Genetic toxicity tests used for regulatory screening must be rigorously validated to ensure accuracy, reliability and relevance. Hence, prior to establishment of an internationally-accepted test guideline, a new assay must undergo multi-stage validation. An in vitro transgene mutagenicity assay based on an immortalized cell line derived from MutaMouse lung (i.e., FE1 cells) is currently undergoing formal validation. FE1 cells retain a lacZ transgene in a λgt10 shuttle vector that can be retrieved for scoring of chemically-induced mutations. This work contributes to validation of the in vitro transgene (lacZ) mutagenicity assay in MutaMouse FE1 cells. More specifically, the work includes an intra-laboratory variability study, and a follow-up study to assess the endogenous metabolic capacity of FE1 cells. The former is essential to determine assay reliability, the latter to define the range of chemicals that can be reliably screened without an exogenous metabolic activation mixture (i.e., rat liver S9). The intra-laboratory variability assessment revealed minimal variability; thus, assay reproducibility can be deemed acceptable. Assessment of metabolic capacity involved exposure of FE1 cells to 5 known mutagens, and subsequent assessment of changes in the expression of genes involved in xenobiotic metabolism; induced transgene mutant frequency (±S9) was assessed in parallel. The results revealed that the FE1 cell line is capable of mobilising several Phase I and Phase II gene products known to be involved in the bioactivation of mutagens. Collectively, the results presented support the contention that the FE1 cell mutagenicity assay can be deemed reliable and reproducible. Consequently, the assay is an excellent candidate for continued validation, and eventual establishment of an OECD (Organization for Economic Cooperation and Development) Test Guideline.

No / An isocratic oil-in-water microemulsion High Performance Liquid Chromatography (MELC) was developed and validated for the determination of terbutaline in urine samples. A solid phase extraction (SPE) method which used Oasis HLB cartridges was optimised to isolate terbutaline from a urine matrix followed by HPLC with fluorescence detection. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The method uses the isocratic oil-in-water micro emulsion to separate the terbutaline from the endogenous urine components. The chromatographic separation was carried out on C18-Spherisorb (250mm×4.6mm) analytical column maintained at 30 °C. The mobile phase was 94.4% aqueous orthophosphate buffer (adjusted to pH 3 with orthophosphoric acid), 0.5% ethyl acetate, 1.5% Brij35, 2.5% 1-butanol and 1.1% Octanesulfonic acid (OSA), all w/w. The terbutaline peak was detected by fluorescence detection, using excitation and emission wavelengths of 267 and 313 nm, respectively. The linearity of response was demonstrated at six different concentrations of terbutaline which were extracted from spiked urine, ranging from 60 to 1000ng/ml. The terbutaline was extracted from urine by a solid phase extraction clean-up procedure on Oasis HLB cartridges, and the relative recovery was >87.64% (n = 5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 20.21 and 61.24ng/ml, respectively. The intra-day and inter-day precisions (in term of % coefficient of variation) were <3.56% and <2.87%, respectively. In the method development the influence of the composition of the microemulsion system was also studied and the method was found to be robust with respect to changes of the microemulsion components.