Eye irritation test is the main test item for cosmetic safety evaluation. Local eye irritation caused by cosmetics usually includes the effects of conjunctiva, cornea, iris and lacrimal gland. Based on the mechanism of eye irritation in vivo, many alternative methods have been developed. Therefore, according to the mechanism of corneal injury, combined with the principle of in vitro methods and the degree of standardized application, Guangmann provides customers with the most professional eye irritation test program according to the different needs of customers.
一．Hen's Egg Test on the Chorioallantoic Membrane ( HET-CAM )
A.Purpose
Test the test substance's ability to cause toxic changes in the chick ambryonal membrane to objectively assess the degree of potential eye irritation of the substance.
B.Experimental Principle
Test of chick ambryonal membrane is an early in vitro evaluation method for eye irritation. It is an alternative method developed using the characteristics of chick ambryonal membrane structure similar to human conjunctiva. The chorioallantoic membrane (CAM) is a respiratory membrane that surrounds the chicken embryo. In this test, the vascular system of the chorioallantoic membrane in the middle stage of hatched embryos is complete, clear and transparent. The test substance is directly contacted with the CAM membrane. After 5 minutes, the chorioallantoic membrane toxicity effect indicators (mainly: bleeding, coagulation) And vascular melting). These indicators reflect changes in the morphology, color, and permeability of blood vessels and vascular networks, as well as the degeneration of chorioallantoic membrane protein and the degree of damage, and then combine to get a score for evaluate the eye irritation of the test substance.
C. Scoring Standard
Table 8-4 HET-CAM score sheet
| Points |
0 |
1 |
2 |
3 |
| Bleeding |
No bleeding |
Mild bleeding: only small blood vessel bleeding and a small amount of bleeding (such as 0.5% TexaponASV, 5min action) |
Moderate bleeding: bleeding of small and large blood vessels, and obvious blood outflow (such as 1.0% TexaponASV, 5min action) |
Severe bleeding: almost all blood vessels are bleeding, and a large amount of blood flows out (such as 5% TexaponASV, acting for 5 minutes) |
| Blood clotting |
No clotting |
Mild coagulation: intravascular and/or extravascular mild coagulation, and/or mild turbidity of the CAM membrane (mild coagulation such as 0.2% NaOH solution for 5 minutes, mild turbidity such as 0.3% acetic acid for 5 minutes) |
Moderate coagulation: intravascular and/or extravascular moderate coagulation, and/or CAM membrane moderate turbidity (moderate coagulation such as 0.3% NaOH solution for 5 minutes, moderate turbidity such as 3% acetic acid for 5 minutes) |
Severe coagulation: intravascular and/or extravascular severe coagulation, and/or severe turbidity of the CAM membrane (severe coagulation such as 0.5% NaOH solution for 5 minutes, severe turbidity such as 30% acetic acid for 5 minutes) |
| Vascular melting |
No vessel fusion |
Mild blood vessel melting: only small blood vessels are melting (such as 0.5% TexaponASV, acting for 5 minutes) |
Moderate vascular melting: melting of small and large blood vessels (such as 1% TexaponASV, acting for 5 minutes) |
Severe vascular fusion: the large blood vessels and all blood bundles are fused (such as 5% TexaponASV, acting for 5 minutes) |
D. Score Drawing
-
a*The blood vessels are clearly visible and normal.
-
a*The blood vessels are clearly visible and normal.
-
-
d* Blood vessel coagulation.
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e* The blood vessels were severely melted and the CAM membrane was cloudy.
E. Irritation Evaluation
Table 1. Evaluation of stimulus score results
| IS |
Prediction classification |
| IS＜1 |
Non-irritant（NI） |
| 1≤IS＜5 |
Slightly irritant（SI） |
| 5≤IS＜9 |
Moderately irritant（MI） |
| IS≥10 |
Strong irritant/Corrosive（Co） |
Endpoint Scoring(ES)：Score of each embryo = the sum of the degree of bleeding, coagulation and vascular melting observed in each embryo.
Endpoint Score (ES) = the sum of the scores of 6 embryos.
Table 2. Evaluation of end point scoring method results
| Irritation classification |
No/light irritation |
Moderately irritating |
Intensity irritation/corrosive |
| Endpoint scoring |
ES≤12 |
12＜ES＜16 |
ES≥16 |
Ⅱ Chorioallantoic Membrane Vascular Assay（CAMVA）
It is a simplified HET-CAM method, which only observes the blood vessel changes (bleeding, vasolysis, and coagulation) after the test substance is exposed to CAM for 30 minutes, and calculates the concentration of the test substance that causes the above-mentioned damage in 50% of the chicken embryos. CAMVA method is suitable for the detection of mild to moderate eye irritation compounds, and can also correctly distinguish irritants from non-irritants.
