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1. CHRONIC TOXICITY
STUDIES-A BRIEF OUTLOOK

2. 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)

3. 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)

4. 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)

5. CHRONIC TOXICITY TESTING:
 Here we cover the following aspects:
1.INTRODUCTION
2.OECD GUIDELINES FOR TOXICITY
3.AIMS OF EXPERIMENT
4.BASIC REQUIREMENTS /INITIAL CONSIDERATIONS
5.PRINCIPLES OF TEST
6.METHOD DESCRIPTIONS
7.PROCEDURE
8.OBSERVATIONS
9.DATA AND REPORTING
10.CONCLUSION.

6. 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 OBSERVATIONS
2.BLOOD ANALYSIS
3.WHOLE BODY GROSS NECROPSY
4.MICROSCOPIC EXAMINATION OF ALL ORGANS AND TISSUES (HISTOPATHOLOGY)

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

8. 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)

9. 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 studies
2.Determine safe dosages to be used in initial human clinical trials
3.Provide info on major toxic effects, indicate target organs and possibility of
accumulation……………….

10. 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
ARTICLE
2.RESULTS of any IN-VITRO / IN-VIVO TOXICITY TESTS
3.AVAILABLE QSAR data and TOXICOLOGICAL data on structurally related substances
4.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………….

11. 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.

12. 6. METHOD DESCRIPTION:
 Includes:
A. Selection of animal species and sex
B. Housing and feeding conditions
C. Preparation of animals
D. Preparation of doses

13. 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)

14. 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

15. 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 ……………

16. 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

17. 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. Homogeneity
ii. Additive concentration
iii. Safety of test substance after it has been added

18. 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…………..

19. 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

20. 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:

21. Continued………………
i. Hematocrit
ii. Hb conc.
iii. Erythrocyte count
iv. TLC
v. DLC
vi. Platelet count
vii. Measure of blood clotting time/potential

22. Continued……………
D. BLOOD BIOCHEMISTRY TEST:
Used to investigate:
a. Toxic effects in tissues
b. 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. Sodium
ii. Potassium
iii. Glucose
iv. TC
v. Urea
vi. BUN
vii. Creatinine
viii. Total protein and albumin

23. 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. Appearance
ii. Vol.
iii. Specific gravity
iv. Ph.
v. Protein
vi. Glucose and blood cells

24. Continued……………………….
F. PATHOLOGY TESTS:
-Involves :
i. G/E
ii. M/E
- Subject all animals in study to a full, detailed, gross necropsy, that includes careful
examination of:
a. Ext. surface of body
b. All orifices
c. Cranial, thoracic and abd. Cavities, and their contents

25. 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………………

26. 9. DATA AND REPORTING:
A. DATA:
-Provide individual statistical data in tabular form, that shows:
i. No. of test animals used
ii. No. of test animals found dead during test/euthanized for ethical reasons
iii. No. showing signs of toxicity
iv. Signs of toxicity
v. Time of onset, duration and severity of toxicity
vi. No. of animals showing lesions, their types and severity

27. Continued…………
B. TEST REPORT:
-Test report must include the following info:
i. Test substance
ii. Vehicle (if appropriate)
iii. Test animals
iv. 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

28. 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 discussion
vii. Conclusion

29. 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 year
B. No toxic effect dosage levels for short to long term repeat dosing………………….

30. 11. REFERENCE/SUBSTANTIAL
BIBLIOGRAPHY:
DETERMINATION OF DRUG TOXICITY (EXPERIMENTAL MODELS IN RODENTS) , BY:
BOGDAN TAMBA
GEORGE FOLTEA
MAGDA LEON
RADU ILIESCU

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