Toxicity is the science dealing with properties, action, toxicity, fatal dose detection or interpretation of result of toxicological analysis & treatment of poison. Toxicity studies helps to avoid adverse effect and enhance the safety of drug. This slide provides the information about toxicity screening on experimental animals.

1. Presented by :
Chaudhari Shubham Sanjay
(Pharmacology)
Roll No. 02
Under Guidance of :
Prof. V. V. Nimbalkar
M. Pharm
(Pharmacology)

2.  Introduction Of Toxicity
 Regulatory Guidelines For Toxicity Studies
 OECD Guidelines For Toxicity Studies
 Acute Eye Irritation
 Dermal Irritation
 Reference

3. Toxicity :
Toxicity is the science dealing with properties, action, toxicity, fatal dose detection or
interpretation of result of toxicological analysis & treatment of poison.
The poison is any substance which causing harmful effect to the body. The drug also a
poison if not given in proper concentration OR The poison is a drug if given in appropriate
concentration

4. ICH : International Council on Harmonization
OECD : Organization for Economic Co-ordination & Development
FDA : Food & Drug Administration
WHO : World Health Organization

5. 1] Acute Oral Toxicity Studies Test
i) 420 – Fixed Dose Procedure
ii) 423 – Acute Toxic Class Method
iii) 425 – Up & Down Procedure
2] Acute Dermal Toxicity {402}
3] Acute Inhalational Toxicity {403}
4] Repeated Dose Studies
i) 407 – Subacute Oral Toxicity
ii) 408 – Chronic Oral Toxicity
iii) 410 – Subacute Dermal Toxicity
iv) 411 – Chronic Dermal Toxicity
v) 412 – Subacute Inhalational Toxicity
vi) 413 – Chronic Inhalational Toxicity
5] Long Term Repeated Dose Studies
i) 451 – Carcinogenicity Studies
ii) 452 – Chronic Toxicity Studies
iii) 453 – Combined Chronic &
Carcinogenicity Study
http://www.oecd-ilibrary.org/environment/test-no-405-acute-eye-irritation-corrosion_9789264185333-en

6.  The substance to be tested is applied in a single dose to one of the eye of experimental animal and
the untreated eye serves as the control.
 The degree of eye irritation/ corrosion is evaluated by scoring lesions of cornea, iris & conjunctiva
at specific interval.
 The duration of study should sufficient to evaluate the reversibility or irreversibility of the effect.
 If animal showing severe distress or pain at any stage of test should be humanely killed.
 Criteria for making decision to humanely kill of severely suffering animals are subject of a
separate guidance document.
S. K. Kulkarni, “Handbook of experimental pharmacology”, 4th Edition 2012, Vallabh prakshan, Pg. no. 180-183

7.  Selection Of Species :
 Healthy young adult Albino Rabbit (12 week)
 Weight : 1.5 Kg to 3 Kg
 Preparations of animals :
 Both eyes of each experimental animal is provisionally selected for testing & should
be examined within 24 hrs. before testing starts.
 Animals showing eye irritation ocular defects or pre- existing ocular injury should
be avoided
S. K. Kulkarni, “Handbook of experimental pharmacology”, 4th Edition 2012, Vallabh prakshan, Pg. no. 180-183

8.  Householding & Feeding :
 Animal placed individually before 24 hours of testing.
 Room temperature should be 20 ℃ (± 3 ℃ )
 Humidity should be 50 – 60 %
 Artificial light should be provided with 12 hours light & 12 hours dark
 Use of topical anesthetics & systemics analgesics
 Application of test sample
 Irrigation
 Test Procedure :
S. K. Kulkarni, “Handbook of experimental pharmacology”, 4th Edition 2012, Vallabh prakshan, Pg. no. 180-183

9.  Use of Topical Anesthetics & Systemics Analgesics :
 This procedure are for reducing the pain and distress in ocular safety testing procedure.
 60 Min. prior to the test substance application (TSA), buprenorphine 0.01 mg/kg is administered by
subcutaneous injection (SC) to provide therapeutic level of systemic analgesia.
 Five min. prior to TSA, one or two drops of topical anaesthetic for ex. 0.5 % proparacaine HCL Or
0.5% tetracaine HCL are applied to each eye.
 The of eye of each animal that is not treated with test article but which is treated with topical anaesthetic
serves as a control
 8 hours after TSA, buprenorphine 0.01 mg/kg (SC) and meloxicam 0.5 mg/kg (SC) are administered to
provide continue therapeutic level of systemic analgesia
 After initial 8 hours, buprenorphine 0.01 mg/kg (SC) should be administered to every 12 hours in
conjunction with meloxicam 0.5 mg/kg (SC) to every 24 hours, until the ocular lesions or sign of pain
and distress are present
 If the several pain or distress should be shown after the test, the rescue dose of buprenorphine 0.03
mg/kg should administered immediately (SC) and repeated as often as every 8 hours if necessary.
S. K. Kulkarni, “Handbook of experimental pharmacology”, 4th Edition 2012, Vallabh prakshan, Pg. no. 180-183

10.  Application of Test Sample :
 The test sample is placed in the conjunctival sac of the one eye of each animal after gently pulling
the lower lid away from eyeball.
 The lid is gently held for one second in order to prevent loss of material.
 The other untreated eye is serves as a control.
 Irrigation :
 The eye of test animal should not washed for 24 hrs. after TSA, except for solid or in case of
immediate corrosive or irritating effect.
S. K. Kulkarni, “Handbook of experimental pharmacology”, 4th Edition 2012, Vallabh prakshan, Pg. no. 180-183

11.  Confirmatory Test :
 If corrosive or severe irritant effect not observed in initial test, the irritant and negative response
confirmed using up to two additional animals.
 If irritant effect is observed in initial test, it is recommended that confirmatory test be conducted
in sequential manner in one animal at a time.
 If result from second animal is sufficient to allow for hazard classification, determination, then no
further test should be conducted.
 Observational Period :
 Should sufficient to evaluate reversibility of the effects observed.
 The expt. should be terminated at any time if severe sign of pain
shown during test.
 Observed for 21 days after TSA to evaluate reversibility.
Fig. : Eye Irritation on Albino Rabbit
S. K. Kulkarni, “Handbook of experimental pharmacology”, 4th Edition 2012, Vallabh prakshan, Pg. no. 180-183

12. Fig. : Acute Eye Irritant Effect On Eye

13.  The test chemical to be tested is applied in a single dose to the skin of an experimental animal,
untreated skin areas of the test animal serve as the control.
 The degree of irritation / corrosion is read & scored at specified intervals.
 Animals showing continuing signs of severe distress and/or pain at any stage of the test should be
humanely killed, and the test chemical assessed accordingly.
S. B. Kasture, “A handbook of experiments in preclinical pharmacology”, Career publication, Pg. no. 108-111

14.  Selection Of Species :
 Healthy young adult Albino Rabbit (12 week)
 Weight : 1.5 Kg to 3 Kg
 Preparations of animals :
 Approximately 24 h before the test, fur should be removed by closely clipping the
dorsal area of the trunk of the animals.
 Care should be taken to avoid abrading the skin, and only animals with healthy,
intact skin should be used.
 Some strains of rabbit have dense patches of hair that are more prominent at certain
times of the year
 Such areas of dense hair growth should not be used as test sites.
S. B. Kasture, “A handbook of experiments in preclinical pharmacology”, Career publication, Pg. no. 108-111

15.  Test Procedure :
 The test chemical should applied on the dorsal / flank region of skin and covered with the gauze
patch.
 In case the chemical is not able to apply directly then the chemical is firstly applied to the gauze
patch and then applied to skin.
 The chemical should uniformly spread to all over the test region should be ensured & then the gauze
patch is stick with the non irritating tape.
 If there is a liquid dose then it applied directly without diluting, while solid dose is diluted by
adding some concentration of water or any other vehicle sufficient for skin contact.
 At the end of exposure period, normally 4 hours, residual test chemical should removed.
 Dose Level :
 0.5 ml for liquid Chemical OR 0.5 gm for solid or paste chemical
S. B. Kasture, “A handbook of experiments in preclinical pharmacology”, Career publication, Pg. no. 108-111

16. Dorsal Region
Flank Region
Fig. : Before test
Dorsal Region
Fig. : After test
Dermal & Ocular Toxicology: Fundamentals and methods, Hobson CRC press, 06-sep-1991, medical, 656 pages

17.  Confirmatory Test :
 If corrosive or severe irritant effect not observed in initial test, the irritant and negative response
confirmed using up to two additional animals For exposure period of 4 hrs.
 If irritant effect is observed in initial test, it is recommended that confirmatory test be conducted
on two animals simultaneously.
 In exceptional case, in which initial test is not conducted, 2 to 3 animals may be treated with
single patch which removed after 4 hours.
 When 2 animals are used, if both showing same response, then no further experiments are need.
 Observational Period :
 Should sufficient to evaluate reversibility of the effects observed.
 Animal should examined for sign of erythema and oedema & response scored at 1,24,48 and 72 hrs
after patch removal
S. B. Kasture, “A handbook of experiments in preclinical pharmacology”, Career publication, Pg. no. 108-111

18. Gauze Patch Control
Negative Response Erythema /Oedema Formation
Dermal & Ocular Toxicology: Fundamentals and methods, Hobson CRC press, 06-sep-1991, medical, 656 pages

19.  S. K. Kulkarni, “Handbook of experimental pharmacology”, 4th Edition 2012, Vallabh
prakshan, Pg. no. 180-183
 S. B. Kasture, “A handbook of experiments in preclinical pharmacology”, Career
publication, Pg. no. 108-111
 Dermal & Ocular Toxicology: Fundamentals and methods, Hobson CRC press,
06-sep-1991, medical, 656 pages
 http://www.oecd-ilibrary.org/environment/test-no-405-acute-eye-irritation-
corrosion_9789264185333-en

20. Thank You…