# Key Properties for the Toxicity Classification of Chemicals:
## 3 Chemical Properties and Safety Assessment Tests Required under the REACH
### Toxicological Information
Moreover, the in vitro OECD TG 460 method is performed by using a confluent monolayer of Madin-Darby Canine Kidney (MDCK) and a fluorescent dye, allowing comparison of the difference in fluorescence between untreated cells and those treated with the evaluated substances. On the other hand, in vitro OECD TG 437 and 438 tests are based on qualitative analysis of the opacity and permeability of bovine and chicken eyes, respectively, and allow to relate it to eye irritation. For the measurement of in vivo ocular irritation, the use of the test OECD TG 405 is recommended and intended preferably with albino rabbit. In each animal, one untreated eye and the other treated with the substance are used to measure and compare the damage after 1 h, 24 h, 48 h, and 72 h, evaluating fully the magnitude and reversibility of the effects observed [25; 30]. Considering mutagenicity effects of substances, several methods can be used to assess the effects under REACH requirements. At the first level, in vitro gene mutation in bacteria based on OECD TG 471 is used frequently. This test is commonly known as the Ames test and allows to determine if a substance can cause genetic mutation in bacteria which could induce it to occur in humans as well [31]. Moreover, in vitro mutagenicity in mammalian cells can be measured through the assessment of cytogenicity (OECD TG 473, in vitro mammalian chromosome aberration test) or micronucle-forming ability (OECD TG 487, in vitro micronucleus test). These tests can determine the capacity of a chemical to interfere with the genetic material of mammalian cells. Moreover, a substance can provoke a change in the genetic material and OECD TG 476 and 490 can be followed to measure this property. Both tests determine gene mutation using _Hprt_ and _xprt_ genes or the Thymidine Kinasegene, respectively [32; 33]. Information obtained from all these in vitro tests in mammalian cells has an impact on identifying other human toxic properties. When there is a positive result in any of the in vitro gene mutation studies in bacteria or mammalian cells, the in vivo mutagenicity must be determined. To do this, REACH recommends carrying out several OECD standard tests (OECD TG 475, 474, 486, 488, 489, 483, and 478). Some of them are based on a chromosomal aberration (chromosome and chromatid) test using different types of cells such as mammalian spermatagoniai and bone marrow cells of rodents. Other tests indicate that the animals must be exposed to the substances to be tested and the different mutations generated are checked. It is remarkable the fact that one of the OECD guidelines to follow, specifically OECD TG 486, has not been updated in at least 20 years, which leads one to think that it will not be carried out as much as other tests [30]. Moreover, the in vivo chromosome aberration test and the in vivo micronucleus test are considered the most adequate tests to follow up on the concern of chromosomal aberrations. In addition, the toxicokinetic profile of a substance allows to describe the complete behavior in a living whole organism. This assessment allows to determine the effects from different exposure routes and the distribution of the substance through the body. In this sense, REACH recommends following a toxicokinetic test based on OECD TG 417, providing information that allows understanding of the mechanism of toxicity of the tested substance. Moreover, this test provides information on mass balance, absorption, bioavailability, tissue distribution, metabolism, excretion, etc. These basic toxicokinetic parameters, together with complementary screens, provide useful information on the toxicokinetics of molecules [34]. The acute oral toxicity of a chemical is the only in vivo test recommended at the first level of tonnage production. The health effects of this event may even occur following ingestion of the substance. Acute oral toxicity can be determined following several methods of OECD guidelines (OECD TG 420, 423, and 425).