Security tests

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EVIDENCE USE AND APPLICATION
 

 

 

Exhibit N ° 491: Short-term exposure. In vitro test method to identify i) Chemicals that induce serious eye damage and ii) Chemicals that do not require classification for eye irritation or serious eye damage.

This in vitro assay is based on the cytotoxicity evaluation performed on a confluent monolayer of SIRC cells (Statens Seruminstitut Rabbit Cornea) cultured in a 96-well polycarbonate box. After five minutes of exposure to a test chemical, cytotoxicity is measured quantitatively as the relative viability of the SIRC cells using the MTT assay. Decreased cell viability is used to predict potential adverse effects that lead to eye damage. Cell viability is assessed by quantitative measurement, after cell extraction, of the formazan blue salt produced by living cells by enzymatic conversion of the vital stain MTT, also known as thiazolyl blue tetrazolium bromide. The obtained cell viability is compared to the solvent control (relative viability) and used to estimate the potential eye hazard of the test chemical. A test chemical is classified as UN GHS Category 1 when concentrations of 5% and 0.05% result in cell viability less than or equal to (≤) 70%. In contrast, a chemical is predicted as a UN GHS No Category when concentrations of 5% and 0.05% result in cell viability greater than (>) 70%.
 

 

 

 

 

 

 

Test No. 432: 3T3 In Vitro NRU Phototoxicity Test

 

This test describes a method to evaluate photocytotoxicity by the relative reduction of the viability of cells exposed to the chemical in the presence versus absence of light.

 

Balb / c 3T3 cells are kept in culture for 24 h for the formation of monolayers. Two 96-well boxes are pre-incubated with eight different concentrations of the test substance for 1 hr. Subsequently, one of the two plates is exposed to the highest non-cytotoxic irradiation dose, while the other plate is kept in the dark. Cytotoxicity in this test is expressed as a concentration-dependent reduction in Neutral Red (NR) vital dye absorption when measured 24 hours after treatment with the test chemical and irradiation. NR penetrates cell membranes by non-diffusion, accumulating in lysosomes. Alterations in the cell surface of the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes result in a decrease in NR uptake and binding. Therefore, it is possible to distinguish between viable, damaged or dead cells. To predict the phototoxic potential, the concentration responses obtained in the presence and in the absence of irradiation are compared, generally at the IC 50 level, that is, the concentration reduces cell viability to 50% compared to untreated controls.

 

 

 

 

 

 

 

 

 

Test No. 442E: In vitro dermal sensitization

In vitro dermal sensitization assays addressing the key event on dendritic cell activation in the skin sensitization signaling pathway.

 

This test is based on the evaluation of key events (TG) of dermal sensitization, after exposure to a test chemical. More specifically, it addresses dendritic cell activation, which is a key event in the adverse outcome pathway (AOP) for skin sensitization. Skin sensitization refers to an allergic response after skin contact with the tested chemical, as defined by the Globally Harmonized System of Classification and Labeling of Chemicals (United Nations GHS). This TG provides three in vitro test methods that address the same key event in AOP: (i) the human cell line activation test or the h-CLAT method, (ii) the U937 or U cell line activation test. -SENS and (iii) Interleukin-8 Reporter Gene Assay or IL-8 Luc assay. All of them are used to support discrimination between skin sensitizers and non-sensitizers according to the UN GHS. The test methods described in this TG quantify the change in the expression of cell surface markers associated with the activation process of monocytes and DC after exposure to sensitizers (e.g., CD54, CD86) or changes in the IL-8 expression, a cytokine associated with DC activation. In the h-CLAT and U-SENS assays, changes in surface marker expression are measured by flow cytometry after cell staining with fluorochrome-labeled antibodies. In the IL-8 Luc assay, changes in IL-8 expression are indirectly measured through the activity of a luciferase gene under the control of the IL-8 promoter. The relative fluorescence or luminescence intensity of the treated cells compared to the solvent / vehicle control are calculated and used in the prediction model, to support discrimination between sensitizers and non-sensitizers. For this project, the first protocol (h-CLAT) will be addressed.

 

 

 

 

 

In vitro sunscreen activity test (test developed by the UTi)

Premature aging caused by radiation is responsible for 90% of the changes that the skin undergoes. UVA radiation with wavelengths 320-400 nm, are the lowest energy, penetrate the epidermis and dermis of the skin and can damage some structural components, such as the elastin and collagen matrix, damage known as UV-induced photoaging. . These processes are cumulative and contribute to the appearance of fine lines, wrinkles and other signs of aging. In this sense, the cosmetic industry is looking for natural ingredients that can be incorporated into cosmetic formulations that protect the skin from the harmful effects of UV rays. However, there is no in vitro biological test that allows the evaluation of the sunlight protective activity of natural ingredients quickly and inexpensively.

 

For this reason, an in vitro test will be developed based on the exposure of human keratinocytes to a source of artificial sunlight, which will allow to determine the protective effect of sunlight of natural products in a fast, sensitive and inexpensive way.