Chapter 6.

Effects
1. General Classification
1.1Immunology.
-This chapter is going to explore the variety of local and systemic
reactions elicited by chemical exposure and the physiological and
immunological basis of those effects. Its necessary to understand how the
immunological system works to understand this chapter (sim de novo, la
vamos nos pro mundo boring da imuno).
- Active immunity involves two types of responses, humoral and cell
mediated, while passive
immunity is composed of the administration of synthetic antibodies,
antivenoms, antitoxins, and therapeutic passive immunization.
1.2. Chemical Allergies
Four types of immunological hypersensitivity.
-Type I. Antibody-mediated = occur as three phases. The initial phase, the
sensitization phase (binding of the antigen to immunoglobulin E (IgE)) . A
second phase, the activation phase (degranulation of mast cells and
basophils, with a subsequent release of histamine and other soluble
mediators soluble mediators). The third stage, the effector phase
(Degranulation of neutrophils and eosinophils). Antigens eg. Airborne
pollens and food ingredients.
-Type II. Antibody-mediated cytotoxic reactions = differ from type I in the
nature of the antigen, the cytotoxic character of the antigen-antibody
reaction, and the type of antibody formed (IgM or IgG). Examples of type II
reactions include complement-mediated (CM) reactions Rh incompatibility
reactions, autoimmune reactions, and drug-induced reactions (this last
one is very important for clinical toxicology)

-Type III. Immune complex reactions = mediated by antigen-antibody

immune complexes. Type III reactions are stimulated by microorganisms
and involve activation of complement. Eg. Serum sickness (systemic) and
arthus reaction (localized)
-Type IV. Delayed type hypersensitivity = involves antigenspecific T-cell
activation.
1.3. Idiosyncratic Reactions
Idiosyncratic reactions are abnormal responses to drugs or chemicals
generally resulting from uncommon genetic predisposition. E.g. the
cardiotoxic action of cocaine is exaggerated in cases of congenital
deficiency of plasma esterases, which are necessary for metabolism of the
drug.
1.4. Immediate versus delayed effects.
Chemical effects are immediate or delayed, depending on the mechanism
of toxicity (ao contrario das reacoes de hipersensibilidade que levam
algum tempo para se desenvolver). Exemplo de imediato: o efeito de
sedativos-hipinoticos que quando em overdose podem levar ate a
depressao respiratoria. Exemplo de tardia: carcinogenicos.
1.5. Reversible versus Irreversible Reactions
In general, the effects of most drugs or chemicals are reversible until a
critical point is reached (carcinogenic effect, teratogenic effect or vital
compromissing). E.g. smoking the consequences can be reversible but
after sometime it will lead to irreversible damage. Reversibility of a
chemical effect may be enacted by administering antagonists, enhancing
metabolism or elimination, delaying absorption, intervening with another
toxicological procedure that decreases toxic blood concentrations, or
terminating the exposure.
1.6. Local Versus Systemic Effects
Depend on the site of exposure. Skin and lungs are generally the first
targets of chemical exposure. Oral exposure requires absorption and
distribution of the agent to lead to systemic effects. Hypersensitivity
reactions types I and IV are precipitated by local activation of immune
responses following a sensitization phase, while drug-induced type II
reactions are elicited through oral or parenteral administration.
1.7. Target Therapeutic Effects
Under physiological conditions, the target action may be more predictable,
particularly in response to a therapeutic dose Nevertheless, these agents
may still produce adverse reactions by the very nature of their ability to
alter physiological processes. The pathologic condition can also influence
the effect. Several factor contribute to the various possible outcomes that
may occur in the presence of comorbid surroundings.

1. The toxicokinetics or the toxicodynamics of the compound may be

altered (as with a drug that exhibits primary renal elimination in the
presence of compromised kidney function).
2. The response of the diseased organ, tissue, or system may be
distorted in response to drug treatment (as with neurologic toxicities
demonstrated with phenothiazine derivatives).
3. The toxicity of a compound mimics the very condition for which the
drug is used (as with the toxic effects of digitalis glycosides for use in
congestive heart failure).
2. Chemical Interactions
2.1. Potentiation
It occurs when the toxic effect of one chemical is enhanced in the presence of
a toxicologically unrelated agent.
0 + 2 > 2 . There is a nontoxic agent (0) and on the target organ may
be a enhancing of the toxicity of another coadministered chemical (2)
2.2 Additive
Two or more chemicals whose combined effects are equal to the sum of the
individual effects are described as additive interactions. Such is the case with
the additive effects of the combination of sedative-hypnotics and ethanol
(drowsiness, respiratory depression). Numerically this is summarized as 2 +
2 = 4.
2.3. Synergistic
By definition, a synergistic effect is indistinguishable from potentiation except
that, in some references, both chemicals must have cytotoxic activity. E.g.
the effect that is experienced with the combination of ethanol and
antihistamines (1 + 2 > 3).
2.4. Antagonistic
The opposing actions of two or more chemical agent. Types of antagonism:
1. Functional antagonismthe opposing physiological effects of
chemicals, such as with central nervous system stimulants versus
depressants.
2. Chemical antagonismdrugs or chemicals that bind to, inactivate, or
neutralize target compounds, such as with the action of chelators in
metal poisoning.
3. Dispositional antagonisminterference of one agent with the
absorption, distribution, metabolism, or excretion (ADME) of another.
Examples of agents that interfere with absorption, metabolism, and
excretion include activated charcoal, phenobarbital, and diuretics,
respectively.

4. Receptor antagonismthe occupation of pharmacological receptors by

competitive or noncompetitive agents, such as the use of tamoxifen in
the prevention of estrogen-induced breast cancer.

Chapter 7. Dose Response

1. General Assumption
1.1Types of Dose-Response Relationships
The result of exposure to the dose can be any measurable,
quantifiable, or observable indicator. The response depends on the
quantity of chemical exposure or administration within a given
time period. Two types:
1. Graded dose response ( tipo uma resposta proporcional a dose)
= relationship of an individual test subject or system to increasing
and/or continuous doses of a chemical
2. Quantal dose response (all-or-none effect) ( tipo tudo ou nada de
reao) = A typical quantal dose-response curve is illustrated in
Figure 7.2 by the lethal dose 50% (LD50) distribution.
* The LD50 is a statistically calculated dose of a chemical
that causes death in 50% of the animals tested (caso vc nao
lembre)

1.2. Concentration-effect and Presence at the Receptor Site

There is a causal relationship between the concentration of the chemical
and the effect, because of this is suggest using the term concentration
effect instead of dose-response. Also there is a relationship between the
presence f the chemical in the site with the effect. That is why it should
measures of concentration at the cellular or organ level.
1.3. Criteria for Measurement
Except for lethality, the selection of a measurable or observable endpoint
is
crucial. A desirable biomarker may be one that accurately reflects the
presence
of the chemical at the molecular site or suggests that the toxic effect
originates
from the target organ. Selection of a measurable endpoint thus depends
on the
suspected mechanism of toxicity, if known, or on empirical
determinations
based on the chemical formula. Some biomarkers are also subjective,
such as
reliance on histological grading, calculation of the degree of anesthesia,
pain,
motor activity, or behavioral change. Thus, the standards for quantifying
the
endpoint are determined and established prior to the experimental setup
2. Lethal Dose 50%
2.1. Definition
It is the statistically calculated dose of a chemical that causes death in
50% of the animals tested. The usefulness of the test provides a
screening method for toxic evaluation, particularly useful for new
unclassified substances. however, is antiquated, requires large
numbers of animals, does not provide information regarding
mechanistic effects or target organ, and does not suggest
complementary or selective pathways of toxicity.
2.2. Experimental Protocol
-At least 10 animals per dose and 10 doses per chemical
- The dose is calculated as milligrams of substance per kilogram of
body weight, mg/kg
- Each point on the graph (Fig. 7.2- O GRAFICO TA LA EM CIMA, esse
graphic de baixo 7.3 y porcentagem acumulada -quantos mortos em
x dose menos a quantidade de mortos em x-1 - por isso o format de
piramide) represents the number of animals that expire within the
group at the end of a predetermined time period (usually 2496
hours).

2.3. Factors That Influence the LD50

- Selection of the species, the route of administration, and the time of day
of exposure and observation.
- The same species must be of the same age, sex, strain, weight, and
breeder.
- the animal care maintenance should be similar in each run, with
attention to light and dark cycles, feeding, and waste disposal schedules.
3. Effective dose 50% (ED50), toxic dose 50% (TD50) and Therapeutic Index
3.1. Relationship to LD50
- ED50 is analogous to LD50 the difference is that the endpoint of ED is the
desirable effect.
- TD50 is similar and is calculated on the basis of the measurement of a toxic
nonlethal endpoint (eg. Respiratory depression).
The relationship between the lethal (or toxic) and the effective dose is
calculated and expressed as the therapeutic index (TI):

3.2. Assumptions Using the TI

This information may be valuable in establishing guidelines for estimating
potential human risk (estimation of the margin of safety of a chemical).
The TI does not provide information on the mechanism of toxicity, or the
pharmacological
action.
4. Inhibitory Concentration 50%
4.1. Definition
The concentration necessary to inhibit 50% of a measured response in an in
vitro system is known as the inhibitory concentration 50%(IC50) (similar to
IC75 and IC25 that are sometimes computed as cutoff values).
4.2. Experimental Determination

IC50 determinations are traditionally

methodology or other in vitro methods

performed

using

cell

culture

4.3. For In Vitro Systems.

Determination of IC50 for in vitro systems is analogous to the calculation of
the LD50 or the TD50 in animal testing. Unlike the LD50 or the TD50,
however, which represents a median lethal or toxic dose, respectively, the
IC50 allows for comparisons of concentrations of chemicals necessary to
inhibit any measurable parameter, such as cell proliferation, protein
synthesis, or DNA synthesis. Thus it becomes a valuable tool for estimating
toxic effects.
Advantages: supplement or replace animal toxicity testing programs. In
addition, in vitro methods are more cost efficient than animal
experimentation. Such testing may prove to be more predictive of assessing
human clinical toxicity than current animal testing protocols
Chapter 8. Descriptive Animal Toxicity Tests
1. Correlation w/ Human Exposure
1.1. Human Risk Assessment
-Animal toxicology testing is useful in determining the potential toxicity of
a compound to humans.
- Together with in vitro tests, animal tests are applied as biological
markers of chemically induced risks, whether synthetic or naturally
occurring.
- Tests are used by regulatory agencies to screen for or predict human
toxic effects in an attempt to establish a significant frame of reference for
monitoring environmental chemical threats, therapeutic adverse
reactions, and commercial toxicity.
1.2. Predictive Toxicology and Extrapolation to Human Toxicity
- Correlation of results of toxicity testing described in animals, with human
exposure, requires careful consideration of the parameters of the animal
tests, including the selection of species with similar physiology.
-The test must be chosen according w the guidelines of the regulatory
agencies.
2. Species Differentiation
2.1. Selection of a Suitable Animal Specie
- Among the mammalian species available the rodent is the most useful
and convenient.
- Because of their dermal sensitivity guinea pigs are used for testing of
local irritants.
- Rabbits have traditionally been employed for testing of ocular irritants as
their eyes are anatomically and physiologically similar to humans.
- Larger species, such as dogs, are useful subjects of specific toxicity
testing involving cardiovascular and pulmonary agents.

- The use of primates for testing behavioral, immunological,

microbiological, and neurological response to chemicals is controversial
but is often necessary for interpretation of human risk assessment.
2.2. Cost-Effectiveness
Animal testing is more costly than in vitro methods, because of housing,
maintenance, animal care facility; daily needs for food, water, bedding;
training of staff; veterinarian; etc
2.3. Institutional Animal Care and Use Committee (IACUC)
The Office of Laboratory Animal Welfare (OLAW) at the National Institutes
of Health (NIH),
which has responsibility for the general administration and coordination of
policy on behalf of the PHS (Public Health Service) provides specific
guidance, instruction, and materials to institutions on the utilization and
care of vertebrate animals used in testing, research, and training.
Pelo que eu entendi cada local que usa animais pra teste tem que ter um
IACUC e esse tem que cumprir todos os requisitos listados pelo OLAW. O
OLAW um braco do PHS resopnsavel por isso.
Other functions of the IACUC include periodic review of the institutions
program, inspection of facilities, preparation of reports, and submittal of
suggestions and recommendations on improving the program.
3. Descriptive Tests
3.1. Required LD50 and Two Routes
The lethal dose 50% (LD50) is required for a substance using at least two
routes of exposure, usually oral and parenteral. Deve-se fazer o teste de
LD50 em animais usando duas vias de administracao e monitora-los.
3.2. Chronic and subcrhonic Exposure
- Acute -> 24-hour exposures
- Subchronic exposure usually involves a 90-day exposure period, with or
without repeated doses.
- Chronic exposure studies are more difficult, cumbersome, and expensive
to conduct. These experiments are reserved for carcinogenicity testing or
accumulation studies. (na maioria das vezes feito em ratos)
3.3.

Type of tests

Chapter 9. In Vitro Alternatives to Animal Toxicity.

1. In vitro methods
1.1. Cell Culture Methods
- Useful for understanding mechanistic toxicology.
- The use of cell culture techniques in toxicological investigations
is referred to as in vitro cytotoxicology, or in vitro toxicology
- The reasons for the increasing interest and success of these
techniques in clinical and research toxicology testing are a
consequence of the considerable advancements realized since
the advent of the technology:
1. Since the growth of the first mammalian cells were described
in capillary glass tubes, cell culture technology has been
extensively refined.
2. The mechanism of toxicity of chemicals in humans and
animals has been elucidated primarily through the use of in vitro
toxicological methods.
3. The necessity to determine the toxic effects of
pharmaceuticals and industrial chemicals, which are developed
and marketed at rapid and unprecedented rates, has made it
compulsory for the development of fast, simple, and effective
alternative test systems.
4. Although the normal rate of progression of any scientific
discipline is determined by the progress within the scientific

community, some areas have received more encouragement

than others.
Specifically, the public objections and disapproval of animal
testing have forced academic institutions, industrial concerns,
and regulatory agencies to direct research initiatives toward the
development of alternative methods of toxicity testing.
1.2. Organ System Cytotoxicity
Homogeneous cell populations isolated from the framework of an
organ present unique opportunities to study the toxic effects on a
single cell type (eg. Liver, lung, kidney etc)
Two methodologies:
1. Primary culture: freshly isolated cells from target organs
(mimetizam melhor o orgao vivo)
2. Continuous cell line: repetitive propagation of primary cultures
(o problema e que com o tempo elas comecam a perder suas
funcoes especializadas e suas capacidade metabolica dedifferentiation). Are either mortal or immonrtal.
1.3. Applications in Clinical Toxicology
Disadvantages of primary cultures:
1. Primary cultures require sacrificing animals to establish the cells.
2. Primary cultures are limited to understanding the mechanisms of
only one of the many possible organ pathologies that could be
determined using animals.
3. Primary systems established from human donors are not
standardized, unless efforts are made to match the age and sex of
the donors.
4. Response of primary cultures to chemical insult may not reflect
the complex nature of chemical-target interaction as inherent in
animal species.
Nevertheless, regulatory agencies in Europe and the United States
have
promulgated the use of select in vitro tests to be incorporated into
routine
industrial toxicity-testing programs. Many of the current systems
currently in
validation programs are in vitro models for local acute toxicology
testing (1).
-

The main goal of these procedures is to replace, reduce, or

refine the skin and
ocular (Draize) test.
As noted above, established continuous cell lines are also employed
to analyze a wide spectrum of mechanisms and effects and are
generally used to detect acute systemic toxicity. In contrast to
animal studies, these systems measure the concentration of a

substance that damages components, alters structures, or

influences biochemical pathways within specific cells. The range of
injury is further specified by the length of exposure (incubation
time) and the parameter used to detect an insult (indicator). Careful
validation of test methods and repetitive interlaboratory
confirmation allows the tests to be predictive for risks associated
with toxic effects in vivo for those concentrations of the effective
substances tested. All established cell lines, however, are prone to
dedifferentiate in culture with time, thus limiting their usefulness for
assessing effects other than general toxicity.
1.4. Relationship to Animal Experiments
Clinical toxicity of chemicals is known to involve various
physiological targets
interacting with a variety of complex toxicokinetic factors.
Chemicals induce injury through an assortment of toxic
mechanisms. As a result, in vitro testing requires several relevant
systems, such as human hepatocytes, heart, kidney, lung, and
nerve cells, as well as other cell lines of vital importance, to obtain
information necessary for human risk assessment. These cell
systems are also used to measure basal cytotoxicitythat is, the
toxicity of a chemical that affects basic cellular functions and
structures common to all human specialized cells.
Basal cytotoxicity of a chemical may reflect the measurement of
cell membrane integrity, mitochondrial activity, or protein
synthesis, since these are examples of basic metabolic functions
common to all cells. The level of activity varies from one organ to
another, depending on its metabolic rate and contribution to
homeostasis, but the processes do not differ qualitatively among
organs. Recent multicenter studies have now shown that human
cell lines predict basal cytotoxicity to a greater extent than animal
cell lines. These in vitro tests are as accurate in screening for
chemicals with toxic potential as lethal dose 50% (LD50) methods
performed in rodents. In addition, validation studies have proposed
that these tests be used to supplement animal-testing protocols
when screening for toxic substances.
2. CORRELATION WITH HUMAN EXPOSURE
2.1. Risk Assessment
Together with animal testing, genotoxicity tests, toxicokinetic
modeling studies, and microarray technology, in vitro cellular test
batteries could be applied as biological markers of chemically
induced risks. Such risks include biomonitoring for environmental
toxicants and biohazards and for the development of specific

antagonists. The batteries of tests are validated for their ability to

screen for or predict human toxic effects, as the measurements
establish a database for monitoring of environmental and clinical
biohazards, and general toxicity.
2.2. Extrapolation to Human Toxicity
Standardized batteries of in vitro tests have been proposed for
assessing acute local and systemic toxicity. This battery of selected
protocols may serve as a primary screening tool before the
implementation of routine animal testing. The correlation of in vitro
with in vivo data functions as a basis for predicting human toxicity.
As it becomes apparent that several batteries of tests reflect the
acute toxicity of defined classifications of substances, significant
numbers of animals in toxicity testing will be reduced.
2.3. Predictive Toxicology
Many relatively common and widely available chemicals have never
been tested in any system. In vitro systems offer the possibility for
use as screening methods for carcinogenic, mutagenic, or
cumulative toxicity. It is possible to reduce or replace toxicity
testing of existing chemicals with risk assessment based on the
results from these batteries. In combination with clinical studies of
dermal and ocular irritancy, and cumulative regressive and
progressive longitudinal studies, in vitro test databases can
contribute to reliable clinically relevant human