CHRONIC TOXICITY STUDIES-A BRIEF OUTLOOK

INTRODUCTION:

• TOXICITY: “Degree ,to which a substance (toxin/poison) can harm humans/animals”

• Toxicity can refer to the effects on:1.Cell (CYTOTOXICITY)2.Organ (Eg: RENAL/LIVER toxicity)3.Whole organism• This explains why scientists use different procedures to assess toxicity

and to provide an estimate of how much of a substance causes a kind of harm

• All substances are potentially toxic, depending on the QUANTITY• Many therapeutic substances can be acutely toxic,but are

beneficial,when used at the appropriate level . Example : Vitamin D , Oxygen,Water (simplest examples)

CONTINUED……………

• A larger quantity of a substance does not automatically imply harm• This is why toxicity determination is primarily focused on DETERMINING

the TYPE and DEGREE of HARM, caused by different amounts of a substance/drug

• Many serious toxic reactions caused by new chemical entities may be detected by ROUTINE TOXICOLOGICAL TESTING

• Experience has shown that predictable “DOSE TIME DEPENDANT” reactions are likely to be revealed in animal experiments

• These tests form the basis of EXPERIMENTAL TOXICOLOGY ,that is applied to every new drug development / unknown substance

• There is NO MEASURE of toxicity, and its effects may occur in short term (acute effects) , / after repeated exposure over a long period (sub-acute,sub-chronic/chronic effects)

CONTINUED…………………….

The following tests are conducted, using rodent models, for toxicity detection:

1. ACUTE TOXICITY TESTS (Single dose)2. SUB-ACUTE TOXICITY TESTS (Daily dose : 14-28 days)3. SUB-CHRONIC TOXICITY TESTS (Daily dose: up to 90 days)4. CHRONIC TOXICITY TESTS (Daily dose: up to 12 months)

CHRONIC TOXICITY TESTING:

Here we cover the following aspects:1.INTRODUCTION2.OECD GUIDELINES FOR TOXICITY3.AIMS OF EXPERIMENT4.BASIC REQUIREMENTS /INITIAL CONSIDERATIONS5.PRINCIPLES OF TEST6.METHOD DESCRIPTIONS7.PROCEDURE8.OBSERVATIONS9.DATA AND REPORTING10.CONCLUSION.

1. INTRODUCTION:

Chronic toxicity test determines toxicity for a substantial portion of a subject’s life According to GHS (GLOBALLY HARMONIZED SYSTEM), it is defined as “ Specific

TARGET ORGAN / SYSTEMIC TOXICITY, arising from a REPEATED EXPOSURE up to 12 MONTHS”

Consists of repeated doses(RDs) ,in ORAL, INHALATION AND DERMAL ADMINISTRATION

End points for RD testing consists of:1.CLINICAL OBSERVATIONS2.BLOOD ANALYSIS3.WHOLE BODY GROSS NECROPSY4.MICROSCOPIC EXAMINATION OF ALL ORGANS AND TISSUES (HISTOPATHOLOGY)

CONTINUED…………….

Data from the aforesaid studies provide information on:1.Cumulative exposure of target organs2.General health hazards, which are likely to occur as a result of repeted low dose exposure to a chemical

2. OECD GUIDELINES FOR TOXICITY:

There are 6 OECD (ORGANIZATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT) TEST GUIDELINES (TG),that describe short-term repeated dose toxicity testing:

1. RD 28 day OT study in Rodents (OECD TG 407)2. RD 90 day OT study in Rodents (OECD TG 408)3. RD Dermal toxicity :21/28 day study (OECD TG 410)4. Sub-chronic Dermal toxicity : 90 day study (OECD TG 411)5. RD inhalation toxicity : 14/28 day study (OECD TG 412)6. Sub-chronic inhalation toxicity : 90 day study (OECD TG 413)

3. AIMS OF THE EXPERIMENT:

Repeat dosing toxicity studies are conducted to:1.Determine side effects ,that may arise from repeated administration of a drug at lower dosages than those used in acute toxicity studies2.Determine safe dosages to be used in initial human clinical trials3.Provide info on major toxic effects, indicate target organs and possibility of accumulation……………….

4. BASIC REQUIREMENTS/INITIAL CONSIDERATIONS:

Info, that assists in selection of the appropriate test concentrations, include:1.IDENTITY, CHEMICAL STRUCTURE and PHYSICOCHEMICAL FEATURES OF TEST ARTICLE2.RESULTS of any IN-VITRO / IN-VIVO TOXICITY TESTS3.AVAILABLE QSAR data and TOXICOLOGICAL data on structurally related substances4.Data, derived from ACUTE INHALATION TOXICITY TESTING

• Dilutions of corrosive / irritating test articles may be tested at concentrations that will yield desired degree of toxicity

• While exposing animals to these materials, the targeted concentrations should be low enough, so that it does not cause marked pain and distress, yet it is sufficient to extend the CRC to levels that reach regulatory and scientific objectives of the test………….

5.PRINCIPLES OF TEST:

For oral/dermal testing:Take test substance

Give dermally/orally in graduated doses, (as per need), to several groups of experimental animals for a period of 12 months

During period of administration/application, animals are observed daily for signs of toxicity

Animals that die during test are NECROPSIED, and the remaining survivors are SACRIFICED and NECROPSIED.

6. METHOD DESCRIPTION:

Includes:A. Selection of animal species and sexB. Housing and feeding conditionsC. Preparation of animalsD. Preparation of doses

A. Selection of animal species and sex: Mammals are selected to be the test animals Animals , with a clearly known ORIGIN, SPECIES and BREED should be used Preferred rodent is rat If other rodents are used, justification should be given for the same Smaller species should be avoided Smaller species can lead to problems, due to increased technical challenges of

dissecting smaller organs Young, healthy adult animals (not exceeding 9 months old) should be used Females should be non-pregnant Weight variation of animals should be minimal (not greater than 20% of mean

weight of each sex)

B. Housing and Feeding conditions:

Animals should be individually identified ,using SUBCUTANEOUS TRANSPONDERS ,to facilitate observations and AVOID CONFUSIONS

All procedures should conform to the local standards of the LABORATORY ANIMAL CARE Temp. in experimental animal room should be in the range of (22-25) degrees Lighting should be artificial,(PHOTOPERIOD of 12 hour darkness and 12 hour light) For feeding, use conventional lab diets, along with an unlimited supply of water Choice of diet depends on the NEED to CO-ADMINISTER a test substance along with it Animals should be group housed in small groups of same sex (individually, if justified) For group caging, use not more than 5 animals per cage.

Terminology: TRANSPONDER : Radio/radar transceiver ,that transmits signals in response to a predetermined signal

C. Preparation of animals:

Healthy animals, who have ACCLIMATIZED to lab conditions for ATLEAST 5 DAYS , and have not been subjected to PREVIOUS EXPERIMENTAL PROCEDURES should be used

Test animals should be characterized as to species, strain, source, sex, weight and / age

Cages should be arranged in such a way that possible effects due to cage placement are decreased ……………

D. Preparation of doses:

Test compound is administered varyingly, depending on the aims of study

Method of oral administration depends on study purpose and physical/ chemical properties of the test material

If necessary, dissolve the test substance in a suitable vehicle Order of choice for test vehicle is: AQUEOUS SOLUTION> EMULSION IN

OIL (Corn oil) > Solution in other vehicles For solution in other vehicles , analyze the toxicity level of the vehicle Determine stability of the test substance ,under conditions of

administrating the same

7. PROCEDURE:A. FOR ORAL TOXICITY STUDIES: Take test animal

administer with test substance daily Route of administration can also be via stomach tube /intubation cannula(other than

normal routes of administration like intraperitoneal ,etc.),in special conditions like “GAVAGE”

Dose can be adjusted as per animal body weight Volume of liquid that is used to administer should not exceed 1 ml/100 g of body

weight (except for aq. Soln. : 2 ml/ 100 g allowed) While administering test substance with food,monitor:i. Homogeneityii. Additive concentrationiii. Safety of test substance after it has been added

Continued…………………….

B. For dermal toxicity studies:- Animals are treated with the test substance for at least 6 hrs./ day Test substance should be applied uniformly over an area approx. 10% of

body weight For highly toxic substances , surface area may be less Area should be covered with a thin and uniform film Between applications , the test substance is held in contact with skin

using a gauze dressing/ non-irritating tape Cover test site in order to retain gauze dressing Ensure that the animal cannot ingest the test substance…………..

8. OBSERVATION:

Observation period should be about 90 days (in sub-chronic test) and about a year (in chronic testing)

Observations can be classified as :A. GENERAL CONDITION AND DEATH RATE:-Made once a day ,at the same time each day-Consider peak levels of anticipated effects after dosing-Record health condition of animals-Observe all animals for MORBIDITY and MORTALITY at least TWICE DAILY

Continued………………

B. WEIGHT,FOOD AND WATER INTAKE ,FOOD INTAKE EFFICIENCY:

-Weigh all animals once a week-Measure rate of food concentrations once a week -If test substance is given via water, measure water consumption weekly

C. HAEMATOLOGICAL TESTS:-Collect blood samples(at the end of test period,/ prior to killing animals, or as a part of it),under appropriate conditions-Observe for the following values:

Continued………………

i. Hematocritii. Hb conc.iii. Erythrocyte countiv. TLC v. DLCvi. Platelet countvii. Measure of blood clotting time/potential

Continued……………

D. BLOOD BIOCHEMISTRY TEST:Used to investigate:a. Toxic effects in tissuesb. Toxic effects in organs-Test is performed on basis of blood samples obtained from each animal prior to /as part of procedure-Observe for the following serum values:i. Sodiumii. Potassiumiii. Glucoseiv. TCv. Ureavi. BUNvii. Creatinineviii. Total protein and albumin

Continued…………………

ix. More than 2 enzymes that indicate hepatocellular effects (AST, ALT,ALP,Sorbitol dehydrogenase)

E. URINE TEST:-Timely urine vol. collection-Check for urine:i. Appearanceii. Vol.iii. Specific gravityiv. Ph.v. Proteinvi. Glucose and blood cells

Continued……………………….

F. PATHOLOGY TESTS:

-Involves :i. G/Eii. M/E Subject all animals in study to a full, detailed, gross necropsy, that

includes careful examination of:a. Ext. surface of bodyb. All orificesc. Cranial, thoracic and abd. Cavities, and their contents

Continued………………..

-Remove adherent tissue from liver, heart, kidney, uterus, testis, epididymis, ovaries, adrenals etc. Determine wet weight after dissection to avoid drying Conduct full histopathology on preserved organs and tissues of all

animals………………

9. DATA AND REPORTING:

A. DATA:-Provide individual statistical data in tabular form, that shows:i. No. of test animals usedii. No. of test animals found dead during test/euthanized for ethical

reasonsiii. No. showing signs of toxicityiv. Signs of toxicityv. Time of onset, duration and severity of toxicityvi. No. of animals showing lesions, their types and severity

Continued…………

B. TEST REPORT:-Test report must include the following info:i. Test substanceii. Vehicle (if appropriate)iii. Test animalsiv. Test conditions : * Rationale for dose level selection *Details of test substance formulation / diet preparation ,achieved conc. , stability and preparation homogeneity *Details of administration of test substance *Actual doses (mg/kg body weight /day ) and conversion factor from diet/ drinking water*Details of food and water quality

Continued…………………v. Results: *Body wt. *Body wt. changes *Food consumption and water concentration *Toxic response data by sex and dose levels , including toxicity signs *Nature, severity and duration of clinical observations (reversible / not) *Ophthalmic exam. Results *Sensory and motor activities *Terminal body wt. ,organ wt. and organ body weight ratios *Necropsy findings *Detailed description of all pathologic findings *Absorption data, if available *Statistical results presentation

vi. Result discussionvii. Conclusion

10. CONCLUSION:

Chemical substance-given to subject-series of interactions and dose related responses-most cases are desired and useful, and in some cases not useful and harmful

There are 4 types of toxicity in general Acute toxicity is involved in LD50 estimation, causing death to 50% of

tested groups of animals Sub-acute, sub-chronic and chronic toxicity studies are used to evaluate:A. Characteristic toxic effects of drugs upon administration (RD) ,for

periods of time, ranging from 2 weeks- 1 yearB. No toxic effect dosage levels for short to long term repeat

dosing………………….

11. REFERENCE/SUBSTANTIAL BIBLIOGRAPHY:DETERMINATION OF DRUG TOXICITY (EXPERIMENTAL MODELS IN RODENTS) , BY:BOGDAN TAMBAGEORGE FOLTEAMAGDA LEONRADU ILIESCU

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