The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.

Mixtures of simazine, atrazine, ametryne, prometryne, deethylsimazine, deethylatrazine, hydroxysimazine, hydoxyatrazine, hydoxyprometryne, N-ethyl-and N-isopropylammeline, N-ethyl-and N-isopropylammelide, melamine, ammeline, ammelide, and cyanuric acid in aqueous solution were separated and determined in a single analysis with a detection limit of 30-400 pmol. The s-triazines were detected by a UV detector after elution from a reversed-phase high-pressure liquid chromatography column using a phosphate buffer-methanol gradient at 2 "C.The s-triazine herbicides are widely used, accounting for 28% of herbicide manufacture in 1974 (Kiihle, 19761, and s-triazines have many other industrial uses [e.g., Cook and Hutter (1981a)l. However, no single method is available for the routine identification and quantification of these compounds, especially the more polar derivatives like ammeline, ammelide, and their N-alkylated derivatives. The available techniques all apply only to limited ranges of compounds. Paper chromatography and thin-layer chromatography require many solvent systems for the desired separations and offer poor and time-consuming quantification [reviews by Fishbein (1970Fishbein ( ,1975; see also Loos and Kearney (1978)l. Gas chromatography has been used extensively, but derivatization is essential for many compounds and there seems to be no universal derivatizing agent; furthermore, derivatization is seldom quantitative, and several columns are required to cover the whole range of compounds (Fishbein, 1970(Fishbein, ,1975Lusby and Kearney, 1978;Muir and Baker, 1978;Stoks and Schwartz, 1979;Muir, 1980). Low-pressure column chromatography, which has poor separative properties and is time consuming, has been used occasionally (Plaisted and Thornton, 1964). High-pressure liquid chromatography (HPLC) of s-triazines overcomes many difficulties arising from low volatility, low solubility, and chemical inertness. Thus, e.g., Smolkovi and Pacikovi (1978) We report here a simple method to identify and quantify chloro-and (methy1thio)-s-triazine herbicides, dealkylatrazines, hydroxyparents, N-alkylammelines, N-alkylammelides, melamine, ammeline, ammelide, and cyanuric acid in aqueous solutions by reversed-phase HPLC of underivatized samples. EXPERIMENTAL SECTIONApparatus. HPLC was done using jacketed stainless steel analytical columns (25 cm X 4.6 mm inner diameter) containing a reversed-phase packing of 5-pm mean particle diameter (LiChrosorb RP-18; Merck, Darmstadt, FRG). The mobile phase was delivered through a dynamic mixer (Altex, Berkeley, CA) by two pumps (Altex Model 110) which were controlled by a gradient programmer (Altex Model 420), and samples were applied to the column by using a high-pressure sample injector with pneumatic actuator (Model 7010/70-01; Rheodyne, Berkeley, CA) connected to an automatic sampler (AS1 45; Kontron, Ziirich, Switzerland). The sample loop of the injector was normally 20 wL. The eluate from the column passed through a UV detector (Uvikon LCD 725; Kontron) coupled to an inte...

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

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