. 2 + 2 = 20)

• E.g. CCl4 & Ethanol induced hepatoxicity

61
Spectrum of undesired effects…

· Interaction of chemicals…

c) Potentiation effect:
• Occurs one chemical dose not have a toxic
effect on a certain organ but when added an-
other chemical makes that chemical much
more toxic (e.g. 0 + 2 = 10)
• E.g. Isopropanol is not hepatoxic, but en-

hance CCl4 induced hepatoxicity

62
Spectrum of undesired effects…

· Interaction of chemicals…
d) Antagonistic effect:
• Occurs when 2 chemicals administered to-
gether interfere with each other’s actions or
one interferes with the action of the other (e.g.
4 + 6 = 8; 4 + (-4) = 0; 4 + 0 = 1)
• Antagonistic effects of chemicals are often
very desirable in toxicology & are the basis of
many antidotes
• There are 4 major types of antagonism: func-
tional, chemical, dispositional, & receptor
63
Spectrum of undesired effects…

g. Tolerance:
It is a state of ↓ed responsiveness to a toxic ef-
fect of a chemical resulting from prior exposure
to that chemical or to a structurally related chem-
ical
2 major MZMs are responsible for tolerance:
• Due to a ↓ed amount of toxicant reaching
the site where the toxic effect is produced →
dispositional tolerance
• Due to a reduced responsiveness of a tis-
sue to the chemical
64
IV. Characteristics of exposure

· Toxic effects in a biological system are not produced by a

chemical agent unless that agent or its metabolic break-
down products reach appropriate sites in the body at a
conc & for a length of time sufficient to produce a toxic
manifestation
· Many chemicals are of relatively low toxicity in the “na-
tive” form but, when acted on by enzymes in the body, are
converted to intermediate forms that interfere with nor-
65
mal cellular biochemistry & physiology
Characteristics of exposure…

· Thus, whether a toxic response occurs is dependent on:

Chemical & physical properties of the agent
Exposure situation
How the agent is metabolized by the system
Conc of the active form at the particular target site(s)
The overall susceptibility of the biological system or
subject

66
Characteristics of exposure…

· The major factors that influence toxicity as it relates to

the exposure situation for a specific chemical:
Route of administration &
Duration & frequency of exposure

67
A. Route & Site of Exposure

· The major routes (pathways) by which toxic agents gain

access to the body:
GIT: ingestion
Lungs: inhalation
Skin: topical, percutaneous, or dermal
Parenteral: other than intestinal canal routes

68
 Route and Site of Exposure…

· Toxic agents generally produce the greatest effect & the

most rapid response when given directly into the blood-
stream (IV route)
An approx. descending order of effectiveness for the
other routes would be:
• Inhalation > IP > SC > IM > intradermal > oral >
Route of exposure

a) Oral route:
Accounts for 80 % of acute toxic episodes
resulting from accidental & intentional ingestion
of poisons
Absorption occurs in all areas of the GIT
dermal
Key factors are lipophilicity & % of substance in
the non-ionized form
Dissolution rate: many Txs recommend dilution,
but this can aid dissolution & absorption in some
instances

69
Route of exposure

a) Oral route:
Accounts for 80 % of acute toxic episodes result-
ing from accidental & intentional ingestion of
poisons
Absorption occurs in all areas of the GIT
Key factors are lipophilicity & % of substance in
the non-ionized form
Dissolution rate: many Txs recommend dilution,
but this can aid dissolution & absorption in some
instances

70
Route of exposure…

• Oral route…
Large amts of solids can form concretions in the
stomach
Food rich in fat delay absorption
Some chemicals can slow own absorption by gas-
tric irritation, closing pyloric sphincter

Oral absorption can allow detoxification of toxins by

1st pass metabolism, or produce toxins from non-
toxic parent compounds

71
Route of exposure…

b) Inhalational route:
Toxins absorbed by lung categorized as:
• Vapor gases: CO, hydrogen sulfide, sulfur
oxides, nitrogen oxides

• Vapor fumes of volatile liquids: chloform,

benzene, carbon tetrachloride, mercury

• Aerosols: lipid-soluble diffusion, water sol-

uble filtration

72
Route of exposure…

c) Dermal route:
Skin is the most accessible organ to toxins & a very efficient
barrier
Poison undergoing percutanous absorption goes through sev-
eral layers of skin to circulation
Absorptions is determined by compound (corrosive ?), length
of exposure, condition of skin (abrasions ?)

73
Route & Site of Exposure…

· The “vehicle” & other formulation factors can markedly

alter absorption after ingestion, inhalation, or topical expo-
sure
· In addition, the route of administration can influence the
toxicity of agents
E.g. an agent that is detoxified in the liver would be
expected to be less toxic when given via the portal cir-
culation (oral) than when given via the systemic circu-
74
lation (inhalation) ????
B. Duration & frequency of exposure

· Toxicologists usually divide the exposure of experi-

mental animals to chemicals into 4 categories:
Acute

Subacute
Subchronic, & Repeated exposures
Chronic

75
Duration & frequency of exposure…

a) Acute exposure:
Exposure to a chemical for < 24 h

It usually refers to a single administration

Examples of exposure routes are, IV, & Sc inj; oral in-

tubation; & dermal application

NB: Acute exposure by inhalation refers to continuous

exposure for < 24 h, most frequently for 4 h

76
Duration & frequency of exposure…

b) Sub-acute: repeated exposure to a chemical for 1 month

or less
c) Sub-chronic: for 1 to 3 months
d) Chronic: for more than 3 months
· These 3 categories of repeated exposure can be by any
route, but most often they occur by the oral route, with
the chemical added directly to the diet

77
Duration & frequency of exposure…

· In human exposure situations, the frequency & duration of ex-

posure are usually not as clearly defined as in controlled ani-
mal studies
But many of the same terms are used
· Workplace/environmental exposures may be:
Acute: occurring from a single incident or episode
Sub-chronic: occurring repeatedly over several wks or
months
Chronic: occurring repeatedly for many months or yrs 78
 Duration & frequency of exposure…

· For many agents, the toxic effects that follow a single expo-
sure are quite different from those produced by repeated
exposure
E.g. Benzene
• The primary acute toxic manifestation is CNS depres-
sion
• But repeated exposures can result in bone marrow toxic-
ity & an ↑ed risk for leukemia
79
Duration & frequency of exposure…

· Acute exposure to agents that are rapidly absorbed is

likely to produce immediate toxic effects
But also can produce delayed toxicity that may or
may not be similar to the toxic effects of chronic ex-
posure

80
Duration & Frequency of Exposure…

· Conversely, chronic exposure to a toxic agent may produce

some immediate effects after each administration in addition
to the long-term, low-level, or chronic effects of the toxic sub-
stance
· In characterizing the toxicity of a specific chemical, it is evi-
dent that information is needed not only for the single-dose &
long-term effects but also for exposures of intermediate dura-
tion
81
Duration & frequency of exposure…

· The other time-related factor that is important in

the temporal characterization of repeated expo-
sures is the frequency of exposure
A chemical that produces severe effects with a
single dose may have no effect if the same
total dose is given in several intervals

82
Duration & frequency of exposure…

· It is evident that with any type of repeated exposure,

the production of a toxic effect is influenced not only
by the frequency of exposure
But may, in fact, be totally dependent on the fre-
quency rather than the duration of exposure

83
Duration & frequency of exposure…

· Chronic toxic effects may occur, therefore, if the chemical:

Accumulates in the biological system
• Rate of absorption exceeds the rate of biotransforma-
tion &/or excretion
Produces irreversible toxic effects
There is insufficient time for the system to recover from
the toxic damage within the exposure frequency interval

84
VI. Variation in toxic responses

a) Selective Toxicity
It means that a chemical produces injury to one
kind of living matter without harming another
form of life even though the 2 may exist in inti-
mate contact
They may be related to each other as parasite
& host or may be 2 tissues in one organism

85
 Variation in toxic responses…
· Selective Toxicity…
Drugs & other chemicals used for selective toxic purposes
are selective for one of 2 reasons
• The chemical is equally toxic to both economic & uneco-
nomic cells but is accumulated mainly by uneconomic
cells
• The chemical reacts fairly specifically with a cytological
or a biochemical feature that is absent from or does not
play an important role in the economic form 86
Variation in toxic responses…

· Selective Toxicity…
Selectivity resulting from differences in distribution
usually is caused by differences in:
The absorption
Biotransformation, or
Excretion of the toxicant

87
Variation in toxic responses…

· Selective Toxicity…
Example1:
• Selective toxicity of an insecticide spray may be
partly due to:
• A larger SA per unit wt that causes the insect to
absorb a proportionally larger dose than does the
mammal being sprayed
• Effectiveness of radioactive iodine in the Tx of hy-
perthyroidism is due to:
• Selective ability of the thyroid gland to accumu-
late iodine
88
Variation in toxic responses…
· Selective Toxicity…
Example 2:
• Selective toxicity caused by differences in com-
parative cytology is exemplified by a comparison
of plant & animal cells
• Plants differ from animals in many ways, e.g
• Absence of a nervous system, an efficient
circulatory system, &
• Presence of a photosynthetic MZM & cell
89
walls
Variation in toxic responses…

· Selective Toxicity…
Example 3:
• The fact that bacteria contain cell walls & hu-
mans do not has been utilized in developing
selective toxic antibiotics, such as penicillin &
cephalosporins, that kill bacteria but are rela-
tively nontoxic to mammalian cells

90
Variation in toxic responses…
· Selective Toxicity…
Example 4:
• Selective toxicity can also be a result of a difference in
biochemistry in the 2 types of cells
• E.g. bacteria do not absorb folic acid but synthesize
it from p-aminobenzoic acid, glutamic acid, &
pteridine, whereas mammals cannot synthesize
folic acid but have to absorb it from the diet
91
Variation in toxic responses…

· Selective Toxicity…
Thus, sulfonamide drugs are selectively toxic to bacteria
b/c the sulfonamides, which resemble p-aminobenzoic
acid antagonize the incorporation of p-aminobenzoic acid
into the folic acid molecule—a rxn that humans do not
carry out

92
Variation in toxic responses…

b) Species differences toxicity:

E.g. Aflatoxin induced liver tumor
• 15 ppb in rats, but 10,000 ppb in mice
Mice express this enzyme constitutively, whereas
rats normally express a closely related form with
much less detoxifying activity toward aflatoxin
epoxide

93
Variation in toxic responses…

c) Individual difference in response:

Even within a species, large inter-individual differ-
ences in response to a chemical can occur b/c of sub-
tle genetic differences
Hereditary differences in a single gene that occur in
more than 1% of the population are referred to as
genetic polymorphism & may be responsible for
idiosyncratic rxns to chemicals
94
Principles & methods of testing
for toxicity
Learning Objectives

· Discuss the studies on general toxicity

· Describe developmental & reproductive toxicity
studies
· Explain the different methods of mutagenicity
studies
· Describe the tests used to study carcinogenic
effect of chemicals

96
Chapter Outline
· Studies on general toxicity:
Acute, sub-acute, sub-chronic & chronic toxicity
studies
· Studies on various types of specific adverse ef-
fects:
Developmental & reproductive toxicity
Mutagenicity
Tests for carcinogens
Immunotoxicity assessment
Irritation & sensitization - local tolerance studies

97
I. Studies on General Toxicity

· Toxicity testing: encompasses a variety

of methods aimed at assessing the poten-
tial harmful effects of substances on bio-
logical systems.
Importance of Toxicity Testing
· Understanding the impact of chemicals on
health and the environment ,for public
safety.
· helps to prevent harmful exposures and in-
 Cont….
Scope of Toxicity Testing
·encompasses a wide array of substances, including
pharmaceuticals, industrial chemicals, and environ-
mental pollutants, necessitating an interdisciplinary
approach that toxicology, chemistry
Fundamental Principles of Toxicity Testing
Reproducibility, Validity, and Ethical Considerations
Reproducibility : ensures that toxicity tests yield con-
sistent results across different laboratories and studies,
·serving as a cornerstone for reliable data interpreta-
 Cont…..

· Validity refers to the accuracy of a test in

measuring what it purports to measure, en-
suring that the results reflect actual biologi-
cal responses rather than artifacts of the
procedure.
· Ethical Considerations :The ethical implica-
tions of toxicity testing necessitate a com-
mitment to minimizing animal testing, pro-
I. Studies on General Toxicity Principles of descriptive animal toxicity
tests
 Two main principles underlie all descriptive animal
toxicity testing
A. The effects produced by a compound in lab
animals, when properly qualified, are
applicable to humans

• On the basis of dose per unit of body surface,

toxic effects in humans are usually in the same
range as those in experimental animals
• On a body weight basis, humans are generally
more vulnerable than are experimental animals
1

· Toxicological testing is best performed with pure

substances since examination with chemical mix-
tures is practically impossible & may give rise to er-
roneous conclusions due to possible chemical inter-
actions

· If it is necessary to determine the toxicity of a cpd

mixture, investigation should start by establishing
the composition of the mixture & determining which
components of the mixture are bioactive

101
Studies on General Toxicity…
· Each substance then must be classified & character-
ized with respect to its physicochemical properties
· The stability of a given substance determines how it
should be administered for testing, particularly if it is
inactivated in the GIT
· In general, the starting point for toxicological testing
of new substances is testing in lab animals

· A chemical may be administrated to animals by oral,

dermal, IV, IM or IP routes

102
Studies on General Toxicity…
· The ideal experimental animal is one that metabo-
lizes & secretes a substance in the same manner
as humans do
· Thus, rodents, hamsters, rabbits, cats & dogs are
the most frequent human surrogates used for toxico-
logical testing
Mainly b/c they are economical, amenable to fre-
quent handling & lacking in the vomiting reflex
· Other animals, such as monkeys, are also used

103
Studies on General Toxicity…
· Metabolic differences b/n humans & surrogate test
animals can result in erroneous conclusions about the
activity of a substance in humans
· Use of younger animals provides the advantage of
obtaining faster results due to their higher rate of me-
tabolism, as well as their being cheaper to purchase &
house
· Age & gender of animals can influence the outcome
of the investigation

104
Studies on General Toxicity…
· The purity of the substance is very important, since impu-
rities may significantly influence toxicological findings
· Before animal testing is started, a literature search must
be done to collect available results of any previous testing
· If the test substance is administrated to experimental ani-
mal by mixing it with food or water, it may be affected by ox-
idation (i.e., through contact with air), or by temperature
variations or by the feeding habits of the experimental ani-
mals

105
Studies on General Toxicity…
· Gastric intubation (gavage), an artificial means of
substance introduction, has advantages with respect
to dosing exactness & reduced variation
· The gavage is performed after the animals have
fasted in order to avoid mixing chemical test sub-
stances with stomach contents
· In general, the chemical dose should not exceed
5% of the test animal’s diet

106
Studies on General Toxicity…

A number of different types of data are used in or-

der to establish the safety of chemical substances
for use. These include:
·Consideration of the chemical structure and any in-
tended biological activity
·In vitro models, such as cell cultures or tissue slices
·Laboratory animals
·Human volunteers
107
Studies on General Toxicity…
· Alternative test methods:
The use of lab-cultured single-cell organisms
Isolated organs or mammalian cell lines

· NB: The advantage of animal testing:

The possibility to examine the results of different
routes of exposure under controlled conditions

108
Studies on General Toxicity…
· However, differences in genetic pool, lifespan, sus-
ceptibility, metabolic pathways & other parameters
of organic life, present serious limitations when
extrapolating the results of animal testing to human
populations
· Animal experiments are usually classified accord-
ing to their duration as:
Acute, sub-acute, chronic or sub-chronic testing

· Chronic & acute toxic effects for a specific toxicant

can differ both qualitatively & quantitatively
109
Fig. Typical tiered testing scheme for the toxicological evaluation of new
chemicals
110
TOXICITY STUDIES

· Acute toxicity (14 Days)

· Sub-acute (repeated doses) toxicity (28
Days) •
· Sub-chronic toxicity (3 Months)
· Chronic toxicity (6 Months to 2 Years)
· Special toxicity (Carcinogenicity)

Acute toxicity studies are conducted to

determine the short-term adverse effects
of drug/chemical when administered in a
single dose, or in multiple doses during a 111
A. Acute toxicity studies
· Objectives:
To determine Maximum Tolerated Dose
(MTD) & No Observable Effect Level
(NOEL)
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 adminis-
tration in humans
To identify potential target organs for
toxicity, determine reversibility of toxic-
ity, & identify parameters for clinical 112
Acute toxicity studies…
· Duration:
A few days to 2 wks after a single dose
· Test system/animal system:
2 species required: mice, rats, or rabbits or dogs
One rodent-mice/rat. One non rodent-usually rab-
bit.
· Dose administration: orally & parenterally.
Oral (by gavage or with food), SC, IP, Intradermal,
Inhalation, Intranasal, Topical (epicutaneous), IV
Various dose levels to groups of both sexes.
Dose selection such that causing than less than
50% but not 0% and more than 50% but not 100%
113
mortality.
Cont…. Acute studies

· Parameters:

Mortality, clinical pathology, gross necropsy,

weight change,
signs of toxicity (Altered Respiration , Weight
loss ,Muscle spasm, Salvation ,
Convulsion , Diarrhea ,Loss of righting re-
flex , Tremor ,Lacrimation
114
B. Sub-acute toxicity studies
· Objectives:
To determine toxicity after repeated administration of the
test material
To help establish doses for sub-chronic studies
· Duration: 14- 28 days
· Test system/animal system
2 species required: mice, rats, rabbits, guinea pigs,
dogs

115
Sub-acutetoxicity studies…
· Dose administration:
3 to 4 doses given by the same routes as previ-
ous toxicity tests
· Parameters:
Mortality
Signs of toxicity
Pathology & histopathology
Weight change
Clinical pathology

116
C. Sub-chronic toxicity testing
· Objectives:
To establish a “no observable effect level"
(NOEL)
To characterize D-R r/ships following repeated
doses
To further identify & characterize specific organs
affected after repeated administration

To predict a reasonable & appropriate dose for

chronic exposure studies (MTD)

117
Sub-chronic toxicity testing…
· Duration:
Commonly 90 days, but varies from 2 wks 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 & the highest producing toxicity
but less than or equal to 10% mortality

118
Sub-chronic toxicity testing…
· Parameters
Mortality
Weight change
Signs of toxicity
Clinical pathology
Pathology and histopathology

119
D. Chronic toxicity testing
· 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’ life-
span
Length depends on intended period of human
exposure

120
Chronic toxicity testing…
· Test system/animal system:
2 species required: Rodents, dogs
· Dose administration:
As in sub-acute/ subchronic toxicity studies
· Parameters:
Mortality
Pathology & histopathology
Weight change
Clinical pathology of all animals (mortalities &
survivors)

121
II. Studies on various types of spe-
cific adverse effects
· Studies on various types of specific adverse ef-
fects
Developmental & reproductive toxicity
Mutagenicity
Tests for carcinogens
Immuno-toxicity assessment
Irritation & sensitization - local tolerance stud-
ies

122
A. Developmental & reproductive
toxicity
· Developmental toxicology:
The study of adverse effects on the developing
organism occurring anytime during the life
span of the organism that may result from
exposure to chemical or physical agents
• Before conception (either parent)

• During prenatal development, or

• Postnatally until the time of puberty

123
Developmental & reproductive toxic-
ity…
· Teratology:
The study of defects induced during develop-
ment b/n conception & birth

· Reproductive toxicology:
The study of the occurrence of adverse effects
on the male or female reproductive system
that may result from exposure to chemical or
physical agents

124
Developmental & reproductive toxic-
ity…
a) General fertility & reproductive performance (segment
I) studies:
They are usually performed in rats with 2 or 3 doses (20
rats per sex per dose) of the test chemical (neither pro-
duces maternal toxicity)
Males are given the chemical 60 days & females 14 days
before mating
The animals are given the chemical throughout gesta-
tion & lactation

125
Developmental & reproductive toxic-
ity…
· Segment I studies…
Typical observations made include:
• The % of females that become pregnant
• The number of stillborn & live offspring,
• The wt, growth, survival, & general condition of
the offspring during the first 3 wks of life

126
Developmental & reproductive toxic-
ity…
b) Teratogenic effects (segment II) studies:
Teratogens are most effective when administered
during the 1st trimester, the period of organogenesis

Thus, the animals (usually 12 rabbits & 24 rats or

mice per group) are usually exposed to one of three
dosages during organogenesis (day 7 to 17 in ro-
dents & days 7 to 19 in rabbits), & the fetuses are
removed by cesarean section a day before the es-
timated time of delivery (gestational days 29 for
rabbit, 20 for rat, & 18 for mouse)
127
Developmental & reproductive toxic-
ity…
· Segment II studies..
The uterus is excised & weighed & then exam-
ined for the number of live, dead, & resorbed fe-
tuses
Live fetuses are weighed
Half of each litter is examined for skeletal abnor-
malities & the remaining half for soft tissue
anomalies
128
Developmental & reproductive toxic-
ity…
c) Perinatal & postnatal toxicities of chemicals
(segment III) studies:

This test is performed by administering the

test compound to rats from the 15th day of
gestation throughout delivery & lactation

Then determining its effect on the birth-

weight, survival, & growth of the offspring dur-
ing the first 3 wks of life

129
B. Mutagenicity

· Mutagenesis is the ability of chemicals to cause

changes in the genetic material in the nucleus of cells
in ways that allow the changes to be transmitted
during cell division
· Mutations can occur in either of 2 cell types, with sub-
stantially different consequences
Germinal mutations
Somatic mutations
130
Mutagenicity…
· Germinal mutations:
Damage DNA in sperm & ova, which can undergo mei-
otic division & therefore have the potential for trans-
mission of the mutations to future generations
A gene change in a body's reproductive cell (egg or
sperm) that becomes incorporated into the DNA of ev-
ery cell in the body of the offspring.
Germline mutations are passed on from parents to off-
spring
If mutations are present at the time of fertilization in
either the egg or the sperm, the resulting combination
of genetic material may not be viable, & the death may
occur in the early stages of embryonic cell division 131
Mutagenicity…
· Germinal mutations…
Alternatively, the mutation in the genetic mate-
rial may not affect early embryogenesis but
may result in the death of the fetus at a later
developmental period, resulting in abortion
Congenital abnormalities may also result from
mutations

132
Mutagenicity…

· Somatic mutations:
Mutations in all other cell types & are not heritable but may
result in cell death or transmission of a genetic defect to
other cells in the same tissue through mitotic division.
An alteration in DNA that occurs after conception. So-
matic mutations can occur in any of the cells of the body ex-
cept the germ cells (sperm and egg) and therefore are not
passed on to children.
133
Mutagenicity…

· B/c the initiating event of chemical carcinogenesis is

thought to be a mutagenic one, mutagenic tests are often
used to screen for potential carcinogens
· Numerous in vivo & in vitro procedures have been de-
vised to test chemicals for their ability to cause mutations

134
Mutagenicity…
· Some genetic alterations are visible with the light
microscope
In this case, cytogenetic analysis of bone mar-
row smears is used after the animals have
been exposed to the test agent

· B/c some mutations are incompatible with normal

development, the mutagenic potential of a chemi-
cal can also be evaluated by the dominant lethal
test
This test is usually performed in rodents

135
Mutagenicity…
· The dominant lethal test…
The male is exposed to a single dose of the
test compound & then is mated with two un-
treated females weekly for 8 wks

The females are killed before term, & the

number of live embryos & the number of cor-
pora lutea are determined

136
Mutagenicity…
· Salmonella/microsome/Ames test:
The test for mutagens that has received the
widest attention that is developed by Ames &
colleagues
This test uses several mutant strains of S. ty-
phimurium that lack the enzyme phosphoribosyl
ATP synthetase, which is required for histidine
synthesis
These strains are unable to grow in a histidine-
deficient medium unless a reverse or back-mu-
tation to the wild type has occurred

137
Mutagenicity…
· Ames test…
Other mutations in these bacteria have been
introduced to enhance the sensitivity of the
strains to mutagenesis

The 2 most significant additional mutations

enhance penetration of substances into the
bacteria & decrease the ability of the bacteria
to repair DNA damage

138
Mutagenicity…
· Ames test…
B/c many chemicals are not mutagenic or car-
cinogenic unless they are biotransformed to a
toxic product by enzymes in the ER, rat liver
microsomes are usually added to the medium
containing the mutant strain & the test chemical

The number of reverse mutations is then quan-

tified by the number of bacterial colonies that
grow in a histidine-deficient medium

139
C. Tests for carcinogens

a) The Standard Test:

The currently accepted design for carcinogenic-
ity testing is to expose rats & mice to the agent
for about 2 years
For each species, 50 male & 50 female animals
per group are dosed with a vehicle or the test
agent in that vehicle
Daily observations are made & if any animals
become moribund during the experiment, they
are killed so that tissues are not lost due to au-
tolysis

140
Tests for carcinogens…

· The Standard Test..

All animals, including those that survive to
the scheduled end of the experiment, are
subjected to autopsy & almost 40 different
tissues are taken from each animal for histo-
logical examination
All observations are recorded, summarized &
analyzed
141
D. Immunotoxicity Assessment
· Objective:
To determine the potential of a test material to
induce immune suppression or immune en-
hancement
· Duration:
Subacute (14 days) or sub-chronic (90 days)
exposure
· Test system/animal system: Rodents
· Dose administration:
Repeated doses administered as in sub-
acute/sub-chronic toxicity studies
142
Immunotoxicity Assessment…
· Parameters:
Level 1:
• Hematology
• Histopathology or lymphoid organs
• Quantity of T- & B-cells (cellularity of lymphoid or-
gans)
• Blastogenesis (mitogen responsiveness; mixed
lymphocyte reaction)
• Quantitation and funciton of natural killer cells
• Macrophage function
• Cytokine production
143
Immunotoxicity Assessment…
· Parameters…
Level 2:
• Kinetics of antibody production to T-depen-
dent antigens
• Quantity of IgM/IgG-producing (plaque-form-
ing) cells
• Delayed hypersensitivity responses to known
sensitizers
• Immune response to infectious agents (e.g.,
Listeria, Streptococcus)
• Immune response to transplantable tumors
144
E. Irritation & sensitization - local
tolerance studies
· Objectives:
To determine the potential of a test material to
provoke ocular irritation, dermal irritation, or
sensitization
· Duration:
Irritation - 1 hr to 3 wks after a single topical or
corneal administration
Sensitization - intradermal or topical induction
doses followed by topical challenges with a non-
irritating dose (6 - 8 wks total)
· Test system/animal system: Rodents, rabbits
145
Irritation & sensitization - local toler-
ance studies…
· Test system/animal system: Rodents, rabbits

· Dose administration
Single patch administration
Multiple doses over 2—4 weeks
Topical (epicutaneous), intradermal, or corneal

146
Irritation & sensitization - local toler-
ance studies…
· 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)

147