OECD Guidelines for the Testing of Chemicals, Section 2
2009; Organization for Economic Cooperation and Development; Linguagem: Inglês
10.1787/20745761
ISSN2074-5761
Tópico(s)Pesticide Residue Analysis and Safety
Resumo2 effects of both endogenous and exogenous factors altering estrogen axis signalling (alterations in production, transport, metabolism and excretion of hormones as well as activation and inhibition of ER). 6.Before performing the REACTIV assay, the laboratory should verify that it has the certifications that may be required by local regulations on the use of transgenic organisms.The REACTIV assay should be performed using the chgh-gfp transgenic line used for the test guideline development, which is commercially available (OECD, REACTIV assay validation report).The use of another transgenic line based on the Choriogenin H promoter driving the expression of GFP or another reporter gene requires a complete OECD validation to adapt the validation criteria, the statistical analysis and the fluorescence thresholds as well as the decision logic.Therefore, other transgenic lines could not be considered as appropriate for the implementation of the REACTIV assay.7.This guideline proposal is based on an international interlaboratory validation study conducted from 2020 to 2022 ( 14).The test has been validated in six laboratories with 18 monoconstituent test substances.Of these: four were tested in six laboratories; another six in five laboratories; another two in four laboratories; another one in three laboratories; another four in two laboratories and another one in one laboratory.8.The endpoint measured is fluorescence in the liver of eleutheroembryos.A very low level of fluorescence is observed in unexposed eleutheroembryos.When transcription of the genetic construct is activated or inhibited following chemical exposure, eleutheroembryos express more or less GFP and, therefore, emit more or less fluorescence.The level of fluorescence of eleutheroembryos exposed to the test chemical is compared to that of eleutheroembryos not exposed to the test chemical.9.The test chemical is tested in the presence and absence of 30 µg/L of testosterone (T).Circulating estrogen and androgen levels remain very low at this eleutheroembryonic life stage.Therefore, circulating T will be mainly exogenous, not endogenous.While endogenous estradiol and T are very low at this stage, CYP19 (aromatase) is still expressed and eleutheroembryos are therefore competent to convert exogenous T to estradiol.Adding T to the test medium allows the detection of substances affecting T availability or antagonising ERs as it is metabolised in vivo into estradiol by the cytochrome P450 enzyme aromatase (CYP19).The concentration of T used for the co-treatment was determined empirically.The chosen concentration (30 µg/L) is the lowest concentration of T inducing a statistically significant increase in fluorescence following a 24 h exposure.The differential gene expression induced by the combination of T and the tested chemical is, therefore, a laboratory induced phenomenon, not observed in the absence of exogenous T at this developmental stage, and thus is only indicative of the capacity of the test item to induce an (anti-)estrogenic activity and is currently not considered predictive of a physiological outcome.It does, however, allow mechanisms of action to be detected that would not be revealed in the absence of an aromatisable androgen such as alterations in aromatase activity or ER antagonism. INITIAL CONSIDERATIONS AND LIMITATIONS10.The assay measures the ability of a chemical to activate or inhibit transcription of the chgh-gfp genetic construct, whether directly through binding to ER or modifying the binding of estrogens to the ER, or indirectly by modifying the amount of estrogen available to activate the
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