CN112033944B - Method for evaluating skin irritation of cosmetics - Google Patents
Method for evaluating skin irritation of cosmetics Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention relates to the technical field of cosmetic safety evaluation, and discloses a method for evaluating skin irritation of cosmetics aiming at the problem that the evaluation index of a cosmetic irritation evaluation method is strong in subjectivity, which comprises the following steps: setting a normal control group and a test group, adding cosmetics to be tested into the test group in a water-soluble mode, taking zebra fish of the normal control group and the test group, taking a picture under a fluorescence microscope, storing the picture, counting the number of neutrophils on the skin of the zebra fish, and carrying out statistical analysis. According to the evaluation method, the evaluation index selection, acquisition, processing and analysis are more objective, the evaluation result can be quantized, and not only can whether a single cosmetic has irritation be evaluated, but also the irritation strength of different cosmetics can be compared; due to the characteristics of low feeding cost, small size, short development period and high single spawning frequency of the zebra fish, the evaluation of a plurality of cosmetics can be simultaneously carried out, the evaluation efficiency is high, the evaluation cost is low, and the laboratory evaluation is convenient.
Description
Technical Field
The invention relates to the technical field of cosmetic safety evaluation, in particular to a method for evaluating skin irritation of cosmetics.
Background
Common methods for evaluating the skin irritation of cosmetics at present comprise a human body closed patch test, an animal experiment method, a chick embryo chorioallantoic membrane test method, a three-dimensional recombinant skin model and the like.
Testing the human body sealing patch: the subject of 18-60 years of age, 30, was selected as eligible and had no skin inflammation or other disease or condition that was not suitable for testing. The test substance was formulated with deionized water to two concentration levels of 1% and 5% by mass fraction. 0.020-0.025 mL of test sample is dripped on a filter paper sheet attached in a spot tester, the filter paper sheet is placed in the spot tester, and a solvent hole and a blank control hole are arranged. The plaque test piece with the sample is applied to the back of the subject with a non-irritating adhesive tape, and is fixed by lightly pressing with the palm of the hand, and the patch is applied for 24h. And (4) after the spot removing tester is removed for 30min, observing the skin reaction by a dermatologist after the indentation disappears, respectively observing once after 24h and 48h of the spot sticking test, recording the reaction result and scoring. The irritation of the test substance was evaluated based on the number of persons who had adverse reactions.
The animal test method is a standard method for skin irritation, and is divided into an acute skin irritation test and multiple skin irritation tests. Acute skin irritation test: the guinea pig or the rabbit without damage to healthy adult skin is used as a test animal, the hairs on two sides of the spine of the test animal are cut off about 24 hours before the test, the epidermis is not damaged, and the hair removing range is about 3cm multiplied by 3cm on the left and the right respectively. Approximately 0.5mL (g) of the test substance is applied directly to the skin and then covered with two layers of gauze (2.5 cm. Times.2.5 cm) and a layer of cellophane or the like, and secured with a non-irritating adhesive tape and bandage. The other side of the skin served as a control. The application time is 4h by adopting a sealing test. And observing the skin reaction of the smearing part 1, 24, 48 and 72 hours after the test object is removed, scoring the skin reaction according to the erythema and eschar formation grade and the edema formation grade, comprehensively evaluating the average value of the integral of the test animal, and judging the skin irritation of the test object according to the highest integral average value of each observation time point of 24 hours, 48 hours and 72 hours and the integral average value. Multiple skin irritation: before the test, the hairs on both sides of the spine of the experimental animal are cut off, the hair removing ranges are respectively 3cm multiplied by 3cm, and the smearing area is 2.5cm multiplied by 2.5cm. About 0.5mL (g) of the test substance is applied to one side of the skin, and when the test substance is prepared by using a non-irritating solvent, the solvent is applied to the other side of the skin as a control, and the test substance is applied for 1 time per day and is continuously applied for 14 days. From the next day, the hair should be cut and the residual test substance removed with water or a non-irritating solvent before each application. The results were observed one hour later and scored according to the erythema and eschar formation and edema formation grades, with the control and test zones treated the same. The result is calculated according to the formula, the average integral of each animal every day is calculated, and the skin irritation of the tested object is judged according to the average integral value.
Chick embryo chorioallantoic membrane test method: selecting fresh, clean and intact chick embryos with the mass of 50 g-60 g. When the eggs are incubated for 9 days, the eggs should be checked, unfertilized, inactive or defective chick embryos are discarded, and severely malformed, cracked or thin shelled chick embryos cannot be used. The fertilized eggs were allowed to stand at room temperature for 24 hours and incubated for 9 days for the test, and each test was conducted with 6 chick embryos, a solvent control, a negative control (0.9% by mass of sodium chloride solution), and a positive control (1% by mass of SDS,0.1mol/L sodium hydroxide). The test is divided into a reaction time method and an end point evaluation method. Reaction protocol: 0.3mL of a transparent liquid test substance is directly dripped on an allantoic membrane by using a pipette or a disposable sterile syringe, the surface of the CAM is covered as much as possible, timing is started, the membrane reaction condition is observed, and the time of each toxic effect within 5min of action is recorded. The stimulation score was calculated by the formula according to the time at which bleeding, clotting and vessel thawing occurred. End point evaluation method: the method is used for detecting solid and turbid liquid test objects in the shapes of particles, granules, voices and the like. 0.3mL of the extruded solid, particulate or granular material (which has been ground to a fine particle) is applied directly to the CAM, ensuring that at least 50% of the CAM surface is covered with the test substance, or a paste-coated film is directly contacted with the CAM film. After 3min of action, the test substance on the CAM membrane is gently rinsed with physiological saline, and the rinsing procedure may quickly mask the slight bleeding change on the CAM membrane, so the results should be observed after the completion of the 30s rinsing. The grades of bleeding, coagulation and vessel thawing are observed and scored, and the skin irritation of the test subject is evaluated as the sum of the scores for the three toxic effects.
Three-dimensional recombination skin model: the skin model was incubated overnight in a basal medium in an incubator at 37 ℃. Adding liquid or solid material onto the surface of skin model, and allowing the material to contact with the surface of skin model for 15min, with DPBS as negative control and 5% SDS as positive control. The reaction was stopped by washing the test with DPBS buffer, and then the skin model was transferred to fresh medium and incubated at 37 ℃ for 42h. After the incubation is finished, the skin model is placed in MTT culture medium for 3 hours, and then the epidermis is taken out and added with acidic isopropanol for full extraction. Centrifuging, collecting supernatant, measuring OD at 570nm, using acid isopropanol as blank, calculating the measured OD, and calculating relative activity (%) based on the OD. The skin irritation of the test substance was evaluated in terms of the cell activity value.
The evaluation indexes of the existing evaluation method such as the number, the physical condition, the dosage, the area, the position, the toxicity effect level, the cell survival rate growth condition, the cell count and the like have strong subjectivity, depend on the experience of an implementer, lead to strong subjectivity of test scoring and have larger subjective interference factors on the result; meanwhile, the animal experiment method does not conform to the 3R (reduction, replacement and optimization) principle and does not conform to the requirements of animal protection and welfare. Among skin model methods, 3D skin models approved in the OECD standard are mostly produced abroad, are expensive, and have excessively high culture and transportation costs.
Therefore, the cosmetic testing method which has the advantages of objective development and evaluation index, low cost, simple operation and more humanization undoubtedly has important significance and value.
Disclosure of Invention
The invention aims to provide a method for evaluating the skin irritation of cosmetics, which can evaluate the irritation of the cosmetics more objectively and has low cost.
The invention provides the following technical scheme:
a method for evaluating skin irritation of a cosmetic, comprising the steps of:
(1) Setting a normal control group: putting zebra fish as a subject in water for culturing;
(2) Setting a test group: taking the zebra fish with the same specification as the steps as a subject, placing the zebra fish in water, adding the cosmetics to be tested into the water in a water-soluble mode, and culturing under the same condition;
(3) And (3) detection: taking the zebra fish of the normal control group and the test group, taking a picture under a fluorescence microscope, storing the picture, analyzing the picture by adopting image processing software, collecting data, and obtaining the number of the neutrophils on the skin of the zebra fish based on a statistical method;
(4) Evaluation of irritation: the quantitative calculation formula of the irritation effect is as follows:
wherein N (test group) represents a statistical value of the number of skin neutrophils of the zebra fish of the test group; n (normal control group) represents the statistical value of the number of the skin neutrophils of the zebra fish in the normal control group.
Preferably, the zebra fish used is transgenic neutrophil green fluorescent zebra fish 2 days after fertilization.
Preferably, the cosmetics to be tested include, but are not limited to, cosmetics containing at least one of glycerin, methylisothiazolinone, ethanol, and butylene glycol.
Preferably, the concentration of the cosmetic to be tested added in the step (2) is within the maximum safe concentration range of the cosmetic to be tested on the zebra fish.
Preferably, in the step (3), the number of the selected zebra fish in each group is more than or equal to 30% of the total number of zebra fish.
As a preference for the process of the invention, the statistical methods in step (3) are analysis of variance and Dunnett's T-test.
Preferably, the zebra fish is cultured in water for more than or equal to 18 hours.
Preferably, the method of the present invention is a method for judging skin irritation of a cosmetic composition, which comprises the steps of: when the significance difference level p of the skin irritation is less than 0.05, the cosmetic to be detected is judged to have irritation on the skin, otherwise, the cosmetic to be detected is judged to have no irritation or no obvious irritation on the skin.
The method for evaluating the skin irritation of the cosmetics takes the zebra fish as a subject, and evaluates the cosmetics by taking the statistical number of skin neutrophils of the zebra fish as an index. Neutrophils play an innate immune role in zebrafish and are the largest number of leukocytes in the body. The inventor researches and discovers that part of cosmetics have the skin irritation effect on zebra fish close to that of a human body, show skin inflammation and cause symptoms such as telangiectasia, hyperpermeability and edema, and when skin irritation components such as methylisothiazolinone, ethanol and the like are contained, the inflammation induces the immune response of neutrophils, and the neutrophils migrate and gather to the skin epidermis. Therefore, the more the stimulation of the cosmetics to be tested on the skin of the zebra fish is, the more the neutrophils are gathered on the epidermis of the skin of the zebra fish, so that the condition of the evaluation index is determined without depending on the subjective experience of an implementer through image analysis software statistics and analysis of variance and Dunnett's T-test, the subjective factors and subjective indexes are excluded, and the stimulation of the cosmetics on the skin of the zebra fish and the skin stimulation of different cosmetics are evaluated through the statistics and analysis of the number of the neutrophils after the zebra fish is stimulated, namely the objective evaluation index.
The invention has the following beneficial effects:
the invention provides a method for evaluating the skin irritation of cosmetics more objectively and with low cost, which comprises the following steps: firstly, a method for objectively evaluating the skin irritation of cosmetics is provided, the evaluation index selection, acquisition, processing and analysis are more objective, the evaluation result can be quantized, and not only can whether a single cosmetic has irritation be evaluated, but also the irritation strength of different cosmetics can be compared;
secondly, the method can be used for evaluating cosmetics containing methylisothiazolinone, ethanol and other components, and is also suitable for evaluating cosmetics with unknown components;
thirdly, due to the characteristics of low feeding cost, small volume, short development period and high single spawning frequency of the zebra fish, a plurality of cosmetics can be evaluated at the same time, the evaluation efficiency is high, and the evaluation cost is low;
finally, the evaluation method is simple and convenient to operate, and facilitates laboratory evaluation.
Drawings
FIG. 1 is a graph showing the neutrophil distribution of zebrafish skin after the treatment of examples 1 to 4 and a normal control group.
The white dots in the figure indicate neutrophils.
Detailed Description
The following further describes the embodiments of the present invention.
The starting materials used in the present invention are commercially available or commonly used in the art unless otherwise specified, and the methods in the following examples are conventional in the art unless otherwise specified.
The safe concentration in the method of the invention refers to the concentration which can not cause death of the zebra fish and damage of organism organs of the zebra fish.
Example 1
A method for evaluating skin irritation of cosmetic containing glycerin 10 wt% and other components with mild and non-irritating effects comprises the following steps:
(1) And (4) determining safe concentration: placing the transgenic neutrophilic granulocyte green fluorescent zebra fish 2 days after fertilization into a water-containing pore plate, adding cosmetics to be tested with different concentrations of 1.39, 2.78, 5.56, 11.1, 22.2 and 44.4 mu L/mL into different pore plates in a water-soluble mode, and after culturing for 18 hours, counting the death and organ damage conditions of the zebra fish, wherein the result is shown in table 1, so that the upper limit of the safe concentration of the cosmetics is determined to be 11.1 mu L/mL;
(2) Setting a normal control group: using 30 tails of transgenic neutrophil green fluorescent zebra fish 2 days after fertilization as a subject, and culturing in a pore plate containing 3mL of water for 18 hours;
(3) Setting a test group: putting 30 zebra fish tails with the same specification as the zebra fish 30 in the step (2) into water, adding a cosmetic to be detected into the water in a water-soluble mode, wherein the concentration of the cosmetic is 11.1 mu L/mL, and culturing for 18 hours under the same condition;
(4) And (3) detection: after the culture is finished, randomly taking 10 tails of the zebra fishes of the normal control group and the test group, placing the zebra fishes under a fluorescence stereo microscope (Nikon AZ 100, japan) for photographing and storing pictures as shown in figure 1, then adopting NIS-Elements D3.10 advanced image processing software to analyze the images, collecting data, and calculating the number of the neutrophils on the skin of the zebra fishes based on a statistical method of variance analysis and Dunnett's T-test statistics;
(5) Evaluation of irritation: the skin irritation was calculated according to the following formula:
wherein N (test group) represents the statistical value of the number of the skin neutrophils of the zebra fish of the test group; n (normal control group) represents the statistical value of the number of skin neutrophils of the zebra fish in the normal control group, and the result is shown in table 2.
Example 2
A method for evaluating skin irritation of a cosmetic, which contains methylisothiazolinone in an amount of 70ppm and has mild and non-irritating other components, comprising the steps of:
(1) And (4) determining safe concentration: placing the transgenic neutrophilic granulocyte green fluorescent zebra fish 2 days after fertilization into a water-containing pore plate, adding cosmetics to be tested with different concentrations of 1.39, 2.78, 5.56, 11.1, 22.2 and 44.4 mu L/mL into different pore plates in a water-soluble mode, and after culturing for 18 hours, counting the death and organ damage conditions of the zebra fish, wherein the result is shown in table 1, so that the upper limit of the safe concentration of the cosmetics is determined to be 5.56 mu L/mL;
(2) Setting a normal control group: transgenic neutrophil green fluorescent zebra fish 30 tails 2 days after fertilization are taken as a subject and placed in a pore plate containing 3mL of water for culture for 18 hours;
(3) Setting a test group: putting 30 zebra fish tails with the same specification as the zebra fish 30 in the step (2) into water, adding a cosmetic to be detected into the water in a water-soluble mode, wherein the concentration of the cosmetic is 5.56 mu L/mL, and culturing for 18 hours under the same condition;
(4) And (3) detection: after the culture is finished, taking 10 tails of the zebra fishes of the normal control group and the test group, placing the zebra fishes under a fluorescence stereo microscope (Nikon AZ 100, japan) for photographing and storing pictures, then adopting NIS-Elements D3.10 advanced image processing software to analyze images and collect data, and calculating the number of the neutrophils on the skin of the zebra fishes based on a statistical method of variance analysis and Dunnett's T-test statistics as shown in figure 1;
(5) Evaluation of irritation: skin irritation was calculated as follows:
wherein N (test group) represents the statistical value of the number of the skin neutrophils of the zebra fish of the test group; n (normal control group) represents the statistical value of the number of skin neutrophils of the zebra fish in the normal control group, and the result is shown in table 2.
Example 3
A method for evaluating skin irritation of cosmetic containing 3% of ethanol and other components with mild and non-irritating effects comprises the following steps:
(1) And (4) determining safe concentration: placing transgenic neutrophilic granulocyte green fluorescent zebra fish 2 days after fertilization into a water-containing pore plate, wherein the water capacity is 3mL, adding cosmetics to be tested with different concentrations into different pore plates in a water-soluble mode, wherein the concentrations are 1.39, 2.78, 5.56, 11.1, 22.2 and 44.4 mu L/mL in sequence, and after culturing for 18 hours, counting the death and organ damage conditions of the zebra fish, wherein the result is shown in table 1, so that the upper limit of the safety concentration of the test is determined to be 22.2 mu L/mL;
(2) Setting a normal control group: using 30 tails of transgenic neutrophil green fluorescent zebra fish 2 days after fertilization as a subject, and culturing in a pore plate containing 3mL of water for 18 hours;
(3) Setting a test group: taking zebra fish 30 tails with the same specification as the zebra fish in the step (2) as a subject, putting the subject in water, adding a cosmetic to be tested into the water in a water-soluble mode, wherein the concentration of the cosmetic is 22.2 mu L/mL, and culturing for 18 hours under the same condition;
(4) And (3) detection: after the culture is finished, taking 10 tails of the zebra fish of the normal control group and the test group, placing the zebra fish under a fluorescence stereo microscope (Nikon AZ 100, japan) for photographing and storing pictures as shown in figure 1, then adopting NIS-Elements D3.10 advanced image processing software to analyze the images, and calculating the number of the neutrophils on the skin of the zebra fish based on a statistical method of variance analysis and Dunnett's T-test statistics;
(5) Evaluation of irritation properties: the formula for the calculation of skin irritation is as follows:
wherein N (test group) represents the statistical value of the number of the skin neutrophils of the zebra fish of the test group; n (normal control group) represents the statistical value of the number of skin neutrophils of the zebra fish in the normal control group, and the result is shown in table 2.
Example 4
A method for evaluating skin irritation of cosmetic containing 5% butanediol and other components with mild and non-irritating effects comprises the following steps:
(1) And (4) determining safe concentration: transgenic neutrophil green fluorescent zebra fish 2 days after fertilization is placed in a water-containing pore plate, the water capacity is 3mL, 30 tails of each zebra fish are added into different pore plates in a water-soluble mode, cosmetics to be tested with different concentrations are added into the different pore plates, the concentrations are 1.39, 2.78, 5.56, 11.1, 22.2 and 44.4 mu L/mL in sequence, after 18-hour culture is carried out, the death and organ damage conditions of the zebra fish are counted, and the result is shown in table 1, so that the upper limit of the safety concentration of the test is determined to be 2.78 mu L/mL;
(2) Setting a normal control group: transgenic neutrophil green fluorescent zebra fish 30 tails 2 days after fertilization are taken as a subject and placed in a pore plate containing 3mL of water for culture for 18 hours;
(3) Setting a test group: taking zebra fish 30 tails with the same specification as the zebra fish in the step (2) as a subject, putting the subject in water, adding a cosmetic to be tested into the water in a water-soluble mode, wherein the concentration of the cosmetic is 2.78 mu L/mL, and culturing for 18 hours under the same condition;
(4) And (3) detection: after the culture is finished, taking 10 tails of the zebra fishes of the normal control group and the test group, placing the zebra fishes under a fluorescence stereo microscope (Nikon AZ 100, japan) for photographing and storing pictures as shown in figure 1, then adopting NIS-Elements D3.10 advanced image processing software to analyze images and collect data, and calculating the number of the neutrophils on the skin of the zebra fishes based on a statistical method of variance analysis and Dunnett's T-test statistics;
(5) Evaluation of irritation properties: the skin irritation effect calculation formula is as follows:
wherein N (test group) represents the statistical value of the number of the skin neutrophils of the zebra fish of the test group; n (normal control group) represents the statistical value of the number of skin neutrophils of the zebra fish in the normal control group, and the result is shown in table 2.
Test results
1. Safe concentration testing
The safe concentration test of the cosmetics of each example is shown in table 1.
TABLE 1 safe concentration results for each cosmetic
As can be seen from the table, the upper limit of the safe concentration of the cosmetic against zebra fish in example 1 was 11.1. Mu.L/mL, the upper limit of the safe concentration of the cosmetic against zebra fish in example 2 was 5.56. Mu.L/mL, the upper limit of the safe concentration of the cosmetic against zebra fish in example 3 was 22.2. Mu.L/mL, and the upper limit of the safe concentration of the cosmetic against zebra fish in example 4 was 2.78. Mu.L/mL, in the test range.
2. Skin irritation test
The results of the skin irritation test for the cosmetics of each example are shown in Table 2.
Table 2 results of the number of skin neutrophils in zebra fish treated in step (3) in each example (n = 10)
P <0.01, p <0.001, compared to normal controls.
As can be seen from table 2 above in conjunction with fig. 1, the skin irritation effect of the cosmetics of examples 2 and 3 was significant, and the skin irritation effect of the cosmetics of examples 1 and 4 was not significant, as judged based on the significant difference in skin irritation water p <0.05 at the test concentrations.
Claims (4)
1. A method for evaluating the skin irritation associated with a cosmetic product, comprising the steps of:
(1) Setting a normal control group: putting zebra fish as a subject in water for culturing;
(2) Setting a test group: taking zebra fish with the same specification as the steps as a subject, putting the zebra fish into water, adding the cosmetics to be tested into the water in a water-soluble mode, and culturing under the same condition;
(3) And (3) detection: taking the zebra fish of the normal control group and the test group, taking a picture under a fluorescence microscope, storing the picture, analyzing the picture by adopting image processing software, collecting data, and obtaining the number of the neutrophils on the skin of the zebra fish based on a statistical method;
(4) Evaluation of irritation: the quantitative calculation formula of the irritation effect is as follows:
wherein N (test group) represents the statistical value of the number of the skin neutrophils of the zebra fish of the test group; n (normal control group) represents the statistical value of the number of the skin neutrophils of the zebra fish in the normal control group;
the zebra fish is transgenic neutrophil green fluorescent zebra fish 2 days after fertilization;
the cosmetic to be detected contains at least one of glycerol, methylisothiazolinone, ethanol and butanediol;
the culture time of the zebra fish in water is more than or equal to 18 hours;
the judgment criteria of the skin irritation of the cosmetics are as follows: when the significant difference level p of the skin irritation is less than 0.05, the cosmetic to be detected is judged to have irritation on the skin, otherwise, the cosmetic to be detected is judged to have no irritation or no obvious irritation on the skin.
2. The method for evaluating the skin irritation of cosmetics according to claim 1, wherein the concentration of the cosmetic to be tested added in step (2) is within the maximum safe concentration range of the cosmetic to be tested on zebrafish.
3. The method for evaluating the skin irritation of cosmetics according to claim 1, wherein the tail number of the zebrafish selected in each group in step (3) is not less than 30% of the total tail number of the zebrafish.
4. The method for evaluating the irritation of cosmetics to the skin according to claim 1 or 3, wherein the statistical method in step (3) is analysis of variance and Dunnett's T-test.
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