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Dr. Pravinkumar WahaneJR II
BJGMC, Pune
Toxicity Studies
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Source, Synthesis & chemistry
Preclinical Studies & Toxicology Studies
Animal models, Efficacy and Safety
Clinical Trials
Drug Development
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Preclinical Safety & Toxicity Testing
Aims at discovering complications or sequelae arising from the pharmacological actions of drug and any unexpected side-effects.
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Primary Goals
Estimate safe starting dose for clinical studies & subsequent dose escalation schemes in humans.
Identify potential target organs for toxicity and study whether such toxicity is reversible.
Assess dose dependence & relationship to exposure
Assess hazards that cannot be evaluated in clinical trials (e.g. carcinogenicity and teratogenicity)
Identify hazards and estimate safe dose range
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Necessity of toxicity studies
Completely novel compound of unique action under consideration
Any chemical modification made to a known drug
Formulations of a compound are considered ( Separately as well as formulated form)
Novel combination of drugs made available as a single formulation
Veterinary drugs → if treatment is given to animals intended for human food
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Requirements
Chemical Criteria
Substance used in toxicity studies should be as pure as material to be given to man
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Materials: Animals
Species: Species difference → single largest difficulty in
interpretation of toxicity studies
Species →health, behaviour, endemic disease and reaction to well studied toxic agent is familiar
Two different species(Rodent & Non Rodent) used
Should differ phylogenetically as widely as possible
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Materials: Animals
Species: Species in which test material is
pharmacologically active due to presence of receptor should be used
Ex. Avoid dogs for studying toxic effects of sulphonamides, monkeys are better
Strains: Use either animals derived from random
breeding in a closed colony or hybrids of two inbred lines
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Materials: Animals
Number, age and sex: Should be sufficient Long term experiments i.e. Carcinogen testing,
large number may be necessary to detect relatively low incidence of the reactions
Young immature animals are preferred generally → rapid growth, but for detection of actions on endocrine and reproductive systems, sexually mature animals are required
Should include groups of both males and females
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Route / Frequency of administration
Two routes to be used, one should be which is intended for clinical use
Aim should be to maintain the appropriate blood and tissue level of the compound under consideration for test duration
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Duration
Depends on type of the test (acute, sub-acute, chronic)
All the important toxic effects of test substances can probably predicted from experiments lasting no more than three months
In the absence of any rational basis for choosing a period, a duration of twice the maximum exposure likely for the human beings is convenient guide. 2 yrs. max
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Toxicity Studies
Systemic Toxicity studies
Reproductive toxicology studies
Local Toxicity studies
Allergenicity/ Hypersensetivity studies
Genotoxicity studies
Carcinogenicity studies
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Systemic Toxicity Studies
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Single dose (Acute)
Short term repeated dose (Subacute)
Repeated dose: 10% life-span (Subchronic)
Repeated dose: >10% life span (Chronic)
Systemic Toxicity Studies
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Single-dose (Acute) Toxicity Studies
Dose – toxicity relationship
Specific toxic effects
Mode of toxic action
Median Lethal dose (LD 50)
Route dependent toxicity
Sex dependent toxicity
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Single-dose (Acute) Toxicity Studies
Rodent species (mice and rats)
Use the same route as intended for humans
Use at least one more route in one of the species. This Ensures systemic absorption of the drug
(unless the intended route in humans is only intravenous)
Recommended limit for oral dosing : Higher value of either 2000 mg/kg or 10 times the normal dose that is intended in
humans
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Single-dose (Acute) Toxicity Studies
At least 5 animals / sex / group
At least 4 graded doses (4 dose-groups)
Observation for toxic effects (if any) : 14 days
Obsevation for mortality :
7 days [parenteral administration]
14 days [oral administration]
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Up and down procedure (Guideline 425)
(End point: mortality)
Preliminary study : 1 animal per step
Starting dose : just below expected LD 50
Dose stepped up / down by a factor of 3.2
Continued till outcome reversed, i.e. mortality <=> survival
Reduction of the number of animals, use of single sex
Preliminary studies, followed by main test (Limit test) Fixed dose procedure (Guideline 420)
(End point: Evident toxicity)
Sighting study : 1 animal per step
Starting dose : expecting some toxicity
Fixed doses of 5, 50, 300 and 2000 mg/kg
Dose stepped up / down till end-point achieved
Acute toxic class (Guideline 423)
(End point: mortality)
3 animals of one sex / step
Initial dose: expecting mortality in some animals
Fixed doses of 5, 50, 300 and 2000 mg/kg
Dose stepped up / down till end point achieved
Modifications (OECD guidelines: 420, 423, 425; yr
2000)
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Other parameters :
Symptoms, signs and mode of death
LD 10 and LD 50 values, preferably with 95 % CI
Genetic effects if any
Establish the:
Minimum lethal dose (MLD)
Maximum tolerated dose (MTD)
Target organ of toxicity (if possible)
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Cytotoxic anticancer agents
MTD determined first in Mice
Findings in Rat confirmed to establish a linear relationship between toxicity and body surface area
MTD can be established in non-rodent species, if: Predictability is known to be poor in Rodents
(e.g. Antifolates) Drug has a novel mechanism of action
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Toxicokinetic studies
Generation of pharmacokinetic data
(ADME):• A part of non-clinical toxicity studies or
• A separate supportive study
Purpose:• Assessment of systemic exposure of test
substance
• Relationship of toxicology to dose and time course
• Choose species & regimen in subsequent toxicity studies
• Designing of subsequent non-clinical toxicity studies
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Rodents
• One rodent species (preferably rat)
• At least 4 graded doses including control
• Minimum of 5 animals of each sex
• Proposed clinical route of administration
• Test substance given daily for 10 consecutive days
Non-Rodents
• One male and one female (Dogs > Primates)
• Starting dose (3 or 5 x extrapolated effective dose) or MTD
(whichever is less)
• Dose escalation every 3rd day, lowered if toxicity seen
• Then test substance given daily for 10 consecutive days
Dose-ranging study
Establishment of MTD for repeated dose studies
Identification of Target organ of toxicity
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Repeated-dose Toxicity Studies
Duration depends on proposed clinical trial
At least 2 species, one non-rodent
Species-specific pharmacokinetics, if any, should
preferably resemble human beings
Route intended for human clinical use
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Repeated-dose Toxicity Studies
Wherever applicable, include a Control group
3 other groups are formed, as:
Highest dose Observable toxicity [MTD]
Lowest dose No observable toxicity [NOAEL]
intended therapeutic dose or multiple of it
Intermediate dose Some symptoms ; not gross toxicity or death placed logarithmically betn doses
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• Rodent : 6-10/sex/group• Non-rodent : 2-3/sex/group
14-28 Day
repeated-dose
(Subacute toxicity
studies)
• Rodent : 15-30/sex/group• Non-rodent : 4-6/sex/group
90-Day repeated-
dose (Subchronic
toxicity studies)
• Rodent : 15-30/sex/group• Non-rodent : 4-6/sex/group
180-Day repeated-dose
(9 months for non rodents?)
(Chronic toxicity studies)
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In all cases: Behavioural : General appearance, activity,
behaviour
Physiological : Body weight, food intake
Biochemical : Hematology, serum and urine analysis
Pathological : Organ weights, gross & microscopic study of viscera & tissues
Parameters to be monitored
• If parenteral drug administration:
Injection site : Gross and Microscopic examination
• In non-rodent species: Electrocardiogram : Initial and Final
Fundus examination
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Reproductive Toxicology
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Reproductive Toxicity
Male Fertility
Female Reproduction & Developmental toxicity
Female Fertility
Teratogenicity
Perinatal development
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Male Fertility Study
One rodent species (preferably rat)
3 dose groups and a control group :
Dose selection : results of previous 14 or 28-day toxicity study
Highest dose : showing minimal toxicity in systemic studies
6 adult male animals per group
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Test substance by intended route of use for :• m
inimum 28 days
• maximum 70 days
Paired with female animals of proven fertility in a ratio of 1:2
Drug treatment of male animals continues during pairing
Pairing continued till detection of Sperm in vagina or 10 days, whichever is earlier
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Pregnant females examined after day 13 of gestation
Males sacrificed at the end of the study
Weights of each testis and epididymis recorded
Sperms from one epididymis examined for motility and morphology
Other epididymis and both testes examined for histology
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Female Reproduction and Developmental Toxicity Studies
For all drugs proposed for women of child bearing age
• Segment I : Female fertility
• Segment II : Teratogenicity
• Segment III : Perinatal development
Segment I, II and III studies in albino mice or rats, and
Segment II study also in albino rabbits as a second test species
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Female Fertility Study (Segment I)
One rodent species (rat preferred)
3 graded doses
Highest dose : doesn’t affect general health of parent animals
(usually the MTD from previous systemic studies)
At least 15 males and 15 females per dose group
Route of administration same as intended for therapeutic use
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Teratogenicity Study (Segment II)
Rodent (preferably rat) and Non-rodent (rabbit)
Drug administered throughout organogenesis
3 dose levels and a Control group:
Highest dose : minimum maternal toxicity
Lowest dose : as proposed clinical dose or its multiple
Route of administration : same as intended for humans
At least 20 pregnant rats (or mice) and 12 rabbits, for each dose
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Observation parameters
Females
GeneralSigns of
intoxication
Body weight
Food intake
Reproductive
Uterus, Ovaries
Products of
conception
Foetuses
General
Number
Gender
Length
Weight
Pathology
Gross (all)
Visceral (half)
Skeletal (half)
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Perinatal Study (Segment III)
Specially if
→ Drug to pregnant / nursing mothers for long periods
→ Indications of possible adverse effects on foetus
One rodent species (preferably rat)
Dosing comparable to multiples of human dose and route
At least 4 groups (including control), 15 females / group
Drug throughout last trimester (from day 15 of gestation)
Dose causing low foetal loss continued throughout weaning
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F1 litter1 male and 1 female from each group
Test substance given
Throughout growth to sexual life
Mating performance and fertility of F1
:
F2
generation
F2 generationgrowth parameters monitored till weaning
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Local Toxicity
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Local Toxicity
Dermal Toxicity
Ocular Toxicity
Vaginal Toxicity
Rectal tolerance test
Parenteral Drugs Tolerance
Inhalation toxicity
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Local Toxicity
If intended for special route (other than oral) in humans
Appropriate site (e.g., skin or vaginal mucous membrane) in a suitable species
Preferably use of 2 species
3 dose levels and untreated and / or vehicle Control
If the drug is absorbed systemically, appropriate systemic toxicity studies also required
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Dermal Toxicity
As cutaneous contamination is always a possibility
Rabbit and Rat
Applied on shaved skin
Concentrations 7 fold higher than the clinical doses
Period of application : 7 to 90 days
Evaluation
› Local signs (erythema, oedema and eschar formation)
› Histological examination of sites of application
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Ocular toxicity studies
2 species, including an albino rabbit with a large conjunctival sac
Initial single dose application: To decide exposure concentrations for repeated-
dose studiesRepeated dose study : Duration subject to clinical use (Maximum of 90
days) 2 different concentrations exceeding human dose
In acute studies, one eye kept as control. A separate control group should be included in repeated-dose studies.
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Ocular toxicity studies
Evaluation
Slit-lamp examination
To detect the changes in cornea, iris and aqueous humor.
Fluorescent dyes (sodium fluorescein, 0.25 to 1.0%)
To detect defects in surface epithelium
Intra-ocular tension monitored by a tonometer
Histological examination (fixation in Davidson’s or Zenker’s fluid)
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Vaginal Toxicity Test
Rabbit or Dog Topical application (vaginal mucosa) as pessary,
cream or ointment 6-10 animals per dose group Higher concentrations / several daily applications
(in multiples of daily human dose)7-30 days, as per clinical useObservation parameters :
General : swelling, closure of introitus Histopathology of vaginal wall
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Rectal Tolerance Test
Preparations meant for rectal administration
In rabbits or dogs
6-10 animals per dose group
Volume comparable to human dose (or the maximum possible volume) to achieve administration of multiples of daily human dose
7-30 days, as per clinical use
Observation parameters Clinical signs :- signs of pain, blood and/or mucus in
faeces, condition of anal region/sphincter Gross examination and (if required) Histological examination of rectal mucosa
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Parenteral Drugs
Intravenous/ intramuscular/ subcutaneous/
intradermal inj.
Injection sites in systemic toxicity studies
examined grossly and microscopically
If needed, reversibility of adverse effects may be
determined on a case to case basis
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Inhalation toxicity studies
1 rodent and 1 non-rodent species
Acute, subacute and chronic toxicity studies according to the intended duration of human exposure
Gases and vapors given in whole body exposure chambers; aerosols are given by nose-only method
Dose (limit dose of 5mg/l) in multiples of human exposure
Particle size of 4 micron (especially for aerosols) with not less that 25% being 1 micron
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Inhalation toxicity studies
3 dose groups and a control
Duration of exposure : maximum of 6 hr/day & 5
days/week
Evaluation :
Respiratory rate, BALF examination, histological
examination of respiratory passages and lung
tissue
Regular parameters of systemic toxicity studies or
assessment of margin of safety
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Allergenicity / Hypersensitivity
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Allergenicity / Hypersensitivity
Any one of the standard tests:
Guinea pig :
Maximization test (GPMT)
Mouse :
Local lymph node assay (LLNA)
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Guinea Pig Maximization Test
Challenge(skin reaction
appears)
Induction : Minimum irritant dose (intradermal injection)
Challenge : Maximum nonirritant dose (topical application)
Doses are determined by a preliminary study
Induction(immune response
develops)
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Main test
A minimum of 6 male and 6 female animals per group
One test group One control group One positive control group (preferable)
If no response : re-challenge 7-30 days after primary challenge
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Induction (Day 0)3 pairs of intradermal injections on either shoulders :• 0.1 ml Freund’s adjuvant alone • 0.1 ml test material (lowest irritant dose)• 0.1 ml test material in Freund’s adjuvant
Day 7 : Topical patch at prepared shoulders (lowest irritant dose)
Challenge (Day 21) Topical patch at prepared flanks (highest non-irritant dose) • Left side: Test agent• Right side: Vehicle
Evaluation [Edema and Erythema] after 48 hr.
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Local Lymph Node Assay
Mice of the same sex, either only males or only females
Drug treatment given on ear skin
3 graded doses (the highest being maximum nonirritant dose) plus vehicle control
A minimum of 6 mice per groupTest material applied on ear skin on 3 consecutive
days On day 5, i.v. 3H-thymidine / bromo-deoxy-uridine
(BrdU)Draining auricular lymph nodes dissected after 5
hrsEvaluation : Increase in 3H-thymidine or BrdU
incorporation
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Genotoxicity
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Genotoxic compounds are presumed to be trans-species carcinogens, need not require long-term carcinogenicity studies
If intended for chronic administration, a chronic toxicity study (up to one year) to detect early tumorigenic effects
ICH Standard Tests are generally conducted
• In vitro test for gene mutation in bacteria• In vitro cytogenetic evaluation of chromosomal
damage :• Mammalian cells or• Mouse lymphoma tk assay
• In vivo test for chromosomal damage using rodent hematopoietic cells
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Ames’ Test ( Bacterial Reverse Mutation
Assay )
S. typhimurium tester strains TA98, TA100, TA102, TA1535, TA97 or
Escherichia coli WP2 uvrA or Escherichia coli WP2 uvrA
In-vitro exposure at a minimum of 5 log dose levels
“Solvent” and “positive control”
2.5 fold (or more) increase in number of revertants in comparison to spontaneous revertants are considered positive
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In-vitro cytogenetic assay
Performed in CHO cells or on human lymphocyte in culture
In-vitro exposure using a minimum of 3 log doses
“Solvent” and “positive control”
[Positive control : Cyclophosphamide / Mitomycin C gives a reproducible and detectable increase in clastogenic effect]
> 50% inhibition of cells is considered significant
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Mammalian cell test systems
Division stimulated with phytohemagglutin
Division arrested in metaphase using a spindle inhibitor
Evaluation by light microscopy
Increased number of aberrations in metaphase chromosomes
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In Vitro Mouse Lymphoma TK Assay
Mouse TK lymphoma cells
Thymidine kinase (TK) enzyme
involved in salvage pathway for
incorporation of thymidine into
cells via phosphorylation
Trifluorothymidine (TFT) also
phosphorylated by TKCells containing
TK are sensitive to toxic effects of
TFTForward mutations from TK+ to TK-
result in loss of TK activityQuntifiication of
mutant cells : Cells with TFT
resistance
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In Vivo Tests for Chromosomal Damage
Mammalian
Bone Marrow
Chromosomal
Aberration
Assay
Rodent
Erythrocyte
Micronucleus
Assay
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In-vivo cytogenetic assay
• One rodent species (preferably rat) • Route of administration same as intended for humans • 5 animals/sex/dose groups • 3 dose levels, “solvent” and “positive” control (Cyclophosphamide)
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In-vivo micronucleus assay
• 1 rodent species (preferably mouse) needed • Route of administration of test substance same as
humans• 5 animals / sex / dose groups • At least 3 dose levels, plus “solvent” and “positive”
control • Positive control : mitomycin C or cyclophosphamide
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Carcinogenicity
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Carcinogenicity
For expected clinical use more than 6 months
For drugs used frequently in an intermittent manner
If concern about the carcinogenic potential due to : Previous demonstration in the product class Structure-activity relationship suggests
carcinogenic risk Preneoplastic lesions in repeated dose toxicity
studies Long-term tissue retention results in reactions
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Carcinogenicity
Where life-expectancy in the indicated population is short (i.e., less
than 2 - 3 years) - no long-term carcinogenicity studies
Where therapy is generally successful, there may be later concerns regarding secondary cancers. So carcinogenicity studies needed
A rodent species (preferably rat). Mouse only if justified
Strain should not have normal incidence of spontaneous tumors
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Carcinogenicity
At least 3 dose levels : Highest dose : sub-lethal, should not reduce life
span of animals by more than 10% of expected normal
Lowest dose : should be comparable to the intended human therapeutic dose or a multiple of it, e.g. 2.5x; to make allowance for the sensitivity of the species
Intermediate dose : placed logarithmically between the other 2
Untreated control and (if indicated) a Vehicle control group
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Carcinogenicity
Life span comparable to human’s over which drug use is intended
Generally, 24 months for rats and 18 months for mice
Atleast 50 animals of each sex
Observation parameters
Physiology, Behaviour, Biochemistry, Pathology
Comprehensive descriptions of benign and malignant tumour development, time of their detection, site, dimensions, histological typing etc
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Carcinogenicity
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Requirements of Clinical Trials and Marketing
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Clinical trials & marketing- Requirements
Systemic Toxicity Studies -oral/parenteral/ transdermal routeDuration of proposed human
administration
Human phases for
which study is proposed
to be conducted
Species Long term toxicity
requirement
Upto 1 week I, II, III 2 2 weeks
1-2 weeks ” ” 4 weeks
2-4 weeks ” ” 12 weeks
> 1 month ” ” 24 weeks
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Systemic toxicity studies – Inhalational route
Duration of proposed human
administration
Human phases for which study is proposed to be conducted
Species Long term toxicity
requirments
Upto 2 wk I, II, III 2 1 month (exposure time 3hr/d, 5d/wk)
Upto 4 wk I, II, III 2 3 months (exposure time 6hr/d, 5d/wk)
> 4 wks I, II, III 2 6 months (exposure time 6hr/d, 5d/wk)
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Local Toxicity Studies
Route Duration of
Clinical trial
Phase Species Duration of Toxicity
study
Dermal
Ocular
Otic
Nasal
Vaginal
Rectal
Upto 2 weeks
I, II 1 4 wk
III 2 4 wk
> 2 weeks I,II,III 2 12 wk
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Thank You