Human eyes are frequently exposed to chemicals accidentally or on purpose due to their external location. Therefore, chemicals are required to undergo the evaluation of the ocular irritancy for their safe handling and use before release into the market. Draize rabbit eye test developed in 1944, has been a gold standard test which was enlisted as OECD TG 404 and OECD TG 405 but it has been criticized with respect to animal welfare due to invasive and cruel procedure. To replace it, diverse alternatives have been developed: (i) Hen Egg Test-Chorio-Allantoic-Membrane (HET-CAM) test (ii) RBC Haemolysis study

Deepak G Wagh, Dr. Sadhana R Shahi

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 www.iajpr.com  Page1227                                                  Indo American Journal of Pharmaceutical Research, 2018                      ISSN NO: 2231-6876                                 THE HEN'S EGG TEST - CHORIO-ALLANTOIC-MEMBRANE (HET -CAM) AND HAEMOLYSIS STUDY AS AN ALTERNATIVE TEST FOR OCCULAR IRRITANCY ANIMAL EXPERIMENTS (Draize Test)  Deepak G Wagh, Dr. Sadhana R Shahi Department of pharmaceutics, Yash Institute of Pharmacy, South city, Opp A.S Club, Waluj Road, Aurangabad, Maharashtra, India.431136.  Corresponding author  Deepak G Wagh Department of pharmaceutics, Yash Institute of Pharmacy, South city, Opp A.S Club, Waluj Road, Aurangabad, Maharashtra, India.431136 deepak.081@gmail.com                     Copy right © 2018 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE INFO  ABSTRACT Article history Received 07/05/2018 Available online 15/06/2018  Keywords Hen Egg Test-Chorio-Allantoic-Membrane (HET-CAM) test and RBC Haemolysis study.  Human eyes are frequently exposed to chemicals accidentally or on purpose due to their external location. Therefore, chemicals are required to undergo the evaluation of the ocular irritancy for their safe handling and use before release into the market. Draize rabbit eye test developed in 1944, has been a gold standard test which was enlisted as OECD TG 404 and OECD TG 405 but it has been criticized with respect to animal welfare due to invasive and cruel procedure. To replace it, diverse alternatives have been developed: (i) Hen Egg Test-Chorio-Allantoic-Membrane (HET-CAM) test (ii) RBC Haemolysis study. Please cite this article in press as Deepak G Wagh et al. The Hen's Egg Test - Chorio-Allantoic-Membrane (Het -Cam) And Haemolysis Study as an Alternative Test for Occular Irritancy Animal Experiments (Draize Test). Indo American Journal of Pharmaceutical Research.2018:8(05).                                                    www.iajpr.com Page1228 Vol 8 Issue 05, 2018.                                                        Deepak G Wagh et al.                                                       ISSN NO: 2231-6876                                INTRODUCTION Originally the Hen Egg Test-Chorio-Allantoic-Membrane (HET-CAM) test was developed for toxicity and irritation studies as an alternative method to replace the Draize-Test.  Hen Egg Test-Chorio-Allantoic-Membrane (HET-CAM) test Ocular irritation of the developed formulation was checked by hen’s egg chorioallantoic membrane test which is a rapid, sensitive, and inexpensive test. Testing with an incubated egg is a borderline case between in vivo and in vitro systems and does not conflict with the ethical and legal obligations. [1,2] The chorioallantoic membrane of the chick embryo is a complete tissue including veins, arteries, and capillaries and is technically very easy to study. It responds to injury with a complete inflammatory process, a process similar to that induced in the conjunctival tissue of the rabbit eyes.  Developed formulation was tested by this method and the result was compared with those obtained using normal saline, which was used as control that is supposed to be practically non-irritant. A means score of 0 was obtained for normal saline [3] The study shows that the formulation is non-irritant to mild irritant and could be regarded as well tolerated. The irritation potential of an ophthalmic medicine can be quantified using this method, by monitoring damage to blood vessels. [4,5,6]  Haemolysis study The blood cells swells when placed in hypotonic solution. While in hypertonic solution shrinkage takes place. Their shapes remain normal in isotonic solution. This principle is used to measure Isotonicity of formulation. One drop of human blood was mixed with one drop of formulation and further incubated for 30 min. and further diluted with saline and observe microscope at resolution of 10X. Similarly 1.8 %and 0.45% and 0.9% Sodium chloride solution were used as hypertonic and  hypotonic solution and isotonic solution  respectively. The above procedure was carried out using this solution instead of formulation and the appearance of cells was compare with appearance of cell in formulation.  According to Hemolysis study we found that no marked changes in RBC’s with our formulation, which is similar to innovator hence we concluded that our formulation is isotonic. Hence in vitro studies with RBC’s conclude that our formulation is same as that of innovator. And it is the safe for ophthalmic use without causing any irritation of discomfort hence no need of Bioequivalence  MATERIALS AND METHOD: RBC received from local pathology lab and Hen eggs (9 days fertilized) were procured from Khadkeshwar Hatcheries Ltd, Aurangabad.  EXPERIMENTAL  Procedure for Hen Egg Test-Chorio-Allantoic-Membrane (HET-CAM) test: Test system: In this test fertilized 4 Hens’s eggs weighing between 10-60 gms were selected. Purchased the 4 hen eggs (i.e 9 days fertilized egg) from Khadkeshwar Hatcheries Ltd, Aurangabad. The eggs are stored in egg boxes (blunt ends upwards).  Selecting the Eggs: Eggs blunt ends are then (on day 10) illuminated with a Candling lamp. (Note: Only eggs with an emergent embryonic vascular system are used for further testing. Eggs that have not been fertilized or have not undergone embryo genesis are rejected)  Preparation Of The Eggs The egg shell is opened along the marked line with the help of spoon and then visible white Membrane is moistened with a few mL of physiological saline.   Test Substance Testing Test substance ( F5 formulation eye drop solution) instilled into egg membrane and same time innovator also instilled into the another egg membrane, simultaneously 0.9% sodium chloride considered as negative control sample and 1.8% Sodium chloride solution considered as Positive control  End Points End points are Haemorrhage, Vasoconstriction, Coagulation and Lysis and checked after 10 min of solution instillation [6]                                                          www.iajpr.com Page1229 Vol 8 Issue 05, 2018.                                                        Deepak G Wagh et al.                                                       ISSN NO: 2231-6876                                Procedure for The Haemolysis Study RBC’s washing and separation: 1. Collected the blood sample (1mL) from healthy human and kept in the centrifuge tube. 2. Immediately added the 20 µL EDTA solution (as an anticoagulants) into above mentioned centrifuge tube and mixed properly. 3. With the help on centrifuge apparatus, centrifuged the step 2 sample for 5 min at 3500 rpm. 4. After completion of centrifugation, blood components were settling down into the bottom and removed the supernant (serum) solution. 5. Added the normal saline solution into blood containing centrifuge tube and centrifuged the samples for 5 min at 3500 rpm.and completed of centrifugation, blood components  6. are settling down into the bottom and supernant (serum) solution discarded and repeated for 3 times  7. RBC’s separated and used for further study.  B) Different solution preparation: 1. During this study prepared the hypertonic solution (1.8% NaCl) and Hypotonic   solution (0.45% NaCl). 2. Collected the normal saline, RLD or Innovator batch sample and In house ophthalmic solution batches (F5 and F5) sample for this study. 3. From above samples pipetted the 0.5 mL (500µL) into test tube and add 10µL of RBC’s sample, Mixed properly.   Magnification of slide: 1. One drop from each test tube containing solution placed  into the glass slide and   covered with cover slip  2. Placed a small drop of immersion oil directly into slide over a coverslip. 3. Rotated the 100x objectives into the immersion oil and focus only with fine focus. 4. Similarly one by one each slide magnified in the 100x objectives[8]  OBSERVATION  Hen Egg Test-Chorio-Allantoic-Membrane (HET-CAM) test  Candling lamp observation for confirmation of emergent embryonic vascular system.          Figure 1 Candling lamp observation for confirmation of emergent embryonic vascular system.                                                           www.iajpr.com Page1230 Vol 8 Issue 05, 2018.                                                        Deepak G Wagh et al.                                                       ISSN NO: 2231-6876                                Observation       Figure 2 Hen Egg Test-Chorio-Allantoic-Membrane (HET-CAM) test.                                                            www.iajpr.com Page1231 Vol 8 Issue 05, 2018.                                                        Deepak G Wagh et al.                                                       ISSN NO: 2231-6876                                OBSERVATION  Haemolysis Study                        Figure 3 Normal RBC.                          Figure 4 Hypertonic Solution RBC.    Figure 5  Hypotonic solutions RBC.            Figure 6 F4 and F5 formulations RBC.    Figure 7 Innovator sample RBC.                                                    www.iajpr.com Page1232 Vol 8 Issue 05, 2018.                                                        Deepak G Wagh et al.                                                       ISSN NO: 2231-6876                                CONCLUSION i) Egg Membrane after instilled the 0.9% sodium chloride (Negative control)                                                       No haemorrhage,  No Vasoconstriction, No lysis and  No coagulation. ii) Egg Membrane after instilled the Innovator sample                   :                                                         No haemorrhage,  No Vasoconstriction, No lysis and  No coagulation iii) Egg Membrane after instilled F5 formulation (Test product):                                                          No haemorrhage,  No Vasoconstriction, No lysis and  No coagulation iv) Egg Membrane after instilled1.8% sodium chloride (positive control):                                                          Haemorrhage observed Coagulation observed  On the basis of above observation, our F5 formulation as test sample does not shows any abnormal effect Same observation on innovator and 0.9% sodium chloride (Negative control) , so F5 formulation is non-irritant hence our test product is safe for ophthalmic or ocular use It can be also concluded that On the basis of above mentioned images of RBC in different solution, we concluded that the innovator sample showing similar i.e. isotonic image with normal RBC ,and in-house formulation (i.e F4,F5 and F6) showing does not any changes in the RBC’s ie also similar to the innovator as well as normal RBC’s.Hence on the basis of statement and images our formulation is isotonic, matching with innovator and our test product is safe for ophthalmic or ocular use.                                                                                      www.iajpr.com Page1233 Vol 8 Issue 05, 2018.                                                        Deepak G Wagh et al.                                                       ISSN NO: 2231-6876                                REFERENCES 1. M. Mandl, b. Kapeller, r. Lieber, k. Macfelda the hen's egg test - chorio-allantoic-membrane (het -cam) assay as an alternative for animal experiments. 2. Luepke np. Hen's egg chorioallantoic membrane test for irritation potential. Food chem toxicol. 1985 feb;23(2):287-91. 3. Batista-Duharte A1,2, Jorge Murillo G3, Pérez UM3, Tur EN3, Portuondo DF2, Martínez BT4, Téllez-Martínez D5, Betancourt JE3, Pérez O6. Int j toxicol. 2016 nov;35(6):627-633. Epub 2016 oct 12.The hen's egg test on chorioallantoic membrane: an alternative assay for the assessment of the irritating effect of vaccine adjuvants. 4. Dr. W.Steiling ITB-Toxikologie In vitro Protocol the hen's egg test on The chorioallantoic  membrane (HET-CAM)  5. Background Review Document Current status of in vitro test methods for identifying Ocular corrosives and severe irritant: Hen egg test- chorioallantoic membrane test method, Wilson CG. Topical drug delivery in the eye. Experimental eye Research 2004;78;737-743. 6. H.Itagakia S.Haginoa S.Katoa T.Kobayashia M.Umeda∗An in vitro alternative to the Draize eye-irritation test: Evaluation of the crystal violet staining method. 7. Dr. W.Steiling ITB-Toxikologie In vitro Protocol the hen's egg test on The chorioallantoic  membrane (HET-CAM). 8. Encyclopedia of pharmaceutical technology 3rd Edition Tonicity chapter by Jaymin C. Shah) and Osmotic pressure on blood cells diagram From Wikipedia, the free encyclopedia).         54878478451180501               

THE HEN'S EGG TEST - CHORIO-ALLANTOIC-MEMBRANE (HET -CAM) AND HAEMOLYSIS STUDY AS AN ALTERNATIVE TEST FOR OCCULAR IRRITANCY ANIMAL EXPERIMENTS (Draize Test)

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THE HEN'S EGG TEST - CHORIO-ALLANTOIC-MEMBRANE (HET -CAM) AND HAEMOLYSIS STUDY AS AN ALTERNATIVE TEST FOR OCCULAR IRRITANCY ANIMAL EXPERIMENTS (Draize Test)

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