Method for evaluating eye irritation of cosmetics

Abstract

The invention provides a method for evaluating eye irritation of cosmetics, and relates to the technical field of detection. The method comprises the following steps: firstly, acquiring a first image of a chick embryo chorioallantoic membrane before contacting a to-be-detected sample; removing the to-be-detected sample contact film after a preset time to obtain a second image; performing color channel separation on the two images, and selecting a green channel image to perform gray value analysis to obtain a first gray value and a second gray value; and finally evaluating the eye irritation level of the sample to be detected according to the gray value difference. According to the method disclosed by the invention, quantitative evaluation on eye irritation is realized by utilizing the optical characteristic of chick embryo blood vessels to green light, the subjective defect that a traditional chick embryo chorioallantoic membrane test depends on visual inspection is overcome, the objectivity, accuracy and repeatability of a detection result are remarkably improved, the operation is simple and convenient, and animal ethical dispute is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a method for evaluating eye irritation of cosmetics. BACKGROUND

[0002] Cosmetics, as consumer products that directly contact the skin and mucous membranes of the human body, its safety evaluation is an important link to protect the health of consumers. The eye tissue has a high sensitivity to external irritants due to its fragile structure and rich nerve endings, and is a high-incidence site of adverse reactions of cosmetics, so the eye irritation evaluation plays an important role in the safety evaluation of cosmetics.

[0003] The traditional eye irritation evaluation mainly relies on the Draize rabbit eye test, which evaluates the irritation of the sample by observing the damage reactions of the cornea, iris and conjunctiva after the sample is dropped into the conjunctival sac of the rabbit eye. However, the Draize rabbit eye test has many problems: first, the method needs to use live animals, which has obvious animal ethics controversy and is contrary to the "3R principle" (replacement, reduction, optimization) advocated by modern toxicology; second, there are limitations in the extrapolation of the test results due to the interspecies differences in anatomical structure and physiological characteristics between rabbit eyes and human eyes; in addition, the scoring results of the method depend on the subjective judgment of the test personnel, and the repeatability and consistency are difficult to guarantee.

[0004] In order to overcome the above defects of the Draize rabbit eye test, researchers have developed various in vitro alternative detection methods. The cell model method uses human corneal epithelial cells or immortalized cell lines for cell viability detection, and the irritation is characterized by the toxicity of the sample to the cells, but this method cannot simulate the barrier function and dynamic microenvironment of the cornea. Organ model such as bovine corneal epithelial model evaluates the penetration and damage ability of the sample by in vitro corneal culture, but the bovine cornea is limited in source and difficult to meet the needs of large-scale detection. The recombinant three-dimensional corneal model is based on human keratinocytes and can simulate the layered structure of the corneal epithelium, but it also cannot completely reproduce the barrier function and dynamic microcirculation of the cornea. The computational simulation method based on quantitative structure-activity relationship or machine learning relies on the known compound database, which is insufficient for new structural components, complex formula products and samples with unknown ingredients.

[0005] The chicken embryo chorioallantoic membrane test has been widely used in the in vitro evaluation of eye irritation due to its similar vascular structure characteristics to the cornea. The existing chicken embryo chorioallantoic membrane test methods mainly include the HET-CAM method and the CAMVA method. The common feature of these methods is that the test personnel observe the bleeding, vascular fusion and coagulation reactions of the chicken embryo chorioallantoic membrane after contacting the test sample by naked eye, and then score according to the observation results, and further determine the irritation grade of the sample. However, due to the differences in observation and understanding of the same reaction phenomenon by different test personnel, the scoring results inevitably have subjectivity, which affects the objectivity and repeatability of the test results. SUMMARY

[0006] The purpose of the present application is to provide a method for evaluating the eye irritation of cosmetics, which can objectively and quantitatively evaluate the eye irritation of cosmetics.

[0007] To achieve the above-mentioned purpose, the present application realizes the following technical solutions: A method for evaluating the eye irritation of cosmetics, comprising the following steps: S1: obtaining a first image of the chicken embryo chorioallantoic membrane before contacting the test sample; S2: contacting the test sample with the chicken embryo chorioallantoic membrane, removing the test sample after a predetermined time of contact, and obtaining a second image of the chicken embryo chorioallantoic membrane; S3: respectively analyzing the first image and the second image for gray value, and obtaining a first gray value and a second gray value; S4: evaluating the eye irritation grade of the test sample according to the difference between the first gray value and the second gray value.

[0008] Further, the step S3 comprises: Separating the first image and the second image for color channel respectively, and obtaining the corresponding monochrome channel images respectively; Analyzing the monochrome channel images for gray value, and obtaining the first gray value and the second gray value.

[0009] Further, the color channel separation is to separate the image into a red channel image, a green channel image and a blue channel image; and the gray value analysis is performed by using the green channel image.

[0010] Further, the first image and the second image have the same shooting area and the same shooting parameters.

[0011] Further, in the step S2, the chicken embryo chorioallantoic membrane is continuously recorded by video during the contact of the test sample with the chicken embryo chorioallantoic membrane.

[0012] Further: In step S2, the preset time is 3-10 minutes.

[0013] Further: In step S2, the sample to be tested is removed by rinsing the surface of the chicken embryo chorioallantoic membrane with physiological saline.

[0014] Further: when the sample to be tested is a liquid, the contact volume is 200-400 μL; when the sample to be tested is a semi-solid or solid, the contact volume is 200-400 mg.

[0015] Furthermore, the chicken embryo chorioallantoic membrane is derived from fertilized chicken embryos that have been incubated for 8-10 days.

[0016] Furthermore, the eye irritation level includes no / mild irritation, moderate irritation, and severe irritation.

[0017] Furthermore, multiple chicken embryo chorioallantoic membranes were used to conduct parallel tests on the same test sample, and the average value of the grayscale difference obtained from each parallel test was used as the basis for evaluating the eye irritation level of the test sample.

[0018] Furthermore, the number of the plurality of chicken embryo chorioallantoic membranes is 6-10.

[0019] Compared with the prior art, the present invention has the following advantages: I. This invention analyzes the grayscale values ​​of images of chicken embryo chorioallantoic membrane before and after contact with the test sample, and quantitatively assesses the eye irritation level of the test sample based on the changes in grayscale values. This overcomes the shortcomings of traditional chicken embryo chorioallantoic membrane test methods that rely on the naked eye observation and subjective scoring by the test personnel. The assessment results are not dependent on the experience and subjective judgment of the test personnel, which significantly improves the objectivity, accuracy and repeatability of the test results.

[0020] Second, this invention uses color channel separation technology to select the green channel image for grayscale value analysis. Since the blood vessels of chicken embryos have special optical properties to green light, when the chicken embryo chorioallantoic membrane undergoes irritation reactions such as hemorrhage, vascular lysis or coagulation, the grayscale value change of the green channel can sensitively reflect these changes, thereby achieving accurate quantitative assessment of eye irritation.

[0021] Third, this invention uses fertilized chicken embryos as experimental materials, which, compared to the Draize rabbit eye test, does not require the use of live animals, meets the requirements of modern toxicology, and avoids animal ethics controversies; compared to the bovine corneal epithelial model, chicken embryo materials are widely available and inexpensive, making them easy to apply on a large scale; compared to computational simulation methods, this invention can directly test complex mixture samples, making it more applicable.

[0022] Fourth, the method of the present invention is simple to operate, requiring only conventional image acquisition equipment and image analysis software, without the need for complex cell culture conditions or expensive special detection instruments, and is easy to promote and apply. Attached Figure Description

[0023] Figure 1 This is a schematic flowchart of a method for assessing the eye irritation of cosmetics according to the present invention; Figure 2 This is a comparison image of chicken embryo chorioallantoic membrane before and after contact with sample 1 in test case 1; Figure 3 This is a comparison image of chicken embryo chorioallantoic membrane before and after contact with sample 2 in test case 2; Figure 4 This is a comparison image of chicken embryo chorioallantoic membrane before and after contact with sample 3 in test case 3; Figure 5 This is a comparison image of chicken embryo chorioallantoic membrane before and after contact with sample 4 in test case 4; Figure 6 This is a comparison image of chicken embryo chorioallantoic membrane before and after contact with sample 5 in test case 5. Detailed Implementation

[0024] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] This invention provides a method for assessing the eye irritation of cosmetics. The method uses chicken embryo chorioallantoic membrane to simulate the cornea. By analyzing the gray value changes of images before and after the chicken embryo chorioallantoic membrane comes into contact with the sample, the eye irritation level of the sample can be objectively and quantitatively assessed.

[0026] The specific steps of the method of the present invention are as follows: S1: Obtain the first image of the chicken embryo chorioallantoic membrane before it comes into contact with the sample to be tested.

[0027] Specifically, first, prepare the chorioallantoic membrane of the chicken embryo. Select fertilized chicken embryos that have been incubated for 8-10 days, preferably fertilized chicken embryos that have been incubated for 9 days. At this time, the vascular network of the chorioallantoic membrane is fully developed, and the membrane structure is intact, making it suitable for eye irritation testing. Place the fertilized chicken embryo with the air cell facing upwards, and carefully peel off the eggshell at the air cell and the white membrane underneath to expose the chorioallantoic membrane. During the operation, care should be taken to avoid damaging the chorioallantoic membrane and its blood vessels.

[0028] Next, acquire the first image of the chicken embryo chorioallantoic membrane before it comes into contact with the sample to be tested. Place the egg with the exposed chorioallantoic membrane under a stereomicroscope and take a photograph using an electron eyepiece or other image acquisition device as the first image. Uniform lighting conditions and shooting parameters, including light source intensity, exposure time, and white balance settings, should be used during shooting to ensure the accuracy and comparability of subsequent image analysis.

[0029] S2: The sample to be tested is brought into contact with the chicken embryo chorioallantoic membrane. After a preset contact time, the sample to be tested is removed, and a second image of the chicken embryo chorioallantoic membrane is obtained.

[0030] Specifically, the sample to be tested is brought into contact with the chorioallantoic membrane of the chicken embryo. A fixed amount of the sample is directly dropped or coated onto the surface of the chorioallantoic membrane. When the sample is liquid, the contact volume is 200-400 μL, preferably 300 μL; when the sample is semi-solid or solid, the contact volume is 200-400 mg, preferably 300 mg. After the sample comes into contact with the chorioallantoic membrane, the positions of the egg and the image acquisition device remain unchanged, and the camera mode can be turned on simultaneously for continuous recording to observe the dynamic changes of the chorioallantoic membrane during the sample contact process and to ensure the consistency of the imaging areas before and after the contact.

[0031] The contact time between the sample and the chicken embryo chorioallantoic membrane is a preset time, which is 3-10 minutes, preferably 5 minutes. A preset time that is too short may result in an insufficient irritant response, while a preset time that is too long may affect the accuracy of the evaluation results due to factors such as sample volatilization or permeation.

[0032] After the preset time has elapsed, remove the sample to be tested. The preferred method of removal is to gently rinse the surface of the chicken embryo chorioallantoic membrane with physiological saline to completely remove any remaining sample. Rinsing with physiological saline effectively removes the sample without causing additional irritation or damage to the chicken embryo chorioallantoic membrane.

[0033] Immediately after removing the sample, acquire a second image of the chicken embryo chorioallantoic membrane. The same shooting parameters and shooting area should be used when capturing the second image as when capturing the first image to ensure comparability between the two images.

[0034] S3: Perform grayscale analysis on the first image and the second image respectively to obtain the first grayscale value and the second grayscale value.

[0035] Specifically, after acquiring the first and second images, grayscale value analysis is performed on both images. Specifically, the first and second images are separated into color channels, that is, the color image is separated into a red channel image, a green channel image, and a blue channel image. Then, the green channel image is selected for grayscale value analysis. The reason for selecting the green channel for analysis is that chicken embryo blood vessels have special optical properties to green light. When the chorioallantoic membrane of the chicken embryo undergoes irritative reactions such as hemorrhage, vascular lysis, or coagulation, the grayscale value change of the green channel is the most significant, and it can sensitively reflect these changes.

[0036] Image analysis software was used to calculate the average grayscale value of the green channel images of the first image and the second image, respectively, and these values ​​were denoted as the first grayscale value. Second grayscale value Then calculate the difference between the first grayscale value and the second grayscale value. The calculation formula is: .

[0037] S4: Evaluate the eye irritation level of the sample under test based on the difference between the first gray value and the second gray value.

[0038] Specifically, based on the difference in grayscale values The size of the sample is used to assess its eye irritation level. This invention classifies eye irritation into three levels: no / mild irritation, moderate irritation, and severe irritation. When the chorioallantoic membrane of a chicken embryo is stimulated, resulting in bleeding or vascular damage, the distribution range of hemoglobin changes, causing a change in the grayscale value of the green channel in the image. The larger the absolute value of the grayscale difference, the greater the vascular change, the more pronounced the irritation reaction, and the stronger the eye irritation of the sample.

[0039] To improve the accuracy and reliability of the evaluation results, this invention uses multiple chicken embryo chorioallantoic membranes for parallel testing of the same sample. The number of chicken embryo chorioallantoic membranes used in parallel testing is 6-10, and each chicken embryo chorioallantoic membrane generates a grayscale value difference. The average of the grayscale value differences obtained from all parallel tests is used as the final basis for evaluating the eye irritation level of the sample under test.

[0040] The following experimental examples further illustrate the method and effects of the present invention.

[0041] Case Study 1: Assessment of Ocular Irritation of Sample 1 like Figure 2As shown, the method of this invention was used to assess the eye irritation of sample 1. A first image was acquired before the chicken embryo chorioallantoic membrane came into contact with sample 1. After color channel separation of the first image, the average gray value of the green channel image was analyzed; the first gray value G1 was 58.503. After 300 μL of sample 1 was contacted with the chicken embryo chorioallantoic membrane for 5 minutes, it was rinsed with physiological saline, and a second image was acquired. The average gray value of the green channel image was analyzed; the second gray value G2 was 56.286. The difference in gray values ​​was calculated. =G2-G1=56.286-58.503=-2.217. Based on this difference in grayscale values, the eye irritation level of sample 1 is determined to be non-irritating / mildly irritating.

[0042] Case Study 2: Assessment of Ocular Irritation in Sample 2 like Figure 3 As shown, the eye irritation of sample 2 was assessed using the method of this invention. The average gray value G1 of the green channel in the first image was 55.711, and the average gray value G2 of the green channel in the second image was 52.763. The difference in gray values... =52.763-55.711= -2.948, the eye irritation level is determined to be no / mild irritation.

[0043] Case Study 3: Assessment of Ocular Irritation in Sample 3 like Figure 4 As shown, the method of this invention was used to assess the eye irritation of sample 3. The average gray value G1 of the green channel in the first image was 50.949, and the average gray value G2 of the green channel in the second image was 44.291. The difference in gray values... =G2-G1=44.291-50.949=-6.658. Based on this difference in grayscale values, the eye irritation level of sample 3 is determined to be moderate.

[0044] Case Study 4: Assessment of Ocular Irritation in Sample 4 like Figure 5 As shown, the method of this invention was used to assess the eye irritation of sample 4. The average gray value G1 of the green channel in the first image was 62.621, and the average gray value G2 of the green channel in the second image was 52.530. The difference in gray values... =G2-G1=52.530-62.621=-10.091. Based on this difference in grayscale values, the eye irritation level of sample 4 is determined to be moderate.

[0045] Case Study 5: Assessment of Ocular Irritation in Sample 5 like Figure 6As shown, the method of this invention was used to assess the eye irritation of sample 5. The average gray value G1 of the green channel in the first image was 60.037, and the average gray value G2 of the green channel in the second image was 38.380. The difference in gray values... =G2-G1=38.380-60.037=-21.657. Based on this difference in grayscale values, the eye irritation level of sample 5 is determined to be severe irritation.

[0046] As can be seen from the above experimental examples, the method of the present invention can objectively distinguish samples with different levels of irritation by the magnitude of the difference in grayscale values. The smaller the absolute value of the grayscale value difference, the lower the eye irritation of the sample; the larger the absolute value of the grayscale value difference, the stronger the eye irritation of the sample. Samples 1 and 2 have relatively small absolute values ​​of grayscale value difference, and are judged to be non-irritating / mildly irritating; samples 3 and 4 have moderate absolute values ​​of grayscale value difference, and are judged to be moderately irritating; sample 5 has the largest absolute value of grayscale value difference, and is judged to be severely irritating. This result indicates that the method of the present invention can effectively and objectively assess the eye irritation of cosmetics.

[0047] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for assessing the eye irritation of cosmetics, characterized in that, Includes the following steps: S1: Acquire the first image of the chicken embryo chorioallantoic membrane before it comes into contact with the sample to be tested; S2: The sample to be tested is brought into contact with the chicken embryo chorioallantoic membrane. After a preset contact time, the sample to be tested is removed, and a second image of the chicken embryo chorioallantoic membrane is obtained. S3: Perform grayscale value analysis on the first image and the second image respectively to obtain the first grayscale value and the second grayscale value; S4: Evaluate the eye irritation level of the sample under test based on the difference between the first gray value and the second gray value.

2. The method according to claim 1, characterized in that, Step S3 includes: The first image and the second image are separated by color channels to obtain their respective monochrome channel images; Perform grayscale analysis on the monochrome channel image to obtain the first grayscale value and the second grayscale value.

3. The method according to claim 2, characterized in that, The color channel separation involves separating the image into a red channel image, a green channel image, and a blue channel image; the grayscale value analysis is performed using the green channel image.

4. The method according to claim 1, characterized in that, The first image and the second image are captured in the same area and with the same shooting parameters.

5. The method according to claim 4, characterized in that, In step S2, during the period when the sample to be tested is in contact with the chicken embryo chorioallantoic membrane, the chicken embryo chorioallantoic membrane is continuously photographed and recorded.

6. The method according to claim 1, characterized in that, In step S2, the preset time is 3-10 minutes.

7. The method according to claim 1, characterized in that, In step S2, the sample to be tested is removed by rinsing the surface of the chicken embryo chorioallantoic membrane with physiological saline.

8. The method according to claim 1, characterized in that, When the sample to be tested is a liquid, the contact volume is 200-400 μL; when the sample to be tested is a semi-solid or solid, the contact volume is 200-400 mg.

9. The method according to claim 1, characterized in that, The chicken embryo chorioallantoic membrane is derived from fertilized chicken embryos that have been incubated for 8-10 days.

10. The method according to claim 1, characterized in that, The eye irritation levels are categorized as no / mild irritation, moderate irritation, and severe irritation.

11. The method according to claim 1, characterized in that, Parallel tests were conducted on the same sample using multiple chicken embryo chorioallantoic membranes. The average of the grayscale differences obtained from each parallel test was used as the basis for evaluating the eye irritation level of the sample.

12. The method according to claim 11, characterized in that, The number of the plurality of chicken embryo chorioallantoic membranes is 6-10.

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