CN107271653A

CN107271653A - A kind of utilization chicken embryo vascular system detection and evaluation conjunctiva excitant and the method for mucous membrane irritation

Info

Publication number: CN107271653A
Application number: CN201710368626.1A
Authority: CN
Inventors: 秦瑶; 程树军; 陈志杰
Original assignee: Guangzhou Huadai Biological Technology Co Ltd
Current assignee: Guangzhou Huadai Biological Technology Co Ltd
Priority date: 2017-05-22
Filing date: 2017-05-22
Publication date: 2017-10-20

Abstract

The invention discloses a kind of detection of utilization chicken embryo vascular system and evaluation conjunctiva excitant and the method for mucous membrane irritation, comprise the steps of：(1) preparation of CAM；(2) conjunctiva irritant test；(3) sticking to mould irritant test；(4) forecast model and classification, the chicken embryo that the present invention passes through standardization is test system, the vascular system of its development in different stages is similar with human body eye conjunctiva and oral submucosa, chicken embryo standardization level is high, steady sources and differences between batches are small, vascular system has biological activity and function, test system is simple, cheap, and without special installation, detection method is quick, sensitive, general.The present invention can be from qualitative and quantitative two aspects detection and the conjunctiva excitant and mucous membrane irritation of evaluation test substance.The inventive method can also replace living animal and human volunteer, be directly used in the detection of the material conjunctiva excitant such as chemicals or cosmetics and mucous membrane irritation, and its rapid screening, classification and mark.

Description

One kind is using the detection of chicken embryo vascular system and evaluates conjunctiva excitant and mucous membrane irritation Method

Technical field

The invention belongs to conjunctiva excitant and mucous membrane irritation technical field, and in particular to one kind utilizes chicken embryo vascular system Detection and evaluation conjunctiva excitant and the method for mucous membrane irritation.

Background technology

With the improvement of living standards, increasing people begins to use personal nursing and cleaning supplies, the former is such as skin With with ophthalmically acceptable product, the latter such as oral cleaning and care product.Most consumers using in these product process all very It is concerned about its safety issue, problem is stimulated including using conjunctiva and cornea that contact eyes generation is not intended to after product；And Stimulation of the oral care product to oral mucosa and gum；Feminine care product is to stimulation of vagina mucosa etc..Therefore, personal nursing The health hazard of product must be detected with the regulatory requirements of cleaning products.Meanwhile, in the R＆D process of these products, The detection and assessment of mucous membrane irritation are also extended through wherein.For example for candidate feed (such as plant extracts, synthesis of chemicals) and Formula, it usually needs test its eye conjunctiva excitant and oral mucosa excitant repeatedly, and want to accurate quantification difference and match somebody with somebody Side or the stimulation degree of product.In addition, for the chemicals of a large amount of unknown toxic characteristics, it is also desirable to which accurate understanding includes conjunctiva thorn Swash the toxicity feature including property and mucous membrane irritation, classification and mark for hazardous chemical.

Chemicals, personal care articles or conjunctiva excitant caused by other eye contact materials, generally refer to these things Matter intentionally or accidentally directly contacts the invertibity inflammatory change of eye conjunctiva and its surrounding mucosa caused by after eyes, and these materials are such as Elution class cosmetics, eye and face apply some make up, ocular drug, spray drinks agricultural chemicals, air freshener, air particle and drift Float, PM2.5 etc..The product that consumer it is generally desirable to purchase meets the requirement of product safety standard, and more wishes production Product can be applied to the special populations such as skin sensitivity, infant without any irritating or gentle.

Personal nursing and cleaning supplies cause the excitant of oral mucosa, generally refer to be used for tooth, oral cavity, lip etc. The product at position, touches the invertibity inflammatory change of caused mucous membrane after oral mucosa, lip, gum and its surrounding tissue, These materials include toothpaste, mouthwash, tooth pad pasting, gutta-percha etc.

Due to the increase of the product frequency of use such as hygiene, personal nursing, the related adverse reaction of above-mentioned mucosal irritation Generation it is more and more universal.On the other hand, requirement also more and more higher of the consumer for Product Safety, it is therefore necessary to existing On the basis of some detection methods, set up some more rapidly with more sensitive detection and appraisal procedure.

The method of existing allogenic material mucous membrane irritation can be divided into animal model, cell method and the class of human trial three.

Traditional animal model detection conjunctiva excitant, is also that a certain amount of tested material is instilled into experimental animal (to be usually Rabbit) eyes in, after intervals, the change of observation animal subject eyes conjunctiva, cornea and iris, so that assess should Whether tested material has eye irritation.Equally, traditional detection oral mucosa excitant, is to bury a certain amount of tested material by operation In the oral cavity cheek pouch for entering experimental animal (being usually Golden Hamster), after intervals, animal is put to death, oral cavity cheek pouch is collected Tissue, observes the pathological change of oral mucosa, to assess whether the tested material has oral mucosa excitant.Examination is used as using animal Test the security of the object detection consumer goods, it has the disadvantage, and cost is high, the cycle is long, be rarely employed modern biotechnology development in science and technology into Really, return animal and bring considerable distress, exist morals and ethically the problem of.In addition, zoopery environmental condition is severe Quarter (animal needs to raise the indoor environment in constant temperature and humidity), animal quality requirement are strict (it is required that meeting wanting for credit rating Ask), detection threshold it is high (testing agency need obtain experimental animal use licensing), and differed too with the actual use situation of people Far, and due to there is species variation between animal and people, the security risk for evaluating human body skin using animal skin makes result Confidence level reduction.

As a large amount of new materials are used for medicine and cosmetics, it would be desirable to which more methods evaluate the peace of these materials Quan Xing.Meanwhile, in order to follow the 3R principles in life and medical research (reducing, optimize and replace zoopery), and existing Application of result for biotechnology is assessed (such as cell culture, organizational project, group are learned and gene technology) in human health risk. The updating program for the toxicology that toxotest geared to the 21st century is core is carried out since before 10 years, in global range. The alternative of substantial amounts of non-animal is applied in the detection and security evaluation of health hazard, and some methods are closed by World Economics In the development institution (OECD) of work, European alternative authentication center (ECVAM) and U.S.'s alternative intersectoral coordination checking The mechanisms such as the heart (ICCVAM) approve and are set to guide detection.

The current irritating method of vitro detection chemical substance conjunctiva mainly has OECD guides 435,437,460,492 Deng wherein 435 be the alternative of early stage accreditation, using in vitro lagophthalmos as test object, gradually by without using experimental animal material The method substitution of material.437 be the ox opacity of the cornea and the permeability test for detecting Eye irritation, using the obsolete material in slaughterhouse as experiment The eye irritation of system detectio chemicals and product, its advantage applies to the detection of nearly all raw material and product, has the disadvantage Domestic material source is difficult, the main damage for assessing cornea, it is impossible to detect the excitant of conjunctiva.460 be using cell monolayer training Foster method, is primarily adapted for use in the detection of the chemicals such as surfactant, and it has the disadvantage not being suitable for detection indissoluble solution, has color Chemicals, be not suitable for extract, formula and the finished product of test dosage forms and complicated yet.492 be tissue engineering comea Method is, it is necessary to the artificial cornea model of commodity in use, and the current country there is no method to produce similar products, also can not effectively import, Secondly artificial cornea model prices are expensive, are not suitable for a large amount of routine testings.

The method of current vitro detection chemical substance mucous membrane irritation is also immature, and for example someone uses the mouth of primary people Transmucosal epithelial cell or the method for cell line culture detect the excitant of oral mucosa, and it has the disadvantage that system is single, culture Cell is without vascular distribution, also without muciparous function, and is not suitable for detection indissoluble solution, coloured chemicals, also not Suitable for test dosage forms and the extract of complicated.The oral mucosa epithelial cell that organizational project is built can not also realize commodity Change supply.

Human volunteer's experiment is the most straightforward approach for evaluating Product Safety.For example human body eye conjunctiva irritant test is needed Product is instilled people's eye conjunctiva, tested whether with excitant.Human test results are the truest, but cost is too high, right There is health hazard in volunteer, the problem of having certain in ethics.

Fertilization chicken embryo vascular system is the ideal method for testing chemicals and consumer product safety, first chicken embryo It is nonmammalian, meets the requirement of animal welfare regulation and the 3R principles of various countries；Next to that organism living, what it grew Different phase is suitable for toxicity or biological activity test；3rd, cost is low, standardization level is high, culture and experiment condition Simply, it is adapted to extensive detection and studies.The existing substantial amounts of research based on chicken embryo is applied to life science, and such as embryo sends out Educate toxotest, promotion and suppress angiogenesis function research, the histocompatbility for assessing biomaterial etc..Utilize chick embryo development During vascular system it is abundant the characteristics of the method that can set up detection and assess toxicity.

During chick embryo development, blood vessel network is the biological process continuously developed, at 4-5 days, and vitelline vessel film is Unilayered vessels film, rete vasculosum is just molded, the form of similar spider web, reacts sensitive to external source material incentive, can be for progress light The experimental studies such as toxicity, angiogenesispromoting effect, embryo's teratogenesis.During chick embryo development 10 days, CAM vascular system is formed, It is three layers of vascular system, blood vessel network has been formed, and the conjunctiva of the similar people of its structure can be used for conjunctiva and mucous membrane irritation Assessment.

Therefore, method side of the present invention to being detected using chicken embryo vascular system and evaluating conjunctiva excitant and mucous membrane irritation Face is conducted in-depth research.

The content of the invention

It is an object of the invention to provide one kind is using the detection of chicken embryo vascular system and evaluates conjunctiva excitant and mucous membrane thorn Swash the method for property, this method is quick, easy, cost is low, standardization level is high, applied widely, can be to conjunctiva excitant Carry out fast and effectively detecting and assessing with mucous membrane irritation material.

The above-mentioned purpose of the present invention is achieved through the following technical solutions：One kind using the detection of chicken embryo vascular system and Conjunctiva excitant and the method for mucous membrane irritation are evaluated, is comprised the steps of：

(1) preparation of CAM

SPF grades of fertilization chicken embryos are chosen, the fertilization chicken embryo with CAM are prepared standby；

(2) conjunctiva irritant test

It is prepared by 2.1 tested materials

Tested material is chosen, after being dissolved with solvent, preparing one group has the dilution of concentration gradient standby, and set positive right According to, negative control and baseline control；

2.2 Screening tests

Tested material is chosen, after being dissolved with solvent, one group of dilution with concentration gradient is prepared again, tested material knot is carried out Film field stimulation Screening tests, it is determined that causing the general concentration range that chicken embryo is reacted；

2.3 conjunctiva excitant RC₅₀It is determined that experiment

Tested material is chosen, after being dissolved with solvent, then one group of dilution with concentration gradient is prepared again, takes step (1) The fertilization chicken embryo of middle preparation, places Teflon " О " shape ring on the chick chorioallantoic membrane of the fertilization chicken embryo, the group is had dense Dilution, positive control, negative control and the baseline control of degree gradient are added separately to Teflon " О " shape ring, constant incubator After incubation, the various change of observation record blood vessel, and compared with blood vessel outside ring, the change of blood vessel is judged and divided according to table 1 Level, vascular reaction classification is positive for vascular reaction more than 1 grade, and conjunctiva excitant RC is calculated by logarithmic chart₅₀Value, even if half There is the tested material concentration of positive reaction in chicken embryo；

Vascular reaction observation and be classified that the phototoxicity of table 1 and conjunctiva are stimulated

Classification	Observation description
		0	Do not observe blood vessel to fade or bleeding, tunica vasculose is normal
1	Blood vessel white is without blood, and tube wall is clear；Or/and capillary injection
		2	The visible tiny blutpunkte of hair cell blood vessel, bleeding area ＜ 20%
3	Obvious angiorrbagia, is dispersed in or hemorrhagic areas ＞ 20%, ＜ 50%
		4	Severe haemorrhage, hemorrhagic areas is more than 50%

(3) sticking to mould irritant test

It is prepared by 3.1 tested materials

Liquid tested material is tested in the undiluted form of stoste, and paste, microgranular or granular product to enter as former state Row experiment uses pure water to be tested after diluting, and sets positive control, negative control and baseline control；

3.2 mucomembranous irritant test

The fertilization chicken embryo prepared in step (1) is taken, Teflon " О " is placed on the chick chorioallantoic membrane of the fertilization chicken embryo Shape ring, Teflon " О " shape ring is added separately to by tested material, positive control, negative control and baseline control, is rinsed after effect Remove tested material, directly observe result, record the degree of every kind of poisonous effect change, every time 6 chicken embryos of experiment, and with blood outside ring Pipe is compared, and the change of blood vessel is judged and is classified according to table 2, and vascular reaction score value is vascular reaction at 1 point and more than 1 point It is positive；

The mucosal irritation vascular reaction grade form of table 2

(4) forecast model and classification

4.1 conjunctiva excitant forecast models

The RC of tested material is calculated from concentration-response relationship₅₀, making conjunctiva according to following condition stimulates judgement：

RC₅₀>=50%, predict " nonirritant ",

10.0% ＜ RC₅₀And ＜ 50%, predict " nonirritant ",

3.0%≤RC₅₀And ＜ 10.0%, predict " weak excitant ",

1.0%≤RC₅₀And ＜ 3.0%, predict " moderate excitant ",

RC₅₀＜ 1.0%, is predicted " severe excitant or corrosivity "；

4.2 oral cavity sticking to mould excitant forecast models

The overall score of 3 kinds of reactions of every chicken embryo is calculated, then calculates the mean total score AES of 6 chicken embryo reactions；

Make oral cavity sticking to mould according to following condition and stimulate and judge：

AES≤6：Predict " no oral mucosa excitant "；

6 ＜ AES and≤12：Predict " slight oral mucosa excitant "；

12 ＜ AES and ＜ 16：Predict " moderate oral mucosa excitant "

AES≥16：Predict " serious oral mucosa excitant ".

In above-mentioned utilization chicken embryo vascular system detects and evaluated the method for conjunctiva excitant and mucous membrane irritation：

Choose in step (1) after the gestational age SPF of 0 day levels fertilization chicken embryo is put into constant incubator incubation 10~14 days, obtain There must be the fertilization chicken embryo of CAM standby.

The species of SPF levels fertilization chicken embryo described in step (1) is preferably Bai Laihang chickens, star miscellaneous 288, White Rock, Chinese ephedra Or Lingnan Yellow chicken.

Step (1)~(4) are carried out preferably in constant incubator, and temperature is preferably 37 DEG C ± 1 when constant incubator is incubated DEG C, relative humidity is preferably 65% ± 5%.

Tested material described in step (2) includes personal care product, bath accessory, oral care implement, household washing production Product, cosmetics or textile extract.

Tested material further preferably includes described in step (2)：(a) single composition chemical substance, including chemicals, medicine Or agricultural chemicals；Or the raw material of (b) based on single composition, including plant extract, crude extract, bioactivity raw material or polypeptide；Or (c) simple formulation, refers to by the semifinished or finished goods of 3~5 kinds of raw material compoundings, including sun-screening agent or antioxidant.

Tested material is chosen in step (2.2), after being dissolved with solvent, one group of dilution with concentration gradient is prepared again, When carrying out tested material conjunctiva field stimulation Screening tests, screening method is preferably included from top to bottom, from bottom to top or upper laxative remedy is entered Row examination.

Step (2.1) and the positives control of step (2.3) preferably sarcosyl SLS, NaOH, acetic acid or bacterium Polysaccharide (PLS) etc., negative control is preferably to dissolve solvent of tested material etc., and baseline control is preferably close or identical with tested material Known product.

The chicken embryo that the present invention passes through standardization is test system, vascular system and the human body eye conjunctiva of its development in different stages Undertissue is similar with oral mucosa, and chicken embryo standardization level is high, steady sources and differences between batches are small, and vascular system has biology Activity and function, test system are simple, cheap, and without special installation, detection method is quick, sensitive, general.

Further, above-mentioned utilization chicken embryo vascular system detection and the method for evaluating mucous membrane irritation, comprise the following steps：

First, chicken embryo blood vessel pilot system

1.1 natural down allantois film preparations

The chicken embryo within 0-9d ages is bought, air chamber is stored in egg rack and is transferred to laboratory upward, should carry out shining egg inspection, nothing Active or defective chicken embryo should be discarded.Chick embryo air sac end is placed in 37.5 DEG C ± 0.5 DEG C of incubation temperature, relative humidity upwards Hatch under conditions of 55%-70% to 10-14d ages.10-14d instar chicken embryos shine egg inspection, in eggshell surface mark air chamber position； Eggshell part is peelled off with the curved tweezer of dentistry sawtooth, exposure white egg film can make egg film with suction pipe dropwise addition 1-2mL0.9%NaCl solution Moistening, 0.9%NaCl solution is poured out.It is careful to remove inner membrance, exposure natural down chorioallantoic membrane with tweezers.

The preparation of 1.2 artificial CAMs

The purchase gestational age SPF of 0 day level fertilizations chicken embryo is put into the perseverance that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% It is incubated in warm incubator.The 3-4 days, by chicken embryo horizontal positioned.The 4-5 days, by chicken embryo to optical test, discard and be not fertilized or send out Educate bad chicken embryo.To the chicken embryo of normal development, an aperture is opened from the non-air chamber end of egg, dust is wiped with cotton ball soaked in alcohol, Extract 2.5-3mL egg white out to reduce the artificial CAM of embry ogenesis from aperture with syringe.Aperture is closed with paraffin. Then the small window of rectangle for opening a length of side 0.8-1cm in egg top end, dust is wiped with cotton ball soaked in alcohol.Will with preservative film Small window closing, for observing chicken embryo vigor daily.By chicken embryo horizontally rotate in temperature be 37 DEG C ± 1 DEG C, relative humidity be 65% ± 5% constant incubator is incubated.The 10-14 days, experiment can be proceeded by.

2nd, conjunctiva irritant test

It is prepared by 2.1 tested materials

2.1.1 water or PBS are that solvent prepares tested material

Prepare before tested material stock solution, first understand the dissolubility of material.For water or the soluble materials of PBS, one is weighed Quantitative tested material is added to glass tube, then adds pure water, distilled water or the PBS of certain volume, mixes it, first match somebody with somebody Storing solution processed, then empirically demands, prepare the dilution of 3-5 various concentrations (w/v) respectively.

2.1.2 oil prepares tested material for solvent

For water and the undissolved materials of PBS, it is solvent that corn oil or olive oil, which may be selected, is prepared into suspension, as far as possible Tested material is set to be uniformly distributed in dissolution system.Storing solution, then empirically demands are first prepared, 3-5 difference is prepared respectively The dilution of concentration (w/v),

2.1.3 positive control, baseline control and negative control

Positive control：0.1%-3% SLS (lauryl sodium sulfate), 0.1-0.5mol/L NaOH or 3% may be selected Acetic acid；

Baseline control：The chemistry with the known results of tester same dosage form, close excitant or close formula may be selected Product, formula or product；

Negative control：Control is used as using 100% solvent.

2.2 examinations are tested

Before formal experiment, first carry out tested material conjunctiva field stimulation and determine experiment, i.e., examination experiment is first carried out, it is determined that causing The general concentration range of chicken embryo reaction.The concentration dilution factor of examination experiment is between 1.2-2.5, and interval between concentration can be with Unequal distance.For different product types, may be selected from top to bottom, method or increase and decrease method selection examination experiment up and down from bottom to top Concentration range.

2.2.1 method from top to bottom

For the product of expected conjunctiva excitant " slight ", examination concentration is from 70%, 50%, 30%, 15% and 10% (w/v) three concentration are selected to be tested from high concentration to low concentration in five concentration.

2.2.2 method from bottom to top

For the product of expected conjunctiva excitant " moderate is serious ", examination concentration is from 30%, 15%, 10%, 5% and Three concentration are selected to be tested from low concentration to high concentration in 2% (w/v) five concentration.If low concentration test result is still Excitant, then be down diluted to 1%, 0.5%, 0.25%, 0.1% etc. again from 2%.

2.2.3 increase and decrease method above and below

For the product that expected conjunctiva excitant is " uncertain ", examination concentration is from can be since 50%, test step by step, on The test result of one step determines the selection of next step test concentrations, increases and decreases up or down respectively.If test result is serious Stimulate, then next concentration selection 30%, if test result is non-stimulated, next concentration selection 70%；The rest may be inferred.

2.3 examination experimentations

The chicken embryo prepared in step 1 is taken, is peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size. Small size (external diameter 9-11mm, internal diameter 8-10mm) Teflon " О " shape ring is placed on chick chorioallantoic membrane surface, 40 μ are drawn with liquid-transfering gun The solution of the above-mentioned each concentration gradients of L, is added in " О " shape ring.Be put into temperature be 37 DEG C ± 1 DEG C, relative humidity be 65% ± 5% constant incubator is incubated 30min ± 1min, after incubation terminates, immediately the various change of observed and recorded blood vessel.And with outside ring Blood vessel compares.3 chicken embryos of each concentration.The change of blood vessel judged and is classified according to table 1, and vascular reaction is classified at 1 grade It is that vascular reaction is positive with more than 1 grade.The concentration that order of severity adjustment according to reaction is formally tested is set.

2.4RC₅₀It is determined that experiment

RC₅₀Determine that test requirements document is tested than examination and use same or less dilution gfactor, optional dilution gfactor 1.2~ Between 2.Diluted concentration 3-5, should cover the concentration range of 2.3 preliminary experiments determination, each 10 chicken embryos of concentration.Take in step 1 The chicken embryo prepared, is peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size.On chick chorioallantoic membrane surface Small size (external diameter 9-11mm, internal diameter 8-10mm) Teflon " О " shape ring is placed, the above-mentioned each concentration gradients of 40 μ L are drawn with liquid-transfering gun Solution, be added in " О " shape ring.The constant incubator that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% is put into incubate 30min ± 1min is educated, is incubated after terminating, immediately the various change of observed and recorded blood vessel.And compared with blood vessel outside ring.Blood vessel Change and judged and be classified according to table 1, vascular reaction classification is that vascular reaction is positive at 1 grade and more than 1 grade.Based on logarithmic chart Calculate excitant RC₅₀Value, even if there is the tested material concentration of positive reaction in half chicken embryo.

Vascular reaction observation and be classified that the conjunctiva of table 1 is stimulated

3rd, mucomembranous irritant test

It is prepared by 3.1 tested materials

Liquid tested material is tested in the undiluted form of stoste；Paste, microgranular or granular tested material or product With tested as former state or with pure water 1:1 dilution；Trickle particle (powdery), powdery should be ground to form before the experiment of solid tested material Tested material can use a small amount of 0.9%NaCl or olive oil/corn oil wetting.Such as tested material is paste, should be coated on tested material Plastic sheeting (such as sealed membrane) surface straticulation, then being covered on CAM films makes tested material directly be contacted with CAM films, effect Time removes film after terminating.

3.2 positive controls, baseline control and negative control

Positive control：1-3% SLS (dodecyl sodium sulfate), granulose PLS may be selected；

Baseline control：The chemical combination with the known results of tester same dosage form, close excitant or close formula may be selected Thing, formula or product.

Negative control：Control is used as using 100% solvent.

3.3 mucosal irritations are tested

The chicken embryo prepared in step 1 is taken, is peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size. Large size (external diameter 13-15mm, internal diameter 11-13mm) Teflon is placed on chick chorioallantoic membrane surface " О " shape rings, it is direct with mucus rifle 100 μ L solids tested materials (toothpaste) are drawn, are added in " О " shape ring.Act on respectively washed off after 30 seconds and 1min it is tested Thing, directly observes result, records the degree of every kind of poisonous effect change, and compared with blood vessel outside ring.6 chickens of each time Embryo.The change of blood vessel judged and is classified according to table 2, and vascular reaction classification is the vascular reaction positive at 1 point and more than 1 point. If observation shows that the scoring of all 6 at least one kind of reactions of chicken embryo is more than moderate (overall score >=12), experiment should repeat one It is secondary.

The mucosal irritation vascular reaction grade form of table 2

4th, forecast model and classification

4.1 conjunctiva excitant forecast models

4.1.1 blood vessel positive reaction is classified

Observe tunica vasculose vascular reaction, hierarchical description such as table 1.

4.1.2 forecast model

The RC of tested material is calculated from concentration-response relationship₅₀.Recommend to carry out statistical analysis, Qi Tazhuan using following 2 kinds of methods Industry statistical method can also be applied.

1. diagram method or logarithm-probit method calculate RC₅₀。

2. with nonlinear regression equation calculation RC₅₀, recommend to use Probit regression models, the model is toxicology, medicine Field of science calculates the conventional statistical model of the dose relationship statistical indicator such as median lethal dose and median effective dose.Statistics is soft Part (for example, SPSS 20.0) is carried out.

4.1.3 conjunctiva excitant is classified

The RC that tested material tested material conjunctiva is stimulated is drawn according to above-mentioned 4.1.2 forecast models₅₀Value, according to following condition judgment Conjunctiva stimulates classification：

If：RC50 >=>=50%, predict " nonirritant ", it is adaptable to similar a surname such as " mildness " " sensitive skin is applicable " Claim

RC50 ＞ 10.0%, and ＜ 50%, are predicted " nonirritant "

RC50 >=3.0%, and ＜ 10.0%, are predicted " weak excitant "

RC50 >=1.0%, and ＜ 3.0%, are predicted " moderate excitant "

RC50 ＜ 1.0%, are predicted " severe excitant or corrosivity "

4.2 oral mucosa excitant forecast models

4.2.1 blood vessel positive reaction is classified

According to table 2, CAM vascular reaction is observed.

4.2.2 forecast model

The overall score of 3 kinds of reactions of every chicken embryo is calculated, then calculates the mean total score (Average of 6 chicken embryo reactions Endpoint Score, AES).

4.2.3 oral mucosa excitant is classified

Overall score is drawn according to above-mentioned forecast model, making oral mucosa excitant according to following condition judges：

If：

AES ＜ 6：Predict " no oral mucosa excitant "；

AES >=6, and≤12：Predict " slight oral mucosa excitant "；

AES ＞ 12, and ＜ 16：Predict " moderate oral mucosa excitant "

AES≥16：Predict " serious oral mucosa excitant ".

In this is detected and evaluated the method for mucous membrane irritation using chicken embryo vascular system：

Chicken embryo described in step (1) is the Bai Laihang chickens for the SPF levels (specific pathogen free microorganism) for being 0-9 days reproduction age (White Leghorn chicken) or star miscellaneous 288 (Shaver Starcross) fertilization chicken embryo, also can select White Rock (White Plymouth Rock), Chinese ephedra (spotted-brown chickens) or Lingnan Yellow chicken (Lingnan Yellow The kind fertilization chicken embryo such as chicken).

Step (1), (two), the detection conjunctiva described in (three) and mucous membrane irritation use 10-14 days instar chicken embryos, When referring to chick embryo development to the 10-14 days, the fine hair merged by chorion somatic mesoderm and chorioallantoic membrane visceral layer mesoderm is urinated Cyst membrane.

The CAM experiment system that step (1), (two), detection conjunctiva and mucous membrane irritation described in (three) are used System can be natural air chamber end CAM system, or artifical-air cell CAM system.

The conjunctiva excitant of tested material described in step (2) refers to that tested substance directly contacts caused eye after eyes The invertibity inflammatory change of conjunctiva and its surrounding mucosa, these materials apply some make up as eluted class cosmetics, eye and face, eye Portion's medicine, spray drinks agricultural chemicals, air freshener, air particle and floating object, PM2.5 etc. may be touched intentionally or accidentally The material of eyes.

The mucous membrane irritation of tested material described in step (3) refers to that tested substance is viscous caused by directly contacting behind oral cavity Film, lip, the invertibity inflammatory change of gum and its surrounding tissue, these materials include toothpaste, mouthwash, tooth pad pasting, gutta-percha, Tooth powder etc..

The preparation of tested material described in step (2) and step (3), selected solvent can be ultra-pure water, PBS or jade Rice bran oil.The mixing of solvent and tested material can be by modes such as grinding, heating, concussion, stirrings, until tested material is mixed completely with solvent It is even.

Being used for described in step (2) and step (3) is loaded and is allowed to fixed Teflon ring, can be bought from market It is non-toxic, not with example reaction and adhesion, can the white ring reused of autoclaving, for the irritating external diameter 9- of conjunctiva 11mm, internal diameter 8-10mm, sample-adding amount are 40 μ L；For a diameter of external diameter 13-15mm, the internal diameter 11-13mm of mucous membrane irritation, plus Sample amount is 100 μ L.

Chicken embryo blood vessel positive reaction described in step (2) and step (3), refers to the suede that chick embryo development is formed for 10-14 days Hair chorioallantoic membrane.The irritative response that may occur, including ghost blood vessel, hyperemia, bleeding, and the face being related to according to differential responses Product, the order of severity are classified, the RC described in classification step (two), (four)₅₀, it is to instigate chicken embryo vascular system to occur positive reaction Tested material diluted concentration.The chemicals and the RC of plant extracts drawn by statistics₅₀Value is accurate to after decimal point 3, The RC of raw material and finished product₅₀Value is accurate to after decimal point 2.

RC described in step 4.1₅₀Meaning except for conjunctiva stimulate degree judgement in addition to, it may also be used for different process Tied between formula, between different suppliers' same materials, between different formulations and product and between different brands similar formulation The irritating comparison of film.

The positive control of eye conjunctiva and oral mucosa irritant test described in step (2), (three) is 5%SLS, also may be used Select other known irritating tester.If detection and assess purpose be used for identify tested material whether have conjunctiva or Mucous membrane irritation, or be only intended to chemical substance mark and excitant classification, then positive control generally select 1-3%SDS, 0.1-0.5mol/L NaOH, 3% acetic acid.If in order to which the comparison between being formulated and doing optimizes, increase benchmark pair may be selected According to.

The baseline control of eye conjunctiva and oral mucosa irritant test described in step (2), (three), the purpose is to for demonstrate,proving The validity of bright test method, the reactivity for checking every batch of chicken embryo, and it is used as the benchmark for detecting and evaluating a certain special formulation With reference to.Baseline control should be that steady sources are reliable, 26S Proteasome Structure and Function is similar to the chemical classification of tested material, physical/chemical is special Property known, result and anticipation reaction degree be well established that.The purpose of such as check and evaluation be in order to develop be applied to baby temperature And formulation product, then compareed on the basis of the corresponding product of optional commercially available known brand and result.

Conjunctiva and mucous membrane irritation classification described in the 4.1 of step (4) and 4.2, the product toxicity available for laws and regulations requirement Classification and mark.A surname for the effects such as nonirritant following concrete numerical value is also suitable gentle product, low stimulation, sensitive skin is used Claim.

Compared with prior art, the invention has the advantages that：

(1) the SPF levels fertilization embryo that the present invention is used, is that standardization level is high, no microbiological contamination, batch quality is steady Fixed active bio system；

(2) present invention uses conjunctiva/mucous membrane irritation that CAM is pilot system detection material, it is adaptable to any Raw material, mixture, the test of formula and product, and do not influenceed by physical behaviors such as sample dissolubility, colors；

(3) present invention is that pilot system make use of different growth phases development degree different using chick chorioallantoic membrane Chicken embryo vascular system detects conjunctiva/mucous membrane irritation of material, and method is sensitive, the degree of consistency with experiment in vivo is high, can Directly replace zoopery to carry out preliminary examination, reduce animal and use；

(4) present invention is used chicken embryo vascular system and the check and evaluation method of foundation, solve monolayer cell culture Dissolvable raw material can only be detected and the limitation of insoluble raw material, product and coloring matter can not be detected, external structure is solved Three-dimensional group weaver cornea and the limitation that skin model is costly, batch is unstable, it is more stable, economical and quick；

(5) method set up of the present invention can replace living animal and human skin, be directly used in chemicals, cosmetics, The irritating regulation experiment of the conjunctivas of the products such as medicine, and mucous membrane irritation prediction；

Brief description of the drawings

Fig. 1 is to be used for the conjunctiva excitant of ophthalmic preparation raw material albucid soluble in embodiment 1, and wherein A figures are blank pair According to CAM；B：Score as 1 grade of vascular reaction；C：Score as 2 grades of vascular reaction；D：Score as 3 grades of blood vessel Reaction；

Fig. 2 is to be used for the conjunctiva excitant of eye cream in embodiment 1, and wherein A figures are the CAMs of blank control；B：Comment It is divided into 1 grade of vascular reaction；C：Score as 2 grades of vascular reaction；D：Positive control ethanol, scores as 3 grades of vascular reaction；

Fig. 3 is to be used for shampoo conjunctiva excitant in embodiment 1, and wherein A figures are the CAMs of blank control；B：Comment It is divided into 1 grade of vascular reaction；C：Score as 2 grades of vascular reaction；D：Positive control ethanol, scores as 3 grades of vascular reaction；

Fig. 4 is to be used for mouthwash mucosal irritation in embodiment 4, and wherein A figures are the CAMs of blank control；B：Scoring For 1 grade of vascular reaction；

Fig. 5 is to be used for baby's massage oil conjunctiva excitant in embodiment 5, and wherein A figures are the CAMs of blank control； B：Score as 1 grade of vascular reaction；

Fig. 6 is to be used for toothpaste mucous membrane irritation in embodiment 6, and wherein A figures are the CAMs of blank control；B：Scoring For 1 grade of vascular reaction.

Embodiment

Embodiment 1 is using the detection of chicken embryo vascular system and evaluates the irritating method of albucid soluble conjunctiva

1st, the preparation of natural down chorioallantoic membrane

7d instar chicken embryos are bought, air chamber is stored in egg rack and is transferred to laboratory upward, according to egg inspection, discard inactive and have scarce Sunken chicken embryo.Chicken embryo is placed in 37.5 DEG C ± 0.5 DEG C of incubation temperature, hatched under conditions of relative humidity 60%-70% to 10d Age.10d checks chicken embryo during age according to egg, in eggshell surface mark air chamber position；Eggshell part is peelled off with the curved tweezer of dentistry sawtooth, exposure White egg film, 1-2mL0.9%NaCl solution, which is added dropwise, moistens egg film, then pours out solution, carefully removes inner membrance with tweezers, Exposure natural down chorioallantoic membrane.

2nd, prepared by tested material

2.1 survey product for water-soluble medicine, weigh a certain amount of commercially available albucid soluble medicine to glass tube, so The pure water of certain volume is added afterwards, is prepared into homogeneous solution.

2.2 positive controls are 3%SLS

2.3 are expected the experience that conjunctiva excitant may be " moderate " according to pharmaceutical raw material, and the concentration for preparing preliminary experiment is 30%th, 15%, 5% and 1% (w/v) four concentration, are tested according to the order from high concentration to low concentration.Formal experiment root Adjusted according to the result of preliminary experiment.

3. conjunctiva stimulation test

3.1 trial tests determine toxicity range

10 days instar chicken embryos are taken, external diameter 9-11mm, internal diameter 8-10mm Teflon " О " shape are placed on chick chorioallantoic membrane surface Ring, the solution of the above-mentioned each concentration gradients of 40 μ L is drawn with liquid-transfering gun, is added in " О " shape ring.Be put into temperature for 37 DEG C ± 1 DEG C, Relative humidity is incubated 30min ± 1min for 65% ± 5% constant incubator, after incubation terminates, immediately observed and recorded blood vessel Various change.And compared with blood vessel outside ring.3 chicken embryos of each concentration.The change of blood vessel judged and is classified according to table 1, Vascular reaction classification is that vascular reaction is positive at 1 grade and more than 1 grade.The concentration that order of severity adjustment according to reaction is formally tested Set.

Preliminary result is as follows.

Concentration	30% (n=3)	15% (n=3)	5% (n=3)	1% (n=3)
					Positive reaction ratio (>=1 grade)	3/3	3/3	3/3	2/3

3.2 conjunctiva excitant RC₅₀It is determined that experiment

According to 3.1 results, the concentration range of formal experimental selection is 15%, 5%, 1% totally 3 concentration, each concentration 10 Chicken embryo.10 days instar chicken embryos are taken, are peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size.Exposure fine hair allantois Film, external diameter 9-11mm, internal diameter 8-10mm Teflon " О " shape ring are placed on chick chorioallantoic membrane surface, and 40 μ L are drawn with liquid-transfering gun The solution of above-mentioned each concentration gradient, is added in " О " shape ring.Be put into temperature be 37 DEG C ± 1 DEG C, relative humidity be 65% ± 5% Constant incubator be incubated 30min ± 1min, after incubation terminates, the various change of observed and recorded blood vessel immediately.As shown in figure 1, And compared with blood vessel outside ring.The change of blood vessel judged and is classified according to table 1, and vascular reaction is classified in 1 grade and more than 1 grade It is positive for vascular reaction.Excitant RC is calculated by logarithmic chart₅₀Value, formal experiment chicken embryo reaction result and RC₅₀Value is as follows：

The irritating RC of conjunctiva₅₀For 8.82%.

3.3 conjunctiva excitants are predicted and classified

According to experimental result, calculated through logarithm-probit method, draw the RC of the sample₅₀For 43.32%, according to conjunctiva The criterion of classification is stimulated, if RC50 ＞ 10.0%, and ＜ 50%, predict " nonirritant ", this experiment draws RC₅₀= 8.82 >=3.0%, and ＜ 10.0%, it is predicted as conjunctiva " weak excitant ".

Embodiment 2 is using the detection of chicken embryo vascular system and evaluates the irritating method of eye cream conjunctiva

1st, the preparation of artificial CAM

The SPF levels fertilization chicken embryo in 0 day embryo age of purchase is put into the perseverance that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% It is incubated in warm incubator.3rd day, by chicken embryo horizontal positioned.4th day, by chicken embryo to optical test, discard and be not fertilized or develop not Good chicken embryo.To the chicken embryo of normal development, an aperture is opened from the non-air chamber end of egg, dust is wiped with cotton ball soaked in alcohol, with note Emitter extracts 2.5-3mL egg white out to form artificial CAM from aperture.Aperture is closed with paraffin.Then on egg top Open a length of side 0.8-1cm small window of rectangle in end top.Small window is closed with preservative film, for observing chicken embryo vigor daily.Will Chicken embryo is put back to the constant incubator that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% and is incubated.14th day, can start into Row experiment.

2nd, prepared by tested material

2.1 survey product for water insoluble paste sample, weigh a certain amount of eye cream (deriving from commercially available homemade goodses) Glass tube is added to, the corn oil of certain volume is then added, is prepared into suspension, makes tested material equal in dissolution system as far as possible Even distribution.

2.2 positive controls are 30% ethanol, and baseline control is similar commercially available import eye cream.

2.3 according to the irritating Empirical rules of eye cream conjunctiva, estimate that the eye cream about exists to the unresponsive maximum concentration of blood vessel 70%, RC scope prepare the concentration of preliminary experiment for 70%, 50%, 30%, 10% totally 4 between 40-60% Concentration (w/v).Formal experiment is adjusted according to the result of preliminary experiment.

3rd, conjunctiva stimulates prediction

3.1 trial tests determine toxicity range

14 days instar chicken embryos are taken, are peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size, the artificial fine hair of exposure Chorioallantoic membrane, external diameter 9-11mm, internal diameter 8-10mm Teflon " О " shape ring are placed on surface, and it is above-mentioned each to draw 40 μ L with liquid-transfering gun The solution of concentration gradient, is added in " О " shape ring.It is put into the perseverance that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% Warm incubator is incubated 30min ± 1min, after incubation terminates, immediately the various change of observed and recorded blood vessel.And with blood vessel phase outside ring Compare.The change of blood vessel judged and is classified according to table 1, and vascular reaction classification is positive for vascular reaction at 1 grade and more than 1 grade Property.The concentration that order of severity adjustment according to reaction is formally tested is set.Preliminary result is as follows.

3.2 conjunctiva excitant RC₅₀It is determined that experiment

According to 3.1 results, the concentration range of formal experimental selection is 50%, 30%, 15% totally 3 concentration, each concentration 10 chicken embryos.14 days instar chicken embryos are taken, are peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size, exposure fine hair urine Cyst membrane, external diameter 9-11mm, internal diameter 8-10mm Teflon " О " shape ring are placed on surface, and it is above-mentioned each dense to draw 40 μ L with liquid-transfering gun The solution of gradient is spent, is added in " О " shape ring.It is put into the constant temperature training that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% Support case and be incubated 30min ± 1min, be incubated after terminating, immediately the various change of observed and recorded blood vessel.As a result as shown in Fig. 2 and with The outer blood vessel of ring compares.The change of blood vessel judged and is classified according to table 1, and vascular reaction classification is blood at 1 grade and more than 1 grade Tube reaction is positive, as a result as shown in Fig. 2 calculating excitant RC by logarithmic chart₅₀Value, even if there is positive reaction in half chicken embryo Tested material concentration.Formal experiment chicken embryo reaction result and RC₅₀Value is as follows：

The irritating RC of sample conjunctiva₅₀For 45.32%.

3.3 conjunctiva excitants are predicted and classified

According to experimental result, calculated through logarithm-probit method, draw the RC of the sample₅₀For 45.32%, according to conjunctiva The criterion of classification is stimulated, if RC50 ＞ 10.0%, and ＜ 50%, predict " nonirritant ", this experiment draws RC₅₀= 45.32 >=3.0%, and ＜ 10.0%, it is predicted as conjunctiva " nonirritant ".Baseline control ＞ 70%.

Embodiment 3 utilizes the detection of chicken embryo vascular system and the irritating method of conjunctiva of evaluation shampoo

1st, the preparation of artificial CAM

The SPF levels fertilization chicken embryo in 0 day embryo age of purchase is put into the perseverance that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% It is incubated in warm incubator.3rd day, by chicken embryo horizontal positioned.4th day, by chicken embryo to optical test, discard and be not fertilized or develop not Good chicken embryo.To the chicken embryo of normal development, an aperture is opened from the non-air chamber end of egg, dust is wiped with cotton ball soaked in alcohol, with note Emitter extracts 2.5-3mL egg white out to form artificial CAM from aperture.Aperture is closed with paraffin.Then on egg top Open a length of side 0.8-1cm small window of rectangle in end top.Small window is closed with preservative film, for observing chicken embryo vigor daily.Will Chicken embryo is put back to the constant incubator that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% and is incubated.11st day, it can start Tested.

2nd, prepared by tested material

2.1 survey shampoo product to be partially dissolved in the thick liquid sample of water, weigh a certain amount of shampoo and add Enter to glass tube, then add the pure water of certain volume, be prepared into suspension, make tested material uniform in dissolution system as far as possible Distribution.

2.2 positive controls are 3%SLS,

2.3 are expected the experience of conjunctiva excitant " weak " according to washing product, prepare the concentration of preliminary experiment for 50%, 30%th, 10% and 2.5% (w/v) four concentration, are tested according to the order from high concentration to low concentration.Formal experiment basis The result adjustment of preliminary experiment.

3. conjunctiva stimulation test

3.1 trial tests determine toxicity range

7 days instar chicken embryos are taken, are peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size.In chick chorioallantoic membrane External diameter 9-11mm, internal diameter 8-10mm Teflon " О " shape ring are placed in surface, and the above-mentioned each concentration gradients of 40 μ L are drawn with liquid-transfering gun Solution, be added in " О " shape ring.The constant incubator that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% is put into incubate 30min ± 1min is educated, is incubated after terminating, immediately the various change of observed and recorded blood vessel.And compared with blood vessel outside ring.It is each dense Spend 3 chicken embryos.The change of blood vessel judged and is classified according to table 1 (be the same as Example 1), and vascular reaction is classified in 1 grade and 1 grade It is positive for vascular reaction above.The concentration that order of severity adjustment according to reaction is formally tested is set.

Preliminary result is as follows.

Concentration	50% (n=3)	30% (n=3)	10% (n=3)	5% (n=3)
					Positive reaction ratio (>=1 grade)	3/3	3/3	3/3	2/3

3.2 excitant RC₅₀It is determined that experiment

According to 4.1 results, the concentration range of formal experimental selection is 10%, 5%, 2.5% totally 3 concentration, each concentration 10 chicken embryos.7 days instar chicken embryos are taken, are peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size.In chick chorioallantoic membrane Surface is placed on chick chorioallantoic membrane surface and places external diameter 9-11mm, internal diameter 8-10mm Teflon " О " shape ring, is inhaled with liquid-transfering gun The solution of the above-mentioned each concentration gradients of 40 μ L is taken, is added in " О " shape ring.Be put into temperature is for 37 DEG C ± 1 DEG C, relative humidity 65% ± 5% constant incubator is incubated 30min ± 1min, after incubation terminates, immediately the various change of observed and recorded blood vessel.Knot Fruit is as shown in figure 3, and compared with blood vessel outside ring.The change of blood vessel judged and is classified according to table 1, and vascular reaction classification exists 1 grade and more than 1 grade is that vascular reaction is positive.As a result as shown in figure 3, calculating excitant RC by logarithmic chart₅₀Value, even if half chicken There is the tested material concentration of positive reaction in embryo.

Formal experiment chicken embryo reaction result and RC₅₀Value is as follows：

Hair washing water sample causes the RC that chicken embryo is reacted₅₀For 5.66%

3.3 conjunctiva excitants are predicted and classified

According to experimental result, calculated through logarithm-probit method, draw the RC of the sample₅₀For 5.66%, pierced according to conjunctiva The criterion of classification is swashed, if RC₅₀>=3.0%, and ＜ 10.0%, predict " weak excitant ".This experiment draws shampoo RC₅₀=5.66% >=3.0%, and ＜ 10.0%, it is predicted as " weak excitant ".

Embodiment 4 utilizes the detection of chicken embryo vascular system and the irritating method of oral mucosa of evaluation mouthwash

1st, the preparation of natural down chorioallantoic membrane

2nd, prepared by tested material

2.1 survey mouthrinse product (certain domestic brand) fluid sample, and directly drawing raw sample is used to test.

2.2 positive controls are 100% ethanol.

2.3 baseline controls are certain import brand mouthwash.

3. mucosal irritation is tested

3.1 experimentation

Step 7 day instar chicken embryo is taken, is peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size.In chicken embryo urine Place external diameter 13-15mm, the Teflon of internal diameter 11-13mm specifications in cyst membrane surface " О " shape rings, 100 μ L are directly drawn with mucus rifle Shampoo, is added in " О " shape ring.Effect washes off tested material after 30 seconds, directly observes result, records every kind of toxicity effect The degree that should change, and compared with blood vessel outside ring.As a result as shown in figure 4,6 chicken embryos of each time.The change of blood vessel according to Table 2 is judged and is classified that vascular reaction classification is that vascular reaction is positive at 1 grade and more than 1 grade.If observation shows whole 6 The scoring of at least one kind of reaction of chicken embryo is more than moderate (overall score >=12), and experiment should be repeated once.

The mucosal irritation vascular reaction grade form of table 2

3.2 mucous membrane irritation forecast models

The overall score of 3 kinds of reactions of every chicken embryo is calculated, then calculates the mean total score (Average of 6 chicken embryo reactions Endpoint Score,AES).

Sample：AES=(1+2+2+1+1+1)=5,

Positive control：AES=(3+3+3+3+3+3)=18,

Baseline control：AES=(1+0+0+0+1+1)=2,

According to the criterion of prediction mucous membrane irritation model, AES ＜ 6：Predict " no oral mucosa excitant "； AES≥ 6, and≤12：Predict " slight oral mucosa excitant "；AES ＞ 12, and ＜ 16：Predict " moderate oral mucosa excitant "；AES ≥16：Predict " serious oral mucosa excitant ".The mouthwash " no oral mucosa excitant " is predicted, but score value is higher than benchmark pair According to product.

Embodiment 5 utilizes the detection of chicken embryo vascular system and the irritating method of conjunctiva of evaluation baby's massage oil

1st, the preparation of natural down chorioallantoic membrane

8d instar chicken embryos are bought, air chamber is stored in egg rack and is transferred to laboratory upward, according to egg inspection, discard inactive and have scarce Sunken chicken embryo.Chicken embryo is placed in 37.5 DEG C ± 0.5 DEG C of incubation temperature, hatched under conditions of relative humidity 60%-70% to 10d Age.10d checks chicken embryo during age according to egg, in eggshell surface mark air chamber position；Eggshell part is peelled off with the curved tweezer of dentistry sawtooth, exposure White egg film, 1-2mL0.9%NaCl solution, which is added dropwise, moistens egg film, then pours out solution, carefully removes inner membrance with tweezers, Exposure natural down chorioallantoic membrane.

2nd, prepared by tested material

2.1 survey massage oil for water insoluble oily liquids sample, draw a certain amount of sample and are added to glass tube, so The corn oil of certain volume is added afterwards, is prepared into suspension, tested material is uniformly distributed in dissolution system as far as possible.

2.2 positive controls are 3%SLS；

2.3 baseline control：Test purpose is that experiment increases similar international product in order to declare that " gentle " and baby use product Compareed on the basis of board product.

2.4 are expected the experience of conjunctiva excitant " slight " according to baby skin protection articles for use, and the concentration for preparing preliminary experiment is 70%th, 50%, 30% and 10% (w/v) four concentration, are tested according to the order from high concentration to low concentration.Formal experiment Adjusted according to the result of preliminary experiment.

3rd, conjunctiva stimulation test

3.1 trial tests determine toxicity range

8 days instar chicken embryos are taken, are peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size.In chick chorioallantoic membrane External diameter 9-11mm, internal diameter 8-10mm Teflon " О " shape ring are placed in surface, and the above-mentioned each concentration gradients of 40 μ L are drawn with liquid-transfering gun Solution, be added in " О " shape ring.The constant incubator that temperature is 37 DEG C ± 1 DEG C, relative humidity is 65% ± 5% is put into incubate 30min ± 1min is educated, is incubated after terminating, immediately the various change of observed and recorded blood vessel.And compared with blood vessel outside ring.It is each dense Spend 3 chicken embryos.The change of blood vessel judged and is classified according to table 1, and vascular reaction classification is anti-for blood vessel at 1 grade and more than 1 grade Should the positive.As a result as shown in Fig. 5, the concentration formally tested according to the order of severity adjustment of reaction is set.

Preliminary result is as follows.

Concentration	70% (n=3)	50% (n=3)	30% (n=3)	10% (n=3)
					Positive reaction ratio (>=2 grades)	1/3	0/3	-	-

3.2 excitant RC₅₀It is determined that experiment

According to result, the concentration range of formal experimental selection is 100%, 70%, 50% totally 3 concentration, each concentration 10 Chicken embryo.8 days instar chicken embryos are taken, are peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size.In chick chorioallantoic membrane table Face is placed on chick chorioallantoic membrane surface and places external diameter 9-11mm, internal diameter 8-10mm Teflon " О " shape ring, is drawn with liquid-transfering gun The solution of the above-mentioned each concentration gradients of 40 μ L, is added in " О " shape ring.Be put into temperature be 37 DEG C ± 1 DEG C, relative humidity be 65% ± 5% constant incubator is incubated 30min ± 1min, after incubation terminates, immediately the various change of observed and recorded blood vessel.As a result such as Shown in Fig. 5, and compared with blood vessel outside ring.The change of blood vessel judged and is classified according to table 1, and vascular reaction is classified at 1 grade It is that vascular reaction is positive with more than 1 grade.Excitant RC is calculated by logarithmic chart₅₀Value, even if there is positive reaction in half chicken embryo Tested material concentration.

Formal experiment chicken embryo reaction result and RC50 values are as follows：

Baby's massage oil samples cause the RC that chicken embryo is reacted₅₀For 90.9%

3.3 conjunctiva excitants are predicted and classified

According to experimental result, calculated through logarithm-probit method, draw the RC of the sample₅₀For 90.9%, stimulated according to conjunctiva The criterion of classification, if RC50 >=50%, is predicted " nonirritant ".Standard accordingly, baby massage oil samples are predicted as " nonirritant ", is declared available for " mildness ".

Embodiment 6 utilizes the detection of chicken embryo vascular system and the method for the mucous membrane irritation for evaluating toothpaste

1st, prepared by artificial CAM

2nd, prepared by tested material

2.1 survey toothpaste (commercially available domestic toothpaste) for water insoluble cream solid easy to foaming, weigh a certain amount of sample Glass tube is added to, the pure water of certain volume is then added, is prepared into suspension, makes tested material equal in dissolution system as far as possible Even distribution.

2.2 positive controls are 3%SLS.Baseline control is certain brand import toothpaste.

3rd, mucosal irritation is tested

3.1 experimentation

Step 7 day instar chicken embryo is taken, is peeled off eggshell to 2 × 2.5cm along the window held successfully with tweezers²Size.In chicken embryo urine Place external diameter 13-15mm, the Teflon of internal diameter 11-13mm specifications in cyst membrane surface " О " shape rings, 100 μ L are directly drawn with mucus rifle Toothpaste mixture, is added in " О " shape ring.Effect washes off tested material after 30 seconds, directly observes result, records every kind of poison The degree of property effect change, and compared with blood vessel outside ring.As a result as shown in fig. 6,6 chicken embryos of each time.The change of blood vessel Judged and be classified according to table 2, vascular reaction classification is that vascular reaction is positive at 1 grade and more than 1 grade.If observation shows complete The scoring of 6, portion at least a kind of chicken embryo reaction is more than moderate (overall score >=12), and experiment should be repeated once.

The mucosal irritation vascular reaction grade form of table 2

3.2 oral mucosa excitants are predicted

The overall score of 3 kinds of reactions of every chicken embryo is calculated, then calculates the mean total score (Average of 6 chicken embryo reactions Endpoint Score,AES)：Sample AES=(1+0+0+1+1+1)=4, baseline control AES=(1+0+0+0+1+1)=3. According to the criterion of prediction mucous membrane irritation model, if：AES≤6：Predict " no oral mucosa excitant "；Sample AES =4 ＜ 6, it is " no oral mucosa excitant " to predict it.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, protection scope of the present invention is included in.Although for example the present invention only list shampoo and Massage oil is conjunctiva stimulated samples, lists toothpaste for mucosal irritation sample.But other materials mentioned in the present invention such as chemistry Product (chloroform, toluene, tributyl chloroethene tin etc.), individual wash shield product (shampoo, hair conditioner, hair dye, Hair-conditioning essential oil, Bath oil, body lotion etc.), home laundry product (liquid detergent, washing powder, soap, liquid detergent, bleaching agent, thimerosal, softener Deng), cosmetics (skin type, sun-proof class, foundation cream, BB frosts, CC refreshing, eye shadow, lip gloss etc.), ophthalmic pharmaceutical (barbiturates, chloroquine, Guanethidine, bretylium tosylate, dilantin sodium, albucid soluble, cocaine, boric acid etc.), plant extracts (crude extract, essence extract And its mixture), bioactivity raw material, polypeptide, other skin contact products (textile such as clothes, underwear extract) etc. also may be used To carry out conjunctiva or mucous membrane irritation detection with the method for the invention.Elution class cosmetics, eye and face apply some make up, eye Portion's medicine, spray drinks agricultural chemicals, air freshener, air particle and floating object, PM2.5 etc. can also use the method for the invention Carry out conjunctiva excitant detection.Toothpaste, mouthwash, tooth pad pasting, gutta-percha etc. can carry out oral mucosa with the method for the invention Excitant is detected, will not enumerate herein.

Claims

1. a kind of utilization chicken embryo vascular system detection and evaluation conjunctiva excitant and the method for mucous membrane irritation, it is characterized in that comprising Following steps：

(1) preparation of CAM

(2) conjunctiva irritant test

It is prepared by 2.1 tested materials

Choose tested material, after being dissolved with solvent, prepare one group have the dilution of concentration gradient standby, and set positive control, Negative control and baseline control；

2.2 Screening tests

Tested material is chosen, after being dissolved with solvent, one group of dilution with concentration gradient is prepared again, tested material conjunctiva thorn is carried out Scope Screening tests are swashed, it is determined that causing the general concentration range that chicken embryo is reacted；

2.3 conjunctiva excitant RC₅₀It is determined that experiment

Tested material is chosen, after being dissolved with solvent, then one group of dilution with concentration gradient is prepared again, system in step (1) is taken Standby fertilization chicken embryo, places Teflon " О " shape ring on the chick chorioallantoic membrane of the fertilization chicken embryo, the group is had into concentration ladder Dilution, positive control, negative control and the baseline control of degree are added separately to Teflon " О " shape ring, and constant incubator is incubated Afterwards, the various change of observation record blood vessel, and compared with blood vessel outside ring, the change of blood vessel is judged and is classified according to table 1, blood Tube reaction classification is positive for vascular reaction more than 1 grade, and conjunctiva excitant RC is calculated by logarithmic chart₅₀Value, even if half chicken embryo There is the tested material concentration of positive reaction；

(3) sticking to mould irritant test

It is prepared by 3.1 tested materials

Liquid tested material is tested in the undiluted form of stoste, paste, oily, thick, microgranular or granular product To be tested after being tested or being diluted using pure water or corn oil as former state, and positive control, negative control and base are set Quasi- control；

3.2 mucomembranous irritant test

The fertilization chicken embryo prepared in step (1) is taken, Teflon " О " shape ring is placed on the chick chorioallantoic membrane of the fertilization chicken embryo, Tested material, positive control, negative control and baseline control are added separately to Teflon " О " shape ring, washed off after effect by Thing is tried, result is directly observed, the degree of every kind of poisonous effect change, every time 6 chicken embryos of experiment, and compared with blood vessel outside ring is recorded Compared with the change of blood vessel is judged and is classified according to table 2, and vascular reaction score value is that vascular reaction is positive at 1 point and more than 1 point；

The mucosal irritation vascular reaction grade form of table 2

(4) forecast model and classification

4.1 conjunctiva excitant forecast model

The RC of tested material is calculated from concentration-response relationship₅₀, conjunctiva excitant is made according to following condition and judged：

RC₅₀>=50%, predict " nonirritant/mildness ",

10.0% ＜ RC₅₀And ＜ 50%, predict " nonirritant ",

3.0%≤RC₅₀And ＜ 10.0%, predict " weak excitant ",

1.0%≤RC₅₀And ＜ 3.0%, predict " moderate excitant ",

RC₅₀＜ 1.0%, is predicted " severe excitant or corrosivity "；

4.2 oral cavity sticking to mould excitant forecast models

AES≤6：Predict " no oral mucosa excitant "；

6 ＜ AES and≤12：Predict " slight oral mucosa excitant "；

12 ＜ AES and ＜ 16：Predict " moderate oral mucosa excitant "

AES≥16：Predict " serious oral mucosa excitant ".

2. utilization chicken embryo vascular system detection according to claim 1 and the side for evaluating conjunctiva excitant and mucous membrane irritation Method, it is characterized in that：The selection gestational age SPF of 0-9 days level fertilizations chicken embryo, which is put into constant incubator, in step (1) is incubated to 10~14 After it, the CAM system reserve at natural air chamber end is obtained.

3. utilization chicken embryo vascular system detection according to claim 1 and the side for evaluating conjunctiva excitant and mucous membrane irritation Method, it is characterized in that：The gestational age SPF of 0 day levels fertilization chicken embryo is chosen in step (1), is formed manually at the 3-4 days by extracting egg white Air chamber, is put into constant incubator and is incubated to after 10~14 days, obtains the CAM system reserve of artifical-air cell.

4. utilization chicken embryo vascular system detection according to claim 1 and the side for evaluating conjunctiva excitant and mucous membrane irritation Method, it is characterized in that：The species of SPF levels fertilization chicken embryo described in step (1) is Bai Laihang chickens, star miscellaneous 288, White Rock, Chinese ephedra Or Lingnan Yellow chicken.

5. the side of conjunctiva excitant and mucous membrane irritation is detected and evaluated according to the utilization chicken embryo vascular system described in claim 1 Method, it is characterized in that：Step (1)~(4) are carried out in constant incubator, and temperature when constant incubator is incubated is 37 DEG C ± 1 DEG C, Relative humidity is 65% ± 5%.

6. utilization chicken embryo vascular system detection according to claim 1 and the side for evaluating conjunctiva excitant and mucous membrane irritation Method, it is characterized in that：Conjunctiva stimulant is the material for intentionally or accidentally touching eyes described in step (2), including elution class Cosmetic, eye and face apply some make up, ocular drug, spray drinks agricultural chemicals, air freshener, air particle and floating object, PM2.5 etc..Mucosal irritation thing is the material in direct contact oral cavity, including toothpaste, mouthwash, tooth pad pasting, gutta-percha, tooth powder etc..

7. utilization chicken embryo vascular system detection according to claim 1 and the side for evaluating conjunctiva excitant and mucous membrane irritation Method, it is characterized in that：Tested material includes described in step (2)：(a) single composition chemical substance, including chemicals, medicine or agriculture Medicine；Or the raw material of (b) based on single composition, including plant extract, crude extract, bioactivity raw material or polypeptide；Or (c) letter Single formula, refers to by the semifinished or finished goods of 3~5 kinds of raw material compoundings, including sun-screening agent or antioxidant.

8. utilization chicken embryo vascular system detection according to claim 1 and the side for evaluating conjunctiva excitant and mucous membrane irritation Method, it is characterized in that：Tested material is chosen in step (2.2), after being dissolved with solvent, one group of dilution with concentration gradient is prepared again Liquid, when carrying out tested material conjunctiva field stimulation Screening tests, screening method includes method or up and down increasing and decreasing from top to bottom, from bottom to top Method carries out examination.

9. utilization chicken embryo vascular system detection according to claim 1 and the side for evaluating conjunctiva excitant and mucous membrane irritation Method, it is characterized in that：Step (2.1) and step (2.3) it is positives control be lauryl sodium sulfate, sodium hydroxide, acetic acid or bacterium Polysaccharide, negative control is the solvent of dissolving tested material, and baseline control is close or identical known product with tested material.