# Key Properties for the Toxicity Classification of Chemicals:
## 4 Chemical's Toxicity Assessment Studies Aligned with Regulatory Requirements
### Toxicological Studies  
The scientific community also carries out toxicological assessments associated with exposure to different substances, irrespective of whether they meet the minimum annual production tonnage. This community is also aligned with this premise of REACH regulations and is working to develop alternative methods for the study of human toxicological properties [192]. Indeed, Table 3 represents the proportions of each measure which are used by the scientific community. As mentioned above, the toxicological information is obtained through in vivo and in vitro tests. As can be observed in the table, in vitro tests are more frequently completed than in vivo ones (62% vs. 38%). This may be due in large part to the greater ease of carrying out this type of test and to following the premise of avoiding animal experimentation. By analyzing the ratios for each type of test (in vivo and in vitro ones), a search for the main endpoints determined for toxicological properties related to human health was carried out. As can be observed in Table 3, cytotoxicity evaluations (69%) are the most frequent ones for in vitro assays and acute oral toxicity (32%) for the in vivo ones. Both are two of the most frequent endpoints to determine when a substance needs to be registered at the first levels of tonnage [25]. Therefore, the use of in vitro assays is widespread because this kind of test promotes reduction in animal testing. The main objective of the in vitro approach is to estimate the effects that chemical substances might provoke on human health. In general, in vitro assays and cell-based assays function as great biological systems for the detection of the toxicity of chemicals for the assessment of their potential biological \begin{table}
\begin{tabular}{c|c} \hline \hline
**In Vitro (38\%)** & **In Vivo (62\%)** \\ \hline Carcinogenicity (11\%) & \\ Toxicokinetics (4\%) & Skin sensibilization (1\%) \\ Irritation (5\%) & Irritation (4\%) \\ Reproductive tests (18\%) & Cytotoxicity (69\%) \\ Oral toxicity (32\%) & Mutagenicity or genotoxicity (21\%) \\ Inhalatory assays (7\%) & Acute toxicity in vitro (5\%) \\ Mutagenicity or genotoxicity (23\%) & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Toxicological information found in the literature (WOS on 11 September 2022). activity and effects on toxicity pathways [193]. Therefore, cell viability, proliferation, and cytotoxicity assays are broadly used for this type of approach on the effect of human health, even though it is not one of the tests required by REACH [194]. In fact, these tests are frequently evaluated on a wide range of different products, such as solvents, drugs, and bioactive compounds, when an initial toxicological profile is to be established [195]. In this sense, one of the most versatile and popular tests is MTT assay, a colorimetric test that allows the determination of small changes in metabolic cell activity, specifically by the mitochondrial reductase enzyme [196, 197]. Other similar in vitro assays for measuring cell viability that are widely used are the resazurin-based method, such as PrestoBlue [198, 199]. Respective of the assay used for this endpoint, the toxicity of the substances may be different depending on the origin and cell type used [200]. Considering the work of scientific cytotoxicity measures are the most frequent endpoint used to explore basic human toxicology, as mentioned previously. Even though cytotoxic assays provide a good approximation of the effects of substances, other in vitro assays can be performed to evaluate toxicological properties. Assessment of damage caused by chemicals to the skin allows to predict their hazard. Conducting in vitro tests of skin corrosion and irritation using reconstructed human epidermis models allows the identification of corrosive substances [201, 202] and most of them follow OECD guidelines recommended by REACH.