toxicity studies

1. W
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L
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2. TOXICITY STUDIES
Dr. K.J.KARTHIKA
FINAL YEAR P.G SCHOLAR5
DEPT. OF RASASHASTRA AND BHAISHAJYAKALPANA
GAVC, TRIPUNITHURA

3. INTRODUCTION
•Toxicology is classically defined as the study of poisons.
•It is concerned mainly with the study of adverse effects of
xenobiotics.
•Casarett 1996 defined it as a science that defines the limits of safety
of chemical agents for human and animal population.

4. HISTORY OF TOXICITY STUDY
•Paracelsus( father of toxicology) determined specific chemicals
responsible for the toxicity of plants and animals.
•“ All substances are poison ,there is none which is not a poison.
The right dose differentiates a poison and a remedy.”- Paracelsus
( dose response relation ship).
•Mathieu Orfila ( father of modern toxicology) Determined
relationship between poison and their biological effects.

5. NECESSITIES
•Toxicological screening is very important for the development of new
drugs and extension of the therapeutic potential of existing molecules.
•Benefit risk ratio can be calculated.
•Prediction of therapeutic index/ safety window (median lethal dose/
median effective dose)

6. TYPES OF TOXICOLOGY STUDIES
•Systemic toxicity studies
- Single dose study (Acute)
- Repeated dose study( Sub acute and chronic)
•Reproductive toxicity studies
- Male fertility
- Female reproduction and Developmental studies
•Local toxicity studies
•Hypersensitivity studies
•Genotoxicity studies
•Carcinogenicity studies

7. Animals used for toxicity studies
•Rats and mice are generally used but for certain classes of
chemicals, such as agrochemicals and pharmaceuticals, the tests
may also be conducted in non-rodent animals such as the beagle
dogs, pigs, marmosets or macaques .
•Some regulatory agencies require that at least two species be used,
one of them to be selected from rodents and the other from non-
rodents.
•In at least one of the species, males and females should be used.
•In the case of rodents, each group should consist of at least five
animals per sex. In the case of non-rodents, each group
should consist of at least two animals per sex.

8. HOUSING AND FEEDING CONDITIONS.
• The temperature in the experimental animal room should be 22ºC
(+ 3ºC).
•The relative humidity should be at least 30% and preferably not
exceed 70% other than during room cleaning the aim should be 50-
60%.
•Lighting should be artificial, the sequence being 12 hours light, 12
hours dark.
•For feeding, conventional laboratory diets may be used with an
unlimited supply of drinking water.
•Animals may be group-caged by dose, but the number of animals
per cage must not interfere with clear observations of each animal.

9. Route of administration:
•Ordinarily, the oral route is sufficient as this is the normal route of
clinical administration. However, some regulatory agencies
suggest in addition a parenteral route of administration.
•In cases where it is proposed to administer the herbal preparation to
a human subject by the parenteral route, it may be sufficient to use
this route alone for animal testing.

10. Biomedical ethics
Before conducting toxicity studies approval form
Institutional Animal Ethics Committee(IAEC) is essential.

11. Guidelines for toxicity / safety profile
evaluation
Of
Ayurveda & Siddha plant drugs(CCRAS)

12. Acute Toxicity Test evaluation:
Acute toxicity refers to those adverse effects occurring following oral
or dermal administration of a single dose of a substance, or multiple
doses given within 24 hours, or an inhalation exposure of 4 hours.
AIM
1. Determination of Therapeutic Index and LD50.
2. Define Intrinsic toxicity of a Chemical.
3. Provide information for the design and dose selection for
prolonged studies.
4. Provide valuable information to the clinician for the prediction,
diagnosis and treatment for acute over dose of chemicals.
5. Classification and labeling of chemicals for regulatory purposes.

13. Sl.No. Test Groups No. of Animals
1. VC
(Control)
10
(5M + 5F)
2. Therapeutic Dose
(TD)
10
(5M + 5F)
3. Average Dose
(TD x 5)
10
(5M + 5F)
4. Highest Dose
(TD x 10)
10
(5M + 5F)
Test dose : Single dose
Route of administration : Oral
Duration : 10 days

14. OBSERVATIONS:
a) Mortality : To be observed on 24, 48 & 72 hours
b) Clinical signs: Monitoring of convulsions, lethargy, sleep, coma,
salivation, diarrhoea
•Cage side examination (daily)
•Skin colour, fur, eyes & mucous membrane (daily)
•Spontaneous and voluntary motor activity on every ½ hour, 1 hour, 2
hours, 4 hours and 24 hours after drug administration.
•0,1,7th day spontaneous motor activity
•Necropsy - In case of animal dies

15. SUB ACUTE TOXICITY
•This study is conducted to determine organs affected by different
dose levels.
•Three dose levels are normally used.
-High dose (elicit definite signs of toxicity but not to kill many of the
animals)
- Low dose (induce no toxic effect.)
-Intermediate dose.
• Doses are generally selected on the basis of information obtained in
acute toxicity studies using both LD50 and the slope of the dose
response curve.
•The duration of sub acute toxicity studies depend on intended
duration of the test substance.

16. Species Animals No. Age
(wks)
Weight
(gms)
Duration of experiment
Male Female TC
Exp.
Imm.
Exp.
Post
Exp.
Mice
(Swiss)
24
6x4*
24
6x4*
4-6 18-20 15 days
oral
50%
15th
day
50%
30th
day
Rat
Wistar
24
6x4*
24
6x4*
4-6 60-90 15 days
oral
50%
15th
day
50%
30th
day
TC-Test compound exposure
Imm Exp – Immediate (48 hours) after last exposure – instant effect
Post exp – Post-exposure (15 days) after last exposure – reversibility
of toxicity if any

17. Sl.No. Test Groups No. of Animals
1. VC
(Control)
12
(6M + 6F)
2. Therapeutic Dose
(TD)
12
(6M + 6F)
3. Average Dose
(TD x 5)
12
(6M + 6F)
4. Highest Dose
(TD x 10)
12
(6M + 6F)
TEST GROUPS
Route of Administration: Oral
Duration: 30 days

18. Pre-experimentation phase
I. Acclimatization of animals
•Period – 7 days (Recording of body weight and food intake
twice in a week)
•Urine qualitative test (Ames multiple sticks)
•Fecal consistency (Filter paper technique)

19. Experimentation phase
I)Test compound exposure multiple dose (once daily for 15 days) and
dosage schedule (as recommended by sponsor)
II)Mortality 6/12/24 hours
III)Body weight (Twice in a week)
IV)Food consumption (Daily)
V)Fecal consistency
VI)Cage side activity
VII)Neurological examination
VIII)Urine qualitative test

20. IX) Haematology - Twice (15th day/30th day) (Hb, RBC, WBC,
Platelet count, differential count)
X) Clinical chemistry – Twice (15th day/30 day) (Blood glucose,
total protein, serum Creatinine, SGOT, SGPT).
XI) Histopathology -Liver, Kidney, Lungs, Spleen, Ovaries, Testis,
Stomach, Intestine

21. General comments
•Metal free pellet diet and water.
•One rat and two mice in each cage
•Test compound, vehicle control, its dosage regimen will be
supplied by sponsor (ISM)
•Scoring of cage side activity, neurological activity according to
OECD guidelines.

22. Observations:
a. Mortality: To be observed on 24, 48 and 72 hrs.
b. Clinical Signs:
• A careful cage side examination will be made daily.
changes in:
- Skin, fur, eyes and mucous membrane.
- Convulsions, lethargy, sleep, coma, salivation, diarrhoea
and date of death.

23. CHRONIC TOXICITY STUDIES
This study is basically to determine the organs affected and to check
whether the drug is potentially carcinogenic or not. This test extends
over a long period of time and it involves large groups of laboratory
animals.
Species No. of Animals Age
(wks)
Weight
(gms)
Duration of
experiment
Male Female TC Exp. Term.
Exp.
Mice
(Swiss)
40
10 x 4*
40
10 x 4*
4 15-18 90 days 100%
Rat
Wistar
40
10 x 4*
40
10 x 4*
4 60-80 90 days 100%
TC-Test compound exposure
Term. Exp – Termination of experiment immediately after last exposure
(48 hours) euthanization of animals for collection of vital organs.

24. Sl.No. Test Groups No. of Animals
1. VC
(Control)
20
(10M + 10F)
2. Low Dose/Now effect
Dose
20
(10M + 10F)
3. Intermediate Dose 20
(10M + 10F)
4. Highest Dose 20
(10M + 10F)
Test groups
Route of drug administration : Oral
Duration of drug administration : 90 days( or more)

25. Expected period of Clinical use Administration period for the
toxicity study
Single administration or repeated
administration for less than one week
2 weeks to 1 month
Repeated administration, between
one week to four weeks
4 weeks to 3 months
Repeated administration, between
one to six months
3 to 6 months
Long term repeated administration
for more than six months
9 to 12 months
As a rule, the test substance should be administered seven
days a week. Administration periods for the toxicity study must be
recorded in each result.
The following table reflects commonly used ranges of
administration periods:

26. Pre-experimentation phase
I. Acclimatization of animals
•Period – 7 days (Recording of body weight and food intake
twice in a week)
•Urine qualitative test (Ames multiple sticks)
•Fecal consistency (Filter paper technique)

27. Experimentation phase
I)Test compound exposure - multiple dose (once daily for 90
days) and dosage schedule (as recommended by sponsor)
II)Mortality 6/12/24 hours
III)Body weight (Twice in a week)
IV)Food consumption (Twice/weekly)
V)Fecal consistency
VI)Cage side activity
VII)Neurological examination

28. •Urine qualitative test (30/60/90 days)
•Haematology - Twice (30/60/90 days) (Hb, RBC, WBC,
Platelet count, differential count)
•Clinical chemistry – Twice (30/60/90 days) (Blood glucose,
total protein, serum creatinine, SGOT, SGPT).
•Histopathology – Liver, heart, brain, lung, kidney, spleen,
sciatic nerve, testes/ ovaries

29. 1. General observations and examinations:
•General signs should be observed daily.
•Body weight: before the start of drug administration, at least once a
week for the first three months of administration and at least once
every four weeks thereafter.
•Food intake: before the start of drug administration, at least once a
week for the first three months of administration and at least once
every four weeks thereafter. If the test substance is administered mixed
in the food, the intake should be measured once a week.
.

30. 2.Haematological examination:
•For rodents, blood samples should be taken before autopsy.
•For non-rodents, blood samples should be taken before the
start of the drug administration, at least once during the
administration period (for studies of longer than one month),
and before autopsy.
•For both haematological and blood chemistry examinations, it
is desirable to include as many parameters as possible.

31. 3.Renal and hepatic function tests:
Since the liver and kidneys are the usual organs of metabolism
and excretion, potentially toxic agents easily affect them; their
functions should be monitored in long term toxicity studies.
For rodents, a fixed number of animals from each group
should be selected and urinalysis should be performed before
the start of drug administration, and at least once during the
administration period.

32. 4.OTHER FUNCTION TESTS:
•If appropriate, ECG and visual, auditory tests should be
performed.
•For rodents, ophthalmological examination should be
performed on a fixed number of animals from each group at
least once during the administration period;
•for non-rodents, examination should be performed on all
animals before the start of drug administration and at least once
during the period of administration.

33. 5. Animals found dead during the examination should be autopsied as
soon as possible.
A macroscopic examination should be made of organs and tissues.
In addition, where possible, organ weight measurements and
histopathological examinations should be performed in an attempt to
identify the cause of death and the nature (severity or degree) of the
toxic changes present.

34. •All survivors should be autopsied at the end of the administration
period or of the recovery period after taking blood samples for
haematological (including blood chemistry) examinations.
• organs and tissues should be examined macroscopically and organ
weights measures.
•Histopathological examinations of the organs and tissues of animals
receiving lower dosage should also be performed
•Histopathological examination of all rodents will further improve the
chances of detecting toxicity.

35. OECD Guidelines

36. Five Organisation for Economic Cooperation and Development
(OECD) Test Guidelines (TGs 402, 403, 420, 423, and 425) describe
acute systemic testing.
1. Fixed Dose Procedure (OECDTG 420)
2. AcuteToxic Class method (OECDTG 423)
3. Up‐and‐Down Procedure (OECDTG 425)
4. Acute DermalToxicity OECDTG 402,
5. Acute inhalation toxicity OECDTG 403.

37. Fixed dose procedure
•5 animals of a single sex are dosed in a stepwise procedure one
animal each,using the fixed doses of 5, 50, 300 and 2000 mg/kg
(exceptionally an additional fixed dose of 5000 mg/kg may be
considered).
•The initial dose level is selected on the basis of a sighting study as
the dose expected to produce some signs of toxicity without causing
severe toxic effects or mortality.
•Further groups of animals may be dosed at higher or lower fixed
doses, depending on the presence or absence of signs of toxicity or
mortality.
•This procedure continues until the dose causing evident toxicity or
no more than one death is identified, or when no effects are seen at
the highest dose or when deaths occur at the lowest dose.

38. AcuteToxic Class method (OECDTG 423)
•Three animals are used for each step. The dose level to be used as the
starting dose is selected from one of four fixed levels, 5, 50, 300 and
2000 mg/kg body weight.
• The starting dose level should be that which is most likely to produce
mortality in some of the dosed animals.
•When available information suggests that mortality is unlikely at the
highest starting dose level (2000 mg/kg body weight), then a limit test
should be conducted.
•When there is no information on a substance to be tested, for animal
welfare reasons it is recommended to use the starting dose of 300
mg/kg body weight. Exceptionally, and only when justified by specific
regulatory needs, the use of additional upper dose level of 5000 mg/kg
body weight may be considered.

39. Animals are observed individually after dosing at least
once during the first 30 minutes, periodically during the
first 24 hours, with special attention given during the
first 4 hours, and daily thereafter, for a total of 14 days,
except where they need to be removed from the study
and humanely killed for animal welfare reasons or are
found dead

40. Up‐and‐Down Procedure (OECDTG 425)
•The main test consists of a single ordered dose progression in which
animals are dosed , one at a time , at a minimum of 48-hour intervals.
• The first animal receives a dose a step below the level of the best
estimate of the LD50 . If the animal survives, the dose for the next
animal is increased by [a factor of] 3.2 times the original dose ; if it
dies, the dose for the next animal is decreased by a similar dose
progression.
•Dosing is stopped when
• 3 consecutive animals survive at the upper bound;
• 5 reversals occur in any 6 consecutive animals tested
• LD 50 is calculated.

41. Six OECDTestGuidelines describe short‐term repeat‐dose toxicity
testing:
Repeated Dose 28‐day OralToxicity Study in Rodents (TG407)
Repeated Dose 90‐Day OralToxicity Study in Rodents (TG 408)
Repeated Dose DermalToxicity: 21/28‐day Study (TG 410)
Subchronic DermalToxicity: 90‐day Study (TG 411)
Repeated Dose InhalationToxicity: 28‐day or 14‐day Study (TG 412)
Subchronic InhalationToxicity: 90‐day Study (TG 413)

42. Repeated Dose 28‐day Oral Toxicity Study in Rodents (TG407)
• The test substance is orally administered daily in graduated doses to
several groups of experimental animals (rat 5 of each sex
), one dose level per group for a period of 28 days.
•A 28 day study provides information on the effects of repeated oral
exposure and can indicate the need for further longer term studies.
•The data derived from using the TG should allow for the
characterization of the test substance toxicity, for an indication of the
dose response relationship and the determination of the No-
Observed Adverse Effect Level (NOAEL).
•Satellite or control groups maintained.

43. Repeated Dose 90‐Day OralToxicity Study in Rodents (TG 408)
•The test substance is orally administered daily in graduated doses
to several groups of experimental animals, one dose level per group
for a period of 90 days.
•During the period of administration the animals are observed
closely for signs of toxicity. Animals which die or are killed during
the test are necropsied and, at the conclusion of the test, surviving
animals are also killed and necropsied.

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