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Safety Assurance by Alternative Methods

In addition to ensuring the safety of cosmetics, Shiseido has also been putting efforts in developing alternative methods of safety testing, given the society's attention to animal protection in recent years. In developing alternative methods, generally, scientists try to recreate each in vivo process in in vitro and/or in silico environments.

Collagen gel test/photo collagen gel test

A three-dimensional cultured skin model prepared with human skin fibroblasts and collagen has been used to assess cytotoxicity. Safety items that have been reported to correlate with cytotoxicity are primary skin irritation, eye irritation and single dose toxicity. In this test, the test substance is applied to the surface of the skin model, thus allowing the testing of water-insoluble materials. After the test substance is applied to the skin model for a certain time period (in photo collagen gel tests, the test substance is applied for a certain time period with and without UV irradiation), an MTT reagent is applied to stain the surviving cells. Then the dye in a specific amount of gel cutout is extracted and the absorbance is measured.
(Reference) Itagaki, H et al., ATLA 27, 348, 1999

Collagen gel test/photo collagen gel test

SIRC-CVS test

This test using SIRC cell is an alternative method for eye irritation testing. After incubation with the test substance for a certain time period, dead cells that have detached from the surface of culture plates are removed by washing, and the surviving cells that are still attached to the culture plates are stained with crystal violet (CV), and the absorbance is measured. The potency of the test substance’s cytotoxicity is evaluated based on the concentration of the test substance at which 50% of the cells are dead (50% inhibitory concentration: IC50). Studies have shown that this IC50 value is highly correlated with in vivo study results that have been reported in published papers. This test is applicable to test substances that disperse evenly in the culture medium. IC50 values obtained from tests using SIRC cells have also been used to estimate single dose toxicity. Studies have shown that the cytotoxicity value correlates with the assessment value for single dose toxicity with respect to most cosmetic ingredients.
(Reference) Hagino et al, Alternatives to Laboratory Animals 38: 139-152, 2010

SIRC-CVS test

3T3-NRU photo toxicity test

This test evaluates phototoxicity using the Balb/c 3T3 cell line. Cells are seeded into two plates and then incubated with the test substance. One of the plates is irradiated with UV, while the other is not exposed to UV. The surviving cells are stained with neutral red. The absorbance is measured to determine the concentration of the test substance at which 50% of the cells are dead (50% inhibitory concentration: IC50). The ratio of IC50s between the irradiated and non-irradiated plates is then calculated to evaluate phototoxicity. This test is applicable only for test substances that disperse evenly in the culture medium. Studies have shown a high degree of correlation with in vivo study results that have been reported in published papers.
(Reference) OECD Guideline for Testing of Chemicals, 432 : in vitro 3T3 NRU phototoxicity test

3T3-NRU photo toxicity test

SH test/photo-SH/NH 2 test

These tests evaluate sensitization potential using THP-1 cells. THP-1 is a human monocytic cell line. After incubation with the test substance for a certain time period, the cells are stained with a fluorescent reagent that detects the fluorescence-labeled SH group to determine the level of cell surface SH-group production (in tests under photo-irradiation conditions: for photo-SH/NH test, the levels of SH-group/NH-group production are measured respectively for cells exposed to UV and cells not exposed to UV), which is one of the factors used to evaluate sensitization potential. As these tests use a regular culture system, they are applicable as long as the test sample disperses evenly in the culture medium. Studies have shown high degree of correlation with in vivo study results that have been reported in published papers.
(Reference) Suzuki et al., Toxicol. In Vitro., 23, 687-696, 2009

SH test/photo-SH/NH2 test

ARE assay/photo-ARE assay

These assays are conducted using the cell line AREc32 (human breast cancer MCF7 cells harboring an ARE-expression vector) to evaluate sensitization potential. After incubation with the test substance for a certain time period (in photo-ARE assays, after incubation with the test substance for a certain time period with and without UV irradiation), the level of luciferase activity induced by oxidative stress is measured,.
Studies have shown high degree of correlation with in vivo study results that have been reported in published papers.
(Reference) Natsch et al., Toxicol. Sci., 102, 110-119, 2008

ARE assay/photo-ARE assay

h-CLAT (human cell line activation test)

h-CLAT(human cell line activation test) evaluates skin sensitization potential using human monocytic cell line, THP-1. After incubation with the test substance for a certain time period (in photo-h-CLAT tests, after incubation with the test substance for a certain time period with and without UV irradiation to the cells), the expression levels of cell surface antigens CD86 and CD54 are measured.

h-CLAT (human Cell Line Activation Test)

To confirm that a product has no negative in vivo effects, various safety parameters must be investigated. One such parameter of particular importance is skin sensitization (allergy) potential. In developing alternative methods, generally, scientists try to recreate each in vivo process in the laboratory. The mechanism of the skin sensitization response, however, is very complex. Shiseido has taken notice of part of that mechanism, Langerhans cell activation in the epidermis, and has demonstrated the effectiveness of examining CD86 expression on THP-1 cells as an indicator in an in vitro system of skin sensitization testing.

Joint research with Kao Corporation that began in January 2003 showed that a modified test system using CD86 and CD54 expression as indicators gave results that were highly inter-laboratory reproducible and highly consistent with the in vivo study results reported in the literature.

Shiseido is working with Kao Corporation to refine this alternative method of skin sensitization testing with the goal of making it an international standard test.
(Reference) Ashikaga et al., Alternatives to Laboratory Animals, 38, 275-284, 2010

ROS assay

This assay evaluates photoreactivity by measuring reactive oxygen species (ROS), which are generated by some test substance after exposure to UV and may damage the skin. This assay is applicable to materials that dissolve in buffers. In ROS assays, the test substance is added with two types of ROS indicator and exposed to UV. The levels of the two types of compounds generated are determined by measuring the color developed by the respective compound at the optimal wavelength. The results are used to evaluate the photoreactivity of the test substance.
Studies have shown a high degree of correlation with in vivo study results that have been reported in published papers.
(Reference) Seto et al., Current Drug Safety, 7, 140-148, 2012

ROS assay

Ames test

This test evaluates genotoxicity using a mutant bacteria. Some bacteria have undergone mutation and, as a result, cannot grow without a specific type of amino acid. Some test substances induces reverse mutations (it is called “reverse mutations” because it is a mutation that “reverses” the mutant phenotype back to the wild-type phenotype) in such special bacteria, making them able to grow without requiring the specific type of amino acid. Given that some substances exhibit certain activities only after being metabolized, the test is also conducted in the presence of a metabolic system. In Ames tests, a test substance and bacteria are mixed with a buffer or a buffer containing metabolic enzymes and incubated on agar media for 2 days. Counts of colonies formed by mutant bacteria are determined and compared with the colony count on the agar plates treated with the control (e.g., distilled water) to evaluate the potential of the test substance to induce genetic mutations.
(Reference) “Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use”[ PDF:281KB ]

Chromosomal aberration test

This test assesses the ability of the test substance to induce structural and numerical chromosomal aberrations in cultured cells. As in the Ames test, chromosomal aberration tests are also conducted in the presence of a metabolic system as some substances exhibit certain activities only after being metabolized. In chromosomal aberration tests, the test substance is added as is or in combination with a metabolic enzymes-containing buffer to plates on which cells have been cultured. After incubation for a certain time period, chromosome specimens are prepared. The specimens are examined microscopically to determine the proportion of cells with structural or numerical chromosomal aberrations for assessing the potential of the test substance to induce chromosomal aberrations.
(Reference) “Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use”[ PDF:281KB ]

In vitro micronucleus test

This test assesses whether a test substance induces chromosomal changes in cultured cells, resulting in the formation of small nuclei called micronuclei within the cell. As in Ames tests and chromosomal aberration tests, micronucleus tests are also conducted in the presence of a metabolic system as some substances exhibit certain activities only after being metabolized. In micronucleus tests, the test substance is added as is or in combination with a metabolic enzymes-containing buffer to culture bottles in which cells have been cultured. After incubation for a certain time period, cell specimens are prepared. The specimens are examined microscopically to determine the proportion of cells with micronuclei for assessing the potential of the test substance to induce micronuclei.
(Reference) “Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use”[ PDF:281KB ]

Expert system (read across)

The Expert system is a prediction method of safety of chemicals by toxicological exerts. Safety information on substances that are structurally similar to the target compound as well as substances that have similar mechanism of toxicity with respect to protein binding, toxicity potential, toxicity alert, percutaneous absorption and gastrointestinal absorption are collected. The collected safety data of similar substances are used to predict the safety of the target compound. The final judgment on safety of the target compound is made by toxicological experts who specialize in skin sensitization, systemic toxicity, etc., based on the correlation between the safety data collected and the mechanism of toxicity of similar materials.

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