Replacing animal-derived components in in vitro test guidelines OECD 455 and 487

Preparation of a universally usable, animal product free, defined medium for 2D and 3D culturing of normal and cancer cells

“…human cells in a non-human medium like FBS-supplemented medium, the transferability of the experimental results is questionable. Furthermore, it is an ethical necessity to replace animal-derived ingredients like FBS in the laboratory to maximize animal welfare [10] , [11] , [12] , [13] .…”

Waterborne Tebuconazole Exposure Induces Male-Biased Sex Differentiation in Zebrafish (Danio rerio) Larvae via Aromatase Inhibition

“…The (anti)­estrogenic and (anti)­androgenic activities were detected using ERα-CALUX and AR-CALUX bioassays with licensed cells (BioDetection Systems B.V., Netherlands) according to the International Organization for Standardization guideline 19040-3, as described by Shuliakevich et al The cell cultivation is described in Text S8. The tebuconazole cytotoxicity was first determined as previously described. , After excluding the cytotoxic effects of the solvent and tebuconazole, the reproductive endocrine activities induced by tebuconazole were evaluated using ERα-CALUX and AR-CALUX assays, as described by Besselink . The protocol details are described in Texts S9 and S10, and the reference concentrations are given in Table S3.…”

“…Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Assay. Zebrafish from the 5−60 dpf exposure group were randomly collected from each tank at 12,19,26,33,40,45,50, and 60 dpf (20 larvae for the first two collections and 5 for all subsequent collections). The target genes, including the sex differentiation genes cyp19a1a, foxl2, dmrt1, and sf-1 and the housekeeping gene rpl8, were detected by RNA extraction and qRT-PCR using two-well technical replicates (Text S3).…”

“…The tebuconazole cytotoxicity was first determined as previously described. 40,41 After excluding the cytotoxic effects of the solvent and tebuconazole, the reproductive endocrine activities induced by tebuconazole were evaluated using ERα-CALUX and AR-CALUX assays, as described by Besselink. 42 The protocol details are described in Texts S9 and S10, and the reference concentrations are given in Table S3.…”

“…Sex Hormones Measurement. Zebrafish at the 5−60 dpf exposure stage were randomly collected at 19,25,30,35,40,50, and 60 dpf. The first four collections included 20 fish per tank each, with 10 fish per tank pooled as a replicate for all subsequent collection.…”