NF VALIDATION Validation of alternative analytical methods Application in food microbiology. Summary report

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1 ACCREDITATION N PORTEE DISPONIBLE SUR 3M Boulevard de l Oise F95029 CERGY PONTOISE Cedex NF VALIDATION Validation of alternative analytical methods Application in food microbiology Summary report EN ISO validation study of the 3M TM Petrifilm TM EB method for Enterobacteriaceae Count Quantitative method This report consists of 33 pages, including 5 appendices. Only copies including the totality of this report are authorized. Competences of the laboratory are certified by COFRAC accreditation for the analyses marked with symbol. Version 0 May 12, 2015 ADRIA DEVELOPPEMENT Creac h Gwen F QUIMPER Cedex Tél. (33) Fax (33) adria.developpement@adria.tm.fr Site web : ASSOCIATION LOI DE 1901 N SIRET N EXISTENCE N TVA FR

2 1 INTRODUCTION Date of first validation, dates of renewal and extension method protocol and principle method 4 2 Main results obtained in the initial validation (1997), the extension studies (2010 and 2015) and the renewal study (2013) Method comparison study (Initial validation in 1997 and extensions in 2010 and 2015) Linearity study Relative accuracy Limit of detection (LOD) and limit of quantification (LOQ) Relative sensitivity Specificity Selectivity Practicability Interlaboratory study Preparation Analyses Results obtained by the collaborating laboratories Calculations and statistical interpretation (Renewal study 2013) Conclusion 24 Appendix 1 method protocol: 3MTM PetrifilmTM Enterobacteriaceae plate 25 Appendix 2 Protocol of the reference method NF ISO (December 2004): Horizontal methods for the detection and enumeration of Enterobacteriaceae. Part 2: colony count method. 26 Appendix 3 Specificity and selectivity: raw data 27 Appendix 4 Mandel graphs (Renewal study 2013) 29 Appendix 5 Statistical calculations (Renewal study 2013) 31 ADRIA Development 2/33 May 12, 2015

3 Quality assurance documents related to this study can be consulted upon request from 3M. The technical protocol and the result interpretation were realized according to the EN ISO (2003). Company 3M Boulevard de l Oise F95029 CERGY PONTOISE Cedex Expert laboratory: ADRIA Développement ZA Creac h Gwen F29196 QUIMPER Cedex Studied method: 3M TM Petrifilm TM Enterobacteriaceae Count (EB) plate Validation standard: NF EN ISO (October 2003): Food microbiology Protocol for the validation of alternative methods method : NF ISO (December 2004): Horizontal methods for the detection and enumeration of Enterobacteriaceae. Part 2: colony count method. Scope of validation: All human and animal feed products, and environmental samples Certification body: AFNOR Certification Test performed according to accreditation ADRIA Development 3/33 May 12, 2015

4 1 INTRODUCTION 1.1 Date of first validation, dates of renewal and extension The 3M TM Petrifilm TM Enterobacteriaceae Count plate was validated on September 10, 1997 (certificate no. 3M01/609/97), according to the requirements for validation studies. Renewals were granted in December 2001, June 2005, July 2009 and July 2013, together with an extension in April 2010 for animal feed products and an extension in February 2015 for environmental samples. 1.2 method protocol and principle The 3M TM Petrifilm Enterobacteria Count plate consists of a cold soluble dehydrated gelling base containing VRBG, combined with an indicator, TTC (2,3,5 Triphenyltetrazolium chloride) to facilitate reading. Enterobacteria appear: red surrounded by a yellow zone and/or red with gas bubbles and/or red surrounded by a yellow zone and with gas bubbles. The protocol is described in Appendix method The reference method used is NF ISO (December 2004): Horizontal methods for the detection and enumeration of Enterobacteriaceae. Part 2: colony count method. The protocol is described in Appendix 2. 2 MAIN RESULTS OBTAINED IN THE INITIAL VALIDATION (1997), THE EXTENSION STUDIES (2010 AND 2015) AND THE RENEWAL STUDY (2013) 2.1 Method comparison study (Initial validation in 1997 and extensions in 2010 and 2015) Linearity study Test performed according to accreditation ADRIA Development 4/33 May 12, 2015

5 Protocol Seven pairs (category / strain) were analyzed at five contamination levels, in order to cover the range of contamination generally encountered. Two replicates were performed per sample. The products were analyzed using both the alternative and reference methods. The following matrices were tested: pork pâté, inoculated with Enterobacter agglomerans 135, isolated from pork liver, milk, inoculated with Hafnia alvei 130, isolated from raw milk, green beans, inoculated with Citrobacter freundii 53, isolated from green beans, salmon fillet, inoculated with Klebsiella oxytoca 179, isolated from a readymade meal, egg, inoculated with Serratia liquefaciens 26, isolated from egg product, dry cat food, inoculated with Escherichia coli 13, isolated from ground steak, process water, inoculated with Escherichia coli 93, isolated from a readymade fish meal. Results Twodimensional graphs for each category are shown in Figure 1 and the regression lines in Figure 2. A summary of the statistical data is provided in Table 1. ADRIA Development 5/33 May 12, 2015

6 Table 1 Summary of the statistical data of the linearity study Matrix R Regression used Rob. F Critical value P% Correlation coefficient Regression line equation Pork paté 2.40 OLS log Alt = log Ref Milk 0.64 GMFR log Alt = log Ref Salmon fillet 2.00 GMFR log Alt = log Ref Green beans 0.17 OLS log Ref = log Alt Egg 0.80 GMFR log Alt = log Ref Dry cat food 0.77 GMFR log Alt = log Ref Process water 1.35 GMFR log Alt = log Ref Statistical interpretation: P > 5% : not significant 1% < P < 5%: significant 0.1% < P < 1%: highly significant P < 0.1% : hyper significant ADRIA Development 6/33 May 12, 2015

7 log( method) log( method) log( method) log( method) 3M Figure 1 Linearity: twodimensional graphs Pork paté Milk log( method) log( method) Salmon fillet Green beans log( method) log( method) ADRIA Development 7/33 May 12, 2015

8 log( method) log(méthode alternative) log( method) log( log(méthode alternative) method) 3M Egg Croquettes Dry cat pour food chat 6,00 5,00 4,00 3,00 2,00 1,00 0,00 log( method) 0,00 1,00 2,00 3,00 4,00 5,00 6,00 log(méthode log( de référence) method) Process Eau de process water 6,00 5,00 4,00 3,00 2,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 log(méthode log( de référence) method) ADRIA Development 8/33 May 12, 2015

9 3M Figure 2 Linearity: regression lines Pork paté OLS 1 regression y = x Milk GMRF regression y = x Salmon fillet GMFR regression y = x Green beans OLS 2 regression y = x Egg GMFR regression y = x ,00 5,00 4,00 3,00 2,00 1,00 0,00 Dry Croquettes cat food pour GMFR chat Régression Regression GMFR y = 1,0922x 0,3266 0,00 1,00 2,00 3,00 4,00 5,00 6,00 Référence 6,00 5,00 Process Eau water de process GMFR Regression Regression GMFR y = 0,9649x 0,1429 4,00 3,00 2,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 ADRIA Development 9/33 May 12, 2015

10 Conclusion The value of P is higher than 5% for the "Pork paté", "Milk", "Green beans", "Egg" and "Process water" matrices, with the nonlinearity test thereby appearing not significant. The value of P is slightly lower than 5% for the "Salmon fillet" matrix, with a P of 4%; it is 0% for the "Process water" matrix. The regression line correlation coefficients show, however, values of and These are high, satisfactory values that could invalidate the robustness of the nonlinearity test Relative accuracy Number and type of samples In the validation and extension studies conducted in 1997, 2010 and 2015, 107, 15 and 30 samples were analyzed respectively, obtaining 86, 13 and 11 exploitable results. These results were exploited in accordance with ISO Product type breaks down as follows: meat products : 24 dairy products : 24 seafood : 10 vegetables : 12 miscellaneous (readymade meals, egg products, cakes, chocolate) : 16 Animal feed : 13 environmental samples : 11 Results A summary of the statistical calculations is provided in Table 2. ADRIA Development 10/33 May 12, 2015

11 Category n R Table 2 Summary of the statistical data of the relative accuracy study Regression used a t (a) b t (b) Critical T P% Ordinate at 0 Meat products GMFR Dairy products GMFR Seafood OLS Vegetables GMFR Miscellaneous GMFR Animal feed GMFR Environmental samples OLS All products GMR Where a: the yintercept of the regression line and b: slope of the regression line Statistical interpretation: P > 5% : insignificant 1% < P < 5%: significant 0.1% < P < 1%: highly significant P < 0.1% : hyper significant Slope at 1 Table 3 Calculation of the bias and repeatability limit Category Bias D method repeatability method repeatability Meat Products Dairy products Seafood Vegetables Miscellaneous Animal feed Environmental samples All products Food category Field of contamination (log UFC/g) Meat Products 1.00 to 5.90 Dairy products 1.00 to 7.88 Seafood 1.30 to 4.80 Vegetables 1.00 to 5.45 Miscellaneous 1.00 to 5.00 Animal feed 1.65 to 6.04 Environmental samples 1.78 to 3.11 All products 1.00 to 7.88 When all products and contamination rates are combined, the bias between the two methods is in favor of the alternative method. ADRIA Development 11/33 May 12, 2015

12 The regression line equation is: log = log The repeatability limit (all products) is for the reference method and for the alternative method. Twodimensional graphs for each category are shown in Figure 3 and the regression lines in Figure 4. Conclusion For the "seafood", "vegetables", "miscellaneous", "animal feed" and "environmental samples" categories, the statistical tests validate these yintercepts close to 0 and slopes close to 1. In all cases, a bias is observed between the alternative method and the reference method, slightly in favor of the Petrifilm Enterobacteria method. Confirmations performed on the Petrifilm test, in the case of overestimation, have revealed the presence of colonies identified as Enterobacteriaceae in most cases. This overestimation is therefore not due to any lack of specificity of the Petrifilm test. These observations make it possible to understand the results of the statistical tests indicating yintercepts other than 0 or slopes other than 1 for meat and dairy product categories, as well as for all categories combined. ADRIA Development 12/33 May 12, 2015

13 log( method) log( log(méthode alternative) method) log( method) log( method) log( method) log( method) 3M Figure 3 Relative accuracy: twodimensional graphics Meat products Dairy products log( method) log( method) Seafood Vegetables log( method) log( method) Miscellaneous Produits Animal d'alimentation feed products animale , ,00 6, , ,00 3, , log( method) 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 log( log(méthode de référence) method) ADRIA Development 13/33 May 12, 2015

14 log (Méthode log( alternative) method) log( method) log(méthode alternative) 3M 6,00 Echantillons Environmental de l'environnement samples Tous All products produits 6,00 5,00 5,00 4,00 3,00 4,00 3,00 2,00 2,00 1,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 log( (Méthode de method) référence) 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 log( method) log(méthode de référence) ADRIA Development 14/33 May 12, 2015

15 3M Figure 4 Relative accuracy: regression lines Meat products GMFR regression Dairy products GMFR regression y = x y = x Seafood OLS 2 regression Seafood GMFR regression y = x y = x Miscellaneous GMFR regression Animal Produits feed d'alimentation products animale GMFR Régression Regression GMFR , y = x ,00 y = 0,9747x 0, ,00 5,00 4,00 3, , ,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 Référence 6,00 4,00 Echantillons Environmental de samples l'environnement OLS2 Regression Regression OLS2 y = 1,0874x 0, ,00 8,00 Tous All produits products y = 0,9618x 0,3691 6,00 2,00 4,00 2,00 0,00 0,00 2,00 4,00 6,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 Référence ADRIA Development 15/33 May 12, 2015

16 2.1.3 Limit of detection (LOD) and limit of quantification (LOQ) Protocol The limit of detection was performed using Serratia liquefaciens 26 culture. Four levels of inoculation were tested, together with level 0. Suspensions were counted by inoculating 1 ml of each suspension 10 times in PCA medium. Results The data is intrinsic to the method. Interpretations are presented in the following tables: A summary is provided in Table 4. Table 4 Summary of the limit of detection and quantification Level targeted Number of "positive" samples Standard deviation So Bias Xo 0 0/6 / / 0.5 1/ / / / Table 5 Formulae Values obtained LC 1.65 So Xo 0.9 LOD 3.3 So Xo 1.7 LOQ 10So Xo 5.2 ADRIA Development 16/33 May 12, 2015

17 cv(<x(y)>) cv(<x(y)>) 3M Relative sensitivity The data is intrinsic to the method. It is obtained from the results of the linearity study. The accuracy profiles obtained for the various matrices are presented in Figure 5. Figure 5 Accuracy profiles obtained for the various matrices 40.0% 35.0% 30.0% 25.0% 20.0% 15.0% 10.0% Pork Pâté paté de porc Egg Coule d'œuf Milk Lait Salmon Pavé de fillet saumon Green Haricots beans verts Dry Croquettes cat food pour chat Process Eau de water process 5.0% 0.0% Méthode de référence method ufc/g ufc/g (x) (x) Specificity Selectivity The raw data is presented in Appendix 3. A specificity study was conducted with 64 target strains and 45 nontargets strains, comparing the results obtained using the Petrifilm Enterobacteria method with those obtained using the reference method. Two strains of Yersinia pseudotuberculosis did not develop either on PEB or on VRBG. The study was completed in 1997 with: 22 strains of Enterobacteriaceae detected among the 23 tested. The strain not recognized by the Petrifilm plate is a strain of Erwinia carotovora ADRIA Development 17/33 May 12, 2015

18 CIP , which was not recognized by the reference method either. (Another strain of Erwinia carotovora was recognized by the Petrifilm plate and not recognized by the reference method). Out of 13 strains not belonging to Enterobacteriaceae: 2 strains of Aeromonas and one strain of Xanthomonas developed, providing typical colonies on the Petrifilm plate and on VRBG. 2.2 Practicability Actual handling time / flexibility of the method with respect to the number of samples for analysis Time to results The handling time per sample is identical for a large or small series of samples Results are obtained in 24 hours (indicated in the instructions) The main advantages of the Petrifilm Enterobacteriaceae method compared to the reference method lie in the response time (24 hours instead of 72 hours for positive samples), the space gained at the incubation stage, ease of handling and ease of waste management. As these are readyforuse media, handling time is significantly reduced in comparison to the reference method. ADRIA Development 18/33 May 12, 2015

19 2.3 Interlaboratory study Preparation The results obtained in the renewal study, conducted in 2001, were used according to ISO This study was performed on semiskimmed pasteurized milk inoculated with Escherichia coli 94. Fifteen laboratories participated in the study and the results of fourteen laboratories were exploited (one laboratory received the samples too late) Analyses The collaborating laboratories and the expert laboratory performed analyses using both the alternative and the reference methods Results obtained by the collaborating laboratories Table 6 Summary of the results obtained by the alternative method and the reference method (in UFC/ml) Lab. method Level 0 Level 1 Level 2 Level 3 method method method method method method method A <1 <1 <1 < B <1 <1 <1 < C <1 <1 <1 < D <1 <1 <1 < E <1 <1 <1 < F <1 <1 <1 < G <1 <1 <1 < I <1 <1 <1 < J <1 <1 <1 < K <1 <1 <1 < L <1 <1 <1 < M <1 <1 <1 < N <1 <1 <1 < O <1 <1 <1 < ADRIA Development 19/33 May 12, 2015

20 Table 7 Summary of the results obtained by the alternative method and the reference method (in log UFC/ml) Level 0 Level 1 Level 2 Level 3 Lab. method method method method method method method method A < < < < B < < < < C < < < < D < < < < E < < < < F < < < < G < < < < I < < < < J < < < < K < < < < L < < < < M < < < < N < < < < O < < < < Calculations and statistical interpretation (Renewal study 2013) Calculations were performed according to amendment 1 of EN ISO (August 15, 2011). DEFINITIONS Accuracy: "closeness of agreement between a test result and the accepted reference value". Trueness: "closeness of agreement between the independent test results obtained in the stipulated conditions of repeatability and reproducibility". Repeatability: "closeness of agreement between successive, independent results obtained with the same method using identical test material, in identical conditions (apparatus, operator, laboratories and shorttime intervals, i.e. the conditions of repeatability)". Repeatability limit (r): "value less than or equal to which the absolute difference between two test results obtained under repeatability conditions is expected with a probability of 95%". ADRIA Development 20/33 May 12, 2015

21 Reproducibility: "closeness of agreement between individual test results performed on identical test material using the same method and obtained by operators in different laboratories using different equipment (i.e. the conditions of reproducibility)". Reproducibility limit (R): "value less than or equal to which the absolute difference between two test results obtained under reproducibility conditions is expected with a probability of 95%". SCRUTINY OF THE MEASUREMENT RESULTS FOR CONSISTENCY Consistency graphs were applied to identify results that are inconsistent compared to the overall results, namely Mandel's h (interlaboratory consistency) and k (intralaboratory consistency) robust statistics. The graphs obtained for the reference method and alternative methods are presented in Appendix 4. The number of values positioned above the thresholds for h and k is shown in the following table: Mandel's values Number of values observed above the threshold method method h > 1% / Lab P Level 2 h > 5% / Lab P Level 2 k > 1% Lab N Level 2 Lab P Level 2 Level 3 k > 5% Lab N Level 2 Lab C Level 1 Lab C Level 1 Lab O Level 1 Lab D Level 3 Lab P Level 2 Level 3 Lab L Level 3 Lab M Level 3 All results were kept for statistical interpretation. ADRIA Development 21/33 May 12, 2015

22 BIAS In order to estimate the bias of the alternative method with respect to the reference method for each level, Dij and t are calculated using the following equations: Dij Y ij, Alt Y ij, Ref t median i ( Dij) / (2 p) Diff If t is higher than 2, the alternative method is significantly biased with respect to the reference method. Table 8 Values obtained per level for t(d) Level Bias D t(d) Conclusion Nonsignificant bias Nonsignificant bias Nonsignificant bias The bias is nonsignificant irrespective of the inoculation level tested. COMPARISON OF THE CHARACTERISTICS OF PRECISION AND TRUENESS OF THE REFERENCE METHOD AND THE ALTERNATIVE METHOD. The statistical calculations are presented in Appendix 5. A summary of the observed values is presented in the table below. Table 9 Level Median method Repeatability standard deviation Reproducibility standard deviation Median method Repeatability standard deviation Reproducibility standard deviation ADRIA Development 22/33 May 12, 2015

23 COMPARISON OF REPEATABILITY STANDARD DEVIATIONS If the ratio of repeatability standard deviations of the alternative method and the reference method is higher than 2, the trueness in repeatability conditions of the alternative method is deemed inferior to that of the reference method. If this ratio is lower than 0.5, trueness of the alternative method is deemed superior to that of the reference method. The values of these ratios are presented in the following table. Table 10 Repeatability standard deviation ratios Contamination method method Ratio level Sr Ref. r Ref. Sr Alt. r Alt. Sr Alt. / Sr Ref Trueness in conditions of repeatability of the alternative method is lower than that of the reference method for level 2 with a fully acceptable repeatability standard deviation (0.123). It is identical for levels 1 and 3. Comparison of reproducibility standard deviations. If the ratio of reproducibility standard deviations of the alternative method and the reference method is higher than 2, the trueness in repeatability conditions of the alternative method is deemed inferior to that of the reference method. If this ratio is lower than 0.5, trueness of the alternative method is deemed superior to that of the reference method. The values of these ratios are presented in the following table. Table 11 Ratio of reproducibility standard deviations Contamination method method Ratio level S R Ref. R Ref. S R Alt. R Alt. S R Alt/S R Ref Trueness in reproducibility conditions of the alternative method is identical to that of the reference method for the three levels tested. ADRIA Development 23/33 May 12, 2015

24 2.4 Conclusion The conclusions of the comparative study of methods are as follows: The Petrifilm Enterobacteriaceae method shows satisfactory linearity. The Petrifilm Enterobacteriaceae method shows satisfactory relative accuracy, with a bias between the two compared methods slightly in favor of the alternative method. The intrinsic data of the Petrifilm Enterobacteriaceae method were also characterized, namely the limit of detection, limit of quantification and relative sensitivity. The major benefit of the Petrifilm Enterobacteriaceae method lies in the time to result, the space gained when incubating the Petrifilm plates, easier waste management, ease of handling and easier reading of the results. The conclusions of the interlaboratory study are as follows: The bias is not significant for the three levels of inoculation tested. Trueness in conditions of repeatability is equivalent to that of the reference method for levels 1 and 3 and is superior for level 2. The standard deviation is low however. It should moreover be noted that the reference method is implemented using two dishes per dilution and the alternative method using one dish per dilution. Trueness in reproducibility conditions is equivalent to that of the reference method for all levels. ADRIA Development 24/33 May 12, 2015

25 Appendix 1 method protocol: 3MTM PetrifilmTM Enterobacteriaceae plate Sample Initial suspension (1/10) Decimal dilutions Inoculation of a Petrifilm plate with 1 ml of suspension 24hour incubation 2 hours at 37 C 1 C Count of typical colonies ADRIA Development 25/33 May 12, 2015

26 Appendix 2 Protocol of the reference method NF ISO (December 2004): Horizontal methods for the detection and enumeration of Enterobacteriaceae. Part 2: colony count method. Initial suspension and decimal dilutions in peptonesaline 1 ml per dish (2 dishes per dilution) VRBG medium added 24hour incubation 2 hours at 37 C 1 C Count of typical colonies: pink to red or purple (with or without a halo) In the absence of typical colonies, confirm 5 whitish colonies Confirmation ADRIA Development 26/33 May 12, 2015

27 Appendix 3 Specificity and selectivity: raw data Positive strain Strain Development of colonies Shigella flexneri cip 8248 Shigella sonnei cip 8249 Salmonella enteritidis cip 8297 Salmonella typhimurium cip 5858 Erwinia carotovora cip 8283 Erwinia carotovora cip Escherichia coli cip Escherichia coli cip Citrobacter diversus cip 8294 Citrobacter freundii cip 5732 Klebsiella pneumoniae cip 8291 Klebsiella oxytoca cip 7932 Enterobacter cloacae adria10 Enterobacter aerogenes cip 6086 Serratia liquefaciens adria8 Hafnia alvei adria 168 Edwarsiella tarda cip 7861 Proteus mirabilis adria 54 Proteus vulgaris adria56 Providencia rettgeri adria 112 Morganella morganii cip A236 Yersinia enterocolitica cip 8027 Kluyvera ascorbata cip 8295 VRBG Typical colonies Development of colonies Petrifilm plate Typical colonies G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G: red colonies without gas surrounded by a yellow halo G: red colonies with gas bubbles ADRIA Development 27/33 May 12, 2015

28 Negative strains Strain VRBG Development of Typical colonies colonies Aeromonas hydrophila cip 5750 Aeromonas hydrophila cip7430 Aeromonas sobria cip 7433 Xanthomonas maltophilia cip 6077 Pseudomonas fluorescens cip 5690 Pseudomonas putida adria 4 Pseudomonas putida adria 8 Pseudomonas aeruginosa cip A22 Acinetobacter spp adria 462 Bacillus circulans ATCC 4513 Lactobacillus plantarum cip A159 Enterococcus faecalis ATCC Staphylococcus aureus cip 658 Development of colonies (*) (*) Petrifilm plate Typical colonies G G G G G G (*) Red colony without acidification halo and without gas G : red colonies without gas surrounded by a yellow halo G: red colonies with gas bubbles ADRIA Development 28/33 May 12, 2015

29 Mandel's robust h values Mandel's robust h values 3M Appendix 4 Mandel graphs (Renewal study 2013) Laboratory h Ref level 1 h Ref level 2 h Ref level 3 h Alt level 1 h Alt level 2 h Alt level 3 h5% h1% h5% h1% B C D E G H I J K L M N O P method robust analysis B C D E G H I J K L M N O P Laboratory h 1%=2.83 1%=2,83 h 5%=1.97 5%=1,97 h Ref level 1 h Ref level 2 h Ref level 3 method robust analysis 4,00 3,00 2,00 1,00 0,00 1,00 2,00 3,00 4,00 B C D E G H I J K L M N O P Laboratory h 1%=2.83 1%=2,83 h 5%=1.97 h Alt level 1 h Alt level 2 h Alt level 3 ADRIA Développement 29/33 May 12, 2015

30 Mandel's robust k values Mandel's robust k values 3M Laboratory k Ref level 1 k Ref level 2 k Ref level 3 k5% K1% k Alt level 1 k Alt level 2 k Alt level 3 B C D E G H I J K L M N O P method robust analysis 4, , , , , , , ,50 0,00 B C D E G H I J K L M N O P Laboratory k 1%=2.57 1%=2,57 k 5%=1.85 5%=1,85 k Ref level 1 k Ref level 2 k Ref level 3 method robust analysis 4, , , , , , , ,50 0,00 B C D E G H I J K L M N O P Laboratory k 1%=2,57 1%=2.57 k 5%=1,85 5%=1.85 k Alt level 1 k Alt level 2 k Alt level 3 ADRIA Développement 30/33 May 12, 2015

31 Appendix 5 Statistical calculations (Renewal study 2013) 3M Level 1 ADRIA Development 31/33 May 12, 2015

32 Level 2 3M ADRIA Development 32/33 May 12, 2015

33 Level 3 3M ADRIA Development 33/33 May 12, 2015