Abstract
As an alternative to the standard Draize eye irritation test, the potential irritancy of compounds was evaluated by observing adverse changes that occur in chorioallantoic membrane CAM) of the hen egg (HECAM) after exposure to a test chemical placed directly on the CAM. The occurrence of hemorrhage, coagulation, and lysis in response to a test compound is the basis for employing this technique to evaluate its potential for in vivo damage to mucous membrane, in particular the eye. Irritancy is scored according to the severity and speed at which damage occurs. In the present study, five different classes of pesticides were screened for irritation potential. There was good correlation between the HECAM assay and the in vivo Draize eye irritation test. The proposed HECAM assay, which reduces the requirement for laboratory animals, could be a painless alternative to the Draize test.
The toxicity of a pesticide is a measure of its capacity or ability to cause injury or illness. Hazard, or risk, of using pesticides is the potential for injury, or the degree of danger involved in using a pesticide under a given set of conditions. Hazard depends on the toxicity of the pesticide and the amount of exposure to the pesticide and is often illustrated with the following equation: Hazard = Toxicity × Exposure. Eyes are very sensitive to many pesticides and, considering their size, are able to absorb large amounts of chemical. Serious eye exposure can result from a splash, spill, or drift or by rubbing the eyes with contaminated hands or clothing. The Draize eye irritation test is one of the most criticized methods because of the injuries inflicted on the test animals. That is why several various tests have been developed to replace the rabbits in detecting the irritation potential of chemicals. The potential irritancy of compounds may be detected by observing adverse changes, which occur in the chorioallantoic membrane (CAM) of the hen egg (HECAM) after exposure to test chemicals (Tavaszi and Budai 2006). The CAM of the developing chicken egg is considered to be a suitable model to study irritation of mucous tissues (Leighton, Nassauer, and Tchao 1985; Spielmann et al. 1997). The HECAM test method was developed by Luepke (1985) and Luepke and Kemper (1986). The CAM is a vascularized respiratory membrane that surrounds the embryonic bird within an egg. The test method is based on the observation that the CAM of an embryonated hen’s egg is similar to the vascularized mucosal tissues of the eye. Developers of the test method assumed that acute effects induced by a test substance on the small blood vessels and proteins of this soft tissue membrane would be similar to the effects induced by the same test substance in the eye of a treated rabbit. Thus, it was proposed that adverse effects on the CAM induced by a test substance would correlate to irritation and/or corrosion in human eyes (National Toxicology Program 2006).
The CAM has been proposed as a model for a living membrane (such as the conjunctiva) because it comprises a functional vasculature. Additionally evaluation of coagulation (i.e., protein denaturation) may reflect corneal damage that may be produced by the test substance. So far, only a small number of approaches using the CAM have been described to assess the biocompatibility of chemicals and medical devices. In present study, three different categories of five pesticides were selected to evaluate irritation potential by using the HECAM test. Bronopol is used as a microbiocide/microbiostat in oil field systems, air washer systems, air-conditioning/humidifying systems, cooling water systems, paper mills, absorbent clays, and metal working fluids. Tetraconazole, fenbuconazole, mancozeb were used as fungicide for different crop protection. Paclobutrazole is used as plant growth regulator.
MATERIALS AND METHODS
The White Leghorn chicken day 9 eggs were obtained from Poultry Research Centre, Chennai. The positive controls, 0.1 N NaOH and 1% sodium dodecyl sulfate (SDS), and vehicle (negative) control, 0.9% NaCl solution, were freshly prepared in distilled water before the start of each assay.
Five technical grade pesticides were used in the present study to evaluate the irritation potential. The details of these pesticides were given in Table 1 (Tomlin 2003). The controls and test solution (in the appropriate solvents) were freshly prepared before each assay at room temperature. The pH was recorded for each solution. Ten grams of each test chemical was dissolved in 0.9% NaCl to obtain a 10% solution prior to application (Kalweit, Gerner, and Spielmann 1987; Vives et al. 1997).
Preparation of Eggs
Each egg (on day 9) was tested with candling light to ensure that all were viable (Leupke 1985; Kalweit, Gerner, and Spielmann 1987; Sterzel et al. 1990; CEC 1991; Spielmann 1995). White Leghorn chicken has been selected because the eggs of this breed yield very consistent and reproducible results, and there do not appear to be any hereditary defects in this breed. Cold lamp was used to ensure optimal illumination of the chorioallantoic membrane. On day 10, the air cell was marked with pencil, using a rotating dentist saw blade to remove the section of shell off. The membrane was carefully moistened with 0.9 % NaCl solution at 37°C. The eggs were replaced in the incubator until ready for assay (maximum of 30 min between opening the eggs and starting the assay). The moistening solution (0.9% NaCl) was poured off from the opened egg and carefully the membrane was removed (without injuring any underlying blood vessels) using tapered forceps.
To summarize, the preparation proceeds as follows:
Identify air chamber
Mark air chamber
Open chamber with rotating dentist saw blade
Remove egg shell
Moisten egg skin (0.9% NaCl)
Carefully remove egg skin
Exposed membrane (CAM)
Test Procedure
The CAM of each egg was applied directly with 0.3 ml of the positive/negative control or test chemical (Kalweit, Gerner, and Spielmann 1987; Vives et al. 1997). Two eggs for controls and three eggs for test substance were used for each assay. The reactions for hemorrhage, coagulation, (Blein et al. 1991; Hagino et al. 1991, 1993, 1999; Bagley et al. 1992; Rougier et al. 1992; de Silva, Rougier, and Dossou 1992; Kojima et al. 1995; Doucet, Lanvin, and Zastrow 1999), and lysis (Luepke and Kemper 1986; Sterzel et al. 1990; CEC 1991; Spielmann 1995; Macian et al. 1996; Gettings et al. 1996; Budai and Varnagy 2000) on the CAM were observed over a period of 5 min (Kalweit, Gerner, and Spielmann 1987, 1990; Sterzel et al. 1990; CEC 1991; Spielmann et al. 1991; Macian et al. 1996; Spielmann et al. 1996; Gilleron et al. 1997; Schlage, Bulles, and Kurkowsky 1999).
The time for each reaction was recorded in seconds and calculated an irritation score (IS) using the formula below (Kalweit, Gerner, and Spielmann 1987, 1990):
where H = hemorrhage; L = vessel lysis; C = coagulation; Sec = start time (seconds).
The irritation threshold (IT) is defined as the highest concentration of the test solution, at which slight reactions occur after 5 min of application. Prior to determination of irritation potential, irritation threshold was calculated (Spielmann et al. 1993; Spielmann 1995). The irritation threshold was determined at different concentrations for tetraconazole (5% and 10%), bronopol (5% and 10%), mancozeb (10%), fenbuconazole (10%) and paclobutrazole (10%). The irritation threshold was determined by applying 0.3 ml of the test chemical to the CAM of three eggs (Luepke 1985; Kalweit, Gerner, and Spielmann 1987; CEC 1991; Hagino et al. 1991; Gettings et al. 1994). After the treatment time of 5 min, the main reaction was scored (either hemorrhage or lysis, or coagulation) according to the following scheme: 0 = no reaction; 1 = slight reaction; 2 = moderate reaction; 3 = severe reaction.
The tests were done in triplicate for each chemical. Mean score of the three eggs was determined (Balls et al. 1995; Steiling et al. 1999).
At the end of each assay the embryos were killed quickly by placing the eggs into a freezer at −20°C.
RESULTS
Irritation Threshold
Tetraconazole (10%) showed slight reaction on CAM and the mean score obtained from three assays was 1.44. The mean score obtained for the 5% solution was less than 1 and hence the test concentration was doubled to the 10% concentration.
Bronopol 10% concentration showed severe reaction, with mean score 3.0. The 5% concentration of bronopol exhibited moderate reaction, with mean score 2.33.
The test chemicals mancozeb, fenbuconazole, and paclobutrazole at 10% concentrations showed no reaction, and the mean irritation threshold and irritation score determined for these test chemicals was 0 (Table 2).
Irritation Score
Prior to the determination of irritation score, the results obtained for irritation threshold was considered. The test chemical concentrations were selected based on the irritation threshold results obtained. All the three end points, hemorrhage, coagulation, and lysis, were observed for bronopol at 173, 15.3, and 11 s, respectively. The mean irritation score for bronopol is 17.5, whereas tetraconazole showed hemorrhage alone at 85.6 s after application of test chemical on the CAM. The test chemicals mancozeb, fenbuconazole, and paclobutrazole showed 0 irritation score (Table 3).
Positive Controls
The positive controls, 0.1 N NaOH and 1% SDS (Reinhardt et al. 1987; Sterzel et al. 1990; Spielmann 1995; Macian et al. 1996; Spielman et al. 1997; Vives et al. 1997), exhibited various end points (hemorrhage, lysis, coagulation) at different times. The mean irritation score for 0.1 N NaOH and 1% SDS were 17.59 and 13.82, respectively.
Solvent Control
Because the test substances were dissolved in 0.9% NaCl (Vinardell and Macian 1994; Spielmann 1995; Gilleron et al. 1996; Macian et al. 1996; Boucet et al. 1999), the solvent was tested for irritation potential to see if any and nonspecific changes in the test system was observed (Table 4).
DISCUSSION
Draize rabbit eye test is the most vital test for determining eye irritation/corrosion of a test substance. In consideration of pain and distress to the animal, search for a painless alternative method was explored. HECAM was proposed to mimic the effects that may occur in the conjunctiva following exposure to a test substance. Chicken embryo models have long been used by embryo toxicologists and virologists (Parish 1985; Luepke and Kemper 1986). Extending the use of chicken embryos, the HECAM test was proposed by Luepke as an alternative in vitro method.
However, the use of a 9-day incubation period is based on chicken embryo development and international regulations did not considered the HECAM as an in vitro test. Later it was proposed that after incubation on day 9, the embryonic differentiation of the chicken central nervous system is sufficiently incomplete so that suffering from pain perception is unlikely to occur. Studies also have suggested that the extra embryonal vascular systems (yolk sac, CAM) are not sensitive to pain. Combined, these studies suggest that on day 9 of incubation, there is no pain perceived by the developing embryo during the HECAM test.
In this scenario, a lot of screening have been carried out by scientists on various categories of test chemicals such as cosmetic, toiletry, and fragrance products (Gettings et al. 1991, 1994, 1996); raw materials used in cosmetics/toiletries and household cleaning products (Bagley et al. 1992); and major ingredients used in cosmetic formulations, which include surfactants and solvents (Kojima et al. 1995). Various broad-spectrum ocular irritants, chemical classes, chemical structures, including alcohols, amines, carboxylic acid, heterocyclic (Gilleron et al. 1996, 1997), were also evaluated.
Therefore, with the advent of the in vitro HECAM assay, considerable work was done on many classes of products, such as solvents, shampoos, surfactants, cosmetics, ocular irritants, etc. However, data on pesticides are rather scanty when compared to other product classes. Pesticides’ ocular irritation potential is routinely evaluated using whole animal model. There is no validated in vitro alternative method to screen the pesticides for eye irritation.
In the present study, a good correlation was found between the HECAM test results and reported data from Draize eye irritation test (Table 5).
For the test chemical tetraconazole, reported in vivo data revealed slight irritant to the eye of the rabbits. Similarly, results obtained from the HECAM test revealed moderate irritancy. Bronopol is a mild irritant to the eye of rabbits in the Draize in vivo test. The HECAM test results for this chemical also showed it as an irritant.
Mancozeb and Fenbuconazole gave nonirritant results in both in vivo and in vitro HECAM tests. For paclobutrazole, reported in vivo results showed that it is a moderate irritant to rabbit eye, whereas according to the HECAM test, it was nonirritant. The HECAM test method has the potential to refine or reduce animal use in eye irritation testing.
Substances that are identified as ocular corrosives or severe irritants would be excluded from testing in vivo, which would reduce the number of rabbits used for ocular testing. The HECAM method also would spare animals the pain and distress of exposure to severe eye irritants.
The HECAM test could reduce the time needed to assess a substance, when compared to the currently accepted in vivo rabbit eye test method. The current cost of a Good Laboratory Practice (GLP)-compliant EPA OPPTS Series 870 Acute Eye Irritation (EPA 1998) or Organization for Economic Cooperation and Development (OECD) Test Guideline (TG) 405 test (OECD 2002) are expected to be more than the cost of an in vitro HECAM test. The results from the present study warrant considering HECAM as a prescreening test for pesticides prior to in vivo rabbit study.
Footnotes
Tables
Acknowledgements
The authors are greatful to the IIBAT management for providing support for this work.