The European Commission has recently published regulation (EU) 2023/464, which amends the Annex to Regulation (EC) No 440/2008, a set of test methods under the Registration, Authorization and Restriction of Chemicals (REACH) regulation. In this amendment, a number of new OECD in vitro test methods are introduced while some old test methods are removed such as the Two-Generation Reproduction Toxicity Study (OECD 416) and the Unscheduled DNA Synthesis Test (OECD 486), which promotes the application of in vitro test methods in the EU. Based on this amendment, significant changes in test methods are summarized in the following based on the testing requirements of the REACH regulation.
1. Serious Eye Damage/Eye Irritation
2. Skin Sensitisation
OECD Test Guideline 497, Defined Approaches on Skin Sensitization, is introduced. With OECD 497, skin sensitisation can be predicted by using 2 in vitro tests (OECD 442C and OECD 442E) and QASR result. If the result of in vitro test is available, category of test chemicals can be made based on QASR result, as shown in the following flowchart. If both in vitro test or QSAR result are not available, there is still a possibility of predicting the category of test chemicals. These test methods can be conducted to significantly reduce the Local Lymph Node Assay (LLNA) testing.
3. Mutagenicity
OECD Test Guideline-488 Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays and OECD 470-Mammalian Erythrocyte Pig-a Gene mutation Assay can be adopted to give more options for in vivo gene mutation experiments. Moreover, unified diagnostic services (UDS) is removed. Currently in the dossier evaluation of the REACH Regulation, Comet or transgenic test is required for gene mutation assays.
4. Reproductive/Developmental Toxicity
OECD 443: Extended One-Generation Reproduction Toxicity Study is introduced to take the place of OECD 416:Two-generation reproduction toxicity study, which has been mandated in REACH earlier. However, the high cost of the test may cause a significant financial pressure for registrants involving high tonnage registration, as some laboratories in Europe have quoted test prices as high as 1 million euros.
5. Endocrine Disrupting Properties
A series of new in vitro test methods for endocrine disrupting properties are newly added, such as OECD Test Guideline 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonistsals; OECD Test Guideline 456: H295R Steroidogenesis Assay; OECD Test Guideline 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonistsals;OECD Test Guideline 493: Performance-Based Test Guideline for Human Recombinant Estrogen Receptor (hrER) In Vitro Assays to Detect Chemicals with ER Binding Affinity, and so on. In vitro testing methods such as OECD Test Guideline 440: Uterotrophic Bioassay in Rodents A shortterm screening test for oestrogenic properties and OECD Test Guideline 441: Hershberger Bioassay in Rats, A Shortterm Screening Assay for (Anti)Androgenic Properties are also introduced. It is anticipated that tests for endocrine disrupting characteristics (EDCs) may be regarded as an important factor in assessing the property of test chemicals.
Other test methods for toxicology and ecotoxicology will not be elaborated here as they are not commonly used and therefore have tiny impact on the selection of tests.
Comments:
As we can see from this amendment, the EU is advancing the in vitro test methods especially the world-leading test methods for skin corrosion/irritation, serious eye damage/irritation and skin sensitisation. These in vitro test methods are expected to expand to other countries and further promote the application of alternative test methods in the worldwide range. In the meanwhile, the EU has amended the CLP regulation and introduced EDCs. Both in vitro and in vivo test methods for EDCs may be combined together to identify the endocrine disrupting properties of test chemicals, which shall attract the attention of enterprises.
The newly added in vivo genotoxicity tests including OECD 488 and OECD 470 provide more available test options. As these tests are novel and expensive, it is arduous to select appropriate laboratory since these tests have stricter requirements. Since OECD 416 has been replaced by extended one-generation reproductive toxicity study, it will not have an influence on REACH registration and there may cause greater financial pressure on relevant registrants as OECD 443 test is costly.
To sum up, when carrying out REACH registration, enterprises should refer to the latest guidelines and regulations and select appropriate testing methods to avoid any potential noncompliance issues.
