Dioxins/TCDD bioassays

Phase 2. In the seeond phase of the study, the partieipants were asked to analyze three extraeted and cleaned sediment samples using the DR CALUX bioassay. Sediments used for extraetion and cleanup were freshwater sediments from the Western Seheldt, The Netherlands. The sediment extracts were prepared by the Royal Institute for Fishery Research (RIVODLO), IJmuiden, The Netherlands, aeeording to the protoeol given here. Dilutions of the supplied sediment extracts were prepared by the partieipants in DMSO and tested for dioxin and/or dioxinlike content. On each 96-well mierotiter plate, a 2,3,7,8-TCDD standard ealibration curve was analyzed. Raw data as well as eonverted data were used for statistieal evaluation. 

Table 1 Intralaboratory repeatability and reproducibility of the dioxin response-chemically activated lucerferase (DR CALUX ) bioassay for sediment extracts 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) TEQ = toxic equivalent.

Several overall conclusions can be drawn based on the statistical evaluation of the data submitted by the participants of the DR CALUX intra-and interlaboratory validation study. First, differences in expertise between the laboratories are apparent based on the results for the calibration curves (both for the curves as provided by the coordinator and for the curves that were prepared by the participants) and on the differences in individual measurement variability. Second, the average results, over all participants, are very close to the true concentration, expressed in DR CALUX 2,3,7,8-TCDD TEQs for the analytical samples. Furthermore, the interlaboratory variation for the different sample types can be regarded as estimates for the method variability. The analytical method variability is estimated to be 10.5% for analytical samples and 22.0% for sediment extracts. Finally, responses appear dependent on the dilution of the final solution to be measured. This is hypothesized to be due to differences in dose-effect curves for different dioxin responsive element-active substances. For 2,3,7,8-TCDD, this effect is not observed. Overall, based on bioassay characteristics presented here and harmonized quality criteria published elsewhere (Behnisch et al., 2001a), the DR CALUX bioassay is regarded as an accurate and reliable tool for intensive monitoring of coastal sediments. 

Several other dioxin and PCB congeners have been tested and were shown to give a response that reflects the differences in the TEF values (Fig. 5.3). However, congeners with a low TEF value showed a relatively low response in the test. This is similary true for 1,2,3,7,8-PeCDD which TEF value was recently adjusted from 0.5 to 1, and which is often a relatively important contributor to the total dioxin content. As a result the test may underestimate the total TEQ content, if calculations were based on the calibration curve for TCDD. However, in general it is evident that the bioassay obeys the TEQ principle and that the result will reflect the total TEQ content of the sample. 

A wide range of environmental samples have been evaluated for dioxin-like compounds with piscine cell line bioassays (Table 5). In nearly all studies some samples have been found to be positive. These results have been converted to bioassay-derived TCDD equivalent concentrations220, and different methods for doing this from the dose-response curves have been developed38 208. When the same samples have been applied to bioassays with mammalian and fish cell lines, the results have been broadly similar104 219 220. However, a few individual samples have sometimes been observed to differ33 220. In most studies, chemical analysis of the... 

Application of this 1/100 safety factor to the NOAEL (1 ng/kg/day or 1000 pg/kg/day) defined in the lifetime rat study (30) that is considered most definitive by essentially all of the regulatory agencies was the basis for the Ontario MOE expert panel to recommend a Maximum Daily Intake for 2,3,7,8-TCDD (or its toxic equivalent of other chlorinated dioxins and furans) of 10 pg/kg B.W./day for humans (38). This more recent approach used by these four non-U.S. groups demonstrate the manner in which carcinogenicits based lifetime exposure control limit recommendations for humans can be realistically derived from the data available from the animal cancer bioassays when interpreted in concert with the data available regarding the likely mechanism of action by which the carcinogenic response occurred in the animal bioassays. 

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