2. Contents
1) Introduction
2) Description of the methods
Pilot studies
Animal Selection
Test substance
Dose selection
Administration of test substance
Measurements
Time Course Studies
3) Supplemental Approaches
4) Data & Reporting
3. INTRODUCTION
OECD : Organisation for Economic Co-Operation & Development
Headquarter: Paris, France
Guideline No: 417 for toxicity studies, adopted on 22 July, 2010
Purpose: to obtain adequate info on ADME, to aid in relating concentration or dose to
observed toxicity, to aid in understanding its mechanism of toxicity
Not designed to special circumstances such as pregnant or lactating women & offspring
Total 115 guidelines for toxicity studies
4. OECD 402 – Acute Dermal Toxicity
OECD 404 – Acute Dermal Irritation/Corrosion
OECD 406 – Skin Sensitization
OECD 407 – Repeated Dose 28-day Oral Toxicity Study in Rodents
OECD 408 – Repeated Dose 90-Day Oral Toxicity Study in Rodents
OECD 410 – Repeated Dose Dermal Toxicity: 21/28-day Study
OECD 411 – Sub-chronic Dermal Toxicity: 90-day Study
OECD 414 – Prenatal Development Toxicity Study
OECD 415 – One-Generation Reproduction Toxicity Study
OECD 416 – Two-Generation Reproduction Toxicity
OECD 420 – Acute Oral Toxicity – Fixed Dose Procedure
OECD 421 – Reproduction/Developmental Toxicity Screening Test
OECD 422 – Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity
Screening Test
OECD 423 – Acute Oral toxicity – Acute Toxic Class Method
OECD 425 – Acute Oral Toxicity: Up-and-Down Procedure
OECD 427 – Skin Absorption: In Vivo Method
OECD 429 – Skin Sensitization: Local Lymph Node Assay
OECD 442 A/B – Skin Sensitization
OECD 443 – Extended One-Generation Reproductive Toxicity Study
OECD 451 – Carcinogenicity Studies
OECD 452 – Chronic Toxicity Studies
OECD 453 – Combined Chronic Toxicity/Carcinogenicity Studies
5. Toxicity type Animal Procedure Observation
Acute Oral Toxicity Rat Fasted 3-4 hr before
test, oral administration
by gavage using a
stomach tube or a
suitable cannula
1 mL/100 g body
weight for 14 days
Changes in skin & fur,
eyes & mucous
membranes. Tremors,
convulsions, salivation,
diarrhea, lethargy,
& coma
Acute Dermal Toxicity Rat Test subs (200 mg/kg)
applied to skin by
removing fur (10 %
body weight), held with
gauge dressing for 24
hour
Changes in skin & fur,
eyes & mucous
membranes. Tremors,
convulsions, salivation,
diarrhea, lethargy,
& coma
6. Skin
Sensitization
Test
Guinea pig Animals (5) exposed to test
substance by intradermal
injection or epidermal
application, following a rest
period of 10-14 days, animals
are exposed to a challenge
dose
Extent & degree of skin reaction
in test animals compared to
control animals
Acute eye
irritation
Albino rabbit 60 mins prior to test substance,
buprenorphine 0.01 mg/kg
administered by s.c. route to
provide therapeutic level of
systemic analgesia & eye not
washed for 24 hour
Ocular lesion, corneal ulceration,
corneal opacity, absence of light
reflex, ulceration of conjunctival
membrane (at 21 days or any
time when pain observed)
7. Inhalation toxicity Rat Inhalation chamber
5 mg/L for aerosols
20 mg/L for vapours &
20,000 ppm for gases
All body organs necropsed
including brain, heart, kidneys,
liver, lung, spleen, ovaries, testes
Reproduction/De
velopmental
toxicity screening
test
Rat Administration by gavage
daily for 7 days a week, 1
ml/100 g body wt.
Dosing begin at 2 weeks
prior to mating
Body wt, blood samples, gross
necropsy
8. Carcinogenicit
y Studies
Rat Orally by gavage in diet or
drinking water, NMT 5 % of
total diet, dosed daily for 24
months
Body wt., tumour dev,
Haematology, gross necropsy
Prenatal
Development
al Toxicity
Study
Rat or Rabbit Daily from 5 days post mating,
orally 1000 mg/kg body wt.
Mortality, moribundity, pertinent
behavioral changes & all signs of
overt toxicity observed by killing
females one day prior to delivery
The fetuses is also examined
9. One generation
reproduction
toxicity study
Rat/mouse Test substance to both sexes
during mating period & only
to females during pregnancy
& nursing period in
diet/drinking water,
1000 mg/kg, 2 weeks prior to
mating, upto 3 week mating
period
Behavioral changes, signs of
toxicity, mortality, body wt,
gross necropsy
Neurotoxicity study
in rodents
Rat Oral by gavage in
diet/drinking water, 1000
mg/kg body wt, dosed daily
for 28 days
Neurological abnormalities,
Haematology, body wt,
ophthalmic evaluation,
histopathology
10. Genetic
Toxicology:
Rodent
Dominant
Lethal Test
Rat/Mice Males are exposed to test
substance & mated to
untreated virgin females, single
administration of test substance
by oral or intraperitoneal
injection
Females are sacrificed & contents of
uteric are examined to determine
number of implants & live & dead
embryos.
Calculation of dominant lethal effect
is based on comparison of live
implants per female in treated group
to live implants per female in the
control group
Bacterial
Reverse
Mutation Test
Bacteria Cultures of bacteria are grown,
at least 5 strains of bacteria, 4
species of S. typhimurium & an
E coli.
Bacteria is exposed to test
substance & incubated at 37 C
for 48-72 hr & number of
revertant colonies is counted
Signs of toxicity, sign of precipitation,
individual plate counts, mean
number of revertant colonies per
plate & std deviation
11. DESCRIPTION OF THE METHODS
1) Pilot Studies
2) Animal Selection
a) Age & Strain
Young, adult animals of same age, weight variation ±20%
b) Number & Sex of animals
4 animals of same sex, both sexes if sex-related differences in toxicity
c) Housing & Feeding Conditions
Housed individually in artificial light, temp 22 degree C, relative humidity 30-70 %
3) Test Substance
Radiolabelled test substance 14C for mass balance & metabolite identification
Unlabelled if specificity & sensitivity is high & mass balance and metabolite identification
can be evaluated
12. 4) Dose Selection
Pilot Study
Single oral dose, dose should be non-toxic, high enough to allow for metabolite
identification in excreta
Main studies
Minimum of two doses
substance of low toxicity, maximum dose of 1000 mg/kg body weight
5) Administration of Test Substance
Dissolved or suspended in the appropriate vehicle
maximum volume administered depends on size of test animals, not exceeding 10mL/kg
body weight for rodents.
Vehicle for IV not interfere with blood integrity or blood flow
Anaesthesia should be used if one cannulates the jugular vein
13. 6) Measurements
Mass Balance
Sum of percent of dose excreted & present in tissues
Absorption
Amount absorbed is Mass balance - % dose in GIT + faeces
In biliary excretion, bile ducts of at least four animals cannulated & single dose of test
administered
Percent absorption = (amount in bile + urine + expired air + carcass without GI tract
contents)/amount administered * 100
Bioavailability
F = ( AUCexp / AUC iv) * (Dose iv / Dose ip)
Exp is experimental route (oral, dermal or inhalation)
14. Tissue Distribution
% of total drug in tissue measured at termination of excretion experiment
if no substance detected, tissue distribution be measured at peak plasma concentration
Tissues collected include liver, GI tract, kidney, spleen, whole blood, target organ tissues
other tissues (e.g. thyroid, erythrocytes, reproductive organs, skin, eye
Organ dissection, homogenization, combustion, autoradiography, etc
Metabolism
Excreta collection for identifying metabolites, 5 % or greater of administered dose
Identification i.e. structural determination of components
15. Excretion
determined by % recovered from urine, faeces & expired air at appropriate time intervals
Collection of excreta terminated at 7 days or after 90 % of the administered dose has
recovered
Sampling at 6, 12 & 24 hour
7) Time Course Studies
Plasma/Blood Kinetics
Cmax, Tmax, half life, AUC; studies to be conducted at 2 or more doses
4 animals of one sex per dose group, different sex if sex-related differences
Blood samples at suitable time points
16. Enzyme Induction/ Inhibition studies in
a) Relationship between biotransformation of test substance & enhanced toxicity
b) Non-linear relationship data between dose & metabolism
c) Toxic metabolite produced by enzyme pathway
SUPPLEMENTAL APPROACHES
provides info about ADME of a chemical in certain species
Use of in-vitro information
Freshly isolated or cultured hepatocytes and subcellular fractions (e.g. microsomes,
from liver used to study metabolites.
Studies with microsomes useful to address potential gender and life-stage differences
17. Use of Toxicokinetic data from Toxicity studies
Analysis of blood, tissue, excreta samples provides data on
bioavailability
changes in plasma concentration in time (AUC, Cmax)
Bioaccumulation potential
Clearance rates
Gender or life-stage changes
Age-related sensitivity due difference in blood-brain barrier
Sub population sensitivity due difference in biotransformation
Extent of exposure of fetuses by transplacental transfer of chemicals
Use of Toxicokinetic Modeling
a) partition coefficients
b) biochemical constants and physiological parameters
c) route-specific absorption parameters
d) in vivo kinetic data for model evaluation
18. DATA & REPORTING
Study report including
Summary
Study design & description of method used
Also mass balance, nature & magnitude of metabolites, tissue residue, rate of clearance,
bioaccumulation potential
Introduction
Study objective, rationale and design, appropriate references, any background history
Materials & Methods
a) Test Substance
Identification by chemical name, chemical purity, physical state, colour, solubility, partition
coefficient, stability, corrosivity, information on isomers
Description of any vehicle, diluents, suspending agents, emulsifiers or other materials
19. b) Test Animals
Species, strain, age, sex, body weight, health status
c) Methods: It includes
Justification for selection of dose levels;
Description of pilot studies used in the experimental design of the follow-up studies;
How the dosing solution was prepared and the type of solvent or vehicle, if any, used;
Number of treatment groups and number of animals per group;
Dosage levels and volume (and specific activity of the dose when radioactivity is used);
Route(s) and methods of administration;
Frequency of dosing;
Fasting period (if used);
Animal handling;
Sample collection and handling;
Analytical methods used for separation, quantitation and identification of metabolites;
20. d) Statistical Analysis
info on method of analysis and computer program
Results
graphic illustrations of the findings,
reproduction of representative chromatographic and spectrometric data,
metabolite identification/quantification and proposed metabolic pathways
molecular structure of metabolites.
Discussion and Conclusion
Provide a proposed metabolic pathway based on the results of the metabolism and disposition of the
test substance;
Discuss any potential species and sex differences regarding the disposition and/or biotransformation
of the test substance;
Tabulate and discuss the identification and magnitude of metabolites, rates of clearance,
bioaccumulation potential, and level of tissue residues of parent, and/or metabolite(s), as well as
possible dose-dependent changes in TK parameters, as appropriate;
Provide a concise conclusion that can be supported by the findings of the study;
