This document discusses guidelines for conducting long-term carcinogenicity studies in rodents. It describes selecting the animal model, dose levels and routes of administration as well as important study parameters like duration, observations, and data collection. Proper experimental design and conduct following good laboratory practices is essential to obtain reliable results. Special attention should be given to animal housing, care, and minimizing exposure to investigators for safety.

1. Cancer Research With Rodents
 Toxicity and pharmacokinetics
 Genotoxicity
 Embryotoxicity, Teratogenicity
 Carcinogenicity / Mutagenicity
 Anti – cancer activity
 Chemopreventive effects / mechanism(s)
 Role of specific gene / protein / pathways, etc
 Other specific disease mechanism(s)

2. MULTISTEP MODEL OF CARCINOGENESIS
NORMAL CELL
INITIATED CELL
PREMALIGNANT LESIONS
(Dysplasia, carcinoma in situ)
CANCER
CARCINOGENESIS
Initiation
Promotion
Progression
CHEMOPREVENTION
Repair
Regression
Inhibition
Chemical carcinogens
Viruses, Radiation
Replication errors
Other factors
Spontaneous remission
Growyh inhibitors
Antipromoters
DNA repair
DNA damage

3. Interactions
Dose of Carcinogen(s)
Duration of exposure
Host Factor(s)
Genetic / Acquired
Susceptibility / Protective
Disease No disease

4. The relationship of biological markers to exposure and disease
Susceptibility
(Genetic / Acquired)
Exposure Internal
dose
Biologically
effective
dose
Early
response
Altered
structure/
function
Disease

6. Long-term assays for carcinogenicity
 The essence is to observe test animals for a major portion of their
lifespan for the development of neoplastic lesions after or during
exposure to various doses of a test substance by an appropriate
route.
 Such an assay requires careful planning and documentation of
the experimental design, good animal care leading to long
survival, a high standard of pathology and unbiased statistical
analysis.
 Obtaining internationally comparable / acceptable and reliable
results.
 Design
 Conduct
 Analysis
 Reporting

7. A detailed written plan should be produced by close
co-operation among planners
 Planning
 Chemists (e.g. Analytical Chemistry)
 Toxicologists (e.g. Biochemistry, Clinical Chemistry, Nutrition,
Pharmacokinetics, Epidemiology)
 Laboratory Animal Specialists (e.g. Animal Health Monitoring,
Animal Husbandry)
 Pathologists (e.g. Histopathology, Hematology, Microbiology)
 Biostatisticians (e.g. Statistics, Computer Programming, Data
Acquisition and Processing)

8.  Review
 Review by Experts (not associated with the project)
 Ethics Committee (e.g. Institutional Animal Ethics)
- Study Director
- Deputy Study Director
- Committee Members
Long term assays for carcinogenicity are time consuming and
costly undertaking requiring adequate physical facilities, and
qualified and experienced personnel as mentioned above.

9. Test Substances
Before initiating a long-term carcinogenicity study, the investigator(s)
should obtain available, relevant information on the characterization,
storage, handling, safe disposal as well as data on control parameters
such as :
 Synonyms and trade names
 Structural and molecular formulae and molecular weight
 Method of analysis including chemical and physical properties of the
pure substance
e.g. description, boiling point, melting point, density, refractive index,
spectroscopy data, solubility in water and organic solvents, volatility,
reactivity, conversion factor (ppm in air / mg per m3), photochemical
properties, extent of ionization, particle size, shape and density, stability
(under various temperature / pH)
 Adequate specifications of commercial products (% of stabilizers /
emulsifiers)
 Impurities and methods of manufacture or route of synthesis
contd………

10. Sources and batch numbers
 Date(s) of receipt
 Methods of storage
 Probable daily exposure level for humans
 Biochemical data (absorption, pharmacokinetics, etc)
Dilemmas: Effect(s) due to impurities / by products / carriers
Testing of ‘technical grade’ vs. ‘highly purified material’
contd,,,,,,

11. Animal species, strain and sex
 Biological response to the test substance is identical to that in
humans
 In absence of such animal species, material be tested in at
least two species
 A particular species may be indicated by metabolic or
pharmacokinetic data, susceptibility to a particular class of
carcinogens or the proposed route of administration
 Criteria:
 availability
 economy
 sensitive to carcinogens
 similarity to humans (metabolic / toxicological /
pathological responses)

12.  Species frequently used:
Rats, Mice and Syrian golden hamsters (respiratory and urinary tract
carcinogenesis)
- short life-span, small size, availability
- existence of considerable biological knowledge on these species
 Other species:
Primates, dogs
- long lifespan, genetic heterogeneity, limited availability, higher
maintenance cost
- not much similarity in metabolic capabilities than rodents
- not recommended for routine testing
Small Fish
Some studies have been employed

13. Animal species, strain and sex
- Well characterized
- Free from interfering congenital defects (spontaneous diseases)
- Consistent low incidence of spontaneous tumors / diseases
- High and specific susceptibility to human carcinogens
 Selection of outbred animals:
 Many laboratories have experience with outbred strains
 Outbred or random-bred strains are more resistant to disease than
inbred animals
Problem: Within single outbred strain there can easily be enormous
differences in the incidence of cancer resulting from a
standard treatment

14.  Strains routinely used in bioassay:
Fischer 344 rats
Sprague-Dawley rats
B6C3F1 mice
ICR Swiss albino mice
CD-1 mice
Outbred hamsters
USA, Japan
Wistar rats
NMRI
Europe
 Selection of Inbred Strains
- genetic heterogeneity will be controlled
- risk that the treated group is comprised entirely of animals resistant to
the test carcinogen is reduced
Problem: Whether one maximum tolerated dose would apply to each
separate strain

15.  Animal Strain
 Strain must be outlined in the experimental protocol, preferably using
international nomenclature
 The genetic status, if not obvious must be defined separately
 The age, weight and sex of the animals must be determined at the start
of the experiment
 The definition of the microbiological status should include the
specifications of pathogens from which the animals are free
 Methods of delivery, transport as well as quarantine or acclimatization
periods of the animals must be specified
 Sex:
Generally equal numbers of animals of each sex should be included

16. Route of administration
Administered by the predominant route of human exposure, unless
pharmacokinetic studies show that other routes are equivalent.
 Oral exposure - mixed in the diet
- in the drinking water
- by gastric intubations
The ultimate choice most often largely depends on the characteristics of
the test material such as volatility, corrosiveness, solubility in water,
stability in the diet, palatability, etc.
The kinetics of uptake, distribution, conversion and elimination are very
important.
In diet / drinking water:  exposure is continuous
 possibility of contamination of the laboratory
environment
 possibility of exposure of personnel
By gavage:  doses administered are known
 local injury due to high concentration
 time consuming
 loss of animals due to faulty intubation

17.  Cutaneous exposure
- simulate a main route of human exposure e.g. for cosmetics
- as a model system for induction of skin tumors
- The kinetics of absorption and metabolism and potential for local or
systemic effects should be considered
 Inhalation exposure
 The respiratory tract is an important route of entry for many substances
hence it has become more and more important despite higher cost
 Particulate matters have different disposition pattern in rodents and
humans : - rodents have small diameter airways
- highly turbinated nasal passages
- variation in bronchial branching patterns
- rodents are obligatory nose breathers
 Requires specially designed exposure chambers and special equipments
for generating, sampling and monitoring the test atmosphere
 Contamination of the animal fur also results in oral intake
 Animals may filter the atmosphere by hiding their noses in their own or in
each other’s fur
 Individual housing needed for reducing this
 Head – nose exposure only may be used contd…..

18. contd…..
 The advantages of continuous exposure for simulating environmental
conditions may be offset by the necessity of watering and feeding during
exposure
 Intake air to chambers need to be filtered through the absolute filters and
exhaust air must be treated to remove the test substances
 Sustain a dynamic flow of sufficient fresh air
 Back-up ventilation system is required
 Air-flow, temp and humidity and chamber concentration and particle size
distribution need to be continuously monitored / recorded
 The method of generating test atmosphere must be reproducible
 Intratracheal instillation
 Possible alternative to inhalation exposure for aerosols
 Widely used in experimental respiratory tract carcinogenesis
 Since humans are more likely to inhale through mouth than rodents, this
approach might imitate more closely inhalation in human
 Instillations are generally given once or at most twice a week to avoid
mechanical damage

19.  Other routes
 Subcutaneous injections:
- different from the exposure in humans
- when compound is poorly absorbed after oral dose or when
contamination of laboratory is to be minimized
 Intraperitoneal, intramuscular and intravenous injections:
- not widely used for testing the potential carcinogenicity
- route(s) do not resemble those in humans
- useful when compound is not absorbed from GI tract of rodents
- daily administration by these routes may be difficult

20. Pharmacokinetics
The study of the dynamics of the fate of chemicals in the body
Pharmacokinetics considerations
design
Interpretation especially when
extrapolating results to human
situations
It can indicate the level of parent compound / product in proportion to
the dose administered
Non-linear dose-response relationships:
 Saturation of uptake, metabolic activation, metabolic detoxification,
depletion of protective molecules, saturation of a repair protein
 If extrapolations are made in the non-linear region it may result in
serious overestimation or underestimation

21.  Information on bioavailability of the compound (dose, time) will be
very useful.
 Pharmacokinetics may also assist in selecting the mode of
administration to be employed.
 Quantify the amount of the compound in tissues and body fluids and
measure the time course of elimination of the compound from blood
(after single and/or multiple doses).
 Pharmacokinetic parameters may be influenced by genetic
variations, exposure to other agents, dietary factors or protective
factors.
 “Worse-case” scenario may be obtained.
Pharmacokinetics

22. Selection of doses for animal bioassays
(a) Number of dose levels:
Two dose levels plus control are often used
(b) High dose:
 MTD: Maximum Tolerated Dose (in each sex)
- Predictions about chronic toxicity are based on previous
subchronic toxicity studies
- Pharmacokinetic approach
 Reduction in life-span and decrease in weight gain are employed
 For non-toxic substances, test compound at about 5% of the diet is
generally employed – no interference with nutrition / caloric intake
(c) Lower dose: determined based on
- pharmacokinetic data
- subchronic toxicity studies
- anticipated human exposure

23. Inception, duration of exposure and observation period
- Few weeks after weaning till major portion of life span
- Prenatal and two-generation studies are not recommended for routine
screening
- 24 months or the entire lifespan
(a) Treatment throughout the entire observation period
(b) Treatment for defined period followed by observation period
Number of animals
 50 males + 50 females in each test group and concurrent
control group is generally accepted as minimum adequate
number
 If interim sacrifice is included then extra animals need to be included

24.  Control groups
 Randomly selected and handled identically to the treated
groups in all respect except exposure to the test material(s)
 Preferably housed in the same room as the treated animals. If
cross-contamination is the problem then housed in a
separate room with identical physical conditions
 In routine carcinogenicity testing, positive controls need not
be used
 Information about historical controls from the colony is of
great importance

25. Methods of randominzation
(a) Unstratified allocation:
 Whether animals can be stratified with respect to litter before allocation
or body weight or birth date
 Generally stratification is not done as a stratified statistical analysis will
need to be done. Most of these variables are generally controlled by
randomization
 After random allocation to various groups; birth date, weight or litter,
etc. may be recorded. During the statistical analysis the true relevance
of these features to cancer incidence may be assessed
(b) Animal placement: Group vs individual
 costs more
 facilitates husbandry
 avoidance of cannibalism
 spread of infection, fighting
 hormonal status
 produces stress
Location / positioning of cages:
Systematic differences between different groups in the height, light or
ventilation of cages should be avoided.

26. Observations
(a) Clinical signs and mortality
(b) Body weight and food consumption
(c) Hematology and Clinical Chemistry
(d) Pathology
 Gross pathology by a trained laboratory animal pathologist
 Check list to ensure that all organs were inspected and sampled. Lesions
to be recorded
 Person to be available during week end for autopsies
 Tissues to be fixed in aqueous neutral phosphate – buffered formaldehyde
solution
 The urinary bladder should be inflated with fixative. All other hollow
organs should be opened either before or after fixation
 The lungs should be fixed by intratracheal infusion of the fixative at
constant pressure
 Clinical signs as well as the results of chemical and hematological
examinations should be available to the pathologist prior to macro /
microscopic examinations
 A terminal blood smear should be prepared from all animals fordiagnosis
of leukemia

27. Data acquisition, processing and storage
 Computerized data storage and retrieved system.
 Raw data, log books, protocols, records and interim and final
reports + slides and paraffin blocks – for 10 years after completion
of the final report.
 Wet tissue storage → should not be shorter than 12 months.

28. Animal Husbandry
 GLP
 Housing conditions
 Animal care facilities
 Intercurrent diseases
 Impurities in diet / air / water / bedding
 Well-ventilated rooms with controlled lighting, temp. and humidity
 Quarantine and acclimatization
 Restricted entry to animal house
 Committee
 Breeding and maintenance under pathogen-free condition
 Maintenance of disease-free animals under conventional, clean conditions
is perhaps even preferable to very strict specific pathogen-free conditions
 Full barrier system may be unnecessarily costly and considerably
hampers biotechnical work
 Cages / racks and other equipments → easy and frequent cleaning
 Contaminated waste, faeces, dead bodies and radioactive waste should
be eliminated according to applicable legal or administrative requirements

29. Diet
 Types of diet
 Nutrients
 Contaminants
 Purified diets
 Storage of diets
 Feeding of animals
 Documentation
Safety Measures
 Each test compound should be regarded as potentially hazardous
(carcinogenic) to humans.
 Major elements of a safety and health plan for the handling of
potential carcinogens and toxic substances include:
(a) Precautions for transport and storage of the test material within the
laboratory
(b) Protective clothing for all personnel who may be exposed to the test
substance or to contaminated apparatus or animals
(c) Disposal of contaminated waste, including faces and dead bodies
contd………

30. (d) Cleaning of contaminated cages, rooms, ventilation systems, mixers
and other equipment:
- protection of maintenance workers and emergency personnel
- reduced pressure in animal rooms
- written and posted instructions for emergencies
- reporting and recording of all accidents
- medical surveillance of all individuals involved in the operation for
evidence of absorption of the test chemical

31. Decontamination and Destruction of Mycotoxins
Aflatoxins B1, B2, G1, G2, etc. (IARC Sci. Pub No.113(1991)
Method 1 : Sodium hypo chlorite
Method 2 : Ammonia
Method 3 ; KMnO4 alkaline
Solid compound Aqueous Solution
Solution in Organic
solvents
Solution in
DMSO,DMF
Add NaOCl
Sonicate
Allow to react > 30 min.
Discard
Make Alkaline Evaporate to dryness
Dil. Ethanol/Methanol
TLC plate
Add Water
Extract with
DCM
Glassware
Spills
Clean and cover
area with NaOCl

32. Destruction of PAH
Pure
compound
Petridish Wastes
Add Water
Ethyl acetate
Sol. In
Organic
Solvent
Sol. In
DMSO/DMF
Sol. In
Water
Spills of
pure
Compound
Spills of Sol.
Add 2- methyl butane
Extract in to
Acetonitrile
Rinse Acetonitrtle
fractions
With 2-methyl butane
Sol. In Oil
Evaporate
to dryness
Add Water &
extract with
Cyclohexane
Absorb on
absorbing
material
Add DMF
to surface
Treat absorbing
material with
KMnO4/H2SO4
Add Acetone
Add KMnO4/H2SO4
Allow to react 1Hrs
or more
Neutralize Discard

33. e.g.
MNU: N-nitroso-N-methyl urea
MNNG: N-nitroso-N’-nitro-N-methyl guanidine
ENU: N-nitroso-N-ethyl urea etc.
Destruction of N-nitrosamines
Pure compound Solution in methanol/ ethanol
Acetone /DMSO/ water
dil with methanol
dil
Add to an equivalent vol. Of
6 mol/L HCl containing Add to an equivalent vol of 6
mol/L HCl containing 35 gm/L
Iron filings
@ 35 gms/L sulfanalic acid
Allow to react 24 Hrs.
Neutralize with KOH
Discard

34. Destruction of N - Nitrosamines
1) Treat with HBr in CH3COOH ( Denitrosation with HBr)
2) Treat with H2SO4 and KMnO4
3) Treat with Ni –Al alloy in NaOH
4) Biological treatment ( 50% Effective)

35. Statistical analysis
(a) Goals of statistical analysis
(b) Sources of bias
(c) Description of the data
(d) Unadjusted testing methods
(e) Mortality – adjusted testing methods
(f) Serial sacrifice studies
(g) Dose – response models
Reporting of results
 Inadequacies of the design and conduct of the experiment must be
presented
 The evidence for carcinogenicity should be fully described along with the
basis for the author’s interpretations
Analysis and reporting
 Validation of data
 Adequacy of the experiment
 Chemically related effects on growth, morbidity and survival
 Categories of tumor
Benign
Malignant

37. IN VIVO SCREENING SYSTEMS FOR THE DETERMINATION OF
CHEMOPREVENTIVE ACTION
SPECIES
Mouse
Rat
Rat
Mouse
Hamster
Mouse
Rat
Mouse
Hamster
Mouse
Rat
CARCINOGEN
4-(Methylnitrosamino) 1-
(3-pyridyl)-1 butanone
(NNK)
MNU or DMBA
Azoxymethane
N-butyl-N-(4-
hydroxybutyl)
nitrosamine
MNU or DEN
B(a)P
N-nitrosomethyl
benzylamine (NMBz)
Nitrosarcosine
DMBA
DMBA / TPA
DEN / Phenobarbitone
TARGET ORGAN
Lung
Mammary Gland
Colon
Bladder
Lung
Forestomach
Esophagus
Esophagus
Oral/Cheek Pouch
Skin
Liver
END POINT (inhibition)
Adenomas / Carcinomas
Adenocarcinoma
Adenomas/Adenocarcinoma
Transitional Cell Carcinoma
Squamous Cell
Carcinoma/Adenocarcinoma
Papillomas
Carcinomas
Papillomas
Carcinomas
Papillomas/Carcinomas
Hepatocellular
Carcinomas/Hepatomas

38. EFFECTIVE RETINOIDS IN EXPERIMENTAL CARCINOGENESIS
MODEL
Rat Mammary
Mouse Urinary
Bladder
Mouse Skin
Hamster Lung
Rat Lung
Esophagus
Colon
Rat Liver
Mouse Liver
Rat Pancreas
CARCINOGEN
MNU
Hydroxybutyl--
BUtylnirosamine
DMBA / TPA
DEN
3-MCA
Nitrososarcosine Ethyl Ester
Azoxymethane
3, Methyl, 4-
dimethylaminobenzene
DEN
Azaserine
EFFECTIVE RETINOID(S)
HPR, Retinyl Acetate (RA)
13-Cis-RA, HPR, RA
13-Cis-RA, HPR
HPR
Retinyl Acetate
None
None
13-Cis-RA
HPR
2-Hydroxy Ethyl-Retinamide
N-4-Propionyloxyphenyl
Retinamide