ANIMAL TOXICITY STUDIES (NON- CLINICAL STUDIES)

Dr. AMEENA KHATOON

Why study toxicology???

Benefit risk ratio can be calculated Prediction of therapeutic index Therapeutic index= Median Lethal dose(LD50) Median Effective dose(ED50) For a safer drug, TI >1

why do we require non clinical studies IN ANIMALS before ADMINISTERED to man??

Pharmacological effects are same in man as in animals. Toxic effect in species will predict adverse effects in man. Giving high doses in animals improves predictability to man. Risk assessment can be made by comparison of toxic doses in test species with predicted therapeutic dose in man.

Surface area ratios of some common laboratory species and man

20 g 200 g 400 g Mouse Rat Guineapig 20 g Mouse 200 g Rat 400 g Guineapig 1.5 kg Rabbit 1.0 0.14 0.08 7.0 1.0 0.57 12.25 1.74 1.0 1.5 kg Rabbit 27.8 3.9 1.08 2 kg Cat 29.7 4.2 2.4 4 kg 12 kg Monkey Dog 64.1 9.2 5.2 124.2 17.8 10.2 70 kg Man 387.9 56.0 31.5

0.04

0.25

0.44

1.0

1.08

2.4

4.5

14.2

2 kg Cat
4 kg Monkey

0.03
0.016

0.23
0.11

0.41
0.19

0.92
0.42

1.0
0.45

2.2
1.0

4.1
1.9

13.0
6.1

12 kg Dog

0.008

0.06

0.10

0.22
0.07

0.24

0.52

1.0
0.32

3.1
1.0

70 kg Man 0.0026

0.018 0.031

0.076 0.16

Types of toxicity studies

Systemic toxicity studies Single dose studies Repeated dose studies Reproductive toxicity studies Male fertility Female reproduction & Developmental studies Local toxicity studies Hypersensitivity studies Genotoxicity studies Carcinogenicity studies

SYSTEMIC TOXICITY STUDIES

SINGLE DOSE / ACUTE TOXICITY STUDIES

Maximum Non Lethal dose(MNLD) determined

Preliminary (pilot study)

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

 The adverse effects occurring within a short time of administration of a single dose of substance. Acute toxicity tests: Animal species Sex Number of animals Route of administration Dose levels Frequency of administration Observation

(a)ACUTE TOXICITY STUDIES

PILOT STUDY
AIM: to select a dose range for subsequent study.

It is done on a small group of mice

Drug is administered in ascending and widely spaced doses, say 10,30,100,300 and 1000mg/kg. Injected mice is observed continuously for 2hrs and then occasionally for further 4hrs and finally overnight mortality is recorded.

PILOT STUDY
(ii) Each dose is given to one animal only and LD50 is estimated from the mean of the logarithms of the smallest effective dose and the largest ineffective dose. (iii) UP AND DOWN/ STAIRCASE METHOD

DEFINITIVE STUDIES (Determination of LD50)

METHOD

Single dose tested in large number of mice of both sexes 2 routes of administration 3-4 dose levels Observation for 7 days after dosing MNLD established Symptoms , signs reported Microscopic and Macroscopic evaluation

Determination of acute lethality

Graphical method (Miller and Tainter): This method is simple and accurate enough in most cases & should always be tried first. The observed percentage mortality is converted into probit referring to the probit table

The values thus obtained are plotted against log dose. The LD50 value and its standard error are determined from the graph, if the line is straight enough.

LD50 determination by graphical method

Group Dose mg/kg 64 71 81 90 100 Log dose 1.81 1.85 1.91 1.95 2.00 Dead/total Dead % 0 20 40 90 100 Corrected% probit

1 2 3 4 5

0/10 2/10 4/10 9/10 10/10

2.5 20 40 90 97.5

3.04 4.16 4.75 6.28 6.96

Corrected formula : for 0%dead: 100(0.25/n) for 100% dead: 100[(n-0.25)/n] Where n is the no. of animals in the group

LD50 determination by graphical method

7.0

6.0

5.0 4.0 3.0 0 1.8 0 1.9 2.0

LD 50

LOG DOSE

Determination of acute lethality

Arithmetical method (karbers method): it is the simplest and rapid though crude method. The interval mean of the number dead in each group of animals(b) is used as well as the dose difference(a). The product of the interval mean and the dose difference is obtained. LD50=100-( axb/n) Where n is the total no. of animals.

LD50 determination by karbers method

Group Dose mg/kg No. of animals Dose diff (a) Dead Mean mortality (b) 1 3 6.5 9.5 Product (axb)

1 2 3 4 5

64 71 81 90 100

10 10 10 10 10

7 10 9 10

0 2 4 9 10

7 30 58.5 95

190.5

LD50=100-(190.5/10)=81mg/kg(approx)

Aim: To determine the maximum tolerated dose. To indicate the nature of toxic reactions, so that suitable chronic studies can be designed. Subacute toxicity studies Animal species (selection of animals) Route of administration Dose levels ( selection of dose) Observation

b) REPEATED DOSE STUDIES (SUB-ACUTE TOXICITY)

b) REPEATED DOSE STUDIES (SUB-ACUTE TOXICITY)

Method one rodent [6-10/sex/group; for 14 days] [15-30/sex/group; for 90 & 180 days] non- rodent [2-3/sex/group; for 14days] [4-6/sex/group; for 90 & 180 days]

b) REPEATED DOSE STUDIES ( CHRONIC TOXICITY)

Chronic toxicity studies Animal species. Dose levels. (The high dose level should ideally be atleast 10 times the expected maximum clinical dose.) Duration. (In rodents: for 6 months to 2yrs In non- rodents: for 1 yr but may be longer) Observation.

REPRODUCTIVE TOXICOLOGY STUDIES

a) MALE FERTILITY METHOD One rodent species(rat) 3 dose groups taken (each with 6 adult males), 1 control
Drug treatment by clinical route for 28-72 days

Mated with females in 1:2 ratio

Females getting pregnant should be examined After 13 days of gestation All male animals sacrificed

Weights of testis, epididymus recorded & examined for their histology Sperms examined for motility & morphology

b) FEMALE FERTILITY
Drug administered in females of established fertility, before mating.

Segment I

Fertility and general reproductive performance study

Teratogenicity
Implantation Embryogenesis

Segment II
Segment III

Peri and post-natal study Fertility and early embryonic development (rat) Embryo- foetal development (rat & rabbit) Post natal development (rat) (post natal survival of offspring), growth parameters, vital senses, behavioral effects
21

LOCAL TOXICITY STUDIES

Required when drug is administered by special route (other than oral) in humans Study design: 2 species along with control used. Dose dependent on dose escalating studies. 3 dose levels.

Types of local toxicity studies

Dermal toxicity Rats & Rabbit studies Local signs (erythema, oedema),
histological examination

Dermal photo- Guinea pig Used in treatment of leucoderma toxicity studies

Examination of erythema & oedema formation

Rabbit or Dog Vaginal toxicity Observation of swelling, histopathology studies of vaginal wall Rabbit or Dog Rectal tolerance Signs of pain, blood or mucous, histology examination of rectal mucosa studies

Ocular toxicity studies

Parenteral drugs

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

For IV/ IM/ SC/ intra-dermal injection Sites of injection examined grossly and microscopically

One rodent and non rodent species Inhalation toxicity Acute , sub-acute and chronic studies studies performed Observation of RR Histological examination of respiratory passages, lung tissue

CARCINOGENECITY 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

CONDUCT OF STUDY
Group sizes of 50 animals/sex at each of 3 dose levels
Control group is of double size

Record for onset of tumor development

Usually carried out for 24 months in rats and 18 months in mice (life span studies)

EVALUATION OF RESULT
Incidence of cancers in control and test Trend towards increasing incidence with increasing doses

Number of animals with single/multiple tumors

Macroscopic changes observed by autopsy

Histopathology of organs and tissues