pravin fianl

Dr. Pravinkumar WahaneJR II

BJGMC, Pune

Toxicity Studies

Source, Synthesis & chemistry

Preclinical Studies & Toxicology Studies

Animal models, Efficacy and Safety

Clinical Trials

Drug Development

Preclinical Safety & Toxicity Testing

Aims at discovering complications or sequelae arising from the pharmacological actions of drug and any unexpected side-effects.

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

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

Requirements

Chemical Criteria

Substance used in toxicity studies should be as pure as material to be given to man

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

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

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

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

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

Toxicity Studies

Systemic Toxicity studies

Reproductive toxicology studies

Local Toxicity studies

Allergenicity/ Hypersensetivity studies

Genotoxicity studies

Carcinogenicity studies

Systemic Toxicity Studies

Single dose (Acute)

Short term repeated dose (Subacute)

Repeated dose: 10% life-span (Subchronic)

Repeated dose: >10% life span (Chronic)

Systemic Toxicity Studies

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


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


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]

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)

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)

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

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

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

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

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

• Rodent : 6-10/sex/group• Non-rodent : 2-3/sex/group

14-28 Day

repeated-dose

(Subacute toxicity

studies)


90-Day repeated-

dose (Subchronic

toxicity studies)


180-Day repeated-dose

(9 months for non rodents?)

(Chronic toxicity studies)

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

Reproductive Toxicology

Reproductive Toxicity

Male Fertility

Female Reproduction & Developmental toxicity

Female Fertility

Teratogenicity

Perinatal development

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

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

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

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

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

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

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)

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

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

Local Toxicity

Local Toxicity

Dermal Toxicity

Ocular Toxicity

Vaginal Toxicity

Rectal tolerance test

Parenteral Drugs Tolerance

Inhalation toxicity

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

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

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.

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)

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

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

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

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

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

Allergenicity / Hypersensitivity

Allergenicity / Hypersensitivity

Any one of the standard tests:

Guinea pig :

Maximization test (GPMT)

Mouse :

Local lymph node assay (LLNA)

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)

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

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.

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

Genotoxicity

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

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

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

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

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

In Vivo Tests for Chromosomal Damage

Mammalian

Bone Marrow

Chromosomal

Aberration

Assay

Rodent

Erythrocyte

Micronucleus

Assay

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)

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

Carcinogenicity

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

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

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

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

Carcinogenicity

Requirements of Clinical Trials and Marketing

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

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)

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

Thank You

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