# Key Properties for the Toxicity Classification of Chemicals:
## 4 Chemical's Toxicity Assessment Studies Aligned with Regulatory Requirements
### Toxicological Studies
In vitro tests of skin corrosion are not usually performed in isolation for the evaluation of toxicity of substances but tend to be combined with other tests such as skin sensitization, absorption, genotoxicity, and eye corrosion [203, 204]. On the other hand, these tests are not as widely used in the scientific community and represent a low proportion of the measures related to toxicological properties of substances. With respect to in vitro mutagenicity or genotoxic evaluation, the use of the bacterial reverse mutation test (Ames test mentioned above) is recommended by REACH and used worldwide. Again, this test is used in a wide range of substances such as drugs, chemicals, solvents, food additives, and pesticides [205, 206, 207]. This test is usually combined with other in vitro tests that allow detection of in vitro mammalian chromosomal aberrations or other chromosome damage [208]. As commented above, genotoxic and mutagenic effects are frequently determined by the scientific community (the second in vitro type of assessment after cytotoxicity, as shown in Table 3) and are usually completed with in vivo assays when there are previous positive results [207]. On the other hand, the correlation between in vitro and in vivo studies is not always good, so other assessment methods are required with the aim to estimate toxicological properties [202]. There are some requirements for human health properties which could be studied both in vitro and in vivo. Skin corrosion/damage has only been tested in vitro and following OECD guidelines (OECD 439). On the other hand, eye damage or irritation can be studied in vivo. REACH studies for eyes damage/irritation uses a rabbit in vivo test (OECD TG 405). There are in vivo studies conducted by some authors that involve eyes and cutaneous damage [209, 210]. Taking into consideration the in vivo models used by the scientific community, _Caenorhabditis elegans_ (C. _elegans_) is a good model to evaluate toxicity assays. This testing model could be an intermediate between in vitro and mammalian studies. The scientific community has studied the toxic effects on reproduction with _C. elegans_ toxicity assays [211]. _C. elegans_ models have been widely used for determining acute LC\({}_{50}\), showing that developmental toxicity in _C. elegans_ could be as predictive as rat or mouse models [212, 213, 214, 215, 216]. _C. elegans_ has a combination of advantageous traits (e.g., short life and reproduction cycle, robust, and easy and cheap to maintain large populations) which allow it to be widely used in toxicology [211, 217, 218, 219]. OECD Guidelines for the Testing of Chemicals have evaluated acute toxicity on Wistar rats that are also widely used by scientific community [220, 221, 222]. However, other animals can be used as in vivo models with the aim to assess acute toxicity such as beagle dogs [223]. In vivo models in _Artemia salina_ (_A. salina_) and zebrafish have been used to evaluate acute toxicity based in the lethality test of [PERSON] et al. (1982) modified for _A. salina_ and the Fish Embryo Toxicity test (FET) for zebrafish (_Danio rerio_) [224; 225; 226]. The zebrafish genome presents a 70% homology with the human genome. The characteristics of the zebrafish model are: small size, external fertilization, short life cycle, reproductive capacity, and rapid development which make it ideal to study embryonic development [227]. Heart from zebrafish shows similar electrocardiographic patterns as humans, which makes it possible to study neurological and cardiac physiology [227]. However, it is important to keep in mind the specific differences between these species. Chemical exposure and neurotoxicity have been studied in in vivo models of _A. salina_ and zebrafish [228]. The great osmoregulation capacity for _A. salina_ make this marine crustacean a good in vivo model [229]. The presence of monoaminergic neurons in the outer medulla and different areas of the brain in Artemia salina characterize its nervous system [230]. It is important to note that the _A.