Determination of Ocular Irritancy Potential of Ophthalmic products using HET-CAM Method

 

Anand Babu K1*, Ram Narayanan R2

1Department of Pharmaceutical Chemistry, College of Pharmacy, Kannur Medical College,

Anjarakandy, Kannur -670612, Kerala.

2Department of Pharmaceutics, Sri Ramachandra Institute of Higher Education and Research,

 Porur, Chennai - 116, Tamil Nadu.

*Corresponding Author E-mail: anandbabu23@rediffmail.com

 

ABSTRACT:

Objective: The research is about the stability of ophthalmic products as they start to produce irritancy effects during their shelf life. The aim of the present study is to assess the stability of certain ophthalmic products by the HET-CAM (Hen’s Egg Test-Chorioallatonic membrane) Method. Method: Incubated Eggs were utilized for performing the HET-CAM assay according to the protocol prescribed by ICCVAM (The Inter agency Coordinating Committee on the Validation of Alternative Methods). Results: Calculation of various irritancy factors like hemorrhage time, vascular lysis time and coagulation time and to calculate HET-CAM score. Statistical interpretation of the data obtained by one-way ANOVA. Conclusion: Results have shown that the few ophthalmic products are slight and moderate irritants. None of the products are strong irritants. However further studies to be conducted to confirm the potential and safety of the products.

 

KEYWORDS: Ophthalmic Products, HET-CAM, ICCVAM, ANOVA.

 

 

INTRODUCTION:

The Draize eye irritation test1 has been used for about 60 years in an attempt to predict human eye injury and it is based on topical instillation of the potential irritant and scoring ocular adverse effects by examination of the rabbit eye2. This test has been criticized based on dose-volume, methods of exposure, use of animals as models, the number of animals, observation and scoring, including the laboratory procedure variability, interpretation of results, and the over prediction of the human response3. However, the Draize eye irritation test continues to be the primary method accepted by the regulatory agencies worldwide4,5.

 

On the other hand, the relevance of some currently used In-vitro tests for a reliable prediction of human eye irritation, partially concerning mild and moderate irritation, is still uncertain. Recently, two alternative methods, i.e., the Bovine Corneal Opacity and Permeability (BCOP) and the Isolated Chicken Eye (ICE) have been scientifically validated6.

 

One of the most robust and successful assays for HET-CAM (Hen’s Egg Test-Chorioallantoic Membrane) of fertilized chicken eggs. Physical property and degree of irritation of different chemical substances were optimized by using the HET-CAM method. During the last couple of years, in Germany as well as France, the HET-CAM has been officially announced as a valid In-vitro assay, at least the prediction of severe irritance. Therefore, in the present study, we investigated the irritation potential of some ophthalmic products in the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM). According to the recently proposed In-vitro strategy approach for eye irritation called “Bottom-Up Approach”, this assay is a part of a group of In-vitro tests that has the greatest potential to distinguish the non-classified substance from irritants. Thus, the present study aims to investigate the irritancy potential of Ophthalmic Products by the HET-CAM test method in the assessment of the irritant potential of low-irritant products.

 

MATERIAL AND METHODS:

Experimental methods:

Eggs were collected from the poultry, Madhavaram, Chennai, AR grade Sodium chloride (spectrum), BOD incubator (scientex), dentist blade and ophthalmic products are procured from the local market.

 

Treatment Group:

Three eggs per group (test substance, negative and positive controls) were used as the treatment groups. Each sample was analyzed three times using three different eggs. Moreover, eggs of the same hen were used to analyze the same sample.

 

CAM preparation:

Eggs not older than 7 days which are clean, fertile, 50-60 g in weight, White Leghorn chicken eggs were selected. Candling of the eggs was done and any eggs that are nonviable and defective were removed. Excessively mishapen eggs or eggs with cracked or thin shells were removed. Shaking, unnecessary tilting, knocking, and all other mechanical irritation of the eggs was avoided when preparing the eggs.

 

The eggs were placed in an incubator with a rotating tray. The eggs were incubated at 38.3±0.2ºC and 58 ± 2% relative humidity when incubating in a stills-air incubator or at 37.8±0.3ºC and 58±2% relative humidity was incubated in a forced-air incubator. Hand rotation of the eggs five times per day until day 8 was done.

 

The Eggs were then candled again on incubation day 8 and nonviable or defective eggs were removed. Eggs were returned to the incubator (without hand rotation) with the large end of the eggs upwards for an additional day.

 

The eggs were removed from the incubator on day 9 for use in the assay. Candling of eggs was again done and any nonviable or defective eggs were removed.

 

The air cell of the egg was marked. The section marked as the air cell was cut with a rotating dentist saw blade and then pared off. Care was taken when removing the eggshell to ensure that the inner membrane is not injured.

 

The inner membrane was moistened with 0.9% NaCl (Normal Saline). To apply the solution a disposable glass pipette can be used. For a maximum of 30 minutes egg should be placed in an incubator.

 

The eggs were removed from the incubator, before its use in the assay and the 0.9% NaCl solution was decanted. Carefully the inner membrane was removed with forceps, ensuring that the inner membrane is not injured.

 

Treatment of eggs with test substances:

Test substances depending upon the physical form, the following-specific application protocols should be followed.

 

Samples which are taken “C001, C002, C003, C004, C005, C006, C007, C008, C009, C010, C011, C012, C013, C014, C015. In this, all the 15 ophthalmic products were presumably confirmed as non irritants, which are available in the market and they were tested by using the In-vitro HET-CAM method.

 

Liquid or diluted test substance or formulation:

0.3 ml of liquid substance or diluted substance was applied directly on to the CAM surface.

 

Formulation of granular test substance or particulate, solid:

0.3ml of solid, particulate, or granular substances (which have been ground to fine dust) was applied directly onto the CAM, ensuring that at least 50% of the CAM surface area is covered. In cases where the weight of the test substance at this volume is greater than 0.3g of the solid, particulate, or granular test substance was used. In either, the weight of the test substance was recorded.

 

OBSERVATIONS:

The reactions were observed on the CAM over a period of 300 seconds. The time for the appearance of each of the noted endpoint was monitored and recorded in seconds. Endpoints observed were hemorrhage, coagulation and vascular lysis. To maximize the livelihood of obtaining reproducible results, reference photographs for all endpoints were made available.

 

RESULTS:

The In-vitro irritation potential of the products analyzed in this study were mentioned in Table 1 and Table 2. All the eye drops were tested by the HET-CAM method, the product C008 was found to be moderately irritating and the products C001, C003, C006, C009, C010, C011, C015 were classified as slight irritants and mentioned in figures below.

 

This classification was done based on the calculated HET- CAM scores by using the formula. Using these values ONE WAY ANOVA7,8,9,10,11 was done by comparing the individual time in seconds obtained for each parameter with that of positive controls (0.1M NaOH). Each trial of hemorrhage time of each sample was compared with the time obtained for the positive control (0.1M NaOH). Likewise, same was done for vascular lysis time and coagulation time. All the three HET-CAM scores were also calculated by ONE WAY ANOVA and it is mentioned in the figure 9 and significance values were calculated and reported.

 

Figure.1  C001  weak irritant         Figure.2 C003 weak irritant           Figure.3  C006 weak irritant            Figure.4 C009 weak irritant

 

Figure.5  C010  weak irritant         Figure.6 C011 weak irritant        Figure.7  C013 weak irritant            Figure.8 C008 moderately irritant

 

Table 1: Results of the HET-CAM Analysis

Code of the Formulation

Hemorrhage Time (s)

Vascular Lysis Time (s)

Coagulation Time (s)

HET CAM Score

Results

C001

224.6

232

243.3

3.426

weak irritant

C002

278.6

286.6

295

0.719

non irritant

C003

225.6

231.6

239

2.548

weak irritant

C004

280.6

281.6

292.3

0.866

non irritant

C005

284.6

287

296.6

0.626

non irritant

C006

224

232.6

240.6

3.479

weak irritant

C007

285

288.6

296

0.653

non irritant

C008

180.3

189.6

223.3

5.212

moderate

C009

222

232.6

257.6

3.538

weak irritant

C010

222

230.6

241

3.528

weak irritant

C011

223

232

241.3

3.535

weak irritant

C012

286

288.6

291

0.644

non irritant

C013

223.6

234

240.6

3.468

weak irritant

C014

288

291

292

0.334

non irritant

C015

222.3

232.3

240.3

3.475

weak irritant

NEGATIVE CONTROL

289.3

298

301.6

0.215

non irritant

POSTIVE CONTROL

40.3

49.6

70.3

12.387

strong irritant

 

0 TO 0.9

NON IRRITANT

1 TO 4

WEAK IRRITANT

 5 TO 8.9

MODERATE IRRITANT

9 TO 21

STRONG IRRITANT

 

Table 2: Significance value for all the results obtained from the analysis

S. No.

Parameter

Trial No. and Corresponding Fig. No. in brackets

P value < 0.005

1.

Hemorrhage time (s)

Trial-01(4)

0.0041

 

 

Trial-02(5)

0.0042

 

 

Trial-03(6)

0.0041

2.

Vascular Lysis Time (s)

Trial-01(7)

0.0043

 

 

Trial-02(8)

0.0044

 

 

Trial-03(9)

0.0044

3.

Coagulation Time (s)

Trial-01(10)

0.0044

 

 

Trial-02(11)

0.0041

 

 

Trial-03(12)

0.0043

4.

HET CAM Score

Trial-01(13)

0.0044

 

 

Trial-02(14)

0.0043

 

 

Trial-03(15)

0.0044

 

 

Average(16)

0.0043

 

Figure. Average of All Three HET CAM Score

 

DISCUSSIONS:

For validation, new toxicological test methods must demonstrate their reliability, which means repeatable and reproducible, and that they are relevant, which means the method is anticipating and has a biological basis for the stated purpose12. Assessment of eye irritation can be done by Draize test – apart from its limitations the only test accepted by the international regulatory agencies and no In-vitro test has successfully been validated to fully replace it for regulatory purposes13.

 

However, to implement this type of approach, it would be necessary that international regulatory authorities accept the scientific validity of assays capable of accurately identifying negative results, without enforcing the need for confirmatory animal results14.

 

CONCLUSION:

Ophthalmic products were analyzed by using the HET-CAM assay showed varied results from moderately irritant to slight and non-irritant. Results suggests that the formulation C008 shown to be a moderate irritant. Similarly the products C001, C003, C006, C009, C010, C011 and C015 were classified as slight irritants and the products C002, C004, C005, C007, C012, C014 were classified as non-irritants. Ophthalmic products basically should be non- irritating as they are meant to be. But these products analyzed have been proven to show irritation. All these should be definitely due to improper fulfillment of the storage condition as they are specially designed products which are to be stored under special condition. However, for water insoluble substances, it might be applicable when combining with another In-vitro method, provided that regulatory acceptance is achieved. Therefore, the number of test animals can be reduced or replaced. At present, no alternative In-vitro test for ocular irritation are available for regulatory purposes. Neverthless many In-vitro methods are applied in-house for laboratories around the world. So, hereby we conclude that in future a battery of tests should be established, as no single assay can fulfill the requirements for risk assessment using an In-vitro method.

 

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Received on 03.05.2020           Modified on 30.06.2020

Accepted on 05.08.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(6):3063-3066.

DOI: 10.52711/0974-360X.2021.00535