Toxic

1. Chapter 3
Principles & methods of testing
for toxicity

2. 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
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3. Chapter Outline
 Studies on general toxicity:
 Acute, sub-acute, sub-chronic & chronic toxicity
studies
 Studies on various types of specific adverse
effects:
 Developmental & reproductive toxicity
 Mutagenicity
 Tests for carcinogens
 Immunotoxicity assessment
 Irritation & sensitization - local tolerance studies
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4. I. Studies on General Toxicity
 Toxicological testing is best performed with pure
substances since examination with chemical
mixtures is practically impossible & may give rise to
erroneous conclusions due to possible chemical
interactions
 If it is necessary to determine the toxicity of a cpx
mixture, investigation should start by establishing
the composition of the mixture & determining which
components of the mixture are bioactive
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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
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5. Studies on General Toxicity…
 Each substance then must be classified &
characterized 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
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6. Studies on General Toxicity…
 The ideal experimental animal is one that
metabolizes & secretes a substance in the same
manner as humans do
 Thus, rodents, hamsters, rabbits, cats & dogs are
the most frequent human surrogates used for
toxicological testing
 Mainly b/c they are economical, amenable to
frequent handling & lacking in the vomiting reflex
 Other animals, such as monkeys, are also used
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7. 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
metabolism, as well as their being cheaper to
purchase & house
 Age & gender of animals can influence the
outcome of the investigation
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8. Studies on General Toxicity…
 The purity of the substance is very important,
since impurities 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 animal by mixing it with food or water,
it may be affected by oxidation (i.e., through contact
with air), or by temperature variations or by the
feeding habits of the experimental animals
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9. 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
substances with stomach contents
 In general, the chemical dose should not exceed
5% of the test animal’s diet
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10. 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
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11. Studies on General Toxicity…
 However, differences in genetic pool, lifespan,
susceptibility, 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
according 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
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12. 12
Fig. Typical tiered testing scheme for the toxicological evaluation of new
chemicals

13. A. Acute toxicity studies
 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) & No
Observable Effect Level (NOEL)
 To identify potential target organs for toxicity,
determine reversibility of toxicity, & identify
parameters for clinical monitoring
 To help select doses for repeated-dose toxicity tests
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14. 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
 Dose administration:
 Oral (by gavage or with food), SC, IP, Intradermal,
Inhalation, Intranasal, Topical (epicutaneous), IV
 Parameters:
 Mortality, clinical pathology, gross necropsy, weight
change, signs of toxicity
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15. B. Sub-acute toxicity studies
 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
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16. Sub-acutetoxicity studies…
 Dose administration:
 3 to 4 doses given by the same routes as
previous toxicity tests
 Parameters:
 Mortality
 Signs of toxicity
 Pathology & histopathology
 Weight change
 Clinical pathology
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17. 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)
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18. 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
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19. Sub-chronic toxicity testing…
 Parameters
 Mortality
 Weight change
 Signs of toxicity
 Clinical pathology
 Pathology and histopathology
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20. 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’
lifespan
 Length depends on intended period of human
exposure
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Chronic toxicity studies
 Long-term or chronic exposure studies are
performed similarly to subchronic studies except
that the period of exposure is longer than 3 months
 In rodents, chronic exposures are usually for 6
months to 2 years
 Chronic studies in non-rodent species are usually
for 1 year but may be longer
 The length of exposure is somewhat dependent on
the intended period of exposure in humans

21. 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)
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22. II. Studies on various types of
specific adverse effects
 Studies on various types of specific adverse
effects
 Developmental & reproductive toxicity
 Mutagenicity
 Tests for carcinogens
 Immuno-toxicity assessment
 Irritation & sensitization - local tolerance
studies
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23. 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
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24. Developmental & reproductive
toxicity…
 Teratology:
 The study of defects induced during
development 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
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25. Developmental & reproductive
toxicity…
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 produces maternal toxicity)
 Males are given the chemical 60 days & females
14 days before mating
 The animals are given the chemical throughout
gestation & lactation
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26. Developmental & reproductive
toxicity…
 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
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27. Developmental & reproductive
toxicity…
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
rodents & days 7 to 19 in rabbits), & the fetuses are
removed by cesarean section a day before the
estimated time of delivery (gestational days 29 for
rabbit, 20 for rat, & 18 for mouse)
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28. Developmental & reproductive
toxicity…
 Segment II studies..
 The uterus is excised & weighed & then
examined for the number of live, dead, &
resorbed fetuses
 Live fetuses are weighed
 Half of each litter is examined for skeletal
abnormalities & the remaining half for soft tissue
anomalies
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29. Developmental & reproductive
toxicity…
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
during the first 3 wks of life
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30. 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
substantially different consequences
 Germinal mutations
 Somatic mutations
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31. Mutagenicity…
 Germinal mutations:
 Damage DNA in sperm & ova, which can
undergo meiotic division & therefore have the
potential for transmission of the mutations
to future generations
 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
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32. Mutagenicity…
 Germinal mutations…
 Alternatively, the mutation in the genetic
material 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
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33. 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
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34. 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 devised to test chemicals for their ability to
cause mutations
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35. Mutagenicity…
 Some genetic alterations are visible with the light
microscope
 In this case, cytogenetic analysis of bone
marrow 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
chemical can also be evaluated by the dominant
lethal test
 This test is usually performed in rodents
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36. Mutagenicity…
 The dominant lethal test…
 The male is exposed to a single dose of the
test compound & then is mated with two
untreated females weekly for 8 wks
 The females are killed before term, & the
number of live embryos & the number of
corpora lutea are determined
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37. 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.
typhimurium 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-
mutation to the wild type has occurred
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38. 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
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39. Mutagenicity…
 Ames test…
 B/c many chemicals are not mutagenic or
carcinogenic 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
quantified by the number of bacterial colonies
that grow in a histidine-deficient medium
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40. C. Tests for carcinogens
a) The Standard Test:
 The currently accepted design for
carcinogenicity 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
autolysis
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41. 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
histological examination
 All observations are recorded, summarized &
analyzed
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42. D. Immunotoxicity Assessment
 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 sub-
acute/sub-chronic toxicity studies
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43. Immunotoxicity Assessment…
 Parameters:
 Level 1:
• Hematology
• Histopathology or lymphoid organs
• Quantity of T- & B-cells (cellularity of lymphoid
organs)
• Blastogenesis (mitogen responsiveness; mixed
lymphocyte reaction)
• Quantitation and funciton of natural killer cells
• Macrophage function
• Cytokine production
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44. Immunotoxicity Assessment…
 Parameters…
 Level 2:
• 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
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45. 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
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46. Irritation & sensitization - local
tolerance studies…
 Test system/animal system: Rodents, rabbits
 Dose administration
 Single patch administration
 Multiple doses over 2—4 weeks
 Topical (epicutaneous), intradermal, or corneal
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47. Irritation & sensitization - local
tolerance 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)
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