Method for evaluating irritation of compound to eyes and application of method

Abstract

The invention provides a method for evaluating irritation of a compound to eyes and application of the method, and belongs to the field of in-vitro detection. According to the method, an irritation screening test based on a TRPV1 ion channel is adopted, the final concentration of a fluorescent probe is 1.0-5.0 [mu] M during detection, the final concentration of cells is 0.8 * 10 < 5 >-1.5 * 10 < 5 > cells / mL, the incubation temperature is 30-40 DEG C, the incubation time is 25-35 min, the accuracy of the detection method is improved, and the method can be used for screening potential eye irritation substances.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro detection and relates to the evaluation of eye irritation, and in particular to a method for evaluating the eye irritation of a compound and its application. Background Art

[0002] The production and use of chemicals pose potential hazards and risks to human health. Therefore, chemical risk assessment and risk management are crucial. Eye irritation is an essential component of chemical risk assessment. The eye, the human organ of vision, possesses a complex structure. When chemicals act on the front of the eye, they can cause damage to the eyeball and mucous membranes. The conjunctiva is the body's most fragile mucous membrane, rich in microvessels and capillaries. The cornea, however, lacks blood vessels but contains numerous nerves, making it the most sensitive area to pain and touch. Therefore, consumer products that come into contact with the eye, including cosmetics, daily cleaning products, and pharmaceuticals, require eye irritation safety evaluation. The importance of eye irritation evaluation is also reflected in its central role in product quality assurance, quality control, and regulatory oversight. Cosmetics, particularly sunscreens, have extremely high safety requirements. Sunscreen products are primarily divided into two categories: physical and chemical. Physical sunscreens primarily contain physical sunscreens such as zinc oxide and titanium dioxide, which protect against UV damage by reflecting or scattering UV rays. These products are therefore considered safer. Chemical sunscreen products contain UV absorbers such as phenylbenzimidazole sulfonic acid, ethylhexyl methoxycinnamate, and homosalate. They absorb UV rays to reduce the damage they cause to the skin. However, excessive addition may cause skin irritation and easily lead to skin problems such as allergies.

[0003] Acute eye irritation evaluation is a key component of the pre-market safety assessment for cosmetics intended for use near the eye. Traditional acute eye irritation testing methods can be categorized into in vivo and in vitro testing, with the in vivo rabbit eye test (Draize test) being the gold standard. With the EU legislation banning animal testing for cosmetics and the growing acceptance of the 3Rs (Reduction, Refinement, and Replacement) principles by the industry and academia, developed countries such as the United States and Japan are accelerating the development of alternative eye irritation testing methods. Currently developed alternative methods include the bovine corneal opacity and permeability test (BCOP), the isolated chicken eye test (ICE), the rabbit corneal epithelial cell test (STE), the reconstituted corneal eye irritation test (RHE), and the chick embryo chorioallantoic membrane test (HET-CAM), which have been validated, evaluated, and applied. However, these single assessments cannot fully address the issue of eye irritation from sunscreen cosmetics, necessitating the development of new methods that can be combined to assess their effectiveness.

[0004] Currently established or under development, in vivo or in vitro eye irritation assessment methods primarily utilize cytotoxicity mechanisms, measuring acute cell damage or death caused by product exposure. In addition to cytotoxicity, other methods rely on inflammatory / immune response mechanisms (such as the chick embryo chorioallantoic membrane assay (HET-CAM), physical barrier damage mechanisms (the bovine corneal opacity and permeability assay (BCOP), and phototoxicity mechanisms (such as the ROS photochemical reaction assay). These objectively observable endpoints are, in fact, late in the irritation response. Conventional in vitro tests cannot reflect the subjective endpoint of stinging. Furthermore, rabbit eye irritation tests, limited by the inability of animals to report stinging, cannot reflect the product's stinging reactivity. While human testing can address these issues, it is expensive and poses ethical concerns. Therefore, establishing in vitro methods to assess the pain hazard of cosmetic products and ingredients based on pain-producing mechanisms could help guide the screening and development of products with lower eye irritation and milder properties, thereby meeting consumer safety needs, particularly the higher mildness requirements for sunscreen products.

[0005] In the process of pain generation and transmission, the starting molecular event of pain generation is the binding of the substance that causes pain to the pain receptor. Among them, the pain receptor mainly distributed in the sensory nerve fibers of the eye is transient receptor potential vanilloid type 1 (human transient receptor potential vanilloid type 1, hTRPV1), which is the molecular basis for the eye's abnormal sensitivity to external stimuli. TRPV1 is a receptor for capsaicin and is mainly distributed in neurons in the dorsal root ganglion and trigeminal ganglion. Along with the sensory nerve fibers of the ophthalmic branch of the trigeminal nerve, TRPV1 is widely distributed in eye structures such as the cornea, iris, conjunctiva, and lacrimal glands. Under the action of various physical and chemical factors, such as temperature, stimuli such as capsaicin, pH, etc., TRPV1 is activated, which can cause Ca 2+ Mg 2+ Plasma influx, which causes eye irritation, and shampoo ingredients in baby bath and shampoo products that cause eye stinging, can all cause eye irritation by stimulating TRPV1 receptors. Therefore, TRPV1 activity testing can reflect the effect and intensity of the test substance on eye irritation.

[0006] Chang Huailong et al. used the SH-SY5Y cell line stably transfected with the hTRPV1 receptor to evaluate the stinging risk of commercially available shampoo products, combining it with the chick embryo chorioallantoic membrane assay (HET-CAM) to assess irritation responses. This method uses a fluorescence microplate reader, which measures fluorescence from all cells. However, when using a large sample size or a short detection window, the microplate reader's accuracy and reproducibility are poor, making the reliability of the method difficult to guarantee (Chang Huailong, Yin Qingfei, Chen Tian. Evaluation of the Stinging Risk of Shampoo Products Based on Stably Transfected hTRPV1 Cell Lines [J]. Flavors & Fragrances, Cosmetics, 2021).

[0007] Prior art CN102137851B discloses a new compound, its isomer or pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same as a TRPV1 antagonist. The compound provided by the invention can be used to prevent or treat irritation of the skin, eyes or mucous membranes. The effect example measures the amount of Ca ion influx into neurons by counting radioactivity. The TRPV1 antagonistic activity of the test compound is calculated as the percentage inhibition of the maximum response of capsaicin at a concentration of 0.5 μM. The disadvantage of this technology is that it uses a radioactive ion detection method, which is costly and complicated to operate, and the accuracy has not been verified, resulting in poor reliability. Summary of the Invention

[0008] This invention addresses the poor accuracy of existing technologies by providing a method for evaluating the ocular irritation of compounds and its application. Based on a TRPV1 ion channel irritation screening test, the invention provides a method for evaluating the ocular sensory irritation of compounds. By clarifying specific detection parameters, the accuracy of the detection method is improved.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a method for evaluating the ocular irritation of a compound, the method comprising performing a TRPV1 ion channel irritation screening test, the method comprising the following steps: S1, culturing cells transfected with TRPV1 virus to obtain TRPV1 cells; S2. Add calcium ion fluorescent probe for incubation, wash, and resuspend to obtain TRPV1 cells bound to the fluorescent probe. The final concentration of the calcium ion fluorescent probe is 4.0 μM, and the final concentration of the cells is 1.0×10 5 / mL, the incubation temperature is 37°C, and the incubation time is 30min; S3, perform fluorescence measurement on TRPV1 cells combined with fluorescent probes, and collect the baseline fluorescence value of calcium ion signals, which is recorded as the baseline fluorescence value; S4. Add the sample to be tested to the TRPV1 cells combined with the fluorescent probe, collect the calcium ion signals of each area within the time interval, and record them as the sample fluorescence value; S5. Calculate the relative fluorescence intensity based on the baseline fluorescence value and the sample fluorescence value, and determine whether the sample to be tested is eye irritating based on the relative fluorescence intensity.

[0010] Preferably, the cells transfected with TRPV1 virus in step S1 are mammalian cells.

[0011] Preferably, the mammalian cells include SIRC cells, SH-SY5Y cells, HEK293 cells, mouse fibroblasts L929, human corneal epithelial cell line HCE-T or human keratinocytes HaCaT.

[0012] Preferably, the mammalian cell is a SIRC cell.

[0013] Preferably, the calcium ion fluorescent probe in step S2 includes Fluo-3 AM or Fluo-4 AM.

[0014] Preferably, the calcium ion fluorescent probe in step S2 is Fluo-3 AM.

[0015] Preferably, the concentration of the sample to be tested in step S4 is determined by a cytotoxicity screening test; the cytotoxicity screening test comprises the following steps: The cells transfected with TRPV1 virus were exposed to different concentrations of sample dilutions. The experimental group, positive control group, negative control group and solvent control group were designed. MTT staining and determination were performed after the exposure. The absorbance value was measured at a wavelength of 570nm, and the relative cell activity of different concentrations was calculated to screen out the appropriate sample concentration.

[0016] Preferably, the relative fluorescence intensity in step S5 is calculated as follows: After the test begins, without adding any sample, record the baseline fluorescence for 50 seconds and calculate the average baseline value; pause the measurement, remove the sample tube, add the sample to be tested, and measure for 204 seconds; calculate the difference between the calcium ion signal and the average baseline value within the 204-second interval as the relative fluorescence intensity.

[0017] Preferably, the criteria for determining whether the sample to be tested is eye irritating in step S5 are: A negative control group, an inhibitor group, and an agonist group were set up; compared with the negative control group, the relative fluorescence intensity of Ca ions in the inhibitor group was significantly decreased (p<0.05); compared with the negative control group, the relative fluorescence intensity of Ca ions in the agonist group was significantly increased (p<0.05); if the relative fluorescence intensity of Ca ions in the test sample was significantly increased compared with the negative control group (p<0.05), it means that the test sample has an eye irritation effect.

[0018] Preferably, the compound comprises a cosmetic and a cosmetic raw material; the cosmetic raw material comprises a sunscreen.

[0019] In another aspect, the present invention provides use of the above-mentioned evaluation method in eye irritation evaluation, including use in eye irritation evaluation of sunscreens.

[0020] In another aspect, the present invention provides an application of the above-mentioned evaluation method in the safety evaluation of a compound on human body.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an irritation screening method based on TRPV1 ion channel activation to evaluate the potential of compounds to cause eye stinging, that is, the sensory pain dimension. By clarifying specific detection parameters, the accuracy of the detection method is improved. It can be used to screen substances with potential eye stinging effects and is particularly suitable for the safety evaluation of products such as sunscreens. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The relative fluorescence intensity results of Example 1; compared with the negative control group, * represents P < 0.05, *** represents P < 0.001.

[0023] Figure 2 This is the fluorescence detection spectrum of Example 1.

[0024] Figure 3 The relative fluorescence intensity results of Example 2; compared with the negative control group, * represents P < 0.05, *** represents P < 0.001.

[0025] Figure 4 This is the fluorescence detection spectrum of Example 2.

[0026] Figure 5 This is the fluorescence detection spectrum of Comparative Example 1.

[0027] Figure 6 This is the fluorescence detection spectrum of Comparative Example 3. DETAILED DESCRIPTION

[0028] Unless otherwise specified, all raw materials and reagents used in the present invention were purchased from commercial suppliers, and experiments were performed according to the operating instructions. Unless otherwise specified, all instruments, equipment, devices, etc. used in the present invention were conventional instruments, equipment, devices, etc., and experiments were performed according to the operating instructions and supporting reagents.

[0029] Experimental methods: (1) BCOP test Negative control sample: sterile ultrapure water; Positive control sample: anhydrous ethanol, purchased from Guangzhou Chemical Reagent Factory, batch number: 20231001 01.

[0030] Preparation of cornea: Select an undamaged cornea and cut it 2-3 mm along the edge of the sclera. Fix the cornea on a preheated cornea holder with the surface facing up. Fill the anterior and posterior chambers with preheated phenol red-free MEM medium and equilibrate in a 32±1℃ incubator for 1-2 h.

[0031] Measurement of basal turbidity: After equilibration, remove the cornea from the incubator and replace the old culture medium in the anterior and posterior chambers with fresh phenol red-free MEM medium. Baseline turbidity values ​​of all corneas were measured using a BASF Opacitometer 3.0.

[0032] Sample addition: Three parallel corneas were used in each group. The anterior chamber fluid was removed, and 0.75 mL of test sample (including negative control, positive control, and test substance sample) was added to the epithelial side of the cornea. The test sample was then exposed in a 32 ± 1°C incubator for 10 min. After exposure, the test sample in the anterior chamber was rinsed at least three times with MEM medium containing phenol red until no sample residue was present. The anterior chamber was then filled with prewarmed MEM medium without phenol red and equilibrated in a 32 ± 1°C incubator for 2 h. The same procedure was used for the positive and negative control groups.

[0033] Measurement of turbidity values ​​after exposure: replace the old culture medium in the anterior and posterior chambers with fresh phenol red-free MEM culture medium, and measure the turbidity values ​​of all corneas again using a BASF Opacitometer 3.0 turbidity meter.

[0034] Permeability test: Remove the culture medium from the anterior chamber and add 1.0 mL of 4 mg / mL sodium fluorescein solution. Place the holder upright back into the incubator and incubate for 90 ± 5 min. Then, collect the culture medium from the posterior chamber and transfer 360 μL / well to a 96-well plate. Measure the absorbance at 490 nm.

[0035] Calculate the corresponding turbidity changes and OD values ​​for the test sample group, positive control group, and negative control group, and correct for them using the negative control group. Calculate the in vitro score using the following formula: In vitro score = average corrected turbidity value + 15 × average corrected OD value. Then, perform a categorical assessment. If the score is greater than 55, the test substance is considered positive for irritation. Specific criteria are shown in Table 1.

[0036] Table 1. Prediction models and classification

[0037] (2) STE test Negative control sample: PBS buffer without calcium and magnesium; Positive control sample: 0.01% SDS; Test substance samples: Take a certain amount of test substance powder and dissolve it in a solvent (anhydrous ethanol diluted 1000 times with sterile ultrapure water) to prepare test substance samples with different mass fractions; Solvent control sample: Anhydrous ethanol was diluted 1000 times with sterile ultrapure water.

[0038] For routine cell culture, cells with good growth and about 80% fusion were taken, digested and prepared into cell suspension, and the cells were counted and the cell concentration was adjusted to 3.0×10 4 / mL, mix thoroughly and plate on 96-well plates, 200 μL per well, culture routinely for 4 days, and change the medium one day before infection when the confluence is >80% under microscopic examination.

[0039] Sample exposure: discard the original culture medium in the wells, add 200 μL of test solution of different concentrations (the mass fraction of the test substance is 5% and 0.5%, respectively) to each well, and perform corresponding treatment on the positive control group, negative control group and solvent control group (no solution is added to the blank group), expose for 5 min, and perform three replicates for each group.

[0040] MTT staining and assay: After exposure, rinse with PBS 2-3 times, then add 200 μL of 0.5% MTT solution to each well and incubate in a 37°C 5% CO2 incubator for 2 h. Aspirate the MTT solution, add 200 μL of isopropyl hydrochloride for 60 min to extract formazan (operate in a dark room at room temperature), and then measure the absorbance at 570 nm using a microplate reader. The relative cell viability (%) of each well is determined according to the following formula:

[0041] Among them, OD570 sample is the absorbance at 570 nm measured by the test group, OD570 blank is the absorbance at 570 nm measured by the negative control group, and OD570 solvent is the absorbance at 570 nm measured by the solvent control group; If the cell viability of both the 5% test substance group and the 0.05% test substance group is not higher than 70%, the irritation of the test substance is determined to be positive. The specific criteria are shown in Table 2.

[0042] Table 2 Reaction classification of STE test method

[0043] (3) Analysis of calcium influx in TRPV1-SIRC cells Negative control: HBSS buffer, purchased from Wuhan Saiweier Biotechnology Co., Ltd., product number G4203-500ML.

[0044] Inhibitor: Manganese chloride, purchased from MCE, product number HY-109521A, dissolved in HBSS at a concentration of 2 mM.

[0045] Agonist: Ionomycin, purchased from MCE, product number HY-13434, dissolved in solvent (anhydrous ethanol diluted 1000 times with sterile ultrapure water) at a concentration of 10 μM.

[0046] Test substance sample: Take a certain amount of test substance, dissolve it in a solvent, and prepare test substance samples with different mass fractions.

[0047] 3.1 Cellular Virus Screening SIRC cells were purchased from Guangzhou Customs Technology Center at a passage number of 25. SIRC cells transfected with TRPV1 virus (purchased from Heyuan Bio) were seeded into 96-well plates and cultured in an incubator for 18-24 h until the cells reached 80% confluency.

[0048] The original culture medium was removed, and 100 μL of test samples of different concentrations were added to each well and exposed in the incubator for 24±0.5 h.

[0049] Add 20 μL of 0.5% MTT solution to each well and incubate at 37°C for 3 ± 0.5 h. Then remove the MTT solution and add 100 μL of DMSO to each well. Oscillate in the dark for 10-15 min, and measure the absorbance at 570 nm.

[0050] The cell activity of the negative control group (Control) was set as 100%, and the relative cell activity of each group was calculated:

[0051] 3.2 TRPV1 channel irritation screening test SIRC cells transfected with TRPV1 virus were collected, the cell suspension was seeded into 12-well plates, and cultured in an incubator for 18-24 h; Fluo-3 AM calcium ion fluorescent probe (purchased from Yisheng; product number 40703ES50) was added and incubated at 37°C for 30 min. The final concentration of the calcium ion fluorescent probe was 4.0 μM and the final concentration of the cells was 1.0×10 5 pieces / mL.

[0052] After incubation, cells were collected and transferred to a 1.5 ml tube, centrifuged at 10,000 g for 1 minute, and the supernatant was discarded. Wash the cells by adding 1 mL of PHF buffer and resuspend by pipetting up and down. Wash twice more and resuspend the cells in a final volume of 500 μL of PHF buffer. Perform the experiment within 1 hour.

[0053] After the test begins, without adding any sample, record the baseline fluorescence for 50 seconds and calculate the average baseline value. Pause the measurement, remove the sample tube, add the sample to be tested (controlled within 15 seconds), and measure for 204 seconds. Calculate the difference between the calcium ion signal and the average baseline value within the 204-second interval as the relative fluorescence intensity.

[0054] The standard for the test substance to have eye irritation: set up a negative control group, an inhibitor group, and an agonist group; compared with the negative control group (NC), the relative fluorescence intensity of Ca ions in the inhibitor group (I) was significantly decreased (p<0.05); compared with the negative control group (NC), the relative fluorescence intensity of Ca ions in the agonist group (A) was significantly increased (p<0.05), indicating that cells overexpressing TRPV1 virus can respond correctly to the compound. Under the premise that the relative fluorescence intensity of Ca ions in the test sample group is increased and significantly different from that in the negative control group (p<0.05), it means that the test substance has potential eye irritation.

[0055] Example 1: Eye irritation evaluation of test substances Test substance: Octocrylene, CAS number: 6197-30-4.

[0056] Here are the results: Table 3 BCOP test results

[0057] The BCOP test results are shown in Table 3. The GHS classification of the test sample "Octocrylene" is not classified and the irritation classification is non-irritating.

[0058] Table 4 STE test results

[0059] The STE test results are shown in Table 4. The eye irritation classification of the test sample "Octocrylene" is non-irritating or slightly irritating, and the GHS classification is not classified.

[0060] The results of calcium influx detection are shown in Table 5. Figure 1 and Figure 2 The relative fluorescence intensity of the sample "Octocrylene" at the test concentrations of 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL was significantly increased compared with the negative control group, indicating that it has potential eye irritation.

[0061] In summary, the test sample "Octocrylene" did not cause eye irritation based on histopathological changes, but caused eye stinging based on TRPV1-SIRC cell calcium influx analysis. Therefore, it can be considered that this sunscreen may cause consumer discomfort.

[0062] Table 5 Calcium influx detection results

[0063] Note: Compared with the negative control group, * represents P < 0.05, *** represents P < 0.001.

[0064] Example 2: Evaluation of eye irritation of test substances Test substance: Ethylhexyl methoxycinnamate, CAS number: 5466-77-3.

[0065] Here are the results: Table 6 BCOP test results

[0066] The BCOP test results are shown in Table 6. The GHS classification of the test sample "ethylhexyl methoxycinnamate" is not classified, and the irritation classification is non-irritating.

[0067] Table 7 STE test results

[0068] The STE test results are shown in Table 7. The eye irritation classification of the test sample "ethylhexyl methoxycinnamate" is non-irritating or slightly irritating, and the GHS classification is not classified.

[0069] The results of calcium influx detection are shown in Table 8. Figure 3 and Figure 4 : The relative fluorescence intensity of the sample "ethylhexyl methoxycinnamate" at test concentrations of 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL did not increase, and was even significantly decreased, compared with the negative control group, indicating that it has no potential eye irritation.

[0070] Conclusion: This raw material does not cause eye irritation based on histopathological changes, and does not cause eye stinging based on TRPV1-SIRC cell calcium influx analysis. Therefore, it can be concluded that this sunscreen will not cause consumer discomfort.

[0071] Table 8 Calcium influx detection results

[0072] Note: Compared with the negative control group, * represents P < 0.05, *** represents P < 0.001.

[0073] In addition, comparative examples 1-6 were set up, and the experimental method was the same as that of Example 1, with the only difference being the calcium influx detection parameters (specifically, the final probe concentration, the final cell concentration, or the incubation time). The specific differences are shown in Table 9.

[0074] Table 9 Test methods and results of different implementations

[0075] The test results of Comparative Example 1 are shown in Tables 10 and Figure 5 , the inhibitor can respond correctly, but the activator cannot respond correctly, making it impossible to correctly screen the activity of the target.

[0076] Table 10 Calcium influx detection results

[0077] Note: *** represents P < 0.001 compared with the negative control group.

[0078] The test results of Comparative Example 3 are shown in Tables 11 and Figure 6 , the inhibitor can respond correctly, but the activator cannot respond correctly, making it impossible to correctly screen the activity of the target.

[0079] Table 11 Calcium influx detection results

[0080] Note: *** represents P < 0.001 compared with the negative control group.

[0081] The test results of all embodiments are summarized in Table 9. It can be seen that when detecting calcium ion influx, the solution of the present invention can make the inhibitor and agonist respond correctly at the same time, and accurately screen the activity of the target.

[0082] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for evaluating the eye irritation of a compound, characterized in that: The evaluation method includes performing a TRPV1 ion channel irritation screening test, and the TRPV1 ion channel irritation screening test method includes the following steps: S1, culturing cells transfected with TRPV1 virus to obtain TRPV1 cells; S2. Add calcium ion fluorescent probe for incubation, wash, and resuspend to obtain TRPV1 cells bound to the fluorescent probe. The final concentration of the calcium ion fluorescent probe is 4.0 μM, and the final concentration of the cells is 1.0×10 5 / mL, the incubation temperature is 37°C, and the incubation time is 30min; S3, perform fluorescence measurement on TRPV1 cells combined with fluorescent probes, and collect the baseline fluorescence value of calcium ion signals, which is recorded as the baseline fluorescence value; S4. Add the sample to be tested to the TRPV1 cells combined with the fluorescent probe, collect the calcium ion signals of each area within the time interval, and record them as the sample fluorescence value; S5. Calculate the relative fluorescence intensity based on the baseline fluorescence value and the sample fluorescence value, and determine whether the sample to be tested is eye irritating based on the relative fluorescence intensity.

2. The evaluation method according to claim 1, wherein: The cells transfected with TRPV1 virus in step S1 are mammalian cells.

3. The evaluation method according to claim 2, characterized in that The mammalian cells include SIRC cells, SH-SY5Y cells, HEK293 cells, mouse fibroblasts L929, human corneal epithelial cell line HCE-T or human keratinocytes HaCaT; the mammalian cells are preferably SIRC cells.

4. The evaluation method according to claim 1, wherein: The calcium ion fluorescent probe described in step S2 includes Fluo-3 AM or Fluo-4 AM; the calcium ion fluorescent probe is preferably Fluo-3 AM.

5. The evaluation method according to claim 1, wherein: The concentration of the sample to be tested in step S4 is determined by a cytotoxicity screening test; the cytotoxicity screening test comprises the following steps: The cells transfected with TRPV1 virus were exposed to different concentrations of sample dilutions. The experimental group, positive control group, negative control group and solvent control group were designed. MTT staining and determination were performed after the exposure. The absorbance value was measured at a wavelength of 570nm, and the relative cell activity of different concentrations was calculated to screen out the appropriate sample concentration.

6. The evaluation method according to claim 1, wherein: The relative fluorescence intensity in step S5 is calculated as follows: After the test begins, do not add any sample, record the baseline fluorescence for 50 seconds, and calculate the average baseline value; pause the measurement, remove the sample tube, add the sample to be tested, and detect for 204 seconds; The difference between the calcium ion signal and the average baseline value within the 204 s interval was calculated as the relative fluorescence intensity.

7. The evaluation method according to claim 1, wherein: The criteria for determining whether the sample to be tested is eye irritating in step S5 are: A negative control group, an inhibitor group, and an agonist group were set up; compared with the negative control group, the relative fluorescence intensity of Ca ions in the inhibitor group was significantly decreased (p<0.05); compared with the negative control group, the relative fluorescence intensity of Ca ions in the agonist group was significantly increased (p<0.05); if the relative fluorescence intensity of Ca ions in the test sample was significantly increased compared with the negative control group (p<0.05), it means that the test sample has an eye irritation effect.

8. The evaluation method according to claim 1, wherein: The compounds include cosmetics and cosmetic raw materials; the cosmetic raw materials include sunscreens.

9. Use of the evaluation method according to any one of claims 1 to 8 in eye irritation evaluation, characterized in that: The application includes the application in the evaluation of eye irritation of sunscreen agents.

10. Use of the evaluation method according to any one of claims 1 to 8 in the safety evaluation of a compound on human body.

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