S.L. 437.54 Art. 6with for PCDD/F if dioxin-like or start of the the analytical homologous groups for be added selected ion There is a all 17 13C- 13C-labelled for which animal origin is mandatory than 10% fat, fat extraction. carried out, on whether must - of the higher chlorinated their (based on range of 30% - and

Specific requirements for GC/MS methods to be complied screening or confirmatory purposes - Addition of 13C-labelled 2,3,7,8-chlorine substituted internal standards (and of 13C-labelled internal dioxin-like PCB standards, PCBs have to be determined) must be carried out at the very beginning analytical method, for example, prior to extraction in order to validate procedure. At least one congener for each of the tetra to octachlorinated groups for PCDD/F (and at least one congener for each of the homologous dioxin-like PCBs, if dioxin-like PCBs have to be determined) must (alternatively, at least one congener for each mass spectrometric recording function used for monitoring PCDD/F and dioxin-like PCBs). clear preference, certainly in case of confirmatory methods, of using labelled 2,3,7,8-substituted internal PCDD/F standards and all 12 internal dioxin-like PCB standard (if dioxin-like PCBs have to be determined).

Relative response factors should also be determined for those congeners no 13C-labelled analogue is added by using appropriate calibration solutions.

- For feedingstuffs of plant origin and feedingstuffs of containing less than 10% fat, the addition of the internal standards prior to extraction. For feedingstuffs of animal origin containing more the internal standards can be added either before extraction or after An appropriate validation of the extraction efficiency should be depending on the stage at which internal standards are introduced and results are reported on a product or fat basis. - Prior to GC/MS analysis, one or two recovery (surrogate) standard(s) be added. Control of recovery is necessary. For confirmatory methods, the recoveries individual internal standards should be in the range of 60% to 120%. Lower or recoveries for individual congeners, in particular for some hepta- and octa-dibenzodioxins and dibenzofurans, are acceptable on the condition that contribution to the TEQ value does not exceed 10% of the total TEQ value PCDD/F only). For screening methods, the recoveries should be in the to 140%.

Separation of dioxins from interfering chlorinated compounds such as PCBs chlorinated diphenyl ethers should be carried out by suitable chromatographic (preferably with a florisil, alumina and/or carbon column). Gaschromatographic separation of isomers should be sufficient (< 25% peak between 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF). Determination should be performed according to EPA Method 1613 revision tetra-through octa-chlorinated dioxins and furans by isotope dilution HRGC/ another with equivalent performance criteria. difference between upperbound level and lower bound level should not for feedingstuffs with a dioxin contamination in the range or above the level. For feedingstuffs with contamination levels well below the level, the difference may be in the range of 25% to 40%. Screening methods of analysis

Introduction analytical approaches can be performed using a screening method: a approach and a quantitative approach. approach response of samples is compared to that of a reference sample at the level of Samples with a response less than the reference are declared negative, those response are suspected positives. Requirements: blank and a reference sample(s) have to be included in each test series, which is extracted and tested at the same time under identical conditions. The reference sample must show a clearly elevated response comparison to a blank, additional reference samples at 0,5 × and 2 × the level of interest should included to demonstrate the proper performance of the test in the range of interest for the control of the level of interest, when testing other matrices, the suitability of the reference sample(s) has to be demonstrated, preferentially by including samples shown by HRGC/HRMS to contain a TEQ level around that of the reference sample or else a blank spiked at this level, since no internal standards can be used in bioassays, tests on repeatability are very important to obtain information on the standard deviation within one test series. The coefficient of variation should be below 30%, bioassays, the target compounds, possible interferences, and maximum tolerable blank levels should be defined. approach quantitative approach requires standard dilution series, duplicate or triplicate measuring as well as blank and recovery controls. The result may be expressed as TEQ, thereby assuming that the compounds responsible correspond to the TEQ principle. This can be performed by using dioxin/furan standard mixture) to produce a calibration curve to calculate level in the extract and thus in the sample. This is subsequently corrected TEQ level calculated for a blank sample (to account for impurities from chemicals used), and a recovery (calculated from the TEQ level in a sample around the limit of interest). It is essential to note that part recovery loss may be due to matrix effects and/or differences between values in the bioassays and the official TEF values set by WHO. 7.2. Requirements for methods of analysis used for screening - GC/MS methods of analysis and bioassays may be used for GC/MS methods the requirements as laid down in point 6 are to be based bioassays specific requirements are laid down in point 7.3 and bioassays in point 7.4.

- Information on the number of false-positive and false-negative large set of samples below and above the maximum level or action level in comparison to the TEQ content as determined by a confirmatory analysis. Actual false negative rates should be under 1 %. The rate of samples should be low enough to make the use of a screening tool advantageous.

- Positive results have always to be confirmed by a confirmatory analysis (HRGC/HRMS). In addition, samples from a wide TEQ-range confirmed by HRGC/HRMS (approximately 2% to 10% of the negative Information on correspondence between bioassay and HRGC/HRMS be made available.

7.3. Specific requirements for cell-based bioassays - When performing a bioassay, every test run requires a series concentration of TCDD or a dioxin/furan mixture (full dose-response R2 > 0,95). However, for screening purposes, an expanded low-level analysing low-level samples could be used. - A TCDD reference concentration (about three times quantification) on a quality control sheet should be used for the outcome bioassay over a constant time period. An alternative could be the relative a reference sample in comparison to the TCDD calibration line since the cells may depend on many factors. - Quality control (QC) charts for each type of reference material recorded and checked to make sure the outcome is in accordance guidelines. - In particular for quantitative calculations, the induction dilution used must be within the linear portion of the response curve. the linear portion of the response curve must be diluted and re-tested. least three or more dilutions at one time are recommended to be tested. - The percent standard deviation should not be above 15% determination for each sample dilution and not above 30% betwee independent experiments. - The limit of detection may be set as three times the standard deviation solvent blank or of the background response. Another approach response that is above the background (induction factor five times the calculated from the calibration curve of the day. The limit of quantification set as five to six times the standard deviation of the solvent blank response or to apply a response that is clearly above the background factor 10 times the solvent blank) calculated from the calibration curve of

* Specific requirements for kit-based bioassays ( ) Manufacturer’s instructions for sample preparation and analyses have to be kits should not be used after the expiration date.

Materials or components designed for use with other kits should not be used. kits should be kept within the specified range of storage temperature specified operating temperature. limit of detection for immunoassays is determined as the sum of the three times the standard deviation, based on 10 replicate analysis of the divided by the slope value of the linear regression equation.

Reference standards should be used for tests at the laboratory to make sure responsiveness to the standard is within an acceptable range.

Reporting of results the used analytical procedure makes it possible, the analytical results contain the levels of the individual PCDD/F and PCB congeners and be lowerbound, upperbound and mediumbound in order to include a information in the reporting of the results and thereby enabling the of the results according to specific requirements. should also include the lipid content of the sample as well the method extraction. recoveries of the individual internal standards must be made available in case are outside the range mentioned in point 6, in case the maximum level and in the other cases upon request.

have yet been submitted of commercially available kit-based bioassays having sufficient reliability to be used for screening for the presence of dioxins at the required levels in foodstuffs and feedingstuffs.