ANIMAL TOXICITY TEST FOR ACUTE, SUBACUTE & CHRONIC TOXICITY.

PRESENTED BY

SINDHU K

MVSC SCHOLAR,

DEPT OF VPT, COVAS.

TOXICITY STUDIES - INTRODUCTION• Toxicology classically has been defined as the study of poisons & concerned with the

adverse effects of xenobiotics.

• Casarett 1996 defined it as a science that defines the limits of safety of chemical agents for human & animal populations.

• Toxicological screening is very important for the development of new drugs and for the extension of the therapeutic potential of existing molecules.

• The US-FDA states that it is essential to screen new molecules for pharmacological activity and toxicity potential in animals (21CFR Part 314).

• Toxicity tests are mostly used to examine specific adverse events or specific end points such as cancer, cardiotoxicity, and skin/eye irritation.

• Toxicity testing also helps calculate the No Observed Adverse Effect Level (NOAEL) dose and is helpful for clinical trails.

HISTORY OF TOXICITY STUDIES

• Paracelsus (Father of Toxicology): determined specific chemicals responsible for the toxicity of plants and animals (dose-response relationship).

• "All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy”

•Paracelsus• Mathieu Orfila, determined the relationship between poisons and their

biological He is referred to as the father of modern toxicology.

SOURCES OF TOXIC SUBSTANCES

• Classified based on their

• chemical nature

• mode of action

• class (exposure class and use class)

• Exposure class: Food, air, water or soil.

• Use class: drugs as drug of abuse, therapeutic drugs, agriculture chemicals, food additives, pesticides, plant toxins and cosmetics

EVIDENCES

The use of sheep brain for the production of rabies vaccine has been phased out in 1992

Classical swine fever virus vaccine is produced in PK-15 Cells instead using large number of rabits

PPR, FMD and sheep pox vaccines are also produced using cell culture vero, BHK-21 and vero cell culture system respectively.

The use of laboratory animals e.g., rabbit and G. pig has been now abandoned for the isolation or typing of mycobacetria with the availability of improved synthetic media.

BIOMEDICAL ETHICS

• Before conducting any toxicological testing in animals or collecting tissue/cell lines from animals, the study should be approved by the Institute Animal Ethics Committee (IAEC) or the protocol should satisfy the guidelines of the local governing body.

NECESSITIES OF TOXICOLOGICAL STUDIESBenefit –risk ratio can be calculated

Prediction of therapeutic index

Therapeutic index= Maximum tolerated dose Minimum curative dose

Smaller ratio, better safety of the drug.

MAGNITUDE OF USE OF ANIMALS

It has been estimated that approximately 20 million animals are being used for testing and are killed annually; about 15 million of them are used to test for medication and five million for products.

China has become one of the biggest countries using lab animals, as is evident in the higher numbers and quality of lab animals (e.g., specific pathogen free, or SPF1; genetically modified) increasingly used in scientific research—16 million a year, compared to 12 million in the 25 European Union countries in 2005 (FELASA 2007)—and the increased publication of animal experiment results in international journals.

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Japan was second only to the US, which used 17.3 million animals in 2005. The third largest use in 2005 was Great Britain, which used 1.9 million animals.

People for the ethical treatment of animals (PETA) reported that the National Centre for Laboratory Animal Sciences (NCLAS) in Hyderabad, supplies approximately 50,000 animals to laboratories every year and to 175 institutions in India, including pharmaceutical companies and educational institutions.

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In India, among rodentia group of animals e.g. mice. rat, G.pig, rabbit, mice is predominantly used in most of cases followed by other rodentia.

The number of monkeys used in research has now drastically reduced in institutions.

Use of some other unique animals like hamster, cotton rat, gerbil is not much common.

There are inbred strains of mice which are used for research and genetically modified laboratory animals are also imported into India by some of institutes.

The information with regard to available laboratory animals resource in different institutions/universities in India is not precisely known.

Standard Methods

Multiple methods have been standardized (certified) by multiple organizations American Society for Testing and Materials (ASTM) Organization for Economic Cooperation and Materials (OECD)

– (Europe based) National Toxicology Program (NTP)

All above standardized protocols available from US EPA, Federal Register and researchers that developed the programs

REGULATORY MECHANISMS IN INDIA

• Institute Animal Ethics Committee (IAEC) • Committee for the Purpose of Control and Supervision of Experiments in

Animals (CPCSEA) • Drugs & Cosmetics Act, 1940, Appendix-I • Department of Animal Husbandry, Dairying & Fisheries, Ministry of

Agriculture, New Delhi-2001 • Department of Biotechnology.• The Prevention of Cruelty to Animals Act, 1960 as amended up to 30th

July 1982 and Animal Welfare Board

RELEVANT TEST MODELS

Pharmacokinetic profilePharmacodynamic responseSpecies, sex, age of experimental animals Susceptibility, sensitivity and reproducibility of test system

In vitro: Isolated organs, tissues cell-cultures

Mechanism of effect in vivo

In vivo toxicological models

IN-VIVO >< IN-VITRO

1.In Vivo Studies In vivo safety pharmacology studies should be designed to define the

dose-response relationship of the adverse effect observed The time course of the adverse effect should be investigated e.g. onset and duration of response

2.In Vitro studies In vitro studies should be designed to establish a concentration-effect

relationship

TYPES OF TOXICITY STUDIES

• Single dose studies/ Acute toxicity studies• Repeated dose studies / sub-acute or Chronic studies• Local toxicity studies• Allergenicity / Hypersensitivity toxicology studies• Genotoxicity studies• Carcinogenicity / Oncogenicity studies

A. SYSTEMIC TOXICOLOGY STUDIES

Preliminary Definitive

• Maximum Non Lethal dose(MNLD) determined

• MTD and MLD determined• Evaluate effects • Target organ of toxicity may be determined

a) SINGLE DOSE STUDIES/ ACUTE TOXICITY

,• Acute toxicity testing- study the effect of a single dose on a particular animal species.

• Acute toxicity testing be carried out with two different animal species (one rodent and one non-rodent).

• In acute toxicological testing, the investigational product is administered at different dose levels, and the effect is observed for 14 days. All mortalities caused by the investigational product during the experimental period are recorded and morphological, biochemical, pathological, and histological changes in the dead animals are investigated.

,• The LD50 was used as an indicator of acute toxicity previously. The

determination of the LD50 involves large numbers of animals, and the mortality ratio is high. (24 h testing)• Graphical method • Arithmetical method (karbers’s) method. when number of animal is

small)

Because of these limitations, modified methods were developed:

1. The fixed dose procedure

2. The acute toxic category method

3. The up-and-down method

Test report

The test report should include the following information:

• Test substance:

• Physical nature,

• Purity

• Physicochemical properties;

• Identification data;

• Source of substance;

• Batch number

CONTD

Vehicle (if appropriate):

• Justification for choice of vehicle (if other than water).

Test animals:

• Species/strain used and justification for choice made;

• Number, age, and sex of animals at start of test;

• Source, housing conditions, diet, etc.;

• Individual weights of animals at the start of the test.

OECD

Organization for Economic Cooperation and Development (OECD) Test Guidelines (TGs 402, 403, 420, 423, and 425) describe acute systemic testing.

• Fixed Dose Procedure (OECD TG 420)

• Acute Toxic Class method (OECD TG 423)

• Up‐and‐Down Procedure (OECD TG 425)

• Acute Dermal Toxicity (OECD TG 402)

• Acute inhalation toxicity (OECD TG 403)

Six OECD Test Guidelines describe short‐term repeat‐dose toxicity testing

• Repeated Dose 28‐day Oral Toxicity Study in Rodents (TG407)

• Repeated Dose 90‐Day Oral Toxicity Study in Rodents (TG 408)

• Repeated Dose Dermal Toxicity: 21/28‐day Study (TG 410)

• Sub-chronic Dermal Toxicity: 90‐day Study (TG 411)

• Repeated Dose Inhalation Toxicity: 28‐day or 14‐day Study (TG 412)

• Sub-chronic Inhalation Toxicity: 90‐day Study (TG 413)

Two mammalian species(one should be non-rodent)Long duration studies (30-180 days)Dose is dependent on dose-escalating studies Drug administered by clinical routeParameters monitored and recorded are:

Behavioral Physiological Biochemical Microscopic observations

b) REPEATED DOSE STUDIES/SUB-ACUTE OR CHRONIC TOXICITY

.• Rodents and non-rodents are used to study the sub-chronic toxicity of a substance.

• Dose: Expected therapeutic level (daily) or expected therapeutic level to increasing dose phase-wise manner.

• The test substance is administered orally for =/>90 days, and regular body weight variations, biochemical and cardiovascular parameters changes, and behavioral changes are observed.

• At the end of the study, the experimental animals are sacrificed. Gross pathological changes are observed, and all the tissues are subjected to histopathological analyses.

• There should be little individual variation between the animals, and the allowed weight variation range is ±20%.

• Used to determine the maximum tolerable dose and nature of toxicity.

.Chronic toxicity studies are conducted with a minimum of one rodent and one non-rodent species. • The test compound is administered over more than 90 days, and the animals

are observed periodically. • A chronic toxicology study provides inferences about the long-term effect of

a test substance in animals, and it may be extrapolated to the human safety of the test substance.

• The report on chronic oral toxicity is essential for new drug entities. There should be little individual variation between the animals, and the allowable weight variation range is ±20%.

DOSE

• High dose: Produce significant retardation of growth or some pathological changes (10 times the expected maximum clinical dose).

• The low dose is about twice the expected maximum clinical dose• Third dose is medium dose fixed midway between the high and

low dose

.• During the study period, the animals are observed for normal physiological functions, behavioral variations and alterations in biochemical parameters at regular intervals (atleast every 14 days).

• At the end of the study, tissues are collected from all parts of the animal and subjected to histological analyses.

TYPES OF LOCAL TOXICITY STUDIES

Dermal toxicity studies

Dermal photo-toxicity studies

Vaginal toxicity studies

Rectal tolerance studies

Rats & RabbitLocal signs (erythema, oedema)histological examination

Guinea pigUsed in treatment of leucodermaExamination of erythema & oedema formation

Rabbit or DogObservation of swelling,histopathology of vaginal wall

Rabbit or DogSigns of pain, blood or mucoushistology examination of rectal mucosa

Ocular toxicity studies

Parenteral drugs

Inhalation toxicity studies

Albino RabbitChanges in cornea ,Iris & aqueous humor, histological examination of eye

For intravenous/ intramuscular/ subcutaneous/ intra-dermal injectionSites of injection examined grossly and microscopically

One rodent and non rodent speciesAcute , sub-acute and chronic studies performedObservation of respiratory rateHistological examination of respiratory passages, lung tissue

D. ALLERGENICITY/HYPERSENSITIVITY TOXICOLOGY STUDIES

Guinea Pig Maximization test

Local lymph node assay

Determination of Maximum non irritant or minimum irritant doseEvaluation of Erythema and oedema

Mice of one sex(either male or female)Drug treatment given on ear skinAuricular lymph node dissection after 5 daysIncrease in 3h-thymidine used for evaluation

E. GENOTOXICITY STUDIESTo detect early tumorigenic effects in cases of chronic illness

In vitro tests:Test for gene mutation in BacteriaCytogenetic evaluation of chromosomal damage in mammalian cells

E.g.; Ames’s Salmonella Assay detects increased number of aberrations in metaphase chromosomes

DNA strand breaks, DNA repair or recombination, Measurements of DNA adducts

IN VIVO TESTS

In vivo test for chromosomal damage using mammalian hematopoietic cells.

Chromosome damage in rodent hematopoietic cellsE.g.; Micronucleus Assay

F. CARCINOGENICITY/ ONCOGENICITY STUDIES

life-time bioassays

carcinogenicity studies are performed on:

drug used for >6 months or frequent intermittent use for chronic diseases

chemical structure of drug indicates carcinogenic potential

therapeutic class of drugs which have produced positive carcinogenicity

NOTE ON STEM CELLS

STEM CELLS NOW EMERGING AS AN ALTERNATIVE TO LABORATORY ANIMALS Drug company interest in stem cell drug testing was demonstrated in July 2008, when GlaxoSmithKline entered into a $25 million-plus agreement with the Harvard Stem Cell Institute.

By testing drugs on specific cells and tissues created from iPS cells, we can even predict a patients individual response to a treatment realizing the vision of personalized medicine.

In the current issue of Stem Cells and Development (2007), Cezar and her colleagues revealed a novel way to test drug toxicity: by monitoring the behaviour of embryonic stem cells exposed to a drug-candidate compound.

Studying how potential drugs affect embryonic stem cells could provide a far more accurate prediction of a drug's potential toxicity than conventional animal models can.

Currently, the most successful development of stem cells as in vitro models for toxicology testing is in human cardiac tissue.

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OECD GUIDELINES FOR ACUTE, SUB-CHRONIC & DERMAL TOXICITY STUDIES

ACUTE• 401 ~ Acute Oral Toxicity• 402 ~ Acute Dermal Toxicity

SUB CHRONIC

• 407 ~ Repeated Dose Oral Toxicity ( Rodents : 28 days / 14 days study ) • 408 ~ Sub-chronic Oral Toxicity Test ( rodents : 90 days study )• 409 ~ Sub-chronic Oral Toxicity Test ( Non-rodents : 90 days study )• 410 ~ Repeated Dose Dermal Toxicity ( 21/28 days study )• 411 ~ Sub-chronic Dermal Toxicity (90 days study )

CHRONIC

• 452 ~ Chronic Toxicity Studies• 453 ~ Combined Chronic Toxicity/Carcinogenicity studies

DEFINITIONS RELATIVE TO DOSES

ACUTE STUDIES ED 50 : The median effective dose = the dose for which half(50%) of the animals exhibit an effect (E) and half of animals exhibit no effectLD 50 : The median lethal dose = the effect may be defined as specific toxic event (tremors) & sometimes defined as lethalityED10 & ED90 are doses at which 10% & 90% of the animals respectively demonstrate effect

GENERAL STUDIES

• LIMIT DOSE : a dose which is considered high enough that if no mortality / significant toxicity is seen in animals receiving this dose, no higher doses are required

eg : Limit doses(EPA/OECD)

1.Acute Oral Toxicity 5000mg/kg

2.Acute Dermal Toxicity 2000mg/kg

3.21-days Dermal Toxicity 1000mg/kg

4.Chronic Studies of Pesticides 1000mg/kg

Note: the limit dose of a non-nutritive material added to diet is generally considered to be 5% (50,000ppm)

SUB-CHRONIC & CHRONIC STUDIES

• ADI: Acceptable Daily Intake (established for food additives/ residues & published by EPA)• NOEL: No Observed Effect Level. Dose at which no effect is

seen• NOAEL: No Observed Adverse Effect Level. Dose at which

no adverse/toxic effect is seen

CHRONIC STUDIES

• MTD: Maximum Tolerated Dose. Is the highest dose that can be tolerated without significant lethality from causes other than tumors.

(for EPA studies MTD for chronic studies with pesticides is a dose which produces an approximate 10-15% decrement in body weight gain)

• HTD: Highest dose tested. Highest dose that can be expected to yield results relevant to humans. This is a proposed new dose which would be selected based on evaluation of results of sub-chronic studies.

VEHICLES USED FOR DOSING

• ORAL: Water, Methylcellulose or Carboxymethylcellulose(0.5-5% aqueous suspension), Oil( corn, peanut, sesame)• DERMAL: Physiological saline, Water, Ethanol, Acetone, Mineral

oil• PARENTERAL: Physiological saline(sterile), sterile water for

injection

CLINICAL SIGNS OF TOXICITY

RESPIRATORY: blockage in the nostrils, changes in rate & depth of breathing, changes in color of body surfaces• Signs like dyspnea, abdominal breathing, gasping, Apnea, Cyanosis,

Tachypnea, nostril discharge = involvement of CNS respiratory center, pulmonary edema, cholinergic inhibition, pulmonary cardiac insufficiency.

MOTOR ACTIVITY

Changes in frequency & nature of movements

Signs like decrease/increase in spontaneous motor activity, Somnolence, Loss of righting reflex, Anesthesia, Catalepsy, Ataxia, Unusual locomotion, prostrations, Tremors & fasiculations = CNS, sensory, autonomic & neuromuscular system

CONVULSIONS

Marked involuntary contractions or seizures of contraction of voluntary muscles

Signs like Clonic convulsions, Tonic convulsions, Tonic-Clonic convulsions, Asphyxial convulsion, Ophisthotonos = CNS, respiratory failure, neuromusculsr & autonomic systems.

REFLEXES

• CORNEAL• PINNAL• RIGHTING• MYOTACT• LIGHT(pupillary)• STARTLE REFLEX

All signs indicates involvement of CNS, sensory, autonomic & neuromuscular systems

OCULAR SIGNS

• Lacrimation, Miosis, Mydriasis, Exophthalmos, Ptosis, Chromodacryorrhea ( red lacrimation ), relaxation of nictating membrane, corneal opacity, iritis, conjunctivitis.

Mainly due to autonomic system involement

CARDIO-VASCULAR SIGNS

• Bradycardia, tachycardia, vasodilation, vasoconstriction, arrhythmias

• Due to autonomic, CNS, cardiac-pulmonary insufficiency, myocardiac infarction, cold environment.

OTHER SIGNS

• Salivation• Piloerection• Analgesia• Muscle tone - hypotonia

- hypertonia• GIT signs: dropping feces, emesis, diuresis(hematuria &

involuntary urination)

AUTONOMIC SIGNS

• Sympatomimetic – piloerection, partial mydriasis.• Sympathetic block – ptosis, diagnostic if associated with sedation.• Parasympathomimetic – salivation, miosis, diarrhoea.• Parasympathomimetic block – mydriasis (maximum), excessive

dryness of mouth.

TOXIC SIGNS OF ACETYLCHOLINESTERASE INHIBITIONMUSCARINIC EFFECTS NICOTINIC EFFECTS CNS EFFECTSBronchoconstriction,Increased bronchosecreation.

Muscular twitching Giddiness, anxiety, headache

Nausia & Vomiting Fasciculation Apathy , confusionDiarrhea Cramping Depression, drowsiness,

seizures,

Bradycardia Muscular weakness AtaxiaHypotension Respiratory depressionMiosis confusionsUrinary incontenance coma

EFFECTS OF DECREASED BODY WEIGHTS ON RELATIVE ORGAN WEIGHTS OF RATS

• Decreases = liver• No change = heart, kidneys, prostrate, spleen, ovaries.• Increases =adrenal gland, brain, epididymides, pituitary,

testes, thyroid, uterus.

Human studies

1. General considerations for clinical studies 2. Specific considerations for clinical studies a. Protocol designb. The study populationc. Statistical analyses3. Sequence of clinical studiesd. Early clinical studiese. Further clinical studies4. Submitting reports of clinical studies to CFSAN (Center for Food

Safety and Applied Nutrition)

PHASES OF DRUG DEVELOPMENT

(ANIMAL MAN)

PHASE III PHASE IVPHASE IPHASE IPHASE IPRECLINICALPRECLINICAL PHASE II

Product Approval (NDA/MAA)

Patient studies

Entry to man(IND / CTA)

NoneNone

Healthy subjects

Safety andtolerability

Healthy subjects

Safety andtolerability

Genetic toxicity(in vivo)

Repeat dose toxicity testing

+Bioanalysis /

Toxicokinetics

Drug Metabolism

Reproductive Toxicity Testing(teratogenicity)

Genetic toxicity(in vivo)

Repeat dose toxicity testing

+Bioanalysis /

Toxicokinetics

Drug Metabolism

Reproductive Toxicity Testing(teratogenicity)

Patients

Small scale efficacy studies

Patients

Small scale efficacy studies

Patients

Large scalemulticentre

studies

Patients

Large scalemulticentre

studies

Chronic (long term) toxicity testing+

Bioanalysis / Toxicokinetics

Reproductive Toxicity Testing (fertility and pre/post natal)

Carcinogenicity studies

Drug Metabolism

Chronic (long term) toxicity testing+

Bioanalysis / Toxicokinetics

Reproductive Toxicity Testing (fertility and pre/post natal)

Carcinogenicity studies

Drug Metabolism

Patients

Large scalepost-marketing

studies

Patients

Large scalepost-marketing

studies

As requiredAs required

Genetic toxicity(in vitro)

Single / repeat dose

toxicity studies+

Bioanalysis / Toxicokinetics

Safety Pharmacology

Drug Metabolism

Lead candidateIdentified

Clin

ical

Non

-clin

ical

MOLECULE

DISCUSSION SESSION

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REFERENCES

• CRC hand book of toxicology

• Google images & Wikipedia

• Online Related materials from slideshare

THANK YOU