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Section C
In Vivo Toxicity Tests
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In Vivo Toxicity Tests
! Acute toxicity tests
! Subchronic or repeat-dose toxicity tests
! Chronic toxicity and carcinogenicity tests
! Testing for specific end points, for example:
- Developmental and reproductive toxicity
- Neurotoxicity
- Immunotoxicity
3
Acute Toxicity Tests: Classic LD50
! Provides lethality data, although testing can determine
other acute effects such as:
- Eye irritation
- Skin irritation and/or sensitization
! Allows toxicity categorization
Test
Category I
Category II
Category III
Category IV
Oral LD50
<50 mg/kg
50–500 mg/kg
500–5,000 mg/kg
>5,000 mg/kg
Dermal LD50
<200 mg/
kg
200–2,000 mg/
kg
2,000–5,000 mg/
kg
>5,000 mg/kg
<0.05 mg/L
0.05–0.5 mg/L
0.5–2 mg/L
>2 mg/L
Inhalation
LD50
4
Acute Toxicity Ratings from Classic Oral LD 50
Tests
Sucrose
29,000
Category IV
Ethanol
10,000
Category IV
3,500
Category III
900
Category III
1,000
Category III
Chloroform
908
Category III
Phenobarbital
150
Category II
Caffeine
192
Category II
DDT
113
Category II
16
Category I
Sodium cyanide
6
Category I
Nicotine
1
Category I
0.1
Category I
TCDD (dioxin)
0.001
Category I
Botulinium tox.
0.00001
Category I
Sodium chloride
Morphine
Aspirin
Strychnine
Tetradotoxin
5
Classic LD50: Value
! Provide data on lethal doses and target organ
- Results can form the basis for dose selection of
subsequent subchronic studies
- May be useful first approximation of acute hazards to
workers
! But, extrapolation to humans is questionable
6
Classic LD50: Major Criticisms
! Requires large numbers of animals to obtain statistically
acceptable numbers
! Tests may be inhumane
! Data are of little practical value
- Could be approximated from experiments with fewer
animals
- Several alternative acute toxicity tests that allow
classification with few animals have been developed
! Limit test
! Up-down procedure
7
Repeat Dose Toxicity Tests: Purpose of
Subchronic Tests
! Studies the effects of non-lethal doses
- A non-toxic dose and two or more doses that cause
different degrees of toxicity
- Typically 28–90 day repeat dosing
- Routes of exposure: oral, dermal, inhalation
! Detects effects resulting from bioaccumulation
! Provides information on target organ-related types of
subchronic toxicity that might occur
- For example: immunotoxicity, neurotoxicity,
cardiotoxicity
- Assessments: include clinical chemistry, gross
observations on behavior and organ, histopathology
8
Repeat Dose Toxicity Tests: Purpose of
Subchronic Tests
! Establishes dose regimens for chronic studies
- Provides data which will allow an estimate to be made
of the MTD (maximum tolerated dose)
! ! MTD = no significant impairment of growth (<
10%) or observable toxicity
Provides data for use in risk assessment
- Can be used as the basis for determining:
! NOEL: no-observable-effect-level
! NOAEL: no-observable-adverse-effect-level
! LOAEL: Lowest-observable-adverse-effect-level
- But with added safety factor
9
Chronic Toxicity Tests
! Purpose: provide data on chronic toxic effects and
carcinogenicity
! Conducted over the greater part of the lifespan of the test
animal
! Preferred for determining the NOEL, NOAEL, LOAEL
! Allows classification of carcinogenic potential for humans
- EPA
- International Agency for Research on Cancer
10
Chronic Toxicity: Carcinogenicity Bioassay
EPA Guidelines for Carcinogenicity Bioassay
Test parameter
Requirement
Species
! Rats
Number of animals per dose
! 50
Doses
At least three plus control:
! High: maximum tolerated dose
! Low: causes no observable
toxicity
! Intermediate
Regimen for dosing
Organs and tissues to be
examined
and mice (inbred)
males, 50 females
! Begin
exposure at six weeks
! End at 24 months of age
! All
animals: external and
histopathological examination
11
Statistical Power of Carcinogenesis Bioassays
12
Testing for Specific Endpoints
! Example: developmental and reproductive toxicity
- Exposure timing
13
Exposure Timing
14
Issues Regarding Bioassays
! Do animal studies predict adverse human health effects
and carcinogenicity?
- Enhance value—PBPK modeling
! Resource intensive
! Development of more effective, rapid, cost-effective
approaches
- Example: transgenic animals whose susceptibility to
tumorigenesis is increased using oncogenes
15