Battery Alternative Methods Study For Eye Irritation Assessment Based On The Risk Decision

Background and ObjectiveChemical stimulate an eye, cause local stimulation, usually involves conjunctiva,cornea, iris and lachrymal stimulating effect. Local eye stimulation most likely caused bystimulation of the cornea, followed by the conjunctiva, iris. The traditional eyes irritateexperiment, using animal test evaluation. Given it been questioned the rationality andethical, this method has not satisfy the current product risk assessment and regulations ofmanagement. Based on the in vivo eye stimulate happen mechanism, at present, hasdeveloped a variety of alternatives to the animal testing method. A single of in vitro methodcan simulate complex eye damage of a role, but still need to continue research how tocombination to use, in order to meet the practical need work. Therefore, it is necessary toaccording to the cornea damage occurs mechanism, combined with the principle of in vitromethods and standardization degree of application of the current stimulation of the manyeyes experiment alternative method of scientific evaluation and applicability. On this basis,according to the testing purpose, a group of in vitro methods strategy to completely replacethe animal testing, meet risk decision and the needs of the safety evaluation.This study is to develop the Chicken Chorioallantoic Membrane-Trypan Blue Staining(CAM-TBS), Red blood cell haemolysis test(RBC)and SIRC cells in Short-term exposure(STE) three eye stimulate experiment alternative methods, and to assess the alone orcombined risk decision system used for scientific and limitations. For the next step, establish eye stimulate experiment in vitro alternative method of combination modelprovides support. In order to meet the alternative of animal experiments and regulatoryrequirements, also for safety evaluation of the product risk decision to provide information.For the future, promoting our country’s eye stimulate the in vitro standardization of theevaluation of the groundwork.Method1CAM-TBSThe experiment using chicken embryos allantoic film takes the structure ofconjunctival similar characteristics, in qualitative observation is developed on the basis ofquantitative detection methods. Through the observation of compounds in the role of CAMcause bleeding, blood vessels dissolved and blood coagulation responses of the damagedegree, determination to absorb a blue of the CAM quantity can be tried quantitativedetection thing damage function, improve the repeatability.2RBCUsually has eye stimulate material also can make protein denaturation or dissolve thecell membrane. Compounds direct contact with the mammals of red blood cells, throughquantitative detection hemolysis red blood cells, and release the cells of the hemoglobindegeneration degree, simulation of the cornea similar damage effect.3STECompound direct role in corneal cause the stimulating effect of the similar to cellstoxic injury to the role. Through testing the cell toxicity indicator may be reaction cornealthe extent of the damage. Experimental results of corneal cells in vitro rabbit (SIRC),short-term exposure cells in the compound, simulation acute corneal stimulation, thecalculation cell activity, growth inhibition degree, and the concentration of the chemicals-effect relations.4Put forward the alternative method of combination strategyRisk assessment based on hazard substances the basic theory and principle, in reference to domestic and foreign eye stimulation test new regulations recognition andbased on the new standard, and puts forward three kinds of alternative methods to givepriority to eye stimulation test risk assessment strategies.Results1CAM-TBSTrypan blue concentration and absorbance OD values of positive correlation betweenthe regression model expression y=0.2968+0.0004xRegression coefficient of the interval estimate:95%CI=[0.0002008，0.0005489],R2=0.8994，showed that trypan blue concentration variables can explain89.94%of thevariation of absorbance OD value.2RBCWilcoxon signed rank sum test to do for paired data, S=1.8182, p=0.9976, theinspection level α=0.05level，differences between the two was not statistically significant.Obtained by the consistency test, kappa=0.6356, confidence interval [0.3930,0.8781]; ingood agreement with the experimental results of the RBC experiment and the Draize rabbiteye.3STEShort-term exposure to the results of Experiment (STE) points SIRC cells comparedwith the Draize rabbit eye experiments classification results after kappa consistency test,kappa=0.6316,95%CI=[0.4040,0.8592], the two classification in good agreement.4Alternative combination method to test strategyThis trial for appropriate "No irritation" to "serious irritation" within the scope of theeye stimulate disaster evaluation."No irritation" to "medium irritation" clearly obvious, thethree kinds of combinational process is shown in figure1.Considering that subjects of physical and chemical properties, may have a irritatingsubstance to go to the CAM-TBS test, if the test results for serious irritation, is directlyclassification for serious irritation; If the test result is not a serious excitant, need to be in cell short-term exposure test or red blood cells hemolysis test. Red blood cells hemolysistest used in surfactant or contain surfactant formula; Other material chooses cells short-termexposure test. According to the test results on anti-deflagration eye irritation thatclassification.Expectations for without irritation material (soluble substances or formula), shouldfirst in cell short-term exposure test, if the test results for without irritation, is directlyclassification for No irritation; Surface active agent or contain surfactant formula red bloodcells need to replenish hemolysis test to confirm or direct test in this way. If the result isstimulating, then for CAM-TBS test, identify and distinguish between the severity of thestimulus.ConclusionThis study is to develop a CAM-TBS, RBC and STE three eye stimulate experimentalternatives, simulated with the damaged cornea conjunctival establish method, based onthe improvement of test materials and test program to explore and establish alternativeexperimental combination. Compare the results of this study and animal Draize rabbit eyesexperiment results show that the level of the three methods of correlation, gradingconsistency and the results are good reproducibility.The three methods can be quantitatively detected compounds’ eye irritation. Eyeirritation classification of different compounds can use different method(s). Eye irritationclassification of compounds, using different approaches to be tested. Severe irritantsubstances classified CAM-TBS, experimental classification method is better; medium/mild irritant substances classified RBC better experiment classification; weak/non-irritating substances classified, STE better experiment classification. CAM-TBS can beused to test a single compound or mixture, the method is not suitable for colored seriousirritant substances classified. RBC and STE commonly used in the detection of dissolvedsubstances, but not for insoluble test substance or mixture, and the former is moresensitive to the surfactant. Considered the advantages and limitations of the three methods, initially proposedthe conclusion of the portfolio strategy: CAM-TBS, experiments with RBC experimentcombinations is more accurately classified serious irritant substances; CAM-TBS,experiment with the STE experiment combined classifier relative consistency of themedium/mild irritant substances classified; combination of RBC experiments and the STEexperiment is weak/no stimulation classification of substances.