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MIN 312/014272 PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK Sponsor 3M Center, 3M Corporate Toxicology, Building 220-2E-02, St Paul, MN 55133-3220, USA. Research Laboratory Huntingdon Life Sciences Ltd., Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, ENGLAND. Report issued: 7 October 2005 Page 1 of 340 MIN 312/014272 CONTENTS Page CONTENTS................................................................................................................................................2 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS................................... 4 QUALITY ASSURANCE STATEMENT...............................................................................................5 CONTRIBUTING SCIENTISTS..............................................................................................................6 S U M M A R Y ................................................................................................................................................7 INTRODUCTION...................................................................................................................................... 8 RELEVANT STUDY DATES..................................................................................................................9 TEST SUBSTANCES............................................................................................................................ 10 EXPERIMENTAL PROCEDURE........................................................................................................ 11 RESULTS................................................................................................................................................ 17 DISCUSSION.......................................................................................................................................... 20 CONCLUSION........................................................................................................................................ 20 REFERENCES......................................................................................................................................... 21 FIGURES 1. Bodyweight - group mean values (g)............................................................................................ 22 TABLES 1. Bodyweight - group mean values (g)............................................................................................. 23 2. Food consumption - group mean values (g/rat)............................................................................24 3. Water consumption - group mean values (g/rat) .........................................................................25 4. Organ weights - group mean values (g) .......................................................................................26 5. Macroscopic pathology - incidence summary............................................................................... 30 6. Microscopic pathology - incidence summary...............................................................................31 :2 : MIN 312/014272 Page APPENDICES 1. Daily dose observations - individual findings............................................................................... 32 2. Bodyweights - individual values (g) ............................................................................................ 34 3. Absolute organ weights - individual values (g)............................................................................. 35 4. Individual pathological findings.................................................................................................... 37 5. pH values for terminal urine - individual values........................................................................... 58 6. PCNA staining in the urinary bladder - individual and mean values.......................................... 59 ADMINISTRATION OF POSF BY INHALATION TO R A T S ......................................................... 60 PROTOCOL AND PROTOCOL AMENDMENTS..............................................................................87 ANALYTICAL PHASE REPORT........................................................................................................121 SCANNING ELECTRON MICROSCOPE EXAMINATION AND X-RAY MICROANALYSIS................................................................................................................. 287 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENTS............................ 338 :3 : MIN 312/014272 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid. The UK Good Laboratory Practice regulations 1999 (Statutory Instrument No 3106) as amended by Statutory Instrument 2004 No. 994. OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98)17. EC Commission Directive, 1999/11/EC of 8 March 1999 (Official Journal No L 77/8), as amended by EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No. L 50/44). These principles of Good Laboratory Practise are accepted by the regulatory authorities and the United States of America and Japan on the basis of Intergovernmental agreements. An expiry date for the test substance was not supplied. The material was assumed to be stable for the duration of the study. No claim of compliance is made with regard to the scanning electron microscope examination and X-ray microanalysis. Terence J. Kenny, B.Sc. (Hons.), Study Director, Huntingdon Life Sciences Ltd. Date :4 : QUALITY ASSURANCE STATEMENT MIN 312/014272 The following have been inspected or audited in relation to this study: Study Phases Inspected Date of Inspection Date of Reporting Protocol Audit 21 August 2001 21 August 2001 Study Preparation Exposure Sampling Clinical signs Test item control disposition Post Mortem SEM X-RAY - EM 13 September 2001 13 September 2001 13 September 2001 13 September 2001 13 September 2001 18 September 2001 27 September 2001 14 September 2001 14 September 2001 14 September 2001 14 September 2001 14 September 2001 18 September 2001 28 September 2001 Report Audit 10-17 December 2001 28 September 2005 17 December 2001 29 September 2005 Protocol: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Study based inspections: Inspections and audits of phases of this study were conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspections and audits of other routine and repetitive procedures employed on this type of study were carried out. These were promptly reported to appropriate Company Management. Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above. The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data. Analytical phases of this study conducted by Exygen Research were subjected to Quality Assurance monitoring and audit according to Exygen's own procedures. Details are presented in the Analytical Phase Report. Tracy Scarfe, FRQA, Group Manager, Department of Quality Assurance, Huntingdon Life Sciences Ltd. :5 : Date CONTRIBUTING SCIENTISTS STUDY MANAGEMENT Terence J. Kenny, B.Sc. (Hons.), Study Director. Rhiannon Davies, B.Sc. (Hons.), Senior Study Supervisor. TOXICOLOGY Derek W. Coombs, B.Sc., MSc., Senior Toxicologist. AEROSOL TECHNOLOGY AND ANALYSIS Ian S. Gilkison, M.A., Ph.D., Section Head, Aerosol Technology and Analysis. PATHOLOGY Samuel McCormick, M.V.B., M.R.C.V.S., Ph.D., F.R.C.Path., Director of Pathology. BIOANALYSIS Richard A. Grazzini, Exygen Research, 3058 Research Drive, State College, PA 16800, USA. SEM MICROSCOPY AND X-RAY ANALYSIS Roy Moate, Plymouth Electron Microscope Unit, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, ENGLAND. :6 : MIN 312/014272 SUMMARY MIN 312/014272 One group of rats (each of 5 males and 5 females) of the Crl:CDBR strain were exposed to POSF, 6 hours a day for 5 consecutive days using a snout-only exposure system. A second group, acting as control, was exposed to air only. The study mean analysed concentration was 316.95 ppm. The following comments are made in summary: Mortality and clinical signs There were no unscheduled deaths or any treatment related signs seen during the course of the study. Bodyweight, food and water consumption Following 5 days of treatment with POSF there was reduced bodyweight gain, food consumption and water consumption in treated males. Organ weights Bodyweight adjusted liver weights were higher in treated animals, with statistical significance (p< 0.05) being attained in females. Absolute lungs and bronchi weights were higher in treated animals with statistical significance being achieved in males and in bodyweight adjusted female lung and bronchi weights. Macroscopic and microscopic pathology There were no treatment-related macroscopic or microscopic findings. Conclusion A no-effect level was not established for this study. :7 : INTRODUCTION MIN 312/014272 The purpose of this study performed at Huntingdon Life Sciences Limited, Huntingdon, England was the assessment of systemic toxic potential in a 1-week inhalation study in rats, by snout-only administration of the test substance POSF, for 6 hours a day, for 5 consecutive days. The test substance was administered by inhalation, a possible route of accidental exposure in man. The rat was the species of choice due to requirement for a rodent species by regulatory agencies and the strain was selected on account of the availability of comprehensive background data, relating to clinical and pathological parameters, at our laboratories. :8 RELEVANT STUDY DATES Approved by: Study Director: HRC Management: Study Sponsor: 17 August 2001 17 August 2001 20 August 2001 Animals arrived at HRC: Exposures commenced: Serum/urine sampling: Terminal kill: Experimental completion date: 5 September 2001 13 September 2001 18 September 2001 18 September 2001 28 September 2002 MIN 312/014272 :9 : TEST SUBSTANCES MIN 312/014272 Sponsor's identification: Perfluorooctanesulfonyl fluoride Other names: POSF, T-7661.1, FX-8B Storage conditions: In a refrigerator (ca 4C) Date received: 14 June 2001 Supplier: Sponsor Batch number: 040227 Expiry date: Assumed to be stable for the duration of the study Purity: >99.5% A small sample (1 ml) was sealed in a suitable contained and stored in Archives at an appropriate temperature. : 10 : EXPERIMENTAL PROCEDURE MIN 312/014272 ANIMALS Twenty rats (10 male and 10 female) aged approximately 6 weeks, of the Crl:CDBR, a caesarean derived strain of Sprague-Dawley origin, were obtained from Charles River (UK) Limited, Manston Road, Margate, Kent, on 5 September 2001. The latest Health Screen Report published by the animal supplier was provided to Huntingdon Life Sciences (HLS). In addition, the additional consignments of animals included a health screen relating to the current status of the breeding colony. These documents were sent to HLS Veterinary Services immediately upon receipt for review and subsequent archiving. Random assignment to experimental groups took place on arrival. The animals were then uniquely identified by numbers tattooed on the tail. The identification of individual rats in the 2 groups together with the target exposure level were as follows: Group 1 (Air control) 2 (POSF) Target exposure level (p p m) 300 Animal numbers Male Female 1-5 11-15 6-10 16-20 ACCOMMODATION The rats were housed 5 of the same sex to a cage in suspended stainless steel cages fitted with mesh front, back and floor with stainless steel sheet sides. Plastic trays lined with absorbent paper were placed below each cage to collect animal excreta and the paper was changed daily. Each cage had a coloured label identifying the group and the numbers of the animals contained within it. The rats were kept in a single room and, additionally, after the start of the exposure period, each group was positioned on an individual cage battery. Exposure took place in the same room. The temperature and relative humidity of the holding room were recorded using a Kent Clearspan recorder. The study holding room temperature and relative humidity were set to be maintained within limits of 21 2C and 55 15% respectively. Recorded ranges were 19.0 to 21.0C and 40 to 72% humidity. Minor deviations from these ranges were of relatively short duration and considered not to have affected the scientific integrity of the study. Lighting was controlled to give 12 hours light (0600 - 1800 hours) and 12 hours dark per 24 hours. : 11 : MIN 312/014272 DIET While in their cages, all rats had access to a weighed quantity of standard quality-controlled laboratory rat food (SDS Rat and Mouse No. 1 SQC modified maintenance diet, Special Diets Services, Witham, Essex). There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test compound could reasonably be expected to be present in the diet. The analytical data have been lodged in Huntingdon Life Sciences Archives. Tap water was available from moulded polypropylene water bottles at all times while the rats were in the cages. The water bottles were rinsed and refilled daily and thoroughly cleaned at intervals during the study. There was no information available to indicate that any substance likely to influence the effect of the test system could reasonably be expected to be present in the drinking water. Results of the routine physical and chemical analyses of water at source (sampling point, Grafham Final Water) as conducted by the supplier, Anglian Water Services Ltd, have been made available to Huntingdon Life Sciences. Anglian Water takes its guidelines on water quality from the EEC directive relating to water for human consumption, viz. Council Directive 80/778/EEC. The analytical data have been lodged in Huntingdon Life Sciences Archives. ADMINISTRATION The test substance was administered for 6 hours a day, for 5 consecutive days. The test material was delivered to an all glass vapouriser and generated as a droplet atmosphere, into a stream of air for administration to the rats by inhalation from snout only exposure chambers. The test substance was metered to the vaporiser from an infusion pump. The vapour/air mixture passed directly into the exposure chamber. The target chamber concentration was achieved by using different liquid feed rates controlled by the infusion pump and using syringes of an appropriate value. The target concentrations for treated rats was 300 ppm. Control rats received air only. The rats were exposed to the control/test atmosphere using ADG snout-only exposure chambers (ADG Developments Ltd, Hitchin, Hertfordshire, England) of a modular construction in aluminium alloy comprising a base unit, 3 sections each having 20 exposure ports, and a top section incorporating a central inlet with a tangential air inlet. All animals (including reserves) were subjected to restraint procedures and exposed to air only (`Sham dosing'), for 5 consecutive days, in order to accustom animals to the restraining procedure prior to study initiation. The rats were restrained once per day, increasing the `Sham dosing' period progressively (0.5, 1, 2, 4 and 6 hours for Days -5 to -1 respectively). Details of administration and analysis of the test atmospheres together with the results obtained are presented in ADMINISTRATION OF POSF BY INHALATION TO RATS appended to this report. : 12 : MIN 312/014272 CLINICAL INVESTIGATIONS Dated and signed records of all activities relating to the day to day running and maintenance of the study, as well as to the group observations and examinations outlined in this procedure were recorded in the Study Daybook. Individual dated and signed records noting nature and severity, date and time of onset and duration and progress of observed clinical signs were maintained for each animal. Mortality Throughout the study, all cages were checked in the morning and again at the end of the normal working day for dead or moribund animals. Clinical signs Dated and signed records of appearance, change and disappearance of clinical signs were maintained. Individual animal records were maintained on the basis of: - any observation, considered to be of possible importance, made at any time during the study; - any observation, considered to be of possible importance, made during transfer to restraining tubes (prior to exposure), during exposure (although severely restricted due to tube restraint), on return to holding cages (after exposure) and as late a possible in the working day. During the acclimatisation period, observations of the animals and their cages were recorded at least once a day. BODYWEIGHT The weight of each rat was recorded a week prior to the start of exposures. During the treatment period, bodyweight was recorded on the day that treatment commenced, daily thereafter, and also prior to necropsy. FOOD CONSUMPTION The quantity of food consumed by each cage of rats was recorded on a daily basis. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each cage in each group and the number of rats surviving in each cage. : 13 : MIN 312/014272 WATER CONSUMPTION The quantity of water consumed by each cage of rats was recorded on a daily basis, commencing 1 week before the start of exposures. Water intake per rat (g/rat/day) was calculated using the total amount of water given to and left by each cage in each group and the number of rats surviving in each cage. TERMINAL STUDIES Pre-terminal urine sampling Individual urine samples were collected from animals within 2 hours following lights on in the animal holding room where possible. For animals failing to produce a specimen during this interval, urine was collected at necropsy. The urine samples were immediately assayed for pH using a microelectrode, and prepared SEM stubs despatched to Plymouth University for calculi and crystal analysis by SEM X-ray element identification. Test substance/metabolite analyses Samples of blood (for serum) were obtained at necropsy by cardiac/aorta puncture while the rats were held under terminal sodium pentobarbitone anaesthesia. The blood samples (up to 4 ml) were collected, run into tubes, allowed to clot at room temperature and the serum separated and frozen prior to despatch to the Sponsor for subsequent analysis at Exygen Research. Necropsy All animals were killed on Day 6 of the study, following 5 days of exposure. Animals were killed by an intraperitoneal injection of sodium pentobarbitone followed by exsanguination from the brachial arteries. All study rats were subjected to a macroscopic post mortem examination. The following procedures applied: All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incisions and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. : 14 : MIN 312/014272 The gastro-intestinal tract was examined as a whole and the stomach, caecum and portions of duodenum, jejunum, ileum, colon and oesophagus were incised and examined. The lungs were removed and all pleural surfaces examined. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded. Sections of liver samples were immediately frozen by immersion in liquid nitrogen and stored at -70C prior to dispatch to the Sponsor for subsequent analysis at Exygen Research. The following organs from all surviving animals were dissected free of fat and weighed. For bilateral organs, left and right organs were weighed together. adrenals kidneys liver lungs heart Preservation of tissues During post mortem examination, samples or the whole of the tissues listed below, from all study animals, were preserved in 10% neutral buffered formalin; except the eyes which were preserved in Davidson's fixative and testes/epididymides were fixed in Bouin's solution and then transferred to 70% alcohol. The lungs were infused with fixative prior to immersion. The nasal cavity was flushed with fixative prior to immersion. abnormalities adrenals aorta (thoracic) brain caecum colon duodenum epididymides eyes femur (longitudinal section through joint) harderian glands head heart ileum jejunum kidneys (with pelvic region)* lachrymal glands larynx (2 levels) liver lungs (section from all lobes including bronchi) lymph nodes: mandibular mesenteric tracheobronchial mammary area (caudal) nasal turbinates oesophagus optic nerves ovaries pancreas pituitary prostate rectum salivary glands (submandibular/ sublingual) sciatic nerves seminal vesicles skeletal muscle (thigh) skin spinal cord (transverse and longitudinal sections at the cervical, lumbar and thoracic levels) spleen sternum stomach testes thymus thyroid with parathyroids tongue trachea (2 levels) urinary bladder* uterus with cervix vagina * To be examined microscopically Histopathology examination The tissues in the list above were examined using a light microscope as annotated. Tissues were embedded in paraffin wax and sections approximately 4 - 5 micrometres were cut, processed and stained with haematoxylin and eosin. : 15 : MIN 312/014272 PCNA Staining for cell proliferation Sections of urinary bladder were taken from all animals for PCNA immunostaining in order to assess the degree of cell proliferation present. A total of 3,000 cells were counted from 3 separate sections (1,000 cells/section) in order to determine the cell proliferation index. Positive PCNA staining cells were counted, and examined by the pathologist to assess the sections and count S-phase positive cells, if practicable. In addition, sections of the duodenum from each animal were taken and stained to act as positive controls for the immunostaining methodology. STATISTICAL ANALYSIS For categorical data, the proportion of animals were analysed using Fisher's exact test for each treated group versus the control. For continuous data, Bartlett's test was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of the Bartlett's test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary. ARCHIVING All specimens, raw data and study-related documents generated during the course of the study at Huntingdon Life Sciences, together with a copy of the final report were lodged in Huntingdon Life Sciences, Archives. Such specimens and records will be retained for a minimum period of five years, from the date of issue of the final report. At the end of the five-year retention period the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Sponsor's knowledge. DEVIATION FROM PROTOCOL Due to some slight tissue damage it was not possible to count 1000 cells/section on a number of urinary bladder sections stained for PCNA analysis. The number of sections affected was low and there was no effect on the outcome of the study. : 16 : RESULTS MIN 312/014272 CHAMBER ATMOSPHERE CONDITIONS Chamber analysed concentration of POSF The data are presented in ADMINISTRATION OF POSF TO RATS BY INHALATION appended to this report. The mean chamber concentrations (ppm) are summarized below: Exposure No. Concentration PPm 1 316.04 2 313.69 3 323.33 4a 5 314.74 Mean 316.95 sd 4.361 cv 1.4 Sd Standard deviation CV Coefficient of variation (%) a No values recorded due to hardware failure, however nominal concentrations were similar for all days therefore the actual exposure levels were deemed to be on target Study mean analysed concentration was 316.95 ppm which was in good agreement with the target concentration of 300. CLINICAL OBSERVATIONS Mortality There were no unscheduled deaths. Clinical signs The data are presented as follows: Appendix 1 - individual findings There were no treatment-related signs. Signs observed post dose included red staining around eyes, brown staining on head and body and wet fur. These signs were seen in a proportion of Test and Control animals during the shamming and treatment periods and were associated with the method of restraint used. : 17 : MIN 312/014272 Bodyweight The data are presented as follows: Figure 1 - group mean values (g) Table 1 - group mean values (g) Appendix 2 - individual values (g) Over the five days of treatment, treated rats gained less weight than controls. On Days 2 and 3 of exposure the male rats lost weight. Food consumption The data are presented as follows: Table 2 - group mean values (g/rat) Over the five days of treatment, a small reduction in food consumption was seen in treated males, the greatest effect being on Days 2 and 3 of exposure. Water consumption The data are presented as follows: Table 3 - group mean values (g/rat) On Days 2 and 3 of exposure water consumption in treated males was reduced. At other times consumption was comparable with that of control rats. TERMINAL INVESTIGATIONS Organ weights The data are presented as follows: Table 4 - group mean values Appendix 3 - individual values Bodyweight adjusted liver weights were higher in treated animals, with statistical significance (p< 0.05) being attained in females. Absolute lungs and bronchi weights were higher in treated animals with statistical significance being achieved in males and in bodyweight adjusted female lung and bronchi weights. : 18 : MIN 312/014272 Macroscopic pathology The data are presented as follows: Table 5 - incidence summary Appendix 4 - individual pathological findings The macroscopic examination performed at termination revealed no changes attributable to treatment with POSF. The incidence and distribution of all the findings were considered to fall within the background range of macroscopic changes. Microscopic pathology The data are presented as follows: Table 6 - incidence summary Appendix 4 - individual pathological findings No treatment related microscopic findings were reported. All microscopic findings were considered to be incidental and of no toxicological importance. Urinary pH The data are presented as follows: Appendix 5 - individual values Differences between the groups were minimal and considered to be of no toxicological significance. Cell proliferation The data are presented as follows: Appendix 6 - individual and mean values. The mean cell proliferation index (% positive cells) value for treated males was greater than the control value, this was principally due to differences at levels 1 and 2 only. However, intragroup differences were large such that a proportion of the individual values for the treated group lay within the range of values seen for the control group. Differences between control and treated males at level 3 and at all levels in females were minimal. It is considered that the differences seen were not of toxicological significance. SEM and X-ray examination of urine and bladder tissue The results of examination together with SEM photomicrographs are presented in the Scanning Electron Microscope Examination and X-ray Microanalysis report appended to this main report. Test substance and metabolite analysis The methods and results of analyses are presented in the ANALYTICAL PHASE REPORT appended to this main report. : 19 : DISCUSSION MIN 312/014272 Administration of a POSF to rats at a dosages of 300 ppm a day over 5 consecutive days produced no mortalities and transient clinical effects which were considered to be treatment-related. There was a reduction in bodyweight gain, food and water consumption amongst treated males principally on Days 2 and 3 of exposure. There were no macroscopic pathology changes. Lung weights were higher in treated animals as were bodyweight adjusted liver weights. The toxicological importance of the organ weight changes is unclear given the absence of macroscopic and microscopic findings. There were no differences in urinary pH or cell proliferation index considered to be of toxicological importance. CONCLUSION A no-effect level was not established for this study. : 20 : REFERENCES MIN 312/014272 BARTLETT, M.S. (1937), Properties of sufficiency and statistical test, Proc. Roy. Soc A 160: 268 -282. FISHER, R.A., Statistical Methods fo r Research Workers, Para. 21.02, Oliver and Boyd, Edinburgh. : 21 : FIGURE 1 Bodyweight - group mean values (g) Group 1 C ontrol G roup 2 3 00 ppm MIN 312/014272 : 22 : MIN 312/014272 : 23 : TABLE 1 Bodyweight - group mean values (g) GROUP COMPOUND DOSAGE (PPM) 1 CONTROL 0 2 POSF 300 SEX: GROUP: DAY -MALE1 2 FEMALE-------12 -7 202 198 148 149 -6 214 211 153 156 -5 223 218 159 159 -4 229 224 162 164 -3 238 232 166 168 -2 244 236 167 170 -1 253 243 173 174 0 254 245 173 175 1 257 249 175 176 2 264 245 175 177 3 266 241 178 177 4 273 250 180 181 5 276 254 180 180 ++ Gain Day 0-5 22 9 7 5 % of Control - 41 - 71 Level of significance - all comparisons made with Vehicle Control: Student's 't' test: ++ p< 0.01 P r in t No: 0001 Printed: 21-SEP-01 Xybion protocol number: MIN 312 TABLE 2 Food consumption - group mean values (g/rat) P r in t No: 0002 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 21-SEP-01 Xybion protocol number: MIN 312 SEX: --------- MALE--------- -------- FEMALE' GROUP: 1 2 1 2 DAY MIN 312/014272 : 24 : -7 29 27 19 21 -6 29 28 20 21 -5 30 28 19 23 -4 30 28 19 21 -3 28 28 18 23 -2 36 26 19 25 -1 19 25 16 13 1 26 24 18 18 2 27 17 17 17 3 27 15 19 18 4 28 22 18 19 5 29 25 18 19 Cumulative Days 1-5 % of Control 137 - 103 75 90 91 - 101 TABLE 3 Water consumption - group mean values (g/rat/) MIN 312/014272 Day -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 Cumulative 1 to 5 % of Control 1M (Control) 30 31 32 33 34 32 33 31 32 31 35 32 161 - Group/dosage 2M 1F (POSF) (Control) 30 21 30 21 32 23 30 25 31 22 30 21 29 22 32 21 21 20 19 21 32 24 32 23 135 110 84 - 2F (POSF) 22 20 23 24 24 21 23 23 21 21 27 23 116 106 : 25 : MIN 312/014272 : 26 : GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 SEX: GROUP: NUMBER: ABSOLUTE VALUES ------- MALE------- 55 TERMINAL BODY WEIGHT (g) N MEAN sd :5 : 274.3 : 14.4 5 252.3 12.7 ADRENALS N MEAN sd : : : 5 0.050 0.005 5 0.054 0.007 HEART N MEAN sd : : : 5 1.173 0.103 5 1.062 0.186 KIDNEYS N MEAN sd : : : 5 2.04 0.14 5 1.94 0.08 LIVER N MEAN sd : : : 5 12.91 1.00 5 12.65 0.82 No differences of statistical significance TABLE 4 Organ weights - group mean values (g) P r in t No: 0004 Printed: 21-SEP-01 Xybion protocol number: MIN 312 BODYWEIGHT ADJUSTED VALUES MALE 55 N MEAN LIVER :5 : 12.29 5 13.27 MIN 312/014272 : 27 : GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 TABLE 4 (Organ weights - continued) SEX: GROUP: NUMBER: ABSOLUTE VALUES ------- MALE------- 55 N MEAN sd : : : LUNGS & BRONCHI +++ 55 1.112 1.828 0.128 0.204 Level of significance - all comparisons made with Vehicle Control: Student's 't' test: +++ p< 0.001 P r in t No: 0004 Printed: 21-SEP-01 Xybion protocol number: MIN 312 MIN 312/014272 : 28 : GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 TABLE 4 (Organ weights - continued) SEX: GROUP: NUMBER: ABSOLUTE VALUES ------FEMALE------ 55 BODYWEIGHTS ADJUSTED VALUES FEMALE 55 TERMINAL BODY WEIGHT (g) N MEAN sd :5 : 179.5 : 8.7 5 180.4 12.5 ADRENALS N MEAN sd : : : 5 0.054 0.004 5 0.056 0.006 HEART HEART N MEAN sd : : : 5 0.814 0.114 5 0.893 0.091 KIDNEYS N MEAN :5 : 0.816 5 0.891 KIDNEYS N MEAN sd : : : 5 1.36 0.11 5 1.44 0.14 N MEAN :5 : 1.37 5 1.44 N MEAN sd : : : LIVER 5 7.97 0.77 5 9.16 1.31 N MEAN LIVER + :5 5 : 8.00 9.12 Level of significance - all comparisons made with Vehicle Control: Student's 't' test: + p< 0.05 P r in t No: 0005 Printed: 21-SEP-01 Xybion protocol number: MIN 312 MIN 312/014272 : 29 : GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 TABLE 4 (Organ weights - continued) SEX: GROUP: NUMBER: ABSOLUTE VALUES ------FEMALE------ 1--- -- 2 --- 55 BODYWEIGHTS ADJUSTED VALUES FEMALE 55 N MEAN sd : : : LUNGS & BRONCHI 5 0.891 0.070 5 1.478 0.159 N MEAN LUNGS & BRONCHI +++ :5 5 : 0.895 1.474 Level of significance - all comparisons made with Vehicle Control: Student's 't' test: +++ p< 0.001 P r in t No: 0005 Printed: 21-SEP-01 Xybion protocol number: MIN 312 GROUP COMPOUND DOSAGE (PPM) TABLE 5 Macroscopic pathology - incidence summary P r in t No: 0006 :1 : CONTROL :0 2 POSF 300 Printed: 21-SEP-01 Xybion protocol number: MIN 312 MIN 312/014272 : 30 : ORGAN AND KEYWORD(S) OR PHRASE ** TOP OF LIST ** KIDNEYS ...................... PELVIC DILATION LN MANDIBULAR ................ CONGESTED ENLARGED LN MESENTERIC ................ ENLARGED SKIN .......................... SCAB(S) STOMACH ...................... ANTRUM WHITE NODULE(S) TEETH ......................... INCISOR(S) PALE UTERUS ........................ FLUID DISTENTION ** END OF LIST ** SEX: --MALE -- -FEMALEGROUP: -1- -2- -1- -2- NUMBER: 5 5 5 5 NUMBER EXAMINED: 5 5 5 5 00 10 NUMBER EXAMINED: 5 5 5 5 10 00 0100 NUMBER EXAMINED: 5 5 5 5 0001 NUMBER EXAMINED: 5 5 5 5 0030 NUMBER EXAMINED: 5 5 5 5 2110 NUMBER EXAMINED: 5 5 5 5 0002 NUMBER EXAMINED: 0 0 5 5 00 11 TABLE 6 MIN 312/014272 : 31 : Microscopic pathology - incidence summary P r in t No: 0011 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 -- N U M B E R - O F - A N I M A L S - A F F E C T E D ---- SEX: --MALE-- -FEMALEGROUP: -1- -2- -1- -2- ORGAN AND FINDING DESCRIPTION NUMBER: 5 5 5 5 ** TOP OF LIST ** KIDNEYS .......................................... --CORTICAL TUBULAR BASOPHILIA --DILATED TUBULE WITH EPITHELIAL HYPERPLASIA --CORTICAL TUBULES WITH HYALINE DROPLETS --CORTICAL INFLAMMATORY CELL INFILTRATE --CORTICAL MINERALISATION --HYPERPLASIA, PELVIC EPITHELIUM --PELVIC DILATATION NUMBER EXAMINED: 5 5 5 5 3333 0101 2200 12 0 1 0100 00 10 00 10 URINARY BLADDER ................................. --REFLUXED SEMINAL COLLOID PLUG ** END OF LIST ** NUMBER EXAMINED: 5 5 5 5 13 00 APPENDIX 1 Daily dose observations - individual findings MIN 312/014272 Group Sign No. 1M Wet fur (Control) Red staining around eyes Brown staining head 2M (POSF) Brown staining muzzle Wet fur Red staining around eyes Brown staining head Brown staining muzzle Only animals showing signs are presented V sign present Animal Day No. -3 -2 -1 1 2 3 4 5 1 V VVV VV 2 VVVVVV V 3 VVVVVV V 4 V VVVVV 5 VVVVVVVV 1 VVVVV 2 VV VV 3 VV 4 VV V 5 VVV 1 2V 3V 5V V 5V 6 VVVVVVVV 7 V VV VVV 8 VVVV VVV 9 VVVV VVV 10 V V V V V V V V 7 VV 9V 6V 7V 8V 6 VV 7V : 32 : APPENDIX 1 (Daily dose observations - continued) MIN 312/014272 Group Sign No. 1F Wet fur (Control) Red staining around eyes Brown staining head 2F (POSF) Brown staining muzzle Wet fur Brown staining head Animal Day No. -3 -2 -1 1 2 3 4 5 11 V V V V V V V V 12 V V V V V V 13 V V V V V V V 14 V V V V V V 15 V V V V V V 11 V V 12 V V V 13 V V V V 14 V V 15 V V V 11 V V 12 V V 13 V V 14 V 15 V 11 V V 16 V V V V V V V V 17 V V V V V V V 18 V V V V V V V 19 V V V V V V V V 20 V V V V V V 17 V 19 V 20 V V V Brown staining muzzle Only animals showing signs are presented V sign present 17 18 20 V V VV : 33 : MIN 312/014272 : 34 : GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 GROUP ANIMAL DAY -7 DAY -6 DAY -5 1M 1 210 228 237 2 194 208 215 3 195 206 217 4 204 209 220 5 206 221 228 2M 6 200 212 215 7 190 203 213 8 207 222 232 9 196 208 210 10 197 210 220 1F 11 152 152 155 12 152 159 164 13 143 151 156 14 140 149 160 15 154 156 160 2F 16 151 158 163 17 153 159 165 18 153 162 163 19 147 153 153 20 142 148 153 2 POSF 300 DAY -4 245 215 223 227 236 222 213 240 219 227 159 168 158 162 162 169 168 170 159 154 APPENDIX 2 Bodyweights - individual values (g) P r in t No: 0007 Printed: 08-OCT-01 Xybion protocol number: MIN 312 DAY DAY DAY DAY DAY DAY DAY DAY DAY -3 -2 -1 0 1 2 3 4 5 251 254 264 263 265 270 275 281 282 222 226 233 233 235 241 246 252 254 233 237 247 248 251 262 263 273 276 240 248 258 261 265 270 271 279 284 245 256 261 264 269 276 275 282 283 233 235 243 240 246 233 221 232 240 217 220 228 228 236 241 233 241 244 248 255 260 264 263 262 259 268 269 228 231 238 243 246 241 240 249 250 236 240 246 252 252 250 252 259 265 163 161 169 167 169 166 170 172 171 172 175 182 182 184 188 191 192 190 160 164 169 169 170 172 172 177 178 165 165 170 172 173 170 174 174 175 170 170 173 176 177 181 181 184 185 175 177 179 182 179 185 185 183 188 171 174 180 183 181 178 186 190 189 174 177 181 185 186 184 184 190 188 161 164 166 166 167 170 170 173 172 159 160 165 160 165 167 163 168 165 APPENDIX 3 Absolute organ weights - individual values (g) P r in t No: 0010 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 08-OCT-01 Xybion protocol number: MIN 312 GROUP ANIMAL TERMINAL BODY WT (g) ADRENALS HEART KIDNEYS LIVER LUNGS & BR 1M 1 2 3 4 5 281.1 249.8 273.6 285.5 281.5 0.052 0.045 0.047 0.057 0.051 1.250 1.012 1.206 1.263 1.133 2.23 1.94 2.02 2.12 1.88 13.56 11.29 12.78 13.87 13.06 1.161 0.908 1.161 1.246 1.082 MIN 312/014272 : 35 : 2M 6 7 8 9 10 239.0 244.1 268.7 247.2 262.7 0.051 0.048 0.058 0.064 0.049 0.973 1.392 1.019 0.963 0.963 1.90 1.90 2.05 1.85 1.99 11.66 11.96 12.81 13.26 13.56 1.600 2.095 1.900 1.901 1.645 MIN 312/014272 : 36 : APPENDIX 3 (Absolute organ weights - continued) GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 GROUP 1F ANIMAL 11 12 13 14 15 TERMINAL BODY WT (g) 169.5 189.3 176.7 174.3 187.9 ADRENALS 0.050 0.059 0.052 0.055 0.053 HEART 0.798 1.008 0.785 0.769 0.709 2F 16 17 18 19 20 188.1 190.5 189.0 171.6 162.8 0.058 0.063 0.050 0.051 0.061 0.994 0.980 0.866 0.845 0.782 KIDNEYS 1.33 1.52 1.23 1.44 1.31 LIVER 7.28 8.96 7.10 8.34 8.15 LUNGS & BR 0.816 1.006 0.891 0.869 0.871 1.57 1.55 1.48 1.38 1.24 9.72 10.10 10.45 7.96 7.55 1.455 1.584 1.690 1.343 1.318 P r in t No: 0010 Printed: 08-OCT-01 Xybion protocol number: MIN 312 APPENDIX 4 Individual pathological findings MIN 312/014272 The initial examination was undertaken by the study pathologist, the results of which were then subjected to a routine peer review by a second pathologist. The diagnoses reported here represent the consensus opinions of both pathologists. Study pathologist: Peer review: Takahito Kambara, B.V.Sc., M.V.Sc., Ph.D., Pathologist Department of Pathology Samuel G. McCormick, M.V.B., M.R.C.V.S., Ph.D., F.R.C.Path., Director of Pathology Department of Pathology : 37 : APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0001 DATE OF DEATH: 18-SEP -01 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS:: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT : 281.1 GRAMS NECROPSY PA THO L O G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA, -MINIMAL, FOCAL MIN 312/014272 : 38 : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0002 DATE OF DEATH: 18-SEP -01 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS:: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 249.8 GRAMS NECROPSY PA THO L O G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA, -MINIMAL, FOCAL URINARY BLADDER : -REFLUXED SEMINAL COLLOID PLUG,-PRESENT MIN 312/014272 : 39 : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0003 DATE OF DEATH: 18-SEP -01 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT : 273.6 GRAMS NECROPSY PA THO L O G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA, -MINIMAL, FOCAL -CORTICAL INFLAMMATORY CELL INFILTRATE, -MINIMAL, FOCAL MIN 312/014272 : 40 : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** APPENDIX 4 : P : (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0004 DATE OF DEATH: 18-SEP-01 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 285.5 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULES WITH HYALINE DROPLETS,-MINIMAL LN MANDIBULAR : -CONGESTED STOMACH : -ANTRUM WHITE NODULE(S); RIDGE, 1MM. MUCOSA, ONE, NEAR TO LIMITING MIN 312/014272 APPENDIX 4 MIN 312/014272 : 42 : (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0005 DATE OF DEATH: 18-SEP-01 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH:: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 281.5 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULES WITH HYALINE DROPLETS,-MINIMAL STOMACH : -ANTRUM WHITE NODULE(S); RIDGE, 1MM. MUCOSA, ONE, NEAR TO LIMITING APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0006 DATE OF DEATH: 18-SEP-01 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 239.0 GRAMS MIN 312/014272 : 43 : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** *** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED *** APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0007 DATE OF DEATH: 18-SEP -01 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS:: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 244.1 GRAMS NECROPSY PA THO L O G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA, -MINIMAL, FOCAL URINARY BLADDER : -REFLUXED SEMINAL COLLOID PLUG,-PRESENT : pp : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** MIN 312/014272 APPENDIX 4 MIN 312/014272 : 45 : (Individual pathological findings - continued) Print No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0008 DATE OF DEATH: 18-SEP-01 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 268.7 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULES WITH HYALINE DROPLETS,-MINIMAL LN MANDIBULAR : -ENLARGED; RIGHT, ONE. STOMACH : -ANTRUM WHITE NODULE(S); RIDGE, 1MM. MUCOSA, ONE, NEAR TO LIMITING URINARY BLADDER : -REFLUXED SEMINAL COLLOID PLUG,-PRESENT APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0009 DATE OF DEATH: 18-SEP-01 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 247.2 GRAMS NECROPSY PA THO L O G Y O B SE RV A T ION S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL -CORTICAL INFLAMMATORY CELL INFILTRATE,-MINIMAL, -CORTICAL MINERALISATION, -MINIMAL, FOCAL FOCAL MIN 312/014272 : 46 : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0010 DATE OF DEATH: 18-SEP-01 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 262.7 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA, -MINIMAL, FOCAL -DILATED TUBULE WITH EPITHELIAL HYPERPLASIA,-MINIMAL, FOCAL -CORTICAL TUBULES WITH HYALINE DROPLETS,-MINIMAL -CORTICAL INFLAMMATORY CELL INFILTRATE, -MINIMAL, FOCAL >NOTE:>FIBROSIS ALSO SEEN IN THE LESION OF TUBULAR HYPERPLASIA URINARY BLADDER : -REFLUXED SEMINAL COLLOID PLUG,-PRESENT MIN 312/014272 : 47 : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0011 DATE OF DEATH: 18-SEP-01 SEX: FEMALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 169.5 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY UTERUS : -FLUID DISTENTION, MODERATE MIN 312/014272 : 48 : *** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED *** APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0012 DATE OF DEATH: 18-SEP-01 SEX: FEMALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 189.3 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY SKIN : -SCAB(S); TAIL, A FEW, 1MM. STOMACH : -ANTRUM WHITE NODULE(S); RIDGE, 1MM. MUCOSA, ONE, NEAR TO LIMITING MIN 312/014272 : 49 : *** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED *** APPENDIX 4 MIN 312/014272 : 50 : (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0013 DATE OF DEATH: 18-SEP-01 SEX: FEMALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 176.7 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL SKIN : -SCAB(S); TAIL, MULTIPLE, 1MM. APPENDIX 4 MIN 312/014272 : 51 : (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0014 DATE OF DEATH: 18-SEP-01 SEX: FEMALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 174.3 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -PELVIC DILATION, MINIMAL; RIGHT. KIDNEYS : -CORTICAL TUBULAR BASOPHILIA, -MINIMAL, FOCAL -HYPERPLASIA, PELVIC EPITHELIUM,-MINIMAL, FOCAL -PELVIC DILATATION,-SLIGHT APPENDIX 4 MIN 312/014272 : 52 : (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER : 0015 DATE OF DEATH : 18-SEP-01 SEX: FEMALE DOSE GROUP: 1 SACRIFICE STATUS:: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH:: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT : 187.9 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL SKIN : -SCAB(S); TAIL, A FEW, 1MM. APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0016 DATE OF DEATH: 18-SEP-01 SEX: FEMALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 188.1 GRAMS MIN 312/014272 : 53 : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** *** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED *** APPENDIX 4 (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0017 DATE OF DEATH: 18-SEP -01 SEX: FEMALE DOSE GROUP: 2 SACRIFICE STATUS:: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT : 190.5 GRAMS NECROPSY PA THO L O G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA,-MINIMAL, FOCAL MIN 312/014272 : 54 : *** ANIMAL HAS NO GROSS OBSERVATIONS RECORDED *** APPENDIX 4 (Individual pathological findings - continued) Print No: 0012 GROUP COMPOUND DOSAGE (PPM) 2 POSF 0 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0018 DATE OF DEATH: 18-SEP-01 SEX: FEMALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 189.0 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY TEETH : -INCISOR(S) PALE; LOWER, RIGHT. : gg : *** ANIMAL HAS NO MICROSCOPIC FINDINGS RECORDED *** MIN 312/014272 APPENDIX 4 MIN 312/014272 : 56 : (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0019 DATE OF DEATH: 18-SEP-01 SEX: FEMALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: 171.6 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA, -MINIMAL, FOCAL -DILATED TUBULE WITH EPITHELIAL HYPERPLASIA,-MINIMAL, FOCAL -CORTICAL INFLAMMATORY CELL INFILTRATE, -MINIMAL, FOCAL >NOTE:>FIBROSIS ALSO SEEN IN THE LESION OF TUBULAR HYPERPLASIA TEETH : -INCISOR(S) PALE; LOWER. APPENDIX 4 MIN 312/014272 : 57 : (Individual pathological findings - continued) P r in t No: 0012 GROUP COMPOUND DOSAGE (PPM) :1 : CONTROL :0 2 POSF 300 Printed: 02-NOV-01 Xybion protocol number: MIN 312 ANIMAL NUMBER: 0020 DATE OF DEATH: 18-SEP -01 SEX: FEMALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE STUDY DAY OF DEATH:: 6 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT : 162.8 GRAMS NECROPSY PA THO LO G Y O B SE RV A T IO N S HISTOPATHOLOGY KIDNEYS : -CORTICAL TUBULAR BASOPHILIA, -MINIMAL, FOCAL LN MESENTERIC : -ENLARGED, MINIMAL; ONE. UTERUS : -FLUID DISTENTION, MODERATE APPENDIX 5 pH values for terminal urine - individual values MIN 312/014272 Group Males Females Animal pH Animal pH number number 1 1 NS 6 INS (Control) 2 8.7 7 a 8.6 3 8.6 8 a 8.8 4 9.1 9 8.9 5 9.3 10 INS 2 11a 9.0 16 a 8.7 (POSF) 12 a 8.9 17 7.4 13 8.3 18 a 9.1 14 a 8.1 19 6.9 15 8.4 20 a 8.8 Samples obtained in animal room following procedure provided by Sponsor, remaining samples obtained immediately prior to or during post mortem examination INS Sample volume < 8 ^l required to obtain accurate pH reading NS No sample obtained 58 : APPENDIX 6 PCNA staining in the urinary bladder - individual and mean values Level 1 Level 2 Level 3 Combined Levels Animal Positive Negative Total C.P.I.* Positive Negative Total C.P.I.* Positive Negative Total C.P.I.* Positive Negative Total C.P.I.* No 1 14 644 658 2.1 15 837 852 1.8 6 248 254 2.4 11.7 576.3 588.0 2.1 2 63 937 1000 6.3 53 947 1000 5.3 37 870 907 4.1 51.0 918.0 969.0 5.2 3 18 982 1000 1.8 20 980 1000 2.0 16 984 1000 1.6 18.0 982.0 1000.0 1.8 4 5 995 1000 0.5 5 995 1000 0.5 4 996 1000 0.4 4.7 995.3 1000.0 0.5 5 6 994 1000 0.6 3 997 1000 0.3 5 995 1000 0.5 4.7 995.3 1000.0 0.5 mean 2.3 2.0 1.8 2.0 6 4 996 1000 0.4 11 989 1000 1.1 6 994 1000 0.6 7.0 993.0 1000.0 0.7 7 65 935 1000 6.5 77 923 1000 7.7 31 969 1000 3.1 57.7 942.3 1000.0 5.8 8 47 953 1000 4.7 49 951 1000 4.9 27 973 1000 2.7 41.0 959.0 1000.0 4.1 9 17 983 1000 1.7 15 985 1000 1.5 7 993 1000 0.7 13.0 987.0 1000.0 1.3 10 98 902 1000 9.8 83 917 1000 8.3 41 959 1000 4.1 74.0 926.0 1000.0 7.4 mean 4.6 4.7 2.2 3.9 ii 3 997 1000 0.3 4 996 1000 0.4 2 998 1000 0.2 3.0 997.0 1000.0 0.3 12 9 991 1000 0.9 16 984 1000 1.6 9 991 1000 0.9 11.3 988.7 1000.0 1.1 13 7 904 911 0.8 6 994 1000 0.6 1 864 865 0.1 4.7 920.7 925.3 0.5 14 6 994 1000 0.6 5 995 1000 0.5 1 999 1000 0.1 4.0 996.0 1000.0 0.4 15 2 998 1000 0.2 7 993 1000 0.7 1 999 1000 0.1 3.3 996.7 1000.0 0.3 mean 0.6 0.8 0.3 0.5 16 1 999 1000 0.1 1 999 1000 0.1 3 997 1000 0.3 1.7 998.3 1000.0 0.2 17 1 999 1000 0.1 0 926 926 0.0 1 999 1000 0.1 0.7 974.7 975.3 0.1 18 6 994 1000 0.6 2 998 1000 0.2 4 763 767 0.5 4.0 918.3 922.3 0.4 19 11 475 486 2.3 4 382 386 1.0 4 416 420 1.0 6.3 424.3 430.7 1.4 20 6 863 869 0.7 2 696 698 0.3 4 557 561 0.7 4.0 705.3 709.3 0.6 mean 0.8 0.3 0.5 0.5 *: cell proliferation index (%positive cells) MIN 312/014272 : 59 : Mean cell proliferation index (%positive cells) Group Sex Mean SD CV(%) 1 Male 2.0 1.95 97.1 2 3.9 2.86 74.2 CV (%) Coefficient of variation (sd x 100 / mean) Group 1 2 Sex Female Mean 0.5 0.5 SD CV(%) 0.34 63.9 0.53 99.8 MIN 312/014272 ADMINISTRATION OF POSF BY INHALATION TO RATS Author Simon Moore : 60 : Administration of POSF by inhalation to rats CONTENTS MIN 312/014272 Page TEST SUBSTANCE AND ADMINISTRATION Test substance.......................................................................................................................................... 62 Administration.......................................................................................................................................... 62 Test atmosphere generation..................................................................................................................... 62 Exposure chambers..................................................................................................................................63 P ro ced u re................................................................................................................................................... 64 Aerosol analysis....................................................................................................................................... 65 Chamber monitoring system ................................................................................................................... 65 Target concentrations...............................................................................................................................66 Exposure chamber conditions.................................................................................................................66 RESULTS Vapour concentration...............................................................................................................................68 D is c u s sio n ................................................................................................................................................. 69 Calculations...............................................................................................................................................70 FIGURES A. Schematic of a rodent inhalation dosing system....................................................................... 71 B. Schematic of a Fourier Transform Infrared Spectrophotometer............................................. 72 TABLES A. Operating conditions of the inhalation exposure system.........................................................73 B. Chamber concentrations of POSF (ppm) - daily mean values............................................... 74 C. Nominal concentrations of POSF(ppm) - individual exposure values.................................. 75 D. Chamber temperature - exposure mean values.........................................................................76 APPENDICES A. Methods of sample collection and analysis for POSF.............................................................77 B. Individual POSF concentration measurements.........................................................................85 : 61 : Administration of POSF by inhalation to rats TEST SUBSTANCE AND ADMINISTRATION MIN 312/014272 TEST SUBSTANCE The test substance, POSF, is a liquid with boiling point of 154C. A consignment of the Test Article (4 x 20 kg, Lot number 040227), was received from the Sponsor on 14 June 2001. The test substance was stored securely in the original containers at room temperature until it was transferred to the atmosphere generation system. The stated purity was >95.5%. Information regarding the purity and stability of the test substance is the responsibility of the Sponsor. ADMINISTRATION The test material was administered to the rats by snout-only exposure chambers described below: The chamber atmosphere was produced by metering the liquid test substance into a glass vapour generator through which dried air was passed at a flow rate of 29 l/minute for administration to the rats, by inhalation from snout only exposure chambers. The target chamber concentration was achieved by metering the test substance from polypropylene syringes mounted on a syringe driver. This atmosphere gave the final chamber concentration of POSF. The setting of the test substance metering system required to obtain the target chamber concentration was determined during preliminary generation trials without animals present and based on the Fourier Transform Infrared (FT-IR) analysis of chamber atmosphere samples. Minor adjustments were made to the test material delivery rates in order to maintain the chamber concentration close to target. Animals assigned to Group 1 (Air control) received an exposure to air only, from the same compressed air source as used for the generation of the test atmospheres. The duration of administration was a single 6-hour exposure, daily, for 5 days. The usage of POSF was determined, for each day of treatment, for the lone test group. TEST ATMOSPHERE GENERATION The vapour delivery system for the dose group comprised a polypropylene syringe located on a syringe driver (Precidor, Model 5003), which delivered the liquid test material to a glass frit contained in a glass vessel via PolyTetraFluoroEthylene (PTFE) tubing. The syringe size and syringe driver settings required to achieve the target concentrations were established during the preliminary phase of the study. Air was passed through the vapouriser at a rate of 29 l/minute. The vapour/air mixture passed out of the vapouriser into the chamber inlet ducting through a 22 mm diameter flexible pipe. All equipment was housed in an extracted cabinet. The air control group received clean air only at a rate of 29 l/minute. : 62 : Administration of POSF by inhalation to rats EXPOSURE CHAMBERS (Figure A) MIN 312/014272 The inhalation exposure system comprised a snout-only inhalation exposure chamber and rats restraining tubes. Accessories included air supply and extract lines, which attached to the top and bottom of the chamber respectively. A filtration system was incorporated into the extract line. A schematic diagram of an exposure system is shown in Figure A. The component parts of the system are described in further detail below: Inhalation chamber ADG snout-only inhalation chamber (ADG Developments Ltd, Hitchin, Hertfordshire, England). This is a modular apparatus of aluminium alloy construction comprising of a base unit, a variable number of animal exposure sections, each having 20 exposure ports, and a top section incorporating a central aerosol inlet surrounded by a tangential air inlet. The chambers used on this study were assembled using 3 rodent exposure sections, identified as levels 1 (top) to 3 (bottom). All exposure sections had 20 ports and formed a 28 cm diameter cylinder with a volume of approximately 47 litres. During dosing each chamber was housed in an enclosed ventilated cabinet. Rats restraining tubes Moulded polycarbonate tubes tapered at one end to allow the snout only to project from the tapered end. The other end is normally closed by insertion of an expanded plastic bung. A push rod passes through the centre of the bung and is adjustable to maintain the position of the rats during restraint. Tubes are attached to a chamber by means of push-fit "O" ring seals located in the exposure ports of the animal exposure sections. The restraining tubes were attached to chamber level two. Chamber level two was used to expose the animals. All exposure ports not in use were sealed with an expanded plastic bung. Air supply and extract Air supplies were provided by a compressor. The air was filtered to remove any residual particulate and was dried (dew point ~2C). A 1 l/minute differential was maintained between the inlet and outlet airflows to provide a small negative pressure within the exposure system. The in-line flowmeters were calibrated daily against high quality tapered tube rotameters measuring the free flow of air at points of attachment of the supply and extract lines to each chamber. The airflows used for each group are detailed in Table A. The calibrated exhaust airflow of each exposure system was passed through a trapping system comprising a Fluosorber carbon vapour filter and a silica gel column before it was passed to atmosphere. The exhaust flows used for each group are detailed in Table A. : 63 : Administration of POSF by inhalation to rats PROCEDURE MIN 312/014272 Two exposure systems were used, one for each group. The procedure followed for each group was similar except that, for the control group, the rats in the control group received air only. Therefore in the following description the comments relating to the sampling and the Fourier Transform Infrared spectrophotometer apply to the test group only. The FT-IR data capture programme was initialised and the path difference was inspected to ensure the value was zero, subsequently, a background scan was recorded and the cell path length was calibrated. A syringe was filled with the test substance, the weight of liquid was recorded and the syringe was mounted on the syringe driver. The syringe was connected to the vapouriser by PTFE tubing. The pump was turned on briefly in order to engage the gearing and to move the liquid along the PTFE tubing almost to the surface of the glass frit. The injection rate to be used was then set. The rats were removed from their cages and placed into restraining tubes, which were then attached to their assigned level of the chamber. Unused exposure ports were sealed with blanking plugs. The rats were removed from their cages and placed into restraining tubes, which were colour coded for the treatment group and numbered for each animal. The tubes were then attached to level 2 of the chamber utilising 5 ports on either side of the 20 port section. Unused exposure ports were sealed with blanking plugs. The inlet and outlet airflows of the exposure system were calibrated using precision made tapered glass tube flowmeters. The connections of the generation and extract systems to the chamber were checked to ensure they were correct and working. The barometric pressure was recorded manually. Generation commenced as the syringe driver was turned on and 15 minutes into the exposure, the chamber sampling system was activated (see below). At intervals of 30 minutes, any reactions by the rats to exposure, together with checks of generation and chamber operational parameters including temperature and inlet and outlet air flowrate were manually recorded. The airflows were measured throughout the exposures using in-line flowmeters. A sample pump continually delivered the chamber atmosphere from the test chamber to the FT-IR system at a flow rate of 1 l/minute. Analysis for the determination of POSF concentration occurred at exactly 30 mimute intervals throughout each exposure. The chamber atmosphere was returned to the chamber downstream of the sampling point. At the end of six hours generation, the syringe driver was turned off and the weight of the remaining syringe contents was recorded. Generation airflow was turned off and the chamber extract allowed to clear the vapour for 5 - 10 minutes (equilibration time t99 is 7 minutes). The data capture program halted automatically after collecting a specific number of samples. At the end of this time, the rats were unloaded from the chambers and returned to their respective holding cages. The chambers were washed with hot water. The IR cell path length was retested to ensure that the initial value prior to the exposure was consistent throughout the exposure. : 64 : Administration of POSF by inhalation to rats ANALYSIS OF THE TEST ATMOSPHERE (FIGURE B) MIN 312/014272 The concentration of POSF in air within the test inhalation chamber was measured using a Fourier Transform Infrared (FT-IR) Spectrophotometer. Definitive details of the FT-IR, its standardisation and validation are given in Appendix A. The Spectrophotometer was located adjacent to the exposure chambers. CHAMBER MONITORING SYSTEM A PC running the AutoQuant 3.01 software was used to monitor and record the system performance during each exposure. The data collection sequence and display were controlled by a personal computer (PC) and all information collected was displayed on a monitor. Simultaneously, the data was stored electronically. This program was composed of three basic stages of operation: an initial setting up (pre-exposure) phase, an exposure monitoring phase and the post exposure data collation and presentation phase. The program and FTIR hardware were loaned by the Sponsor for the duration of the study. Raw data printed as a hard copy. Setting-up phase In the initial phase, prompted by the program screen display, the instrument response was checked, followed by a background scan. An ultra pure nitrogen cylinder was used for this purpose. The pathlength was then calibrated using a certified cylinder of ethylene. A regulated flowrate of 2 l/minute was used for the nitrogen and ethylene cylinders, the analyser drew 1 l/minute of the gas stream and the remainder was vented to waste. The flow meters used in conjunction with the cylinders were monitored using a calibrated in-line tapered tube gas flowmeter. The study details including the daily pathlength, number of scans and samples and frequency of samples are entered and stored into the data capture program prior to the exposure. Exposure monitoring phase This phase was started 15 minutes later than the start of atmosphere generation. The test chamber's environment was monitored during a 30-minute cycle when the analysed concentration was recorded. The data were displayed on screen, printed and stored on the local hard drive of the computer and an external zip disc. A total of twelve sample points were recorded, each of which represented 32 coadded scans. : 65 Administration of POSF by inhalation to rats Post exposure phase MIN 312/014272 At the end of the exposure, the data collected during exposure was collated and printed. The mean values, together with standard deviation were calculated for each parameter recorded. The first set of chamber concentration data was included in the calculation of the mean and standard deviation because chamber conditions stabilised within the 15 minutes from the start of exposure (equilibration time, t99was 7 minutes) before the analysis was started. Midac Grams 32 version 4.11 software was solely used to generate a hard copy of the infrared data. The IR cell path length calibration of the FT-IR was reassessed to ensure that the initial value prior to the exposure was consistent throughout the exposure. Details of the analytical methodologies used are given in Appendix A. TARGET CONCENTRATIONS The target concentrations of POSF was: Group 2 Designation Test Group Concentration (PPm) 300 The target concentrations were selected in consultation with the Sponsor, following the review of available data. EXPOSURE CHAMBER CONDITIONS Chamber analysed concentration of POSF The test atmosphere was sampled from one point within the test atmosphere chamber. This was continually drawn through a transfer line, which was therefore in equilibrium with the mean concentration from the test chamber. The chamber atmosphere was returned to the exposure chamber downstream of the sampling point. Every 30 minutes, the software for automated analysis and data logging activated the FT-IR. The methodology is presented in Appendix A. Chamber spatial distribution All the animals and the analytical sampling point were situated on level two of the three level exposure chamber. : 66 : Administration of POSF by inhalation to rats Nominal concentration of chamber atmospheres MIN 312/014272 The chamber nominal concentrations were calculated from the amount of liquid used over the six-hour exposure period, the mass of the liquid and the exposure mean airflow. The formulae used were as follows: V Concentration = -------- x 1,000,000 ppm Va + V (1) V = -W---x--R---x--T-x--7-6-0--m---m--H--gM Atm (2) where V= W= M= R= T= Atm = Va = gaseous volume of POSF (L) mass of POSF (g) molecular weight of POSF (502.14 g/mole) Gas constant (0.08205 L atm mmol-1 K-1) temperature (K) atmospheric pressure (mmHg) volume of air (L) Airflow and temperature These parameters were recorded manually, as described above under the Test Atmosphere generation and Procedure sections. : 67 : Administration of POSF by inhalation to rats RESULTS MIN 312/014272 VAPOUR CONCENTRATION Analysed concentration of POSF The data are presented as follows: Daily mean values Individual values Table B Appendix B The study mean concentration (the mean of daily mean values) for each group exposed to POSF are presented below: Group 2 (Test Group) Chamber concentration (ppm) Target Analysed 300 317 Analysed concentration was in good agreement with target concentration. The coefficient of variation of the daily mean was 1.4 % for Group 2. The uncertainties relating to the analysed and room air concentrations were estimated by the data capture program based on the least squares statistics. Nominal concentration of POSF The data are presented in Table C and is summarised below: Group 2 (Test Group) Nominal concentration (ppm) 283 A/N ratio (%) 111.5 , ,^T ( Analysed concentration A/N = I ------ ------------------------ lx 100 ( Nominal concentration ) For Group 2, the nominal concentration for each exposure was calculated from the following parameters: The mass of POSF delivered into each vapour generator; The mean chamber temperature; The barometric (atmospheric) pressure; The molecular weight of POSF; The gas constant; The chamber airflow; The exposure duration. The equations used for the calculation of the nominal concentration are detailed in Table C. : 68 : Administration of POSF by inhalation to rats MIN 312/014272 The nominal concentration for each exposure period was calculated, for the test group from the mass of POSF delivered into the generator. The daily ratios of analysed to nominal concentration (A/N), expressed as a percentage, were between 109 and 115%, with a coefficient of variation of only 2.1%. Possible reasons for this unexpectedly high A/N ratio are discussed below. Chamber Temperature The daily mean chamber temperatures are presented in Table D. The chamber temperatures were similar for both groups on each day of the study. DISCUSSION Control of the POSF vapour delivery to the exposure chambers was excellent, with a coefficient of variation of less than 1.5%, and a study mean concentration, which was within 6% of the target value for the test group. As no analytical data was available for exposure 4 due to a computer problem, it seems reasonable (using the consistent A/N ratio) that the animals were dosed with a concentration around ca. 313 ppm. The ratio between the average analysed and the nominal chamber concentrations (A/N ratio) showed a definite discrepancy between these two values, with a mean of 111.5%. The coefficients of variation for the analysed and nominal values are 1.4% and 1.5% respectively, which shows that the discrepancy is consistent throughout the five day long exposure. The estimate of the nominal chamber concentration was lower than the analysed value and this is attributed to a combination of uncertainties as follows: - POSF reference spectra and subsequent AutoQuant method calculations. These have errors of 5% associated with them (responsibility of the Sponsor); - The cylinder of ethylene standard has certified errors of 2%; - Uncertainties in airflow measurements. A 1 l/minute difference in the airflow would produce a 3% change in the nominal concentration; - The test material has a stated purity of >95.5% POSF. Thj other components of the test material were various perfluoroalkyl sulfonyl fluoride5-\<4.5%) and their absorbance may interfere with that of POSF; - The effect of using ultra pure nitrogen for the background scans (rather than the control chamber atmosphere) is unknown. : 69 Administration of POSF by inhalation to rats CALCULATIONS MIN 312/014272 In order to minimise the cumulative errors, which result from repeated rounding of numbers, much of the data in this report has been calculated continuously using unrounded numbers and only rounded for printing. Consequently, these rounded numbers may include rounding errors in the last significant figure, possibly leading to small apparent discrepancies with other data in the report. : 70 Administration of POSF by inhalation to rats FIGURE A Schematic of a rodent inhalation dosing system MIN 312/014272 Key a Polypropylene syringe b Syringe driver c Test compound feed line d Glass Vapouriser e Air supply (29 l/minute) f Sinter diameter (114 mm) g Exposure chamber h Observation port i Blanking plugs j Rodent restraining tubes (standard sections with 20 exposure ports on levels 1 to 3 k Exhaust plenum l Filtration m Air extract (30 l/minute) : 71 Administration of POSF by inhalation to rats FIGURE B MIN 312/014272 Schematic of a Fourier Transform Infrared spectrophotometer Key a Nitrogen cylinder b Ethylene cylinder c Flowmeter regulated to 1 l/minute d Test compound sample line from chamber e 10 cm IR cell f FT-IR spectrophotometer g Flowmeter regulated to 1 l/minute h Diaphragm pump i Sample line return to chamber : 72 : Administration of POSF by inhalation to rats TABLE A Operating conditions for the inhalation exposure system MIN 312/014272 P aram eter Target concentration of POSF (ppm) Chamber airflows (l/minute) Elutriator output (chamber inlet) Chamber extract Vapour generator settings Test material feed Syringe size (ml) Syringe pump setting (Speed, mm/min): Exposure 1 Exposures 2-5 N/A Not applicable 1 (Air control) 0 Group 2 (Test Group) 300 29 29 30 30 N/A Precidor 5003 syringe pump N/A 50 N/A 0.145 N/A 0.140 : 73 : Administration of POSF by inhalation to rats TABLE B MIN 312/014272 Chamber concentration of POSF (ppm) - daily mean values Exposure No. 1 2 3 4b 5 Mean sd CV (%) b sd CV Analysed Concentration PPm 316.04 313.69 323.33 Error a 0.85 0.83 0.82 Room Air PPm 0.398 0.373 0.289 Error 0.004 0.004 0.003 Concentration minus Room Air (PPm) 315.65 313.32 323.04 314.74 0.83 0.428 0.004 314.31 316.95 0.83 0.372 0.004 316.58 4.361 0.009 0.0598 0.0007 4.414 1.4 1.1 16.1 19.9 1.4 Error value reported by Autoquant software. No analytical data collected for exposure 4 due to computer problem with data capture program Standard deviation Coefficient of variation (sd x 100/mean) 74 : Administration of POSF by inhalation to rats TABLE C MIN 312/014272 Nominal concentrations of POSF (ppm) - individual exposure values Group 2 (Test Group) - Target concentration 300 ppm Exposure No. Duration (min) Barometric pressure (mmHg) POSF usage (g) Chamber airflow (1/min) 1 360 753 64.6 30.0 2 360 763 63.0 30.0 3 360 757 63.5 30.0 4 360 762 63.3 30.0 5 360 766 64.8 30.0 Chamber concentration Nominal c Analysed (ppm) 289 (ppm) 316 278 313 283 323 280 a 285 314 Mean of Means sd CV (%) 360 0.0 0.0 760 5.2 0.7 63.8 0.81 1.3 30.0 0.0 0.0 283 4.4 1.5 317 4.4 1.4 No analytical data collected due to computer problem with data capture program Nominal concentrations were calculated from the following equations: y6 Concentration (ppm) = ------------- x 10 Va + V A/N ratio (%) 109.1 112.6 114.2 a 110.3 111.5 2.32 2.1 V- W x R x TX 760 mm Hg ------------ M Atm where V= W= M= R= T= Atm = Va = gaseous volume of POSF mass of POSF molecular weight POSF (502.14 g/mole) gas constant (0.08205 l atm mol'1K '1) temperature (K), = temperature (C, see Table D) + 273 atmospheric pressure (mmHg) volume of air (litres) passing through the chamber during the exposure A/N Analysed/nominal concentration ratio expressed as a percentage sd Standard deviation CV Coefficient of variation (sd x 100/mean) : 75 Administration of POSF by inhalation to rats TABLE D Chamber temperature - exposure mean values Temperature (oC) Exposure Group 1 Group 2 (Control) (Test Group) 1 20.0 20.2 2 20.0 20.0 3 20.0 20.0 4 19.9 20.0 5 19.9 19.9 Mean 20.0 20.0 sd 0.06 0.11 CV (%) 0.3 0.5 sd standard deviation CV Coefficient of variation (sd x 100/mean) MIN 312/014272 : 76 Administration of POSF by inhalation to rats APPENDIX A Methods of sample collection and analysis for POSF MIN 312/014272 SAMPLE COLLECTION Chamber concentration The atmosphere sample was continually drawn from the test chamber to the FT-IR system at a regulated flow rate of 1 l/minute using a laboratory pump positioned downstream of the IR cell. The concentration of POSF was determined at exactly 30 minute intervals throughout each exposure. The sampled atmosphere was returned to the exposure chamber downstream of the sampling point. The airflow to the spectrophotometer was monitored throughout each of the exposures using a calibrated in-line tapered tube gas flowmeter. Gas sampling lines were PTFE tubing (0.6 cm diameter). METHOD OF ANALYSIS Chamber atmosphere samples were analysed by extractive FT-IR. The method of sample analysis is detailed, together with a summary of the method validation, in the Inhalation Analytical Procedure at the end of this Appendix. : 77 : Administration of POSF by inhalation to rats APPENDIX A MIN 312/014272 (Methods of sample collection and analysis for POSF - continued) CALCULATIONS FT-IR analysis The samples of chamber atmosphere were passed through the IR cell, which was calibrated using nominal vapour standards. The method for calculating the concentration of POSF from the mass used to prepare each vapour standard using the nominal feed rate is given below in equations 1 and 2. V Concentration = -------- x 1,000,000 ppm Va + V (1) W x R x T 760 mm Hg V = ------------x-------------- M Atm (2) where V= W= M= R= T= Atm = Va = gaseous volume of POSF (ml) mass of POSF (g) molecular weight of POSF (502.14 g/mole) Gas constant (0.08205 ml atm mmol-1K-1) temperature (K) atmospheric pressure (mmHg) volume of air (ml) : 78 : Administration of POSF by inhalation to rats APPENDIX A MIN 312/014272 (Methods of sample collection and analysis for POSF - continued) COMPOUND SPECIFIC INHALATION ANALYTICAL PROCEDURE FOR POSF The analysis of POSF (Perfluorooctane sulfonyl fluoride) in air sample substrate The method outlined in this document has been validated and is considered fit for the purpose of monitoring conditions in an Inhalation Toxicology study. This document details the basic procedures for the analysis of POSF sampled by extractive FT-IR from test atmospheres. The resulting samples, of approximate concentration 240 to 360 ppm, are analysed using the MIDAC I-Series spectrophotometer and AutoQuant software. Study specific amendments and additions will be detailed within a supplementary document. NOTE Throughout this document, the symbol indicates that the relevant information is not available at present, but will be included in a Study specific supplement. EFFECTIVE FROM: 29 August 2001 Test substance POSF, perfluorooctane sulfonyl fluoride has the following formula: C8F 17SO2F. Appearance Clear liquid Storage Ambient temperature Reagents Air In House Compressed Ethylene Sponsor Calibration gas (certified 2%) Nitrogen Sponsor Calibration gas (certified >99.9995%) Liquid nitrogen BOC Cooling Medium : 79 : Administration of POSF by inhalation to rats APPENDIX A MIN 312/014272 (Methods of sample collection and analysis for POSF - continued) Equipment Balance and Data printer Sartorius R160P with YDP-01 Flow meter Gilmont Instruments 0-4 Lpm FT-IR Midac Corporation I2001 series (serial no. 151) Computer Compaq LTE5400 Sotware Midac Corporation AutoQuant Version 3.01 Midac Grams 32 Version 4.11 Chamber ADG 3 level snout only Syringe Driver Precidor Type 5003 UV Quartz Cells Midac Corporation ZnSe Gas Cell General laboratory glassware Accessories also employed are: a vacuum pump; cylinder regulators, power connectors; regulated exhaust pumps. Consumables Syringes Aldrich 50 ml polypropylene Method of sample extraction A volume of POSF is dispensed from the test drum into a 50ml syringe. The syringe driver is set to give a concentration of 300ppm in an airstream of 30 l/minute airflow, mixed thoroughly in the vapouriser and fed directly into the chamber. The test atmosphere is drawn along the sample line at a flow rate of 1 l/minute directly through the IR cell. Production of a Background Scan A flowrate of ll/minute ultra-pure nitrogen (cylinder No. C0C7700219 (nitrogen >99.9995 2%) is drawn though the IR cell. After a period of 3-5 minutes, an FT-IR single beam spectrum is generated approximately every 2 seconds and continuously collected for 32 scans to produce a more definitive spectrum. : 80 : Administration of POSF by inhalation to rats APPENDIX A MIN 312/014272 (Methods of sample collection and analysis for POSF - continued) Storage of standards and samples Stability experiments were not required for on-line analysis. Calibration and Quantification For each sample measurement, the absorbance determines the amount present in the sample using the equation below (Beer's Law): Where A a b Amount (p p m ) = ------axb absorbance at a given frequency of POSF in the sample spectrum absorption coefficient (absorptivity) of POSF in the sample spectrum, which is derived from the reference calibration transfer standard spectrum path length of the cell derived from calibration data Calibration of Standards Calibration is performed daily before each exposure (see "MIN/312 Operating Procedure"). A certified concentration of ethylene (cylinder No. C0C9700874 (ethylene = 2020ppm 2%) is passed through the sampling system prior to sample collection to check instrument response and determine the calibrated cell path-length. Cell path-length is a critical input to the analytical method used to perform each spectral analysis. The ethylene value is read from the test certificate on the calibration cylinder. A test atmosphere of ethylene at a flowrate of 1 l/minute is drawn through the IR cell. After a period of 3-5 minutes, a FT-IR single beam spectrum is generated approximately every 2 seconds and continuously collected for 32 scans to produce a less noisy spectrum. This produces a calibrated path length. This value is then entered into the POSF method file and manually recorded. QC Path length Limits: -The calibration will be repeated by the user if no peak is present in the 1400-650 cm'1 region or if the path length value is outside the range 0.100 to 0.115 m. If the calibration still does not conform, all of the calibration set up parameters will be checked. QA Instrument Calibration Check: The calibration is reverified and subsequently recorded at the end of the exposure. Instrument calibration checks are within 5%. Repetition of two four hour periods showed that the final calibration gave the path length values differed by less than 2% from the initial calibration value of the ethylene cylinder (cylinder No. C0C9700874 (ethylene = 2020ppm 2%). The 2% value is inside the 5% tolerance limits allowed as quality check standards. The temperature of the test gas stream and the barometric pressure will be monitored manually. : 81 : Administration of POSF by inhalation to rats APPENDIX A MIN 312/014272 (Methods of sample collection and analysis for POSF - continued) FT-IR analyser specification The Midac I2000 series FT-IR spectrophotometer has a wavelength accuracy of 0.5cm 1. A MCT detector monitors the absorbance of various chemical bonds. AutoQuant version 3.01 (MIDAC, Irvine, CA) software is used for all infrared data acquisition and quantification. The length of the IR cell or path length (approximately 10 cm) dictates the concentration range that will give a linear relationship between absorbance and concentration. The optimum flow rate for the IR cell is 1 l/minute, which is set by a flow meter and all the analysis is conducted at that flow rate to avoid differences of cell pressure. Further details can be obtained from the operating procedure stored proximal to the FT-IR system. The analyser draws 1 l/minute from the gas stream and the remainder is returned to the exposure system exhaust. All exhaust lines are monitored by precision stainless steel and glass in-line rotameters. Raw Data Raw data is defined as the first hard copy output obtained immediately following data acquisition by the FT-IR system. This will be signed and dated immediately after printing. An electronic backup copy of this is saved to the local zip drive. If a run time hard copy was not obtained through a printer fault, the electronic copy from the zip drive directory may be used to generate a hard copy, which will be signed and dated. Exposure data is stored in the file format YYMMDD.txt. Computer hardware: Computers and VDUs are maintained by the Sponsor. Printer is maintained by the IT department. FT-IR System hardware: The Sponsor has supplied the hardware. Arrangements for servicing and maintenance would be made in consultation with the Sponsor. Software: Documentation is the responsibility of the Sponsor. Safety The COSHH assessment provides details regarding the toxicity of the test material POSF. It is considered that the standard handling procedures used within the department are suitable to prevent exposure to the test material. In case of a total power failure, switch off the FT-IR and test compound generation equipment immediately. : 82 : Administration of POSF by inhalation to rats APPENDIX A MIN 312/014272 (Methods of sample collection and analysis for POSF - continued) Summary of method validation The raw data for the method validation is located in study MIN/312. Comparison of test blanks and test samples showed that the analyte was well resolved from any potential interfering peak. Repeatability The variability of the POSF level in a dynamic (exposure) system is determined by the airflow and feed rate through the syringe driver being set correctly and checked every 30 minutes (as the flow is pressure dependent). Statistical analysis shows that, with these checks in place, over a four hour exposure in a 3-level ADG snout only exposure system, the Coefficient of variation is typically less than 7.0 at 300 ppm. This figure includes the variability of generation as well as the repeatability of measurement. Specificity This Fourier Transform infrared technique monitors the absorbance of POSF in the region 1400-650 cm-1, specifically between 1400 and 1000 cm-1. Linearity Linear regression analysis of the FT-IR spectrophotometer response to standards was conducted during preliminary trials. This was used to demonstrate that the FT-IR response is linear over the concentration range. Feed rates were selected to achieve a nominal concentration of 240 to 360 ppm (see data file 010912.txt, 010912a.txt and reference sequences 010912 MIN312 and 010912a MIN312) utilising a total extract flow rate of 30 l/minute. Least squares regression analysis with a unweighted linear regression of the predicted concentration from the nominal against FT-IR analysed concentration (240 to 360 ppm) produced a correlation coefficient of 0.999301 and relative errors less than 1.0% in the range 360 to 240 ppm. The Limit of Quantification (LOQ) for POSF will be set by the lowest acceptable nominal feed rate, however, the LOQ and Limit of Detection (LOD) are potentially as low as 12.17 and 4.01 ppm respectively (calculated statistically using the standard deviation obtained for a nominal feed rate to obtain a concentration of 240 ppm). Uncertainty of POSF analysis Uncertainties were said (by the Sponsor) to be less than 5% for the AutoQuant method calculations. The POSF reference spectrum is from the 3M quantitative library and was generated at 3M using EPA guidelines. Other principal uncertainties were: ethylene path length Calibration (2%); interference from alkyl sufonyl fluorides (<4.5% v/v, but interference unknown). : 83 : Administration of POSF by inhalation to rats APPENDIX A MIN 312/014272 (Methods of sample collection and analysis for POSF - continued) MIN/312 - STUDY SPECIFIC SUPPLEMENT TO THE INHALATION ANALYTICAL PROCEDURE FOR POSF (PERFLUORO-OCTANE SULFONYL FLUORIDE) This supplement details additions and amendments to the procedure to be used for the FT-IR assay of POSF obtained from air samples collected on the above study. The assay, incorporating the additions and amendments, is suitable for the analysis of POSF, in air, at concentrations within the range of 240 to 360ppm. Details given in this supplement supersede those in the compound specific IAP. EFFECTIVE DATE : 4 September 2001 Analytical standard Name Batch number Purity Expiry date Supplier FX-8B, POSF, T-7661.1, Perfluoro-octane sulfonyl fluoride 040227 >95.5% (Perfluoroalkyl sulfonylfluorides <4.5%) Not Supplied Sponsor Fourier Transform - Infrared Spectrophotometer The analysis is performed using a FT-IR analyser supplied by the sponsor. Preparation of standard solutions Provide nominal feed rates to the chamber in the range 240 to 360 ppm. Calibration and Quantification Calibration of the pathlength by an ethylene standard (cylinder No. C0C9700874 (ethylene = 2020ppm 2%). Quantification using the 3M quantitative library generated at 3M using EPA guidelines for generating reference spectra (v). : 84 : Administration of POSF by inhalation to rats APPENDIX B Individual POSF concentration measurements MIN 312/014272 Exposure 1 Date and Time 13/09/01 09:24 Concentration (ppm) Error 272.86 0.71 13/09/01 09:54 345.50 0.88 13/09/01 10:24 323.61 0.85 13/09/01 10:54 315.21 0.83 13/09/01 11:24 316.94 0.83 13/09/01 11:54 310.42 0.84 13/09/01 12:24 312.39 0.84 13/09/01 12:54 316.65 0.86 13/09/01 13:24 318.48 0.87 13/09/01 13:54 316.82 0.87 13/09/01 14:24 322.98 0.88 13/09/01 14:54 320.68 0.88 Mean 316.04 0.85 sd 16.272 0.047 CV (%) 5.1 5.5 2 14/09/01 09:25 276.10 0.72 14/09/01 09:55 320.49 0.81 14/09/01 10:25 321.14 0.82 14/09/01 10:55 314.85 0.81 14/09/01 11:25 310.00 0.81 14/09/01 11:55 312.98 0.83 14/09/01 12:25 317.30 0.85 14/09/01 12:55 315.09 0.85 14/09/01 13:25 321.14 0.87 14/09/01 13:55 315.14 0.86 14/09/01 14:25 319.44 0.87 14/09/01 14:55 320.64 0.87 Mean 313.69 0.83 sd 12.375 0.043 CV (%) 3.9 5.1 sd standard deviation CV Coefficient of variation (sd x 100/mean) Room Air (ppm) 0.171 0.245 0.309 0.365 0.402 0.413 0.445 0.464 0.475 0.488 0.497 0.5 0.398 0.1066 26.8 0.166 0.233 0.297 0.332 0.357 0.405 0.419 0.439 0.448 0.463 0.457 0.465 0.373 0.0986 26.4 Error 0.003 0.003 0.003 0.003 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.005 0.004 0.0007 17.6 0.003 0.003 0.003 0.003 0.003 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.0005 15.0 Concentration Room Air (ppm) 272.69 345.26 323.31 314.85 316.53 310.01 311.95 316.19 318.00 316.33 322.48 320.18 315.65 16.240 5.1 275.93 320.25 320.84 314.52 309.65 312.58 316.88 314.65 320.69 314.68 318.99 320.18 313.320 12.3137 3.9 : 85 Administration of POSF by inhalation to rats APPENDIX B MIN 312/014272 (Individual POSF concentration measurements - continued) Exposure a 3 Date and Time 15/09/01 07:55 15/09/01 07:59 15/09/01 08:02 15/09/01 08:06 15/09/01 08:10 Concentration (ppm) c c c c c Error 0.18 0.18 0.19 0.2 0.03 Room Air (ppm) 0.208 0.224 0.233 0.245 0.005 Error 0.002 0.002 0.002 0.002 0.000 Concentration Room Air (ppm) 15/09/01 08:13 15/09/01 08:15 0.273 c 0.07 c 0.65 0.012 0.021 0.000 0.000 15/09/01 08:17 318.76 0.75 0.077 0.001 318.69 15/09/01 08:25 350.94 0.82 0.098 0.001 350.84 15/09/01 08:55 319.95 0.78 0.18 0.002 319.77 15/09/01 09:25 318.60 0.79 0.233 0.002 318.36 15/09/01 09:55 323.86 0.82 0.284 0.003 323.58 15/09/01 10:25 327.15 0.84 0.314 0.003 326.84 15/09/01 10:55 318.38 0.84 0.345 0.003 318.04 15/09/01 11:25 318.32 0.83 0.359 0.003 317.96 15/09/01 11:55 321.12 0.85 0.375 0.003 320.75 15/09/01 12:25 321.74 0.85 0.379 0.003 321.36 15/09/01 12:58 322.46 0.86 0.404 0.004 322.05 15/09/01 13:28 318.70 0.86 0.417 0.004 318.29 Mean 323.33 0.82 0.289 0.003 323.04 sd 9.101 0.034 0.117 0.0009 9.154 CV (%) 2.8 4.2 40.5 35.1 2.8 5 17/09/01 09:07 259.59 0.68 0.189 0.003 259.40 17/09/01 09:37 329.96 0.83 0.262 0.003 329.70 17/09/01 10:07 333.40 0.84 0.333 0.004 333.06 17/09/01 10:37 330.72 0.85 0.387 0.004 330.33 17/09/01 11:07 322.77 0.81 0.303 0.003 322.46 17/09/01 11:37 316.42 0.82 0.458 0.004 315.96 17/09/01 12:07 313.36 0.83 0.492 0.004 312.87 17/09/01 12:37 313.05 0.84 0.508 0.005 312.54 17/09/01 13:07 312.63 0.85 0.528 0.005 312.11 17/09/01 13:37 316.11 0.86 0.544 0.005 315.57 17/09/01 14:07 315.02 0.87 0.561 0.005 314.46 17/09/01 14:37 313.82 0.87 0.570 0.005 313.25 Mean 314.74 0.83 0.428 0.004 314.31 sd 18.963 0.051 0.1292 0.0008 18.931 CV (%) 6.0 6.1 30.2 19.5 6.0 sd standard deviation CV Coefficient of variation (sd x 100/mean) No data collected for exposure 4 due to computer problem with data capture program Problem with data capture value excluded from mean and standard deviation : 86 : Administration of POSF by inhalation to rats PROTOCOL AND PROTOCOL AMENDMENTS MIN 312/014272 Study N um ber : M IN/312 CONFIDENTIAL Huntingdon Life Sciences PROTOCOL P E R F L U O R O O C T A N E S U L F O N Y L F L U O R ID E (P O S F ; T -7661.1) PR E LIM IN A R Y T O X IC IT Y STUDY BY IN H A LA TIO N A D M IN IST R A T IO N TO CD RATS FO R 1 W EEK Sponsor 3M Center 3M Corporate Toxicology Building 220-2E-02 St Paul MN 55133-3220 USA Research Laboratory Huntingdon Life Sciences Ltd Woolley Road Alconbury Huntingdon Cambridgeshire PE284HS ENGLAND Total number of pages: 20 Final Protocol Huntingdon Life Sciences Ltd, registered in England: 815730 Page / : 87 : MIN 312/014272 Study Number : MIN/312 CONTACT DETAILS Huntingdon Life Sciences Sponsor's Monitoring Scientist John Butenhoff Final Protocol : 88 : Page U Study Number : MIN/312 MIN 312/014272 Huntingdon Life Sciences PROTOCOL APPROVAL PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK T.J. Kenny, B.Sc (Hons.). Study D irector, H untingdon L ife Sciences Ltd. D ate The signature o f the Study D irector confirm s this protocol as the w orking docum ent for the study. A ny changes m ade subsequent to the date o f the Study D irector's signature w ill be docum ented in formal amendments. S C.J. H ardy, B.Sc., Ph.D ., M L B iol., C.Biol., Dip. R. C. Path. (Toxicology) M anagement, Huntingdon Life Sciences Ltd. D ate joim B utennon Sponsor, D ate Please sign both copies o f this page, retain one fo r your records and return one to the Study D irector a t Huntingdon Life Sciences. Final Protocol Page Hi : 89 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK E nquiry N um ber: 23923A Number of pages for internal distribution: 17 This workiwg docum ent is approved for circulation and use: Study Director" D ate Primary location of study H untingdon R esearch Centre H untin g d o n C am b rid g esh ire PE28 4HS Building Number: All procedures to be perform ed at the above site unless otherwise detailed below. Location of specific tasks H isto lo g y : Routinely done at Huntingdon Research Centre, Huntingdon, Cambridgeshire, PE28 4HS, but m ay be perform ed at Eye Research Centre, Eye, Suffolk, IP23 7PX for logistical reasons. SEM X-ray analysis Test substance/metabolite analysis : University o f Plymouth. : 3M. Final Protocol Page 1 90 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences CONTENTS 1. INTRODUCTION 2. STUDY SCHEDUUE AND STRUCTURE 2.1. Duration of treatment 2.2. Scheduled time plan 2.3. Identity of treatment groups 3. TEST SUBSTANCE AND ATMOSPHERE GENERATION 3.1. Test substance 4. ANIMAL MANAGEMENT 4.1. Animals - supply, acclimatisation and allocation 4.2. Animals - housing, diet and water supply 5. INHALATION PROCEDURES 5.1. Pre-study characterisation 5.2. Test atmosphere generation 5.3. Test atmosphere administration 5.4. Test atmosphere monitoring 6. SERIAL OBSERVATIONS 6.1. Clinical observations 6.2. Mortality 6.3. Bodyweight 6.4. Food consumption 6.5. Water consumption 7. NECROPSY AND HISTOLOGY 7.1. Pre-terminal urine sampling 7.2. Test substance/metabolite analyses 7.3. Method of kill " 7.4. Macroscopic Pathology 7.5. Organ weights 7.6. Fixation 7.7. Histology 7.8. PCNA staining for cell proliferation (Table 1) 8. PATHOLOGY 8.1. Light microscopy 8.2. Extension of initial examination 9. DATA TREATMENT 9.1. Statistical analysis 10. REPORTING 11. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 11.1. Quality Assurance 11.2. Archiving Page 3 4 4 4 5 5 6 6 6 7 8 8 9 9 10 10 10 11 11 11 11 12 12 12 12 12 13 13 13 13 15 15 15 15 15 16 16 16 17 Final Protocol Page 2 : 91 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 1. IN T R O D U C T IO N M anagem ent of study Study Director Monitoring Toxicologist In the temporary absence of the Study Director, the scientific responsibilities will be taken over by the Monitoring Toxicologist; other items of routine study management should be referred to the following person in the first instance. T.J. Kenny D.W. Coombs R. Davies O b jectiv e Assessment of systemic toxic potential in a 1 week inhalation study in CD Rats. Good L aboratory Practice The study will be conducted in compliance with principles of Good Laboratory Practice Standards as set forth in: The UK Good Laboratory Practice Regulations 1999 (Statutory Instrument No 3106). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. EC Commission Directive 1999/11/EC of 8 March 1999 (Official Journal No L 77/8). A nim als (Scientific Procedures) A ct 1986 compliance The in-life experimental procedures to be undertaken during the course of this study are subject to the provisions of the United Kingdom Animals (Scientific Procedures) Act 1986 (the Act). The Act, administered by the UK Home Office, regulates all scientific procedures in living animals which may cause pain, suffering, distress or lasting harm and provides for the designation of establishments where procedures may be undertaken, the licensing of trained individuals who perform the practical techniques and the issue of project licences for specified programmes of work. This study will comply with all applicable sections of the Act and the associated Codes of Practice for the Housing and Care of Animals used in Scientific Procedures and the Humane Killing of Animals under Schedule 1 to the Act, issued under section 21 of the Act. The number of animals used will be the minimum that is consistent with scientific integrity and regulatory acceptability, consideration having been given to the welfare of individual animals in terms of the number and extent of procedures to be carried out on each animal. Final Protocol Page 3 : 92 : MIN 312/014272 : 93 : MIN 312/014272 : 94 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 3.1. T est substance Sponsor's identification Perfluorooctanesulfonyl Fluoride (POSF) FX-8B, Lot 040277. Storage conditions At ambient temperature or in a refrigerator, unless otherwise directed by the Sponsor. Any such decision will be documented in the study data and included in the final report. Sponsor's responsibilities Documentation of methods of synthesis, fabrication or derivation. Stability data. Certificate of analysis. 4. A N IM A L M A N A G E M E N T 4.1. A nim als - supply, acclim atisation and allocation 4.1.1. Animals Species Strain Age ordered Weight range ordered Supplier Rat. Crl:CD BR. 42 2 days. To be within an 11 g range for each sex. Charles River (UK) Limited. 4.1.2. A cclim atisation Duration Husbandry conditions : At least 7 days before commencement of treatment. : Refer to Section 4.2. 4.1.3. A llocation to treatm ent groups Allocation Method Cage distribution 4.1.4. Identification : On arrival. : Random. : To equalise environmental influences between groups and to minimise the likelihood of cross contamination between groups. Numbering Method Cage labels Unique for each animal within study. Tail tattoo. Uniquely identifying the occupants. Final Protocol Page 6 : 95 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 4.1.5. Anim al replacem ent 10 spare animals will be ordered to replace any individuals rejected during the acclimatisation period. Replacement before treatment : Ill-health. Abnormalities. Bodyweight range extremes. Replacement during treatment : None scheduled. 4.2. A nim als - housing, diet and w ater supply 4.2.1. E nvironm ental control Rodent facility Restricted entry - to minimise entry of external biological and chemical agents. Air supply Filtered, not recirculated. Temperature Maintained within the range of 19-23C. Relative humidity Maintained within the range of 40-70%. Monitored continuously or daily. Excursions outside these ranges documented in the study data. Lighting Alarm systems 12 hours light : 12 hours dark. Activated on ventilation failure and when temperature/humidity limits exceeded. Electricity supply Public supply with automatic stand-by generators. 4.2.2. Anim al accom m odation Animals per cage Cage material Cage flooring Five of the same sex, unless reduced by mortality or isolation. Polycarbonate or stainless steel. Stainless steel grid. The cages will be suspended above absorbent paper. The latter will be changed at appropriate intervals each week; cages, cage-trays, food hoppers and water bottles will be changed at appropriate intervals. Precise details of caging will be included in the final report. Final Protocol Page 7 : 96 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 4.2.3. Diet and w ater supply Copies of all certificates of analysis are stored in the archives. Diet supply Diet name : Rat and Mouse No. 1Maintenance Diet. Fish meal is not present in this diet. Diet type Availability : Pelleted diet. : Non-restricted. Certification : Before delivery each batch of diet is analysed by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier's analytical certificates are scrutinised and approved before any batch of diet is released for use. This diet contains no added antibiotic or other chemotherapeutic or prophylactic agent. W ater supply Supply Regulatory agency Availability Public drinking water. U.K. Department of the Environment. Non-restricted via polyethylene or polycarbonate bottles with sipper tubes. Certification 4.2.4. Contam inants assay Certificates of analysis are routinely received from the supplier. It is the Sponsor's responsibility to advise Huntingdon Life Sciences of any specific contaminants likely to prejudice the outcome of the study. Analyses for such contaminants may be performed if requested by the Sponsor. (PFOS, a metabolite of POSF may be present in fish meal used in some animal diets). 5. IN H A LA TIO N PR O C ED U R ES 5.1. P re-study characterisation Before commencement of treatment the system will be characterised at the target exposure vapour concentrations without animals in order to: demonstrate reproducibility of vapour concentration. demonstrate homogeneity of vapour concentration and distribution between levels in the chamber. Final Protocol Page 8 : 97 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 5.2. T est atm osphere generation Equipment Method All glass vaporiser. The test substance (liquid) will be metered to the vaporiser (through which dried air is passed) from an infusion pump. The vapour/air mixture produced passes directly into the exposure chamber. Concentrations : Different concentrations of the test substance will be produced using different liquid feed rates controlled by the infusion pump and using syringes of an appropriate volume. 5.3. T est atm osphere adm inistration Route Exposure system : Inhalation -snout only exposure. : ADG exposure chamber, modular construction in aluminium alloy comprising a base unit, a variable number of sections each having 20 exposure ports, and a top section incorporating a central aerosol inlet with a tangential air inlet. A separate chamber will be used for each group. During exposure, the rats will be held in restraining tubes with their snouts protruding from the ends of the tubes into the exposure chambers. The restraining tubes will be attached to chamber level 2. Animal exposure ports not in use will be closed with blanking plugs. Each exposure system will be housed in a separate extract cabinet to avoid possible cross-contamination between groups. Controls (Group 1) Sham dosing Air only. The animals on study will be acclimated to the method of restraint, normally over a 5 day period immediately preceding the first test substance exposure. Duration of daily exposure 6 hours, for 5 consecutive days. Airflow 30 1/minute. Final Protocol : 98 : Page 9 MIN 312/014272 Study Number MIN/312 Huntingdon Life Sciences 5.4. T est atm osphere m onitoring Equipment : Fourier-transformed infrared spectroscopy (FTIR). Supplied by Sponsor. 5.4.1. Test atm osphere sam pling S am pling : Samples of the test atmosphere will be drawn from each test chamber through the FTIR instrument. 5.4.2. Test atm osphere analysis C o ncentration Chemical analysis: m eth o d o lo g y FTIR (at frequent intervals to demonstrate adequate control of temporal variation). An FTIR analyser, with suitable methodology will be supplied by the Sponsor and this will be validated at Huntingdon Fife Sciences. This methodology will be followed in principle, although specific conditions may be modified at the discretion of the Head of Section, Aerosol Technology and Analysis. SERIAL OBSERVATIONS The precise times of all examinations will be included in the final report. 6.1. Clinical observations Animals and their cages Inspected at least twice daily for evidence of reaction to treatment or ill-health. Deviations from normal recorded at the time in respect of Nature and severity. Date and time of onset. Duration and progress of the observed condition. Physical examination Once each week for all animals. In addition, detailed observations will be made daily, on days of exposure according to the following frequency: 1. Pre-exposure observation. 2. Observation severely restricted due to tube restraint. 3. As each animal is returned to its home cage. 4. As late as possible in the working day. The above schedule will be amended, as necessary, in the light of signs observed. Final Protocol Page 10 : 99 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 6.2. M ortality Debilitated animals Observed carefully, may be isolated to prevent cannibalism. Premature sacrifice Animals may be killed on humane grounds or if considered in extrem is. Animals found dead, killed in e xtre m is or on humane grounds A necropsy is performed as soon as possible. Animals found outside the normal workday will be preserved in a refrigerator (approximately 4C) provided for this purpose. 6.3. Bodyw eight Bodyweight recording : Week-1. Day that treatment commences. Daily. At necropsy. More frequent weighing may be performed to aid the monitoring of the condition of animals displaying ill-health. These data will be retained in the archives. 6.4. Food consum ption Food consumption recording : W eek-1. Daily. Food supplied Food spilled Food remaining : Daily. : Recorded at cage cleaning. : Recorded daily. W ater consum ption Water consumption recording : Week-1. Daily. Water supplied Water remaining : Daily. : Recorded daily. Final Protocol Page 11 100 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 7. N E C R O P S Y AND H IS T O L O G Y 7.1. P re-term inal urine sam pling Individual urine samples will be collected within 2 hours following lights on in the animal holding room. The urine samples will be immediately assayed for pH using a microelectrode, centrifuged to obtain the sediment and the prepared SEM stubs despatched to the Principal Investigator at Roy Moate University of Plymouth for calculi and crystal analysis by SEM Xray element identification. If fresh void urine is not obtained from a proportion of the animals within the time allowed, then a sample will be removed from the urinary bladder at necropsy. 7.2. T est substance/m etabolite analyses Samples of blood (for serum) will be taken at necropsy by cardiac/aorta puncture, and run into tubes allowed, to clot at room temperature (up to 4 ml), and the serum separated and frozen prior to despatch to the Sponsor. The frozen samples will be despatched to the Sponsor's Principal Investigator for analysis. Principal Investigator : Lisa Clemen. Address : 3M Environmental Laboratory 935 Bush Avenue, Building 2-3E-09 St. Paul, Minnesota 55133-3331, USA Tel No. : 651-778-6018 (0) 651-778-6176 (F). Any further processing and analysis of the samples and data collation will be the responsibility of the Sponsor. A copy of the Sponsor's report will be included as an addendum to the study report. All raw data relevant to the analyses of test substance and metabolites in plasma will be retained by the Sponsor. 7.3. M ethod of kill Method Intra-peritoneal sodium pentobarbitone. Followed by exsanguination. Sequence To allow satisfactory inter-group comparison. 7.4. M acroscopic Pathology (Table 1) Complete Checks Photography All animals. Retained tissues. Unusual or suspected treatment-related findings; at the discretion of the necropsy supervisor or Study Director. Final Protocol Page 12 : 101 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 7.5. O rgan w eights (Table 1) Data collection Data presentation For bilateral organs, left and right organs will be weighed together unless otherwise specified on the Pathology Procedures Table. ^ Organ weights are not routinely recorded for animals killed or dying prematurely. Absolute. Adjusted for terminal bodyweight. 7.6. Fixation (Table 1) Standard Others Fresh frozen liver 7.7. H istology (Table 1 and Section 8.1) Processing Routine staining Special staining Zinc Formalin. Eyes: In Davidson's fluid. Urinary bladder: Bouins. Inflate lungs with fixative and slightly distend the urinary bladder with fixative. At terminal kill and after organ weighing, a sample of liver will be removed from all animals, immediately frozen by immersion in liquid nitrogen and stored at -70C prior to despatch to the Sponsor. All animals killed or dying prematurely. All terminal animals. Urinary bladder: section sagittally, using one half for SEM Xray microprobe analysis, and the other half for light microscopy and cell proliferation index. Kidney pelvis: sections to be present for light microscopy. 4-5 pm sections stained with haematoxylin and eosin. None. 7.8. P C N A sta in in g fo r cell p ro life ra tio n (T ab le 1) Sections of urinary bladder will be taken from all animals for PCNA immunostaining in order to assess the degree of cell proliferation present. A total of 3,000 cells will be counted from 3 separate sections (1,000 cells/section) in order to determine the cell proliferation index. Positive PCNA staining cells will be counted, and the examining pathologist will assess the sections and count S-phase positive cells, if practicable. In addition sections of the duodenum from each animal will be taken and stained to act as positive controls for the immunostaining methodology. Final Protocol Page 13 102 MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences TABLE 1-Pathology procedures Tissue and regions to be examined Abnormalities Adrenals Aorta - thoracic Brain Caecum Colon Duodenum Epididymides Eyes Femur (longitudinal section through ioinfl Harderian glands Head Heart Ileum Jejunum Kidneys Lachrymal glands Larynx (2 levels) Liver Lungs (section from all lobes including bronchi) Lymph nodes - mandibular - mesenteric - tracheobronchial Mammary area - caudal Nasal turbinates (3 levels) Oesophagus Optic nerves Ovaries Pancreas Pituitary Prostate Rectum Salivary glands (submandibular/sublingual) Sciatic nerves Seminal vesicles Skeletal muscle - thigh Skin Spinal cord (transverse and longitudinal sections at the cervical, lumbar and thoracic levels) Spleen Sternum Stomach Testes Thymus Thyroid with parathyroids Tongue Trachea (2 levels) Urinary bladder (with kidney pelvis) Uterus with cervix Vagina Necropsy Weigh Fix * ** * * * * * * * * * a) * * * ** * * ** ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Histology Pathology Light microscopy ** a) Including nasal cavity, paranasal sinuses and nasopharynx. * Organs weighed, samples fixed or sections examined microscopically. PCNA * * Final Protocol Page 14 103 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 8. P A T H O L O G Y 8.1. L ight m icroscopy C ategory Premature deaths Terminal sacrifice A nim als All from all groups. All animals. Tissues All specified in Table 1. All specified in Table 1. Peer Review : Carried out by a reviewing pathologist to internationally accepted standards. 8.2. Extension of initial exam ination At the discretion of the pathologist, further processing and staining techniques may be used to evaluate individual lesions. Details of these techniques will be documented and retained in the archives. Light microscopy may be extended, following consultation with the Sponsor, as follows: Any such requirement will be documented in an amendment to the protocol. SEM of urinary bladder epithelium for assessment of possible necrosis. 9. DATA T R EA TM EN T 9.1. Statistical analysis D ata-types The following data types will be analysed at each timepoint separately:- bodyweight, food consumption and water consumption, over appropriate study periods, organ weights, both absolute and adjusted for terminal bodyweight where appropriate, pathological findings, for the number of animals with and without each finding. M ethods For categorical data, the proportion of animals will be analysed using Fisher's Exact test for each treated group versus the control. For continuous data, Bartlett's test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett's test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary. Final Protocol Page 15 104 MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 10. R E P O R T IN G Study progress Draft final report Authorised final report Periodic verbal and written updates on study progress will be provided by the Study Director. Routine synopsis reports will be sent after 1 week and at termination of the in-life phase. For review by the Sponsor. After approval from the Sponsor. Routinely reports are supplied on A4 paper. The following numbers of reports are supplied. Type of report Printing Draft report Authorised final Photographic report (if any) Double-sided Double-sided Single-sided Single-sided N um ber of copies Bound Unbound 02 10 01 10 Any additions or corrections to an authorised final report will be documented as a formal addendum/amendment to the final report. In the absence of ongoing communications, Huntingdon Life Sciences reserves the right to finalise, sign and issue the final report from this study six months after issue of the draft. In such an event, all materials will be transferred to the archive. Any subsequent requests for modifications, corrections or additions to the final report will be the subject of a formal report amendment (or new study, as appropriate) and will be subject to additional cost. : 105 : MIN 312/014272 Study Number : MIN/312 Huntingdon Life Sciences 11.2. A rchiving All raw data, samples and specimens arising from the performance of this study will remain the property of the Sponsor. Types of sample and specimen which are unsuitable, by reason of instability, for long term retention and archiving may be disposed of after the periods stated in Huntingdon Life Sciences Standard Operating Procedures. All other samples and specimens and all raw data will be retained by Huntingdon Life Sciences in its archive for a period of five years from the date on which the Study Director signs the final report. After such time, the Sponsor will be contacted and his advice sought on the return, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will continue to retain the materials subject to a reasonable fee being agreed with the Sponsor. Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy of the final report in its archive indefinitely. Final Protocol 106 Page 17 Study Number : MIN/312 Protocol Amendment Number : 1 MIN 312/014272 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK Total number of pages: 4 Number of pages for internal distribution: 4 Study Director T.J. Kenny, B.Sc (Hons.). The signature o f the Study D irector authorises the im plem entation o f this am endm ent to protocol. In this amendment, deleted statements are struck through and new statements are underlined. Any changes to the study design after the date o f this authorising signature w ill be docum ented in a further formal amendment. AMENDMENT APPROVAL For Huntingdon Life Sciences Ltd Authorised by: (Study Director) ( , For the Sponsor Approved by: Q d ^ 2^ D ate: Z H / h s \ \ tn h 'Tft ) Date:. ? -7 /V *^> r Z - # o i Page 1 : 107 : Study Number : MIN/312 Protocol Amendment Number : 1 MIN 312/014272 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK Reasons for amendments To include building num ber w here study is to take place. To update the timeplan. To include hazard class. To clarify necropsy table as to which tissues to process. Amendments Primary location of study H untingdon Research Centre H untingdon C am b rid g esh ire PE284HS Building Number: Y14 2.2. Scheduled time plan Animals to arrive Treatm ent to commence Terminal sacrifice to commence H istopathology to be completed Draft report to be issued : 5 September 2001 : T3 S eptem ber 2001 : 18 Septem ber 2001 : October Novem ber 2001 (estim ated) : November 2001 (estim ated) Page 2 108 : MIN 312/014272 Study Number : MIN/312 Protocol Amendment Number : 1 Huntingdon Life Sciences 3. TEST SUBSTANCE AND ATMOSPHERE GENERATION In order for Huntingdon Life Sciences to com ply with the Health and Safety at W ork etc. A ct 1974, and the Control o f Substances Hazardous to H ealth Regulations 1999, it is a condition o f undertaking the study that the Sponsor shall provide Huntingdon Life Sciences w ith all inform ation available to it regarding known or potential hazards associated with the handling and use o f any substance supplied by the Sponsor to H untingdon Life Sciences. The Sponsor shall also com ply w ith all current legislation and regulations concerning shipm ent o f substances by road, rail, sea or air. Such inform ation in the form o f a com pleted H untingdon Life Sciences test substance data sheet m ust be received by Safety M anagem ent Services at Huntingdon Life Sciences before the test substance can be handled in the laboratory. A t the discretion o f Safety M anagem ent Services at H untingdon Life Sciences, other docum entation containing the equivalent inform ation m ay be acceptable. Inform ation received w ill be used to set the Huntingdon Life Sciences H azard Class, w hich determ ines safety precautions taken in the workplace. H untingdon Life Sciences Hazard Class: : 109 : Page 3 MIN 312/014272 : 110 : Study Number : MIN/312 Protocol Amendment Number : 2 MIN 312/014272 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK Total number of pages: 4 Number of pages for internal distribution: 4 Study Director T J. Kenny, B.Sc (Hons.). The signature o f the Study D irector authorises the im plem entation o f this am endm ent to protocol. In this am endm ent, deleted statements are struck through and new statem ents are underlined. A ny changes to the study design after the date o f this authorising signature w ill be docum ented in a further formal amendment. AMENDMENT APPROVAL For Huntingdon Authorised b (Study Dir For the Sponsor Approved by: j A ^ I Datera 1( Ju= /~ Q efti Dat e: l [ S p ] r 2 &Q I Page 1 111 Study Number : MIN/312 Protocol Amendment Number : 2 MIN 312/014272 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK Reasons for amendments : Clarification o f nam e and address o f Principal Investigator for SEM X -ray analyses Change to fixative follow ing discussion with Sponsor. Addition/clarification o f QA and Archiving procedures. Amendments 7. NECROPSY AND HISTOLOGY 7.1. Pre-terminal urine sampling Individual urine sam ples w ill be collected within 2 hours follow ing lights on in the anim al holding room. The urine sam ples w ill be im m ediately assayed for pH using a m icroelectrode, centrifuged to obtain the sediment and the prepared SEM stubs despatched to the Principal Investigator at Roy M oate, at University o f Plym outh, Drake Circus. Plym outh, PL4 8AA, for calculi and crystal analysis by SEM X -ray elem ent identification. If fresh void urine is not obtained from a proportion o f the anim als w ithin the tim e allowed, then a sample w ill be rem oved from the urinary bladder at necropsy. A co p y o f th e P rin cip al In v estig ato r's report w ill be included as an addendum to th e study report. A ll raw data relevant to the analyses w ill be returned to Huntingdon Life Sciences for archiving. Page 2 : 112 : MIN 312/014272 Study Number : MIN/312 Protocol Amendment Number : 2 Huntingdon Life Sciences Fixation (T able 1) Standard O thers Fresh frozen liver : Zinc Formalin. 10% neutral buffered formalin : Eyes: In D avidson's fluid. U rinary bladder: Bouins. Inflate lungs w ith fixative (10% NBF1 and sliehtlv distend the urinary bladder w ith fixative (B ouin'sl. Testes and epididym ides: Initially in B ouin's fluid : A t term inal kill and after organ weighing, a sam ple o f liver w ill be rem oved from all animals, im m ediately frozen by im m ersion in liquid nitrogen and stored at -70C prior to despatch to the Sponsor. 11. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 11.1. Quality Assurance The follow ing w ill be inspected or audited in relation to this study. Protocol Audit Study based inspections Report Audit Authorised protocol and any amendments. Critical phases o f this study w ill be inspected. The draft report and study data w ill be audited after issue o f the draft report to the Sponsor. Test substance/metabolite assay This phase o f the study and associated reports w ill be conducted and produced according to local S O P 's and subject to th e S ponsor's ow n Q A procedures. SEM X-ray calculi and crystal analysis The facility undertaking the analysis w ill be inspected hv and the data and the Principal Investigator's report w ill be audited by H untingdon Life Sciences Q uality A ssurance D epartm ent QA findings w ill be reported to the Study D irector and Com pany M anagem ent prom ptly on com pletion o f each action, except for process based inspections, which w ill be reported to appropriate Com pany M anagem ent only. Page 3 113 MIN 312/014272 Study Number : MEV/312 Protocol Amendment Number : 2 Huntingdon Life Sciences 11.2. Archiving All raw data, samples and specimens arising from the perform ance o f this study w ill rem ain the property o f the Sponsor. Raw data, sam ples and specimens arising from the test substance/m etabolite assay conducted by the Sponsor w ill be archived by the Sponsor. Raw data, sam ples and specimens arising from the SEM X -rav calculi and crystal analysis will be archived by H untingdon Life Sciences. Types o f sam ple and specim en which are unsuitable, by reason o f instability, for long term retention and archiving m ay be disposed o f after the periods stated in Huntingdon Life Sciences Standard Operating Procedures. A ll other samples and specim ens and all raw data will be retained by H untingdon Life Sciences in its archive for a period o f five years from the date on w hich the Study D irector signs the final report. A fter such time, the Sponsor will be contacted and his advice sought on the return, disposal or further retention o f the materials. If requested, H untingdon Life Sciences w ill continue to retain the m aterials subject to a reasonable fee being agreed w ith the Sponsor. Huntingdon Life Sciences w ill retain the Q uality Assurance records relevant to this study and a copy o f the final report in its archive indefinitely. 114 : Page 4 MIN 312/014272 Study Number : MIN/312 Protocol Amendment Number : 3 H u n tin g d o n Life Sciences PERFLUO RO OCTANESULFO NYL FLUORIDE (POSF; T-7661.1) PRELIM INARY TOXICITY STUDY BY INHALATION ADM INISTRATION TO CD RATS FOR 1 W EEK Total number of pages: 3 Number of pages for internal distribution: 3 Study Director T.J. Kenny, B.Sc (Hons.). The signature o f the Study Director authorises the implementation o f this amendment to protocol. In this amendment, deleted statements are struck through and new statements are underlined. Any changes to the study design after the date o f this authorising signature will be documented in a further formal amendment. AMENDMENT APPROVAL For Huntingdon Authorised (Study Director) ces Ltd For the Sponsor Approved by: <^7^0**- 7. Date: V p Date: * ~1 (2C o 2 'Z07-' : 115 : Page 1 MIN 312/014272 Study Number : MIN/312 Protocol Amendment Number : 3 H u n tin g d o n Life Sciences PERFLUO RO OCTANESULFO NYL FLUORIDE (POSF; T-7661.1) PRELIM INARY TOXICITY STUDY BY INHALATION ADM INISTRATION TO CD RATS FOR 1 W EEK Reasons for amendm ents : The Sponsor has changed the Principal Investigator A m endm ents 116 : Page 2 MIN 312/014272 Study Number : MIN/312 Protocol Amendment Number : 3 H u n tin g d o n Life Sciences 7.2. T est substance/m etabolite analyses Samples o f blood (for serum) will be taken at necropsy by cardiac/aorta puncture, and run into tubes allowed, to clot at room temperature (up to 4 ml), and the serum separated and frozen prior to despatch to the Sponsor. The frozen samples will be despatched to the Sponsor's Principal Investigator for analysis. Principal Investigator : Lisa Clemen. Address : 3M Environmental Laboratory 935 Bush Avenue, Building 2-3E-09 St. Paul, M innesota 55133-3331, USA Tel No. : 651-778-6018(0) 651-778-6176 (F). Subsequent to this amendment the duties o f Principal Investigator will be transferred to: Principal Investigator Address Dr Richard Grazzini Exveen Research 3058 Research Drive State College PA 16801 USA Tel No. Fax No. 001 814 231 8032 Any further processing and analysis o f the samples and data collation will be the responsibility o f the Sponsor. A copy o f the Sponsor's report will be included as an addendum to the study report. All raw data relevant to the analyses of test substance and metabolites in plasma will be retained by the Sponsor. : 117 : Page 3 MIN 312/014272 : 118 : MIN 312/014272 Study Number : MIN/312 Protocol Amendment Number : 4 H u n tin g d o n Life Sciences PERFLUO RO OCTANESULFO NYL FLU O R ID E (POSF; T-7661.1) PRELIM INARY TOXICITY STUDY BY INHALATION ADM INISTRATION TO CD RATS FOR 1 W EEK Reasons for amendments : Assignment of liver samples for test substance/metabolite analyses Addition of analytical method details. Amendments 7.2. Test substance/metabolite analyses Samples of blood (for serum) will be taken at necropsy by cardiac/aorta puncture, and run into tubes allowed, to clot at room temperature (up to 4 ml), and the serum separated and Samples of liver will be collected at necropsy in accordance Section 7.6 of the Protocol and frozen prior to despatch to the Sponsor. The frozen samples will be despatched to the Sponsor's Principal Investigator for analysis. Principal Investigator Address : Lisa Clemen. : 3M Environmental Laboratory 935 Bush Avenue, Building 2-3E-09 St. Paul, Minnesota 55133-3331, USA Tel No. : 651-778-6018 (0) 651-778-6176 (F). 119 : Page 2 MIN 312/014272 Study Number : MIN/312 Protocol Amendment Number : 4 H u n tin g d o n Life Sciences Subsequent to this Protocol Amendment 3 the duties of Principal Investigator will be transferred to: Principal Investigator Address Dr Richard Grazzini Exygen Research 3058 Research Drive State College PA 16801 USA Tel No. Fax No. 001 814 231 8032 001 814 231 1580 All samples will be analysed for POSF. PFOS and PFOA. Samples will be analysed for POSF according to Exygen method ExM-023-071A in its current revision. Samples will be analysed for PFOS and PFOA according to Exygen method ExM-023-071 in its current revision. Any further processing and analysis of the samples and data collation will be the responsibility o f the Sponsor. A copy o f the Sponsor's report w ill be included as an addendum to the study report. All raw data relevant to the analyses of test substance and metabolites in plasma will be retained by the Sponsor. Page 3 120 MIN 312/014272 ANALYTICAL PHASE REPORT STUDY TITLE Perfluorooctanesulfonyl Fluoride (POSF; T-7661.1) Prelim inary Toxicity Study by Inhalation A dm inistration to CD Rats for 1 W eek DATA REQUIREM ENTS Analytical M ethod R equirem ents STUDY DIRECTOR Terry J. Kenny H untington Life Sciences, Ltd. STUDY COMPLETED ON M arch 10, 2004 PERFORM ING LABORATORY / TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Corporate Toxicology Building 220-2E-02 St. Paul, M N 55133-3220 PROTECT Protocol Number: M IN/312 Exygen Study Number: 023-080 T otal Pages: 165 : 121 : MIN 312/014272 Exygen Study No.: 023-080 f i n r m T a R O A T n n v p p a r T i r i ? m i u p i t a m ^ i ? c T iATi?iv/ri?ivTT' XAV/JUV/V/iTAAJU11li 1V/AJL7i n i .UjlTAJL/1^i Exygen Study N um ber 023-080, the analytical phase of the study entitled Perfluorooctanesulfonyl Fluoride (POSF; T-7661.1) Prelim inary Toxicity Study by Inhalation Adm inistration CD Rats for W eek," conducted for 3M Corporate Toxicology, was perform ed in com pliance w ith O ECD Principles o f G ood Laboratory Practice (as revised in 1997), EN V /M C/CH EM (98)17 by Exygen Research. R ichard A. (firazzmV Principal Investigator Exygen Research Study D irector H untingdon Life Sciences Ltd John Butenhoff Sponsor Representative 3M Corporate Toxicology >r D ate z s r U x L & t t .f D ate D ate Exygen Research : 122 : Page 2 of 166 MIN 312/014272 Exygen Study No.: 023-080 O T J JT----------------------- V^ ^ SST T A V^ ri? QT.AAM.T. lA7^lVITvAiUi?1i\^JJrLr E x y g en R e se a rc h 's Q u ality A ssurance U nit re v iew ed E x y g en S tu d y N u m b e r 023-080, the analytical phase of the study entitled, "Perfluorooctanesulfonyl Fluoride (POSF; T7661.1) Prelim inary Toxicity Study by Inhalation A dm inistration to CD Rats for 1 W e e k " . A ll re v ie w e d ph ases w ere in sp ected fo r co n d u ct acco rd in g to E xygen R e se a rc h 's Standard O perating Procedures, the Study Protocol, and all applicable G ood Laboratory Practice Standards. All findings were reported to the Study D irector and to management. Phase Date Inspected Date Reported to Exygen PI and Management Date Reported to Study Director and Test Facility Management 1. Protocol Review 09/23/02 11/13/02 11/21/02 12/09/02 2. Extraction, Fortification 09/23/02 11/13/02 11/14/02 12/09/02 3. Extraction, Fortification 09/24/02 11/13/02 11/21/02 12/09/02 4. Raw Data Review 11/04,05/02 11/13/02 11/14/02 12/09/02 5. Raw D ata Review 11/25,26/02 12/03/02 12/18/02 01/20/03 6. D raft Analytical Report Review 11/07,10/03 11/17/03 03/10/04 7. Final Analytical Report Review 03/08/04 03/08/04 03/10/04 03/10/04 M iw a N abetani Q uality A ssurance A uditor H a y L t o ' o<yD ate Exygen Research Page 3 of 166 : 123 : MIN 312/014272 Exygen Study No.: 023-080 CKRTTFirATTON OF ATTTTITTMTTP T T V This report, for Exygen Study N um ber 023-080, is a true and com plete representation of the raw data for the study. Subm itted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: R ichard A. GraZzini President Exygen Research ..4 l J i 4 .. / M D ate Exygen Research Facility M anagem ent ohn M. Flaherty Vice President Exygen Research Study D irector, H untingdon Life Sciences, Ltd D ate H untingdon Life Sciences, Ltd Sponsor Representative, 3M: John Butenhoff 3M Corporate Toxicology Exygen Research D ate D ate Page 4 of 166 124 : MIN 312/014272 Exygen Study No.: 023-080 a i u m , i i r m i xCinH TT\X7 TT'*'W"V^.Tm'W"W-lT Am r iujg ix i u a i m. t Perfluorooctanesulfonyl Fluoride (POSF; T-7661.1) Prelim inary Toxicity Study by Inhalation A dm inistration to CD Rats for 1 W eek PROTOCOL NUMBER: M 3N /312 EXYGEN STUDY NUMBER: 023-080 TYPE OF STUDY: A naly tical SAM PLE M ATRIX: Rat Liver and Serum TEST SUBSTANCES: Perfluorooctanesulfonyl Fluoride (POSF), Perfluorooctanesulfonate (PFO S) and Pentadecafluorooctanoic A cid (PFOA) SPONSOR: 3M Corporate Toxicology Building 220-2E-02 St. Paul, M N 55133-3220 STUDY DIRECTOR: Terry J. Kenny H untingdon Life Sciences Ltd W oolley Road A lco n b u ry H untingdon Cambridgeshire, England PE28 4HS SPONSOR REPRESENTATIVE: John Butenhoff 3M Corporate Toxicology Building 220-2E-02 St. Paul, M N 55133-3220 PERFORM ING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TTMFTART F- Study Initiation Date: 08/17/01 Experim ental Term ination Date: 09/28/02 Study Com pletion D ate: 03/10/04 Exygen Research Page 5 of 166 : 125 : MIN 312/014272 Exygen Study No.: 023-080 P B O T P O T D l?D C n\T\T l?T T he Study D irector for this project w as T erry J. K enny at H untingdon Life Sciences Ltd. The follow ing personnel from Exygen Research were associated with various phases of the study: Name Richard A. Grazzini Em ily D ecker Paul Connolly Karen Risha Lawrence Ord R ickey Kelley Chas Sim ons Carisa Kelley Joe G allagher Chris Pfleegor X iaom ing Zhu T itle President Scientist Technical Lead-LC/M S Scientist Sample Custodian Sample Custodian Technical Lead-GC/M S T ech n ician Scientist Scientist T ech n ician Exygen Research : 126 : Page 6 of 166 MIN 312/014272 Exygen Study No.: 023-080 TABLE OF CONTENTS T IT L E P A G E ................................................................................................................... G O O D L A B O R A T O R Y P R A C T IC E C O M PL IA N C E S T A T E M E N T .... Q U A L IT Y A S S U R A N C E S T A T E M E N T ............................................................ C E R T IF IC A T IO N O F A U T H E N T IC IT Y ............................................................. S T U D Y ID E N T IF IC A T IO N ...................................................................................... P R O JE C T P E R S O N N E L ............................................................................................ T A B L E O F C O N T E N T S ............................................................................................ L IS T O F T A B L E S ......................................................................................................... L IS T O F F IG U R E S ....................................................................................................... L IS T O F A P P E N D IC E S ............................................................................................. 1.0 S U M M A R Y ............................................................................................................ 2.0 O B JE C T IV E ............................................................................................................ 3.0 IN T R O D U C T IO N ................................................................................................. 4 .0 T E S T S Y S T E M ...................................................................................................... 5.0 R E F E R E N C E M A T E R IA L ................................................................................ 6 .0 D E S C R IP T IO N O F A N A L Y T IC A L M E T H O D ....................................... 6.1.1 P F O S a n d P F O A E x trac tio n P ro c e d u re .................................................. 6 .1 .2 P O S F E x tra c tio n P ro c e d u re ........................................................................ 6 .2 P rep aratio n o f S tandards and F o rtificatio n S o lu tio n s ......................... 6.3 C h ro m a to g ra p h y .................................................................................................. 6 .4 In stru m en t S e n s itiv ity ....................................................................................... 6.5.1 D escription o f LC/M S/M S Instrum ent and O perating C onditions 6 .5.2 D esc rip tio n o f G C /M S In stru m en t and O peratin g C o n d itio n s........ 6.6.1 P F O A a n d P F O S Q uan titatio n and E x am p le C a lc u la tio n ................ 6.6 .2 P O S F Q u an titatio n and E x am p le C a lc u la tio n ....................................... 7 .0 E X P E R IM E N T A L D E S IG N ............................................................................. 8.0 R E S U L T S ................................................................................................................. 9 .0 C O N C L U S IO N S ................................................................................................... 10.0 R E T E N T IO N O F D A T A A N D S A M P L E S ............................................... .. 1 ..2 ..3 ..4 ..5 ..6 ,,7 ..8 ..9 10 11 11 11 12 12 13 13 14 14 15 15 15 17 17 19 20 20 21 21 Exygen Research 127 Page 7 of 166 MIN 312/014272 Exygen Study No.: 023-080 T able I. T1 /TlOJ Tl U f/'ATT' T1'AA TDTT1 /TMTTT Page S u m m ary o f P F O S in C ontrol R at L iv e r S a m p le ......................................................23 T a b le H. S u m m ary o f P F O S in C ontrol R at S eru m S a m p le ................................................... 23 T ab le HI. S u m m ary o f P F O A in C ontrol R at L iv e r S a m p le .................................................... 23 T a b le IV . S u m m ary o f P F O A in C ontrol R at S eru m S a m p le ..................................................23 T a b le V. S u m m ary o f P O S F in R a t L iv er C o n tro l S a m p le .................................................... 24 T ab le V I. S u m m ary o f P O S F in R a t S erum C ontrol S a m p le ..................................................24 T a b le VH. S u m m ary o f P F O S F o rtificatio n R eco v eries in R a t L i v e r .................................... 25 T ab le V m . S u m m ary o f P F O S F o rtificatio n R eco v eries in R a t S e ru m ...................................25 T ab le IX . S u m m ary o f P F O A F o rtificatio n R eco v eries in R a t L iv e r.................................... 25 T a b le X . S u m m ary o f P F O A F o rtificatio n R eco v eries in R a t S e ru m ................................. 26 T ab le X I. S u m m ary o f P O S F F o rtificatio n R eco v eries in R a t L i v e r .................................... 26 T a b le XU. S u m m ary o f P O S F F o rtificatio n R eco v eries in R a t S e ru m ...................................26 T a b le XDI. S u m m ary o f P F O S R esid u es in R at L iv e r S a m p le s ................................................ 27 T a b le X IV . S u m m ary o f P F O A R esid u es in R at L iv e r S a m p le s.................................................28 T ab le X V . S u m m ary o f P O S F R esid u es in R at L iv e r S a m p le s .................................................29 T ab le X V I. S u m m ary o f P F O S R esid u es in R at S eru m S a m p le s .............................................. 30 T a b le X V H S u m m ary o f P F O A R esid u es in R a t S erum S a m p le s............................................31 T a b le X V III. S u m m ary o f P O S F R esid u es in R a t S erum S a m p le s ............................................32 Exygen Research : 128 : Page 8 of 166 MIN 312/014272 Exygen Study No.: 023-080 F ig u re 1. T y p ic a l C a lib ra tio n C u rv e f o r P F O S ................................ Page F ig u re 2. T ypical C alib ratio n C u rv e fo r P F O A .......................... F ig u re 3. T y p ical C alib ratio n C urve fo r P O S F ...................... Figure 4. Figure 5. Figure 6. Figure 7. C hrom atogram R epresenting a 0.1 ng/m L standard for PFO S and PFO A . ....37 C h ro m ato g ram R ep resen tin g a 25 p g /m L stan d ard fo r P O S F .................... ....38 Chrom atogram Representing a Rat Liver Control Sample for PFOS and P F O A (E xygen ID 0 2 0 1 6 8 4 C ontrol A , Set: 0 9 2 6 0 2 A ) ............... ...39 Chrom atogram Representing Control Rat Liver Sample for POSF ;Vm Exy^cu---------------T lINu. A uNzAuNzOor n/ / , Oa e, i u y z u u z u a ).................................... F ig u re 8. Figure 9. Chrom atogram Representing Control Rat Serum for PFOS and PFOA, (E xygen ID: 0 2 0 1 6 8 2 C ontrol A , Set: 0 9 2 0 0 2 A )............................... ...41 C hrom atogram R epresenting Control R at Serum for P O SF (Exygen ID: 0 2 0 1 6 8 2 , Set: 0 9 2 4 0 2 G 8 )............................................... Figure 10. C hrom atogram R epresenting C ontrol R at L iver fortified w ith 10 ng/g of P F O S and P F O A (E xygen ID: 0 2 0 1 6 8 4 S pk A , Set: 0 9 2 6 0 2 A ).................... ...4 3 F igure 11. C hrom atogram R epresenting C ontrol R at L iver fortified at 5 pg/g w ith P O S F (E xygen ID: 0202 8 7 7 S p k A , Set: 0 9 2 0 0 2 G 8 ).............................. ...4 4 Figure 12. C hrom atogram R epresenting C ontrol R at Serum fortified w ith 10 ng/m L o f P F O S an d P F O A (E xygen ID: 0 2 0 1 6 8 2 S pk A , Set: 0 9 2 0 0 2 A )............... ...4 5 Figure 13. C hrom atogram R epresenting C ontrol R at Serum fortified w ith 5 pg/m L o f P O S F (E xygen ID: 020 1 6 8 2 S pk A , Set: 0 9 2 4 0 2 G 8 )............................... ...4 6 Figure 14. C hrom atogram Representing R at L iver Sam ple for PFO S and PFO A (E xygen ID: 0 2 0 2805, S p o n so r ID: E 01-1 6 4 3 -3 3 8 5 1 Set: 0 9 2 6 0 2 A )......... ...4 7 F igure 15. C hrom atogram R epresenting R at L iver Sam ple for P O SF (Exygen ID: 0 2 0 2 8 0 0 , S p o n so r ID: E 0 1 -1 6 4 3 -3 3 8 4 6 Set: 0 9 2 0 0 2 G 8 )........ ...48 F igure 16. C hrom atogram R epresenting R at Serum Sam ple for PFO S and PFO A (E xygen ID: 0202 7 8 5 , S p o n so r ID: E 01-1 6 4 3 -3 3 8 3 1 Set: 0 9 2 0 0 2 A R )...... ...4 9 Figure 17. C hrom atogram R epresenting R at Serum Sam ple for P O SF (Exygen ID: 0 2 0 2 7 8 0 , S p o n so r ID: E 0 1 -1 6 4 3 -3 3 8 2 6 Set: 0 9 2 4 0 2 G 8 )........... ...50 Exygen Research Page 9 of 166 : 129 : MIN 312/014272 : 130 : MIN 312/014272 Exygen Study No.: 023-080 1.0 SUMMARY Exygen Resarch extracted and analyzed rat liver and serum sam ples for the determination of perfluorooctanesulfonate (PFOS) and pentadecafluorooctanoic acid (PFOA) according to E x y g en M e th o d E x M -023-071 (Appendix B ) an d fo r the determ in atio n o f perfluorooctanesulfonyl fluoride (POSF) according to Exygen M ethod ExM -023-071 A (Appendix C). T he lim it o f quantitation fo r PFO S and PFO A in rat liver w as 10 ng/g and 10 ng/m L in rat serum . The lim it of quantitation for PO SF in rat liver was 2.5 pg/g and 2.5 pg/m L for rat serum. The LO Q for each matrix was determ ined in a m ethod validation studies perform ed at Exygen (Exygen Studies: 023-074 and 023-075). H ow ever, the LOQ for PFO A and PFOS in each matrix was determ ined using a 1 m L sam ple aliquot for serum and urine and 1.0 g sam ple w eight for liver. D ue to lim ited sam ple size, only 0.1 m L was used for serum and 0.1 g for liver, w hich increased the LO Q for each m atrix by a factor of 10. T he L O Q fo r P O S F in serum w as d eterm in ed using 0.1 m L sam p le a liq u o t and in som e cases only 0.05 m L was used w hich increased the L O Q for P O S F in serum by a factor o f 2. PFO S in the rat liver sam ples ranged from non-detected levels to 67,600 ng/g. PFO A in the rat liver sam ples ranged from non-detected levels to 6110 ng/g. There was no PO SF detected in any of the rat liver sam ples. PFOS in the rat serum sam ples ranged from non detected levels to 13,900 ng/mL. PFO A in the rat serum sam ples ranged from non detected levels to 8850 ng/m L. There was no P O SF detected in any of the rat serum sam ples. The average percent recoveries i standard deviations for PFOS in rat liver and serum sam ples were 91% 6% and 109% 19%, respectively. The average percent recoveries standard deviations for PFO A in rat liver and serum sam ples w ere 134% 4% and 112% 7%, respectively. The average percent recoveries standard deviations for POSF in rat liver and serum sam ples were 85% 6% and 72% 19%, respectively. 2.0 OBJECTIVE The objective o f this phase was to determ ine levels of perfluorooctanesulfonyl fluoride (PO SF), perfluorooctanesulfonate (PFO S), and pentadecafluorooctanoic acid (PFO A ) in sp ecim en s o f ra t liv er an d seru m sam ples according to P ro to co l M IN /3 1 2 (Appendix A). 3.0 INTRODUCTION T his report details the results o f the analysis for the determ ination o f PFO S and PFO A in rat liver and serum sam ples, using the analytical m ethod entitled, "M ethod of A nalysis for Exygen Research Page 11 of 166 : 131 : MIN 312/014272 Exygen Study No.: 023-080 the D eterm ination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) Ta n d P e n ta d e c a f lu" o r o o c ta n o ic A c id f P F O A l-------------\ --------------------/ in T?A*` ***a*t Q pm m1VTwPxr, `- 'V iv u u uQuriurl TWTri^lTli-e^o- ,, Coiniivral UlOU u 11/ results for the determ ination of PO SF in rat liver and serum sam ples, using the analytical m ethod entitled, M ethod of A nalysis for the D eterm ination of Perfluorooctanesulfonyl Fluoride (PO SF) in Rat U rine, Serum , and Liver by G C/M S." T he study w as initiated on A ugust 17, 2001, when the study director signed protocol num ber M IN/312. The analytical experim ental start date was Septem ber 20, 2002, and the analytical experim ental term ination date was Septem ber 28, 2002. 4.0 TEST SYSTEM The control rat liver and serum used for the matrix blanks and m atrix fortifications were received frozen on dry ice on June 12, 2002 and A ugust 7, 2002 from Pel-Freez Biologicals, Rogers, Arkansas. Tw enty rat liver and tw enty rat serum sam ples were received frozen on dry ice at Exygen fro m 3M E n v iro n m en tal L aboratory on A u g u st 1, 2 0 0 2 and lo g g ed in by Exygen personnel and placed in frozen storage. Sam ple log-in and chain of custody inform ation can be found in the raw data package associated w ith this study. Storage records w ill be kept at Exygen Research and a true copy o f the storage records can be found in the raw data package associated w ith this study. 5.0 REFERENCE MATERIAL The analytical standard PFO A was received at Exygen on July 3, 2002 from SigmaA ldrich and stored refrigerated. The analytical standard PFOS was received at Exygen on M ay 15, 2000 from 3M E nvironm ental T echnology and Services and stored frozen. The analytical standard PO SF was received at Exygen on June 6, 2002 from Sigm a-Aldrich and stored ambient. The available inform ation for the reference m aterial is listed below. Compound PFOA PFOS POSF Exygen Inventory No.. SP1328 SP249 SP1187 TCR No. NA SD018 15721HQ P u rity (%) 99.9 86.9 98.6 Expiration Date 07/03/03 08/31/06 06/06/03 Exygen Research Page 12 of 166 : 132 : MIN 312/014272 Exygen Study No.: 023-080 The m olecular structures o f test substances are given below: N am e: PFOS Chem ical Name: Perfluorooctane sulfonate M olecular W eight: 499, as shown N am e: PFOA Chem ical Name: Perfluorooctanoic acid M olecular W eight: 413, as shown N am e: POSF Chem ical Name: Perfluorooctanesulfonyl fluoride M olecular W eight: 502, as shown 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical m ethods ExM -023-071 and ExM -023-071A were used for this study. The m ethods were versioned after the analysis of the sam ples were com pleted to allow for different sam ple sizes and to make m inor typographical corrections. The revised m ethods are included with this report. 6.1.1 PFOS and PFOA Extraction Procedure A 100 p L aliquot o f the serum and 0.1 g of the liver w ere used for the extraction procedure. A fter fortification of appropriate sam ples, the serum sam ples were brought up Exygen Research Page 13 of 166 : 133 : MIN 312/014272 Exygen Study No.: 023-080 to 20 m L w ith T ype I W ater and the liver sam ples w ere brought up to 10 m L w ith Type I *%/**-W ater. T h e s---e---r--u---m--------s--a---m--- rn--l--e--s-- WPTP vnrtPYpH fr\r ~ 1 a mi m iunuuitva unu u iv 1u v u a c u i l j J l1t^/O-, W t l C hom ogenized with a tissuem izer for ~ 1 minute. An aliquot of one m illiliter was transferred from each sam ple and 5 m L of acetonitrile was added and the sam ples were shaken for ~ 20 minutes. The sam ples were centrifuged and the supernatant was decanted onto a conditioned SPE colum n. Then the sam ples were eluted w ith 2 m L of methanol. Each sam ple w as analyzed by LC/M S/M S electrospray. 6.1.2 POSF Extraction Procedure A 100 p L aliquot o f the serum and 0.1 g o f the liver w ere used for the extraction procedure. A fter fortification of appropriate sam ples, the sam ples were heated to 60C80C for - 3-4 m inutes and then analyzed by GC/M S. 6.2 Preparation of Standards and Fortification Solutions A stock standard solution of PFOA was prepared on July 8, 2002 and a stock standard solution of PFO S was prepared on A ugust 2, 2002 as specified in Exygen m ethod ExM 023-071. The stock standard solutions were prepared at a concentration of 100 pg/m L by dissolving 10 m g of each standard (corrected for purity and salt content) in methanol. F rom this solution, a 1.0 pg/m L fortification standard solution w as prepared by taking 1 m L of each stock and bringing the volum e up to 100 m L w ith methanol. A 0.1 pg/m L fortification standard w as prepared by taking 10 m L of the 1.0 pg/m L m ixed fortification standard and bringing the volum e up to 100 m L with methanol. A set of standards containing PFO A and PFO S w as prepared dilution o f the 0.1 pg/m L and various calibration solutions in the follow ing manner: Initial Cone. (pg/m L )1 0.1 0.1 0.1 0.005 0.002 l 0.001 of PFOA and PFOS V olum e (mL) 5 2 1 10 10 10 D iluted to (mL) 100 100 100 100 100 100 Final Cone. (ug/mL) 0.005 0.002 0.001 0.0005 0.0002 0.0001 A stock standard solution of PO SF was prepared on June 11, 2002 as specified in Exygen m ethod ExM -023-071A . The stock standard solution was prepared at a concentration of 1990 pg/m L by dissolving ~ 0.2 g o f the standard (corrected for purity) in m ethanol. From this solution, a 1000 pg/m L calibration standard solution was prepared by taking ~ 25 m L o f the stock and bringing the volum e up to 50 m L w ith m ethanol. Exygen Research Page 14 of 166 134 : MIN 312/014272 Exygen Study No.: 023-080 A set o f standards containing PO SF was prepared by dilution o f the 1000 pg/m L: Initial Cone. (pg/m L )1 1000 1000 1000 1000 1000 1000 1of POSF Volum e (mL) 5 2.5 1 0.5 0.25 0.1 D iluted to (mL) 10 10 10 10 10 10 Final Cone. (ng/mL) 500 250 100 50 25 10 T he stock standard solutions and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. D ocum entation of standard preparation can be found in the raw data associated w ith this report. 6.3 Chromatography Q uantification of PFO A and PFOS was accom plished by LC/M S/M S electrospray. The retention tim e o f P FO A w as ~ 8.6 and ~ 8.8 m in for PFO S. Peaks w ere detected in the control liver sam ple corresponding to the analyte retention tim e of PFO S, but the amounts detected were only significant enough to alter several fortification recoveries and the rest w ere less than the low est calibration standard (0.0001 pg/m L). Q uantification of PO SF was accom plished by GC/M S. The retention tim e of PO SF was ~ 3.5 min. Peaks w ere not detected in the control liver sam ple corresponding to the analyte retention tim e of POSF. 6.4 Instrument Sensitivity The sm allest standard am ount injected during the chrom atographic run had a concentration o f 0.0001 pg/m L of PFO A and PFO S and 25 pg/m L for POSF. 6.5.1 Description of LC/MS/MS Instrument and Operating Conditions Instrum ent: PE SCIEX API 365 Biom olecular M ass A nalyzer Interface: SCIEX Turboion Spray Liquid Introduction Interface Com puter: Dell OptiPlex GX 110 S o ftw are: P E S ciex A n aly st v ersion 1.1 W indow s NT, version 4 Exygen Research Page 15 of 166 : 135 : MIN 312/014272 Exygen Study No.: 023-080 HPLC: H ewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser H P A utosam pler H P Colum n Oven H P L C C o lu m n :G en esis Cg (Jones C h rom atography), 2.1 m m x 50 m m , 4 p Colum n Temp.: 35 C Injection V ol.: 15 p L M obile Phase (A): 2 m M A m m onium A cetate in W ater M obile Phase (B): M ethanol Flow Rate: 0.3 mL/min. Tim e 0 o r\ .\j 5.0 9.0 9.5 14.0 14.5 20.0 %A 90 nr\ y \j 10 10 0 0 90 90 %B 10 10 90 90 100 100 10 10 Ions monitored: A n aly te PFOA PFOS M ode negative negative Transition M onitored 413 -> 369 499 -> 99 Approxim ate R eten tio n T im e (min') 8.6 8.8 Tune File Parameters C o n tro ls IS-Iospray D P-Declustering Potential FP-Focusing Potential EP-Entrance Potential CE-Collision Energy C X P-Collision Cell Exit Potential D F-Deflector CEM -Channel Electron M ultiplier Set -4200.0 -51.0 -230.0 10.0 -70 -5 300.0 2800.0 Exygen Research Gas Flows Nebulizer Gas Curtain Gas Collision Gas TIS Tem perature Set 12 13 4 350C Page 16 of 166 : 136 : MIN 312/014272 Exygen Study No.: 023-080 6.5.2 Description of GC/MS Instrument and Operating Conditions instrum ent! H ewlett-Packard model 6890 Series Gas Chrom atograph/m odel 5973 m ass selective detector Colum n: R estek R TX -502.2, 60 m x 0.25 m m ID , 1.4 p m df Oven Temperature: H old at 40C for 4 m in., ram p 20C/m in. to 100C, hold for 0 min. Injector Temperature: 220C Transfer Line Temperature: 220C Carrier Gas: H elium GC H ead Pressure: 5 psi for 0.25 m in., ram p 80 psi/m in. to 60 psi, hold 0.5 min., ram p -8 0 psi/m in. to 30 psi, hold at 30 psi for rem ainder of the analytical run. Injection M ode: Splitless Injection Liner: 4 m m ID straight glass Injection Purge Delay: 2 min. Purge Flow to Split Vent: 10 mL/m in. Injection Volume: 1 mL Electron M ultiplier Voltage: From A TU N E + 200V Scan Mode: SIM M onitored Ions: D w ell Time: m /z 's 69 (quantitation ion); 131, 219 (q u alifier ions) 75 ms Integrator: R etention Tim es: H ew lett-Packard C hem Station software ~3.5 m inutes Total run time: ~7 m inutes 6.6.1 PFOA and PFOS Quantitation and Example Calculation Fifteen m icroliters of sam ple or calibration standard were injected into the LC/M S/M S. T he peak area w as m easured and the standard curve w as generated (using 1/x fit w eighted linear regression) by A nalyst softw are using six concentrations of standards. The concentration w as determ ined from the equations below. Equation 1 calculated the am ount of analyte found (in ng/m L, based on peak area) using the standard curve (linear regression param eters) generated by the A nalyst software program . Then Equation 2 calculated the am ount of analyte found in ng/g for liver and ng/m L for serum. E q u atio n 1: A nalyte found (ng/mL) = (Peak area - intercept) x AF W here: AF = Aliquot Factor slope Exygen Research Page 17 of 166 : 137 : MIN 312/014272 : 138 : MIN 312/014272 Exygen Study No.: 023-080 6.6.2 POSF Quantitation and Example Calculation O ne m illiliter o f headspace o f the sam ple or calibration standard w as injected into the G C /M S . T he p e a k area w as m easu red and the stan d ard c u rv e w as gen erated (using 1/x fit w eighted linear regression) by H P Chem station software using five concentrations of standards. The concentration was determ ined from the equations below. Equation 1 calculated the am ount of analyte found (in pg/m L, based on peak area) using the standard curve (linear regression param eters) generated by the H P Chemstation softw are program . T hen E quation 2 calculated the am ount of analyte found in pg/g for liver and pg/m L for serum. E q u atio n 1: A nalyte found (pg/m L) = (Peak area - intercept) x D F W here: D F = D ilution Factor slope E quation 2: A nalyte found (ppm , pg/g for liver and pg/m L for serum) = anal, found (ppm ) x standard volum e added tm Ll SV (m L) or SW (g) W here: SW = Sam ple W eight SV = Sam ple Volum e For sam ples fortified w ith known amounts o f PO SF prior to analysis, Equation 3 calculated the percent recovery. E quation 3: Recovery (% ) = ((anal, fo u n d (ppm ) - avg. anal, in Ctrl ip p m ri x l0 0 % am ount added (ppm) An exam ple of a calculation using an actual sam ple follows: R at liver sam ple Exygen ID 0202877 Spk A (Set: 092002G 8), fortified at 5 pg/g w here: peak area intercept slope dilution factor 276118 4400 3230 1 ppm added (fort level) 5 standard volum e added (mL) sam ple w eight (g) 0.005 0.10403 Exygen Research Page 19 of 166 : 139 : MIN 312/014272 Exygen Study No.: 023-080 From equation 1: A n alvj t-e---f-o--u--n--d-\iO" cipr-/m---T--3J [276118-44001 x 1 3230 From equation 2: Analyte found (ppm) 84.12 pg/m L 84,12 (ug/m Lf x 0.005 (m i 3 0.10403 (g) From equation 3: % Recovery = 4.04 (ig/g (4.04 ppm 1 x 100% 5 ppm Note: This exam ple calculation was done using rounded num bers, and therefore m ay be slightly different from the values shown in the R A W DATA. 7.0 EXPERIMENTAL DESIGN Each set of sam ples (liver or serum ) consisted of one m atrix blank, tw o matrix blanks fortified at know n concentrations, and ~ 20 sam ples. Each set of sam ples (liver or serum) for G C/M S analysis consisted of one matrix blank, one m atrix blanks fortified at known concentrations, tw o field sam ples fortified at a know n concentration and ~ 10 sam ples. E ach sam ple w as extracted using the appropriate m ethod and then analyzed in duplicate for PFO A and PFO S and extracted in duplicate for POSF. 8.0 RESULTS T he P F O S fo u n d in the co n tro l rat liv e r and seru m sam ples are listed in Tables I-II. T he P F O A fo u n d in the co n tro l rat liv er an d serum sam ples are listed in Tables III-IV . The P O S F fo u n d in the co n tro l ra t liv er an d serum sam ples are listed in Tables V -V I. Peaks were detected in the control liver sam ple corresponding to the analyte retention time of PFO S, but the am ounts detected were only significant enough to alter several fortification recoveries. In d iv id u al reco v eries fo r P F O S in th e ra t liv er a n d serum sam p les are d etailed in Tables VII-VIII. T h e average p ercen t recoveries stan d ard d e v iatio n s fo r P F O S in rat liv e r and serum sam ples were 91% 6% and 109% 19%, respectively. Individual recoveries for P F O A in th e ra t liv er an d serum sam ples are d etailed in Tables IX -X . T he average percent recoveries standard deviations for PFO A in rat liver and serum sam ples were Exygen Research Page 20 of 166 : 140 : MIN 312/014272 Exygen Study No.: 023-080 standard deviations for PO SF in rat liver and serum sam ples were 85% 6% and 72% 19%, respectively. P F O S in the ra t liv er sam p les ranged from n o n -d etected lev els to 67,600 ng/g. Individual resu lts are listed in Table XIII. P F O A in the ra t liv er sam p les ran g ed fro m non-d etected lev els to 6110 ng/g. Ind iv id u al results are listed in Table XIV. T h ere w as no P O S F d ete c te d in any o f the ra t liv er sam ples. Ind iv id u al resu lts are listed in Table XV. PFO S in th e ra t seru m sam ples ran g ed fro m n o n -d etected levels to 13,900 ng/m L . Individual resu lts are liste d in Table XVI. P F O A in the ra t serum sam p les ra n g e d from n o n d etected levels to 8850 ng/m L . Individual resu lts are listed in Table XVII. T h ere w as no P O S F d e tec ted in any o f th e ra t seru m sam ples. Individual resu lts are listed in Table XVIII. 9.0 CONCLUSIONS The rat liver and serum sam ples were successfully extracted and analyzed for PFOS, PFO A and PO SF according to the appropriate analytical method. 10.0 RETENTION OF DATA AND SAMPLES W hen the final analytical report is com plete, all original paper data generated by Exygen Research w ill be shipped to the sponsor. This does not include facility-specific raw data such as instrum ent logs, how ever exact copies of tem perature logs will be submitted. E x a c t co p ies o f all raw d ata, as w ell as a signed copy o f th e final analytical rep o rt and all original facility-specific raw data, w ill be retained in the Exygen R esearch archives for the period of tim e specified in O ECD Principles of G ood Laboratory Practice (as revised in 1997), E N V /M C /C H E M (98)17. R etained sam ples o f reference substances are archived by the sponsor. Exygen Research : 141 : Page 21 of 166 MIN 312/014272 Exygen Study No.: 023-080 TABLES Exygen Research : 142 : Page 22 of 166 MIN 312/014272 : 143 : MIN 312/014272 Exygen Study No.: 023-080 Table V Summarv nf p n sp in Rat i ivor nntrril Comnln . , ----------------------------j v *" t v i. v v i i u v i u u n i | J i v Sponsor Exygen _________ jP_______________ ID Lot #21824 0202877 Ctrl Lot#21824_______0202877 Ctrl Set Number 092002G8 092302G8 POSF Found (pg/g) ND ND Table VI. Summary of POSF in Rat Serum Control Sample Sponsor Exygen Set POSF ___________ ID___________Number Found (pg/mL) Lot #07024 0201682 Ctrl Lot #07024______ 0201682 Ctrl 092402G8 ND 093002A____________ ND ND - N ot D etected (A rea less than the low est concentration of the calibration standards (25 |ig/m L )) Exygen Research : 144 : Page 24 of 166 MIN 312/014272 Exygen Study No.: 023-080 Table VII. Summary of PFOS Fortification Recoveries in Rat Liver Sponsor ID Exygen ID Set Number Amt Lot#14124 0201684 Spk A Lot#14124 0201684 Spk A* Lot#14124 0201684 Spk B Lot#14124 0201684 Spk B* E01-1643-33846 0202800 Spk C E01-1643-33846 0202800 Spk C* 092602A 092602A 092602A 092602A 092602A 092602A 10 10 50 50 5000 5000 AVERAGE STANDARD DEVIATION RELATIVE STANDARD DEVIATION % 90 87 92 84 98 97 91 6 6 Table VIII. Summary of PFOS Fortification Recoveries in Rat Serum Sponsor Exygen Set Amt ID ID Number Added (ng/mL) Lot#07024 0201682 Spk A Lot #07024 0201682 Spk A* Lot #07024 0201682 Spk B Lot #07024 0201682 SpkB* E01-1643-33826 0202780 Spk C E01-1643-33826 0202780 Spk C* 092002A 092002A 092002A 092002A 092002A 092002A 10 10 50 50 5000 5000 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 127 116 127 109 85 87 109 19 17 Table IX. Summary of PFOA Fortification Recoveries in Sponsor Exygen Set Amt ID ID Number Added (ng/g) Lot #14124 0201684 Spk A Lot #14124 0201684 Spk A* Lot #14124 0201684 Spk B Lot#14124 0201684 SpkB* E01-1643-33846 0202800 Spk C E01-1643-33846 0202800 Spk C* 092602A 092602A 092602A 092602A 092602A 092602A 10 10 50 50 5000 5000 AVERAGE STANDARD DEVIATION RELATIVE STANDARD DEVIATION % Recovery 130 130 134 135 140 135 134 4 3 D uplicate Injection Exygen Research Page 25 of 166 : 145 : MIN 312/014272 o u i m n a i j uifrnwn ^c rr i1 7'>u__i_i:nkic__a_ii.i:u__i_itn_e__c__u__v__e__n_e_s_ in im-_ixl L i_v__e r Sponsor Exygen Set Amt j P ____________ ID .. Number Added (pg/g) Lot#21824 0202877 Spk A E01-1643-33854 0202808 Spk B E01-1643-33854 0202808 Spk C Lot#21824 0202877 Spk A E01-1643-33856 0202810 Spk B E01-1643-33856 0202810 Spk C 092002G8 092002G8 092002G8 092302G8 092302G8 092302G8 5 5 5 5 5 5 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 81 78 79 91 93 87 85 6 8 Table XII. Summary of POSF Fortification Recoveries in Rat Serum Sponsor Exygen Set Amt jjj____________ ID______ Number Added (pg/mL) Lot #07024 0201682 Spk A E01-1643-33827 0202781 Spk B E01-1643-33827 0202781 Spk C Lot#07024 0201682 Spk A E01-1643-33841 0202795 Spk B E01-1643-33841 0202795 Spk C 092402G8 092402G8 092402G8 093002A 093002A 093002A 5 5 5 5 5 5 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD DEVIATION: % Recovery 86 92 89 66 51 49 72 19 27 Exygen Research Page 26 of 166 : 146 : MIN 312/014272 Exygen Study No.: 023-080 Table XIII. Summary of PFOS Residues in Rat Liver Samples Sponsor Exygen Set PFOS ID ID Number E01-1643-33846 0202800 092602A ND E01-1643-33846* 0202800 092602A ND E01-1643-33847 0202801 092602A ND E01-1643-33847* 0202801 092602A ND E01-1643-33848 0202802 092602A ND E01-1643-33848* 0202802 092602A ND E01-1643-33849 0202803 092602A ND E01-1643-33849* 0202803 092602A ND E01-1643-33850 0202804 092602A ND E01-1643-33850* 0202804 092602A ND E01-1643-33851 01=1643=33851* m02m02Q8n0<5 092602A ncn^m a 33600 34000 E01-1643-33852 E01-1643-33852* 0202806 0202806 092602A 092602A 28200 28900 E01-1643-33853 0202807 092602A 44100 E01-1643-33853* 0202807 092602A 47000 E01-1643-33854 E01-1643-33854* 0202808 0202808 092602A 092602A 60600 52800 E01-1643-33855 E01-1643-33855* 0202809 0202809 092602A 092602A 51800 52200 E01-1643-33856 E01-1643-33856* E01-1643-33857 0202810 0202810 0202811 092602A 092602A 092602A 84.2 65.0 38.0 E01-1643-33857* 0202811 092602A 48.4 E01-1643-33858 0202812 092602A 40.1 E01-1643-33858* 0202812 092602A 39.1 E01-1643-33859 0202813 092602A 44.7 E01-1643-33859* E01-1643-33860 0202813 0202814 092602A 092602A 44.5 31.2 E01-1643-33860* 0202814 092602A 31.8 E01-1643-33861 E01-1643-33861* 0202815 0202815 092602A 092602A 56700 61700 E01-1643-33862 0202816 092602A 56200 E01-1643-33862* 0202816 092602A 43700 E01-1643-33863 0202817 092602A 65600 E01-1643-33863* 0202817 092602A 67600 E01-1643-33864 0202818 092602A 53100 E01-1643-33864* 0202818 092602A 60100 E01-1643-33865 0202819 092602A 44200 E01-1643-33865* 0202819 092602A 44000 * Duplicate Injection ND = Not Detected (Area less than lowest calibration standard of 0.0001 pg/mL) Exygen Research Page 27 of 166 : 147 : MIN 312/014272 Exygen Study No.: 023-080 Table XIV. Summary of PFOA Residues in Rat Liver Samples Sponsor Exygen Set PFOA ID ID Number E01-1643-33846 0202800 092602A ND E01-1643-33846* 0202800 092602A ND E01-1643-33847 E01-1643-33847* E01-1643-33848 E01-1643-33848* E01-1643-33849 E01-1643-33849* E01-1643-33850 E01-1643-33850* E01-1643-33851 01-1643-33851* 0202801 0202801 0202802 0202802 0202803 0202803 0202804 0202804 0202805 0202805 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A ND ND ND ND ND ND ND ND 3400 3280 E01-1643-33852 E01-1643-33852* E01-1643-33853 E01-1643-33853* E01-1643-33854 E01-1643-33854* E01-1643-33855 E01-1643-33855* E01-1643-33856 E01-1643-33856* E01-1643-33857 0202806 0202806 0202807 0202807 0202808 0202808 0202809 0202809 0202810 0202810 0202811 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 3080 3100 4680 4590 5930 6110 4120 4070 ND ND ND E01-1643-33857* E01-1643-33858 E01-1643-33858* E01-1643-33859 E01-1643-33859* E01-1643-33 860 EOI-1643-33860* EOl-1643-33861 E01-1643-33861* E01-1643-33862 E01-1643-33862* E01-1643-33863 E01-1643-33863* E01-1643-33864 E01-1643-33864* E01-1643-33 865 E01-1643-33865* * Duplicate Injection 0202811 0202812 0202812 0202813 0202813 0202814 0202814 0202815 0202815 0202816 0202816 0202817 0202817 0202818 0202818 0202819 0202819 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A 092602A ND ND ND ND ND ND ND 54.9 57.6 31.5 35.7 41.6 42.6 78.2 75.7 43.7 41.4 ND = Not Detected (Area less than lowest calibration standard of 0.0001 pg/mL) Exygen Research Page 28 of 166 : 148 : MIN 312/014272 Exygen Study No.: 023-080 Table XV. Summary of POSF Residues in Rat Liver Samples Sponsor ID Exygen ID Set Number POSF E01-1643-33846 0202800 092002G8 ND E01-1643-33846* 0202800 092002G8 ND E01-1643-33847 0202801 092002G8 ND E01-1643-33847* 0202801 092002G8 ND E01-1643-33848 0202802 092002G8 ND E01-1643-33848* 0202802 092002G8 ND E01-1643-33849 0202803 092002G8 ND E01-1643-33849* 0202803 092002G8 ND E01-1643-33850 0202804 092002G8 ND E01-1643-33850* 0202804 092002G8 ND E01-1643-33851 0202805 092002G8 ND E01-1643-33851* 0202805 092002G8 ND E01-1643-33852 0202806 092002G8 ND E01-1643-33852* 0202806 092002G8 ND E01-1643-33853 0202807 092002G8 ND E01-1643-33853* 0202807 092002G8 ND E01-1643-33854 0202808 092002G8 ND E01-1643-33854* 0202808 092002G8 ND E01-1643-33855 0202809 092002G8 ND E01-1643-33855* 0202809 092002G8 ND E01-1643-33856 0202810 092302G8 ND E01-1643-33856* 0202810 092302G8 ND E01-1643-33857 0202811 092302G8 ND E01-1643-33857* 0202811 092302G8 ND E01-1643-33858 0202812 092302G8 ND E01-1643-33858* 0202812 092302G8 ND E01-1643-33859 0202813 092302G8 ND E01-1643-33859* 0202813 092302G8 ND E01-1643-33860 0202814 092302G8 ND E01-1643-33860* 0202814 092302G8 ND E01-1643-33861 0202815 092302G8 ND E01-1643-33861* 0202815 092302G8 ND E01-1643-33862 0202816 092302G8 ND E01-1643-33862* 0202816 092302G8 ND E01-1643-33863 0202817 092302G8 ND E01-1643-33863* 0202817 092302G8 ND E01-1643-33864 0202818 092302G8 ND E01-1643-33864* 0202818 092302G8 ND E01-1643-33865 0202819 092302G8 ND E01-1643-33865* * Duplicate Aliquot 0202819 092302G8 ND ND = Not Detected (Area less than lowest calibration standard of 25 pg/mL) Exygen Research Page 29 of 166 : 149 : MIN 312/014272 Exygen Study No.: 023-080 Table XVI. Summary of PFOS Residues in Rat Serum Samples Sponsor ID Exygen ID Set N um ber PFOS E01-1643-33826 0202780 092002A ND E01-1643-33826* 0202780 092002A ND E01-1643-33827 0202781 092002A ND E01-1643-33827* 0202781 092002A ND E01-1643-33828 0202782 092002A ND E01-1643-33828* 0202782 092002A ND E 0 1 -1643-33829 0202783 092002A ND E01-1643-33829* 0202783 092002A ND E01-1643-33830 0202784 092002A ND E01-1643-33830* 0202784 092002A ND E01-1643-33831 pni.ig43.33g3i * 0202785 0202785 092002AR n\JoS oiun\J\nJLouAL XDJ.V 13200 12700 E01-1643-33832 0202786 092002AR 8870 E01-1643-33832* 0202786 092002AR 8610 E01-1643-33833 0202787 092002AR 9100 E O l-1643-33833* 0202787 092002AR 9750 E01-1643-33834 0202788 092002AR 8480 E01-1643-33834* 0202788 092002AR 8240 E01-1643-33835 0202789 092002AR 9860 E01-1643-33835* 0202789 092002AR 9900 E01-1643-33836 0202790 092002A ND E01-1643-33836* 0202790 092002A ND E01-1643-33837 0202791 092002A ND E01-1643-33837* 0202791 092002A ND E01-1643-33838 0202792 092002A ND E01-1643-33838* 0202792 092002A ND E01-1643-33839 0202793 092002A ND E01-1643-33839* 0202793 092002A ND E 01-1643-33840 0202794 092002A ND E01-1643-33840* 0202794 092002A ND E01-1643-33841 0202795 092002AR 11300 E01-1643-33841* 0202795 092002AR 10600 E 0 1 -1643-33842 0202796 092002AR 13800 E01-1643-33842* 0202796 092002AR 13000 E01-1643-33843 0202797 092002AR 10300 E01-1643-33843* 0202797 092002AR 10800 E01-1643-33844 0202798 092002AR 13900 E01-1643-33844* 0202798 092002AR 12600 E01-1643-33845 0202799 092002AR 10800 E01-1643-33845* * Duplicate Injection 0202799 092002AR 9610 ND = Not Detected (Area less than lowest calibration standard of 0.0001 pg/mL) Exygen Research Page 30 of 166 : 150 : MIN 312/014272 Exygen Study No.: 023-080 Table XVII. Summary of PFOA Residues in Rat Serum Samples Sponsor ID Exygen ID Set N um ber PFOA E01-1643-33826 0202780 092002A ND E01-1643-33826* 0202780 092002A ND E01-1643-33827 0202781 092002A ND E01-1643-33827* 0202781 092002A ND E01-1643-33828 0202782 092002A ND E01-1643-33828* 0202782 092002A ND E01-1643-33829 0202783 092002A ND E01-1643-33829* 0202783 092002A ND E01-1643-33830 0202784 092002A ND E01-1643-33830* 0202784 092002A ND E01-1643-33831 F .n i-i6 4 3 -3 3 R 3 i* 0202785 0202785 092002AR Q920Q2.AR 8760 8850 E01-1643-33832 0202786 092002AR 5960 E01-1643-33832* 0202786 092002AR 6020 E01-1643-33833 0202787 092002AR 7750 E01-1643-33833* 0202787 092002AR 7760 E01-1643-33834 0202788 092002AR 7170 E01-1643-33834* 0202788 092002AR 7140 E01-1643-33835 0202789 092002AR 5970 E01-1643-33835* 0202789 092002AR 5990 E01-1643-33836 0202790 092002A ND E01-1643-33836* 0202790 092002A ND E01-1643-33837 0202791 092002A ND E01-1643-33837* 0202791 092002A ND E01-1643-33838 0202792 092002A ND E01-1643-33838* 0202792 092002A ND E01-1643-33839 0202793 092002A ND E01-1643-33839* 0202793 092002A ND E 01-1643-33 840 0202794 092002A ND E01-1643-33840* 0202794 092002A ND E01-1643-33841 0202795 092002A 81.5 E01-1643-33841* 0202795 092002A 88.9 E01-1643-33842 0202796 092002A 39.9 E01-1643-33842* 0202796 092002A 40.9 E01-1643-33843 0202797 092002A 53.8 E01-1643-33843* 0202797 092002A 50.5 E01-1643-33844 0202798 092002A 88.7 E 0 1 -1643-33844* 0202798 092002A 99.9 E01-1643-33845 0202799 092002A 58.2 E01-1643-33845* * Duplicate Injection 0202799 092002A 58.7 ND = Not Detected (Area less than lowest calibration standard of 0.0001 pg/mL) Exygen Research Page 31 of 166 : 151 : MIN 312/014272 Exygen Study No.: 023-080 Table XVIII. Summary of POSF Residues in Rat Serum Samples Sponsor ID Exygen ID Set Number POSF E01-1643-33826 E01-1643-33826* E01-1643-33827 E01-1643-33827* E01-1643-33828 E01-1643-33829 E01-1643-33829* E01-1643-33830 E01-1643-33831 E01-1643-33831* E01-1643-33832 E01-1643-33833 E01-1643-33833* E01-1643-33834 E01-1643-33835 E01-1643-33835* E01-1643-33836 E01-1643-33836* E01-1643-33837 E01-1643-33837* E01-1643-33838 E01-1643-33838* E01-1643-33839 E01-1643-33839* EO I-1643-33840 E01-1643-33840* E01-1643-33841 E01-1643-33841* E01-1643-33842 E01-1643-33842* E01-1643-33843 E01-1643-33843* E01-1643-33844 E01-1643-33844* E01-1643-33845 E01-1643-33845* * D uplicate A liquot 0202780 0202780 0202781 0202781 0202782 0202783 0202783 0202784 0202785 0202785 0202786 m no7A 0202787 0202787 0202788 0202789 0202789 0202790 0202790 0202791 0202791 0202792 0202792 0202793 0202793 0202794 0202794 0202795 0202795 0202796 0202796 0202797 0202797 0202798 0202798 0202799 0202799 092402G8 092402G8 092402G8 092402G8 092402G8 092402G8 092402G8 092402G8 092402G8 092402G8 092402G8 AW fLnT /Ui mL Ur Oo 092402G8 092402G8 092402G8 092402G8 092402G8 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A 093002A ND ND ND ND ND ND ND ND ND ND ND nu ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND = N ot D etected (A rea less than low est calibration standard of 25 pg/m L) Exygen Research Page 32 of 166 : 152 : MIN 312/014272 Exygen Study No.: 023-080 FIGURES Exygen Research : 153 : Page 33 of 166 MIN 312/014272 Exygen Study No.: 023-080 Figure 1. Typical Calibration Curve for PFOS 092002A R .rdb (P FO S ): Linear Regression ("1 / x" weighting): y = 8 35 .52 6 x + 3 4.5 29 9 (r = 0 .9 9 64 31 7 ) 4532 4400 4200 4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 Concentration, ng/mL Exygen Research Page 34 of 166 : 154 : MIN 312/014272 Exygen Study No.: 023-080 Figure 2. Typical Calibration Curve for PFOA 092002A R .rdb (P FO A ): Linear Regression ( 1 / x weighting) 18352 833 138 0.9973981 3e4 0e4 5e4 0e4 5e4 0e4 5e4 0e4 5e4 0e4 5e4 0e4 5e4 0e4 5e4 0e4 5e4 0e4 5000 Concentration, ng/mL Exygen Research Page 35 of 166 : 155 : MIN 312/014272 : 156 : MIN 312/014272 Exygen Study No.: 023-080 Figure 4. Chromatogram Representing a 0.1 ng/mL standard for r r u a ana r r wa 09/23/02 Simple Nmc `092002AB-3011 Sampl. ID 'Stand. 09/23/02 Exygen Research 157 Page 37 of 166 MIN 312/014272 : 158 : MIN 312/014272 Exygen Study No.: 023-080 Figure 6. Chromatogram Representing a Rat Liver Control Sample ior r r u a ana rr irxygen 10 uzuioo** i^oniroi a , aei 092602A) 0.361 no/ml. 09/27/02 SempleName: "09262A SempleID: `Ctrl Masses): *413.0/360.01 I Comment *0201684Contrai A* 09/27/02 \rvN J 1 10.6728 A Exygen Research 159 Page 39 of 166 MIN 312/014272 : 160 : MIN 312/014272 Exygen Study No.: 023-080 Figure 8. Chromatogram Representing Control Rat Serum for PFOS d llU I F V /iij AJL/ l / ^ U l O O / y V / U I l l l U l O C l* j 09/20/02 SamptaName: -0fi2002A-106' SampleID: 'Ctrl' Vat Exygen Research 161 Page 41 of 166 MIN 312/014272 : 162 : MIN 312/014272 163 MIN 312/014272 : 164 : MIN 312/014272 Exygen Study No.: 023-080 Figure 12. Chromatogram Representing Control Rat Serum fortified m m iw u s u il i u i r r u o a m i r r u A o x y g e n m ; u u io o Spk A, Set: 092002A) QC 0.250 09/20/02 Sample Name: '082002A-1 ID: 'Spk' I Pe* Name *PFQA' Um (m ): -413.0/389.0an Comment'0201882 Spk A 10nQ/mL' Annotabc S u p la Indaxi 14 Saapla Type i QC Concentrations 0.250 na/aL FU: 'ml aerum.vrftf 09/20/02 Exygen Research 165 Page 45 of 166 MIN 312/014272 : 166 : MIN 312/014272 : 167 : MIN 312/014272 168 : MIN 312/014272 169 MIN 312/014272 170 : MIN 312/014272 Exygen Study No.: 023-080 APPENDIX A Study Protocol MIN/312 (Exygen Study No. 023-080) and Amendments and Deviations Exygen Research : 171 : Page 51 of 166 MIN 312/014272 : 172 : MIN 312/014272 : 173 : MIN 312/014272 : 174 MIN 312/014272 Exygen Study No.: 023-080 Stndr Number : MIN/312 Huntingdon Life Sciences PERFLUO RO OCTANESULFO NYL FLUO RIDE (POSF; T-7661.1) PRELIM INARY TOXICITY ST U D Y BY INHALATION ADM INISTRATION TO CD RATS FO R I WEEK Enquiry N um ber 23923A N um ber of pages for internal distribution: 17 This working-document is approved for circulation and use: Date ( J Primary location o f study Huntingdon Research Centre Huntingdon Cambridgeshire PE284HS Building Number: All procedures to be performed at the above site unless otherwise detailed below. Location o f specific tasks Histology SEM X-ray analysis Test substance/metabolite analysis : Routinely done at H untingdon Research Centre, Huntingdon, Cambridgeshire, PE28 4HS, but may be performed at Eye Research Centre, Eye, Suffolk, IP23 7PX for logistical reasons. : University o f Plymouth. : 3M. Final Prnfnml Exygen Research Page 55 of 166 : 175 : MIN 312/014272 Exygen Study No.: 023-080 Study Number : MLN/312 Huntingdon Life Sciences CONTENTS 1. INTRODUCTION 2. STUDY SCHEDULE AND STRUCTURE 2.1. Duration of treatment 2.2. Scheduled time plan 2.3. Identity o f treatment groups 3. TEST SUBSTANCE AND ATM OSPHERE GENERATION 3.1. Test substance 4. ANIMAL MANAGEMENT 4.1. Animals --supply, acclimatisation and allocation 4.2. Animals - housing, diet and water supply 5. INHALATION PROCEDURES 5.1. Pre-study characterisation 5.2. Test atmosphere generation 5.3. Test atmosphere administration 5.4. Test atmosphere monitoring 6. SERIAL OBSERVATIONS 6.1. Clinical observations 6.2. Mentality 6.3. Bodyweight 6.4. Food consumption 6.5. W ater consumption 7. NECROPSY AND HISTOLOGY 7.1. Pre-terminal urine sampling 7.2. Test substance/metabolite analyses 7.3. Method o f kill 7.4. Macroscopic Pathology 7.5. Organ weights 7.6. Fixation 7.7. Histology 7.8. PCNA staining for cell proliferation (Table 1) 8. PATHOLOGY 8.1. Light microscopy 8.2. Extension o f initial examination 9. DATA TREATMENT 9.1. Statistical analysis . 10. REPORTING . 11. QUALITY ASSURANCE AND ARCHIVING PROCEDURES 11.1. Quality Assurance 11.2. Archiving Page 3 4 4 4 5 5 6 6 6 7 8 8 9 9 .10 10 10 11 11 11 11 12 12 12 12 12 13 13 13 13 15 15 15 15 15 15 16 16 17 Exygen Research : 176 : Page 56 of 166 MIN 312/014272 : 177 : MIN 312/014272 Exygen Study No.: 023-080 Study Number : MIN/312 Huntingdon Life Sciences Animal model Route Treatment groups and dosages Group Compound Exposure level (mg/1) Dosale (nrmri CD Rats, accepted by regulatory agencies, background data available. Inhalation. Air Control Perfluorooctanesuifonyl Fluoride (POSF) STUDY SCHEDULE AND STRUCTURE 2.1. Duration o f treatment Minimum period 1 week (five days). The treatment period may be extended, with the Sponsor's consent, to incorporate any additional observations considered necessary; documented in an amendment to protocol. Throughout the necropsy period treatment will continue and serial observations will be recorded at appropriate intervals (Section 6). Data for any additional complete weeks before commencement of necropsies will be included in the final report. Scheduled time plan Animals to arrive Treatment to commence Terminal sacrifice to commence Histopathology to be completed Draft report to be issued September 2001 September 2001 September 2001 October 2001 November 2001 (estimated) (estimated) Exygen Research : 178 : Page 58 of 166 MIN 312/014272 Exygen Study No.: 023-080 Study Number :MIN/312 2 3 . Identity o f treatment groups (to be selected from 20 animals ordered) Treatment Huntingdon Life Sciences Num ber o f animal.! Perfluorooctanesulfonyl Fluoride (POSF) Group 1 2 Cage numbers M ale Female 13 24 Animal numbers Male F em ale 1-5 11-15 6-10 16-20 3. TEST SUBSTANCE AND ATMOSPHERE GENERATION In order for H untingdon Life Sciences to comply with the Health and Safety at W ork etc. Act 1974, and the Control of Substances Hazardous to Health Regulations 1999, it is a condition of the study that the Sponsor shall provide Huntingdon Life Sciences w ith all information available to it regarding known or potential hazards associated with the handling and use of any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply with all current legislation and regulations concerning shipment o f substances by road, rail, sea o r air. Such information in the form o f a completed Huntingdon Life Sciences test substance data sheet must be received by Safety Management Services at Huntingdon Life Sciences before the test can be handled in the laboratory. A t the discretion o f Safety Management Services at Huntingdon Life Sciences, other documentation containing the equivalent information m ay be acceptable. Information received will be used to set the Huntingdon Life Sciences Hazard Class, which safety precautions taken in the workplace. Huntingdon Life Sciences Hazard Class: Exygen Research : 179 Page 59 of 166 MIN 312/014272 : 180 : MIN 312/014272 : 181 : MIN 312/014272 : 182 : MIN 312/014272 : 183 : MIN 312/014272 : 184 : MIN 312/014272 : 185 : MIN 312/014272 : 186 : MIN 312/014272 Exygen Study No.: 023-080 Study Number : MINA)12 Huntingdon Life Sciences 7 5 . O rgan weights (Table 1) Data collection Data presentation For bilateral organs, left and right organs will be weighed together unless otherwise specified on the Pathology Procedures Table. Organ weights are not routinely recorded fo r anim als killed or dying prematurely. Absolute. Adjusted fo r terminal bodyweight. 7.6. Fixation (Table 1) Standard Others Fresh frozen liver 7.7. Histology (Table 1 and Section 8.1) Processing Routine staining Special staining Zinc Formalin. Eyes: hi Davidson's fluid. Urinary bladder Bourns. Inflate lungs with fixative and slightly distend the urinary bladder with fixative. A t term inal kill and after organ weighing, a sample o f liv er will be removed from all animals, immediately frozen by inmxsrsion in liquid nitrogen and stored at -70C prio r to despatch to the Sponsor. A ll animals killed o r dying prematurely. All terminal animals. Urinary b lad d e r section sagittally, using one half fo r SEM Xray m icroprobe analysis, and the other h alf for light m icroscopy and cell proliferation index. Kidney pelvis: sections to be present for light microscopy. 4-5 pm sections stained with haematoxylin and eosin. None. 7.8. PCNA staining fo r cell proliferation (Table 1) Sections of urinary bladder will be taken from all animals for PCNA immunostaining in order to assess the degree o f cell proliferation present. A total o f 3,000 cells will be counted from 3 separate sections (1,000 cells/section) in order to determine the cell proliferation TM w Positive PCNA staining cells will be counted, and the examining pathologist will assess the sections and count S-phase positive cells, if practicable. In addition sections of the duodenum from each animal will be taken and stained to act as positive controls for the methodology. Piviral OmfivAnl Exygen Research Page 67 of 166 : 187 : MIN 312/014272 Exygen Study No.: 023-080 Study Number : MIN/312 TABLE 1-Pathology procedures Adrenal Huntingdon Life Sciences Pathology Light microacopy PCNA Femur (longitudinal section through joint) Larynx (2 levels) Lungs (section from all lobes including bronchi) Nasal turbinates (3 levels) Salivary glands (submandibular/rabUngual) cervical, lumbar and thoracic levels) Thyroid with parathyroids Trachea (2 levels) Urinary bladder (with kidney pelvis) Uterus with cervix Including nasal cavity, paranasal sinuses and nasopharynx. Organs weighed, samples fixed or sections examined microscopically Exygen Research : 188 : Page 68 of 166 MIN 312/014272 Exygen Study No.: 023-080 Study Number : MIN/312 H untingdon Life Sciences PATHOLOGY 8.1. Light microscopy Category Animals Premature deaths All from all groups Terminal sacrifice All animals. All specified in Table 1. Peer Review Carried out by a reviewing pathologist to internationally accepted standards. 8.2. Extension o f initial examination may be used to evaluate individual lesions. Details o ftb ese techniques will be documented and retained the archives Light microscopy may be extended, following consultation with the Sponsor, as follows: Any such requirement will be documented in an amendment to the protocol. SEM o f urinary bladder epithelium for assessment of possible necrosis. DATA TREATMENT Statistical analysis Data-types The following data types will be analysed at each timepoint separately > bodyweight, food consumption and water consumption, over appropriate study periods. organ weights, both absolute and adjusted for terminal bodyweight where appropriate Methods For categorical data, the proportion of animals will be analysed using Fisher's E xact test for each treated group versus the control. For continuous data, Bartlett's test will first be applied to test the homogeneity o f variance between the groups. Using tests dependent on the outcome o f B artlett's test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary. Final Protocol Exygen Research Page 69 of 166 : 189 : MIN 312/014272 Exygen Study No.: 023-080 Study Number : MIN/312 Huntingdon Life Sciences REPORTING Study progress Draft final report Authorised final report Periodic verbal and written updates on study progress will be provided by the Study Director. Routine synopsis reports w ill be sent after 1 week and at termination o f the in-life phase. For review by the Sponsor. After approval from the Sponsor; Routinely reports are supplied on A4 paper. The following num bers o f reports are supplied Type of report Draft report Authorised final Photographic report (if any) Double-sided Double-sided Single-sided Single-sided N um ber o f copie Bound Unbound Any additions or corrections to an authorised final report will be documented as a formal addendum/amendment to the final report. In file a nf inn aat llitsve , fslullcfihh natnt beess.vi<egemnnnctaae',nnaaoAd.lllfl.iionsmoaesnniuanmg.etfo--e4triwh--ni)a.eg.llsfciwon.vmai.1.ll1ml_r_tbeu_rpe._n_-oi-t_-crr--ttaa--tnf-i-rs_o-o---Cf-.n__m-e-__s-r_-,r-_--t-e_-hH---d-1i`--u-s-_-t-i-n-o-s-_-t-t_--iu--d,n--1-di*g1-ej>yd'*aos'"rni*cxthLmtiivfoeen. tS.mh.Ac.siineyaynMf_ctsSesueIurDs.bt.isarssceaseqsuuwqeuceuricvveyomeneif_sstrtmhretmeeqheqeueUduenTrersiasatggsitfLmshtttofmoIttronor m odifications', correcons o r additions t--o t-^-h--e---f-i-nal *r"e1pTMo"rt w ill bUVe t1h*1e 3sUuWbjJeCcWt IoUlf ia formal reoort amendm ent (( norr nn eewtu sirttun/d4y,r, a- -s appropriate) a__n_dl w__imlli be subject to add. i.t.ional. c o s t * QUALITY ASSURANCE AND ARCHIVING PROCEDURES 11.1. Quality Assurance The following will be inspected o r audited in relation to this study. Protocol Audit Study based inspections Report Audit Authorised protocol and any amendments. Critical phases o f this study will be inspected. The draft report and study data will be audited after issue o f the draft report to the Sponsor. Test substance/metabolite assay This phase o f the study and associated reports will be conducted and produced according to local SOP's and subject to the Sponsor's ow n QA procedures. QpconAmmnpflilnnefdtiiinontn-gt srotwff ill be aeaanchh arcet*pioonrt,ede--x--t-c-o-e--pt--ht effo_S_rt_u_p_dr_*yo__c-D_-e_--s_i-r_s--ei-.-cb--ta-o--s-re-1-d-a--n-i-dn-rstCupueocimotgi.powannusye, iwHMh-a{i.JnclUah_gUweipmiUlelynbOteD*prroepmorprttleJyd on to appropriate Company Management only Final Protocol Exygen Research Page 70 of 166 : 190 : MIN 312/014272 Exygen Study No.: 023-080 Study Number : M1N/312 Huntingdon Life Sciences U Z Archiving All raw data, samples and specimens arising from the performance o f this study will rem ain the property of the Sponsor. Types o f sample and specimen which are unsuitable, by reason o f instability, for long term retention and archiving m ay be disposed o f after the periods stated in Huntingdon Life Sciences Standard Operating Procedures. All other samples and specimens and all raw data will be retained by Huntingdon Life Sciences in its archive for a period o f five years from the date on which foe Study Director signs foe final report. After such time, foe Sponsor will be contacted and his advice sought on foe return, disposal or further retention o f the materials. I f requested, Huntingdon Life Sponsor. Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy o f foe final report in its archive indefinitely. Exygen Research Final Protocol : 191 : Page 71 of 166 MIN 312/014272 : 192 : MIN 312/014272 : 193 : MIN 312/014272 Exygen Study No.: 023-080 Study Num ber : MIN/312 Protocol Am endment Number : 1 Huntingdon Life Sciences 3. TEST SUBSTANCE AND ATMOSPHERE GENERATION In order for Huntingdon Life Sciences to comply with the Health and Safety at W ork etc. A ct 1974, and die Control o f Substances Hazardous to Health Regulations 1999, it is a condition o f undertaking the study that the Sponsor shall provide Huntingdon Life Sciences with all information available to it regarding known or potential hazards associated with the handling and use o f any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply with all current legislation and regulations concerning shipment o f substances by road, rail, sea or air. Such information in the form o f a completed Huntingdon Life Sciences test substance data sheet must be received by Safety Management Services at Huntingdon Life Sciences before the test substance can be handled in the laboratory. At the discretion o f Safety Management Services at Huntingdon Life Sciences, other documentation containing the equivalent information may be acceptable. Information received will be used to set the Huntingdon Life Sciences Hazard Class, which determines safety precautions taken in the workplace. Exygen Research : 194 : Page 74 of 166 MIN 312/014272 Exygen Study No.: 023*080 Study Number : MIN/312 Protocol Am endment Number Huntingdon Life Sciences TABLE 1-Pathology procedures Abnormalities Adrenals Aorta - thoracic C o lo n Epididymides Femur (longitudinal section through joint) Necropsy Histology Pathology Light microscopy Jejunum Kidneys (wife pelvic region) Larynx (2 levels] Lungs (section from all lobes including bronchi) Lymph nodes - mandibular tracheobronchial Mammary area -caudal Nasal turbinates (3 levels) Oesophagus Pituitary Salivary glands (submandibular/sublingual) Sciatic nerves Spinal cord (transverse and longitudinal sections at the cervical, lumbar and thoracic levels) Thyroid wife parathyroids Trachea (2 levels) Urinary bladder Uterus with cervix Including nasal cavity, paranasal sinuses and nasopharynx. Organs weighed, samples fixed or sections examined microscopically Exygen Research : 195 : Page 75 of 166 MIN 312/014272 Exygen Study No.: 023-080 Study Number : MIN/312 Protocol Am endment N um ber : 2 Huntingdon Life Sciences PERFLUO RO OCTANESULFO NYL FLUO RIDE (POSF; T-766I.1) PRELIM INARY TOXICITY STUDY BY INHALATION ADM INISTRATION TO CD RATS FOR 1 WEEK Total num ber of pages: 4 n m u ra r o i pages io r in te rn a i a isrriD u n o n : 4 Study Director T.J. Kenny, B.Sc (Hons.). The signature o f the Study Director authorises the implementation o f this amendment to protocol, in this amendment, deleted statements are struck through and new statements are underlined. Any changes to the study design after the date o f this authorising signature will be documented in a further formal amendment. AMENDMENT APPROVAL F or H untingdon IlfEyScientes Ltd Authorised (Study Diie For the Sponsor Approved by y Date: X I ] Exygen Research : 196 : Page 76 of 166 MIN 312/014272 Exygen Study No.: 023-080 Study Number Protocol A m endm ent Num ber Huntingdon Life Sciences PERFLUO RO OCTANESULFO NYL FLUO RIDE (POSF; T-7661.1) PRELIM INARY TOXICITY STUDY BY INHALATION ADM INISTRATION TO CD RATS FOR I WEEK Reasons for amendm ents : Clarification of name and address o f Principal Investigator for SEM X-ray analyses Change to fixative following discussion with Sponsor. Addition/clarification o f QA and Archiving procedures. Am endments 7. N E C R O PSY AND H ISTO LO G Y 7.1. Pre-term inal u rine sampling Individual urine samples will be collected within 2 hours following lights on in the animal holding room. The urine samples will be immediately assayed for pH using a microelectrode, centrifuged to obtain the sediment and the prepared SEM stubs despatched to the Principal Investigator at Roy Moate, at University o f Plymouth, Drake Circus, Plymouth. PL4 8AA. for calculi and crystal analysis by SEM X-ray element identification. I f fresh void urine is not obtained from a proportion o f the animals within the time allowed, then a sample will be removed from die urinary bladder at necropsy. ' A a r e v p f the Principal Investigator's report will be included as an addendum to the smHy report. All raw data relevant to the analyses will be returned to Huntingdon Life Sciences for archiving Exygen Research : 197 : Page 77 of 166 MIN 312/014272 : 198 : MIN 312/014272 Exygen Research 199 : Page 79 of 166 MIN 312/014272 Exygen Study No.: 023-080 ...SEP. 19.2002,10:12AM .MEDICAL 220 2E 02 55r. utunvj 11 oe INHAlftiiun iua u FAX:014I0I9228S P. 001 Poe-*Fax Note 7871 TtUrt r^merr EfTMH nt fZT# FAX HEADER SHEET WU -3 Huntingdon Life Sciences Workingforabetterfttture TOTALNUMBER o f PAGES INCLUDINGTHIS PAOBi I f (h (nnimjJwioD b antb& etory p le a * conta u t <w Tali +44 (0) 1480 892224 ATTEVTinW n il. COMPANY FAXNUMBER. n . *nuMm m v j u m RUfea X yci Research, DO]8142721039 MESSAGE; FROM; T erry Kenny daTB 18$September2002 J B ntenhoff (F ax 001 S51 733 1773) R ej P e r flu o r a a c ta n en lflm y l flnorfde (PO SF; T-7661 4V w, f " >3 4-Wtekfollowed by3; recovery D ear Dr Graasni, ' pro eo l amendment 3 for the preliminary jtudy MIN/3 J2. ^ v e u d y aeth er with a copy o f Best regaras Terry Kenny Direct dial: +44 1480 892070 Local facsim ile m achine: +44 1480 893238 MuuaoBd Exygen Research " &*(*. T.h*44) M <92000Pa: +4*rt lean BOOrtSJ Page 80 of 166 : 200 : MIN 312/014272 : 201 : MIN 312/014272 Exygen Research : 202 : Page 82 of 166 MIN 312/014272 : 203 : MIN 312/014272 OCT. 24. 2003 8:43AM MEDICAL 220 2E 02 Studv Number MLN/312 Protocol Amendment Number : 4 Exygen Study No.: 023-080 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF; T-7661.1) PRELIMINARY TOXICITY STUDY BY INHALATION ADMINISTRATION TO CD RATS FOR 1 WEEK Totil number of pages: 3 Number of pages tor internal distribution; 3 Study Director : T J. Kenny, B.Sc (Hons.). The signature of the Study Director authorfaea the implementation of this smendment to protocol. In this amendment, deleted statements ire struck through and new statements ire underlined. Any changes to the itudy design liter the date of this authorising signature will be documented in i further formal amendment. AMENDMENT APPROVAL ForHuntfngdoi Authorised by: (Study Di Ltd For the Sponsor Approved by: Pte: S 3303. Date: / ( S * * 6 * " i * i 2 . 0 & 1 9:4amRECEIVED TIME OCT.24 PRINT TIME OCT.24. Pagel 9 : SIAM Exygen Research Page 84 of 166 : 204 : MIN 312/014272 : 205 : MIN 312/014272 RECEIVED TIME OCT.24. 9:4801 PRINT TIME OCT.24. Page 3 9:5001 Exygen Research Page 86 of 166 : 206 : MIN 312/014272 Exygen Study No.: 023-080 Precise Research. Pro ven Results. PRO TO CO L DEVIATION Deviation Number: 1 Date of Occurrence: 09/2 7-28 /02 Exygen Study Number: 023-080 Protocol N u m b e r M IN /312 DESCRIPTIO N OF DEVIATION Am endm ent'#4 ExM-023-071 Revision 1 Section 4 .5 .3 .e - accepted recoveries of 134%, 1 3 5 % 140% and 135% fo r fortifications for PFOA in Set 092602A. Deviation issued. ACTIONS TAKEN for .example - deviation Issued. SOP revision, etc Recorded No negative im pact on study. A IIAMAPPAA CPTT OHMN SQTTIU D Y Date M anagem ent Signature (Sponsor) U:\Forms\PROTOCOL DEVIATION.doc Exygen Research -0/ Date Date 7*6-F*** ~2&&0 Date . Date Exygen Q A U R eview r h , Wj x/ \ r) ( o % 9/25/2002/5 3 0 5 8 Research D rive State College, PA 1 6 8 0 1 , USA T: 80 0.281.3219 F: 814.272.1019 exygen.com Page 87 of 166 : 207 : MIN 312/014272 Exygen Study No.: 023-080 Precise Research. \ Proven Results. Page PROTO CO L DEVIATION Deviation Number. 2 Date of Occurrence: 0 9 /2 4 /0 2 , 0 9 /3 0 /0 2 of 1 Exygen Study Number: 023-080 Protocol Number: M IN /3 12 DESCRIPTION OF DEVIATION Am endm ent #4 ExM-Q23-071A Revision 2 Section 4 .4 .2 - used 0.05 mL fo r serum sample Exygen ID 0202782 in Set 092402G8. A m e n d m e n t #4 E x M -023-071A Revision 2 S ection 4 .5 .4 .f - a c re n te d rprovprip; n f for fortifications .for POSF in Set 093002A. anH Deviation issued. ACTIONS TAKEN for exam ple - deviation Issued. S O P revision, etc Recorded By: No negative im pact on study. D IMPACT ON STUDY Date:Ml% b Investigator Signature M anagem ent SSilgannaattuure (Sponsor) fy U:\Forms\PROTOCOL DEVIATION.doc Exygen Research Date Date X ' F 3 -2 . > 0 't Date Date Exygen QAU Review \ h . /i'i b 3> 9/25/2002/5 3058 Research Drive State College, PA 16801, USA T: 800.281.3219 F: 8 1 4 .2 7 2 .1 0 1 9 exygen.com Page 88 of 166 : 208 MIN 312/014272 Exygen Study No.: 023-080 APPENDIX B /Ai .nu oa liijr fi iinc oa li Ai T/ fi av ful iinu/uI *. Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum and Urine (Exygen Method No. ExM-023-071) Exygen Research : 209 : Page 89 of 166 MIN 312/014272 Exygen Study No.: 023-080 . TITLE Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), . Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid CPFOA) in Rat Liver, Serum and Urine Revision 1 AUTHORS ' John Flaherty, Karen Risha, and Emily Decker DATE ISSUED November 20,2002 SPONSOR 3M Medical Department Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, M N 55144-1000 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 METHOD NUMBER ExM-023-071 Revision 1 TOTAL NUMBER OF PAGES 43 / --V. ' Exygen Research : 210 : Page 90 of 166 MIN 312/014272 : 211 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod N o: ExM -023-071 Revision 1 TABLE OF CONTENTS TITLE MANAGEMENT APPROVAL................................................................................................ 2 TABLE OF CONTENTS............................................................................................................ 3 LIST OF T A B LE S.......................................................................................................................4 LIST OF FIGURES 1. SUM M ARY................................................................................. ......... .............................7 2. EXPERIMENTAL COMPOUNDS 3. CHEMICALS AND SUPPLIES...................................................................................... 9 3.1. Chemicals............................................................................................................ 9 3.2. Standards........................................................................................................... 9 3.3. Equipment and Supplies.....................................................................................9 3.4. S o l u t io n s ........................................................................................................... 10 3.5. Preparation of Standard Solutions........................................................ 10 3.3.1. otocxsolution 3.5.2. Fortification Solutions 3.5.3. Calibration Standards............ .......................................................................11 4. M ETHOD.......................................................................................................................... 12 4.1. Flow Diagram....................................................................................................12 4.2. Sample Processing........................................................................................... 12 4.3. BatchSetu p...................................................................................................... 13 4.4. Sample Extraction..........................................................................................13 4.4.1. Liver Extraction..........................................................................................13 4.4.2. Serum and U nne Extraction..................................................................... 13 4.4.3. SPE Column Conditioning....................................................................... 14 4.5. Quantitation......................................................... ...........................................14 4.5.1. LC/MS/MS System and Operating Conditions.........................................14 4.5.2. Calibration Curve Procedures..................................................................... 15 4.5.3. Sample Analysis.........................................................................................16 4.6. Acceptance Criteria..................................................................................... 17 4.7. Performance Criteria.................................................................................. 17 4.8. Time Required f o r Analysis........................................................................18 5. CALCULATIONS........................................................................................................... 18 6. SAFETY.............................................................................................................................19 Exygen Research Exygen Research : 212 : Page 3 of 43 Page 92 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen Method No: ExM-023-071 Revision 1 LIST OF TABLES Table 1. Recovery Summary of PEHS in R at liv e r and Serum............. Table 2. Recovery Summary of PFOS in Rat Liver, Serum and U rine. Table 3. Recovery Summary of PFOA in Rat Liver, Serum and Urine. 20 21 22 Exygen Research Exygen Research : 213 : Page 4 of 43 Page 93 of 166 MIN 312/014272 : 214 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision 1 LIST OF FIGURES (continued) Figure 25. Representative Chromatogram of a Control Liver Sample Fortified at 10 ng/g with P F O S ............................................................................................. 36 Figure 26. Representative Chromatogram o f a Control Liver Sample Fortified at 10 ng/g with PFOA............................................................................................. 37 Figure 27. Representative Chromatogram of a Control Liver Sample Fortified at 50 ng/g with P F H S ............................................................................................. 37 Figure 28. Representative Chromatogram o f a Control Liver Sample Fortified at 50 ng/g with P F O S ............................................................................................. 38 Figure 29. Representative Chromatogram o f a Control Liver Sample Fortified at 50 ng/g with PFOA............................................................................................. 38 Figure 30. Representative Chromatogram of a Control Serum Sample Fortified at 10 ng/mL with PFHS..........................................................................................39 Figure 31. Representative Chromatogram of a Control Serum Sample Fortified at 10 ng/mL with PFOS..........................................................................................39 Figure 32. Representative Chromatogram of a Control Serum Sample Fortified at 10 ng/mL with PFO A .........................................................................................40 Figure 33. Representative Chromatogram o f a Control Serum Sample Fortified at 50 ng/mL with PFHS.................. .......................................................................40 Figure 34. Representative Chromatogram o f a Control Serum Sample Fortified at 50 ng/mL with PFOS..........................................................................................41 Figure 35. Representative Chromatogram of a Control Serum Sample Fortified at 50 ng/mL with PFO A .........................................................................................41 Figure 36. Representative Chromatogram o f a Control Urine Sample Fortified at 10 ng/mL with PFOS..........................................................................................42 Figure 37. Representative Chromatogram o f a Control Urine Sample Fortified at 10 ng/mL with PFO A .........................................................................................42 Figure 38. Representative Chromatogram o f a Control Urine Sample Fortified at 50 ng/mL with PFOS..........................................................................................43 Figure 39. Representative Chromatogram of a Control Urine Sample Fortified at 50 ng/mL with PFO A .........................................................................................43 Exygen Research Exygen Research : 215 : Page 6 o f 43 Page 95 of 166 MIN 312/014272 Exygen Study No.: 023-080 /- E xygen M ethod No: ExM -023-071 Revision 1 1. SUMMARY . This report details a method of analysis for residues of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum and Urine. Residues o f PFHS, PFOS and PFOA are extracted from each matrix with acetonitrile. The acetonitrile extract is added to water and loaded onto a conditioned C l 8 solid phase extraction (SPE) cartridge. Analyte residues are eluted with 2 mL o f methanol. Quantification o f PFHS, PFOS and PFOA is accomplished by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using multiple reaction monitoring (MRM). The proposed limit o f quantitation (LOQ; the lowest fortification specified by the method which gives adequate recovery according to EPA guidelines) for this method is 10 ng/g (parts-per-billion) each for PFHS, PFOS and PFOA. The theoretical limit of detection (LOD) will be based on the signal to noise ratio and will be at least greater than 3 times the level of noise, based on the instrumentation system used. For all analytes, the lowest analytical standard corresponds to 0.1 ng/mL. This method was developed using rat liver, serum and urine. Typical percent recoveries standard deviations (at 10 and 50 ng/g) are shown below: Representative calibration curves are shown in Figures 1-3. Representative chromatograms are shown in Figures 4 to 39. Exygen Research Page 7 of 43 Exygen Research : 216 : Page 96 of 166 MIN 312/014272 Exygen Study No.: 023-080 E xygen M ethod N o: ExM -023-071 Revision 1 EXPERIM ENTAL COM POUNDS The structures for PFHS, PFOS and PFOA are given below. PFHS Chemical Name Molecular weight Perfluorohexanesulfonate 399, as shown PFHS is supplied as the potassium salt (C6F13SO3IC ), molecular weight = 438 PFOS Chemical Name Molecular weight Perfluorooctanesulfonate 499, as shown PFOS is supplied as the potassium salt (CgFnSOj'iC) molecular weight = 538 PFOA Chemical Name Molecular weight Pentadecafluorooctanoic Acid 413, as shown Exygen Research Exygen Research : 217 : Page 8 of 43 Page 97 of 166 MIN 312/014272 : 218 : MIN 312/014272 Exygen Study No.: 023-080 Exygen Method No: ExM-023-071 Revision 1 Notes: 1. In order to avoid contamination, the use o f disposable labware (containers, tubes, pipettes, etc.) is highly recommended. 2. Teflon or Teflon-lined containers or equipment should not be used. 3. It may be necessary to check the solvents (acetonitrile, methanol) for the presence of contaminants (especially PFOA) by LC/MS/MS before use. Certain lot numbers have been found to be unsuitable for use. 4. Use disposable micropipettes or pipettes to aliquot standard solutions when preparing standards and samples for extraction. 5. Equivalent materials may be substituted for those specified in this method. 3.4. Solutions (1) 2 mM ammonium acetate in water is prepared by weighing 0.134 g o f ammonium acetate and dissolving in 1 L of water. (2) Hypercarb filtered type I water is prepared by filtering type I water through a Hypercarb guard column using a HPLC pump at -2-3 mL/min. Before use, wash the guard cartridge with -2 5 mL of HPLC grade acetonitrile, then - 25 mL of type I water, then begin collecting the filtered type I water eluate for use in the extraction. Repeat the wash after filtering -2 L o f water. Note: The aforementioned example is provided for guidance, alternative volumes may be prepared as long as the appropriate ratios o f the solvent to solute are maintained. 3.5. Prparation of Standard Solutions Analytical standards are used for three purposes: 1. Calibration Standards - These standards are prepared in methanol and are used to calibrate the response of the detector used in the analysis. 2. Laboratory Control Spikes - These fortifications are prepared at concentrations corresponding to the LOQ and 5x LOQ and are used to determine analytical recovery. Laboratory control spikes are prepared in control matrix. 3. Matrix Spikes - These fortifications are prepared by spiking into the field samples at a known concentration. Matrix spikes are used to evaluate the effect o f the sample matrix on analytical recovery and are prepared at the client's request. The analyst may vary the absolute volumes of the standards as long as the correct proportions of solute to solvent are maintained. Exygen Research Page 10 of 43 Exygen Research : 219 : Page 99 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen Method No: ExM-023-071 Revision 1 3.5.1. Stock solution Prepare individual stock solutions at 100 pg/mL for PEHS, PFOS and PFOA by weighing out 10 mg of each analytical standard (corrected for purity and if necessary, salt content) and dilute to 100 mL with methanol in separate 100-mL volumetric flasks. The stock solutions (in 125-mL LDPE bottles) are to be stored in a refrigerator at 2C to 6C and are stable for a maximum period of one year from the date of preparation. 3.5.2. Fortification Solutions a. Prepare a mixed fortification standard at 1.0 pg/m L (1000 ng/mL) of PFHS, PFOS and PFOA by adding 1.0 m L o f each of the 100 pg/mL stock solutions into a 100-mL volumetric flask and bring up to volume with methanol. b. Prepare a mixed fortification standard at 0.1 pg/m L (100 ng/mL) of PFHS, PFOS and PFOA by diluting 10.0 mL o f the 1.0 pg/m L mixed fortification solution to 100 mL with methanol in a volumetric flask. Example: one hundred microliters of the 0.1 pg/mL solution spiked into 1 g o f liver or 1 m L of serum/urine is equivalent to a 10 ppb (10 ng/mL or ng/g) fortification. Store all fortification standard solutions in a refrigerator (in 125-mL LDPE bottles) at 2C to 6C for a maximum period of one year from the date of preparation. Note also that additional concentrations may be prepared if necessary. 3.5.3. Calibration Standards LC/MS/MS calibration standards containing PFHS, PFOS and PFOA are prepared at 0 ,1 ,0 .2 ,0 .5 ,1 .0 ,2 .0 and 5.0 ng/mL in methanol via dilution of the 0.1 pg/m L mixed fortification solution (section 3.5.2.b). The following is a typical example; additional concentrations may be prepared as needed. Initial Cone. (ng/mL) 100 100 100 5.0 2.0 1.0 Volume (mL) 5.0 2.0 1.0 10.0 10.0 10.0 Diluted to (mL) 100 100 100 100 100 100 Final Cone. (ng/mL) 5.0 2.0 1.0 0.5 0.2 0.1 The standards may be used for a period o f one year (in 125-mL LDPE bottles) when stored refrigerated (at 2C to 6C). Exygen Research P ag e 11 o f 43 Exygen Research : 220 : Page 100 of 166 MIN 312/014272 : 221 : MIN 312/014272 : 222 : MIN 312/014272 : 223 : MIN 312/014272 : 224 : MIN 312/014272 Exygen Study No.: 023-080 E xygen M ethod No: ExM -023-071 Revision 1 Typical calibration curves for PFHS, PFOS and PFO A can be found in Figures 1 3. 4.5.3 Sample Analysis a. Inject the same aliquot (between 10 to 50 |_tL) of each standard, sample, recovery, control, etc. into the LC/MS/MS system. b. Standards corresponding to at least five or more concentration levels (starting with the LOQ level or below) must be included in an analytical set. c. An entire set of calibration standards should be injected at the beginning of a set followed by calibration standards interspersed approximately every 5-10 samples (to account for a second set o f extracted standards). As an alternative, an entire set o f calibration standards mav be included at the beginning and at the end of a sample set. In either case, calibration standards m ust be the first and last injection in a sample set. d. The concentration o f each sample/fortification/control is determined from the standard curve, based on the peak area o f each analyte. The standard responses should bracket responses of the residue found in each sample set. Results may be quantitated up to 10% outside the curve by extrapolation. If necessary, dilute the samples to give a response within the standard curve range. e. Fortification recoveries falling within 70 to 130% are considered acceptable. f. Samples must be stored refrigerated between 2C to 6C until analysis. g. Samples in which either no peaks are detected or peaks less than the lowest concentration o f the calibration standards are detected at the corresponding analyte retention time will be reported as ND (not detected). Samples in which peaks are detected at the corresponding analyte retention time that are less than the LOQ and greater than or equal to the lowest concentration of the calibration standards will be reported as NQ (not quantifiable). The analysis performed during the method development included fortifications at 10 and 50 ng/g o f PFHS in rat liver, 10 and 50 ng/m L o f PFHS in serum, 10 and 50 ng/g o f PFOS and PFOA in rat liver and 10 and 50 ng/mL of PFOS and PFOA in serum and urine. Typical chromatograms can be found in Figures 4-39. Exygen Research Page 16 o f 43 Exygen Research : 225 : Page 105 of 166 MIN 312/014272 Exygen Study No.: 023-080 E xygen M ethod N o: ExM -023-071 Revision 1 4.6. A c c e p t a n c e C r it e r ia The following criteria must be met to ensure the presence of PFHS, PFOS and PFOA: 1. The chromatogram must show a peak o f a daughter ion at 80 amu from a parent of 399 amu for PFHS, a daughter ion at 99 amu from a parent of 499 amu for PFOS, and a daughter ion at 369 amu from a parent of 413 amu for PFOA. 2. M ethod blanks must not contain analyte at levels greater than the LOQ. If a blank contains the analyte at levels greater than 10 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 3. Recoveries of control spikes and matrix spikes (if any) must be between 70-130% o f their known values. If a control spike falls outside the nn1n(\m 1acceptable limits, the entire set of samples should be re-extracted. Any u--i--a-Liia ayixc _iu__u_l__s_i_u_c___/__v__-_u__v__7_0___s__ii_u__muu u__c__c__v_a_iiu__a_u*.r_uj to__y_. me ana_ly,,st t*o determine if re-extraction is warranted. 4. Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number of calibration standards that could be excluded must not exceed 20% of the total number o f standards injected. 5. The correlation coefficient (R) for calibration curves generated must be >0.9925 (R2 >0.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 6. Retention times between standards and samples must not drift more than 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 4.7. Performance Criteria The following two criteria must be performed once as a system suitability test, before the commencement of analysis, when using an instrumentation set-up that has not been used for this method. First Criterion: Run a standard solution on LC/MS/MS corresponding to the estimated LOQ (10 ng/mL) in matrix and obtain a signal to noise ratio for the analyte transition of at least 9:1, compared to a reagent blank. If this criterion cannot be met, optimize and change instrument operating parameters (or increase the injection volume, if appropriate). Exygen Research P age 17 o f 43 Exygen Research : 226 : Page 106 of 166 MIN 312/014272 : 227 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No; ExM -023-071 Revision 1 c. Use Equation 3 to calculate the amount of analyte found (in ppb) Equation 3: Analyte found (ng/g or ng/mL) = (analyte found Cng/mLl x FV fmLl sample weight (g) or sample volume (mL) FV = final volume For reporting purposes, samples in which either no peaks are detected or peaks less than the lowest concentration o f the calibration standards are detected at the corresponding analyte retention time will be reported as ND (not detected). Samples in which peaks are detected at the corresponding analyte retention time that are less than the LOQ and greater than or equal to the lowest concentration of the calibration standards will be reported as NQ (not quantifiable). 6. SAFETY The analyst should read the material safety data sheets for all standards and reagents before performing this method. U se universal precautions when handling standards and reagents, including working in fume hoods and wearing laboratory coats, safety glasses, and gloves. : 228 : MIN 312/014272 : 229 : MIN 312/014272 : 230 : MIN 312/014272 : 231 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision 1 Figure 1. Calibration Curve for PFHS Compound 1 name: PFHS Coefficient of Determination: 0.996465 Calibration c u n 1916.26 * x + 9.14134 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 9.98e3-i x Response- ng/ml Exygen Research Exygen Research : 232 Page 23 of 43 Page 112 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision 1 Figure 2. Calibration Curve for PFOS Compound 2 name: PFOS Coefficient of Determination: 0.997009 Calibration curve: 3211.30 * x + 48.9285 Response type: External Std, Area Curve type: Linear, Origin: Bclude, Weighting: 1/x, Aris trans: None 1.62e4n Response- i ng/m L Exygen Research Exygen Research : 233 Page 24 of 43 Page 113 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision 1 Figure 3. Calibration Curve for PFOA Compound 1 name: PFO A Coefficient of Determination: 0 .995945 Calibration curve: 31270,9 * x 3675.36 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: 1/x, Ahs trans: None ng/mL Exygen Research Exygen Research : 234 : Page 25 of 43 Page 114 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision l Figure 4. Representative Chromatogram of a 0.1 ng/mL Standard Containing PFHS 0052102-#, 0.1 nmL PFHS 1 mMVJlv>4Mi-t f i u ttw 1.00 2.00 3.00 4.00 3.00 100 7.00 100 9.00 1100 11.00 12.00 Figure 5. Representative Chromatogram of a 0.1 ng/mL Standard Containing PFOS CQ6350W, 0.1 ngftnL PFOAand PFOS Exygen Research Exygen Research : 235 : Page 26 of 43 Page 115 of 166 MIN 312/014272 Exygen Study No.: 023-080 E xygen M ethod No: ExM -023-071 Revision 1 Figure 6. Representative Chromatogram of a 0.1 ng/mL Standard Containmg PFOA 0060002-0,0.1 IW/mtPFOA100 PF08 070602A-112an (Mu, 22) 1001 Figure 7. Representative Chromatogram of a 0.5 ng/mL Standard Containing PFHS C062102-4,05 n^mL PFHS Exygen Research Exygen Research : 236 : Page 27 of 43 Page 116 of 166 MIN 312/014272 : 237 : MIN 312/014272 : 238 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision 1 Figure 12. Representative Chromatogram of a 5.0 ng/mL Standard Containing PFOA C0625Q2-1,5,0 nmL PFOA and PF08 W-Jul-2Q02 20:10:56 LCflIAS/MS #7 MRU of2 Chanrwia E&413> 988 Figure 13. Representative Chromatogram of a Reagent Blank Sample Analyzed for PFHS Exygen Research 7*1.18 Page 30 of 43 Exygen Research : 239 : Page 119 of 166 MIN 312/014272 Exygen Study No.: 023-080 E xygen M ethod N o: ExM -023-071 Revision 1 Figure 14. Representative Chromatogram of a Reagent Blank Sample Analyzed for PFOS Figure 15. Representative Chromatogram of a Reagent Blank Sample Analyzed for PFOA Exygen Research Exygen Research : 240 : Page 31 o f 43 Page 120 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision 1 Figure 16. Representative Chromatogram of a Control Liver Sample Analyzed for PFHS 24-Jun-20Q219:17:57 atLG/MS/MS H MRM 1Channel ES- 3 1.>W8 0 Figure 17. Representative Chromatogram of a Control Liver Sample Analyzed for PFOS 1.00 00 3.00 4.00 ICO &00 7.00 BOO 8.00 iioo Exygen Research Page 32 o f 43 Exygen Research : 241 : Page 121 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision l Figure 18. Representative Chromatogram of a Control Liver Sample Analyzed for PFOA 10O7OaO2A-n0Sm(Mn, 2x2) th M'JuWQOa 21:35d7 LC/MS/MS #7 MRMor2Chamal ES' 413 >35# Z\2M Aim Figure 19. Representative Chromatogram of a Control Serum Sample Analyzed for PFHS 24-*lurv2002 23:38:22 MRMoiL1CC/UhwS/wMISES#7' 3#9>0O Exygen Research Exygen Research : 242 : Page 33 o f 43 Page 122 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision 1 Figure 20. Representative Chromatogram of a Control Serum Sample Analyzed for PFOS 070802A*1z2 Sm(JAi, 2x2) 1.00 2.00 3.00 4.00 5.00 .00 7.00 8-00 9.00 10.00 11.00 Figure 21. Representative Chromatogram of a Control Serum Sample Analyzed for PFOA 07M02Ar1223m (N*v2x2) 10Oi Exygen Research Exygen Research : 243 : Page 34 of 43 Page 123 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No: ExM -023-071 Revision 1 Figure 22. Representative Chromatogram of a Control Urine Sample Analyzed for PFOS 07OO02A-134Sfn(Mn, 2x2) OMui-2oaiM:03:2a LCM$MSt7 MRMof 2 Ctwwgto ES* Figure 23. Representative Chromatogram of a Control Urine Sample Analyzed for PFOA Exygen Research Exygen Research : 244 : Page 35 o f 43 Page 124 of 166 MIN 312/014272 : 245 : MIN 312/014272 : 246 : MIN 312/014272 : 247 : MIN 312/014272 : 248 : MIN 312/014272 : 249 : MIN 312/014272 : 250 : MIN 312/014272 : 251 : MIN 312/014272 Exygen Study No.: 023-080 Analytical Method: Method of Analysis for the Determination of Perfluorooctanesulfonyl Fluoride (POSF) in Rat Urine, Serum, and Liver by GC/MS (Exygen Method No. ExM-023-071A, Revision 2) Exygen Research : 252 : Page 132 of 166 MIN 312/014272 Exygen Study No.: 023-080 TITLE Method of Analysis for the Determination of Perfluorooctanesulfonyl Fluoride (POSF) in Rat Urine, Serum, and Liver by GC/MS AUTHORS John Flaherty and Alan Sensue DATE REVISED August 30, 2002 SPONSOR 3M Center 3M Corporate Toxicology Building 220-2E-02 St. Paul, MN 55133-3220 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 METHOD NUMBER ExM-023-071A, Revision 2 TOTAL NUMBER OF PAGES 34 Exygen Research : 253 : Page 133 of 166 MIN 312/014272 : 254 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 TABLE OF CONTENTS MANAGEMENT APPROVAL............................................................................................2 TABLE OF CONTENTS...................................................................................................... 3 LIST OF TABLES................................................................................................................ 4 LIST OF FIGURES SUMMARY................................................................................................................... 6 EXPERIMENTAL COMPOUNDS 3. CHEMICALS AND SUPPLIES 3.1. Chemicals............................................. ...........................................................,..,8 3.2. Standards 3.3. Equipment and Supplies 3.4. Preparation of Standard Solutions.............................................. ....................... 8 3.4.1. Stock solution.............................................. ;;.......................................... 9 ^ . 9 F rv rfi n r a t in n / P n liK r u ti fn nn sn-n >!fVm tirvr*c 3.4.3. Calibration Standards 4. METHOD................................................................................................ ..................n 4.1. F lo w D ia g ra m ...................................................................................................................... n 4.2. Sample Processing.............................................................................................n 4.3. Batch Set up...................................................................................................... n 4.4. Sample Analysis Preparation............................................................................1 2 4.4.1. Liver Analysis........................................................................................ 1 2 4.4.2. Serum / Urine Analysis...........................................................................12 4.5. Quantitation......................................................................................... ..............n 4.5.1. GC/MS System and Operating Conditions............................................... 13 4.5.2. Autosampler Operating Conditions........................................................14 4.5.3. Calibration Curve Procedures............................................................... 1 5 4.5.4. Sample Analysis......................................................................................1 5 4.6. Acceptance Criteria........................................................................................... 16 4.7. Performance C atena......................................................................................... 1 7 4.8. Time Required for Analysis.............................................................................. 1 7 5. Calculations................................................................................................................ 1 7 6 . S a fe ty ................................................................................................................................................1 9 Exygen Research Exygen Research : 255 : Page 3 o f 34 Page 135 of 166 MIN 312/014272 : 256 : MIN 312/014272 : 257 : MIN 312/014272 Exygen Study No.: 023-080 E x y g e n M eth o d N o. E x M -0 2 3 -0 7 1A R ev . 2 1. SUMMARY This report details a Method o f Analysis for the Determination of 3Perfluorooctanesulfonyl Fluoride (POSF) in R at Urine, Serum, and Liver bv GC/MS. Analysis o f headspace gas is used to detect POSF in rat urine, serum, and liver. Quantification o f POSF is accomplished by gas chromatography / mass spectrometry (GC/MS) analysis. The proposed limit o f quantitation (LOQ; the lowest fortification specified by the method which gives adequate recovery according to EPA guidelines) for this method is 2.5 pg/mL (for serum and urine) or 2.5 pg/g (for liver) for POSF. This method was developed using rat urine, rat serum, and rat liver. Typical *percen,t recoverites are shown in T a b' l e s "I7. --IT7. a n d--m `a*ivj/t vuvinuu*u ,,u,,auuiu_i_a_u_u_i_i curve is shown in Figure 1. Representative chromatograms are shown in F ieures 2 to 12. 6 : 258 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 2. EXPERIMENTAL COMPOUNDS The structure for POSF is given below. POSF Chemical Name: Perfluorooctanesulfonyl fluoride (POSF) Chemical Formula: C8F 17SO2F CAS #: 307-35-7 Molecular Weight: 502 O C F 3( C F 2) 6C F 2- S - F o Exygen Research Exygen Research : 259 : Page 7 o f34 Page 139 of 166 MIN 312/014272 : 260 : MIN 312/014272 : 261 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 3.4.3. Calibration Standards GC/MS analysis calibration standard levels are determined by spiking a small amount (typically 5 pL) into a sealed vial which contains an appropriate amount o f control matrix (100 pL for rat serum and rat urine or 0.1 g rat liver), evaporating the standard, and injecting 1 mL o f headspace. Examples o f calibration standard levels are shown as follows: : 262 : MIN 312/014272 Exygen Study No.: 023-080 E x y g e n M eth o d N o. E x M -0 2 3 -0 7 1A R ev . 2 4. METHOD 4.1. Flow Diagram The flow diagram o f the method is given below, followed by a detailed description o f each step. Method Flow Diagram Weigh ~0.1 g o f liver or measure 100 pL o f serum (or urine) into headspace vial . (seal immediately) .i . Fortify samples designated as matrix spikes and/or laboratory control Knit i* ' Heat to 80C for ~4 minutes for manual injection (heat to 60C for ~3 minutes if using a LEAP Combi-PAL autosampler) .+ Inject 1 mL headspace 4 GC/MS Analysis 4.2. Sample Processing No sample processing is needed for serum or urine samples. However, frozen serum or urine samples must be allowed to completely thaw to room temperature before use. For liver samples, remove approximately 0.1 grams (while still frozen) and place into a 12 mL headspace vial. Immediately seal (cap) the vial. Place vials (containing the liver sample) into a freezer if time to analysis exceeds one day. 4.3. Batch Set up a. A batch o f samples should not contain more than 20 field samples. b. Each batch o f samples analyzed must include at least one control (method blank using control matrix) and one matrix control fortified at a known concentration (typically between the LOQ and lOx LOQ levels). Exygen Research Page II of 34 Exygen Research : 263 : Page 143 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071 A Rev. 2 c. A t least one field sample in each batch must also be separately fortified at a known concentration. Additional samples in the batch may also be fortified based upon the sponsor's requirements. d. Samples will be analyzed in duplicate. 4.4. Sample Analysis Preparation 4.4.1. Liver Analysis . a. Remove ~0.1 g o f liver sample and place into a 12 mL headspace vial. Tightly cap the vial. Fortify sample (if necessary) via syringe. b. Heat to 80C for ~4 minutes for manual injection or heat to 60C for ~3 minutes if using a LEAP Combi-PAL autosampler. c. Inject 1 mL headspace. d. Analyze samples using GC/MS. 4.4.2. Serum / Urine Analysis a. Measure 100 pL o f serum (or urine) sample into a 12 mL headspace vial. Tightly cap the vial. Fortify sample (if necessary) via syringe. b. Heat to 80C for ~4 minutes for manual injection or heat to 60C for ~ 3 minutes if using a LEAP Combi-PAL autosampler. c. Inject 1 mL headspace. d. Analyze samples using GC/MS. Exygen Research Exygen Research : 264 : P age 12 o f 34 Page 144 of 166 MIN 312/014272 : 265 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 4.5.2. Autosampler Operating Conditions Study Plan No. ExP-023-075 Exygen Study No. 023-075 Instrument: Injection / Volume: Incubator / Agitator Temp: Incubator / Agitator Time: Incubator / Agitator Speed: Syringe Temp: Syringe Fill Speed: Syringe Inject Speed: Syringe Pullup Delay: Pre-Injection Delay: Post-Injection Delay: LEAP Technologies Combi-PAL autosampler Headspace / lmL 60C 3 min. 400 RPM - 2 sec. on, 18 sec. off 70C 50 pL/sec. 750 pL/sec. 1 sec. 100 msec. 200 msec. Exygen Research Exygen Research : 266 : Page 14 o f 34 Page 146 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 4.5.3. Calibration Curve Procedures a. Prepare calibration standards by spiking an appropriate volume o f standard solution into a sealed (capped) headspace vial which contains an appropriate amount o f control matrix (100 pL for rat serum and rat urine or 0.1 g rat liver). After the vial is conditioned (heated) for (approximately) four minutes (at 80C), manually inject the same amount o f headspace (1 mL) o f each calibration standard (ranging from the lowest level standard to the highest level prepared), into the GC/MS. If using a LEAP Combi-PAL autosampler for injection, the vial is conditioned (heated) for (approximately) three minutes at 60C (the syringe is heated to 70C). b. Use weighted linear standard curves for quantitation. Linear standard curves are generated for each analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using HP Chemstation software. Any calibration standard found to be a statistical outlier may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that may be excluded must not exceed 20% o f the total number o f standards injected. c. The correlation coefficient (R) for calibration curves generated must be 0.9925 (R2 0.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should b e re-analyzed. A typical calibration curve for POSF can be found in Figure 1. 4.5.4. Sample Analysis a. Prepare samples by weighing (for liver) or measuring (for serum and urine) the sample into a headspace vial. If necessary, spike an appropriate volume of standard solution into the sealed (capped) headspace sample vial. After the vial is conditioned (heated) for approximately four minutes (at 80C), manually inject the same aliquot (1 mL) o f each standard, sample, recovery, control, etc. into the GC/MS system. If using a LEAP Combi-PAL autosampler for injection, the vial is conditioned (heated) for (approximately) three minutes at 60C (the syringe is heated to 70C). b. Standards corresponding to at least five or more concentration levels (starting w ith the LOQ level or below) must be included in an analytical set. c. A n entire set o f calibration standards should be injected at the beginning o f a set followed by a batch o f samples. However, a batch may not consist of Exygen Research p age 15 o f 34 Exygen Research : 267 : Page 147 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 more than twenty samples (excluding spikes and duplicates) until a new calibration curve must be generated. d. The concentration o f each sample/fortification/control is determined from the standard curve, based on the peak area o f each analyte. The standard responses should bracket responses o f the residue found in each sample set. Results may be quantitated up to 10% outside the curve by extrapolation. If necessary, a smaller sample aliquot may be used if compound response exceeds the calibration curve. e. Fortification recoveries falling within 60 to 140% are considered acceptable. Recoveries outside these limits may be accepted with sponsor approval. f. Rat serum and urine samples must be stored frozen at -10C, or refrigerated between 2C to 6C one day prior to analysis, and rat liver must be stored frozen at -10C until analysis. g. Field samples in which no peaks are detected at the corresponding analyte retention time will be reported as ND (not detected). Samples in which peaks are detected at the corresponding analyte retention time that are less than the LOQ will be reported as NQ (not quantifiable). 4.6. Acceptance Criteria The following criteria must be m et to ensure the presence o f POSF: 1. The instrument must have adequate sensitivity (s/n > 10) at the LOQ level. 2. Method blanks must not contain POSF at levels greater than the LOQ. I f a blank contains the analyte at levels greater than the LOQ, analysis will cease until the source o f the contamination is found. 3. Recoveries o f control spikes and matrix spikes should be between 60 140% o f their known values. I f a control spike falls outside the acceptable limits, another spike should be prepared and analyzed. Any matrix spike outside 60-140% should be evaluated by the analyst to determine if re analysis is warranted. 4. Any calibration standard found to be a statistical outlier m ay be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 5. The correlation coefficient (R) for calibration curves generated must be 0.9925 (R2 0.985). I f calibration results fall outside these limits, then Exygen Research Page 16 o f 34 Exygen Research 268 : Page 148 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 4.7. Performance Criteria The following two criteria must be performed as a system suitability test, before the commencement o f analysis when using an instrumentation set-up that has not been used for this method. First Criterion: Prepare and inject a GC/MS standard corresponding to the estimated LOQ to determine if the instrument has adequate sensitivity. If this criteria cannot be met, optimize by changing instrument operating parameters. Second Criterion: r\nl--?--n--n-- a Sfit n f cttanHarHc n f"*ftV-A nr trtnrp AWnVnUAVAWrrUfrUaIt4iWU `IVmtmwloe, nfi-onnwi oatf. UI kUV1/toU.V.Y. +l Ui l ^t LOQ, up to the highest concentration level to be included in the analysis. Generate a calibration curve for the analyte and obtain a linear regression with a coefficient o f determination (RJ) o f at least 0.985 for the analyte. Once this criterion is met, samples may be analyzed. 4.8. T ime R equired for Analysis One person can take a set o f 20 samples through the sample preparation procedure in approximately 2 hours. The GC/MS analysis o f the set (containing 6 standard injections, 20 field samples, 1 matrix blank, 1 laboratory control spike, and 1 matrix spike) will take approximately 5 hours. 5. CALCULATIONS a. U se Equation 1 to calculate the amount o f POSF found (in pg/m L, based on peak area) using the standard curve (1/x weighted linear regression parameters) generated by the HP Chemstation software program. Equation 1: A nalyte found (jig/m L ) = (P eak A rea - Intercept! x D F S lo p e D F = factor by w hich the final volum e w as diluted, if necessary. Exygen Research Page l7 o{34 Exygen Research : 269 : Page 149 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 b. Equation 2 was used to calculate the amount o f POSF found (in pg/mL or Pgfe)- Equation 2: . Total Analyte Found (pg/mL or pg/g) = (Analyte Found ug/mLi x Standard Volume Added (mLl SW (g) or SV (mL) SW **sample weight, SV = sample volume c. Equation 3 was used to calculate the percent recovery for samples fortified with known amounts o f POSF prior to extraction. Recovery (%) = Total Analyte Found (ug/mL*l - Analyte Found in Samnle (ng/mL*) x 100 Analyte Added (pg/mL*) * = (or pg/g) For reporting purposes, field samples in which no peaks are detected at the corresponding analyte retention time will be reported as ND (not detected). Samples in which peaks are detected at the corresponding analyte retention time that are less than the LOQ will be reported as NQ (not quantifiable). : 270 : MIN 312/014272 Exygen Study No.: 023-080 E x y g e n M eth o d N o. E x M -0 2 3 -0 7 1A R ev . 2 6. SAFETY The analyst should read the material safety data sheets for all standards and reagents before performing this method. U se universal precautions when handling standards and reagents, including working in fume hoods and wearing laboratory coats, safety glasses, and gloves. Exygen Research Exygen Research : 271 : P age 19 o f 34 Page 151 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 Table I. Recovery Summary of POSF in Rat Serum Summary of Rat Serum Recoveries Sample Sample Injected Sample Area Std Injected Trial______ Amount (pig)______ Counts Amount (pg) Std Area Counts % Recovery #1 0.1 603475 0.1 536407 113 #2 0.1 506452 0.1 536407 9 4 .4 #1 0.01 46925 0.01 51030 9 2 .0 : 272 : MIN 312/014272 : 273 : MIN 312/014272 : 274 : MIN 312/014272 Exygen Study No.: 023-080 E xygen M ethod No. ExM -023-071A Rev. 2 Figure!. Calibration curve for POSF Wpott-- tf,0 0 e + 0 0 5 4 s.oo+oos140**0653 . OC4-0 05- 3.04-0Q>< \^ - l.? 7 + 0 O 3 c^sef o f Bat (r*3 ) - 0.934 curv m * wrehod U tm ti CiXHBCBM\3VHBSBCDS\P08fM C a l i U r a t l o n T a b i l a s t U p d * ta d t T u* J u l 03 O l i >23 20 0 3 Exygen Research Exygen Research : 275 : Page 23 o f 34 Page 155 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod N o. ExM -023-071A Rev. 2 Figure 2. Representative Chromatogram of a 0.10 ug Standard Injection of POSF TU 02301 073023 030101Cpam tor Ai3 3003 >11 502Jisq o ln d : Ll \ - l\StMfAUl\ tl < \ .D < J u l U a n u o iag JUntaebod laosrinnunc i o c /x a 1SKl"o*o4 ?InSfo" I! B p i, a t s ohpMa / tiot kpoSetod. i n j . , s u , u a t v io l Vt*l ifciabrt 1 M T S K g T f r 'i w I * 1in r h ! ' i W T s t \ i t i r a - Exygen Research Exygen Research 276 : Page 24 o f 34 Page 156 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 Figure 3. Representative Chromatogram of a 0.01 jig Standard Injection of POSF AOmpce.ettsifmcodr lOMZIHUC pi. S am Mice In fo t t t I !.<\011-071\RIBAi\a7030JaS\03021S. 3 3003A il 2iOO gm u la g Xcqltathca C/W*M 12POST, h*ad*pau io j SjtL o 2Stg/ta, sed M d, mL v i a 2 l V isi sue ncrraoror rue h"'k dd- h-ajfu. ih Exygen Research Exygen Research : 277 : Page 25 o f 34 Page 157 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen Method No. ExM-023-071A Rev. 2 Figure 4. Representative Chromatogram of a Control Sample (100 pL Rat Serum) *QepaUaweorrt |i 5j*Vjf>ai-mVljn\*T_ojf_laa\OT3gaai`B VHSi5aha?ol HNnauttaoSoba*.r.,i' 3 w/ ;' 7 w t" 1` :#*?!*?*3?,*s =* -4-"*-3--- U W *t ran """*Sso03 *td' WL v i . l H 'IIU U U IJ J- Exygen Research Exygen Research : 278 : Page 2 6 of 34 Page 158 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 Figure 5. Representative Chromatogram of a Control Sample (100 pL Rat Urine) m*0B4UeO i L \0 2 3 -0 7 1 \ M C * I5 l\0 7 0 3 020 9 \T O 3 0 2 2 2 .0 AcquirediHstaewwn* 5 31 ** p n .u o lcg A stathed 5 f t S * ! **' 5< J** h*dpe l a j . , s td , U a t v ia l 71a l Rlottinafaer:i C tatro t, l i o J S t a t Orla' " S3~ Exygen Research Exygen Research : 279 : Page 27 o f 34 Page 159 of 166 MIN 312/014272 280 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 Figure 7. Representative Chromatogram of a Fortified Rat Serum Control Sample (0.1 pg Injection of POSF) Aojuirad fLil 0J0'9VTQuaoa,nOpeaaaeo* I \033 -471 \sw n lx \(n 0 3 3 J u l 300} u n am ualm g tUx^<ethcd 802 Inaecum ne OC/H8 M Muni 0SS, Hit. kaadapaoa i i r t 8 t d , U bL v ia l Uif>*C5> r n>#*on i 9Bu)OT.i Ko# 250S/L sed. + 0 ||I. r u __a__s_m_ V ial H urten 3 Exygen Research Exygen Research : 281 : Page 29 of 34 Page 161 of 166 MIN 312/014272 : 282 : MIN 312/014272 Exygen Study No.: 023-080 Exygen M ethod No. ExM -023-071A Rev. 2 Figure 9. Representative Chromatogram of a Fortified Rat Liver Control Sample (0.1 pg Injection of POSF) *OmCjH4*wUmirtodr ` MLi\0-071.\aAKDM\070303S\ro30aXO.E * 1 , 3 4 *" u , i 9 5 V U i a tu f e 3 o f stOM3/ t Std. 7 i . I j j r a c i l v r Exygen Research Exygen Research : 283 : Page 3 1 o f34 Page 163 of 166 MIN 312/014272 Exygen Study No.: 023-080 Exygen Method No. ExM-023-071A Rev. 2 Figure 10. Representative Chromatogram of a Fortified Rat Serum Control Sample (0.01 pg Injection of POSF) m* O perator <MLi\<s3*a7i\Kuara\7ua2a\roe3u.o aafla *9Mgulxad 3 OUI 3003 Xacexuaute i 2 109 pa tu in g Ae$fa>tbad J03 S u p l* t a Miao Info it t POST. U C keadapac SfiL o f JSpj/mL * td . ln * j li / i serdi.feUnbalcnva i a l viol a n te rt 2 S-M9MSva' V SMumna abthth ahrm jtoaifei tifiB Exygen Research Exygen Research : 284 : Page 32 o f 34 Page 164 of 166 MIN 312/014272 Exygen Study No.: 023-080 E xygen M ethod No. E xM -023-071A Rev. 2 Figure 11. Represeutative Chromatogram of a Fortified Rat Urine Control Sample (0.01 pg Injection of POSF) &?*!S3ammiS S o I M* WW.*1l MM w__;: S3'" * a**Ui .3,1S*". .U*ln9 5M *ed. lOC^L r a t o rin a rrenmir "*JMlw w ^ * *** ^ ^ ^ ^ -i -HTi Exygen Research Exygen Research 285 : Page 33 o f 34 Page 165 of 166 MIN 312/014272 Exygen Study No.: 023-080 E x y g en M e th o d N o. E x M -0 2 3 -0 7 1A R ev. 2 Figure 12. Representative Chromatogram of a Fortified Rat Liver Control Sample (Q.01 pg Injection of POSF) M l >L t\4 2 3 -O n \ l)U > * 3 X V 0 3 U a * \V 0 3 0 3 1 J.B O patatoc i a AaqoUed ; 3 J u l 3003 2:33 cm u alag acquath o d 502 z t tx tm n t I . QO/Mfi f | S a lila h u m * POSF, la b haariapaoa ia j. , Sad, I3m t. v ia l M lac la te SffL o f 3 5 3 9 / a t S td , -+ c a t liv a c V ia l sober1 4 .if. s s m o ---------reemuraur------ ------------------- -- mo m PCBP Met MM 2M 30 m 10 M iM W 13 t d ' a ..* 3 " a m w / uo ai ih "4Ja"4W " Exygen Research Exygen Research : 286 : Page 34 of 34 Page 166 of 166 MIN 312/014272 SCANNING ELECTRON MICROSCOPE EXAMINATION AND X-RAY MICROANALYSIS Plymouth Electron Microscope Unit Scanning Electron Microscope Examination and X-Ray Microanalysis. Study Number: MIN312 For: H untingdon Life Sciences H untingdon Cambs PE284HS Plym outh Electron M icroscope U nit U niversity o f Plym outh D rake Circus Plym outh PL4 8AA 01752 233092 : 287 : MIN 312/014272 Materials and Methods 1. S E M ex am in atio n o f urinary b lad d er tissu e sam ples: Subsam ples (approxim ately 0.5 x 0.5cm ) w ere cut from fixed tissue sam ples (supplied in 70% ethanol) and critical point dried (protocol attached). D ry sam ples w ere m ounted on specim en stubs w ith colloidal silver adhesive, sputter coated w ith gold and exam ined in a JEOL 5300 SEM at 15kV. 2. SEM /X -Ray m icroanalysis o f urine samples: Sam ples as supplied w ere carbon coated and exam ined in a JEOL 6100 SEM at 15kV. X -ray data w ere collected and analysed w ith an O xford Instrum ents ISIS X -R ay A nalysis system. : 288 : MIN 312/014272 Specimen Preparation for Scanning Electron Microscopy Critical Point Drying (CPD) 1. F ix fresh ly e x c is e d sam ple ap p ro p ria te ly (eg. 2 .5 % G lu ta ra ld e h y d e in 0 .1 M p h o sp h ate b u ffer) 2. W ash in buffer - 3x 15 m inutes 3. D ehydrate in ethanol series 30% -1 5 m inutes 50% -1 5 m inutes 70% -1 5 m inutes 90% -1 5 m inutes 100% -1 5 m inutes 100% -1 5 m inutes 4. F ill CPD cham ber w ith 100% ethanol and cool to 10C or less 5. Place specim ens in C PD cham ber fV\. PW1I Va Uc Va Mari\lAu fJl.r1vWnVH \fxTrIitMV*i ll iln^ Un iiHU rV`Ma 1i 4UvV\1n1 VUV/VIV*V 7. E xhaust alcohol from chamber. 8. L eav e sam ple 30 m in u tes in ch a m b e r to infltrate w ith c a rb o n dioxide. 9. E xhaust rem aining ethanol 10. H e a t c h am b e r to critical po in t 11. V ent carbon dioxide gas from cham ber 12. R em o v e sam ple fro m ch am b e r and m o u n t for S E M exam ination. : 289 : MIN 312/014272 Results 1. S E M E x am in atio n o f U rinary B la d d e r E pithelium . Sample 5 - no low pow er im age w as collected due to poor im age signal from the sample. : 290 : MIN 312/014272 : 291 : MIN 312/014272 : 292 : agili!pi? ^ MIN 312/014272 : 293 : MIN 312/014272 : 294 : MIN 312/014272 : 295 : MIN 312/014272 : 296 : MIN 312/014272 : 297 : MIN 312/014272 : 298 : MIN 312/014272 : 299 MIN 312/014272 : 300 : MIN 312/014272 : 301 : MIN 312/014272 : 302 : MIN 312/014272 : 303 : MIN 312/014272 : 304 : MIN 312/014272 : 305 : MIN 312/014272 : 306 : s MIN 312/014272 : 307 : MIN 312/014272 : 308 : MIN 312/014272 309 : MIN 312/014272 : 310 : MIN 312/014272 2. SEM/X-Ray analysis of Urine samples Representative images are provided to illustrate typical crystal structure and deposit morphology. X-Ray analysis spectra from each sample, to show general elemental composition, and composition of specific crystals and deposits (as labelled) are provided. : 311 : MIN 312/014272 : 312 : MIN 312/014272 : 313 : MIN 312/014272 G2 17F : 314 : MIN 312/014272 G2 18F : 315 : MIN 312/014272 : 316 : O perator: Plymouth EM Unit Client: HLS Job: MIN/312 control stub (28/09/01 09:50) cps MIN 312/014272 Energy (kev) : 317 : O perator: Plymouth EM Unit Client: HLS Jo b : MIN/312 cps G1 2M (28/09/01 09:54) MIN 312/014272 20 Energy (keV) : 318 : O perator: Plymouth EM Unit Client: HLS J o b : MIN/312 G1 3M (28/09/01 09:56) MIN 312/014272 Energy (kev) : 319 : O perator: Plymouth EM Unit Client: HLS Job : MIN/312 G1 4M (28/09/01 10:04) 100 MIN 312/014272 20 Energy (kev) : 320 : O perator: Plymouth EM Unit Client: HLS Jo b : MIN/312 G1 5M (28/09/01 10:19) MIN 312/014272 Energy (kev) : 321 : O perator: Plymouth EM Unit Client: HLS Job : MIN/312 cps G2 7M (28/09/01 10:23) MIN 312/014272 Energy : 322 : Operator: Plymouth EM Unit Client: HLS Job : MIN/312 cps G2 7M crystal (28/09/01 10:58) MIN 312/014272 Energy (kev) : 323 : O perator: Plymouth EM Unit Client: HLS Job : MIN/312 cps G2 8M (28/09/01 11:00) MIN 312/014272 Energy (kev) : 324 : O perator: Plymouth EM Unit Client: HLS J o b : MIN/312 G2 9M (28/09/01 11:13 MIN 312/014272 Energy (kev) : 325 : MIN 312/014272 O perator: Plymouth EM Unit Jo b : MIN/312 cps G1 11F 'light area' (28/09/01 11:31) 250 200 Energy (kev) : 326 : Operator: Plymouth EM Unit Client: HLS Job : MIN/312 G1 12F (28/09/01 11:35) cps MIN 312/014272 Energy (keV) : 327 : MIN 312/014272 Operator: Plymouth EM Unit Client: HLS J o b : MIN/312 G1 13F light deposit (28/09/01 11:54) cps Energy (keV) : 328 : MIN 312/014272 O perator: Plymouth EM Unit Client: HLS J o b : MIN/312 cps G1 15F crystal (28/09/01 12:09) MIN 312/014272 Energy (kev) : 330 : Operator : Plymouth EM Unit Client : HLS Job : MIN/312 G2 16F light area (01/10/01 16:15) MIN 312/014272 Energy (kev) : 331 O perator: Plymouth EM Unit U llC llt . n L O Jo b : MIN/312 cps G2 16F crystal (01/10/01 16:17) MIN 312/014272 40 20- Energy (kev) : 332 : MIN 312/014272 O perator: Plymouth EM Unit /'i: i_11 o V s l ld l l . I IL-O J o b : M N/312 cps G2 17F light deposit (01/10/01 16:21) 60 20 Energy (kev) : 333 : O perator: Plymouth EM Unit lji o I s l l G l l l . I IL .O Jo b : M N/312 cps G2 18F crystal (01/10/01 16:24) 40 20 10 MIN 312/014272 Energy (keV) : 334 : O perator: Plymouth EM Unit V / iic iii . n L o Jo b : MIN/312 cps G2 18F platelet (01/10/01 16:26) MIN 312/014272 Energy (kev) MIN 312/014272 O perator: Plymouth EM Unit O lid II . I IL O Job : MIN/312 cps G2 19F light deposit (01/10/01 16:34) Energy (keV) : 336 : Operator: Plymouth EM Unit Client: HLS Jo b : MIN/312 G2 20F crystals 01/10/01 16:32) cps MIN 312/014272 20 Energy (keV) : 337 : MIN 312/014272 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2001 : 338 : MIN 312/014272 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2003 THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 88/320 EEC LABORATORY TEST TYPE Huntingdon Life Sciences Huntingdon Research Centre Woolley Road Alconbury Huntingdon Cambs. PE28 4HS Analytical Chemistry Clinical Chemistry Ecosystems Environmental Fate Environmental Toxicity Toxicology Phys/Chem Tests DATE OF INSPECTION 07thApril 2003 A general inspection for com pliance with the Principles of Good Laboratory Practice w as carried out at the above laboratory as part o f UK GLP Com pliance Program m e. At the tim e o f the inspection no deviations were found o f sufficient magnitude to affect the validity o f non-clinical studies perform ed at these facilities. Dr. R oger G. A lexander Head, UK G LP M onitoring A uthority : 339 : MIN 312/014272 HUNTINGDON RESEARCH CENTRE GLP COMPLIANCE STATEMENT 2005 : 340 :
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