Research For Some Alternative Methods Of Dermal Toxicity In Cosmetic Safety Evaluation

Skin is the nature barrier to protect body from harmful chemicals, which is also a sensitive organ could be damaged. Such as environment pollutant, occupational exposure and cosmetic utilization possibly result in skin damage or even systemic toxicity. Cosmetic is closely related with human life for protecting health and improving life. So, it is important to assessment the safety of cosmetic in order to safeguard the consumer health and promote the cosmetic industry.The traditional safety evaluation methods for estimating cosmetic toxicity usually apply guinea pig and rabbit as subject animals, witch demand a lot of animals, time consuming, high cost and could not satisfy the requirment about the quickly increasing compound. Except that, those methods have some disadvantages like low sensibility and species difference. Especially, those are discrepancy the protection of animal welfare. Therefore, according to the"4R"principal, developing quicly, high performance and sensitive alternative methods has become a main tendency and a hot pot on international recently. The EU had produce a ban to apply animal test on cosmetic evaluation, as well as the member states couldnot import production which has been tested by animal experiment since March 2009. China is a large cosmetic producer and comsumer. Therefore, it is urgent to establish cosmetic alternative test methods to correspond international request.This article plan to make a study on alternative methods for some dermal toxicity on the base of EU research, in order to provid basement for evolving correlation technology study and formulating the guideline for cosmetic safety evaluatin about alternative method technology in our country.1. Improvement of Local lymph node assay (LLNA) for cosmetics safety evaluationPURPOSE To improve the local lymph node assay (LLNA),establishing an alternative method to detect cosmetics for both sensitization and irritation. METHOD Select one negative control: 4-aminobenzoic acid;three sensitiver: 2,4-dinitrochlorobenzene (DNCB), hexyl cinnamic aldehyde(HCA), 2-aminophenol(2-APC); and one irritative compound: potassium hydroxide (KOH),sodium dodecyl sulfate (SDS). According to the normal LLNA, each solution was given to the female Balb/c mouse, measuring the thickness of each ear. On the sixth day, humane killed the mice and weighed each auricle. The bilateral draining auricular lymph nodes were excised and weighed. Prepare the single cell suspensions, count lymphocyte and detect the proliferation of lymph cell with Cell Counting kit-8(cck-8). RESULT Groups of KOH and DNCB(above 0.5%) may cause significant increase in ear thickness and weight(p<0.05), which could be considered as irritants, whereas 2-APC and HCA were negative. In the allergic test, three sensitizers showed positive, but sensitivity were differences among each index. HCA, DNCB and 2-APC could all augment the weight of lymph node obviously(p<0.05); DNCB and HCA could increase lymphocyte count, whereas HCA was only observed in the high dose. HCA and DNCB were able to cause proliferation of lymphocyte above the middle dose. During the allergic test, KOH always seemed negative. CONCLUSION The reformed LLNA using auricle thickness and weight as observed marker for irritation, and using lymph nodes weight and proliferation of lymphocyte as observed marker, could evaluate both sensitization and irritation at the same time, which have potential to become an alternative method in the future.2. Establishment of reconstituted human engineered skinmodel for evaluating cosmetic skin corrosion / irritation PURPOSE Constructing reconstituted human engineered skin model, to establish an alternative approach for evaluating cosmetics skin corrosion/irritation. METHORD Using human dermal tissue , separate the epidermis and dermis to obtain cells respectively for primary culture. Construct reconstituted skin model with rat tail collagen. Then, culture the model under air-liquid face. Skin models were affected by Chemicals for 3min and 1h in corrosivity test, and 15min in irritation test following with 42h culture respectively. Then, the cell active was detected by MTT essay and IL-1αreleases test (ELISA). RESULT In corrosion test, the cell active was declined <50% (3min) and 15% (1h) after giving three corrosive chemicals. However, the cell active wasn't change for two un-corrosive chemicals. In irritation test, three of four irritative chemicals caused cell active less than 50%, and another one made the IL-1αrelease more than 60pg/ml in ELISA test. It seemed that all of four chemicals were irritations. CONCLUSION It is succeed in constructing reconstituted human engineered skin model, which could become an alternative method for predicting skin corrosion and irritation.3. Research for an in vitro method to evaluate sensitization using THP-1 cellsPURPOSE Look for an in vitro test method to evaluate sensitization using THP-1 cells by cell-surface marker: CD54 and CD86. METHOD Applying human monocytic series leukemia leucoma cell line THP-1, which was induced to immature dendritic cell (iDC) using GM-CSF and IL-4. Supply chemicals to non-induced and induced THP-1 cells respectively. Detect the fluorescence express of cell sueface marker CD54 and CD86 using flow cytometry. And then, estimate the skin sensitization. RESULT After THP-1 cells were suffered by the sensitizer DNCB, HCA and EN, the antibody fluorescence express seemed strenthened by DNCB and EN. It is more than 140% compared to the control group. However, there was no obviously fluorescence enhancement by HCA. In induced iDC, there were no significant fluorescence express increase in both surface antibody suffered by chemicals. CONCLUSION The test method using THP-1 cells by detecting CD54 and CD86's express, could examine moderate and strong sensitizers, not weak sensitizers.4. Evaluating the percutaneous absorption and mutagenicity of Nanosize Titanium Dioxide with human engineered skin modelPURPOSE To try to find out whether the nanosize TiO2 could penetrate skin and induce gene mutation to underlayer cells. In order to provid evidence for evaluating the safety of nanosize TiO2. METHOD Construct reconstituted human engineered skin model firstly, then culture them on the tanswell superior cabin, cultureing L5178Y cells underlayer. Nanosize TiO2 was suffered on the surface of skin models. Evaluating the gene mutation of L5178Y cells using microwell plate Tk mutation assay. RESULT The relative suspension growth rate(RSG) trended descending according to the increasing density for both cells. The RSG for indirect contact cells was generally greater than those for direct contact ones. When the dose increaced, the plate efficiency(PE) also presented decrease, however, there were no significant difference for triflorothymidine (TFT) mutation frequency(MF) between groups. CONCLUSION Nanosize TiO2 could penetrate engineered skin models; whereas, the models displayed partially barrier function for nanoparticals. It doesn't cause gene mutagenic for L5178Y cells on the underside of skin models.