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Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Page 1 of 12 Appendix G: Amendments and Deviations (Copies) Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Page 2 of 12 Study Plan Deviation Study No.: 01 G 00 0 02 Date: 8.5.00 Deviation No.: 1 S ubject: Fabric conditioning Page: 8 Paragraph: 5,3.2 Change: In addition to the description in the study plan (paragraph 5.3.2, page 8) the fabrics were pre-conditioned w ith deionisized w ater (spray application). Reason: Improvement o f ability to take either saline (groups A, C, E, G, I) or liquid test article (groups B,D,F.H) into the fabrics. Original: study director copy: QUA .. y ..-..........2 . . . date / . r . ^ date 6 8 . Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Page 3 of 12 Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Page 4 of 12 Date: No.: Subject: November 26, 2001 1 Blood serum analysis for PFOS, PFHS and M570 on 52 blood samples at CENTRE, State College, PA, U.S.A. Page: 10 Paragraph: 5.10 Line: 4,5 Change: Reason: Since PFOS was found in the liver samples, PFOS, PFHS and M570 will be analyzed in 52 blood samples according to the attached tabulation at CENTRE Analytical Laboratories, Inc. (attn. Dr John Flaherts & Mrs. Kim Doolittle), Research Drive State College, PA 16801, U.S.A. Bottle-neck in the bio-analytical department at Fraunhofer ITA Signature Approval: Fraunhofer ITA Study Director ?. M . ? ( C -I Date C. Dasenbrock Monitor of Sponsor cc: Fraunhofer ITA QAU 2 - _M zco 4 M jfc Date M.B. Ketkar Fraunhofer ITEM Fraunhofer ITA ^) Amendment No. 1 to Study 01 G 00 0 02 28 Day Repeated Dermal Contact Study of 3M Articles in Sprague-Dawiey Rats Page 2 of 2 Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles In Sprague-Dawley Rats Animal and Serum Sample Identification Fraunhofer ITA Study N: 01 G 00 0 02 Day of shipment on dry ice: November 26, 2001. Each plastic bag containing the requested sample groups (2M/2F) is marked in its I s1 line with "01 G 00 002 Serum", in its 2nd and 3rd line with the group number / consecutive animal number within the group and the day of sample taking, e.g. "Gr. 15/03,04 D14" for males and "Gr. 16/03,04 D14" for females. The same information is given on each of the tubes containing the rat blood serum. Repent-Dose Dermal Toxicity/Absorption Study of Several Fabric Protectors in Rats Serum Analysis of Collected Samples The following serum samples will be analyzed in duplicate for PFOS, PFHS and M570 with one matrix spike per group at Day 28: Group ID Sample D4 = Day 4 D14 = Day 14 D28 = Day 28 D42 = Ree. Day 14 A 01 A 02 C 05 C 06 E 09 E 10 G 13 G 14 H 15 H 16 1 17 I 18 Contro! USA Fabric Protector Cloth (Red Can) T-7338 OUS Fabric Protector Cloth (Red Can) T-7339 Carpet Protector Cloth (Mill Applied) T-7341 Carpel & Uphol. Protector Liquid (K-Satl) T-7342 Carpet & Uphol. Protector Cloth (K-Salt) T-7343 None None None None None None None None 2M=Gr. 15/ 01, 02 2M=Gr. 15/ 03, 04 2F=Gr. 16/ 01,02 2F=Gr. 16/ 03, 04 2M=Gr. 17/ 01, 02 2M=Gr, 17/ 03, 04 2F=Gr. 18/01,02 2F=Gr. 18/ 03, 04 Grand Total Number o f Samples 2M=Gr. 01/ 05,06 2F=Gr. 02/ 05, 06 2M=Gr. 05/ 05,06 21 =Gr. 06/ 05, 06 2M=Gr. 09/ 05,06 2F=Gr.l0/ 05,06 2M=Gr. 13/ 0 5 ,06 21 =Gr. 14/ 05, 06 2M=Gr. 15/0 5 ,0 6 2F=Gr. 16/ 05, 06 2M=Gr. 17/0 5 ,0 6 2F=Gr. 18/ 05, 06 2M=Gr. 01/ 07, 08 2F=Gr. 02/ 0 7 ,08 None None 2M=Gr. 15/ 07, 08 2F=Gr. 16/ 07, 08 2M=Gr. 17/ 07, 08 2F=Gr. 18/ 07,08 Totals 8 4 4 4 16 16 52 Page 5 of 12 Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Page 6 of 12 tW g Precise RRpesearch. Proven Results. PROTOCOL AMENDMENT Amendment Num ber 2 Effective Date: 1 /1 1 /0 2 Page Exygen Study N um ber 023-068 Protocol N u m b e r 01G 00 0 02 DESCRIPTIO N O F A M EN D ED SECTION Section 5 .1 0 Blood and Liver Samples AMENDED TO The rat serum samples-.will be analyzed according to th e method en titled "Extraction Of Potassium P erfluorooctanesulfonate Or O th er Fluorochem ical Compounds From Serum For Analysis Using HPLC-Electrospray/Mass Spectrom etry" (3M m ethod num ber E TS -8-4.1) w ith the m odifications listed on th e a ttach ed pages. RATIONALE To cla rify th e an alytical m ethod to be used for th e analysis o f th e ra t serum samples. No negative im p ac t. IMPACT ON STUDY ncipal rhyestigator Slgnatu M Study Director Signature A (hv-axttli 4 (i m r) Sponsor RepreserUatiye Signature lanagement Signature U :\F o im \P R O T O C O C _ A M E N D M E N T J .doc Exygen QAU Review tm s . Date r-IW ate S ~^7. Date & ZW Z Date ll> v M u JA th i$1* 2/11/2002/5 Drive Slate College, PA 16801, U SA T: 800.281.3219 LF: 814.272.1019 exygen.com Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Page 7 of 12 Protocol No. 01G00 0 02 Amendment #2 ANALYTICAL METHOD MODIFICATIONS Stock Solution stock solution (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. Fortification Solutions methanol. b- 0-1 Qg/mL Fortification Solution - Pipette 10.0 mL of the 1 0 ug/ml fortification solution into a 100-mL volumetric flask and bring up to volume with methanol. ^ c' PS/mL Fortification Solution - Pipette 10.0 mL of the 0.1 ug/mL fortification solution into a 100-mL volumetric flask and bring up to volume with methanol Store all fortification standard solutions (in 125-mL LDPE bottles) in a refrigerator at 2C to 6C for a maximum period of 6 months from the date of preparation. Calibration Standards Prepare six LC/MS/MS calibration standards in methanol via dilution of the 0.1 pg/mL and 0.01 pg/mL fortification solutions. This is a typical example; additional concentrations may be prepared as needed. i nitial Cone. (pg/mL) 01 0.1 0.1 0.01 0.01 0.01 Volume (mL) 5 2 5 2 Diluted to (mLl T10o0o 100 100 100 100 100 Final f n n , r,, /mT n0.n00o5c 0.002 0.001 0.0005 0.0002 0.0001 Exygen Research Page 1 of 5 Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Page 8 of 12 date",," " p i t a . Protocol No. 01G00 002 Amendment #2 !0l"`i0nS <in 125' mL , DPE bottles) i,, a C f r P 'riod of 6 ">" >' 'to the 3. The sample volume for the red blood cells samples will be 100 uL The " f d:5 M added * the sample will be 0.5 mL and !he ^ o u n lo f Thc , M sd!Uni carbonate/sodium bicarbonate buffer added will be 1 0 mL The samples will only be filtered with 0.2 pm nylon mesh filter if necessary. ' 4. The samples will be analyzed under the following conditions: LC/MS/MS System and Operating Conditions (Turbolonspray) Mass Spec: Interface: PE SCIEX API 3000 Biomoiecular Mass Analyzer SCIEX Turboion Spray Liquid Introduction Interface Harvard infusion pump Computer: Dell UitraScan PI 110 Software: PE SciexAnalyst LI Windows NT HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven C* <J" eS Chr0matogn,I>hl')' 2-l ram x 50 rara. 4(l Injection Voi.: 10 pL Mobile Phase (A): 2 mM Ammonium Acetate in ASTM type I water Mobile Phase (B): Methanol Flow Rate: 0.3 mlAnin. Time % A % B 0 60 40 1.0 0 100 7.0 0 100 7.5 60 40 11.0 60 40 t may be necessary to adjust the HPLC gradient in order to optimize ^ r u m e n t performance Columns with different dimensions (e g., 2.1 mm x 30 mm) and columns from different manufacturers (Keystone Exygen Research Page 2 of 5 Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Page 9 of 12 Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] Page 10 of 12 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Protocol No. 01G 00 0 02 Amendment #2 be excluded from the calibration curve. However, the total number of calibration standards that may be excluded must not exceed 20% of the total number of standards injected. c. The correlation coefficient (r) for calibration curves generated must be >0.9925 (r2>0.985). If calibration results fall outside these limits, then appropriate steps should be taken to adjust instrument operation, and the relevant set of samples must be reanalyzed. Sample Analysis Inject the same volume used for the calibration standards (between 10 to 20 pL) of each sample, fortification, control, etc. into the LC/MS/MS. Each sample must be analyzed in duplicate. a. Standards corresponding to at least six concentrations must be included in an analytical set. b. Inject an entire set of standards (six) at the beginning of the run and inject standards interspersed about every 5-10 samples. All sample injections must be bracketed by standard injections. c. Each set of samples analyzed (not to exceed 25) must include at least one reagent control (extraction solvents only), at least one matrix control, and two matrix control samples fortified at known concentrations and carried through the procedure to verify recovery. Each set must also include at least one methanol blank and one ASTM Type I H20 blank. d. The concentration of each sample, fortification, control, etc. is determined from the standard curve based on the peak area of the analyte in all standards injected during a set. The standard responses must bracket responses of the residue found in the sample set. If necessary, dilute the samples and re-ana]yze to give a response within the standard curve range. e. Fortification recoveries within 80 to 120% are acceptable for fortifications at the LOQ (10 ng/mL) and at levels greater than the LOQ. Failure to meet these criteria requires an investigation of cause and a full reanalysis of the affected samples. f. Samples in which no peaks are detected at the corresponding analyte retention times will be reported as ND (not detected). Samples in _ which peaks are detected at the corresponding analyte retention times but are less than the response of the lowest standard (0.0001 pg/mL) will be reported as NQ (not quantifiable). Exygen Research Page 4 of 5 Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] Page 11 of 12 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats Protocol No. 01G 00 0 02 Amendment #2 g. Background Levels of analyte found in control/blank samples that correspond to values below the LOQ, but are still quantifiable, will be used to correct fortification recoveries. h. If samples are not loaded on the instrument to be analyzed the day they are extracted, samples must be stored refrigerated at approximately 2C to 6DC until analysis and analyzed preferably within a week. Fraunhofer ITEM Final Report 01 G 00 0 02 [Appendices G] Page 12 of 12 28 Day Repeated Dermal Contact Study of 3M Test Articles in Sprague-Dawley Rats EV ;enRESEARCH k . Precise Ree:search. Proven Results. PROTOCOL DEVIATION Deviation Number: 1 Date of Occurrence; 1/15-16,17/02 Exygen Study Number: 023-068 Protocol Number: 01GG0 0 02 . DESCRIPTION OF DEVIATION Protocol Amendment 2 - Sample Analysis.e-Accepted recovery of 79% for PFOS for 0113831 Spk A in Set 011502A. For 0113831 Spk A and 8 and their duplicate injections in Sets 011502AD, Q11602A, Q11602B, accepted recoveries for PFHS of 53%, 56%, 56%, 60%, 70%, 70%, 69%, 70%, 67%, 68%, 68%, and 68%. In Set 011702A for 0113831 Spk B and its dup. in j., accepted recoveries for PFHS of 73% and 74%. In Sets 011602A and 011602B, accepted recoveries for PFHS of 72% (0114158 5pk C and dup. inj.), 78% and 77% (0114162 Spk D and dup. in j.), 76% (0114166 Spk and dup. in j.), 74% (0114170 Spk C and dup, in j.), and 78% and 77% (0114174 Spk D and dup. inj.) Protocol deviation issued. ACTIONS TAKEN for example - deviation issued. SOP revisinn gin Recorded By: No negative impact on study. IMPACT ON STl IDY Date: Windipal friK/estigatpr Signature f . . A H w v i J IjW ) Study Director Signature Sponsor Representative Signature Management Signature 1* R/ Hl^/ z Date ' / Date *> Or? Date Date Exygen QAU Review 2/8/2002/4 VJ^30,3505E8 Research Drive State College, PA 16801, USA T: 800.281.3219 F: 814.272.1019 exygen.com