# Key Properties for the Toxicity Classification of Chemicals:
## 3 Chemical Properties and Safety Assessment Tests Required under the REACH
### Toxicological Information  
Humans may contact a wide variety of chemicals, either by direct contact or by release into the environment. The use of chemical substances requires examining the potential toxicity and, ultimately, their possible impact on the contribution to human diseases. Knowing the effects of chemicals allows making decisions about the use of chemical products. In most cases, it is required to carry out a battery of tests to evaluate the potential hazard of a chemical substance and thus, be able to make evidence-based decisions on whether to use the substance. REACH guidance establishes the requirements for the study of toxicological properties related to human health depending on the amount of annual production of the substance. As can be observed in Table 1, REACH regulates the need to obtain toxicological information on substances through different tests with increasing complexity depending on annual substance production. In the first range of measurement, REACH regulations require the obtainment of test information based on acute oral toxicity, skin or eye irritation, and mutagenicity. It is important to note that in the registration of a substance with higher tonnage ranges, the performance of in vivo tests is determined by the lack of clear results in in vitro ones in terms of risk assessment or classification of the substance [25]. It could be the case that some studies are only conducted in vitro. Thus, at higher substance production ranges, other endpoints are required. Specifically, in vivo studies on acute and subchronic toxicity at repeated doses, in vivo mutagenicity and genotoxicity, and assessment of reproductive effects are included. All these necessary tests are presented in Table 1 and most of them are based on standard tests recommended by OECD guidelines and are determined by REACH as stated preference methods. Therefore, the information required for the chemical risk evaluation is clearly defined. To explore the damage caused by chemicals on the skin, a wide range of standard tests can be performed in accordance with OECD guidelines. Until a few years ago, the assessment of the possible corrosive effect of chemicals on the skin involved the use of laboratory animals (OECD TG 404); however, nowadays the use of other standard tests prevails. Thus, OECD TG 430, 431, 435 and 439 are recommended to evaluate in vitro skin corrosion and irritation using different methods. Therefore, test OECD TG 430 is based on the rat skin transcutaneous electrical resistance (TER) method, OECD TG 431 is based on the reconstructed human epidermis (RHE) one, and OECD TG 435 contributes an in vitro membrane barrier test method that allows to identify corrosive substances. The OECD TG 439 test includes several validated methods that allow skin irritation assays to be assessed. Moreover, several standard methods are recommended by REACH to assess skin sensitization and some of them used laboratory animals such as guinea pigs and murine (OECD TG 429, 442A, 442B, and 406). Thus, with the aim to improve animal welfare, other mechanistically based in vitro and _in chemico_ test methods (OECD TG 442C, 442D, and 442E) have taken the lead in the assessment of skin sensitization [30]. These standard methods allow measurement of different events that lead to skin sensitization and quantify the possible allergic response, such as the inflammatory responses generated in keratinocytes and gene expression related to specific cellular signaling routes. All these methods should be used for risk assessment associated with first-tier substances (1-10 tons) [30]. In the case of eye irritation assessment, several in vitro and in vivo tests based on OECD guidelines are recommended by REACH. The in vitro tests OECD TG 491 and 492 are based on cytotoxicity measurements by the MTT assay using a confluent monolayer of _Statens Seruminstitut_ Rabbit Cornea (SIRC) cells and reconstructed human cornea-like epithelium (RhCE), respectively.