Document e5q0R5nZ7aNow070yx6XZrvRe

A R 2% -0 % \ Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044 T: (215) 443-8710 F: (215) 443-6587 PROTOCOL 418-008 SPONSOR'S STUDY NUMBER: 6295.9 STUDY TITLE: Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats. PURPOSE: The purpose of this study is to test for toxic effects/ disturbances resulting from PFOS treatment of Crl:CDBR VAF/Plus male and female rats before cohabitation through mating, gestation and lactation. This study evaluates ICH Harmonised Tripartite Guideline stages A through F of the reproductive process and should detect effects on the estrous cycle, tubal transport, implantation, gestation, parturition, lactation and maternal behavior in female rats, on the development of the offspring of the treated male and female rats, and permit detection of functional effects (e.g., effects on libido or epididymal sperm maturation) that may not be detected by histological examinations of male rat reproductive organs. Because manifestations of effects induced during this period may be delayed in the offspring, observations will be continued through production of F2 generation litters. TESTING FACILITY: Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044-1297 Telephone: (215) 443-8710 Telefax: (215)443-8587 STUDY DIRECTOR: Raymond G. York, Ph.D., DABT Associate Director of Research SPONSOR: 3M Toxicology Services 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 000061 Protocol 418-008 Page 2 STUDY MONITOR: Marvin T. Case, D.V.M., Ph.D. Telephone: (612)733-5180 Telefax: (612) 733-1773 ALTERNATE STUDY MONITOR: Andrew M. Seacat, Ph.D. Telephone: (612)575-3161 Telefax: (612)733-1773 REGULATORY CITATIONS: Study Design as Modification of: U.S. Food and Drug Administration (1994). International Conference on Harmonisation; Guideline on detection of toxicity to reproduction for medicinal products. Federal Register, September 22,1994, Vol. 59, No. 183. U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule. 21 CFR Part 58. Japanese Ministry of Health and Welfare (1997). Good Laboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997, European Economic Community (1989). Council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles o f good laboratory practice. Official Journal of the European Communities: Legislation. 32 (No. L 315; 28 October): 1-17. REGULATORY COMPLIANCE: This study will be conducted in compliance with the Good Laboratory Practice (GLP) regulations cited above. All changes or revisions of this protocol shall be documented, signed by the Study Director and the Sponsor, dated and maintained with the protocol. The Quality Assurance Unit (QAU) will audit the protocol, the raw data and the report, and will inspect critical phases of the study in accordance with the Standard Operating Procedures of Argus Research Laboratories, Inc. The final report will include a statement signed by the Study Director that the report accurately reflects the raw data obtained during the performance of the study and that all applicable GLP regulations were followed in the conduct of the study. Should significant deviations from GLP regulations occur, each will be described in detail, together with how the deviation might affect the quality or integrity of the study. 000062 Protocol 418-008 Page 3 STUDY SCHEDULE: See ATTACHMENT 1 to the protocol. TEST ARTICLE AND VEHICLE: Identification: Test Article: Name: Physical Description: Lot/Batch Number: Specific Gravity: Purity: Expiration Date: PFOS. Light-colored powder. 217. - 0.6. 98.9%. May, 2000. Information on the identity, composition, strength and purity of the test article is on file with the Sponsor. Vehicle: 0.5% Tween 80 in Reversed Osmosis Membrane Processed Deionized Water (R.O. Deionized Water). Supplier and lot identification of Tween 80 to be documented in the raw data. Neither the Sponsor nor the Study Director is aware of any potential contaminants likely to be present in the vehicle that would interfere with the results of this study. Therefore, no analyses other than those mentioned in this protocol will be conducted. Safety Precautions: Gloves, mask, appropriate eye protection and a uniform/lab coat are to be worn during formulation preparation and dosage administration. The Material Safety Data Sheet (MSDS) is attached to the protocol (ATTACHMENT 2). Storage: Bulk Test Article: Vehicle Components: Prepared Vehicle: Prepared Formulations: Room temperature. Room temperature. Room temperature. Frozen (-20C). All test article shipments to the Testing Facility should be addressed to the attention of Julian Gulbinski, Manager of Formulations, at the previously cited address and telephone number. 000063 Protocol 418-008 Page 4 Shipments should include information concerning storage conditions and shipping cartons should be labeled appropriately. The recipient should be notified in advance of shipment. FORMULATION: Frequency of Preparation: Formulations (suspensions) will be prepared daily at the Testing Facility. Detailed preparation procedures are attached to this protocol (ATTACHMENT 3). Adjustment for Puritv: The test article will be considered 100% pure for the purpose of dosage calculations. Testing Facility Reserve Samples: The Sponsor will reserve a sample (1 g) of each lot of the bulk test article used during the course of this study. The Testing Facility will reserve a sample (5 mL) of each lot of the vehicle components used during the course of this study. Samples will be stored under the previously cited conditions. ANALYSES: Samples additional to those described below may be taken if deemed necessary during the course of the study. Bulk Test Article Sampling: No analyses of the bulk test article will be conducted during the course of this study. Information on the stability of the bulk test article is on file with the Sponsor. Analyses of Prepared Formulations: Stability: Stability data for prepared formulations bracketing the range of concentrations and conditions of this study are on file with the Sponsor and will not be determined during the conduct of this study. Suspensions will be prepared daily at the Testing Facility. 000064 Protocol 418-008 Page 5 Homogeneity Analyses: Homogeneity of the test article in prepared suspensions will be verified during the course of this study. A syringe will be used to withdraw samples (5 mL each) from the top, middle and bottom of the highest concentration on the first day of preparation. Each sample (5 mL) will be divided into two aliquots, one of 2 mL and one of 3 mL. One aliquot (2 mL) will be shipped for analysis; the other aliquot (3 mL) will be retained at the Testing Facility as a backup sample. Backup samples will be stored under the previously cited conditions and discarded at the Testing Facility upon the request of the Sponsor. Concentration Analyses: Concentration of the prepared test article suspensions will be verified during the course of this study. A syringe will be used to withdraw samples (5 mL each) from each concentration during the first and sixth week of dosage administration. Each sample (5 mL each) will be divided into two aliquots, one of 2 mL and one of 3 mL. One aliquot (2 mL) will be shipped for analysis; the other aliquot (3 mL) will be retained at the Testing Facility as a backup sample. Backup samples will be stored under the previously cited conditions and discarded at the Testing Facility upon the request of the Sponsor. Shipping Instructions: Samples to be analyzed will be shipped (frozen on dry ice) to: Kris J. Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone: (612) 778-6018 Telefax: (612) 778-6176 Both the recipient and the Study Monitor will be notified in advance of sample shipment. DISPOSITION: Prepared formulations will be discarded at the Testing Facility. All remaining bulk test article will be returned to the Study Monitor at the previously cited address. 000065 Protocol 418-008 Page 6 TEST SYSTEM: Species/Strain and Reason for Selection: The CrLCDOBR VAF/Plus (Sprague-Dawley) rat was selected as the Test System because: 1) this strain of rat has been demonstrated to be sensitive to reproductive and developmental toxins and has been widely used throughout industry for reproductive and developmental toxicity evaluations; 2) historical data and experience exist at the Testing Facility0-35; and 3) the test article is pharmacologically active in the species and strain. Number: Initial population acclimated: 195 virgin male and 205 virgin female rats. Population selected for study: 175 male rats (35 per dosage group) and 175 female rats (35 per dosage group). Ten mated female rats will be assigned to Caesarean-sectioning on day 10 of presumed gestation; the. remaining female rats will be permitted to deliver litters. 250 F1 generation pups (25 per sex per dosage group) will be selected at weaning on day 21 postpartum for continued postnatal observation. Body Weight and Aae: Male rats will be ordered to weigh from 300 g to 325 g each at receipt, at which time they will be expected to be at least 60 days of age. Female rats will be ordered to weigh from 200 g to 225 g each at receipt, at which time they will be expected to be at least 60 days of age. Actual body weights will be recorded the day after receipt and will be documented in the raw data. The weight ranges will be included in the final report. Sex: Both Fo and F1 generation male and female rats will be evaluated. Only Fo generation male and female rats will be given the test article. Source: Charles River Laboratories, Inc., Raleigh, North Carolina. The rats will be shipped in filtered cartons by air freight and/or truck from Charles River Laboratories, Inc., to the Testing Facility. OC0066 Protocol 418-008 Page 7 Identification: Fo Generation: Rats are permanently identified using Monel self-piercing ear tags (Gey Band and Tag Co., Inc., No. MSPT 20101). Male and female rats are assigned temporary numbers at receipt and given unique permanent identification numbers when assigned to the study before administration of the first dosage of the test article. F1/F2 Generations: Pups will not be individually identified during lactation; ail parameters will be evaluated in terms of the litter. At weaning, each rat selected for continued observation will be identified with a Monel self-piercing ear tag. ANIMAL HUSBANDRY: All cage sizes and housing conditions are in compliance with the Guide for the Care and Use of Laboratory AnimalsTM. Housing: Fo Generation Rats/F1 Generation Litters: Fo generation rats will be individually housed in stainless steel wire-bottomed cages except during the cohabitation and postpartum periods. During cohabitation, each pair of rats will be housed in the male rat's cage. Beginning no later than day 20 of presumed gestation, Fo generation female rats assigned to natural delivery will be individually housed in nesting boxes. Each dam and delivered litter will be housed in a common nesting box during the postpartum period. F1 Generation Rats/F2 Generation Litters: After weaning, the F1 generation rats will be individually housed before cohabitation, housed in pairs (one male rat per female rat) during cohabitation, and individually housed after cohabitation. The same type of caging will be used as described for the Fo generation rats. Beginning no later than day 20 of presumed gestation, F1 generation female rats will be individually housed in nesting boxes. Each dam and delivered litter will be housed in a common nesting box during the postpartum period. 0CCC67 Protocol 418-008 Page 8 Nesting Material: Bedding material (bed-o'cobs) will be supplied to female rats assigned to natural delivery. Bedding will be changed as often as necessary to keep the animals dry and clean. Analyses for possible contamination are conducted annually and documented in the raw data. Room Air. Temperature and Humidity: The animal room is independently supplied with at least ten changes per hour of 100% fresh air that has been passed through 99.97% HEPA filters (Airo Clean room). Room temperature will be maintained at 64F (18C) to 79F (26C) and monitored constantly. Room humidity will also be monitored constantly and maintained at 30% to 70%. Light: An automatically controlled 12-hour light: 12-hour dark fluorescent light cycle will be maintained. Each dark period will begin at 1900 hours EST. Diet: Rats will be given Certified Rodent Diet #5002 (PMI Nutrition International) available ad libitum from individual feeders. Water: Water will be available ad libitum from individual bottles attached to the cages or from an automatic watering access system. All water will be from a local source and passed through a reverse osmosis membrane before use. Chlorine will be added to the processed water as a bacteriostat; processed water is expected to contain no more than 1.2 ppm chlorine at the time of analysis. Water is analyzed monthly for possible bacterial contamination and twice annually for possible chemical contamination. Contaminants: . Neither the Sponsor nor the Study Director is aware of any potential contaminants likely to be present in the certified diet, the drinking water or the nesting material at levels that would interfere with the results of this study. Therefore, no analyses other than those routinely performed by the feed supplier or those mentioned in this protocol will be conducted. 0CCG68 Protocol 418-008 Page 9 RANDOMIZATION AND COHABITATION: Fo Generation: Upon arrival, rats will be assigned to individual housing on the basis of computergenerated random units. After acclimation, male and female rats will be selected for study on the basis of physical appearance and body weights recorded during acclimation. The rats will be assigned to dosage groups based on computer-generated (weight-ordered) randomization procedures. Within each dosage group, consecutive order will be used to assign rats to cohabitation, one male rat per female rat. The cohabitation period will consist of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ will be considered to be at day 0 of presumed gestation and assigned to individual housing. Female rats not mated within the first 7 days of cohabitation will be assigned alternate male rats that have mated (same dosage group) and will remain in cohabitation for a maximum of seven additional days. The first ten female rats per dosage group with a confirmed date of mating will be assigned to Caesarean-sectioning on day 10 of presumed gestation. The remaining female rats will be permitted to naturally deliver litters. A table of random units will be used to assign five rats per group to a pharmacokinetic sample collection at scheduled sacrifice after completion of the cohabitation period (male rats siring litters with dams allowed to naturally deliver a litter) or on day 21 postpartum (female rats allowed to naturally deliver litters). F1/F2 Generation Puds: Day 1 of lactation (postpartum) is defined as the day of birth and is also the first day on which all pups in a litter are individually weighed (pup body weights will be recorded after all pups in a litter are delivered and groomed by the dam). On day 4 postpartum, a table of random units will be used to select pups to be culled, and litters will be reduced to eight pups each. Whenever possible, the same number of male and female pups per litter will be continued on study. At weaning of the F1 generation pups on day 21 postpartum, a table of random units will be used to select 25 male and 25 female pups per group, resulting in a total of 250 F1 generation rats (125 per sex) chosen for continued evaluation. At least one male pup and one female pup per litter, when possible, will be selected. OC0069 Protocol 418-008 Page 10 AnMiMifiTRATIQN: Route and Reason for Choice: The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one of the possible routes of human exposure. Method and Frequency: Dosages will be adjusted for the most recently recorded body weight and given at approximately the same time each day. Fo Generation Male Rats: Male rats will be given the test article once daily beginning 28 days before cohabitation (maximum 14 days) and continuing through the day before sacrifice. Male rats will be sacrificed after completion of the cohabitation period. Fo Generation Female Rats: Female rats will be given the test article once daily beginning 28 days before cohabitation (maximum of 14 days) and continuing through day 9 of presumed gestation (rats assigned to Caesarean-sectioning), day 24 of presumed gestation (rats assigned to natural delivery that do not deliver a litter) or day 20 postpartum (rats that deliver a litter). F1 Generation: F1 generation pups will not be directly given the test article, but may be possibly exposed to the test article during maternal gestation (in utero exposure) or via maternal milk during the lactation period. Rationale for Dosage Selection: Dosages will be selected by the Sponsor on the basis of previous studies conducted with the test article. 000070 Protocol 418-008 Page 11 Dosage Levels. Concentrations and Volumes: Dosage Group Number of Rats Per Sex Dosage (mg/kg/day) Concentration (mg/mL) Dosage Volume (mUkg) Argus Batch Number I 35 0 (Vehicle) II 35 0.1 0 0.02 5 B-418-00BA(Day.Month.Year) 5 B-418-006-B(Day.Month.Year) III 35 0.4 0.08 5 B-418-008-C(Day.Month.Year) IV 35 v 35 1.6 3.2 0.32 0.64 5 B-418-008-0(Day.Month.Year) 5 B-41 B008-E(Day.Month.Year) The test article will be considered 100% pure for the purpose of dosage calculations. TESTS. ANALYSES AND MEASUREMENTS - Fo GENERATION: Viability - Male and Female Rats: All Periods: At least twice daily. Clinical Observations and/or General Appearance - Male and Female Rats: Acclimation Period: At least once. Dosage Period: Twice daily. Prior to dosage administration and once approximately one hour postdosage. Maternal Behavior: Days 1,4, 7, 14 and 21 postpartum. Any observed abnormal behavior will be recorded daily. Clinical observations may be recorded more frequently than cited above, rf deemed appropriate by the Study Director and/or Study Monitor. Body Weights - Male Rats: Acclimation Period: At least once. Dosage Period: Weekly. Sacrifice: Terminal weight. 0CCG71 Protocol 418-008 Page 12 Body Weights - Female Rats: Acclimation Period: At least once. Dosage Period: Weekly to cohabitation. Daily during presumed gestation and on Days 1, 4, 7 and 14 postpartum (rats assigned to natural delivery). Sacrifice: Terminal weight. Feed Consumption Values - Male Rats (recorded and tabulated): Dosage Period: Weekly. Feed Consumption Values - Female Rats (recorded and tabulated): Dosage Period: Weekly to cohabitation. Daily during presumed gestation. Days 1,4, 7 and 14 postpartum (rats assigned to natural delivery). Feed consumption not tabulated after day 14 postpartum, when it is expected that pups will begin to consume maternal feed. Feed Consumption Values - Male and Female Rats: Feed consumption values may be recorded more frequently than cited above if it is necessary to replenish the feed. During cohabitation, when two rats occupy the same cage with one feed jar, replenishment of the feed jars will be documented. Individual values will not be recorded or tabulated. Estrous Cycling and Mating: A table of random units will be used to select 15 female rats per group for evaluation of estrous cycling by examination of vaginal cytology for 14 days before the start of the cohabitation period. During cohabitation, all female rats will be evaluated daily until spermatozoa are observed in a smear of the vaginal contents and/or a copulatory plug is observed in situ. Duration of Gestation: The duration of gestation is calculated from day 0 of presumed gestation to the day the first pup is observed. OC0072 Protocol 418-008 Page 13 Fertility Parameters: Fertility Index (percentage of matings that result in pregnancies). Gestation Index (percentage of pregnancies that result in birth of live litters). Number of offspring per litter (live and dead pups). Number of implantation sites. General condition of dam and litter during the postpartum period. Viability Indices (percentage of pups bom that survive 4 and 7 days). Lactation Index (percentage of pups bom that survive 21 days). Caesarean-Sectioning Observations: Rats will be Caesarean-sectioned on day 10 of presumed gestation. Placentae that appear abnormal (size, color or shape) will be noted in the raw data. The rats will be examined for number and distribution of: Corpora Lutea. Implantation Sites. Viable and Nonviable Embryos. (A viable embryo is oval or crescent shaped, pink, firm and enclosed in an amniotic sac filled with clear fluid. A nonviable embryo is amorphous, small, pale pink to tan or deep red to black, soft and enclosed in an amniotic sac filled with clear, cloudy, or opaque fluid.) Natural Delivery: Female rats will be evaluated for: Clinical Observations During Parturition. Duration of Gestation (day 0 of presumed gestation to the time the first pup is observed). Length of Parturition (time of delivery of last pup minus the time of delivery of the first pup divided by N-1 pups in each litter). Litter Size (defined as all pups delivered). 00G73 Protocol 418-008 Page 14 Pup Viability at Birth. METHOD OF SACRIFICE - Fo GENERATION: Rats will be sacrificed by carbon dioxide asphyxiation. Embryos will be discarded after examination. NECROPSY - Fo GENERATION: Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation (a table of random units will be used to select one control group rat of each sex from which all tissues examined at necropsy will be retained, in order to provide control tissues for any possible histopathological evaluations of gross lesions). Unless specifically cited below, all other tissues will be discarded. Male and Female Rats Assigned to Pharmacokinetic Sample Collection: At scheduled sacrifice after completion of the cohabitation period (male rats siring litters with dams allowed to naturally deliver a litter) and on day 21 postpartum (female rats allowed to naturally deliver a litter), five rats per group will be assigned to a pharmacokinetic sample collection. In addition to the appropriate evaluations described below, blood samples (approximately 4 mL per rat) will be collected from the inferior vena cava into serum separator tubes and centrifuged. The resulting serum (approximately 2 mL) will be immediately frozen on dry ice and maintained frozen (-70C) until shipment to the Sponsor for analysis. The liver will be excised, weighed, and a sample section (lateral lobe) will be frozen and retained at -70C until shipment to the Sponsor for analysis. After completion of sample collection, serum and liver section (lateral lobe) samples will be shipped (frozen on dry ice) to Kris J. Hansen, Ph.D., at the previously cited address for analysis. Both the recipient and the Study Monitor will be notified in advance of sample shipment. Scheduled Sacrifice of Male Rats: After completion of the cohabitation period, male rats will be sacrificed and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. The following organs will be excised and weighed and retained for possible histologic evaluation: testes, epididymides, prostate and seminal vesicles (weighed with and without fluid). The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin for possible histopathological evaluation. The remaining organs will be retained in neutral buffered 10% formalin. 0C0C74 Protocol 418-008 Page 15 Scheduled Sacrifice - Female Rats Assigned to Caesarean-Sectioning. On day 10 of presumed gestation, female rats will be sacrificed, Caesarean-sectioned, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Uteri of apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites<5). Uteri of nonpregnant rats and all ovaries will be retained in neutral buffered 10% formalin for possible future evaluation. Scheduled Sacrifice - Female Rats Assigned to Natural Delivery: Rats that do not deliver a litter will be sacrificed on day 25 of presumed gestation and examined for gross lesions. Uteri will be stained with 10% ammonium sulfide to confirm the absence of implantation sites(5). After completion of the 21-day postpartum period, female rats will be sacrificed, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. The number and distribution of implantation sites will be recorded. Dams with No Surviving Puds: Dams with no surviving pups will be sacrificed after the last pup is found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Postpartum data for these dams will be excluded from summary tables. Rats Found Dead or Moribund: Rats that die or are sacrificed because of moribund condition or abortion will be examined for the cause of death or moribund condition on the day the observation is made. The rats will be examined for gross lesions. Testes, epididymides, prostate and seminal vesicles of male rats will be excised and individual organ weights will be recorded (seminal vesicles weighed with and without fluid). The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. The remaining organs will be retained in neutral buffered 10% formalin. Pregnancy status and uterine contents of female rats will be recorded. Aborted fetuses and/or delivered pups will be examined to the extent possible. Ovaries will be retained in neutral buffered 10% formalin. Uteri of apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites(5). OC0075 Protocol 418-008 Page 16 TESTS. ANALYSES AND MEASUREMENTS - F1 GENERATION: V ia b ility : Preweaning Period: Litters will be observed for dead pups at least twice daily. The pups in each litter will be counted once daily. Postweaning Period: Twice daily. Clinical Observations and/or Generai Appearance: Preweaning Period: Once daily. Postweaning Period: Once weekly. Maternal Behavior: Days 1, 4, 7, 14 and 21 postpartum. Any observed abnormal behavior will be recorded daily. Clinical observations may be recorded more frequently than cited above, if deemed appropriate by the Study Director and/or the Study Monitor. Body W eights: Preweaning Period: Days 1 (birth), 4, 7, 14 and 21 postpartum. Postweaning Period: Weekly. Presumed Gestation Period: Days 0, 7, 10, 14, 17 and 20 (female rats only). Lactation Period: Days 1, 4, 7, 10 and 14 (female rats only). Sacrifice: Terminal weight. Feed Consumption Values (recorded and tabulated): Preweaning Period: Not recorded. Postweaning Period: Weekly except during cohabitation. Presumed Gestation Period: Days 0, 7 ,1 0 ,1 4 ,1 7 and 20 (female rats only). Lactation Period: Days 1, 4, 7 ,1 0 and 14 (female rats only). 0CCC76 Protocol 418-008 Page 17 Feed consumption values may be recorded more frequently if it is necessary to replenish the feed. During cohabitation, when two rats occupy the same cage with one feed jar, values will be documented when feed jars are filled. These intervals will not be tabulated. Preweanina Developmental Observations: The number of pups meeting the criterion is recorded on each day of testing. Testing continues until the day the criterion is attained by all pups in the litter. Surface Righting Reflex (ability to right in 5 seconds): From day 1 postpartum. Pinna Unfolding: From day 2 postpartum. Eye Opening: From day 12 postpartum. Acoustic Startle Response: From day 13 postpartum. Air Righting Reflex: From day 14 postpartum. Pupil constriction is evaluated once, on day 21 postpartum. Postweanina Developmental Observations: Sexual Maturation: Female rats will be evaluated for the age of vaginal patency, beginning on day 28 postpartum. Male rats will be evaluated for the age of preputial separation, beginning on day 39 postpartum. Passive Avoidance Testing: Beginning at 24 1 day postpartum, one male rat and one female rat from each litter, where possible, will be evaluated in a passive avoidance test for learning, short-term retention and long-term retention. The passive avoidance apparatus consists of a two-compartment chamber with hinged Plexiglas lids. One compartment is fitted with a bright light and Plexiglas floor. The other compartment is fitted with a grid floor to which a brief (1 sec) pulse of mild electric current (1 mA) can be delivered. The two compartments are separated by a sliding door. On each test trial, the rat is placed into the "bright" compartment, the sliding door is opened and the light is turned on. The rat is allowed to explore the apparatus until it enters the "dark" compartment. The sliding door is then immediately closed, the light is turned off and the brief pulse of current is delivered to the grid floor. The rat is then removed from the apparatus and placed into a holding cage for 30 seconds before the start of the next trial. Trials are repeated until the rat remains in the "bright" 0C0C-77 Protocol 418-008 Page 18 compartment for 60 seconds on two consecutive trials (the criterion for learning) or until 15 trials have been completed. The latency to enter the dark compartment or the - maximum 60-second interval is recorded for each trial. Each rat is tested twice. The test sessions are separated by a one-week interval, and the criterion is the same for both days of testing. Dosage groups are compared for the following dependent measures: The number of trials to the criterion in the first session-this measure will be used to compare groups for overall learning performance. The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the first test session-this measure will be used to compare groups for activity levels and exploratory tendencies in a novel environment. The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 2 in the first test session-this measure will be used to compare groups for short-term retention. The number of trials to the criterion in the second test session-this measure will be used to compare groups for long-term retention. The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the second session-this value is another indication of long-term retention. Watermaze Testing: Beginning at approximately 70 days postpartum, one male rat and one female rat from each litter will be evaluated in a water-filled M-maze for overt coordination, swimming ability, learning and memory. Each rat is tested in a watertight 16-gauge stainless steel modified M-maze. The maze is filled with water to a depth of approximately nine inches, and the water is monitored for temperature (range of 21 C 1C). On each test trial, the rat will be placed into the starting position (base of the M-maze stem farthest from the two arms) and required to swim to one of the two goals of the M-maze, in order to be removed from the water. On the first trial, the rat is required to enter both arms of the maze before being removed from the water. The initial arm chosen on trial 1 is designated the incorrect goal during the remaining trials. Rats that fail to make a correct goal choice within 60 seconds in any given trial are guided to the correct goal and are then removed from the water. A 15-second intertrial interval will separate each trial. Each rat is required to reach a criterion of five consecutive errorless trials to terminate the test session. The maximum number of trials in any test CCC078 Protocol 418-008 Page 19 session is 15. Latency (measured in seconds) to choose the correct goal or the maximum 60-second interval is recorded for each trial, as is the number of errors (incorrect turns in the maze) during each trial. Each rat is tested twice. The test sessions are separated by a one-week interval, and the correct goal and the criterion are the same for both test sessions. Dosage groups are compared for the following dependent measures: The number of trials to criterion on the first day of testing-this measure will be used to compare groups for overall learning performance. The average number of errors (incorrect turns in the maze) for each trial on the first day of testing-this measure will also be used to compare groups for overall learning performance. The latency (in seconds) to reach the correct goal on trial 2 of the first day of testing-this measure will be used to compare groups for short-term retention. The number of trials to criterion on the second day of testing-this measure will be used to compare groups for long-term retention. The average number of errors for each trial on the second day of testing-this measure will also be used to compare groups for long-term retention. The latency (in seconds) to reach the correct goal on trial 1 of day 2 of testing-this is another indicator of long-term retention. Reproductive Capacity: At approximately 90 days of age, the F1 generation rats within each dosage group will be assigned to cohabitation, one male rat per female rat, based on computer-generated random units or random unit tables, with the exclusion of sibling matings. The cohabitation period will consist of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ will be considered to be at day 0 of presumed gestation and assigned to individual housing. Female rats that do not mate within the first 7 days of cohabitation will be assigned alternate male rats from the same dosage group that have mated. Female rats will be allowed to naturally deliver and maintain litters through a 21-day postpartum period. Mating Performance: As cited above for Fo generation rats. CCGC79 Protocol 418-008 Page 20 Duration of Gestation: As cited above for Fo generation rats. Fertility Parameters: As cited above for Fo generation rats. F2 Generation Litter Data: Viability, clinical observations and body weights for F2 generation pups will be recorded as cited above for F1 generation litters. METHOD OF SACRIFICE - F1 GENERATION RATS/F2 GENERATION PUPS: As previously cited for Fo generation rats. NECROPSY - F1 GENERATION RATS: Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation (a table of random units will be used to select one control group rat of each sex from which all tissues examined at necropsy will be retained, in order to provide control tissues for any possible histopathological evaluations of gross lesions). Unless specifically cited below, all other tissues will be discarded. Scheduled Sacrifice - F1 Generation Male Rats: Rats will be sacrificed after completion of the 14-day cohabitation period. A gross necropsy of the thoracic, abdominal and pelvic visceral will be performed. Testes and epididymides of male rats will be excised and individual organ weights will be recorded. The epididymides will be retained in neutral buffered 10% formalin. The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. Scheduled Sacrifice - F1 Generation Female Rats: Female rats will be sacrificed after completion of the 21-day postpartum period. The number and distribution of implantation sites will be recorded. Rats that do not deliver a litter will be sacrificed on day 25 of presumed gestation and uteri will be stained with 10% ammonium sulfide to confirm the absence of implantation sites(S). A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Female rats without a confirmed mating date that do not deliver a litter will be sacrificed on an estimated day 25 of presumed gestation. occoso Protocol 418-008 Page 21 F1 Generation Rats Found Dead or Moribund: Rats that die or are sacrificed because of moribund condition or abortion will be examined for the cause of death or moribund condition on the day the observation is made. The rats will be examined for gross lesions. Testes, epididymides, prostate and seminal vesicles of male rats will be excised and individual organ weights will be recorded (seminal vesicles weighed with and without fluid). The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin. The remaining organs will be retained in neutral buffered 10% formalin. Pregnancy status and uterine contents of female rats will be recorded. Aborted fetuses and/or delivered pups will be examined to the extent possible. Ovaries will be retained in neutral buffered 10% formalin. Uteri of apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites(5). F1 Generation Dams with No Surviving Puds: Dams with no surviving pups will be sacrificed after the last pup is found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Postpartum data for these dams will be excluded from summary tables. F1/F2 Generation Puds Found Dead on Dav 1 Postpartum: Pups that die before examination of the litter for pup viability will be evaluated for vital status at birth. The lungs will be removed and immersed in water. Pups with lungs that sink will be identified as stillborn; pups with lungs that float will be identified as livebom, and to have died shortly after birth. Pups with gross lesions will be preserved in Bouin's solution for possible future evaluation. Should postmortem autolysis preclude these evaluations, it will be noted in the necropsy data. F1/F2 Generation Puds Found Dead or Moribund on Days 2 to 21 Postpartum: Pups found dead or sacrificed due to moribund condition will be examined for gross lesions and for the cause of the moribund condition or death. Pups with gross lesions found on days 2 to 4 postpartum will be preserved in Bouin's solution for possible future evaluation; gross lesions of pups found on days 5 to 21 postpartum will be preserved in neutral buffered 10% formalin. Should postmortem autolysis preclude these evaluations it will be noted in the necropsy data. o c0051 Protocol 418-008 Page 22 F1/F2 Generation Puds Not Selected for Continued Observation: F1 and F2 generation pups culled on day 4 postpartum will be sacrificed and examined for gross lesions; pups with gross lesions will be preserved in Bouin's solution. Necropsy will include a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. All F1 generation pups culled on day 21 postpartum will be sacrificed and examined for gross lesions; gross lesions will be preserved in neutral buffered 10% formalin. Necropsy will include a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Scheduled Sacrifice - F2 Generation Puds: On day 21 postpartum, pups will be sacrificed and examined for gross lesions. Necropsy will include a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. GOOC52 Protocol 418-008 Page 23 PROPOSED STATISTICAL METHODS*6-12*: Averages and percentages will be calculated. Litter values will be used where appropriate. Additional procedures and/or analyses may be performed, if appropriate. Type of Test* I. Parametricb II. Nonparametricc A. Bartlett's Test0 A. Kruskal-Wallis Test (75% ties) Significant at p<.0.05 Not Significant Significant at p<;0.05 Not Significant Nonparametric Analysis of Variance Dunn's Test Significant at ps0.05 Not Significant B. Fisher's Exact Test (>75% ties) Dunnett's Test III. Test for Proportion Data Variance Test for Homogeneity of the Binomial Distribution a. Statistically significant probabilities are reported as either p * 0.05 or psO.01. b. Used only to analyze data with homogeneity of variance. c. Proportion data are not included in this category. d. Test for homogeneity of variance. CGG0S3 Protocol 418-008 Page 24 DATA ACQUISITION. VERIFICATION AND STORAGE: Data will be hand- and/or computer-recorded. Records will be reviewed by the Study Director and/or appropriate management personnel within 21 days after generation. All original records will be stored in the archives of the Testing Facility. All original data will be bound and indexed. A copy of all raw data will be supplied to the Sponsor upon request. Preserved tissues will be stored at the Testing Facility at no charge for one year after mailing of the draft final report, after which time the Sponsor will be contacted to determine the disposition of these materials. RECORDS TO BE MAINTAINED: Protocol and Amendments. Test Article, Vehicle and/or Reagent Receipt, Preparation and Use. Animal Acquisition. Randomization Schedules. Mating History. Treatment (if prescribed by Staff Veterinarian). General Comments. Clinical Observations and/or General Appearance. Blood Sample Collection, Processing and Shipment. Body Weights. Feed Consumption Values. Caesarean-Sectioning Observations. Natural Delivery Observations. Litter Observations. Reflex and Physical Development and Behavioral Observations - F1 Generation Pups. Gross Necropsy Observations. Organ Weights (if required). Photographs (if required). Study Maintenance (room and environmental records). Feed, Water and Bedding Analyses. Packing and/or Shipment Lists. KEY PERSONNEL: Executive Director of Research: Mildred S. Christian, Ph.D., ATS Director of Research: Alan M. Hoberman, Ph.D., DABT Associate Director of Research and Study Director: Raymond G. York, Ph.D., DABT Director of Laboratory Operations: John F. Barnett, B.S. Manager of Study Coordination: Valerie A. Sharper, M.S. Manager of Animal Operations and Member, Institutional Animal Care and Use Committee: Dena C. Lebo, V.M.D. Manager of Regulatory Compliance: Kathleen A. Moran, M.S. Consultant, Veterinary Pathology: W. Ray Brown, D.V.M., Ph.D., ACVP 0G00S4 Protocol 418-008 Page 25 FINAL REPORT: A comprehensive draft final report will be prepared on completion of the study and will be finalized following consultation with the Sponsor. The report will include the following: Summary and Conclusion. Experimental Design and Method. Evaluation of Test Results. Appendices: Figures, Summary and Individual Tables Summarizing the Above Data, Protocol and Associated Amendments and Deviations, Study Director's GLP Compliance Statement, Reports of Supporting Data (if appropriate) and QAU Statement. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE STATEMENT: The procedures described in this protocol have been reviewed by the Testing Facility's Institutional Animal Care and Use Committee. All procedures described in this protocol that involve study animals will be conducted in a manner to avoid or minimize discomfort, distress or pain to the animals. The Sponsor's signature below documents the fact that information concerning the necessity for conducting this study and the fact that this is not an unnecessarily duplicative study may be obtained from the Sponsor. No alternative (in vitro) procedures were available for meeting the stated purposes of the study. 000035 Protocol 418-008 Page 26 REFERENCES: 1. Christian, M.S. and Voytek, P.E. (1982). In Vivo Reproductive and Mutagenicity Tests. Environmental Protection Agency, Washington, D.C. National Technical Information Service, U.S. Department of Commerce, Springfield, VA 22161. 2. Christian, M.S. (1984). Reproductive toxicity and teratology evaluations of naltrexone (Proceedings of Naltrexone Symposium, New York Academy of Sciences, November 7, 1983), J. Clin. Psychiat. 45(9):7-10. 3. Lang, P.L. (1988). Embryo and Fetal Developmental Toxicity (Teratology) Control Data in the Charles River Cri:CDBR Rat. Charles River Laboratories, Inc., Wilmington, MA 01887-0630. (Data base provided by Argus Research Laboratories, Inc.) 4. Institute of Laboratory Animal Resources (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 5. Salewski, E. (1964). Frbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Arch. Pathol. Exp. Pharmakol. 247:367. 6. Snedecor, G.W. and Cochran, W.G. (1967). Variance test for homogeneity of the binomial distribution. Statistical Methods, 6th Edition, Iowa State University Press, Ames, pp. 240-241. 7. Sokal, R.R. and Rohlf, F.J. (1969). Bartlett's test of homogeneity of variances. Biometry, W.H. Freeman and Co., San Francisco, pp. 370-371. 8. Snedecor, G.W. and Cochran, W.G. (1967). Analysis of Variance. Statistical Methods, 6th Edition, Iowa State University Press, Ames, pp. 258-275. 9. Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1129. 10. Sokal, R.R. and Rohlf, F.J. (1969). Kruskal-Wallis Test. Biometry, W.H. Freeman and Co., San Francisco, pp. 388-389. 11. Dunn, O.J. (1964). Multiple comparisons using rank sums. Technometrics 6(3):241-252. 12. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, pp. 96-104. 0C00S6 PROTOCOL APPROVAL: FOR THE TESTING FACILITY George E. Dearlove, Ph.D., DABT Associate Director of Research Protocol 418-008 Page 27 ____ i9~jj --F> Date Date jLsOm<*U arbara J. Pai on, B.A. Chairperson, Instatiutional Animal Care and Use Committee Date ^ FOR THE SPONSOR Marvin T. Case, D.V.M., Ph.D. Study Monitor ff.fl.A v /' f ^ Date cccc$7 G P rim edica Argus Research Laboratories, Inc. 905 Sbeehy Drive, Building A Horsham, PA 19044 Telephone: (215) 443-8710 Telefax: (215) 443-8587 PROTOCOL 418-008 COMBINED ORAL (GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: 6295.9 Amendment 8 - 1 8 May 1999 1. Concentration Analyses (page 5 of the protocol): The concentration samples were sent to the Sponsor. Sample analyses will be conducted at the discretion of the Sponsor and no report will be sent to the Testing Facility. Reason for Change: This change was made at the request of the Sponsor to clarify the protocol. 2. Male and Female Rats Assigned to Pharmacokinetic Sample Collection (page 14 of the protocol): The liver and serum samples were sent to the Sponsor. Samples will be analyzed at the discretion of the Sponsor. Reason for Change: This change was made at the request of the Sponsor to clarify the protocol. 3. F1/F2 Generation Puds Not Selected for Continued Observation (page 22 and Amendment 5 of the protocol): The stomach contents of these pups were sent to the Sponsor for analysis. Stomach contents will be analyzed at the discretion of the Sponsor. 0C00S8 Reason for Change: Protocol 418-008 Amendment 8 Page 2 This change was made at the request of the Sponsor to clarify the protocol. Dena C. Lebo, V.M.D. Date Chairperson, Institutional Animal Care and Use Committee a-<- Marvin T. Case, D.V.M., Ph.D. Study Monitor Date 0C00S9 3M Environmental Laboratory Protocol - Analytical Study Potassium Perfluorooctanesulfonate in Two Generation Rat Reproduction In-vivo study reference number: Argus 418-008 Study number: FACT 052798.1 Test substance: Potassium perfluorooctanesulfonate (PFOS) Name and address of Sponsor: Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 Name and address of testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 Experimental start date: Expected termination date: December 31, 1998 Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry Author: Lisa Clemen Kris Hansen Study Director Date Marvin Case Date Sponsor Representative 000090 i 1,0 P u r p o s e The analytical portion of this dosing study is designed to evaluate levels of potassium perfluorooctanesulfonate (PFOS), or a metabolite of PFOS designated by the study director, in the liver of the parent and subsequent generations of the test system, or in the serum as necessary. The in life portion of this study was conducted at Argus Research Laboratories. 2.0 Regulatory Compliance________________________________________________________ This study is conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report. 3.0 Test Materials__________________________________________________________________ 3.1 Test, control, and reference substances and matrices 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance matrix: Rat liver and serum 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rat liver and serum 3.2 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report 3.2.2 Analytical reference substance matrix: Argus Research Laboratories; traceability information will be included in the final report 3.2.3 Analytical control matrix: 3.2.3.1 Rat liver - Argus Research Laboratories; traceability information will be included in the final report 3.2.3.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report 3.3 Number of test and control samples. Liver samples for testing were received from 40 test animals and 10 control animals. Serum samples will be tested at the discretion of the Study Director. 3.4 Identification of test and control samples: The samples are identified using the Argus Research Laboratories identifiers, which consist of a letter followed by the Argus project number, the animal number, the group designation, and the draw date. 3.5 Purity and strength of materials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor. 000091 2 3.6 Stability of test material: Characterization of the stability of the test material is the responsibility of the Sponsor. 3.7 Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at -20 10 C. 3.8 Disposition of test and/or control substances: Biological tissues and fluids are retained per GLP regulation. 3.9 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 4.0 Experimental - Overview____________________________________________________ Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-008 are received for analysis of fluorine compounds. At the discretion of the Study Director, a series of analytical tests will be performed on select tissues. Initially, all liver samples will be analyzed for PFOS by electrospray/mass spectrometry (ES/MS). On the basis of findings from these analyses, additional sample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment will be written. 5.0 Experimental - Analytical Methods___________________________________________ 5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/Mass Spectrometry 5.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC- Electrospray/Mass Spectrometry 6.0 Data Analysis___________________________________________________________________ 6.1 Data transformations and analysis: Data will be reported as the concentration (weight/weight) of PFOS per tissue or sample, or of PFOS per unit of tissue or fluid. 6.2 Statistical analysis: Statistics used may include regression analysis of the serum concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. O 0CC92 3 7.0 Maintenance of Raw Data and Records 7.1 The following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.1.2 Study correspondence 7.1.3 Shipping records 7.1.4 Raw data 7.1.5 Electronic copies of data 7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: 7.2.1 Training records 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 7.2.5 Appropriate specimens. 8.0 References__________________________________________________________________ 8.1 3M Environmental Laboratory Quality System Chapters 1, 5 and 6 8.2 Other applicable 3M Environmental Laboratory Quality System Standard Operating Procedures 9.0 Attachments________________________________________________________________ 9.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/Mass Spectrometry 9.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC- Electrospray/Mass Spectrometry 000093 4 Potassium Perfluorooctanesulfonate CAS Number-2759-39-3 A tt a c h m e n t F: P reparatory and A nalytical M ethods 3M Environmental Laboratory Report No. Tox-012 Laboratory Request Number (LRN)-U2006 Proprietary and Confidential 000094 3M Environmental Laboratory Method Extraction of Potassium perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry Method Number: FACT-M-1.0 Author: Lisa Clemen Approved By: Laboratory Manager I/aaA ^ k fb---------- Group Leader _ A C ll/frtA -_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Technical Reviewer Adoption Date: Revision Date: S '/.c /F f' Date Date shi-h? Date 1.0 Scope and Application__________________________________________________________ 1.1 Scope: This method is for the extraction of Potassium Perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Microsoft 7.0.1/95 FACT-M-1.0 Extraction of PFOS from Liver Page 1 of 8 00OC95 2.0 S u m m a r y o f M e t h o d 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver using ion pairing reagent and 5.0 mLs of ethyl acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs of extract is removed to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 |im filter into glass autovials. 3.0 Definitions______________________________________________________________________ 3.1 None. 4.0 W arnings and Cautions_________________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions when handling animal livers, they may contain pathogens. 5.0 Interferences___________________________________________________________________ 5.1 There are no known interferences at this time. 6.0 Equipment ____________________________ ____ __________________________________ 6.1 The following equipment is used while carrying out this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 6.1.6 Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR Nitrogen Evaporator, Organomation Balance 7.0 S upplies and Materials__________________________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Glass, type A, volumetric flasks 7.6 40 mL glass I-CHEM vials 7.7 Plastic sampule vials, Wheaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL 7.9 Labels FACT-M-1.0 Extraction of PFOS from Liver Page 2 of 8 000096 7.10 Syringes, capable of measuring 10 pL to 50 pL 7.11 Glass, type A, volumetric pipettes 7.12 Graduated pipettes 7.13 Electronic pipettor, Eppendorf or equivalent 7.14 Timer 7.15 Disposable plastic 3 cc syringes 7.16 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.17 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards________________________________________________________ 8.1 Reagents 8.1.1 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 1ON: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-QTM water, mix until all solids are dissolved. Store in a 1 L nalgene bottle. 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) IN. Dilute 10N 1:10. Measure 10 mL of the 1ONNaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: Weigh approximately 169 grams of TBA into a 1 L volumetric containing 500 L Milli-QTM water. Adjust to pH 10 using approximately 64 mL 10N NaOH and dilute to volume with Milli-QTM water. Add NaOH slowly while adding the last 1 mL of NaOH because the pH changes abruptly. Store in a 1 L nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8.1.4 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker or equivalent), (Na^Oj/NaHCOj) 0.25M: Weigh approximately 26.5 g of sodium carbonate (NaiCOj) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and dilute to volume with Milli-QTM water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.7 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received frozen from testing laboratory. 8.1.9 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. FACT-M-l.O Extraction of PFOS from Liver Page 3 of 8 000097 8.2.2 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.3 Bring to volume with methanol for a stock standard of approximately 1000 ppm Gig/mL). 8.2.4 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.2.5 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 9.0 Sample Handling_____________________________________________ 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. 10.0 Q uality Control____________________________________________________________ _ 10.1 Matrix Spikes 10.1.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver. 10.1.3 Expected concentrations will fall in the mid-range of the initial calibration curve. 10.2 Continuing Calibration Checks 10.2.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.2.2 One check is prepared per group of ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.2.3 Prepare each continuing calibration check from the same liver homogenate used to prep the initial curve. 10.2.4 The expected concentration will fall within the mid-range of the initial calibration curve. 11.0 C alibration and Standardization______________________________________________ 11.1 Prepare Liver Homogenate to Use for Standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 If 40 g is not available, use appropriate amounts of liver and water in keeping with a 1:5 ratio. 11.1.3 See section 13.0 to calculate the actual density of liver. FACT-M-1.0 Extraction of PFOS from Liver Page 4 of 8 000098 11.1.4 Add 1 mL of homogeneous solution to a 15 mL centrifuge tube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total of sixteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.5 Two 1 mL aliquots serve as matrix blanks. Use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total of fourteen samples. Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm - pL Approx, final cone, of PFOS in liver - Blank 4 0.010 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.000 ppm 11.1.1 See section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 Extract spiked liver homogenates following 12.14-12.24 of this method. Use these standards to establish each initial curve on the mass spectrometer. 12.0 P rocedures_____________________________________________________________________ 12.1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 12.2 Cut approximately 1 g of liver using a dissecting scalpel. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 12.10 Cap the sample and vortex for 15 seconds. FACT-M-1.0 Extraction of PFOS from Liver Page 5 of 8 000099 12.11 Pipette 1 mL homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. (See Worksheet for documenting the remaining steps.) 12.12 Spike liver homogenates with the appropriate amount of PFOS standard as described in section 11.1 or Table 1. 12.13 Pipette two 1 mL aliquots of Milli-QTM water to centrifuge tubes. These will serve as instrument blanks. 12.14 Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.15 Using a volumetric pipette, add 5 mLs ethyl acetate. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 Centrifuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifuge to approximately 3500 rpm. 12.18 Remove 4 mLs of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.19 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.20 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.21 Vortex mix for 30 seconds. 12.22 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.23 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.24 Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to page of study notebook. 13.0 D ata A nalysis and Calculations_______________________________________________ 13.1 Calculations: 13.1.1 Calculate the density of liver (mg) in 1.0 mL homogenate using the following equation: g of Liver x Average weight of ten 1 mL aliquots (mg) (g of Liver + g of Water) FACT-M-1.0 Extraction of PFOS from Liver Page 6 of 8 oooioo 13.1.2 Calculate actual concentrations of PFOS in calibration standards using the following equation: uL of Standard x Concentration (ng /mL) = Final Concentration (p.g/g or mg/kg) mg Liver /1 mL homogenate of PFOS in Liver Average weight of liver in solution as determined in 13.1.1, by weighing ten 1 mL homogenates of approximately 40 mg liver in 200 mL of Milli-Q water. 14.0 M ethod Performance _____________________________________________ __________ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 15.0 P ollution P revention and W aste Management__________________________ _ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records_______________________________________________________________________ 16.1 Complete the extraction worksheet and tape into the study notebook. 17.0 T ables, D iagrams, Flowcharts, and Validation Data_______________________ 17.1 The validation report associated with this method is FACT-M-1.0 & 2.0-V-l. 18.0 References__________________________________________________________________ 18.1 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 19.0 A ffected Documents________________________________________________________ 19.1 FACT-M-2, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date FACT-M-1.0 Extraction of PFOS from Liver Page 7 of 8 CO G Extraction Worksheet for FACT-M-1 Study # - Sample Number set # H,0 Blank Liver Blank PFOS approx. 0.5 ppm actual #W ppm - - PFOS approx. 5 ppm actual ppm #W - PFOS approx. 50 ppm actual ppm #W - Date and Initials for Std. - 1 - -- - -- -- -- -- - -- -- -- -- -- -- -- -- -- -- -- -- -- - -- 1Studv numb ;r where the original work.sheet is located._____ Blank Liver Homoeenate: Std # Liver Extraction Method Liver amount = g Date & Initials Vortex 15 sec. Pipette 1 mL o f Liver Solution Pipette 1 mL o f t0.5 M TBA, pH 10. Std. # Pipette 2 mL o f 0.25 Na2CCh/0.25M NaHCO-? Buffer Std. # Pipette 5 mL o f Ethyl Acetate TN-A- Shake 20 min. Centrifuge 20-25 min. Centrifuge Speed Remove a 4 mL aliouot of organic laver Put on Nitrogen Evaporator to dryness Evaporator Temperature Add 1.0 mL o f Methanol TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um SRI filter into a 1.5 mL autosample vial MS/MSD/___Cont. Checks: Spiked______ uL of a _____ppm std (_____________ ) for a final concentration of ppm. MS/MSD used sample_____________ . Cont. Checks used same homogenate as for std curve. FACT-M-1.0 Extraction of PFOS from Liver Page 8 of 8 000102 3M Environmental Laboratory Method Analysis of Fluorochemicals in Liver Extracts U sing HPLC-Electrospray/M ass Spectrometry Method Number: FACT-M-2.0 Author: Lisa Clemen Approved By: 5 -i Laboratory Manager ri-zj/iir f s lA . ---- -------- Group Leader A CiwiOk Technical Reviewer Adoption Date: Revision Date: fOj/\ s'h c / j Date 5"A- O f t ? ? Date sh ih x Date 1.0 Scope and A pplication ___________________________________ _ 1.1 Scope: This method is for the analysis of extracts of liver or other tissues for fluorochemical surfactants rosing HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 1 of 8 000103 2.0 Summary of M ethod_____________________________________________________________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 D efinitions______________________________________________________________ _ 3.1 None. 4.0 W arnings and Cautions_______________________________________________________ __ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences___________________________________________________________________ 5.1 Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 E quipment_______________________________________________________________________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 Supplies and M aterials__________________________________________________________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 Reagents and Standards_____________________________________________________ _ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 2 of 8 000104 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one liver blank, and seven liver standards are prepared during the extraction procedure. See FACT-M-1. 9.0 Sample Handling_____________________________________________________________ _ 9.1 Fresh liver standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be refrigerated until analysis can be performed. 10.0 Quality Control_______________________________________________________________ 10.1 Matrix Blanks and Method Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g. peak area, retention time and peak shape, etc.) will be assessed for each run. 11.0 Calibration and Standardization______________________________________________ 11.1 Analyze the extracted liver standards prior to and following each set of extracts. The mean of two standard values, at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software. FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 3 of 8 000105 11.2 The r2value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 Procedures_____________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A scan is usually collected along with the SIRs. Save method. 12.1.3 Typically the sample list begins with the first set of liver standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 4 of 8 000106 12.3 Instrument Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eye piece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 LC constant flow mode flow rate 10 - 500 uL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10 Click on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations_______________________________________________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 5 of 8 0C0107 13.1.3 Calculate actual concentration of PFOS anion in total liver (mg): f ug PFOS anion calc, from std curve' V g o f liver used for analysis , x Total mass o f liver (g) 1000 u g /1 mg 14.0 M ethod Performance_________________________________________________________ 14.1 The method detection limit is equal to at least three times the baseline noise in the matrix blank. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 15.0 P ollution Prevention and W aste M anagement_______________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers. All containers are located in the laboratory. 16.0 Records________________________________________________________________________ 16.1 Store chromatograms in the study folder. Each chromatogram should have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate media. Record in study notebook the file name and location of backup electronic data. 17.0 T ables, D iagrams, Flowcharts, and Validation Data_________________________ 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-1.0 & 2.0-V-l. 18.0 References________________________________________________ 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 6 of 8 000108 19.0 Affected Documents 19.1 FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 7 o f 8 OC0109 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 8 of 8 O C O ilO 3M Environmental Laboratory Method Extraction of Potassium perfluorooctanesulfonate or other Anionic Fluorochemical surfactants from Serum for Analysis Using HPLC-Electrospray/M ass Spectrometry Method Number: FACT-M-3.0 Author: Lisa Clemen Approved By: Adoption Date: ^ j i z j <{ 2 Revision Date: |0|/\ Laboratory Manager Group Leader A Technical Reviewer Date 4 /z 3^14 Y Date mIu / m Date 1.0 Scope and A pplication 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. Microsoft 7.0.1/95 FACT-M-3.0 Extraction of PFOS from Serum Page 1 of 8 O O O lll 2.0 Summary of M ethod __________________________________________________________ 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other anionic fluorochemical surfactants from serum using an ion pairing reagent and 5.0 mL of ethyl acetate. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four mL of extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 D efinitions 3.1 None. __________________________________________________________________ 4.0 W arnings and Cautions__________________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal serum, it may contain pathogens. 5.0 Interferences____________________________________________________________________ 5.1 There are no known interferences at this time. 6.0 E quipment_______________________ ________________________________________________ 6.1 The following equipment is used while carrying out this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR Nitrogen evaporator, Organomation Balance, ( 0.100 gm) 7.0 Supplies and Materials_______________________________ _____________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Glass, type A, volumetric flasks 7.5 40 mL glass I-CHEM vials 7.6 Polypropylene centrifuge tubes, 15 mL 7.7 Labels 7.8 Syringes, capable of measuring 10 pL to 50 pL 7.9 Glass, type A, volumetric pipettes 7.10 Graduated pipettes FACT-M-3.0 Extraction of PFOS from Serum Page 2 o f 8 7.11 Electronic pipettor, Eppendorf or equivalent 7.12 Timer 7.13 Disposable plastic 3 cc syringes 7.14 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.15 Crimp cap autovials Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards_________________________________________________________ 8.1 Reagents 8.1.1 Sodium hydroxide (J.T Baker or equivalent), (NaOH) ION: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.1.2 Sodium hydroxide (J.T Baker or equivalent), (NaOH) IN. Dilute 10N 1:10. Measure 10 mL of 10N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: Weigh approximately 169 grams of TBA into a 1 L volumetric containing 500 L Milli-QTM water. Adjust to pH 10 using approximately 64 mL of 10N NaOH and dilute to volume with Milli-QTM water. Add NaOH slowly while adding the last mL of NaOH because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH =10. Adjust as needed using IN NaOH solution. 8.1.4 Sodium carbonate/sodium bicarbonate buffer (J.T. Baker or equivalent), (Na2C 0 3/NaHC03) 0.25M: Weigh approximately 26.5 g of sodium carbonate (Na2C 0 3) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli-QTM water. Store in a 1 L nalgene bottle. 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 Other fluorochemicals, as appropriate. 8.1.7 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.8 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.9 Serum, frozen liquid from Sigma. 8.1.10 Control serum received with each sample set. 8.1.11 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. FACT-M-3.0 Extraction of PFOS from Serum Page 3 of 8 O G O IJ 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. 8.2.2 Prepare other fluorochemical standards, as appropriate. 8.2.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (jig/mL). 8.2.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.2.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 9.0 Sample Handling_________________________ 9.1 All sera are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality Control__________________________________________________________ 10.1 Matrix Blanks and Method Blanks 10.1.1 Two 1.0 mL aliquots of the serum are extracted following this procedure and used as matrix blanks. See section 11.1.2. 10.1.2 Two 1.0 mL aliquots of Milli-QTM water are extracted following this procedure and used as method blanks. 10.2 Matrix Spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using serum chosen by the analyst, usually control serum received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.3 Continuing Calibration Checks 10.3.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.3.2 One check is prepared per group of ten samples. For example, if a sample set = 34, four checks are prepared and extracted. FACT-M-3.0 Extraction of PFOS from Serum Page 4 of 8 0C0114 10.3.3 Prepare each continuing calibration check from the same serum used to prep the initial curve. 10.3.4 The expected concentration will fall within the mid-range of the initial calibration curve. 11.0 Calibration and Standardization______________________________________________ 11.1 Prepare Serum Standards 11.1.1 Transfer 1 mL of serum to a 15 mL centrifuge tube. 11.1.2 If the majority of serum sample volumes are less than 1.0 mL, extract standards using serum volumes in the standards equal to the serum volumes in samples. Do not extract below 0.50 mL of serum. Record the serum volume on the extraction sheet. 11.1.3 Mix or shake between aliquots while preparing a total of sixteen aliquots of serum in 15 mL centrifuge tubes. 11.1.4 Two 1 mL or appropriate aliquots serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total of fourteen samples. 11.1.5 Refer to the validation report FACT-M-3.0-V-1 and FACT-M-4.0-V-1 which lists the working ranges for calibration curves. Table 1 Approximate Spiking Amounts for Standards and Spikes Using 1.0 mL of Serum Working Standard (Approx. Cone.) 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm pL Approx, final cone, of PFOS in serum - Blank 20 0.010 ppm 5 0.025 ppm 10 0.050 ppm 20 0.100 ppm 5 0.250 ppm 10 0.500 ppm 15 0.750 ppm 11.1.4 See section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 Extract spiked serum standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. FACT-M-3.0 Extraction of PFOS from Serum Page 5 of 8 000115 12.0 Procedures 12.1 Obtain frozen serum samples and allow to thaw. 12.2 Vortex mix for 15 seconds then remove 1.0 mL or appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Return serum samples to freezer after extraction amount has been removed. 12.4 Record the serum volume on the extraction worksheet. The final methanol volume will equal the initial serum volume. 12.5 Label the tube with the study number, serum ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike serum with the appropriate amount of PFOS standard as described in section 11.1 or Table I for the calibration curve standards. Also spike matrix spikes and continuing calibration standards. 12.7 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.8 Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.9 Using a volumetric pipette, add 5 mL ethyl acetate. 12.10 Cap each sample and put on the shaker for 20 minutes. 12.11 Centrifuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifuge to approximately 3500 rpm. 12.12 Transfer 4 mL of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.13 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.14 Add 1.0 mL or appropriate volume of methanol to each centrifuge tube using a graduated pipette. (This volume equals the initial volume of serum used for the extraction.) 12.15 Vortex mix for 30 seconds. 12.16 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. 12.17 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.18 Cap and hold for HPLC-electrospray/mass spectrometry analysis. Extracts may be stored at 4 C until analysis. 12.19 Complete the extraction worksheet, attached to this document, and tape to page of study notebook. FACT-M-3.0 Extraction of PFOS from Serum Page 6 of 8 OC 013.6 Extraction Worksheet for FACT-M-3 Study # - Sample Number set # H , 0 Blank Serum Blank PFOS approx. 0.5 ppm actual ppm #W - PFOS approx. 5 ppm actual ppm #W - PFOS approx. 50 ppm actual ppm #W - Date and Initials for Std. or Comments - - - - - - 1 - -- -- -- - -- - ---- -- -- -- -- -- -- -- -- -- ---- -- -- -- ' Study numb er where the original worl(sheet is located. Blank Serum Std # Serum amount = Serum Extraction Method : e Date & Initials Vortex 15 sec. Pipette Serum Volume mL Pipette 1 mL of 0.5 M TBA, pH 10. Std. # Pipette 2 mL o f 0.25 Na^COV0.25M NaHCCh buffer Std. # Pipette 5 mL o f ethyl acetate TN-A- Shake 20 min. Centrifuge 20-25 min. Remove a 4 mL aliauot of organic layer Centrifuge speed: Put on Nitrogen Evaporator to drvness Evaporator #: Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. Filter using a 3cc B-D syringe with a 0.2um SRI filter into a 1.5 mL autosamole vial MS/MSD/___Cont. Checks: Spiked_____ uL of a _____ ppm std (_____________ ) for a final concentration of ________ppm. MS/MSD used sample______________. Cont. Checks used same serum as for std curve. FACT-M-3.0 ExtractionofPFOS fromSerum Page 8of 8 0001.1.7 3M Environmental Laboratory Method Analysis of Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry Method Number: FACT-M-4.0 Author: Lisa Clemen Approved By: Laboratory Manager Adoption Date: H / h I ^ 8 Revision Date: V/ Date Group Leader d St. CJinvtn Technical Reviewer Date Date 1.0 Scope and Application 1.1 Scope: This method is for the analysis of extracts of serum or tissue for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report. Word 7.0.1/95 FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 1 of 8 000118 2.0 Summary of M ethod 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 D efinitions_____________________________________ _________________________________ 3.1 None. 4.0 W arnings and Cautions__________________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.Q Interferences___________________________________________________________________ 5.1 Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment_______________________ ;____________________________________________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 Supplies and M aterials_______ __________________________________________________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 Reagents and Standards________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 2 of 8 0C0119 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may . be provided by a Milli-Q TOC Plus system. 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one serum blank, and seven serum standards are prepared during the extraction procedure. See FACT-M-3. 9.0 Sample Handling_________________________________________________________________ 9.1 Fresh serum standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be refrigerated at 4 C until analysis can be performed. 10.0 Quality C ontrol_______________________________________________________________ 10.1 M atrix Blanks and Method Blanks 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 System suitability (e.g., peak area, retention time, peak shape, etc.) will be assessed for each run. 11.0 Calibration and Standardization______________________________________________ 11.1 Analyze the extracted serum standards prior to and following each set of extracts. The mean of two standard values, at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software. FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 3 of 8 000120 11.2 The r2value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 P r o c e d u r e s_________________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A scan is usually collected along with the SIRs. Save method. 12.1.3 Typically the sample list begins with the first set of serum standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 p.L injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 4 of 8 000121 12.3 Instrument Set-up 12.3.1 Refer to AMDT-EP-31 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eye piece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode flow rate 10 - 500 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.1 OClick on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 D a t a A n a ly sis and C a l c u l a t io n s_________________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 5of 8 000122 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, anion in serum (pg/mL): ug of PFO calc, from std. Curve x Dilution Factor x Final Volume (mL) Initial Volume of serum (mL) 14. Q M ethod Performance_____ ____________________________________________________ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 15. Q Pollution Prevention and W aste M anagement_____________________ _ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records________________________________________________________________________ 16.1 Store chromatograms in the study folder. Each chromatogram must have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 T ables. Diagram s. Flowcharts, and V alidation Data_________________________ 17.1 Attachment A: FACT-M-4 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-3.0 & 4.0-V-l. 18.0 References_____________________________________________________________________ 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" 19.0 A ffected Documents___________________________________________________________ 19.1 FACT-M-3.0, "Extraction of Fluorochemical Anions from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 6 of 8 OC 01.23 20.0 R e v is io n s Revision Number. Reason For Revision Revision Date FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 7 of 8 000124 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone ug/mL - Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone (ug/mL): Calculated by dividing the initial volume from the concentration FACT-M-4.0 Analysis of Serum Extract Using ES/MS Page 8 of 8 000125 Potassium Perfluorooctanesulfonate CAS Number-2759-39-3 3M Environmental Laboratory Report No. Tox-012 Laboratory Request Number (LRN)-U2006 A ttachment G: R esults of Confirmatory Dose A nalyses Proprietary and Confidential OC0126 000127 FACT-TOX-012 Argus# 418-008 Study: Product NumberfTest Substance): Matrix: Method/Reviskm: Analytical Equipment System Number Instrument Sofovare/Version: Date o f Extraction/Analyst: Date o f Analysis/Analyst: Date o f Data Reduction/Analyst: Argus 418-008, Two-Generation Reproduction Study o f PFOS in Rats PFOS (T-6295.9) Tween Dosing Vehicle Filename: ETS-8-4 I AETS-8-5.1 R-Squared Value: M adeline04l098 MassLj-nx 3.2 Slope: Y-Intercept: 9/13/99 IAS. 9/23/99 IAS 9/15/99 IAS, 9/24/99 IAS 9/16/99 IAS, 9/27/99 IAS Sam ple D ata See list to right See Attachments See Attachments See Attachments T w een D osing C onfirm ation Group Dose Sample f Method Blk H 2 0 Bfk-1 Method Blk H 2 0 BIk-2 Matrix Blk Rabbit L h erB lk -l Matrix Btk QC Rabbit Liver Bfk-2 B-418-008-B (302ppb MS. exl9/13/99) B-418-008-C (50.3ppb MS. ext9/13/99) B-418-008-0 (ISIppbMS. txt9/!3/99) B-418-008-E (302ppb MS. 19/13/99) B-418-008-E (302ppb MS ext9/23/99) Group 1 B -418-008-A, 12/27/98 (ext 9/13/99) Control Diluted 1/1 0.0 mg/kg/day 0 mg/mL Group 2 B -418-008-B, 12/21/98 (ext 9/13/99) 0.1 mg/kg/day Diluted 1/40 0.02 mg/mL Group 3 0.4 mg/kg/day B -4 18-008-C, 12/21/98 (ext 9/13/99) Diluted 1/1000 0.08 mg/mL Group 4 1.6 mg/kg/day B -418-008-D, 12/21/98 (ext 9/13/99) Diluted 1/1000 032 mg/mL Group 5 3.2 mg/kg/day B418-008-E , 12/21/98 (ext 9/13/99) B -418-008-E 12/21/98 (ext 9/23/99) 0.64 mg/mL Diluted 1/1000 Limit o f Q uantitation Limit (LOQ) - PFOS 30 ng/g Method Detection Limit (MDL): PFOS * IS ng/g PFOS * Porfluorooctanesul foliate Expected Cone PFOS ng/mL NA NA NA NA 302 50.3 151 302 302 0.00 PFOS Cone. n g/m L NA NA NA NA 778 130 461 852 865 0.0 Dilution Factor NA NA NA NA NA NA NA NA NA 1 20000 512 40 80000 70.1 1000 320000 303 1000 640000 640000 565 1000 581 1000 NR - Sample not received nor reported. NA " Not Applicable Date Entered/Analyst: Date Verified/Analyst: 9/27/99 GML Total PFOS ng/raL NA NA NA NA NA NA NA NA NA 0.0 20489 70050 302550 564560 580860 PFOS-Bckgmd Cone. ng/mL NA NA NA NA 266 60 159 288 284 NA PFOS % Recovery Accuracy NA NA NA NA 88% 119% 105% 95% 94% <LOD NA 102% NA 88% NA 95% NA 88% NA 91% Filenames Grp 1 Grp 2 Grp 3 Grp 4 Grp 5 MS, MSD PFOS 091599013 091599014 091599015 091599016 092799149. 091599020-023.092799159-160 FACT-M-2.0 Excel Version 5.0^5 TOX-Ol2-dose008 10/15/99 to a\* FACT-TOX-012 Argus# 418-008 Study: Product NumberfTest Substance): Matrix: Method/Revision: Analytical Equipment System Number. Instrument Sofhvare/Veraon: Date o f Extraction/Analyst: Date o f Analysts'Analyst: Date o f Data Reduction/Analyst: Argus 418-008, Tno-Generation Reproduction Study o f PFOS in Rata PFOS (T-6295.9) Tween Dosing Vehicle Filename: ETS-8-1.1 &ETS-8-5.1 R-Squared Value: M ad e lin e 04I098 Slope: MassLynx 3.2 Y-Inlerccpt: 9/13/99 IAS. 9/23/99 IAS 9/15/99 IAS, 9/24/99 IAS 9/16/99 IAS, 9/27/99 IAS Sample Data See Attachments See Attachments See Attachments See Attachments Tween Dosing Confirmation Group Dose ll Method Blk Method Blk Matrix Blk Matrix Blk QC Sam ple# 0 0 H 2 0 Blk-I H 2 0 Btk-2 Rabbit Liver Bik-I Rabbit Liver Blk-2 B-4I8-008-B (302ppb MS, ext9/13/99) B-418-008-C (50.3ppb MS. ext9/I3/99) B-I18-008-D (15Ippb MS, (9 /1 3 /9 9 ) B-4I8-008-E (302ppb MS, ext9/l3/99) B-4I8-008-E (302ppb MS ext9/23/99) Group 1 Control 0.0 rogfleg/dav Group 2 0.1 mg/kg/day 0.02 tng/mL Group 3 0.4 mg/kg/day 0.08 mg/mL Group 4 1.6 mg/kg/day B -l 18-008-A, 12/27/98 (ext 9/13/99) Diluted 1/1 B-I18-008-B, 12/21/98 (ext 9/13/99) Diluted 1/40 B -l 18-008-C, 12/21/98 (ext 9/13/99) Dituted 1/1000 B-4I8-008-D, 12/21/98 (ext 9/13/99) Diluted 1/1000 032 mg/mL Group 5 3.2 mg/kg/day 0.64 mg/mL B -418-0 0 8 -E 12/21/98 (ext 9/13/99) B-J18-00S-E, 12/21/98 (ext 9/23/99) Limit o f Quantitation Limit (LOQ) * PFOS 30 ng/g Method Detection Lit.Jt (MDL): PFOS " 15 ng/g PFOS " Perfluorooctanesulfonate Eipected C one PFOS ng/mL NA NA NA NA 302 50 HI 302 302 0.00 PFOS Cone n g/m L NA NA NA NA 266 60 159 288 284 0.00 PFOS % Recovery Accuracy NA NA NA NA 88% 119% 105% 95% 94% <LOD 20000 20489 102% 80000 70050 88% 320000 302550 95% 640000 640000 564560 580860 88% 91% NR " Sample not received nor reported. NA - Not Applicable Date Entered/Analyst: Date Verified/Analyst: 09/27/99 GML 0012 00 Q FACT-M-2.0 Excel Version 5.0/95 TOX-O12-doseOOS 10/15/99 * to AM