CHEK Proficiency study 671 CMI, MI and free formaldehyde in body lotion Date 30 November 2017 Version 1 Status Final Report number CHEK-17-671 page 0
Colophon Number 671 Name CMI, MI and free formaldehyde in body lotion Contact Author Netherlands Food and Consumer Product Safety Authority (NVWA) CHEK working group Paterswoldseweg 1 9726 BA Groningen The Netherlands PO Box 43006 3540 AA Utrecht The Netherlands T +31 088 223 33 33 chek@vwa.nl www.nvwa.nl/chek ISBN 1380-51 Mariëlle van Vondel Authorization Krista Bouma (operational manager) page 1
Content 1 Introduction 1 2 Time-table 1 3 Sample preparation 1 4 Statistical results 2 4.1 CMI in body lotion samples uniform level 3 4.2 MI in body lotion samples uniform level 4 4.3 Free formaldehyde in body lotion samples uniform level 5 4.4 Summary preservatives in body lotion 6 5 Methods of analysis 6 6 Remarks from participants 8 7 List of invited participants 8 8 Explanation of graphical presentations 9 9 Tables and graphical presentations 10-17 page 2
1 Introduction Proficiency study number 671 as directed by the CHEK working group concerned the investigation of the CMI (5-chloro-2-methyl-4-isothiazolin-3-one), MI (2-methyl-4- isothiazolin-3-one) and free formaldehyde contents of two body lotion samples. To get an impression of the performance of the quantitative determination of CMI, MI and free formaldehyde in body lotion 23 laboratories were invited to join this proficiency study. 2 Time-table Homogeneity test of samples 27 September 2017 Distribution of samples 9 October 2017 Deadline for the production of results 10 November 2017 Final report 30 November 2017 3 Sample preparation 600 gram body lotion was obtained from the local market, containing parabens. After mixing of the bulk sample 0.90 g Kathon CG (1.482%) and 5.44 gram formaldehyde (36%) were added and thoroughly mixed for 5 minutes using a planet mixer. After homogenisation, the sample was divided in approximately 60 sub-samples of about 10 gram, prepared and packed in plastic sachets. Homogeneity The homogeneity was checked by the laboratory of the Netherlands Food and Consumer Product Safety Authority, Groningen by randomly selected samples for the determination of the CMI, MI and free formaldehyde contents (see table II). The average contents were 4.87 mg/kg CMI, 9.50 mg/kg MI and 0.389% m/m free formaldehyde. Stability The stability was checked by the laboratory of the Netherlands Food and Consumer Product Safety Authority, Groningen in CHEK proficiency study 600. Samples were regarded as sufficient stable for the purpose of a proficiency study. The sample preparation for this study was similar. Samples are therefore expected to be stable and the stability was not checked. Instructions Samples were stored in a refrigerator (+4 C) until shipment. To each of the participants two samples coded A and B were sent. Participants were instructed to store the samples in a refrigerator. The samples should be analysed directly after that they have been taken out of the refrigerator. If there are water drops inside the sachets, then they should be incorporated in the test samples body lotion. Then samples should be mixed to a homogeneous sample before investigation. page 1
4 Statistical results The results of the laboratories and the Z-scores are given in table I. Figures I to IX give graphical presentations of the results. One-way analysis of variance (ANOVA), preceded by checking for normality and outlier checking of the results (Cochran/Grubbs) is the statistical procedure for obtaining the estimates of within-laboratory and between laboratory variability. Performance The performance of a determination is assessed as following: satisfactory = maximum allowable RSD R 2 questionable = 2< maximum allowable RSD R <3 unsatisfactory = maximum allowable RSD R 3 page 2
4.1 CMI in body lotion uniform level 4.1.1 CMI in body lotion samples A and B 18 labs: Results of laboratory 1 are removed by hand. 18 labs Unit Average 9.47 mg/kg Repeatability standard deviation (s r ') 0.15 mg/kg Repeatability rel. standard deviation (RSD r ') 1.6 % Reproducibility standard deviation (s R ') 1.05 mg/kg Reproducibility rel. standard deviation (RSD R ') 11.1 % Horwitz acceptable value for (RSD R ') 11.4 % Repeatability r' 0.42 mg/kg Reproducibility R' 2.93 mg/kg 4.1.2 Summary CMI in body lotion samples A and B The results of this and earlier performed studies are summarised in the table below. Prof. study Number of labs Average mg/kg RSD R % Acceptable RSD R % 671 18 9.47 11.1 11.4 651 13 9.53 11 11 627 15 10.7 9.3 11 600 13 11.4 19 11 560 10 9.06 18 11 4.1.3 Conclusions CMI in body lotion samples A and B The performance of the determination of CMI in body lotion is satisfactory. The results of laboratory 1 are removed by hand because of far outlying results. The results are drawn from a normal distribution. No Cochran or Grubbs outliers are rejected in the datasets of sample A and B. The results of laboratory 17 are marked as stragglers by the Cochran test indicating bad repeatability but are not rejected. The absolute Z-score of laboratory 1 is greater than 3 and suggests poor performance in terms of accuracy. page 3
4.2 MI in body lotion uniform level 4.2.1 MI in body lotion samples A and B 21 labs: Results of all laboratories 21 labs Unit Average 4.77 mg/kg Repeatability standard deviation (s r ') 0.116 mg/kg Repeatability rel. standard deviation (RSD r ') 2.4 % Reproducibility standard deviation (s R ') 0.47 mg/kg Reproducibility rel. standard deviation (RSD R ') 9.7 % Horwitz acceptable value for (RSD R ') 12.6 % Repeatability r' 0.326 mg/kg Reproducibility R' 1.30 mg/kg 4.2.2 Summary MI in body lotion samples A and B The results of this and earlier performed studies are summarised in the table below. Prof. study Number of labs Average mg/kg RSD R % Acceptable RSD R % 671 21 4.77 9.7 12.6 651 17 5.15 13 13 627 17 4.79 13 13 600 14 4.72 19 13 560 10 2.99 23 14 4.2.3 Conclusions MI in body lotion samples A and B The performance of the determination of MI in body lotion is satisfactory. The results are drawn from a normal distribution. No Cochran or Grubbs outliers are rejected in the datasets of sample A and B. The results of laboratory 17 are marked as stragglers by the Cochran test indicating bad repeatability but are not rejected. page 4
4.3 Free formaldehyde in body lotion uniform level 4.3.1 Free formaldehyde in body lotion samples A and B 18 labs: Results of all laboratories. 15 labs: Results of laboratories 1, 17 and 19 are rejected by the Cochran test 18 labs 15 labs Unit Average 0.306 0.311 % m/m Repeatability standard deviation (s r ') 0.013 0.0038 % m/m Repeatability rel. standard deviation (RSD r ') 4.2 1.2 % Reproducibility standard deviation (s R ') 0.034 0.026 % m/m Reproducibility rel. standard deviation (RSD R ') 11.3 8.4 % Horwitz acceptable value for (RSD R ') 4.8 4.8 % Repeatability r' 0.036 0.010 % m/m Reproducibility R' 0.096 0.073 % m/m 4.3.2 Summary free formaldehyde in body lotion samples A and B The results of this and earlier performed studies are summarised in the table below. Prof. study Number of labs Average % m/m RSD R % Acceptable RSD R % 671 15 0.311 8.4 4.8 651 16 0.302 21 4.8 627 13 0.285 21 4.8 600 11 0.348 7.1 4.7 560 6 0.218 14 5.0 4.3.3 Conclusions free formaldehyde in body lotion samples A and B The performance of the determination of free formaldehyde in body lotion is satisfactory. The results approach a normal distribution. The results of laboratories 1, 17 and 19 are marked as outliers by the Cochran test indicating bad repeatability and are rejected. No Grubbs outliers are rejected in the datasets of sample A and B. The absolute Z-score of laboratories 3 and 17 are greater than 3 and suggest poor performance in terms of accuracy. page 5
4.4 Summary preservatives in body lotion The results of this proficiency study are summarised in the table below. Number of accepted results Average Unit RSD R [%] CMI 18 9.47 mg/kg 11.1 MI 21 4.77 mg/kg 9.7 Free formaldehyde 15 0.311 % m/m 8.4 5 Method of analysis 5.1 Methods of analysis CMI/MI Lab Extraction Technique Detector 1 Methanol HPLC MS 2 Methanol/acetic acid (0.4%) HPLC DAD 3 Acetonitrile HPLC DAD 4 Methanol/acetic acid (0.4%) (80:20) HPLC DAD 5 - HPLC UV 7 - HPLC - 8 Methanol LC MS/MS 9 Methanol/acetic acid (0.4%) (80:20) HPLC DAD 10 Methanol/acetic acid (0.4%) (1:4) HPLC DAD 11 Methanol/acetic acid (0.4%) (80:20) HPLC UV 12 - HPLC DAD 13 - HPLC - 14 Water HPLC DAD 15 - HPLC DAD 16 Methanol/water (20:80) HPLC DAD 17 Methanol/acetic acid (0.4%) (80:20) HPLC DAD 18 Methanol/acetonitrile/acetic acid (0.1%) (10:10:80) HPLC DAD 20 - HPLC DAD 21 Methanol/acetic acid HPLC DAD 22 Methanol/water HPLC UV 23 Methanol/acetic acid HPLC UV page 6
5.2 Methods of analysis free formaldehyde Lab Extraction Technique Detector 1 Photometric 2 Tetrahydrofuran/water (90:10) HPLC DAD 3 Photometric 4 Photometric 5 - HPLC UV 6 6mM disodium hydrogen phosphate HPLC UV 7 - HPLC - 8 Methanol HPLC DAD 11 Formaldehyde/water HPLC UV 12 - HPLC DAD 13 - HPLC - 15 Photometric 16 NMR 17 Photometric 18 Acetonitrile HPLC DAD 19 Tetrahydrofuran HPLC DAD 20 - HPLC DAD 22 Photometric page 7
6 Remarks from participants Lab 3 Lab 4 Lab 9 Determination of Formaldehyde: according to ASU K84.00-7:1991/09. We reported the photometry because we had problems with the determination of formaldehyde with HPLC-DAD after derivatisation (reagent: 2,4- dinitrophenylhydrazine). Peak purity was tested as part of our identification procedure. However, using a DAD enabled to detect that the MI peak was not pure in both samples. Identification using retention time variation complied with our internal acceptance criteria. Lab 10 The samples were not analysed for free formaldehyde. Lab 13 Sample packaging unsuitable. Not resealable. Not to be opened without loss. Pretty little sample material, more would have been nice. Lab 14 We can't quantify CMI and free formaldehyde in our lab. Lab 16 Results corrected by recovery. 7 List of invited participants Europe Austria - 1 participant Croatia - 1 participant Cyprus - 1 participant Estonia - 1 participant France - 3 participants Germany - 6 participants Hungary - 1 participant Ireland - 1 participant Portugal - 1 participant Slovenia - 1 participant Spain - 1 participant Switzerland - 1 participant Outside Europe Iran - 1 participant Taiwan - 1 participant United Arab Emirates - 2 participants page 8
8 Explanation of graphical presentations Z-score As a criterion for evaluation of the performance of an individual laboratory a so-called Z-score is used. The Z-score is given by the following equation: Z x Where: x = an (average) laboratory result. = the average result of all laboratories (calculated exclusive outliers). = an assigned precision standard or fixed target value. As a fixed target value the maximum allowable standard deviation is used at a given concentration-level (according to Horwitz). The Z-score for an individual laboratory can be compared with those of previous proficiency studies to determine whether the laboratory performance has improved. Because Z is standardised, it is comparable for all analytes, testmaterials and analytical methods. In general, an absolute value of Z greater than three suggests poor performance in terms of accuracy. When overall performance in a specific interlaboratory test is graded as "good", values of Z <1 would be very common and values of Z >3 would be very rare. It is possible to classify these scores: Satisfactory = 1 > Z 2 Questionable = 2 < Z < 3 Unsatisfactory = Z 3 Saw-tooth plot The results of the samples are presented in a so-called saw-tooth plot. In this figure individual results of two (nearly) identical individual results are plotted. The average of the sample, the 2s- and 3s-intervals of the target values and the 2s- and 3sintervals after removal of outliers are also included (group s). For split-level samples 2s- and 3s-intervals are presented for each sample. Youden-plot Based on Youden statistics the calculated variance is split in a variance caused by systematic and random errors of the individual laboratories. It is necessary that the samples are similar. Generally the points form an elliptical pattern with the major axis of the ellipse running diagonally at an angle of 45 to the X-axis. The lengths of the perpendiculars drawn from the points to the 45 line are directly related to the random errors. Systematic errors will be presented along the 45 line. The perpendiculars intersect the 45 line at various distances from the point through which the 45 line was drawn. These distances are directly related to the systematic errors. page 9
9 Tables and graphical presentations Table I; Proficiency study 671: CMI, MI and free formaldehyde in body lotion Lab CMI [mg/kg] MI [mg/kg] Free formaldehyde [% m/m] Samples/year Sample A Sample B Z-score Sample A Sample B Z-score Sample A Sample B Z-score 1 126.8 (3) 110.4 (3) 101.02 4.67 4.34-0.45 0.262 (1) 0.287 (1) -2.45-2 9.58 9.47 0.05 4.63 4.58-0.28 0.357 0.348 2.81 30 3 7.19 7.28-2.07 4.22 4.17-0.96 0.258 0.259-3.53 50 4 9.79 10.07 0.43 5.07 4.91 0.36 0.315 0.317 0.35 150 5 10.9 10.9 1.32 5.68 5.64 1.47 0.276 0.276-2.35-6 - - - - - - 0.316 0.321 0.52 1 7 8.59 8.40-0.90 4.49 4.46-0.50 0.328 0.337 1.46 150 8 8.5 8.4-0.94 4.1 4.0-1.20 0.332 0.334 1.50-9 9.83 9.66 0.25 4.92 4.98 0.29 - - - 70 10 7.73 7.84-1.56 3.91 4.03-1.33 - - - - 11 9.65 9.53 0.11 5.04 4.93 0.35 0.301 0.307-0.46-12 11.5 11.4 1.83 5.66 5.70 1.50 0.302 0.295-0.83 100 13 9.0 9.0-0.44 4.5 4.5-0.45 0.323 0.326 0.92 1000 14 - - - 4.99 5.08 0.43 - - - 800 15 9.63 9.48 0.08 4.81 4.85 0.09 0.333 0.333 1.50 50 16 9.10 9.26-0.27 4.74 4.78-0.02 0.288 0.295-1.30 ca. 200 17 10.1 9.5 0.30 5.4 4.9 0.62 0.206 (1) 0.244 (1) -5.79 10-15 18 - - - 4.44 4.45-0.55 0.323 0.325 0.89 400 19 - - - - - - 0.372 (1) 0.314 (1) 2.17-20 9.66 9.72 0.20 4.61 4.62-0.26 0.322 0.325 0.85 100-300 21 10.8 10.5 1.09 4.80 4.70-0.04 - - - 60 22 9.30 9.40-0.11 4.70 4.80-0.04 0.281 0.272-2.32 500 23 10.3 9.99 0.62 5.20 5.52 0.97 - - - 15-20 μ 9.47 4.77 0.311 1.08 0.60 0.015 (1) = Cochran outlier (2) = Grubbs outlier (3) = Results removed by hand page 10
Table II; Homogeneity of body lotion samples Sample no 1st analysis CMI [mg/kg] 2nd analysis 1st analysis MI [mg/kg] 2nd analysis Free formaldehyde [% m/m] 1st analysis 2nd analysis 1 4.70 4.94 9.04 9.26 0.394 0.385 2 4.55 4.61 9.67 9.97 0.383 0.396 3 4.81 4.65 9.91 9.53 0.399 0.380 4 6.05 4.74 11.6 9.23 0.383 0.401 5 4.48 4.92 9.54 9.50 0.384 0.397 6 4.60 4.76 9.21 9.39 0.381 0.389 7 4.69 6.69 9.36 9.11 0.396 0.379 8 4.76 4.27 9.34 9.23 0.384 0.395 9 4.89 4.83 9.50 9.20 0.401 0.391 10 4.79 4.74 9.23 9.28 0.384 0.382 average 4.83 4.92 9.64 9.37 0.389 0.390 t-test t samples -0.32 1.11-0.14 t critical,95% 2.26 2.26 2.26 Homogeneity S between 0 0 0 S within 0.560 0.550 0.00917 0.614 1.08 0.0179 S R 1.05 0.47 0.026 S between / 0 0 0 S between /S R 0 0 0 Based on the t-test there is no significant difference between the averages of 1st and the 2nd analysis for CMI, MI and free formaldehyde. The ratio of the sampling standard deviation and the target value and S R is lower than the recommended value of 0.3. Samples are regarded as sufficient homogeneous for the purpose of the proficiency study. S within = within samples standard deviation. S between = between samples standard deviation. σ = target value calculated from the Horwitz equation or assigned precision standard. S R = target value (reproducibility standard deviation) calculated from the proficiency study. page 11
Graphical presentations CMI in body lotion N.B. Results of laboratory 1 are not presented because of high values Figure Ia; Saw-tooth plot CMI in body lotion, calculated with target s. Figure Ib; Saw-tooth plot CMI in body lotion, calculated with group s (exclusive laboratory 1). page 12
Figure II; Youden plot CMI in body lotion. Figure III; Z-score CMI in body lotion. page 13
Graphical presentations MI in body lotion Figure IVa; Saw-tooth plot MI in body lotion, calculated with target s. Figure IVb; Saw-tooth plot MI in body lotion, calculated with group s. page 14
Figure V; Youden plot MI in body lotion. Figure VI; Z-score MI in body lotion. page 15
Graphical presentations free formaldehyde in body lotion Figure VIIa; Saw-tooth plot free formaldehyde in body lotion, calculated with target s. Figure VIIb; Saw-tooth plot free formaldehyde in body lotion, calculated with group s (exclusive laboratories 1, 17 and 19). page 16
Figure VIII; Youden plot free formaldehyde in body lotion. Figure IX; Z-score free formaldehyde in body lotion. page 17