Animal toxicity study requirements for clinical trial

1. ANIMAL TOXICITY
STUDY REQUIREMENT
FOR CONDUCT OF
CLINICAL TRIAL
May 9, 2019
1

2. Toxicology: The study of poisons & is concerned with
the adverse effects of xenobiotics.
Animal toxicology studies also called non-clinical
toxicity studies.
Chemical agents that cause toxicity include-
 Drugs
 Insecticides/herbicides
 Plant toxins
 Animal toxins
 Chemical weapons
 Radioactive elements
2

3. • Preclinical studies are to be conducted according to
Schedule Y of Drugs and Cosmetic Act 1940.
3

4. Objective
Identify toxic substance prior to clinical use
Quality assessment of the drug
Quantity assessment of the drug (plasma and
tissue levels)
To study cumulative toxicity
Estimate safe starting dose for clinical studies
& subsequent dose escalation schemes in
humans.
6

5. • Assess hazards that cannot be
evaluated in clinical trials (e.g.
carcinogenicity and teratogenicity)
• Different types of dose identification can
be done. E.g. lethal dose LD50,
maximum tolerated dose MTD, minimum
effective dose and (ED50)
7

6. GENERAL PRINCIPLE
8
SOP
Test substances and
instruments should
be properly
standardized.

7. TYPES OF TOXICITY STUDIES
9
• single dose of a substance, or multiple doses given within 24hrs or
an inhalation exposure of 4hrs, observed for 14 days
ACUTE TOXICITY
several days to 1 month.
SUBACUTE TOXICITY
repeated or spread over an intermediate time range (1-3 months).
SUB CHRONIC TOXICITY:
• Exposures (repeated or continuous) over a long (> 3 months) even up
to 2 years
CHRONIC TOXICITY:

8. 11

9. Single-dose (Acute) Toxicity Studies
16
2 Rodent
species
Ensures systemic absorption of the drug
(unless the intended route in humans is
only intravenous)
Recommended limit
• 2000 mg/kg or
• 10 times the normal dose that is intended in
humans
Use the
same route
as intended
in humans
Whichever
is higher

10. • At least 5 animals / sex / group
• At least 4 graded doses (4 dose-groups)
• Observation for toxic effects (if any) : 14 days
• Observation for mortality :
 7 days [parenteral administration]
 14 days [oral administration]
17

11. Other parameters :
Symptoms, signs and mode of death, signs of
intoxication, effect on body weight
Gross and histopathological changes
LD 10 and LD 50 values, (preferably with 95 % CI)
Genetic effects if any
Establish the:
Maximum tolerated dose (MTD)
Minimum lethal dose (MLD)
Target organ of toxicity (if possible)
18

12. Cytotoxic anticancer agents
• MTD confirm
• To establish a linear relationship between
toxicity and body surface area
• MTD can be established in non-rodent
species, if:
o Rodents are poor predictors of human toxicity
e.g. antifolate
o Cytotoxic drugs acts by a novel mechanism of
action
19

13. Repeated dose toxicity studies
(Sub acute/ sub chronic toxicity)
21

14. Aim:
To identify Target Organ Toxicity
To establish MTD for subsequent studies
Animal- at least 2 mammalian species(1
rodent & 1 non- rodent)
• Rodent- must be between 6-8 weeks
• Non-rodent should be between 4-9 months
Younger and still growing animals are
preferred in initiation of subchronic studies
22

15. Rodents
• One rodent species (preferably rat)
• At least 4 graded doses including control
• Two groups : control and treatment
• Minimum of 5 animals of each sex
• Proposed clinical route of administration
• Test substance given 7 days a week
Non-Rodents
• One male and one female
• Starting dose (3 to 5 times the extrapolated effective dose) or
MTD (whichever is less)
• Dose escalation every 3rd day, lowered if toxicity seen
• Then test substance given 7 days a week
Dose-ranging study/ Repeated dose toxicity
study
23

16. Repeated-dose Toxicity Studies
• Wherever applicable, include a Control group
• Drug is given on 14, 28, 90 & 180 days,
• 3 other groups are formed :
 Highest dose Observable toxicity [MTD]
 Lowest dose No observable toxicity
[NOAEL]
should be comparable to intended
therapeutic dose or multiple of it
 Intermediate dose Some symptoms ; not gross
toxicity or death placed
logarithmically between two doses
25

17. 26
Methodology
• Rodent : 6-10/sex/group
• Non-rodent : 2-3/sex/group
14-28 Day repeated-dose
(Sub acute toxicity studies)
• Rodent : 15-30/sex/group
• Non-rodent : 4-6/sex/group
90-Day repeated-dose (Sub
chronic toxicity studies)
• Rodent : 15-30/sex/group
• Non-rodent : 4-6/sex/group
180-Day repeated-dose
(Chronic toxicity studies)

18. Parameters
to be
monitored
Physiological
Organ
Pathology:
Gross and
Microscopic
ECG and
fundoscopy
(non-rodent)
Behavioral
Biochemical &
microscopic
observations of urine
and blood
27

19. Number of animals required for
repeated-dose toxicity studies
14-28 days 84-182 days
Group Rodent (Rat) Non-rodent
(Dog or
Monkey)
Rodent (Rat) Non-rodent
(Dog or
Monkey)
M F M F M F M F
Control 6-10 6-10 2-3 2-3 15-30 15-30 4-6 4-6
Low dose 6-10 6-10 2-3 2-3 15-30 15-30 4-6 4-6
Intermediate
dose
6-10 6-10 2-3 2-3 15-30 15-30 4-6 4-6
High dose 6-10 6-10 2-3 2-3 15-30 15-30 4-6 4-6
28

20. Lethality testing &
calculation
• It is acute toxicity testing in which death is considered a
single end-point
• LD50-
• Intraperitoneal LD50 ̴ 30% of oral
• Iv ̴ 10% of oral
29

21. SPECIAL TOXICITY
STUDIES
• Reproductive
• Local toxicity
• Allergenicity/hypersensitivity
• Genotoxicity
• Carcinogenicity
• Mutagenicity
30

22. Reproductive Toxicity
• Male Fertility
• Female Reproduction & Developmental
toxicity
o Female Fertility
o Teratogenicity
o Perinatal development
31

23. Reproductive toxicology study
FERTILITY STUDY
Rats
Studies are on fertility,
reproductive capability,
estrous cycles, mating
behavior, conception rate
and early stages of
gestation
TERATOLOGY STUDY
Rats and rabbits
Females are treated only
during organogenesis
period since embryo is
most susceptible to
induction of birth defects
at this time.
PRENATAL/ POST
NATAL TESTING
Pregnant females are
treated during last
quarter of pregnancy,
through lactation until
weaning. And then
sacrificed to detect
internal anomalies.
Purpose- determine the
effect of drugs in late
fetal development, labor,
delivery, lactation,
neonatal viability and
growth
32

24. 33

25. • 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
34

26. Reproductive Toxicity of Lead Acetate in Adult male Rats: Histopathological and
Cytotoxic Studies
Haouas Z, Zidi I, Sallem A, Bhouri R, Ajina T, et al. (2015) Reproductive Toxicity of Lead Acetate in
Adult male Rats: Histopathological and Cytotoxic Studies. J Cytol Histol 6: 293. doi:10.4172/2157-
7099.1000293
35
Figure 3: Photomicrographs of TUNEL-stained testicular sections from controls (A) and rats
exposed to 2 g/l of lead acetate (B, C, and D). Magnification 400x. Arrows indicated TUNEL-
positive cells (spermatogonia; permatocytes I; spermatids) (A, B, C, D: Scale bar 20 µm)

27. Test substance by intended route of use
for
• minimum 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
36

28. Pregnant females examined after day
13 of gestation
Males sacrificed at the end of the
study
Weight of each testis and epididymis
recorded
Sperms from one epididymis
examined for motility and
morphology
Other epididymis and both testes
examined for histology
37

29. 38
Female Reproduction and
Developmental Toxicity Studies

30. • 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
39

31. 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
40

32. Pups : Physiology, Behavior, Pathology (sex-wise distribution
noted)
Body weight
Food intake
Clinical signs
of intoxication
Reproduction
and
Parturition
Pathology
Gross &
Micro
Females allowed to litter,
medication continued till weaning of pups
Drug treatment continued during mating and gestation period
Drug administration : 28 days (males) & 14 days (females)
before mating
41

33. 42
In Utero Exposure of Biochanin-A Alters Female Reproduction in Rat.
M.G.S. Soujanya1, K. Prathap Reddy2, V. Sridevi1, P. Ramachandra Reddy1* and P. Sreenivasula Reddy2
1Department of Biochemistry, Yogi Vemana University, Vemanapuram, Kadapa, A.P., India
2Department of Zoology, Sri Venkateswara University, Tirupati, A.P., India
Received date: April 11, 2016; Accepted date: April 22, 2016; Published date: April 24, 2016

34. 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
43

35. Observation parameters
Females
General
Signs of
intoxication
Body weight
Food
intake
Reproductive
Uterus,
Ovaries
Products of
conception
Fetuses
General
Number
Gender
Length
Weight
Pathology
Gross (n)
Visceral (n/2)
Skeletal (n/2)
44

36. The Impact of Proximol (Cymbopogon proximus) Intake on Pregnant Albino
Rats and their Fetuses During Gestation Period
International Journal of Morphology June 2017; 35(2):500-505
DOI: 10.4067/S0717-95022017000200019
45
Fig 1. Photographs of the uterus of pregnant rats at the 20th day of
gestation. A) Control. Showing normal symmetrical distribution
of fetuses in the two uterine horns. B) Treated. Asymmetrical
distribution of fetuses in the two uterine horns. Left uterine horn
showing visible embryonic resorption sites (arrow).

37. Fig 2. Photographs of fetuses at the 20 th day of gestation. A) Control. Fetus
exhibited normal morphology and normal length. B-D) Treated. Showing:
clubfoot (arrow, B), hematoma at the back (arrow, C), and fore limb without
digits (arrow, D).
46

38. Perinatal Study
• 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
• Second generation (F2) selected at weaning and studied.
• Observed parameters are same as teratogenicity study 48

39. Local Toxicity
• Dermal Toxicity
• Ocular Toxicity
• Vaginal Toxicity
• Rectal tolerance test
• Parenteral Drugs Tolerance
• Inhalation toxicity
49

40. 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
50

41. Dermal Toxicity
• As cutaneous contamination is always a possibility
• Rabbit and Rat
• Applied on shaved skin (>or = 10% of Body surface area)
• Concentrations several 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
51

42. Fig. 6. Effect of CPD photoreactivation on UV-induced erythema and
hyperplasia in the skin of b -act-CPD photolyase transgenic mice. The
depilated back of b -act-CPD-1 transgenic mice was exposed to UV-B light
for four consecutive days (1.5 MED per day) and were either given 3 h of
photoreactivating light or kept in the dark. As a control, non-UV-exposed
animals were used. Animals were killed 3 days after the last exposure and,
except for the photoreactivation step, had been kept in the dark throughout
the experiment. ( A ) Appearance of the dorsal skin of non-UV-exposed (left),
UV-exposed (middle) and UV- exposed/photoreactivated animals (right),
showing clear erythema 52

43. Phototoxicity
• Tested using armstrong/ harber test in guinea pig
• Drugs used in leucoderma
• Pretest with 8 animals done to ascertain highest non-irritant
dose
• Test performed on 10 animals and 5 controls
• Induction dose – 0.3ml/patch for 2 hours±15 minutes followed
by 10 J/cm2 of UV light
• Repeated on 0, 2, 4, 7, 9 and 11 days
• Animal is then challenged with the same concentration on
days 22 – 24 followed by 10 J/cm2 of UV light
• Observations: erythema, edema formation 24 and 48 hours
later.
• Positive control- musk ambrett/ psoralin should be used.
53

44. 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 studies
Repeated dose study :
o Duration subject to clinical use (Maximum of 90 days)
o 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
54

45. Ocular toxicity studies
• Evaluation
• Slit-lamp examination
o To detect the changes in cornea, iris and aqueous
humor.
• Fluorescent dyes (sodium fluorescein, 0.25 to 1.0%)
o To detect defects in surface epithelium
• Intra-ocular tension monitored by a tonometer
• Histological examination (fixation in Davidson’s or Zenker’s
fluid)
55

46. 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 use
• Observation parameters :
 General : swelling, closure of introitus
 Histopathology of vaginal wall
56

47. 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 feces, condition of anal
region/sphincter
 Gross examination and (if required)
 Histological examination of rectal mucosa
57

48. 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
58

49. 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
• Evidence of exposure of test substance of particle size of 4
micron (especially for aerosols) with not less that 25% being 1
micron should be provided
59

50. Inhalation toxicity studies
 3 dose groups and a control
 Duration of exposure : maximum of 6 hr/day & 5 days/week
Evaluation :
 Respiratory rate, Bronchial lavage fluid examination,
histological examination of respiratory passages and lung
tissue
 Regular parameters of systemic toxicity studies or assessment
of margin of safety
60

51. Allergenicity /
Hypersensitivity
Any one of the standard tests:
• Guinea pig :
o Maximization test (GPMT)
• Mouse :
o Local lymph node assay (LLNA)
61

52. 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)
62

53. 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
63

54. 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.
64

55. 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 group
Test 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 hrs
Evaluation : Increase in 3H-thymidine or BrdU incorporation
65

56. GENOTOXICITY
66

57. • Genotoxic compounds are presumed to be trans-species
carcinogens, may 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 tic assay
• In vivo test for chromosomal damage using rodent
hematopoietic cells
67

58. 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
68

59. 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
69

60. In-vivo cytogenetic assay
Clastogenic & Aneugenic effects in metaphase chromosomes (min
100)
Bone marrow : Giemsa staining
Sacrificed 2 hours after colchicine administration
Dosing on day 1 followed by i.p. colchicine administration at 22
hours
• 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)
70

61. In-vivo micronucleus assay
↑micronuclei in polychromatic erythrocytes [M-PCE] (min 1000)
Bone marrow : Smeared with May Gruenwald / Giemsa stain
Sacrifice of animals 6 hours after the last injection
Dosing : day 1 and 2 of study
• 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
71

62. CARCINOGENICITY
72

63. 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
73

64. 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
74

65. 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
75

66. 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, Behavior, Biochemistry, Pathology
 Comprehensive descriptions of benign and malignant
tumor development, time of their detection, site,
dimensions, histological typing etc
76

67. Animal Toxicity
requirements for clinical
trials and marketing of a
new drug
77

68. Route of
administration
Duration of
proposed human
administration
Human Phase(s)
for which study is
proposed to be
conducted
Long term toxicity
requirements
Systemic Toxicity Studies
Oral or Parentral
or Transdermal
Single dose or
several doses in one
day, Upto 1wk
I,II,III 2species,2wk
> 1 wk but Upto
2wk
I,II,III 2species;4wk
(2 weeks)
Upto 2 weeks Marketing
permission
2 species; 4 week
> 2 wk but Upto
4wk
I,II,III 2species;12wk
> 2 wk but Upto
4wk
I,II,III 2 species equal to
duration of human
exposure
Marketing
permission
2 species; 12 week78

69. 79
Route of
administration
Duration of proposed
human
administration
Human Phase(s) for
which study is
proposed to be
conducted
Long term toxicity
requirements
Systemic Toxicity Studies
Oral or
Parentral or
Transdermal
> 4 weeks up to 12
weeks
I,II,III 2 species equal to
duration of human
exposure
Marketing
permission
2 species; 24 week
> 12 weeks up to 24
weeks
I,II,III 2 species equal to
duration of human
exposure
Marketing
permission
2 species; 24 week; non-
rodent 36weeks
> 24 weeks I,II,III 2 species; 24 week; non-
rodent 36weeks
Marketing
permission
2 species; 24 week; non-
rodent 36weeks

70. Route of
administration
Duration of
proposed
human
administration
Human Phase(s) for which study
is proposed to be conducted
Inhalation
(general
anesthetics,
aerosols)
Upto 2 wk I,II,III 2species;1month
; (Exposure time
3h/d, 5d/wk)
Upto 4wk I,II,III 2species;12week
, (Exposure time
6h/d, 5d/wk)
> 14wk I,II,III 2species;24week
, (Exposure time
6h/d, 5d/wk)
80

71. Local Toxicity Studies
Dermal Upto 2 week I,II 1species ;4 week
III 2species ; 4 week
> 2 week I,II,III 2species ;12week
Ocular or Otic
or Nasal
Upto 2 week I,II 1species ;4week
III 2species ;4week
> 2 week I,II,III 2species ;12week
Vaginal or
Rectal
Upto 2 week I,II 1species ;4week
III 2species ;4week
> 2 week I,II,III 2species ;12week
81

72. Laboratory parameters to be
included in toxicity studies
• Hematological parameters
• Urinalysis Parameters
• Blood Biochemical Parameters
• Gross and Microscopic Pathology
82

73. Hematological parameters
Haemoglo
bin
Total
RBC
Count
Haemat
ocrit
Differen
tial
WBC
Count
Total
WBC
Count
Reticulo
cyte
Platele
t
Count
Termina
l Bone
Marrow
Examin
ation
ESR
(Non-
rodents
only)
General
Blood
Picture
Coagulation
Parameters
(Non-
rodents
only):
Coagulation Parameters: Bleeding Time, Coagulation Time, Prothrombin
Time, Activated Partial Thromboplastin Time. 83

74. Urinalysis Parameters
Colour &
Appearance
24-hour urinary
output
Specific Gravity Sugar & Acetone
Albumin & Bile
pigments
Reaction (pH) Urobilinogen
Occult Blood
Microscopic
examination of
urinary sediment.
84

75. Blood Biochemical Parameters
Glucose
Cholesterol &
Triglycerides
HDL & LDL
(Non-rodents only)
Alkaline
Phosphatase
(ALP) & GGT
(Non-rodents only)
SGPT (ALT) &
SGOT (AST)
Bilirubin
Blood Urea
Nitrogen &
Creatinine
Albumin &
Globulin
Sodium,
Potassium,
Phosphorus,
Calcium
85

76. Gross and Microscopic Pathology
Brain*: Cerebrum,
cerebellum, Midbrain
Liver*
(Rectum)
Urinary bladder
Uterus*
Epididymis
Ovary
Testis*
Skin
Mammary gland
Mesenteric
lymph node
Skeletal muscle
(Middle Ear)
Eye
(Spinal Cord)
(Parathyroid)
Thyroid
Spleen*
(Trachea)
Lung*
Stomach
Oesophagus
AortaHeart*
(Pancreas)
Adrenal*
Thymus
Kidney*
Colon
Terminal ileum
JejunumDuodenum
* Organs marked with an asterisk should be weighed.
() Organs listed in parenthesis should be examined if indicated by the nature of the
drug or observed effects. 86

77. Observations
that should be
carried out
Clinical
signs of
intoxication
Mating
behavior
Body
weight
Food
intake
Post-partum health &
gross pathology of
affected organ
Progress of
gestation/
parturition periods
Length of
gestation
Parturition
Growth
parameter
Survival
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78. Limitations of toxicity
studies
• There may be variation of toxicity in different species
• Various types of allergic reaction e.g hypersensitivity reactions,
genetically determined A/E that my occur in human may not be
detected in animal studies
• Toxicity studies are time consuming (2-5 yrs)
• Large number of animals may be needed to obtain valid
preclinical data
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79. • Extrapolation of TI & toxicity data from animal to human
is not predictive of all toxicities
• For statistical reason, rare adverse effect are unlikely to
be detected in preclinical studies
• Subjective responses nausea, tinnitus, loss of libido d/t
new drug are difficult to discover
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80. Pharmacokinetic Studies
• Once a compound is cleared of toxicological studies PK
studies are performed
• Mice, rats & non human primate like monkeys
• Done to provide data on how a drug is
o Absorbed
o Its bioavailability on oral & iv
o Volume of distribution
o Biotransformation and
o Excreted from the body & elimination half life
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81. How to calculate LD50
1.Mathematical/Arithmetical method:
• Karber’s method, simple, rapid but crude method
• Does not involve plotting of DRC
2.Graphical method:
• Accurate & preferred method
• Percentage mortality is converted into probit
• Values thus obtained are plotted on DRC
• Log dose on X-axis, probit Scale on Y-axis
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82. • MM=diff of 2 adjacent no.of dead animal/2
• E.g =(A+B)/2=(0+2)/2=1
• ∑(DD×MM)=84.5,divided by no.of animal (n=10)
• Hence 84.5/10=8.45, finally value is subtracted from minimum
dose which produces 100% mortality i.e 20 mg/kg
• So LD50=20-8.45=11.55mg/kg
Exp
Group
dose
mg/kg
Dose
diff.DD
No.of
Animal
n
No.of
dead
animal
Mean
mortality
MM
DD×MM
1 3 - 10 0 (A) - -
2 5 2 10 2(B) 1 2
3 10 5 10 3 2.5 12.5
4 12 2 10 5 4 8
5 15 3 10 7 6.5 19.5
6 20 5 10 10 8.5 42.5
∑(DD×MM) 84.5
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83. Graphical method
5 LD50=antilog(1d)
probit
0
1d log dose
Fig. Determination of LD50
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84. For Phase I studies: -
• Single dose toxicity studies
• Dose Ranging studies
• Repeated dose systemic studies of appropriate duration
to support the duration of proposed human exposure.
o Male Fertility study
o In-vitro Genotoxicity tests
• Relevant local toxicity studies
• Allergenicity/hypersensitivity tests
• Photo-allergy or dermal photo-toxicity test
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85. Phase II Clinical Trials
• Non-clinical safety data (listed previously) already
submitted while obtaining the permissions for phase I trial,
with appropriate references.
• Directly starting phase II trial – complete details of the non-
clinical safety data needed for obtaining permission for
phase-I trial
• Repeat dose systemic toxicity studies of appropriate
duration to support the duration of proposed human
exposure.
• In-vivo genotoxicity tests
- segment II reproductive/developmental toxicity study
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86. Phase III Clinical Trials
• Summary of non-clinical safety and Phase I and Phase II
trials data already submitted while obtaining the
permissions
• Directly starting Phase III trial – complete details of the
non-clinical safety data needed for obtaining permission for
Phase-I trial and Phase II
• Repeat dose systemic toxicity studies of appropriate
duration to support the duration of proposed human
exposure
• Reproductive/developmental toxicity studies. (female
reproduction or teratogenic toxicity)
• Carcinogenicity studies ( when there is a cause for concern
or when the drug is to be used for more than 6 months)
96

87. Phase IV Clinical Trials
• Summary of all the non-clinical safety data already
submitted while obtaining the permissions or Phase I, II
and Phase III trials with appropriate references
• Directly initiate Phase IV trial- complete details of the
non-clinical safety data needed for obtaining permission
for Phase-I, II trial and Phase II I trial
97

88. Application Of Good
Laboratory Practices (GLP)
• The animal studies be conducted in an accredited
laboratory. Toxicology studies, also be conducted in an
accredited laboratory.
98

89. References
• Schedule Y (March 19, 2019, page 188 – 199)
• Bikash Medhi
99

90. THANK YOU
100

91. 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
101