Document ZBJ78rg7K6rGO55NL74q0n7DO

FLUOROCARBONS AND HUMAN HEALTH: STUDIES IN AN OCCUPATIO NAL C O HO RT A TH ESIS SUBM ITTED TO TH E FACULTY O F TH E GRADUATE SCHOOL O F TH E UN IVER SITY O F M INNESOTA BY FRANK DAVIS GILLILAND IN PARTIAL FULFILLMENT O F TH E REQUIREM ENTS FOR THE DEGREE OF DO CTOR O F PHILOSOPHY/ENVIRO NM ENTAL HEALTH OCTOBER, 1992 3M MN03112178 ACKNOW LEDG EM ENTS I am indebted to Dr. Jack Mandel whose competent research and career advice were invaluable. Not only did Dr Mandel guide me to this research project; he directed me to the NIO SH occupational medicine fellowship that has enriched my clinical medicine knowledge and supported my research efforts over the last three years. His generosity with his time and patience are deeply appreciated. Dr. Timothy Church. Dr. William Toscano. Dr. Ian Greaves, and Dr. Thomas Sellers served on my committee. They deserve a special thanks for their efforts. I wish to thank the Division of Environmental and Occupational Health at the University of Minnesota and the Occupational Medicine Section at SL Paul Ramsey Medical Center for the superb training opportunities I have had over the past three years. Dr. William Lohman, Dr. Samuel Hall, and Paula G eiger at SL Paul Ramsey Medical Center provided much appreciated support during the arduous task of residency and doctoral training. Several members of the Division of Environmental and Occupational health staff were instrumental in the successful completion of this research effo rt Sarah W olgamot and Maralyn Zappia provided excellent administrative support Gavin W att, Mindy Geisser, Richard Hoffbeck, and other members of Colon Cancer Control Study provided outstanding computer and statistical support. Dr. Larry Zobel and Dr. Jeffrey Mandel of the 3M Corporation's Medical Department provided advice and support Their help was an essential element in the success of this project Stan Sorenson, Dr. Roger Perkins, and other 3M Medical Department members shared their invaluable experience and knowledge. I would also like to acknowledge the support of the Dow Chemical Corporation over the last two years of my training. Last, but not least, this work could not have been accomplished without the loving support of Susan, my wife. Her understanding and excellent editorial comments are greatly appreciated. 3M MN03112179 ABSTRACT Perfluorooctanoic add (PFOA) has been reported to be a nongenotoxic hepatocardnogen and reproductive hormonal toxin in rats. Although PFOA is the major component of total fluorine in humans, little information is available concerning human toxidties. The health effects of PFOA were assessed in two studies conducted in occupationally exposed workers. The associations between PFOA and reproductive hormones, hepatic enzymes, lipoproteins, hematology parameters, and leukocyte counts were studied in 115 male employees. Serum PFOA was positively assodated with estradiol and negatively assodated with free testosterone (TF) but was not significantly assodated with luteinizing hormone. The negative assodation between IT 7and PFOA was stronger in older men. Thyroid stimulating hormone and PFO A were positively associated. PFOA and prolactin were positively assodated in moderate drinkers. The effect of adiposity on serum glutamyl oxaloacetic and glutamyl pyruvic transaminase decreased as PFOA increased. The induction of gamma glutamyl transferase by alcohol was decreased as PFO A increased. The effect of alcohol on HDL was reduced as PFOA increased. A positive assodation between hemoglobin, mean cellular volume, and leukocyte counts with PFOA was observed. These results suggest that PFOA affects male reproductive hormones and that the liver is not a significant site of toxidty in humans at the PFOA levels observed in this study. However, PFO A appears to modify hepatic and immune responses to xenobiotics. A retrospective cohort mortality study of 2788 male and 749 females workers employed between 1947*1984 at a PFOA production plant was conducted. Overall, there w ere no significantly increased cause spedfic SMRs. Among men, ten years of employment in PFOA production was assodated with a significant three fold increase in prostate cancer mortality compared to no employment in production. Given the small number of prostate cancer deaths and the natural history of the disease, the assodation between production work and prostate cancer must be viewed as hypothesis generating and should not be over interpreted. If the prostate cancer mortality excess is related to PFOA, the results of the two studies suggest that PFO A may increase prostate cancer mortality through endocrine alterations. 3M MN03112180 TABLE O F CONTENTS 1. INTR O D UCTIO N....................................................................................................... 1 2. REVIEW O F TH E LITER A TU R E.......................................................................... 4 2.1 Introduction................................................................................................ 4 2 Organic Fluorochemicals ........................................................................ 4 2.3 Physical Properties__________________________ ______ ________ 6 2.4 S y n th e s is .................................................................------ ....------- .....7 2.5 Sources O f Organic Fluoride Exposure............... .................................8 2.6 Toxicokinetics of P FO A ...........-------- ...............-------.......-- ...-- 11 2 .7 Toxicodynamics of PFO A -----------.......--------- ------------------------------ - 16 2.7.1 M ale Reproductive Toxidties------------------------------------------ 16 2.7.2 Fem ale Reproductive T o x id tie s------------------------------------- 20 2.7.3 Thyroid Toxidties----------------------------------------------------------- 20 2.7.4 Hepatic Toxid ties----------------------------------------- ................. 21 2.7.5 Nongenotoxic Carcinogenesis---------------------------------------- 23 2.7.6 Immunotoxidty------------------------------- ...........----- ......-- .... 23 2.7.7 Mechanisms of A ction----------------------------------------------------24 2.8 Occupational Fluorine Exposures At Chem ol'rte............................. 26 2.9 Epidemiological Studies-------------------------------------------------------- 27 2.10 Sum m ary............................. .........2 8 3. M E TH O D S ............................................................................................................- .2 9 3.1 Introduction........ ............................ ............ .....------ .....--------- ............. 29 3 .2 Retrospective Cohort Mortality Study......... ......................................... 30 3.2.1 Definition O f The Cohort------------------------------------------------ 30 3.2.2 Study Databases And F iles-------------------------------------------- 31 3.2.3 Data Editing .------------------------------------------------------------------31 3.2.4 Validation Of The Historical Cohort Inform ation-------------- 32 3.2.4.1 Assessment O f Completeness O f A scertain m ent____________ 32 3.2.4.2 Validation O f Cohort Inform ation............................33 3.2.5 Vital Status Ascertainm ent__________________________ 33 3.2.6 Validation of Vital Status Ascertainment............................... 34 3.2.7 Analysis--------------------------------------------------- -----------------34 3.3 Cross Sectional Study O f PFOA Exposed W o rk e rs ....................... 36 3.3.1 Population Definition And Recruitment_______ _________ 36 3.3.2 Data Collection...__ _-- ______________ 37 3.3.2.1 Study Logs And Files________________________37 3.3.2.2 Q uestionnaire...........................____ .............-- 37 3.3.2.3 Laboratory Procedures____________ 37 3.3.2.3.1 Height and W eig h t----------------------------- 37 3.3.2.3:2 Blood___________ 38 3.3.2.3.2.1 Drawing And H andling______38 3.3.2.3.2J2 Assays____________________ 38 3.3.2.3.2.3 Quality Assurance_________ 40 3.3.3 Analysis____________ - ____________________________ 40 4. R E S U L T S .................................................................................................................43 l 3M MN03112181 4.1 Cross Sectional Perfluorocarbon Physiologic Effects Study...........43 4.1.1 Participant Characteristics....................................................... 43 4.1.2 Total Serum Fluorine..... ...........................................................44 4.1.3 Hormone Assays....................................................................... 45 4.1.4 Hormone R atio s........................................................................ 49 4.1.5 Cholesterol, Low Density Lipoprotein, High Density Lipoprotein, And Triglycerides......................................................... 51 4.1.6 Hepatic Param eters...................................................................52 4.1.7 Hematology Param eters.......................................................... 54 4.1.8 Summary O f Results.............................. 56 4.2 The 1990 Chemolite Retrospective Cohort Mortality S tudy----------58 4.2.t Standardized Mortality Ratios (S M R s )---------------------------- 59 4.2.1.1 SM Rs For W om en..................................................... 59 4.2.1.2 SM Rs For M e n .......................................................... 59 4.2.2 Standardized Rate Ratios (S R R s)......................................... 60 4.2.3 Mantel- Relative Risks (R R M H ).............. ..................... .......6 1 4.2.4 Proportional Hazard Regression Model Relative Risk Estim ates___________________ ______ ________________ 61 4.2.4.1 Proportional Hazard Models For M ale W orkers.....................................................................................61 4.2.4.2 Proportional Hazard Models For Fem ale W orkers.....................................................................................63 4.3 Physiologic Effects T ab les...................................................................... 64 4.4 Mortality Tab les.............. .'........................................................................157 4.5 Figures___________________________ .......-- .................................190 5. D ISC U SSIO N .......................................................................................................... 198 5.1 Physiologic Effects Study___________________________________198 5.1.1 Introduction............................................................................... 198 5.1.2 H orm ones......------------------------------------------- ..1 9 8 5.1.3 Cholesterol, Triglycerides, and Lipoproteins...-- ---------- 202 5.1.4 Hepatic Param eters------ --------- .......-- -------------- ----------- 203 5.1.5 Hematology Counts and Param eters.................................. 206 5.1.6 Total Fluorine............................................................................ 209 5.1.7 Methodological Considerations.-- ------------------------------- 210 5.1.7.1 Selection B ias___ _______ 210 5.1.7.2 Information Bias----------------------- 211 5.1.7.3 Confounding B ia s ........................... ......................2 1 4 5.1.7.4 Analytic Model Specification Bias---------------------216 5.2 1990 Chemolite Mortality S tu d y.-- ..................................................... 217 5.2.1 Introduction.-- .........................................................................217 5.2.2 Participant Characteristics............... .................................... 217 5.2.3 Mortality R esults..................................................................... 218 5.2.4 Methodological Considerations.............________ 220 5.2.4.1 Information Bias.......................................................220 5.2.4.2 Confounding and Selection Bias..........................221 5.2.4.4 Analytic Model Specification Bias........................223 6. SUMMARY, CO NCLUSIO NS AND R EC O M M EN D A TIO N S............ ........... 225 6.1 Cross-Sectional Study of the Physiologic Effects of P FO A ..........225 ll : k 3M MN03112182 6.2 Retrospective Cohort Mortality Study O f The Chemolite Workforce, 1947-1990....................... 226 REFER EN CES------------------------------------------------------------------------------------------230 APPENDIX 1 ____________________________________________________ .....255 APPENDIX 2 ............................................................................................................... 259 APPENDIX 3 .............................. 281 3M MN03112183 LIST O F TABLES Table 4.1.1 Age Distribution In Five Y ear Age G roups....................................... 64 Table 4.1.2 Distribution O f Alcohol And Tobacco U s e..................................... . 65 Table 4.1.3 The Joint Distribution O f Tobacco And Alcohol U s e ...................... 66 Table 4.1.4 Distribution O f Age By Smoking And Drinking Status.....................67 Table 4.1.5 Pearson Correlation Coefficients Between Total Serum Fluorine, Age, Body Mass Index (Bm i),............................................. 68 Table 4.1.6 Body Mass Index Distribution.............................................................69 Table 4.1.7 Body Mass Index By Smoking And Drinking Status....................... 70 Table 4.1.8 The Distribution O f Age, Alcohol And Tobacco Use By Body Mass Index_____....___ ...-- ................................................... 71 Table 4.1.9 Toted Serum Fluoride Distribution......................................................72 Table 4.1.10 Total Serum Fluoride By Body Mass Index, Age, Smoking And Drinking Status_____ ...................................-- ...7 3 Table 4.1.11 Age Distribution By Total Serum Fluorine C ateg ory.....................74 Table 4.1.12 Distribution O f Tobacco Use By Total Serum Fluoride C ategory................................................................................................ 75 Table 4.1.13 Distribution O f Alcohol Use By Total Serum Fluoride C ategory................................................................................................ 76 Table 4.1.14 Body Mass Index Distribution By Toted Serum Fluorine Category.................................................................................................77 Table 4.1.15 Coefficient O f Variation For Seven Hormone Assays................... 78 Table 4.1.16 The Observed Versus Expected Number O f Workers With Hormone Assays Outside The Assay Reference Range..................................................................................................... 79 Table 4.1.17 Pearson Correlation Coefficients Between Serum H orm on es_________ 80 Table 4.1.18 Pearson Correlation Coefficients Between Total Serum Fluoride, Age, Body Mass Index (Bm i), Daily Alcohol Use, Daily Tobacco Consumption, And Serum Hormones.............................................................................................. 81 Table 4.1.1.9 Bound Testosterone (Tb) By Body Mass Index, Age, Smoking, Drinking Status And Toted Serum Fluoride...................82 Table 4.1.20 Linear Multivariate Regression Model O f Factors Predicting The Bound Testosterone (Ng/DI) Among 112 M ale W orkers................................................ 83 Table 4.1.21 Free Testosterone (Tf) By Body Mass Index, Age, Smoking And Drinking Status And Total Serum Fluoride_____________________ ...............________________ ..... 84 Table 4.1.22 Linear Multivariate Regression Model O f Factors Predicting The Free Testosterone Value (N g /D I)____________ 85 Table 4.1.23 Participant Estradiol By Body Mass Index, Age, Smoking Drinking Status And Total Serum Fluoride__________________ 86 Table 4.1.24 Linear Multivariate Regression Model O f Factors Predicting The Estradiol Value (Pg/DI) Among 113 M ale W orkers.................................................................... 87 3M MN03112184 Table 4.1.25 Lutenizing Hormone (Lh) By Body Mass Index, Age, Smoking And Drinking Status, And Total Serum Fluorine................................................................................................. 88 Table 4.1.26 Linear Multivariate Regression Model #1 O f Factors Predicting The Lutenizing Hormone* Value (Mu/M I) Among 113 Male Workers.................................................................. 89 Table 4.1.27 Follicle Stimulating Hormone (Fsh) By Body Mass Index, Age, Smoking And Drinking Status, And Toted Serum F lu o rin e ............................................................................................. 90 Table 4.1.28 Linear Multivariate Regression Model O f Factors Predicting The Follicle Stimulating Hormone Value (M u/M I) Among 113 Male Workers_____________ 91 Table 4.1.29 Thyroid Stimulating Hormone (Tsh) By Body Mass Index, Age, Smoking And Drinking Status, And Total Serum Fluorine____________________________ 92 Table 4.1.30 Linear Multivariate Regression Model O f Factors Predicting The Thyroid Stimulating Hormone* Value (Mu/M I) Among 113 M ale W orkers------------------------------ 93 Table 4.1.31 Prolactin By Body Mass Index, Age, Smoking, Drinking Status, And Total Serum Fluorine.....-- .................------ ...........9 4 Table 4.1.32 Linear Multivariate Regression Model O f Factors Predicting The Prolactin Value (Ng/MB) Among 113 M ale W orkers.................................................................................................. 9 5 Table 4.1.33 Pearson Correlation Coefficients Between Hormone Ratios And Total Fluoride, Age, Body Mass Index, Alcohol And Tobacco Consum ption--------------------------- 96 Table 4.1.34 Pearson Correlation Coefficients Between Prolactin Hormone Ratios And Total Fluoride, Age, Body Mass Index, Alcohol And Tobacco Consumption...... ................. .......... 97 Table 4.1.35 Pearson Correlation Coefficients Between Thyroid Stimulating Hormone Ratios And Total Fluoride, Age, Body Mass Index, Alcohol And Tobacco Consumption............... 98 Table 4.1.36 Pearson Correlation Coefficients Between Follicle Stimulating Hormone Ratios And Toted Fluoride,Age, Body Mass Index, Alcohol And Tobacco Consumption.............. 98 Table 4.1.37 Pearson Correlation Coefficients Between Pituitary Glycoprotien Hormone Ratios And Toted Fluoride, Age, Body Mass Index, Alcohol And Tobacco Consumption_______ 99 Table 4.1.38 Linear Multivariate Regression M odell O f Factors Predicting The Bound-Free Testosterone Ratio Among 112 M ale Workers_______________________________________ 100 Table 4.1.39 Linear Multivariate Regression M odel2 O f Factors Predicting The Bound-Free Testosterone Ratio Among 112 M ale Workers.............................................................................. 101 Table 4.1.40 Linear Multivariate Regression Model O f Factors Predicting The Estradiol-Bound Testosterone Ratio Among 112 Male Workers................................................................ 102 v L 3M MN03112185 Table 4.1.41 Linear Multivariate Regression Model O f Factors Predicting The Estradiol-Free Testosterone Ratio Among 112 Male W orkers................................................................ 103 Table 4.1.42 Linear Multivariate Regression Model O f Factors Predicting The Estradiol-Lh+ Ratio Among 112 M ale W orkers................................................................................................. 1 0 4 Table 4.1.43 Linear Multivariate Regression Model O f Factors Predicting The Bound Testosterone-Lh+ Ratio Among 112 M ale Workers...............................................................................105 Table 4.1.44 Linear Multivariate Regression Model O f Factors Predicting The Free Testosterone-Lh+ Ratio Among 112 M ale Workers____________________________________ 106 Table 4.1.45 Linear Multivariate Regression Model O f Factors Predicting The Bound Testosterone-Prolactin Ratio Among 111 Male Workers.................................................................107 Table 4.1.46 Linear Multivariate Regression Model O f Factors Predicting The Free Testosterone-Prolactin Ratio Among 111 Male Workers.................................................................108 Table 4.1.47 Linear Multivariate Regression Model O f Factors Predicting The Estradiol-Prolactin Ratio Among 111 M ale Workers............................................... ...--------------------------- 109 Table 4.1.48 Linear Multivariate Regression Model O f Factors Predicting The Prolactin-Fsh<> Ratio Among 111 M ale W orkers................................................................................................ 110 Table 4.1.49 Linear Multivariate Regression Model O f Factors Predicting The Prolactin-Lh** Ratio Among 111 Male W orkers------------------- 111 Table 4.1.50 Linear Multivariate Regression Model O f Factors Predicting The Prolactin-Tsh+ Ratio Among 111 M ale W orkers________________ _______________________________ 112 Table 4.1.51 Linear Multivariate Regression Model O f Factors Predicting The Bound Testosterone-Tsh+ Ratio Among 112 M ale Workers...............................................................................113 Table 4.1.52 Linear Multivariate Regression Model O f Factors Predicting The Free Testosterone-Tsh+ Ratio Among 112 M ale W orkers....................................................................1 1 4 Table 4.1.53 Linear Multivariate Regression Model O f Factors Predicting The Estradiol-Tsh-t- Ratio Among 112 M ale W orkers._______________________________________________115 Table 4.1.54 Linear Multivariate Regression Model O f Factors Predicting The Bound Testosterone-Fsh+ Ratio Among 112 M ale W orkers_______________________________________116 Table 4.1.55 Linear Multivariate Regression Model O f Factors Predicting The Free Testosterone-Fsh+ Ratio Among 112 M ale W orkers.________________________________ _____ 117 Table 4.1.56 Linear Multivariate Regression Model O f Factors Predicting The Estradiol-Fsh+ Ratio Among 112 M ale W orkers________________________________________________ 118 vi 3M MN03112186 Table 4.1.57 Linear Multivariate Regression Model O f Factors Predicting The Bound Tsh-Fsh+ Ratio Among 112 M ale W orkers.................----- ..................-- .-- ................................... 119 Table 4.1.58 Linear Multivariate Regression Model O f Factors Predicting The Tsh-Lh+ Ratio Among 112 M ale W orkers................................................................................................ 120 Table 4.1.59 Linear Multivariate Regression Model O f Factors Predicting The Bound Lh-Fsh+ Ratio Among 112 M ale W orkers................................................................................................ 121 Table 4.1.60 Pearson Correlation Coefficients Between Total Serum Fluoride, Age, Body Mass Index (Bmi), Daily Alcohol Use, Daily Tobacco Consumption, And Lipoproteins................ 122 Table 4.1.61 Linear Multivariate Regression Model O f Factors Predicting The Cholesterol Among 111 M ale Workers............... 123 Table 4.1.62 Linear Multivariate Regression Model O f Factors Predicting The Low Density Lipoprotien Among 111 M ale Workers--------------------------------------------------------- 124 Table 4.1.63 Linear Multivariate Regression Model O f Factors Predicting The High Density Lipoprotien (Hdl) Among 111 M ale W orkers................ 125 Table 4.1.64 Linear Multivariate Regression Model O f Factors Predicting The Triglycerides Among 111 M ale W orkers.............126 Table 4.1.65 Pearson Correlation Coefficients Between Total Serum Fluoride, Age, Body Mass Index (Bmi), Daily Alcohol Use, Daily Tobacco Consumption, And Hepatic Param eters.......-- .-- ..........------------------------------- ----------------127 Table 4.1.66 Pearson Correlation Coefficients Between Hepatic Enzymes, Serum Hormones, And Lipoproteins.......................... 128 Table 4.1.67 Pearson Correlation Coefficients Between Hepatic Param eters____________________________________________ 129 Table 4.1.68 Serum Glutamic Oxaloacetic Transaminase (S g o t), Glutamic Pyruvic Transaminase (Sgpt),Gam m a Glutamyl Transferase (Ggt), And Alkaline Phosphatase (Akph) By Total Serum Fluorine-------------------------------------- 130 Table 4.1.69 Serum Glutamic Oxaloacetic Transaminase (Sgot) By Body Mass Index, Age, Smoking And Drinking Status------------131 Table 4.1.70 Serum Glutamic Pyruvic Transaminase (Sgpt) By Body Mass Index, Age, Smoking And Drinking Status-------------------- 132 Table 4.1.71 Gam m a Glutamyl Transferase (Ggt) By Body Mass Index, Age, Smoking And Drinking Status.................................... 133 Table 4.1.72 Alkaline Phosphatase (Akph) By Body Mass Index, Age, Smoking And Drinking Status.......................................................... 134 Table 4.1.73a Linear Multivariate Regression Model 1 O f Factors Predicting The Serum Glutamic Oxaloacetic Transaminase (Sgot) Among 111 M ale W orkers..................... 135 Table 4.1.73b Linear Multivariate Regression Model 2 O f Factors Predicting The Serum Glutamic Oxaloacetic Transaminase (Sgot) Among 111 Male W orkers.......................136 VII 3M MN03112187 Table 4.1.73c Linear Multivariate Regression Model 3 O f Factors Predicting The Serum Glutamic Oxaloacetic Transaminase (Sgot) Among 111 M ale Workers......................137 Table 4.1.74a Linear Multivariate Regression Model 1 O f Factors Predicting The Serum Glutamic Pyruvic Transaminase (Sgpt) Among 111 M ale Workers................................................. 138 Table 4.1.74b Linear Multivariate Regression Model 2 O f Factors Predicting The Serum Glutamic Pyruvic Transaminase (Sgpt) Among 111 M ale W orkers---------------------------- ..---------- 139 Table 4.1.74c Linear Multivariate Regression Model 3 O f Factors Predicting The Serum Glutamic Pyruvic Transaminase (Sgpt) Among 111 M ale Workers.................................................. 140 Table 4.1.75a Linear Multivariate Regression Model 1 O f Factors Predicting The Gam m a Glutamyl Transferase (Ggt) Among 111 M ale Workers.............................................................. 141 Table 4.1.75b Linear Multivariate Regression Model 2 O f Factors Predicting The Gam m a Glutamyl Transferase (Ggt) Among 111 M ale Workers.............................................................. 142 Table 4 .1 .75c Linear Multivariate Regression Model 3 O f Factors Predicting The Gam m a Glutamyl Transferase (Ggt) Among 111 M ale W orkers.............................................................. 143 Table 4.1.76 Linear Multivariate Regression Model 1 O f Factors Predicting The Alkaline Phosphatase (Akph) Among 111 M ale W orkers..................................................................................... 144 Table 4.1.77 Pearson Correlation Coefficients Between Total Serum Fluoride, Age, Body Mass Index (Bmi), Daily Alcohol Use, Daily Tobacco Consumption, And Hematology Param eters.-- ................................................................................... 145 Table 4.1.78 Linear Multivariate Regression Model O f Factors Predicting The Hemaglobin Among 111 M ale W orkers............146 Table 4.1.79 Linear Multivariate Regression Model O f Factors Predicting H ie Mean Corpuscular Hemoblobin (Mch) Among 111 Male Workers.................................................................147 Table 4.1.80 Linear Multivariate Regression Model O f Factors Predicting The M ean Corpuscular Volume (Mcv) Among 111 M ale Workers------------------------------------------------ .....-- ........1 4 8 Table 4.1.81 Linear Multivariate Regression Model O f Factors Predicting H ie W hite Blood Cell Count (W bc)* Among 111 M ale W orkers..................................................._________ 149 Table 4.1.82 Linear Multivariate Regression Model O f Factors Predicting The Polymorphonuclear Leukocute Count (Poly) Among 111 M ale Workers__________________________ 150 Table 4.1.83 Linear Multivariate Regression Model O f Factors Predicting The Band Count (Band) Among 111 M ale W orkers.................................................................................................151 Table 4.1.84 Linear Multivariate Regression Model O f Factors Predicting The Lymphocyte Count (Lymph) Among 111 M ale Workers.......................................................................................152 v iii 3M MN03112188 Table 4.1.85 Linear Multivariate Regression Model O f Factors Predicting The Monocyte Count (Mono) Among 111 M ale Workers.......................................................................................153 Table 4.1.86 Linear Multivariate Regression Model Of Factors Predicting The Eosinophil Count (Eos).......................................... 154 Table 4.1.87 Linear Multivariate Regression Model Of Factors Predicting The Platelet Count (Plate) Among 111 M ale W orkers................................................................................................ 155 Table 4.1.88 Linear Multivariate Regression Model O f Factors Predicting The Basophil Count (Baso) Among 111 M ale W orkers..................................................................................156 Table 4.2.1 Characteristics O f 749 Fem ale Employees, 1947-1989................ 157 Table 4.2.2 Characteristics O f 2788 M ale Employees, 1947-1990...................158 Table 4.2.3 Vital Status And Cause O f Death Ascertainment Among 749 Fem ale Employees, 1947-1990---------------------------- 159 Table 4.2.4 Vital Status And Cause O f Death Ascertainment Among 2788 M ale Employees, 1947-1989_______________ 159 Table 4.2.5 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) Among 749 Fem ale Employees, 1947-1989.................. 160 Table 4.2.6 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Duration O f Employment Among Female Em ployees,..........................................................................................161 Table 4.2.7 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Latency Among Fem ale Employees, 1947- 1989.......................................................................................................162 Table 4.2.8 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Any Employment In The Chemical Division Among Fem ale Employees, 1947-1989.........................................163 Table 4.2.9 Numbers O f Deaths And Standardized Mortality Ratios (Sm rs), Based On U .S. W hite M ale Rates................................... 164 Table 4.2.10 Numbers Of Deaths And Standardized Mortality Ratios (Sm rs), Based On Minnesota White M ale Rates, Among 2788 M ale Employees, 1947-1989................................... 165 Table 4.2.11 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Latency, Based On Minnesota W hite M ale Rates, Among Male Employees, 1947-1989...........................166 Table 4.2.12 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Latency, Based On Minnesota W hite M ale Rates, Among Male Employees, 1 9 4 7 -1 9 8 9 ............_ 167 Table 4.2.13 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Latency, Based On Minnesota W hite M ale Rates, Among Male Employees, 1947-1989...........................168 Table 4.2.14 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Duration O f Employment, Based On Minnesota W hite M ale Rates, Among M ale Employees, 1947-1989............................................................................................ 169 Table 4.2.15 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Duration O f Employment, Based On ix 3M MN03112189 Minnesota W hite M ale Rates, Among Mate Employees, 1947-1989............................................................................................ 170 Table 4.2.16 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Duration O f Employment, Based On Minnesota W hite M ale Rates, Among M ale Employees. 1947-1989............................................................................................ 171 Table 4.2.17 Numbers O f Deaths And Standardized Mortality Ratios (Smrs), Based On Minnesota W hite M ale Rates, Among 1339 M ale Employees Ever Employed In The Chemical Division, 1947-1989____________________________ 172 Table 4.2.18 Numbers Of Deaths And Standardized Mortality Ratios (Smrs), Based On Minnesota W hite M ale Rates, Among 1449 M ale Employees Never Employed In The Chemical Division, 1947-1989-------------------------------------- 173 Table 4.2.19 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Latency, Based On Minnesota W hite M ale Rates, Among M ale Employees Never Employed In The Chemical Division, 1947-1989----------------------------------------- 174 Table 4.2.20 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Latency, Based On Minnesota W hite M ale Rates, Among M ale Employees Ever Employed In The Chemical Division, 1947-1989..........................................................175 Table 4.2.21 Numbers Of Deaths And Standardized Mortality Ratios (Smrs) By Duration O f Employment, Based On Minnesota White M ale Rates, Among M ale Employees Ever Employed In The Chemical Division, 1947-1989............... 176 Table 4.2.22 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Duration O f Employment, Based On Minnesota W hite M ale Rates, Among M ale Employees Ever Employed In The Chemical Division, 1947-1989................177 Table 4.2.23 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Duration O f Employment, Based On Minnesota W hite M ale Rates, Among M ale Employees Never Employed In The Chemical Division, 1947-1989............ 178 Table 4.2.24 Numbers O f Deaths And Standardized Mortality Ratios (Smrs) By Duration O f Employment, Based On Minnesota W hite M ale Rates, Among M ale Employees Never Employed In The Chemical Division, 1 9 4 7 -1 9 8 9 ........... 179 Table 4.2.25 Age Adjusted Standardized Rate Ratios (Srrs) For All Cause, Cancer, And Cardiovascular Mortality By Duration O f Employment, Among M ale Employees, 1947-1989.................................................. ......_______ ________ .180 Table 4.2.26 Age Adjusted Standardized Rate Ratios (Srrs) For All Cause, Cancer, Lung Cancer, Qi Cancer, And Cardiovascular Mortality By Ever/Never Employed In The Chemical Division, Among M ale Employees, 1947- 1989...................................................................................................... 181 x 3M MN03112190 Table 4.2.27 Age Stratified, Years O f Follow-Up Adjusted Rate Ratios (Rrmh) For All Cause, Cancer, And Cardiovascular Mortality By Ever/Never Employed In The Chemical Division, Among M ale Employees, 1947-1989.......................... 182 Table 4.2.28 Age Stratified, Years O f Follow-Up Adjusted Rate Ratios (Rrmh) For All Cause, Cancer, And Cardiovascular Mortality By Duration O f Employment In The Chemical Division, Among M ale Employees, 1947- 1989...................................................................................................... 183 Table 4.2.29 Proportional Hazard Regression Model O f Factors Predicting The All Cause Mortality Among 2788 M ale W orkers................................................................................................ 184 Table 4.2.30 Proportional Hazard Regression Model O f Factors Predicting The Cardiovascular Mortality Among 2788 M ale W orkers...................................................................................... 184 Table 4.2.31 Proportional Hazard Regression Model O f Factors Predicting The Cancer Mortality Among 2788 M ale Workers................................................................................................ 185 Table 4.2.32 Proportional Hazard Regression Model O f Factors Predicting The Lung Cancer Mortality Among 2788 Male Workers................................................................................................ 185 Table 4.2.33 Proportional Hazard Regression Model O f Factors Predicting The Gi Cancer Mortality Among 2788 M ale W orkers................ 186 Table 4.2.34 Proportional Hazard Regression Model O f Factors Predicting The Prostate Cancer Mortality Among 2788 M ale W orkers.....................................................................................186 Table 4.2.35 Proportional Hazard Regression Model O f Factors Predicting The Pancreatic Cancer Mortality Among 2788 M ale Workers...................................................................................... 187 Table 4.2.36 Proportional Hazard Regression Model O f Factors Predicting The Diabetes Mellitus Mortality Among 2788 M ale Workers................................................................ 187 Table 4.2.37 Proportional Hazard Regression Model O f Factors Predicting The All Cause Mortality Among 749 Fem ale W orkers________________________________________________188 Table 4.2.38 Proportional Hazard Regression Model O f Factors Predicting The Cardiovascular Mortality Among 749 Fem ale Workers......................................................--------------------- 188 Table 4.2.39 Proportional Hazard Regression Model O f Factors Predicting The Cancer Mortality Among 749 Fem ale W orkers........................................ 189 xi 3M MN03112191 LIST O F TABLES Figure 1. Free Testosterone Versus Total Serum Fluorine................................ 190 Figure 2. Bound Testosterone Versus Total Serum Fluorine............................. 191 Figure 3. Estradiol Versus Total Serum Fluorine..................................................192 Figure 4. Lutenizing Hormone Versus Total Serum Fluorine------------------------ 193 Figure 5. Follicle Stimulating Hormone Versus Total Serum Fluorine............. 194 Figure 6. Prolactin Versus Toted Serum Fluorine..................................................195 Figure 7. Thyroid Stimulating Hormone Versus Total Serum Fluorine............. 196 Figure 8. Bound Testosterone To Free Testosterone Ratio Versus Total Serum Fluorine--------------------- .........-- .............-- .............-- 197 3M MN03112192 Li2 i s :-U SI1Q N Fluorine was first isolated as an element in 1880 by M oisser1. Five years later he synthesized the first fluorocarbons through uncontrolled reactions of carbon with elemental fluorine. It was not until the late 1930s that the controlled synthesis of fluorocarbons became possible. In the 1940s, Frigidaire and DuPont developed chlorofluorocarbons, the first commercially available fluorocarbons, for use in refrigeration During the sam e period periluorocarbons, a subclass of perfluorinated organic fluorocarbons with unique properties, were first synthesized to m eet the special needs of the Manhattan project2. The electrochemical fluorination method for perfluorocarbon production made commercial production of perfluorocarbons possible and opened the door to widespread use of perfluorocarbons 3-4. Fluorocarbons are wide ranging in their structures and uses. M any commercial applications have been developed for chlorofluorocarbon compounds including refrigeration, degreasing, aerosol dispensing, polymerization, polymer foam blowing, drugs, and reactive intermediates or catalysts. Perfluorocarbons (PFCs) have extensive applications because of their unique physical and chemical properties. These applications indude use as artifidal blood substitutes, computer coolants, polymers such as teflon, surfactants, lubricants, foaming agents, ski waxes, and in an extensive spedalty chemical industry which produces grease and oil repellent coatings for paper and doth, polymers, insectiddes, and a variety of consumer products. Perfluorocarbons are currently being tested as replacements for chlorofluorocarbons in industrial processes and products. For many years fluorocarbons were generally thought to be nontoxic. Perfluorocarbons were considered to be particularly nontoxic because they were chemically and physically inert and showed low acute toxicity in animals 4. Recent epidemiological and experimental studies have assodated exposure to chlorofluorocarbons, a subclass of fluorocarbons previously dassified as nontoxic, with direct and indirect adverse human health effects. Subsequently, researchers and regulators turned their attention to the study of other fluorocarbons. The discovery that one perfluorocarbon, perfluorooctanoic add 1 3M MN03112193 (PFOA), was present in measurable quantities in residents of several U .S. cities s*7, the recognition that some perfluorocarbons including PFOA have long half lives in the humans 8 and the observations that PFOA produced toxic effects in animals, including hepatotoxidty, endocrine toxicity, immunotoxidty, and carcinogenesis 9, has led to a re-evaluation of the toxic potential of perfluorocarbons, particularly PFOA, in humans. Despite widespread exposure to perfluorocarbons, little is known about their effects on human health, it was apparent that additional studies designed to explore their physiologic effects and potential adverse health outcomes and conducted in an occupational cohort with high exposure to PFCs, were necessary. The 3M Chemolite Plant located in Cottage Grove, Minnesota is one of a few PFC production facilities in the world. Biological monitoring data from studies of the Chemolite workforce showed that employees have had high levels and long durations of exposure to PFOA 8* 10. Ib is occupational cohort provided the opportunity to study the effects of PFOA on humans. The specific goals and objectives of this study were: GOAL-1) To quantify the human effects of perfluorooctanoic ad d on the following physiologic parameters: a) Hormones: free and bound testosterone, estracflol, lutenizing hormone, thyroid stimulating hormone, prolactin, and follide stimulating hormone. b) Serum lipids and lipoproteins: cholesterol, low density lipoprotein, high density lipoprotein, and triglycerides. c) Hematologic parameters: hemoglobin, mean corpuscular volume, white blood cell count, polymorphonudear leukocyte count, band count, lymphocyte count, monocyte count, platelet count, eosinophil count, and basophil count. 2 3M MN03112194 d) Hepatic enzymes: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, gamma glutamyl transferase, and alkaline phosphatase. OBJECTIVE 1: to conduct a cross-sectional study of production workers to estimate the relationships between total serum fluoride, a surrogate assay for prefluorooctanoic add, and physiologic parameters. GOAL 2)To quantify the mortality in an occupational cohort with long term exposure to perfluorooctanoic ad d production. OBJECTIVE 2 : to conduct a retrospective cohort occupational study to assess the mortality experience of workers using expected mortality based on Minnesota mortality rates. 3 3M MN03112195 2. REVIEW O F TH E LITERATURE 2.1 introduction The presence of small amounts of fluoride in human blood was recognized in 1 8 5 6 11. More than 100 years later, Taves 5>6 presented evidence that fluorine exists in two major forms in humans and animals; in a free ionic state and in a covalently bound organic state. Prior to this report, it was assumed that fluorine existed primarily as inorganic ionic fluoride in biological systems. Taves' observations have since been confirmed by several other investigators12'16. The discovery that organofluorine compounds constitute the majority of fluorine found in humans focused research on characterizing these undefined compounds. Guy identified a perfluorinated compound, perfluorooctanoic ad d (PFO A), as a major constituent of the serum organic fluorine fraction 7* 17 Perfluorooctanoic a d d (PFOA) is the only organic fluorine compound to be identified in human seru m 18. The recognition of human and animal toxidties assodated with perfluorochemicals 9- 19, has renewed interest in understanding the human health effects of perfluorocarbons (PFC ), particularly PFOA. - 2 Organic Fluorochemicals Organic fluorochemicals, otherwise referred to as fluorocarbons, are compounds composed of fluorine, carbon and other elements such as oxygen, nitrogen and sulfur. Perfluorocarbons have structures analogous to hydrocarbons, except the hydrogens are exhaustively replaced by fluorine 20. A limited number of organic fluorochemicals occur in nature 21*23, however no PFCs occur naturally 24,2S. The first report of the synthesis of a fluorocarbon was published in 1890 when Moissan claimed to have purified carbon tetrafluoride. it is likely he isolated fluorographite, how ever1. Pure carbon tetrafluoride was not obtained until 1930 26. W ork by Ruff and the Belgian chemist, Swarts, in the late 19th and early 20th centuries laid the foundation of organic fluoride chemistry. Midegiy and Henne extended Swarts' work and reported the synthesis of dichlorodifluormethane, 4 3M MN03112196 t I CI2F2, in 1930 27. This chlorofluorocarbon with the trade name Freon 12 is an inert, non-toxic refrigerant which was vastly superior to other refrigerants available in the 1930s. After commercial production of Freon 12 began in 1936, it rapidly became a major industrial chem ical4 26. A number of cholorfluoromethanes and chlorofluoroethanes have been produced on a commercial scale in many regions of the world. These chlorofluorocarbons have been used in large amounts as aerosol propellants and degreasers, in addition to their use as refrigerants. Currently, their production is being reduced as a result of their ozone depleting properties 28,29. In 1937, Simons and Block developed a method to produce laboratory quantities of perfluorocarbons, such as C 3F8, C 4F 10, cycioCsF-jo and c y d o C e F i2 **3- The analysis of these compounds led to the understanding that many of the structures of saturated hydrocarbons could be replicated in the form of perfluorocarbons. Research in the area of perfluorocarbons was stimulated by two developments. First, Plunkett cfiscovered the polymer, polytetrafluoroethylene, or T e flo n 1. Second, the development of perfluorocarbon chemistry was stimulated by the U.S. effort to develop atomic weapons during World W ar II under the Manhattan Project. The 235U isotope of uranium was required for the development of atomic bombs. One method of uranium isotope separation was gaseous diffusion. The only volatile uranium compound available for use in this diffusion process eras uranium hexafluoride, UF6 , an extremely reactive gas. Materials were needed for use as coolants, lubricants, sealers and buffer gases in equipment exposed to this highly reactive g a s 1' 2,26. Perfluorocarbons prepared by Simons were found to be inert to UF6. This cflscovery led to a research effort directed toward understanding the properties of a variety of perfluorocarbons and developing commercial methods for preparation of perfluorocarbons. The development by Simons of the electrochemical fluorination (ECF) w as a major milestone in the fiuorochemicai industry. Since World W ar II there has been much interest and work in this new branch of organic chemistry based on perfluorocarbons. The use of Simons' EC F method has allowed the production of a wide variety of perfluorocarbons including perfiuorinated alkanes, alkenes, ethers, esters, amides, sulfonamides and compounds with cyclic and ring structures 2. The In e rt perfluorocarbons are compounds made up of only carbon and fluorine. This dass 5 L 3M MN03112197 of compounds ranges from carbon tetrafluoride to complex multiple ring structures such as perfluorodecalin. Perfluorinated surfactants indude carboxylic adds, sulfonic adds, and their derivatives. These compounds form the basis of an extensive fluorochemical industry. A variety of perfluorinated polymers and elastomers exist. The most widely used are polytetrafluoroethylene and Kel-F, a elastomer of vinyldiene fluoride and hexafluoropropylene. 9.3 Physical Properties Perfluorooctanoic ad d is a straight chain eight carbon carboxylic a d d with a molecular weight of 414.16. The melting point of POFA is 59-60'C . Its boiling point is 189`C at standard conditions 30. Perfluorooctanoic add is produced as a complex mixture of branched chain isomers. In practice, all eight carbon carboxylic ad d isomers are refered to as PFOA. The ammonium salt of PFOA (APFOA) is the common industrially used form of PFOA. It is a white crystalline powder that easily becomes airborne and sublimes at 130*C. Perfluorocarbons have unique chemical and physical properties 20,26,31,32. The importance of perfluorination in produdng these properties cannot be overemphasized. Perfluorocarbons are not just another hydrocarbon-like molecule. Chemically, perfluorocarbons are remarkably in ert They are stable to boiling in strong adds and bases. Very few oxidizing or redudng agents react appredabiy with perfluorocarbons. Perfluorocarbons that contain other organic molecules such as nitrogen, oxygen and sulfur will partidpate in reaction at the site of these molecules. For instance, perfluoroctanoyl sulfonic a d d will react and form the sulfonamide derivative. The amide portion of this molecule can then be conjugated with many other organic compounds. The perfluorinated portion of these larger molecules remains non-reactive. Perfluorocarbons are heat stable. They (ran be heated to greater than 250*C without breakdown. At high temperatures, greater than 400`C , some compounds will breakdown. For example, PTFE, breaks down to perfluoroisobutylene (PFIB), an extremely toxic g a s 1. Because most perfluorochemicals are heat stable they are used in high temperature applications. 6 L 3M MN03112198 The inert perfluorocarbons are excellent insulators. Polymers, such as PTFE, and inerts PFCs, such as perfluorohexane, are used in electrical applications because of their superior dielectric properties. Their heat stability and insulation properties make perfluorocarbon materials the insulators of choice 20. Perfluorinated surfaces are the most non-wettable and non-adhesive surfaces known 20126. Fluorochemicai surfactants are some of the most potent surface active agents yet discovered 31. Very low concentrations of fluorochemical surfactants effectively reduce the surface tension at interphase boundaries. Most perfluorocarbons are poorly soluble in both aqueous and organic solutions. They form a group of fluorophiiic compounds, however some perfluorocarbons with functional groups such as the salts of PFOA, are highly w ater soluble 3132. Perfluorocarbon liquids dissolve oxygen avidly. This unique property is the basis for the use of perfluorocarbons as blood substitutes 33. Perfluorinated carboxylic and sulfonic adds are some of the strongest organic adds known 31. The pKa of PFOA is 2.5 34. Thus, when in physiologic solutions, they exist in primarily anionic forms. The anionic forms have a strong propensity to form complex ion p a irs *. In the p ast some investigators have assumed that the chemical and physical properties of many fluorocarbons is synonymous with lade of activity in biologic systems 35136. However, abundant evidence exists that their chemical and physical inertness does not imply biologic inertness18>* 3738. 2ASynthesis Synthesis of fluorocarbons has been accomplished using four major methods; electrochemical fluorination (ECF). direct fluorination, teleomerization, and catalytic methods using high valence heavy metals. The E C F was developed by Simons in 1941 3 The Simons process is the oldest commercial technique and remains a commercial method to obtain many perfluorocarbons. A solution of * personal communication from James Johnson, 3M Corporation 7 L 3M MN03112199 organic substrate is electrolyzed in anhydrous H F at a low voltage, high current, nickel anode. The products of these electrolysis cell reactions are largely perfluorinated. The spectrum of material produced by the EC F process is defined by the starting material. Commercial products from this process include perfluoroalkanes, perfluoroaikyl ethers, perfluoroalkenes, perfluoroalkyl esters, perfluorotriaikyl amines, perfluorocarboxylic add s and perfluorosulfonic adds 2. Products of EC F often indude a significant proportion of complex isomers and fragmentation products. For example, EC F production of PFOA from straight chain octanoic add produces 30% complex branch chain isomers 39. The mixture of products from each EC F run is unpredictably variable. These Isomeric mixes are difficult to separate and purify 33. Workers produdng PFCs using EC F may be exposed to a complex mixture that changes composition over time. Direct fiuorination is another method used to produce perfiuorocarbons. it is not subjected to the impurity problems associated with the EC F process. Direct fiuorination reacts fluorine gas with hydrocarbon substrate. Because fluorine gas is extremely reactive, direct fiuorination is a technically difficult process and has only recently been pilot tested for commerdal production of fluorocarbons. World production of fluorocarbons is limited to a handful of com m erdal plants. The 3M Corporation operates PFC production plants in Minnesota, Illinois, Alabama and Antwerp, Belgium. A plant in Italy owned by a Japanese and Italian consortium produces limited amount of fluorocarbons. Perfiuorocarbons are also produced in Germany and have been produced, in the past, in the former Soviet Union. 2.5 Sources O f Organic Fluoride Exposure G u y 17 presented possible candidates for the organic fluorine constituents of human blood based on observation made during the isolation of PFOA from serum. The organic fluorine was not likely to be a macromolecule such as a protein or nudeic add , because of its solubility in organic solvents such as ether or chlorofomVmethanol. It was not covalently bound to albumin since it was removed on charcoal at pH 3 at room temperature. The sdubility characteristics suggested that multiple compounds existed with different polarities. The major 8 3M MN03112200 compound was a polar lipid like molecule that was identified as PFOA. O ther less polar compounds appeared to be present. This data suggests that fluorocompounds other than PFOA were bound to albumin. These compounds were not esters of C i 3 - 18 fatty adds and were less polar than PFOA. Perfluorooctanyl sulfonamide (PFOS) and its derivative compounds fit this description and may be constituents of the organic fluorine fraction. Although exposure is probably low, the properties of PFOS suggest that it may accumulate to measurable levels. In contrast to ionic fluoride, little has been reported concerning the organic fluorine content of w ater and beverages. The fluorine content of ground w ater is essentially all in ionic form. Some fluorochemicals, such as the perfluorinated carboxylic add surfactants and their salts, are soluble in w ater. Such w ater soluble compounds may locally contaminate surface and ground w ater near industrial plants that use these compounds. Other perfluorinated compounds such as the alkanes, alkenes, and ethers are fluorophilic and are insoluble in aqueous solutions. Although data on the oral organic fluorine intake Is limited, it is unlikely that w ater and beverages are significant sources of organic fluorine in humans. The diet as a source of the organic fluorine found in human serum has been the subject of speculation 5*6118t ^ Non-perfluorinated fluorocompounds have found in biological systems. Marais showed that fluoroacetate was the compound responsible for toxicity from the poisonous plant Dichapetalum cymosum 41. Other investigators have found plant spedes that synthesize fluoroacetate, fluorodtrate, and monofluorinated fatty adds. Peters reported that a few toxic plants produce fluoroacetate 42. Fluoroacetate and fluorodtrate have been found in beans grown in high fluoride s o il23. Peters 21 and Lovelace et al. 22 have reported the occurrence of fluorodtrate In a few plants and foods. In animals, the metabolic activation of fluoroacetate into (-)-erythro-fluorocitrate blocks the transport of dtrate into the mitochondria and dtrate breakdown by aconitase 42< 43. O ther omega-fatty adds with even numbers of carbon atoms are highly toxic as a result of oxidation that produces fluoroacetate. Fluorodtrate also undergoes rapid defluorination in rat liver in the presence of glutathione (QSH) 44 Given the low environmental levels, the infrequent occurrence, the toxicity, and the rapid 9 3M MN03112201 metabolism of these compounds in mammalian species, it is unlikely that these monofluorinated compounds contribute substantially to the organic fluorine content in humans. Taves measured the organic and inorganic fluorine in 93 food items 45. No significant organic fluorine was found in the tested foods. Ophaug and Singer tested a market basket of food. They concluded that there was no significant organic fluorine content in food. Although food and beverages generally do not contain PFCs, it is possible that they may be contaminated by fluorochemical packaging materials. W ater and grease repellent coatings In packaging material could leach into food items in small quantities. This could occur when materials that are not designed for microwave use are used in microwave ovens. Studies have not been reported that quantify human exposures from food packaging sources. Perfiuorocarbons are contained in many consumer products. Fluorocarbon surfactants such as PFOA, PFOS, and ifs derivatives are present in window deaning products, floor waxes and polishes, fabric and leather coatings and carpet and upholstery treatments 20. Additionally these compounds are used to coat food wraps and are incorporated into plastic food storage bags. Fluorocarbons are the basis for a new generation of cross country ski waxes. Teflon and Teflon related products are widely used as lubricants, electrical insulators, heat and chemical stable gaskets and linings and in non-stick cookware. Ruoroalkanes such as perfluorohexane are being evaluated as CFC replacements. If perfluorohexane or other fluorocarbons are used as replacements for CFC's, consumer exposure from aerosols and other products will increase dramatically. PFC's have several experimental medical uses inducting use as blood substitutes, x-ray and magnetic resonance imaging contrast agents 46, vitreous replacement and in liquid ventilation therapeutic methods 47. Recently, a potent fluorocarbon insecticide has been marketed to control fire ants 48. Perfiuorocarbons have a variety of industrial uses. Teflon and other polymers are used where heat stable and chemically inert liners, gaskets and lubricants are necessary. In addition, they are used as electrical insulators both in solid and 10 3M MN03112202 liquid form and used as inert non-conductive liquid coolants in electrical devices such as Cray supercomputers. Perfluorinated surfactants are important fire suppression materials. Perfluorocarbons have been used to control the metal vapors in electroplating processes and to prevent the release of toxic gases\ from landfills20. Perfluorocarbons are being considered to replace CFC's in many processes such as refrigeration, polymer foam blowing and building insulation. New applications are being continually developed for these unique compounds, making increased exposure to workers probable. 2.6 Toxicokinetics of PFOA Since Taves and Guy's observations, perfluorocarboxylic adds, peffluorosulfonic adds and their derivatives have been the subject of numerous toxicokinetic and toxicodynamic studies in animals. These studies have focused primarily on two compounds, PFOA, and perfluorodecanoic add (PFDA). Perfluorooctanoic ad d or its salts are well absorbed by ingestion, inhalation or dermal exposure. Absorption has been studied primarily in rats, although a number of other spedes have been studied. Five male and five fem ale rats were exposed to airborne APFOA for one hour. In this experiment the nominal air concentration of ammonium perfiuorooctanoate was 18.6 mg/i. No animals died during the inhalation exposure or the 14 day post exposure observation period. Pooled serum samples contained 42 ppm of organic fluorine for males and 2 ppm for females. Inorganic fluoride content was 0.02 ppm for males am i 0.01 ppm for fem ales 9. Kennedy and H a ll38 studied the inhalation toxicity in male rats of ammonium perfluoroctonate using both single dose and repeated dose schedules. They found a LC50 of 980 mg/m3 for a 4 hour exposure placing PFOA in the moderately toxic by inhalation category. Following ten repeated doses at levels of 1 .0 ,7 .6 , and 8 4 mg/m3 blood ammonium PFOA levels were obtained. At the 1.0 mg/m3 level PFOA levels were 13 ppm, at the 7.6 mg/m3 level PFOA levels were 4 7 ppm and at 84 mg/m3 level PFOA levels were 108 ppm. Therefore it appears that PFOA is well absorbed by inhalation. It should be noted that the exposures were to APFOA dust, the likely form for occupational exposure. 11 3M MN03112203 Ammonium perfluorooctonoate in food and PFOA administered by gavage in propylene glycol or com oil vehicles are well absorbed in rets. In an acute oral LD50 study 9, rets displayed a dose dependent spectrum of toxicities indicating that PFOA was absorbed after ingestion. PFOA levels were not measured in this study. In a subacute oral toxicity study, rats were fed PFOA for 90 days 9. Serum concentration of organic fluorine showed a dose response relationship in both sexes. A marked gender difference in organic fluorine levels was observed. Males had organic fluorine 50 times higher than fem ales at each dose level. Studies have since demonstrated excellent oral absorption of PFOA in a variety of species including rets, mice, guinea pigs, dogs, hamsters and monkeys 9* 19. Of most immediate, relevance to humans have been stucRes in a small number of rhesus monkeys 9. In a 90 day oral toxicity study, monkeys w ere given 3 ,1 0 , and 30 mg/kg/day doses of APFOA. In monkeys at the 3 mg/kg/day dose, mean serum PFOA w as 50 ppm in males and 58 ppm in fem ales. At the sam e dose, males had 3 ppm and fem ales 7 ppm in liver samples. At 10 mg/kg/day doses, male monkeys had a mean serum PFOA of 63 ppm and fem ales 7 5 ppm. Liver levels were 9 and 10 ppm for males and fem ales, respectively. Because ail but 1 monkey died at the 30 and 100 mg/kg/day dose levels, only 1 serum sample from a male monkey in the 30 mg/kg/day dose group was available, in this monkey the serum level of PFOA was 145 ppm. In the 30 and 100 mg/kg/day dose group mean liver levels were greater than 100 ppm. Thus, the oral route of absorption may be a significant contributor to the body burden of PFO A in exposed workers. Dermal absorption of PFOA has been studied in rats and rabbits. Ammonium perfluorooctanoate is a fine white powder that may come into contact with skin and be absorbed. In rets dermally exposed to ammonium perfluoroctonate at 4 dose levels, PFOA was absorbed in a dose dependent fashion 37. In single dose dermal exposure experiments using rabbits, PFOA appeared to be absorbed. Levels of fluorine were not measured, but dose dependent toxic changes were noted 9 In a multi-dose experiment, ten male and ten fem ale rabbits were injected dermally with a 100 mg/kg dose of PFOA on a five day a w eek schedule for two weeks. Total serum fluorine levels were increased in a dose-dependent fashion. Dose-dependent changes in weight were noted 49 From these studies, 12 3MJVIN03112204 it appears that dermal exposure to the salts of PFOA are absorbed in animals, in the past, Chemoilte workers have been exposed to large dermal doses of ammonium perfluoroctonate. It appears that dermal exposure may have played a significant role in the absorption of PFOA in these workers. Upon recognition that PFOA could be absorbed dermaily, work practices were changed and engineering controls were adopted that reduced dermal exposures. The role that dermal exposures currently play in PFOA absorption at Chemoilte has not been well studied. Once absorbed, PFOA enters the plasma probably by diffusing as a neutral ion pair. In plasma, PFOA is strongly bound to proteins in the serum with more than 97.5 percent in bound form 50. It is likely that albumin is the major site for high affinity binding 8'7*S0'54]. There does not appear to be a sex difference in protein binding 50154 Hanhijarvi et al. have suggested that protein binding is saturable in rats 5S. Using human serum, Ophaug and Singer39 found that PFOA w as 99% protein bound at PFOA levels up to 16 ppm total fluorine, however. Guy suggested that perfluorocarboxyiic adds bind to albumin in a sim ilar fashion to fatty adds 24. This hypothesis is consistent with the results of several studies. Taves observed that the organic fraction of serum co-migrated with albumin during electrophoresis 6. Dialysis and uritrafiltration studies observed the retention of organic fluorine during dialysis and uitrafiitration 7* 17>56. Belisie and Hagen reported that PFOA appeared to be strongly protein bound in human serum 51. Extraction of PFOA from addified water is quantitatively complete using hexane. W hen PFOA is extracted from plasma, recovery is only 35 percent. Plasma appeared to complex PFOA and PFDA. The partitioning of the bound into organic phase during extraction was more difficult and necessitated the use of more polar solvents. Kievens 53 suggested that CF2 and CF3 groups complex with polar groups that are present in the amino adds in proteins such as albumin. In protein predpitation studies using bovine serum albumin, PFOA bound to albumin at an estimated 28 binding sites per molecule s2. Nordby and Luck studied the predpitation of human albumin by PFOA. Under addic pH conditions, PFOA produced reversible predpitation of albumin 57 by binding to high affinity sites. These studies do not rule out significant binding to other plasma proteins or erythrocyte components, in studies using serum protein electrophoresis, the protein bound organic fluorine was distributed in a diffuse 13 3M MN03112205 pattern 6- 17 suggesting that PFOA protein binding may be nonspecific. The large amount bound to albumin may reflect the abundance of albumin in plasma and serum. In rats, PFOA is distributed to all tissues studied except adipose tissue. The highest concentrations of PFOA are in the serum, liver, and kidneys. Ylinen et al. 94 studied the disposition of PFOA in male and fem ale rats after single and 28 day oral dosing. After a single dose of 50 mg/kg, PFOA was concentrated in the serum. Twelve hours after dosing 40% of the PFOA dose w as found in the serum of m ales and 10% in fem ales. Males retained 3.5% of the dose in serum after 14 days. PFOA was retained in the liver for much longer than in serum. In females, the half-life of PFOA in liver was 60 hours compared to 2 4 hours in serum. In males the half-life was 210 hours in liver and 105 hours in serum. It is noteworthy that PFOA was not found in adipose tissue in detectable quantities. After 28 days of PFOA treatment, PFOA was distributed to the following sites in decending amounts: serum, liver, lung, spleen, brain, and testis. Again, no PFOA was found in adipose tissue. The distribution of PFO from serum to the tissues occurred in a dose dependent manner for fem ales. In male rats, the concentrations of PFOA in testis and spleen followed a dose dependent trend. The levels in male ratserum and liver was the sam e for the 10 mg/kg and 30 mg/kg dose group. Johnson and Gibson 58159 studied the distribution of 14C labeled ammonium perfluorooctonoate after a single iv dose in rats. Their findings were similar to those of Ylinen et al. The primary sites of distribution were the liver, kidneys, and plasma. Other sites, including adipose tissue, had less than 1% of the administered dose. The level of PFOA in the testis of male rats was not reported. As discussed previously, the 90 day oral toxicity study in rhesus monkeys showed that the relative amounts of PFOA in serum and liver was different in monkeys compared to rats. In the low dose group of monkeys (3 and 10 mg/kg/day) serum had 5 to 10 times the PFOA levels found in liver. However, at higher dose levels, the PFOA levels were equally distributed. Additionally, no sex differences were noted In the monkeys liver and serum PFOA levels. There is no evidence that perfluorinated compounds including PFO A are biotransformed by living organisms. Several studies have examined whether 14 3M MN03112206 PFOA is conjugated or incorporated into tissue constituents such as triglycerides or lipids. Ylinen et a!, found no evidence in W istar rats for metabolism or incorporation of PFOA into lipids 34 Although the lipid content in PFOA treated rats was different than that in untreated rats, Pastoor et al. did not find evidence for PFOA incorporation into lipids or of metabolism 60 Vander Heuval et al. showed that PFOA was not incorporated into triacylglycerols, phospholipids, or cholesterol esters in the liver, kidney, heart, fat pat, or testis of male or fem ale rats 61. No evidence has been found that PFOA is conjugated in phase II metabolism 61. Kuslikis et al. studied the formation of activated coenzyme A (CoA) derivatives of PFOA using rat liver microsomes. They found no evidence for the formation of a CoA derivative. Sex related differences in the toxicokinetics of PFOA have been reported for rats. The mechanism of PFOA excretion appears to be species-dependent since these gender differences are not seen in mice, monkeys, rabbits, or dogs 9>62. The half-life of PFOA in fem ale rats has been estimated to be less than one day " , whereas the half-life of PFOA in males is five to seven days " . it is of note that PFDA does not exhibit this gender difference " . It is hypothesized that the sex differences in sensitivity to the toxidties of PFOA are as a result of the slower excretion of PFOA in male rats compared to fem ale rats. Investigators have reported that rate have an estrogen-dependent active renal excretion mechanism for PFOA which can be inhibited by probenecid " 54 As noted previously, females have a much shorter half-life than maie rats. The half-life in males can be reduced by castration or estrogen administration. It can be reduced to the female half-life by a combination of castration and estrogen treatm ent Estrogen administration alone is almost as effective as the combination of castration and estradiol treatment in reducing the PFOA half-life. This treatm ent increased the renal excretion of PFOA in maie rats to those observed in fem ale rats. O ther investigators have reported that the gender difference in half-life depends on a testosterone mediated increase in PFOA tissue binding 64. This hypothesis is consistent with the gender difference in tissue half-life discussed previously Johnson has suggested that the primary method of excretion in intact m ales is via the hepatobiliary route " " . He reported that cholestyramine enhanced the fecal elimination of carbon 14 labeled PFOA in male rats. These data suggest there was biliary excretion with enterohepatic circulation of PFOA, particularly in 15 3M MN03112207 male rats. However, in a male worker with high serum PFOA levels who was treated with cholestyramine, little if any change in excretion of PFOA was noted. In this study PFOA was excreted slowly in the urine. In humans, the half-life of PFOA appears to be extremely long and is not sex dependent. Ubel and Griffith 8 reported kinetic data for one highly exposed worker. At the tim e he was removed from exposure his serum organic fluorine was 66 ppm, 80 percent of which was PFOA. O ver the next 18 months his organic fluorine level decreased to 39 ppm. Urinary excretion of PFOA fell from 387 micrograms/24 hours to 80 micrograms/24 hours. The decline in organic fluorine levels was consistent with two compartment kinetics, with a calculated half-life of 2 to 5 years. Additional unpublished biological monitoring data from three Chemolite workers is consistent with the 2 to 5 year haif-life. In the Chemolite workforce, male and fem ale workers employed in jobs with similar PFOA exposure have increased PFOA levels. Since men and women with similar exposures have similar levels, a large gender difference in PFOA toxicokinetics is unlikely. Therefore, the relevance of the rat data in assessing the effects of PFOA in humans is questionable. 2.7 Toxlcodvnamics of PFOA 2.7J Male-Eteproclufitiysloxidlies Both PFOA and PFOA have been found to produce significant toxidties in the reproductive systems of male rodents 1963,6S. The testis has been reported as the target organ of toxicity for both PFOA and P F D A 19*68 Additional evidence exists suggesting that these compounds affect the function of the hypothalamicpituitary-gonad axis (H P G )19*6S. Perfluorodecanoic add , but not PFOA, has been shown to produce degenerative changes in rat seminiferous tubules that could progress to tubular necrosis. Van Rafelghem et at. reported that a single ip dose of 50 mg/kg of PFDA, produced degenerative changes in rat seminiferous tubules 8 days after injection 68 Similar but lesser changes were noted in the seminiferous tubules of hamsters and guinea pigs treated in the sam e manner. They reported no such change in 16 3M MN03112208 treated mice. Bookstaff and Moore 85 did not observe similar changes in rats treated with 20-80 mg/kg of PFDA. They used a different strain of rats in their experiments which is less susceptible to testicular toxicants than those used by Van Rafelghem et al. Thus, the effects of perfluorocarboxylic adds on seminiferous tubules may be limited to a specific compound, PFDA, in a spedfic strain of rats. The effects observed by Van Rafelghem et al. in other spedes were not consistent and did not demonstrate a dose-response relationship, in monkeys treated orally with PFOA, no compound related histopathologic changes in the seminiferous tubules were noted 8. In a two year rat feeding study, PFOA treated animals were observed to have increased numbers of Leydig ceil tumors*. M ale and fem ale rats were fed PFOA containing diets resulting in a mean intake of 1.5 and 15 mg/kg/day. A statistically significant increase in Leydig cell adenomas of 0% , 7% , and 14% in the control, low dose, and high dose groups, respectively, was observed at the end of the two year study. The result was statistically significant as a result of the unexpectedly low number of adenomas in control animals. Historically, CD rats experience a lifetime mean Leydig cell incidence of 6.3 percent with a range of 2 to 12 percent The high dose group incidence is outside the expected range and may represent a compound related effect Although the evidence was not definitive, it suggested that PFOA may alter the histology as well as the function pf Leydig ceils in rats. Perfluorooctanoic acid was not mutagenic in the standard teste including the Ames assay using five spedes of Salmonella typhimurium and in Saccharomyces cerevisiae 9. Mammalian cell transformation assays using C 3 H 10 T 1/2 cells were also negative 67. These data suggest that PFOA is not a genotoxic xenobiotic. The increase in Leydig (tell tumors may be the result of an epigenetic mechanism. The observation that rats fed PFOA for 2 years had an increased inddence of Leydig cell adenomas prompted researchers to examine the hormonal effects of PFOA in male re ts 19. Adult male CD rets were treated orally with PFO A in doses of 1 to 50 mg/kg. Serum estradiol levels were elevated in the rets treated with more than 10 mg/kg of PFOA. In the highest dose group estradiol was 2.7 times * Report: 3M Riker Laboratories. Two Year Oral Toxicfty/Carcinogenicity Study of FC143 in Rats #281CR0012,1983 17 3M MN03112209 greater than the estradiol levels In pair fed control group rats. Serum testosterone levels were significantly decreased in a dose dependent manner when compared with ad libitum feed control animals. No significant differences were observed between the high dose rats and their pair fed controls, however. No significant differences were noted in serum luteinizing hormone (LH) levels. Additionally, the accessory sex organ relative weights of the highest group were significantly less than those of their pair-fed controls. In order to clarify the site of PFOA action, C o o k 19 conducted a set of challenge experiments in PFOA treated rats. The results of these experiments demonstrate that the altered testosterone levels were PFOA related. Human chorionic gonadotropin (hCG) challenge ran be used to identify abnormalities in the steriodogenic pathway. Human chorionic gonadotropin binds to the LH receptors on Leydig cells and stimulates sex steroid hormone synthesis Abnormalities in Leydig ceil function can be detected by challenging Leydig cells with hCG and measuring steroid hormone production. Similarly, abnormalities in pituitary secretion of gonadotropins ran be identified using a gonadotropin releasing hormone (GnRH) challenge that stimulates LH release Hypothalamic dysfunction can be identified using a naloxone challenge to stimulate GnRH release 70. In rats treated with PFOA for 14 days at the sam e dose level as the initial experiment, the Leydig cell production of testosterone was significantly blunted after hCG challenge in the highest dose group compared to ad libitum fed controls. A small, non-significant blunting of th e testosterone production in response to GnRH and naloxone was observed. Following GnRH and naloxone stimulation, LH levels were not significantly different in the treatm ent and control animals. The hCG challenge showed that the decrease in testosterone in PFOA treated rats resulted from altered steroidogenesis in the Leydig ra il. The results from the GnRH and naloxone stimulation were not definitive. The results were compatible with an effect at the pituitary level as well as at the Leydig cell level. Cook et al. examined the site at which testosterone steroidogenesis w as affected by PFOA. Progesterone, 17 alpha-hydroxyprogesterone and androstenedione were measured after hCG challenge. Progesterone and 17 alphahydroxyprogesterone were unaffected. Androstenedione levels were significantly decreased in PFOA treated rats compared to controls. Given that the conversion of 17 alpha-hydroxyprogesterone to androstenedione by C 17/20 lyase is 18 3M MN03112210 necessary for testosterone synthesis, these results suggest that decreased testosterone is the result of a block in this conversion step, in hCG stimulated rat Leydig ceils, the 17 alpha hydroxylase/C-17/20 lyase is inhibited by estradiol. Taken together, these data are consistent with the hypothesis that the elevated estradiol levels associated with PFOA treatment inhibit the C -17/20 iyase enzyme and thereby depress testosterone levels. Cook et ai. suggested that the blunted response of LH to low testosterone may be mediated, in part, by elevated estradiol levels. A subtle hypothalamic or pituitary effect m ay also be present, however. The mechanism for the estradiol elevation was not stucfied. Perfluorodecanoic add alters reproductive hormones in male rats in a fashion similar to PFOA. In male rats treated with doses of PFDA ranging from 20 to 80 mg/kg, given as a single ip dose, PFDA decreased plasma androgen levels in a dose dependent fashion 65. Both plasma testosterone and 5-alpha dihydrotestosterone were significantly reduced. Compared to a d libitum fed control rat values, mean plasma testosterone was decreased by 88 percent in PFDA treated animals and DH T was decreased by 82 percent. These changes were reflected in accessory sex organ weight and histology. The changes in accessory sex organs after PFDA administration were found to be reversed by testosterone replacement. The PFDA decrease in androgens was the result of decreased responsiveness of Leydig cells to LH. There was no evidence for altered metabolism of testosterone. Additionally, plasma LH concentrations did not increase appropriately in the face of low plasma testosterone concentrations. This suggests that PFDA may alter the normal feedback mechanisms of the HPG axis. It is of interest to note that 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), which, like PFOA, is a nongenotoxic rat carcinogen, a peroxisome proliferators, and an inducer of P-450 system, has been shown to produce hormonal effects in male rats similar to those observed for PFOA and PFDA. Moore et a l.71 studied the effect of TCDD on steroidogenesis in rat Leydig cells. Exposure of cell to TC D D resulted in depression testosterone and 5-aipha-DHT concentrations without altering LH concentration or testosterone metabolism. Moore concluded that TCDD treatment inhibits the early phase of the synthetic pathway and the mobilization of cholesterol to cytochrome P450scc- However, Moore et ai. 19 3M MN0311221 observed decreased estradiol. TC D D has been shown to increase the estrogen mediated feedback inhibition of LH secretion 72 Additionally, in stucfies using M CF-7 breast tumor cells, the antiestrogenic effect of TC DD was m ediated by alterations in the cytochrome P450 metabolism of estradiol73. The decreased testosterone in rats could be mediated by the effect of TC D D on Leydig M ils directly, by alterations in testosterone metabolism, or through increased negative feedback at the pituitary or hypothalamic level. Recently, reports from occupational studies of TC D D exposed workers have associated TC D D exposure with hormonal alterations In human males. Egeland et a l.74 reported that men with high TC DD levels had significantly depressed serum testosterone levels. The changes in testosterone were not associated with altered LH values. Estradiol values were not reported. They concluded that dioxin has a similar effects in men and male rodents. The obvservations that PFOA, PFDA, and TCDD have overlapping spectrums of rodent toxidties suggests that peroxisome proliferators, inducers of the P-450 system and non-genotoxic carcinogens may also alter the hypothalamic -pituitary-gonad function in male animals. 2 .7 .2 F em ale R eproductive T o xicities In the two year rat feeding study, fem ale rats treated with PFO A were observed to have an increased number of mammary fibroadenomas compared to control animals. All mammary carcinomas occurred in control animals. Hyperplasia of the ovarian stroma was observed, but specific hfstopathological studies were not reported * . No information is available concerning the effect of PFO A and PFDA on HPG axis in women or fem ale animals. 2 .7 .3 Thyroid T o xicities Altered thyroid hormone dynamics have been observed in rats exposed to PFDA 75"7B. A single ip dose of PFDA in rats results in a rapid and persistent decrease in thyroxin (T4) and T 3 7B. Gutshall reported that the decrease in thyroid hormones occurred as early as eight hours after treatm ent and persistent for at least 90 days n . These changes were associated with a hypothyroid-like state in * Report: 3M Rlker Laboratories. Two Year Oral Toxicfty/Carcinogenicity Study of FC143 in Rats #281CR0012,1983. 20 the treated rats. The alterations in serum thyroid levels occurred at dose levels that did not produce a hypothyroid syndrome 78. Animals with depressed T 4 levels were found to be metabolically euthyroid 77 Replacement of T 4 resulted in normal food intake, but did not reverse the hypothyroid-like syndrome of hypothermia and bradycardia 75. This suggests that PFDA has a marked effect on cellular metabolism that is independent of its effect on thyroid homeostasis. The low T4 was thought to be a result of two mechanisms. First, PFDA readily displaces T4 from albumin which results in increased metabolic turn over of the hormone. Second, the response of the hypothalamic-pituitary-thyroid (H PT) axis appeared to be depressed as assessed by thyrotropin releasing hormone simulation testing 7S. in these studies, the animals had increased levels of thyroid responsive hepatic enzyme activities suggesting that the PFDA treated rats were not functionally hypothyroid. The histological appearance of the thyroid glands were unremarkable, although treated rats had significantly lower thyroid weights. TSH levels were not studied. No similar studies are available for PFOA. PFOA has been noted to produce a transient weight loss in treated rate 30. The hypothyroid-like syndrome observed in PFDA treated rats has not been studied in PFOA treated rate, however. Since the thyroid hormone effects of PFDA do not cause the hypothyroid-like state in rats, PFOA m ay alter the HPT axis without producing this syndrome. 2.7.4 Hepatic Toxidties The primary site of PFOA toxicity in rodents is the liver. Peroxisome proliferation (PP), induction of enzymes involved in B-oxidation of fatty acids, and induction of cytochrome P450 occur after a single PFOA dose. Marked hepatomegaly has been noted coincident to the PP and enzyme induction. Increased liver size was the result of a combination of both hypertrophy and hyperplasia Cell hypertrophy predominated after an initial burst of cell proliferation. The initial hyperplasia is evidenced by large hepatocytes and markers of DNA synthesis 80. Areas of increased necrosis in the periportal regions have been observed 81. The relationship between hepatic enlargement, peroxisome proliferation, and increased B-oxidation Is undear. Xenobiotic induced changes in one spedfic peroxisomal enzyme are not necessarily linked to changes in other peroxisomal 21 3M MN03112213 enzymes or hepatic enlargem ent82. Studies have suggested that xenobiotic induced hepatomegaly and PP may be related to the endocrine status of experimental animals or to oxidative stress 83'86. Adrenal and thyroid hormones may play a role in peroxisomal proliferation. 80188 Studies of dofibrate, a PP, have shown that endocrine manipulation can modify its hepatic effects, in adrenalectomized and thryoidectomized rats, clofibrate-induced hepatomegaly was reduced compared to the effect in control rats 881M . Conversely, in thyroidectomized or hypophysectomized rats, dofibrate induced peroxisomal B-oxidation enzymes were increased compared to normal rats * . Thottassery et al. compared the PFOA-induced hepatomegaly in normal rats, adrenalectomized rats and adrenalectomized rats with cortisol replacement . They found that hepatomegaly was cortisol dependent and was primarily a result of hepatocyte hypertrophy. Hyperplastic responses were also cortisol dependent and were noted in periportal regions of the liver. Peroxisomal proliferation did not depend on cortisol and was observed in centrilobular regions. They concluded that PFOA-induced hepatomegaly and peroxisome proliferation were separate processes. In oral feeding studies, PFOA and other PP were reported to cause increased hepatomegaly in males compared to fem ales. This difference could be reduced by exogenous estradiol administration or castration and eliminated by castration and estradiol administration 54. These observations may be explained by an estrogen dependent renal excretion mechanism or a testosterone mediated increase in tissue binding * 87. 88 Issemann and Green have cloned a mouse PP activated receptor, m PPAR, a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors that is activated by peroxisome proliferators 89. This receptor directly mediates the effects of peroxisome proliferators (PPs). Tugwood has shown that PPs activated PPAR recognizes a specific response unit on the Acyl-CoA oxidase gene promoter In a manner sim ilar to the steroid hormone receptor90. The action of PFOA and other PPs m ay be m ediated by a family of cytosolic receptors that regulate gene transcription in a m anner sim ilar to other nuclear hormone receptors. 22 3 M _ M N 0 3 1 12214 2 .7 .5 N ongenotoxic C arcin o gen esis In initiation, selection, and promotion experiments in rats, PFOA produced an increased number of hepatocellular carcinomas 91 92 Several mechanisms for PFOA associated nongenotoxic carcinogenesis have been suggested. Perfiuorooctanoic a d d is an archetypal member of a unique sub d ass of PPs that are not metabolized. Reddy has argued that the structurally diverse peroxisome proliferated (PP) are a distinct dass of nongenotoxic carcinogens 89 Reddy proposed that PPs induce oxidative stress which results in increased tumor formation. According to this theory, the observed increase in hydrogen peroxide formation assodated with increased B-oxidation is not assodated with an increase of similar magnitude in detoxifying catalase activity 86. Oxidative attack by hydrogen peroxide and other reactive oxygen species on cell constituents and membranes leads to DNA damage and increased cell proliferation. Increased proliferation in concert with DNA damage produces increased cell transformation and malignandes. Studies testing the theory that PFOA induces HCC by increasing oxidative stress have lead to conflicting results. Takagi et al. observed an increase in 8hydroxydeoxyguanosine in liver DNA from rats exposed to PFOA. They conduded that rat hepatocytes were under increased oxidative stress 93. Handler et al. found no increase in hydrogen peroxide production in intact livers exposed to PFOA 94. Lake et al. foiled to find an assodation between hepatic tumor formation and peroxisome proliferation 95. Thottassery et al. observed that the PFOA induction of B-oxidation was independent of adrenal hormone status. A PFOA assodated increase in catalase activity depended on cortisol80. Therefore, the hormonal status in animals used in experiments could confound studies of oxidative stress and account for the conflicting results. 2 .7 .6 Im m u notoxidtv In the 90 day monkey feeding study, bone marrow and lymphoid tissue were a site of histopathology 9. Treated monkeys in the highest two dose groups were observed to have moderate hypocellularity of the bone marrow. Spedfic 23 3M MN03112215 histopathological findings were not reported. Atrophy of lymphoid follicles In lymph nodes and the spleen were noted in the same treatm ent groups. No follow-up studies of these observations have been reported. Studies in PFOA treated rats have not shown histological changes in the immune system 9. 9 7 .7 M ech an ism s o f A ction The mechanism of toxicity of perfiuorinated surfactants m ay be mediated by their effect on cell membranes. Olson and Andersen "s u g g e s te d that PFOA may alter membrane function through changes in fatty add composition and oxidation status. Levitt and Uss hypothesized that the effect of perfiuorinated surfactants is mediated by their alteration of membrane organization or fluidity 97. Shindo 32 reported that misdbility of fluorocarbon and hydrocarbon surfactants depends strongly on carbon chain length. A carbon chain length greater than eight carbons is necessary for immisdbility, Perfluorocarbon surfactants with eight or fewer carbon atoms are misdble with hydrocarbon surfactants with carbon chain lengths up to nine. These observations could have important implications for biological systems that contain fluorocarbon surfactants. Cellular membranes are a phase boundary, usually between a lipid phase and an aqueous phase. Surfactants will segregate to this phase boundary. Two immisdble surfactants may form two coexistent monolayers on the inside and outside of the membrane whereas misdble surfactants will form only one such monolayer. The presence of two monolayers will maximally reduce the surface tension at the boundary, whereas a single monolayer will affect surface tension to a lesser degree. Changes in surface tension may alter membrane fluidity and affect its function in such processes as signal recognition and transduction, it is interesting to note that the change in misdbility in Shindo's experimental system occurred for fluorocarbon surfactants with carbon chain lengths greater than eight. This change in misdbility depended on hydrocarbon surfactant chain length as well. The effects of PFOA and PFDA on experimental membrane systems and cellular membranes have been Investigated. Inoue studied the differential effects of octanoic add and perfluorooctanoic ad d on experimented cell membrane 24 3M MN03112216 properties 98. The phase transition temperature of dipalmitoylphosphatidylcholine vesicles decreased linearly as PFOA increased in concentration up to one mM and then reach a plateau. This suggested that PFOA m ay form aggregates in the membrane above a critical concentration. Such a phase separation is observed to occur in micelles 32. The partition coefficient between w ater and the membranes for PFOA, K 8910, was larger than the coefficient for ionized octanoic add , K - 1 3 5 , possibly because of the difference in hydrophobidty between hydrocarbon and fluorocarbon chains in aqueous solution. H ie differences between the toxicokinetics and toxicodynamics of PFOA and PFDA may be the result of their differing miscibilities with ceil membrane surfactants. Levitt and Lies investigated the effect of PFOA and PFDA on the plasma membranes of cells from F4 human B-lymphoblastoid cell line using the dye merocyanine 540 (M C 5 4 0 )97 The dye binds to phospholipids that are loosely packed on the outer M il membrane, but does not bind to highly organized lipids and does not penetrate the membrane of healthy cells " . A large decrease in MC540 cell surface binding was observed after treatm ent with sub-lethal concentrations of PFOA and PFDA but not other non-perfiuorinated fatty adds. Albumin or serum reduced the change in M C540 binding. This effect may be a result of the strong protein binding of PFOA and PFDA by albumin *. These observations suggest that PFOA and PFDA either interact directly with M C540 lipid binding sites or alter the structure of the lipids in the membranes. in experiments examining functional changes in the lymphoblastoid cell lines, Levitt and Liss observed that PFOA and PFDA could cause direct dam age to cells resulting in the release of membrane bound <ll proteins and immunoglobulins in soluble form 9S. PFDA was significantly more potent than PFOA in solublizing proteins and killing cells. This may be the result of different misdbilities in the cell membrane of these compounds. However, neither PFOA nor PFDA reduces the ability of surface immunoglobulins to migrate and undergo capping after antigen recognition 97. In the PFOA concentration ranges that decreased M C540 binding, PFOA did not affect immunoglobulin migration and capping. Capping involves the cytoskeletal mediated polar migration of immunoglobulins within the plane of the membrane 10. Apparently, the PFOA 25 3M_MN03112217 and PFDA associated membrane changes do not affect membrane characteristics that are important for receptor migration. The membrane effects of PFDA have been studied in greater detail. Piicher et al. reported that a single injection of PFDA in rats significantly reduced the apparent number of B adrenergic receptors in cardiac c e lls 101. This change in number of receptors was reflected in the diminished response of adenylyl cyclase (AC) to epinephrine in PFDA treated rat cardiac ceils. The intrinsic properties of AC were not altered. The action of PFOA was on the epinephrine receptor. The fatty ad d composition of the treated rat cardiac ra il membranes was significantly altered 101. Palmitic (16:0) ad d was elevated 13 percent, eicosotrienoic (20:3 w 6) was elevated 71 percent, and docosahexaenoic add (22:6 w 3) was elevated 18 percent Arachldonic add (20:4) w as reduced by 18 percent. Several other investigators have reported changes in membrane function following PFDA exposure. W igler and Shaw 102 demonstrated that PFDA inactivated a membrane transport channel for 2-aminopurine in L 5178 Y mouse lymphoma cells. In vitro experiments reported by Olson et a l.103 showed that erythrocytes exposed to PFDA exhibited decreased osmotic fragility and increased fluidity. Taken together, these studies indicate that perfluorinated surfactants exert their effects on ceil membranes. The effects appear to be limited to the outer portion of the membranes as the result of differential partitioning within the membrane or 1binding to spedfic membrane constituents. Although PFOA and PFDA can be cytotoxic as a result of their detergent action on membranes, their membrane effects at lower doses are not related to their detergent action. From available data, it appears that functional membrane changes may be limited to spedfic receptor mediated functions. 2 J Occupational Fluorine Exposures At Chemolite In workers employed in fluorochemical production plants, blood organic fluorine has tor outweighed ionic fluoride A 12* 14,51- 56. More than 98 percent of the total fluorine in these groups has been reported to be organic fluorine. Therefore, the use of total fluoride levels, which consist predominantly of organic fluorine compounds, is a valid surrogate for organic fluorine in occupationally exposed groups. In workers at the Chemolite plant, PFOA has been identified in the serum 26 3M MN03112218 of these workers and was estimated to account for 90 percent of organic fluorine found in the serum samples 8. in this cohort of workers, total fluorine is a good surrogate measure for PFOA. Industrial hygiene measurement of fluorochemicals have been conducted at the Chemolite plant since the 1970s 8. These measurements indude area samples, persona! breathing samples and surface wipe samples, in 1977, a comprehensive effort at evaluating fluorochemical exposures was conducted at the Chemolite p lant During certain operations breathing zone PFOA concentrations were as high as 165 ppm. After extensive engineering control alterations, the plant was serially re-surveyed. In general, airborne exposures were below the recommended limit of 0.1 mg/m3. However, there was evidence of surface contamination in production buildings 8. In 1986, airborne PFOA, as well as breathing zone samples were less than 0.1 mg/m3 based on 8 hour time weighted averages. Levels as high as 1.5 mg/m3 were measured in breathing zone samples during certain dean-up and maintenance zone samples. Perfiuorobutyric a d d was aiso found, but in much lower concentrations. Spray dryer operators had consistently higher exposures, even following extensive equipment improvements. * It appears that airborne exposure to PFOA was low for most workers. Spray dry operators and workers involved in d ean up and maintenance activities have higher intermittent exposures. Although personal protection devices are required in high exposure jobs, worker compliance has not been evaluated. The role that surface contamination plays in worker exposure has not been d efin ed *. The mute of PFOA exposure in worker has not been dearly identified. 2.9 Epidemiological Studies A retrospective cohort mortality study of employees at the Chemolite Plant In the period of 1948-1978 was conducted by Mandel and Schuman 8 O f the 3,686 male employees who were employed for at least 6 months, 159 deaths were identified. There was no excess mortality in the employees as compared to all ] personal communication from Stan Sorenson, 3M Corporate Medical Department personal communication from Stan Sorenson, 3M Corporate Medical Department 27 3M MN03112219 cause or cause specific mortality in the U.S. white male population. The subcohort of all chemical division workers did not show any ail cause or causespecific excess in mortality. Starting in 1976 medical surveillance examinations were offered to Chemolite employees in the Chemical division 8. Approximately 90 percent of the workers participated in the program. No health problems related to the exposure to fluorocarbons were encountered in participants. Serially conducted surveillance examinations have failed to reveal any relationship between blood levels of organic fluorine and clinical pathology * . 2.10 Summary Animal studies have suggested that there are five areas of toxicity associated with PFOA exposure. These include hepatotoxicity, immune system alterations, reproductive hormone alterations, Leydig cell adenomas, and non-genotoxlc hepatocarcinogenicity. Toxicity studies have primarily used rodents. There is considerable variability between strains of rats for some of the toxic endpoints such as Leydig cell adenomas. AdcBtionaJly, some of the effects seen in rats have not been seen in other rodent species such as mice, hamsters or guinea pigs. The limited data available on PFOA exposed rhesus monkeys and occupationally exposed workers suggests that any extrapolation of the results from rodent experiments to humans requires more information about the mechanism of PFOA toxicity. From this data it does not appear that the liver is a major site for PFOA toxicity in humans. O f greater human health concern are the potential effects on the immune system and the reproductive hormones. In the past, workers have been found to have significant blood levels of PFOA. Many workers have levels above one ppm. These blood levels are 50-1000 times background levels in the general population. These levels may be high enough to produce toxidties in occupationally exposed humans. A confident estimate of risk cannot be made until further information on the adverse health effects of PFOA exposure in humans is obtained. * personal communication from Larry Zobel; 3M Corporation Medical Department 28 1 3M MN03112220 3. METHODS 3.1 Introduction The effects of perfluorooctanoic add (PFOA) exposure on human health were studied in employees of the 3M Chemoiite plant (hereafter referred to as Chemolite) located in Cottage Grove, Minnesota. Two studies were conducted to investigate of the human health effects assodated with PFOA exposure. First, mortality assodated with occupational PFOA exposure was studied using a retrospective cohort design. Second, a cross sectional study design was used to estimate the relationships between PFOA exposure and selected physiologic parameters. A retrospective cohort study was designed to examine mortality among workers. All workers ever employed at the Chemolite plant for greater than six months were induded in the cohort. All causes and cause-spedfic mortality w ere compared to expected mortality. Expected mortality was calculated by applying sex and race specific quinquennial age, calendar period, and cause-spedfic mortality rates for the United States and Minnesota populations to the distribution of observed person-time 104> 10S. Age adjusted standardized rate ratios were calculated106. A relative risk (RR) for PFOA exposed workers compared to unexposed workers was calculated using proportional hazard regression m odels107. The RR were stratified by gender and adjusted for age at first employment, duration of employment and calendar period of first em ploym ent Any significant differences between observed and expected cause-specific mortality were to be explored using nested case control studies. Case studies were completed tor causes of death with 5 or more deaths and standardized mortality rates greater than 1.5. Each deceased individual's record was examined tor commonalties In job history Information including age at first employment, calendar period of employment, years in the Chemical Division, and duration of employment. Selected physiologic effects of PFOA exposure were studied using a cross sectional study design. The relationships between total serum fluorine and biochemical parameters including reproductive hormones, hepatic biochemical parameters, lipid and lipoprotein parameters, and hematologic parameters, were 29 3M MN03112221 explored. A sample of the work force employed on November 1 .1 9 9 0 was invited to participate. All employees in high exposure jobs were asked to participate. A sample of workers employed in low exposure jobs was frequency matched to the age and sex distribution of the high exposure group. Each participant completed a questionnaire which included medical history and information concerning alcohol, tobacco, and medication use. The questionnaire is provided in Appendix 3-1. Blood was drawn for determination of hematologic and biochemical parameters. Total serum fluorine, free (FT) and bound testosterone (BT), estradiol (E), thyroid stimulating hormone (TSH ), follicle stimulating hormone (FSH), prolactin (P) and luteinizing hormone (LH) were assayed. The PFOAhormone dose-response relationship for each hormone was estimated using linear regression techniques to adjust for the effects of age, sex, body mass, alcohol consumption, tobacco use, and other potential confounders. The PFOAhormone dose-response relationship was further explored by fitting linear multivariate models to hormone ratios. All unique ratios between the seven hormones were defined. Twenty-one hormone ratios were calculated for each participant. The prevalence of hormone values outside the laboratory reference range for men was compared to the expected prevalence assuming a normal distribution for assay values. 3^B elm spective Cohort Mortality Study 3.2.1 Definition-Qf-Thfi..CoJiQil The Chemolite facility opened in 1947. Individuals who were employed at the Chemolite plant between January 1 ,1 9 4 7 and Decem ber 3 1 ,1 9 8 3 were identified from company records. Workers with fewer than six months employment were excluded. In October 1951 large scale commercial PFOA production facilities became operational (Abe 1982). Because large scale PFOA production did not begin until 1951, a second cohort with potentially significant PFOA exposure was defined as those workers employed between October 1,1951 and Decem ber 31, 1983. Subjects with greater than six months employment were included in this second PFOA cohort. 30 3M MN03112222 The cohort was initially assembled in 1979. Subsequently, the cohort was updated to include new employees through 1983. Personnel records for employees working prior to 1979 were coded for demographic Iterns and work history by trained abstractors. Computerized corporate personnel databases were utilized to provide information for workers employed in the 1979 to 1983 period. Abstracted work history included year of first employment, year of last employment, years employed at Chemolite, and months worked in the chemical division. Individual job histories were not abstracted because job titles were defined by wage grades and did not correspond to specific jobs or locations within the plant. 3.2.2 study Databases And Fifes A Chemolite cohort database was created on a VAX computer using Ingres software. Data stored on magnetic tape were transferred to the VAX. Duplicate records were identified and removed. Missing data were identified. The Ingress update function was used for data editing. Final analytic files for the Monson program, SAS programs, and custom programs were constructed using the Ingress report writer. 3.2.3 Data Editing The Ingres relational database allowed extensive internal consistency checks to be made. All dates were checked for plausibility. Those records with implausible, inconsistent, or improperly formatted dates were edited and corrected if information was available. Records of workers with few er than six months employment were flagged and excluded from the analytic data set. A random check of 50 of the 364 workers with fewer than six month employment found no errors in classification of employment length. Extensive attempts w ere made to obtain all missing data items. Sources of information included plant personnel records, corporate personnel databases, benefit records, archived corporate records, plant medical records, and death certificates. No individual employees or next-of-kin were contacted. Four employees were excluded from the cohort as a result of missing demographic data items. 31 3M MN03112223 a 2.4 Validation Of The Historical Cohort tin i* ]iiiF -u w r 3 2.4.1 A ssessm ent O f C o m p leten ess O f A scertain m ent The cohort was initially defined from personnel records stored at the Chemolite plant. Complete records were maintained on all workers ever employed at the plant. Hourly and salaried workers were included in these files, as w ere all transferred, terminated and retired former employees. Records for workers first employed in the 1947-1978 period were abstracted from documents, coded and computerized. A corporate computerized database was used to update the cohort through Decem ber 1 ,1 9 8 3 . Since insufficient induction tim e had lapsed between 1983 and 1989, no new employees or work history information was added to the cohort database for the post 1983 period for this study. Verifying the ascertainment of all eligible cohort members w as problematic. The assumption that the personnel records represented a complete roster was difficult to check because of a lack of independent information. Several sources were used to exclude major errors in the enumeration of the cohort The historical plant hiring pattern based on seniority dates was compared with the distribution of dates of first em ploym ent Qualitatively, dates of major plant expansion corresponded to peaks in the cSstribution of dates of first employment and to seniority dates. Large increases in hiring due to new plant openings were reflected in peaks in the distribution of starting dates in the cohort A sample of 25 annuity beneficiaries retired from the Chemolite plant were obtained from the corporate personnel office. M l 25 were found to be included in the enumerated cohort Several plant personnel record systems were randomly sampled. Separate files were maintained for active workers, retirees, transferred and terminated workers, and workers whose employment at Chemolite ended prior to 1960. A sample of records for current employees with start dates prior to Decem ber 3 1 ,1 9 8 3 was compared to the cohort. All 12 records from the 1945-1960 period for start dates were found in the cohort database. O f 30 records sampled from the 1961 -1969, 28 (93% ) were included in the cohort Fifty two records had starting dates in the 1970-1978 period. O f these 52 records, forty seven (90% ) w ere found in the 32 3M MN03112224 database, in the 1979-1980 period 18 of 44 (41 % ) records were in the database. Lastly, in the 1981 through 1983 period, 36 of 37 records were in the database (97% ). The low ascertainment for workers first employed in the 1979-1980 period was further examined. O f the 34 workers not in the cohort database, 16 (47% ) were first employed in the 7/79-1/80 period. These omissions occurred in the transition period between document abstracting and electronic updating of the cohort. Using seniority lists, 44 workers currently employed were hired between 1979 and 1980. They represent approximately 1% of the total number of individuals in the workforce and less than 0.5% of the total person tim e at risk for the cohort. Records for retired workers were sampled from files containing all workers retired from Chemolite. Forty seven of the 48 (98% ) sampled records were present in the database. A sample of the files containing the personnel records of employees completing employment before 1960 was randomly drawn. Of the 67 selected records, 65 (97% ) were in the database. Finally, files containing records of all transferred, terminated, or disabled employees were randomly sampled. O f the 120 sampled records, 116 (97% ) were present in the cohort database. 3.2.4.2 Validation Of Cohort Information information in the edited database was compared to information in the personnel records. A random sample of 25 records was drawn from the personnel files. Database names, social security numbers (SSN), dates of birth (DO B), and dates of employment were verified against record information. The sole error occurred in coding the last digit of one SSN. All other information vires correctly entered into the database. The reliability of ICD8 coding of death certificates for underlying cause of death was evaluated by resubmitting a sample of death certificates for coding by the same nosologist The sample consisted of 25 death certificates from 1970 -1989. No change in the major categories of cause of death was noted. All earner deaths were coded concordantly. Within cardiovascular causes of death, two certificates were discordant 12.5 Vital Status-Asceitala.m5.at 33 3M MN03112225 The vital status was ascertained from the Social Security Administration (SSA) and the National Death Index (N D I). All individuals with unknown vital status were traced successfully and vital status determined. Vital status determination in the 1979-1989 period was obtained through the N D I. Death certificates were requested from the appropriate state health departments for those individuals identified as, or presumed to be, deceased. A professional nosologist coded the death certificates for underlying cause of death according to International Classification of Diseases, 8th revision (IC D 8). Information concerning the date and cause of two deaths which occurred outside the United States was obtained from family members or other available sources. Date of death and the ICD8 code for the underlying cause of death were entered into the database. 3 .2 .6 V alidation of V ita l S tatus A scertainm ent The vital status determination procedures for the cohort was evaluated. Corporate benefit records were utilized as an independent source for vital status among the retirees. Vital status from the database was compared to vital status in corporate records. A list of ail retirees in the 1947-1984 cohort was sent to 3M benefits departm ent These individuals were matched to retirees who had received 3M death benefits. 3M records were not complete for periods prior to 1975. In the pre-1983 period, 4 deaths in retirees were identified by 3M records. Vital status was correctly ascertained by the SSA matching procedurefor only one of these retirees. In the 1983-1989 period, 34 deaths in retirees were identified in 3M records. The NDI matching procedure ascertained all 3 4 of these deaths. The NDI was not available for 1990. 3M records indicate that 8 retirees died during 1990. The incomplete SSA ascertainment in the period 1975 to 1983 resulted in extending the ND I search to indude 1979 to 1983. All 3M identified deaths were also identified in the subsequent ND I search covering the 1979 to 1983 period. SL2,7Analysis Analytic methods employed in this study were appropriate for cohort studies. The relative risk was estimated by calculating an adjusted standardized mortality ratio 34 3M MN03112226 (S M R )10S. This study used both national and Minnesota mortality rates for comparisons. Mortality for men in the Chemolite cohort was compared to expected national and Minnesota mortality, adjusted fo rag e, calendar period, sex and race. The use of mortality rates in the rural counties surrounding the plant were not considered to be stable for many causes of death and were not used. Since less than one percent of plant employees are non-white, white male and female rates were used for comparison. For women, only U .S. rates were used because cause- and calendar period-specific Minnesota rates were not available. SMRs were calculated for all cause, all cancer, and cause-specific mortality. The effects of disease latency, duration of employment, duration of follow-up, and work in the Chemical Division were examined using stratified SM R analyses. Three additional methods of analysis were used to assess the validity of the SM R contrasts. The three methods were: standardized rate ratios (S R R )106, Mantel Haenszel adjusted relative rates (RRm h ) 108>and proportional hazard regression adjusted R R 107. Limited exposure data were available from plant records. Exposed workers were defined as all workers who worked for 1 month or more in the chemical division. Exposed and unexposed workers' all cause, all cancer, and cause-specific mortality was compared using stratified SMRs, S R R s 1oe, and stratified Mantel Haenszel analysis108> 109. Additionally, the same summary measures were calculated contrasting the rates for workers with at least ten years duration of employment and those with less than ten years em ploym ent The relative risk (R R ) and 95% C l for the RR for deaths from all causes, cancer, cardiovascular diseases, and selected specific causes were estimated using a proportional hazard model (PH) 107* 109. The tim e to event or censoring was defined as tim e from first employment to event or Decem ber 3 1 ,1 9 8 9 . In PH models for specific causes of death, deaths from other causes were censored at the time of death. Exposure was quantified by months of chemical division employment Covariates included in the models were age at first employment, year of first em ploym ent and duration of em ploym ent The analyses were stratified by gender. The appropriateness of the proportional hazard assumptions were tested using stratified models with graphical analysis of log (-iog(survh/aI)) 35 3M_MN03112227 versus follow-up time relationships and models that tested the significance of a product term between exposure and log(fol!ow-up tim e )109,110. 3.3 Cross Sectional Study O f PFOA Exposed W orkers 3 3 1 Population Definition And Recruitment Medical screening of workers employed at the Chemolite plant occurs every two years. The general medical screening program included a medical questionnaire (Appendix 3-1), measurement of height, weight and vital signs, pulmonary function evaluation, urinalysis, serum assays, and hematology indices. This screening program offered an opportunity to assess the physiologic effects of PFOA exposure in workers engaged in commercial production of a limited spectrum of PFCs. O f particular interest were the effects of PFOA, the primary fluoicchemicai found in the serum of Chemolite workers. (Griffith and Ubel, 1980). Participation in the Physiologic Effects Study required the subjects' willingness to undergo hormonal and biochemical testing and to have an additional 15 ml of blood diawn for total fluorine assay. In the cross-sectional study, exposure classification was based on the potential for PFOA exposure in a workers job am i plant location. All workers engaged in any facet of PFOA production in the previous five years were considered to have potentially high PFO A exposure. The jobs considered to have high exposure potential included all jobs in the production buildings (bldg 6 and 15), all maintenance workers who were assigned to the PFOA production areas, and ail management jobs requiring physical presence in the production building. Plant records and job history information was used to assign exposure status to individual workers. A random sample of workers in jobs with low exposure potential was frequency matched to the age and sex distribution of the high exposure workers. W orkers with low exposure potential were defined as those assigned to jobs not involved in the production of PFCs for at least five years. A roster of workers meeting the low exposure potential was defined from plant records and knowledge of plant personnel about the location of high exposure jobs. A gender stratified sample from the group of workers in low exposure jobs with an age (5 year strata) distribution sim ilar to the exposed group was identified and invited to participate. If a worker in a job with 36 3M MN03112228 low exposure declined to participate, another worker in the sam e age and sex stratum was randomly selected and invited to participate. In ail cases informed consent was obtained. Participation in this study was voluntary. 3.3.2 Data Collection 3.3.2.1 Study Loos And Files A roster of participants was maintained by the plant occupational health nurses. A log for biological sample information was completed by the laboratory technician. The date and time of ample coolection was recorded. Quality assurance samples were recorded on a separate log. Results reported on paper records were maintained as mecBcal records. Results for other tests were transmitted electronically to a computerized database and coded as SAS datasets. All records were stored with employee medical records or in the corporate medical offices for confidentiality purposes. Printed laboratory results and questionnaire data were entered into a SAS dataset. 3.3.2.2 Questionnaire Each participant completed a medical questionnaire prior to reporting to the plant rnedical office. (Appendix 3-1) Items included demographic information, symptoms, illness history and diagnoses, and medication usage. Detailed questions concerning tobacco use and alcohol use were included. W orkers were not re-contacted to obtain missing information or to correct inconsistencies. Responses were not validated. Two plant occupational health nurses collected the questionnaires and returned them to the corporate medical departm ent In the corporate medical office, data were coded and entered into a SAS data base. 3.3.2.3 Laboratory Procedures 13.2.3,1 Height andWeight 3M MN03112229 Upon reporting to the plant medical office, participants had their height and weight determined by an occupational health nurse. Height and weight were measured once on the same calibrated scale. a.3.2.3.2 Blood 3.3.2.3.2.1 Drawing And Handling Four vacutainers of blood were drawn from a single venipuncture by a laboratory technologist. Two 15 mi red top vacutainers of blood were drawn and allowed to dot. One 10 ml purple top vacutainer was drawn for hematology studies. A spedaliy prepared fluorine free 15 ml vacutainer was used to collect blood for total serum fluorine determination. Venipunctures were scheduled to occur at the same time of day and on the same shift for each worker. Ail blood w as drawn between 6:30 and 8:00 a m . Workers in the Chemical Division of foe Chemolite plant rotate shifts on a weekly basis. Blood was drawn after a worker was assigned to foe day shift for at least 3 days. Ail specimens were refrigerated at the plant prior to transport to the appropriate laboratory. Clotted red top vacutainer spedm ens were centrifuged for 12 minutes to separate serum from ceils before transport to the contract laboratory, in order to render the total serum fluorine spedm ens non-infectious, serum for total fluorine assays was ether extracted in the corporate medical department prior to sending the samples to the 3M Chemical Division analytic laboratories. 3.3.2.3.2.2 Assays Serum samples were analyzed for total serum fluorine, hepatic biochemical parameters, cholesterol, lipoproteins, and seven hormones. Assayed biochemical parameters included serum glutamic oxaloacetic transaminase (SG O T), serum glutamatic pyruvic transaminase (SG PT), gamma glutamyl transferase (G G T), and alkaline phospatase (AKPH). The following hormones were assayed: bound testosterone, free testosterone, estradiol, prolactin, luteinizing hormone (LH), follicle stimulating hormone(FSH), and thyroid stimulating hormone (TSH ). EDTA preserved whole blood samples underwent routine hematologic analysis induding 38 3M MN03112230 complete blood count with erythrocyte indices and leukocyte differential cell count (CBC). Analyses were done without knowledge of the subject status or purpose of the study. Total serum fluorine was determined in 3M 's Chemical Division analytic laboratory using the sodium biphenyl extraction method (Venkateswarlu, 1982). H ie accurate determination of total fluorine in the parts per million (ppm) range required specialized equipment, procedures, and personnel. Assays were completed in a dedicated laboratory following tested protocols. Upon receipt of extracted serum samples divided aliquots w ere frozen at >70 degrees centigrade. After all samples had been received, batches of 15 samples were assayed on successive working days. Each batch included high and low quality control samples. Each sample was assayed twice. If the difference in assayed values was greater than 1 ppm, the sample was re-assayed. The total serum fluorine value was reported as a mean value and a rounded integer value. Serum glutamic oxaloacetic transaminase (SG O T), serum glutamatic pyruvic transaminase (SG PT), gamma glutamyl transferase (G G T), and alkaline phospatase (AKPH) were assayed by the United Health Services Laboratory In Apple Valley, Minnesota using clinical colorimetric assays. CBCs were determined using automated Coulter counters. Light microscopy w as utilized for differential counts. Estradiol, prolactin, thyroid stimulating hormone (TSH ), luteinizing hormone (LH), and follicle stimulating hormone (FSH ) were assayed by the United Health Services laboratory using radioimmunoassay (RIA) and enzym e linked immunosorbent assay (ELISA). FSH, LH, and prolactin were assayed using Abbott laboratories IM X microparticle enzyme linked immunoassays. TSH was assayed using London Diagnostics chemiluminescense immunometric assay. Estradiol was determined using Diagnostic Products Corporation's Coat-a-count assay. 39 3M MN03112231 Testosterone was assayed by the Mayo Clinic dinicai laboratories. Total testosterone was determined by R1A using proprietary immunoglobulins. Free and bound testosterone was determined using equilibrium dialysis.111. 3.3 2.3.2.3 Quality Assurance Two methods were used to assess the accuracy and reliability of the laboratory assays. The laboratories routinely followed quality assurance programs. Three standards were run with each batch, if the control values were outside two standard deviations of the intra assay mean value for each standard, the assay was repeated. If 10 controls were outside 1 standard deviation of the m ean, the assay was flagged for review. The reliability of each of these assays was assessed. For each assay, five specimens were randomly selected and split into two aliquots. The aliquots were labeled with different identifiers ensuring that the assays w ere carried out in a blinded fashion. Both aliquots were submitted on the sam e day to the laboratory. The coefficient of variation was calculated for each hormone. 3,3,3.Analysis There were two analytic strategies. First, assay results were treated as continuous parameters and modeled using regression methods. Models were fit to assess the relationship between assay results and total fluorine, body mass index, alcohol consumption, and smoking. Second, hormonal assay results were dichotomized into those within the reference range and those outside the reference range. The hormone assay categories were based on published sex specific normal reference values for each assay. The purpose of this dichotomization was to evaluate the possibility that highly susceptible individuais may be affected at lower levels of exposure and not follow the adjusted doseresponse curve. The relationships between total serum fluorine and the assayed param eters were estimated by fitting linear multivariate regression models to the data. The dinicai parameters and ratios of selected parameters were first modeled as functions of nominally categorized exposure and covariates. Dependent variables that were 40 3M_MN03112232 not normally distributed were appropriately transformed. Total serum fluorine was categorized into mutually distinct categories. Cutoff values for the categories were chosen to assure adequate numbers in each category while maintaining the fullest range of exposure values possible. Accordingly, total serum fluorine level categories were defined as the following: less than 1 ppm, greater than 1 ppm tolessthan 4 ppm, 4 ppm to 10 ppm, greater than 10 ppm to 15 ppm, amt greater than 15 ppm. If insufficient numbers of events occurred within incBviduai categories, the number of categories was reduced by combining adjacent categories. Additionally, models were fitted with total serum fluorine entered as a continuous variable using linear, square, square root transformations. Age, body mass index (BM I), alcohol use and tobacco use were included in the model as potential confounders. Age was included in the models as both a categorical variable and a continuous variable. Age was grouped into four ten year age categories. Age was treated as a continuous variable using linear, square, square root, and log transformations. BMI was entered in the models as a categorical variable and as a continuous variable. BMI categories were less than 25 kg/m2, 25-30 kg/m2 , and greater than 30 kg/m2 . Additionally, BMI was dichotomized into obese, greater than 28 kg/m2 , and non-obese, less than or equal to 28 kg/m2 . The continuous variable was entered as linear, square, log, and square transformations. Alcohol use was categorized into 3 categories: less than 1 drink per day, greater than one to 3 drinks per day, and non response to the questionnaire item. Smoking was categorized as current nonsmokers and current smokers. A nonresponse category was not included since only two individuals were in this category. These two individuals were excluded from analyses that required smoking history. Smoking was quantified as cigarettes smoked per day. Linear, square and square root transformations of cigarettes per day w ere used in regression models. The choice of the final model was somewhat subjective. For each dependent variable, other covariates were included in the final model if they were potential confounders. O ther potential confounding hormones and biochemical parameters were included in the models if they produced significant changes in effect estim ates. r 41 3M MN03112233 Total serum fluorine and confounding covariates were entered into models as continuous variables. Significant nonlinear dose-response relationships were evaluated by comparing model fit and param eter estimates using categorical variables and continuous variables. Square, square root, exponential, and logarithmic transformations were used if the transformed variables produced models of superior predictive power as assessed by model fit All two way interactions between total serum fluorine and the included covariates were evaluated. Interaction terms were Included in the final model if the param eter estimate for the interaction term w as significant at the alpha =.10 level. The potential for susceptibility to confound the relationship between PFOA exposure and the assayed parameters was examined by comparing the observed prevalence of assay results outside of the reference range with the expected prevalence. The prevalence of abnormal assays was based on published reference values for the adult male US population. Reference ranges for test parameters were defined as being within 2 standard deviations above or below the mean value for the parameter. The laboratory maintains laboratory and assay specific reference range for each assay. Given that the distribution of values is approximately normal, about 2.5% of individual values are expected to foil above the upper limit and 2.5% below the lower lim it it follows that the prevalence for a high test is .025. The prevalence for a low value is .025. Using these prevalences, an expected number of tests outside of the reference range can be defined. A priori hypotheses based upon animal and in vitro studies defined the expected direction of the effect The calculation of an observed to expected ratio allowed the estimation of the relative prevalence for a test outside of the normal range in the study subjects as compared to the general population. The 95% C l for the ratio w as calculated assuming that the expected number is a constant and the observed number is a random variable with a Poisson distribution. 42 3M MN03112234 A. RESULTS 4.1 Cross Sectional Perfluorocarbon Physiologic Effects Study In October 1990, at the time of the cross sectional study, the workforce at Chemolite consisted of 880 salaried am i hourly employees. There were 50 men and 2 women in high exposure potential jobs. Since there were only 2 women in this group, the study was restricted to males. Forty-eight (96% ) of the 50 male workers in high exposure potential jobs agreed to participate. The exact number of low exposure workers invited to participate in the study w as not recorded. However, few individuals in this group refused to participate. Thus, it is estimated that over 60% of low exposure workers participated. 4.1.1 Participant Characteristics Since frequency matching for age was used to select study participants, the overall age distribution reflected the age distribution of workers in high exposure potential jobs (Table 4.1.1). Ages ranged from 24 to 59 years, with a median age of 37 years and a mean age of 39.2 years. Table 4.1.2 presents the alcohol and tobacco use profile of the study participants. The light drinkers category included 22 participants who reported no alcohol use. Consumption of one to three ounces of ethanol per day was reported by 20 (18.7% ) participants. No participants reported drinking greater than three ounces of ethanol per day. Eight workers (7.0% ) did not complete this item of the questionnaire. There were 28 (24.8% ) smokers who smoked an average of 21.7 cigarettes per day. Smoking status was not available for two workers (1.8% ). The association between smoking and alcohol consumption is presented in Table 4.1.3. Thirteen (15.3% ) of 85 nonsmokers and seven (25.0% ) of 28 smokers reported moderate drinking (p=.24). Table 4.1.4 cfisplays the age distribution for alcohol and tobacco use categories. There were no significant differences in mean ages among smoking or drinking categories. 43 3M MN03112235 Total fluorine was not significantly correlated with age, BMI, alcohol, or tobacco use (Table 4.1.5). BMI and age w ere correlated (r.26, p=.005). Alcohol use and tobacco use were not significantly correlated (r=.08; p>.7). BMI ranged from 18.8 to 40.5 kg/m2 with a median value of 26.3 kg/m2 and a mean of 26.9 kg/m2 (Table 4 .1 .6 ). Half of all workers had BMIs between 25 and 30 kg/m2 . The mean BMI in smokers was not significantly different from that of nonsmokers (Table 4.17). The mean BMI for moderate drinkers was not significantly different from the BMI of light drinkers. Smoking status and BMI were not significantly associated (Table 4.1.8). There was a significant linear relationship between BMI and age (B -.1 0 SE(B)=.035). This relationship w as not substantially altered after adjusting for smoking stab s, alcohol use, and total serum fluorine level. 4.1.2 Total Serum Buorine The total serum fluorine values ranged from zero to 26 with a median value of two ppm, a mean of 3.27 ppm and a standard deviation of 4.68 ppm (Table 4.1.9). The inter-assay coefficient of variation was 66% calculated from repeated assays on different days. ' Twenty-three (20.0% ) of 115 workers had total serum fluorine values less than one ppm. This group included eight workers values reported as zero ppm (below the limits of detection). Eighty-eight workers (76.5% ) had levels less than or equal to three ppm. Six (5.2% ) of 115 workers had values between 10 and 15 ppm and five (4.4% ) had values greater than 15 ppm. All workers with levels greater than ten had worked in Building 15, the primary PFC production area at the Chemolite Plant. There w ere no significant differences in total serum fluoride mean values among the BMI, age, alcohol use and tobacco use categories (Table 4.1.10). No statistically significant differences in mean age between total fluorine categories were observed (Table 4.1.11). 44 3M MN03112236 Participants with less than one ppm total fluorine smoked the least (16.3) number of cigarettes per day (Table 4.1.12). Those with one ppm to three ppm total fluorine smoked the greatest number of cigarettes per day (24.5). This difference was statistically significant (p<.005). As estimated in a regression model, the linear relationship between total fluorine and smoking status, adjusted for age and BMI, was small in magnitude (B=0.10, SE(B)=0.062, p=.09). Smokers average total serum fluorine was estimated to be 0.1 ppm higher than nonsmokers. The number of cigarettes smoked per day was weakly correlated with total serum fluorine (Table 4.1.5). Drinking status was not associated with total fluorine (Table 4.1.13). Overall, eight (7.0% ) participants did not respond to this question. Four had less than one ppm total serum fluorine. Table 4.1.14 presents the distribution of BMI in the total fluorine categories defined previously. BMI mean values were not significant cfifferences among the total serum fluorine categories. The linear relationship between BMI and total fluorine, adjusted for age, smoking, and alcohol use, was w eak and not significant (B-.016 SE(B ).069, p>.5). 4.1.3 Hormone Assays The intra-assay coefficient of variation (CV) for the bound and free testosterone, estradiol, TSH , LH, prolactin, and FSH assays are provided in Table 4.1.15. The estradiol assay had the highest CV, 18.3% . The prolactin assay had the lowest C V of 3.1% . Table 4.1.16 presents the observed and expected number of hormone assays out of the assay reference range, the observed to expected (O /E ) ra tio , and the 95% confidence limits. The O /E ratio was significantly greater than one for estradiol, free testosterone, bound testosterone and prolactin. The O /E ratios for LH, FSH, and TSH were not significantly (Afferent from one. The Pearson correlation coefficients among the seven hormones assayed in study participants are presented in Table 4.1.17. As expected, estradiol was 45 3M MN03112237 correlated with free testosterone (r.40. p=.0001} and bound testosterone (r=.32, p=.0006). Bound testosterone was correlated with free testosterone (r=.74 p.0001), LH (r.28, p=.003) and FSH (r.16, p.04). LH and FSH were significantly correlated (r.63, p=.0001). FSH and TSH were significantly correlated (r=.23, p=.01). As shown in Table 4.1.18. total fluorine was significantly correlated with prolactin (r=.19, p=.045) and TSH (r=.26, p=.005). Age was negatively correlated with estradiol (r=-.25, p=.01), free testosterone (r--.45, P -.0 0 0 1 ), bound testosterone (r=-.24, p=.01), and prolactin (r--.19, p=.01). Age was positively correlated with FSH (r=>.33, p=.0003). As expected, BMI was negatively correlated with free and bound testosterone( r--.26, p.005 and r--.36, p=.0001 respectively). BMI was correlated positively with LH (r=.20, p -.0 3 ). Alcohol consumption was significantly correlated with FSH (r-.24 p -.0 1 ). Bound testosterone ranged from 141 to 1192 ng/dl with a mean of 572 ng/dl and a median of 561 ng/dl (Table 4.1.19). The standard deviations w ere large. The mean bound testosterone values were not significantly different among the total serum fluorine groups. As expected, the mean bound testosterone decreased significantly as BMI increased. The mean bound testosterone values were significantly different among the age categories (p.016). There was a significant nonlinear relationship between total serum fluorine and bound testosterone (BT) in the final regression model (Table 4.1.20). Bound testosterone, which was positively associated with both LH and estradiol, decreased as both age and BMI increased. Alcohol and cigarette use were weakly associated with BT. There was a significant interaction between age and total serum fluorine. There was a negative association between bound testosterone and total serum fluorine in young workers than in older workers, in workers greater than 45 years of age, total serum fluorine w as associated with a slight increase in BT. The relationship between bound testosterone and total serum fluorine is presented for four different sets of covariate value (Figure 2 ) . Dose-response curves for bound testosterone were plotted for young, lean individuals aged 30 with BMIs of 25, young obese individuals aged 30 with BMIs of 35, middle aged lean individuals aged 50 with BMIs of 25, and middle aged 46 3M MN03112238 obese individuals aged 50 with BMls of 35. Each of the relationships is for nonsmoking, tight drinking men with the sample mean LH value (5.4 m ll/l) and mean estradiol value (33.4 pg/ml). in 30 year old workers, bound testosterone decreased as total serum fluorine increased in both BMI groups. The doseresponse relationship for 40 year old workers was approximately flat (not shown). In workers greater than 50 year of age, BT increased as total serum fluorine increased. Total serum fluoride was not significantly associated with free testosterone (FT) (Table 4.1.21). Within BMI categories, free testosterone was highest in the less than 25 kg/m2 group and lowest in the greater than 30 kg/m2 category. The difference in mean FT among BMI categories was statistically significant (p=.03). There was a significant nonlinear dose-response relationship between total serum fluorine and FT in the final regression model (Table 4.1.22). As total serum fluorine increased, free testosterone decreased. There was a significant interaction between age and total serum fluorine. Figure 4.2 illustrates the modifying effect of age on the total serum fluorine free testosterone relationship. The covariate vectors (nonsmoker, light drinker, mean LH and estradiol, age=30 and BM I=25 or 25, age=50 and BM I=25 or 35) were the same as used Figure 1. Lean or obese 50 year old men had low free testosterone (less than 9 ng/dl) for all values of total serum fluorine. In 30 year olds, free testosterone decreased asymptotically toward the 50 year old values. In this model, a 50 year old, obese, moderate drinker with any total serum fluorine level (the lower limit of assay sensitivity was approximately 1 ppm total serum fluorine) had free testosterone below nine ng/dl. As shown in Table 4.1.23, the estradiol means in the three BMI groups w ere not significantly different (p -.8 8 ). As the age of participants increased, mean estradiol levels decreased. In the greater than 30 to 40 year age group, mean estradiol was 36.8 pg/ml compared to 25.9 pg/ml in the greater than 50 to 60 year age group. The age group means were significantly different (p -.0 1 8). There was a nonsignificant positive association between mean estradiol and total serum fluorine. 47 3M MN03112239 As shown in Table 4.1.24, estradiol and total serum fluorine were positively associated in the final regression model. Total serum fluorine followed a nonlinear relationship with estradiol. No interaction terms were statistically significant. As expected, free testosterone and estradiol were positively associated (B=.85 p=.0007). The relationship between total serum fluorine and estradiol is illustrated in Figure 3. The plotted curves depict the relationship for lean (25 kg/m2 ) and obese (35 kg/m2) male workers who were 30 years old with sample mean free testosterone and who were nonsmokers and light drinkers. As total serum fluorine increased over the observed range, estradiol increased quadraticaJIy. In obese men (BM I=35 kg/m2 ) aged 30, estradiol exceeded 44 pg/ml when total serum fluorine was between 15 and 20 ppm. The highest estradiol levels were in young, obese smokers who consumed 1 to 3 ounces of ethanol per day. LH was not significantly associated with serum fluorine, but was negatively associated with BMI (p=.003) and positively associated with smoking (p=.025), age, and B T . There was no association between total serum fluorine and FT. (Table 4.1.25, Table 4.1.26, and Figure 4). FSH was not significantly related to total serum fluorine levels but w as positively associated with age (p=.014) (Table 4.1.27, Table 4.1.28). The final regression model for FSH is illustrated in Figure 5. The relationship was essentially flat over the total fluorine range. TSH was positively associated with total serum fluorine in both univariate and multivariate analyses (Table 4.1.29, Table 4.1.30 and Figure 7). TSH was not significantly related to age, BMI, alcohol use, smoking, and other hormones. Prolactin was positively associated with total serum fluorine and smoking (Table 4.1.31, Table 4.1.32). Moderate drinkers had a different prolactin-total serum fluorine relationship compared to light drinkers and nonrespondents. Figure 6 illustrates the relationship of prolactin with total serum fluorine and the modifying effect of alcohol use. Total serum fluorine was weakly associated with prolactin in light and moderate drinkers. However, in moderate drinkers (1-3 oz/day), there was a positive association between prolactin and total serum fluorine. 48 3M MN03112240 4.1.4 Hormone Ratios The univariate distributions of the 21 ratios are provided in Appendices 4.1 and 4.2. A table is presented for each of the 21 ratios showing the number of participants, mean ratio value with the standard deviation, median ratio value, and the range of ratio values in each of the previously defined categories of BMI, age, alcohol use, tobacco use, and total serum fluorine Correlations between total serum fluorine (ppm), age (years), BMI (kg/m2 ), alcohol use (oz/day), and cigarette consumption (dgarettes/day) and all possible ratios between E, free testosterone TF, TB, and LH are displayed in Table 4.1.33. The estradiol to bound testosterone ratio (E/TB) and estradiol to free testosterone ratio (E/TF) were significantly correlated with BMI (r=.32, p=.001 and r=.27, p=.004 respectively). The estradol to luteinizing hormone ratio (E/LH) was negatively correlated with age (r=-.26, p=.005), and positively correlated with BMI (r=.18, p=.06). The bound testosterone to luteinizing hormone ratio (TB/LH) followed a different pattern as compared to E/LH. The correlation coefficient between TB/LH and age was -.32 (p.001) while the coefficient between TB/LH and BMI was -.14, (p=.13). The free testosterone to luteinizing hormone ratio (TF/LH) had the strongest correlation with age (r-.40, p*=.0Q01) but was not .significantly correlated with BMI. The bound testosterone to free testosterone ratio (TB/TF) followed a unique pattern. TB /TF was positively correlated with age (r.24, p=.01), and negatively correlated with BMI (r=-.16,p=.08). Prolactin ratios with bound testosterone (TB/P), free testosterone (TF/P), estradiol (E/P), follicle stimulating hormone (FSH /P), luteinizing hormone (P/LH), and thyroid stimulating hormone (P/TSH ) are presented in Table 4.1.34. None of the prolactin-hormone ratios were significantly correlated with total serum fluorine or BMI. Ail except P/TSH were significantly correlated with cigarette consumption. Table 4.1.35 presents the Pearson correlation coefficients for the bound testosterone to thyroid stimulating hormone (TB/TSH) ratio, the free testosterone to thyroid stimulating hormone (TF/TSH ), and the estradol to thyroid stimulating 49 3M MN03112241 hormone (E/TSH ). Total serum fluorine and TF/TSH were negatively correlated (r=-.18,p=.05). All three ratios were significantly and negatively correlated with age. TB/TSH and TF/TSH were negatively correlated with BMI, (r--.24 p=.01 and r=-.23, p=01 respectively). The Pearson correlation coefficients for the bound testosterone to follicle stimulating hormone (TB/FSH) ratio, the free testosterone to follicle stimulating hormone (TF/FSH ), and the estradiol to follicle stimulating hormone (E/FSH ) are provided in Table 4.1.36. Age was the only covariate that wms significantly correlated with the three ratios. The correlation coefficients for selected ratios between pituitary glycoprotein hormones, TSH , LH, and LH, are presented in Table 4.1.37. The thyroid stimulating hormone to follicle stimulating hormone (TSH /FS H ), the thyroid stimulating hormone to luteinizing hormone (TSH/LH), and the follicle stimulating hormone to luteinizing hormone (FSH/LH) are provided. Age w as significantly correlated with the FSH/LH ratio and the TSH /FSH ratio. Alcohol consumption was correlated with both TSH /FSH and TSH/LH. As shown in the final regression models, the TB /TF ratio increased as toted serum fluorine increased (Tables 4.38 and 4.39). Alcohol consumption, cigarette consumption, estradiol, prolactin, and TSH were not significantly related to the TB/TF ratio in either model. These covariates do not substantially alter the estimated relationship between total serum fluorine and TB /TF ratio when induded in the regression model. The quadratic increase of the TB /TF ratio over the observed range of total serum fluorine is illustrated in Figure 4.8. The covariates used were: nonsmoker, less than one m ince of alcohol consumed per day, 30 years of age, and a BMI of 30 kg/m2. Table 4.1.40 presents the full regression model for the estradiol to bound testosterone ratio (E/TB). Total serum fluorine was not significantly associated with the E/TB ratio. BMI was a determinant of the E/TB ratio. Free testosterone was negatively related to the E/TB ratio. 3M MN03112242 The full regression model for estradiol to free testosterone ratio (E/TF) is displayed in Table 4.1.41. There was a significant positive dose-response relationship between the EfTF ratio and total serum fluorine. Although the doseresponse relationship for free testosterone was modified by age, the doseresponse relationship for the ratio was not modified by age. As shown in Tables 4 .1 .4 2 ,4 .1 .4 3 and 4.1.44, total serum fluorine was not significantly associated with E/LH and TB/LH, but was positively association with the TF/LH ratio (0=-.O5, p=.09). Bound testosterone and FSH were associated with the TF/LH ratio (B -.00 3, p=.0001) and (8=-.33, p=.0001). Cigarette consumption and free testosterone were strongly and significantly related to the TB /P ratio (8=1.49, p=.02 and 8=3.93, p=.008 respectively) (Table 4.1.45). Cigarette consumption and bound testosterone were significantly related to the TF/P ratio (B=.04, p=.03 and B=.002, p=.03 respectively) (Table 4.1.46). Only cigarette consumption was significantly related to the E/P ratio (B=.10, p=.005) (Table 4.1.47). Tables 4.48 through 4.50 present full regression models for the ratios of prolactin to FSH (P/FSH ), prolactin to LH (P/LH), and prolactin to TSH (P/TSH ). In each of the three regression models total serum fluorine was positively and significantly associated with the prolactin-hormone ratio. Moderate drinkers had a significantly different ratio total serum fluorine dose-response relationship compared to the relationships in light drinker and nonrespondents. The full regression models for the glycoprotein hormone ratios are presented in Table 4.1.51 to 4.1.59. As shown in table 4.1.52, total serum fluorine was significantly related to TF/TSH (B=-.28, p=.03) and bound testosterone and FSH were significantly related to the TF/TSH ratio (B=.01, p=.006 and 8.68, p.04 respectively). Total serum fluorine was not significantly associated with the other glycoprotein hormone ratios. AJL.5 Cholesterol. Low Density Lipoprotein. High Density Lipoprotein. And Irialycerides 51 3M MN03112243 Table 4.1.60 provides the correlation coefficients for serum lipids, specifically cholesterol, low density lipoprotein (LDL), and high density lipoprotein (HDL), with total serum fluorine, age, BMI, alcohol consumption, and cigarette consumption. Total serum fluorine was not significantly correlated with cholesterol, LDL, HDL, or triglycerides. Cholesterol and triglycerides were correlated with age ( r-.2 5 , p=.008 and r-.1 9 , p=.04, respectively), and BMI (r=.19, p*.04 and r=.27, p=.004, respectively). Cigarette smoking was positively and significantly correlated with cholesterol (r=.35, p=.0001), LDL (r-.2 8 , p=.002), and triglycerides (r=.19, p.04). HDL was not significantly correlated with any variable, although the correlation with alcohol consumption was suggestive ( r-.1 8 , p -.0 6 ). Total fluorine was not significantly associated with cholesterol, LDL or triglycerides (Tables 4.1.61, Table 4.1.62, Table 4.1.64). Smoking, age, and QGT were positively and significantly associated with cholesterol. Smoking and prolactin were positively and significantly associated with L D L Smoking and free testosterone were positively associated and bound testosterone was negatively associated with triglycerides. The final regression model for H D L displayed in Table 4.1.63, presents a different picture. HDL decreased as total fluorine increased in moderate drinkers. In light drinkers, there was a negligible change in HDL as total fluorine increased. - Self-reported moderate alcohol consumption was positively associated with H D L Additionally, bound testosterone was positively associated with H D L while free testosterone was negatively associated. 4.1.6 Hepatic Parameters: Serum Glutamic Oxaloacetic Transaminase (SG O TL Serum Glutamic Pyruvic Transaminase fSGPTL Alkaline Phosphatase (AKPH). Gamma Glutamvl Transferase fGG~n. Table 4.1.65 presents the correlation coefficients between the hepatic parameters, SGOT, SG PT, G GT, AKPH, and total serum fluorine, age, BMI, alcohol consumption, and cigarette consumption. The hepatic parameters were not significantly correlated with total serum fluorine. SG O T was not significantly correlated with any of the participant characteristics. SG P T and G G T were correlated significantly only with BMI (r=.20, p=.02 and r=.27, p=.004 52 3M MN03112244 respectively). AKPH was significantly correlated with age, BMI, alcohol consumption, and cigarette consumption. The correlation coefficients between the hepatic parameters and cholesterol, LDL, HDL, triglycerides, estradiol, TF, TB, and prolactin are displayed in Table 4.1.66. SG O T and AKPH were significantly correlated with prolactin. S G P T was correlated with cholesterol and triglycerides. G G T was correlated with cholesterol, triglycerides, and free testosterone. As expected, SG O T, SG PT, and GGT were highly correlated (Table 4.1.67). AKPH was only correlated with GGT. The SGOT, SGPT, G G T, and AKPH mean values w ere not significantly different among the five total serum fluorine categories (Table 4.1.68). SG O T and SG PT mean values were not significantly different for BMI, age, alcohol use, and smoking (Tables 4.1.69 to 4 .1 .7 2 ). Mean G G T was significantly higher in the greater than thirty BMI group (p.03). As shown in Table 4.1.72. mean and median AKPH values were significantly higher in smokers compared to nonsmokeis (p=.012). Tables 4.1.73 A, B, and C present three linear multiple regression models for SGOT. In non-obese workers (BM I=25), SG O T decreased as total fluorine increased. In obese workers (BM!= 35), the association between total serum fluorine and SG O T was in the opposite (Erection. Model 2 included G G T as a covariate (Table 4.1.73 B). The association between total fluorine and SG O T, as well as the effect modification by BMI, were present after adjusting for G G T. When SG PT was included in the regression model (Table 4.1.73 C ), the association between total fluorine and SG O T was w eak and nonsignificant. The effect modfication by BMI was no longer present. AKPH had little effect on the regression estimates when included in the model. Three linear multipie regression models for SG PT are provided in Tables 4.1.74 A, B, and C. In non-obese workers (BM 1-25), SG PT decreased as total fluorine increased. However, in obese workers (B M I- 35), the association between total serum fluorine and SG PT was in the opposite direction. Uttle change occurred in the estimates after adjusting for G GT. As seen in Table 4.1.74 C , the association was significant, although weaker in strength, after adjusting for SG O T. The effect 53 3M MN03112245 modification by BMI was present When AKPH was included in the model, effect estimates did not change significantly. The final regression models for G G T, provided in Tables 4.75 A, B, and C, present a different picture. G G T decreased as total fluorine increased in moderate drinkers. In light drinkers, G G T decreased less steeply as total fluorine increased. Controlling for SG O T and SG PT (model 2 and 3) did not significantly alter the relationship between total fluorine and G GT. Moderate alcohol consumption was positively associated with G G T. Table 4.1.76 presente the final regression model for AKPH. In nonsmokers, total serum fluorine was negatively associated with AKPH. As the number of cigarettes smoked per day increased to more than five per day, AKPH increased as total serum fluorine increased. 4.1.7 Hematology Parameters: Hemoglobin.W hite Blood-Count Polymorphonuclear Leukocyte Count-Band Count. Eosinophil C o u n t Lymphocyte C ount. Monocyte Count. Platelet Count. And Basophil Count. Table 4.1.77 presents the correlation coefficients between the nine hematology parameters and total serum fluorine, age, BMI, alcohol use, and cigarette consumption. The only param eter that was significantly correlated with total serum fluorine was lymphocyte count (r=.19, p=.04). Monocyte count was correlated with BMI (r=-.22, p=.04) and alcohol consumption, (r=-.21, p=.03). Ail the parameters, except the basophil and band counts, w ere strongly associated with cigarette consumption. Alcohol consumption was correlated with hemoglobin, (r-.20, p=.04), and band count (r=.26, p=.005). The final regression models for hemoglobin and the erythrocyte indices, mean corpuscular hemoglobin (M CH) and mean corpuscular volume (M C V). are presented in Tables 4 .1 .7 8 ,4 .1 .7 9 , and 4.1.80 respectively. Total serum fluorine was significantly associated with hemoglobin.. The association hemoglobin and MCV were modified by smoking. In smokers who smoked seven or more cigarettes per day, hemagiobin and M CV increased significantly as total fluorine increased. In nonsmokers, hemagiobin and MCVdecreased as total fluorine 54 3M MN03112246 increased. The association of total fluorine with MCH was modified by smoking and by alcohol use. The increase in MCH as total fluorine increased was enhanced with increased smoking. In light drinkers, total serum fluorine had a weak association with MCH. In moderate drinkers, M CH increased as total fluorine increased. There was a positive association of both MCH and M CV with alcohol consumption. None of the estimated associations are of clinically significant magnitude over the range of total fluorine values. The white blood cell count (WBC) increased significantly in nonrespondents as total fluorine increased above 2ppm, increased less in moderate drinkers, and increased the least in light drinkers (Table 4.1.81). As expected, cigarette smoking intensity was positively associated with W BC. PMN increased significantly in alcohol use nonrespondents as total fluorine increased and increased less steeply in moderate drinkers (Table 4.1.82). In light drinkers, total serum fluoride above 10 ppm was associated with a decreased in PM N. Cigarette smoking was positively associated with PM N. As shown in Table 4.1.83, the final regression models for band count provides little evidence that total fluorine was associated with band count Moderate alcohol use was estimated to reduce the band count Smoking was positively associated with band count The negative association between total fluorine and lymphocyte count was modified by adiposity, alcohol consumption, and cigarette smoking (Table 4.1.84). The decrease in lymphocyte count was sm aller as BMI increased. The decrease in lymphocyte count associated with total fluorine above 3 ppm was greater in moderate drinkers compared to nonrespondents. As cigarette consumption increased, the decrease in lymphocyte count increased. The positive association between total fluorine and monocyte count (M O NO ) was modified by adiposity (Table 4.1.85). As BMI increased, the association with MONO was w eaker Cigarette smoking and LH w ere positively associated with MONO. Alcohol consumption was negatively associated with M ONO. The association between total fluorine and eosinophil count (EO S) w as negative for nonsmokers, but was positive as more than ten cigarettes per day were smoked 55 3M MN03112247 (Table 4.1.86). As smoking increased, the PFO A associated decrease in BASO was smaller (Table 4.1.88). The association between total fluorine and platelet count (PLAT) was modified by adiposity and cigarette smoking intensity (Table 4.1.87.). In lean participants (BM!=25), PLAT increased as total fluorine increased, in obese participants (BMI=40), the PLAT decreased as total fluorine increased. As smoking increased, the rate of increase in PLAT associated with total fluorine above 10 ppm decreased. 4.1.8 Summary O f Results The serum fluorine levels in Chemolite workers w ere 20-100 tim es higher than expected in workers not directly involved in PFOA production. All workers with levels above 10 ppm fluorine work in PFOA production areas. Smoking was associated with a small increase in serum fluorine. Age was not associated with serum fluorine levels. The two women employed in the PFOA production areas had total serum fluorine levels similar to men. Alcohol use, smoking, age, BMI, and hormones had the expected associations with peripheral leukocyte counts, hematology parameters, cholesterol, HDL, LDL, ' and hepatic enzymes. The main hormone resuite are: 1. The number of male workers with hormone values outside of the laboratory reference range was greater than expected for estradiol, free testosterone, bound testosterone, and prolactin. 2. Total serum fluorine was negatively associated with free testosterone and positively associated with estradiol. No association was noted between total serum fluorine and LH. 3. E/TF and TB /TF, but not E/TB, were positively associated with total serum fluorine. 4. E/LH and TB/LH were not associated with total serum fluorine. However, the relationship between total serum fluorine and TF/LH was suggestive. 56 3M MN03112248 5. TSH was positively associated with total serum fluorine. TF/TSH was negatively associated with total serum fluorine; TB/TSH and E/TSH were not. 6. Prolactin and total serum fluorine were positively associated in moderate drinkers, but not in light drinkers. 7. P/FSH, P/LH, P/TSH were positively associated with total serum fluorine. TBIP, I F IP, and E/P were not associated with total serum fluorine The main hepatic param eter resuits are: 1. The increase in SG O T and SG PT levels associated with adiposity was enhanced by total serum fluorine. 2. The induction of G G T by alcohol was decreased as total serum fluorine increased. 3. The induction of AKPH by smoking was increased by increasing levels of total serum fluorine. The main cholesterol and lipoprotein results are: 1. Cholesterol and triglyceride levels were not associated with total serum fluorine. 2. LDL was not associated with total serum fluorine. 3. The positive association between moderate alcohol use and HDL levels was reduced as total serum fluorine increased. The main hematology param eter and peripheral leukocyte count results are: 1. The effect of smoking on hemoglobin and M CV was enhanced by total serum fluorine. 2. Total serum fluorine was negatively associated with all peripheral leukocyte counts except PMNs and MONOs, which were positively associated. 3. The associations between cell counts and total serum fluorine were modified by smoking, drinking, and adiposity. 57 3M MN03112249 a 2 The 1990 Chemoflte Retrospective Cohort Mortality Study A total of 3,537 individuals who were employed at the Chemolite plant between January 1 ,1 9 4 7 and Decem ber 3 1 ,1 9 8 3 were identified from company records. The cohort consisted of 2,788 (79% ) male and 749 (21% ) fem ales employees (Tables 4.2.1 and 4.2.2). The majority of women (67.3% ) never worked in the Chemical Division. O f the 19,309 person years (PY) observed for women, 68.8% occurred in those who w ere never employed in the Chemical Division. The mean follow-up for women was 25.8 years in the overall cohort, 24.6 years in the Chemical Division (CD) cohort, and 26.4 years in the non-CD cohort The distribution of follow-up periods was similar in the women's CD and non-CD cohorts. The women's mean age at first employment was 27.6 years. Sixty-eight percent were less than 30 years old at employment; 9.7% were older than 40 at first employment at Chemolite. The CD cohort was slightly older than the non-CD cohort. The CD and non-CD distributions of latency times w ere not statistically different (p=.66). The mean duration of employment for women was 8 .7 years and ranged from six months to 41.4 years. The distribution of years of .employment was significantly different for CD and non-CD women (p<.0001). O f non-CD women, 11.9% were employed for more than twenty years. O f 245 women in the CD cohort, 51 (21.1% ) were employed for more than twenty years. As shown in Table 4.2.2, the 2,788 men who were ever employed for more than six months at Chemolite contributed a total of 71,117.7 PY which vims about equally divided between the CD and non-CD cohorts. The mean follow-up for the overall male cohort was 25.5 years. The distribution of follow-up periods and distribution of year of first employment was similar in the mate CD and non-CD cohorts. The average age at death was higher in the male non-CD group, 58.1 years, compared to the CD group, 54.2 years. The duration of employment for men (mean 13.6 years, median 9.8 years) Was longer than for women. The distribution of years of employment was significantly different for C D and non-CD men (p<.0001). O f non-CD men, 25.5% were employed for longer than twenty 58 3M MN03112250 years. Of men in the CD cohort, 38.0% were employed for longer than twenty years. Vital status was obtained for 100% of the women's cohort (Table 4.2.3). Among the 749 women there were 50 deaths; 11 in the CD cohort and 39 in the non-CD cohort Vital status was obtained tor 100% of the men's cohort. Among the 2788 men there were 348 deaths; 148 deaths in the CD group and 200 in the non-CD group. Six individuals who had employment records that were missing information were excluded from the cohort and their vital status was not ascertained. Death certificates were obtained for 99.5% of deaths. Two deaths occurred outside the U .S. and causes of death were ascertained by other means. 4.2.1 Standardized Mortality Ratios fSM R sl 4.2.1.1 SMRs For Women The numbers of deaths, the SMRs and 95% confidence intervals (C l) among women in the 1947-1989 foliow-up period are shown in Table 4.2.5. The SMRs for ail causes of death (S M R = .7 5,95% Cl .56-.99), and cancer (SM R =.71,9 5 % Cl .42-1.14) were significantly lower than expected in comparison to national rates. No association was found with duration of employment or latency for deaths from all causes, cancer, and carcBovascular diseases (Tables 4.2.6 and 4.2.7). SMRs for CD women and non-CD women are displayed in Table 4.2.8. The estimated SM R for the CD cohort of women were less than expected. In CD women, the all causes SM R was .46 (95% C l .23,.86) and the cancer SM R was .31 (95% Cl .07,1.05). The SM Rs for the non-CD women were closer to unity. 4.2.1.2 SMRs For Men The number o f male deaths, the expected number of male deaths based on U.S. national white male rates, and age and calendar period adjusted SM Rs with associated 95% CIs are presented in Table 4.2.9. The SM R for all causes (.73, 95% Cl .66,.81), for cardiovascular diseases (SM R =.71,9 5 % C l 60,.48), for all gastrointestinal (G l) diseases (.50,95% C l .26,.87) and for ail respiratory diseases (.50,95% C l .27,.86) were significantly less than one. None of the 59 3M MN03112251 cause-specific SM Rs were large nor were the estimates significantly different from one. As shown in Table 4.2.10, the results were sim ilar when the expected numbers of male deaths was based on Minnesota white male rates. Table 4.2.11, Table 4.2.12, and Table 4.2.13 present adjusted SM Rs and 95% C l for males based on Minnesota mortality rates for three latency intervals 1 0 ,1 5 , and 20 years respectively. The three latency intervals the all causes SM R ranged from .75 to .77. For all cancers, SM Rs ranged from 1.06 to 1.12 and were nonsignificant. Among men there was no association between any cause of death and duration of employment (Table 4.2.14, Table 4.2.15, and Table 4.2.16). Table 4.2.17 and 4.2.18 display the SM Rs and 95% C l for CD and non-CD male workers. The all causes SMRs were .69 (.59,.79) for the non CD group and .86 (.72,1.01) for the CD group. The SM Rs for prostate cancer, based on a comparison with Minnesota population rates, were 2.03 (95% C l .55,4.59) in the CD group and .58 (95% Cl .07,2.09) in the non-CD cohort. There were 4 observed deaths from prostate cancer compared to 2 expected in the CD group. The latency analysis for non-CD and CD men are presented in Tables 4.2.19 and 4.2.20. There was no associations between any cause of death and latency in either group. As shown in Table 4.2.21 and 4.2.22, male CD cohort members with more than 10 or more than 20 years of employment had SM Rs that were less than one for all causes of death, all malignancy, cardiovascular diseases and all respiratory diseases. Among male non-CD cohort members with more than ten years of employment or more 20 years of employment, the SM Rs for all causes, cardiovascular disease and all respiratory diseases were significantly less than expected (Table 4.2.23 and 4.2.24). There was no association of any cause of death with duration of employment at Chemolite in either CD or nort-CD groups. 2 .2 Standardized Rate Ratios fSRRs) Age adjusted standardized rate ratios (SRRs) were calculated for all causes, all cancer, and cardiovascular diseases mortality comparing men employed at the 60 3M MN03112252 plant tor ten years or more to men employed tor less than ten years. The SRRs are presented in Table 4.2.25. The 95% CIs for all causes, all cancer, and all cardiovascular diseases were wide and include one. Confounding variables such as year of first employment and length of follow-up were not controlled in this analysis due to small numbers and unstable rates within the large number of strata Table 4.2.26 presents the age adjusted SRRs for all causes, ail cancers, lung cancer, G l cancer, and all cardiovascular diseases mortality comparing men ever employed in the CD with men never employed in the CD . All SRRs were slightly greater than one, however, none was statistically significant. 4.2.3 Mantel- Relative Risks (RRMH) Age stratified RRm H. contrasting the rates in men ever employed in the CD compared to the rates in men never employed in the CD , were calculated for all causes, all cancer, and ail cardiovascular diseases mortality and are displayed in Table 4.2.27. The estimated RR for CD employment versus non-CD employment did not follow a monotonic pattern and the 95% C Is include one for each of the three endpoints. Table 4.2.28 presents the RRMH tor men employed for less than ten years to those employed for more than ten years. The all causes RRMH (2 .1 6 ,9 5 % C l 1.52,2.70) in the 30 to 39 year age at first employment strata was reflected in both the RRMH for all cancers (1 .7 5 ,9 5 % C l.95,3.21) and cardiovascular diseases (3 .5 3 ,9 5 % C l 1.68,6.21). The RRm H were not adjusted for important time covariates such as the year of first employment. 4-2-4 Proportional Hazard Regression Model Relative Risk Estimates 4.2.4.1 Proportional Hazard Models For M ale Workers Table 4.2.29 to 4.2.36 show the final proportional hazard (PH ) model for death from ail causes, cardiovascular diseases, all cancers, lung cancer, G l cancer, prostate cancer, pancreatic cancer, and diabetes among the 2788 m ale workers 61 3M MN03112253 ever employed at Chemolite for greater than six months. There was no evidence for violation of the PH assumptions or for significant nonlinear associations between the independent variables and mortality. As expected, age at first employment was positively associated with ail causes of death. The RR for a one year increase in age at first employment was 1.082 (95% C l 1.069,1.094). Year of first employment and duration of employment were negatively associated with all causes mortality. The risk of death associated with months in the Chemical Division was small and nonsignificant For cardiovascular diseases mortality, the RR for a one year increase in age at first employment was 1.126 (95% Cl 1.069,1.094). Y ear of first employment was negatively associated with cardiovascular diseases mortality. Tim e in the CD was not associated with death from cardiovascular diseases. Age at first employment was positively associated with cancer mortality. The RR for a one year increase in age of employment was 1.08 (95% C l 1.06,1.10). Duration of employment was negatively associated with cancer. The RR was .972 (9 5 % C l.96,.99) for a one year increase in em ploym ent There was no association of cancer mortality with employment tim e in the CD. The final prostate cancer mortality proportional hazard model for male cohort members Is shown in Table 4.2.34. Tim e in the Chemical Division w as positively and significantly associated with prostate cancer mortality. The relative risk for a one year increase In CD employment time was 1.13 (95% C l 1.01,1.43). Age at first employment was positively associated with prostate cancer mortality risk. A one year increase in age at first employment was associated with a RR of 1.09 (95% Cl .99,1.19). The RR for lung cancer mortality w as 1.07 (95% C l .03,1.12) for a one year increase in age of employment. Months in the chemical division was not significantly associated with lung cancer mortality. Table 4.2.33 shows the final proportional hazard (PH) model for all G l cancer mortality. The estimated RR for a one year increase in age at first employment was 1.14 (95% Cl 1.09,1.19). Y ear of first employment, duration of employment and time employed in the CD were not associated with G I cancer risk. Age at first employment was positively associated with pancreatic cancer mortality. The other covariates were weakly associated with pancreatic cancer risk and were 62 3M MN03112254 not significantly different from one. A one year increase in age at first employment was positively associated with diabetes mortality (RR = 1 .1 0 ,9 5 % Cl 1.01,1.19). 4 .9 4-2 Proportional Hazard Models For Female Workers Table 4 .2 .3 7 ,4 .2 .3 8 and 4.2.39 show the final PH model for death from all causes, cardiovascular diseases and all cancers among the 749 fem ale cohort members. Age at first employment was positively associated with ail causes mortality. The RR for ail causes of death among women employed for two to ten years (3.72) and among women employed for greater than ten years (2.33) were significantly greater than the all causes mortality in women employed for less than two years. Tim e in the CO was not related to mortality. The RR for death from cardiovascular diseases associated with a one year increase in age at first employment was 1.13 (1.07,1.18). The year at first employment, duration of employment, and time in the CD were not significantly associated with fem ale cardiovascular diseases mortality. The RR for death from cancer was associated with age at first em ploym ent A one year increase in age at first employment increase the RR for death from cancer (1.09 (1.04,1.14). The year at first employment, duration of employment, and time in the chemical division were weakly and non-significantly associated with fem ale cancer mortality. 63 3M MN03112255 4.3 Physiologic Effects Tables TA B LE 4.1.1 A G E D IS T R IB U T IO N IN F IV E Y E A R A G E G R O U P S 3M C H E M O L IT E PLANT, C O TTA G E G R O V E . M IN N E S O T A AGE NUMBER PERCENT 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 TOTAL MEAN SD MEDIAN RANGE 3 18 26 22 18 9 13 6 115 39.2 8.91 37 24-59 2.6 15.7 22.6 19.1 15.7 7.8 11.3 5.2 100.0 64 3M MN03112256 TA BLE 4 .1 2. D IST R IB U T IO N O F A LC O H O L A N D TO B A C C O U S E 3M C H E M O L IT E PLANT, C O TTA G E G R O V E , M IN N E S O T A USE STATUS NUMBER PERCENT TOBACCO USE CURRENT SMOKER NONSMOKER MISSING VALUES TOTAL ALCOHOL USE <102 ETHANOL/DAY* 1-3oz ETHANOL/DAY MISSING VALUES TOTAL ` Includes 22 nondrinkers 28 85 2 115 87 20 8 115 24.3 73.9 1.8 100.0 75.6 17.4 7.0 100.0 65 3M MN03112257 TABLE 4 .1 .3 T H E J O IN T D IS T R IB U T IO N O F TO B A C C O A N D A LC O H O L U S E 3M C H E M O L ITE PLANT. C O TTA G E G R O V E , M IN N E S O T A ALCOHOL USE <loz/day 1-3oz/day missing TOTAL SMOKER TOBACCO USE NONSMOKER MISSING TOTAL 19(67.9% ) 7(25.0% ) 2 (7.1%) 28 (100%) 67(78.8% ) 13(15.3% ) 5 (5.9%) 85(100% ) 1 (50.0%) 0 (0%) 1 (50.0% ) 2 (100%) 87(75.6% ) 20(17.4% ) 8(7.0% ) 115(100% ) 66 3M MN03112258 TABLE 4 .1 .4 D IS T R IB U T IO N O F A G E B Y S M O K IN G A N D D R IN K IN G S TA TU S . 3M C H E M O U T E PLANT, C O TTA G E G R O V E , M IN N E S O T A A G E (yea rs) N MEAN SD M EDIAN RANG E TE S T# A lc o h o l <ioz/d 1-3oz/d missing 87 39.9 9.31 20 37.5 6.95 8 36.6 8.70 Tobacco smoker 28 40.4 7.59 nonsmoker 85 39.0 9.35 missing 2 32.5 3.53 TOTAL 115 39.2 8.91 37 24-59 37 27-51 *p -2 9 35 27-54 *p.17 39 28-54 37 24-59 *p-.47 32 30-35 37 24-59 ` Student t test, Prob>T, reference groups <1 oz/day, smoker 67 3M MN03112259 TABLE 4.1.5 PEARSON CORRELATION COEFFICIENTS BETWEEN TOTAL SERUM FLUORINE. AGE, BODY MASS INDEX (BM1), DAILY ALCOHOL USE, AND DAILY TOBACCO CONSUMPTION. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLUORINE AGE BMI ALCOHOL TOBACCO TOTAL FLUORINE to " ") 1 m m m AGE (years) BUOtgAn^) .004 .0002 1 2.6 D-.005 m1 m m* ALCOHOL (oz/day) -.007 -.14 .08 1 TOBACCO (cigs/day) .006 .15 -.04 .08 1 68 3M MN03112260 TABLE 4.1.6 BODY MASS INDEX DISTRIBUTION 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA BMI (kfl/m 2) >15-20 >20-25 >25-30 >30-35 >35-45 TOTAL MEAN BUI SO MEDIAN BMI RANGE NUMBER 1 40 57 15 2 115 26.9 3.4 26.3 18.8-40.5 PERCENT 0.9 34.8 49.5 13.0 1.8 100.0 69 3M MN03112261 TABLE 4.1.7 BODY MASS INDEX BY SMOKING AND DRINKING STATUS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA B M I(kg /m 2) N MEAN SD MEDIAN RANGE TEST# Alcohol <1oz/d 1-3oz/d missing Tobacco smoker nonsmoker missing Total 87 20 8 28 85 2 115 26.9 ^ 272 25.9 354 3.10 3.64 26.6 3.63 27.0 2.99 262 2.87 26.9 3.45 26.1 18.8-405 27.0 222-33.7 *p.71 26.1 212-30.4 265 18.8-282 26.6 21.4-33.7 p-57 262 24.1-282 262 182-405 Student t test, t test p-value, reference groups <loz/day, smoker 70 3M MN03112262 TABLE 4.1.8 THE DISTRIBUTION OF AGE, ALCOHOL AND TOBACCO USE BY BODY MASS INDEX 3M CHEMOLITE PLANT. COTTAGE GROVE, MINNESOTA TOBACCO USE SMOKER NONSMOKER MISSING TOTAL <25 11 (26.8%) 29(70.7%) 1 (2.5%) 41 (100%) B M I mg/kg2 25-30 15 (26.3%) 41 (71.9%) 1 (1.8%) 57(100%) >30 2(11.8%) 15 (882%) 0 (0%) 17(100%) ALCOHOL USE <1 oz/tiay 1-3oz/day MISSING TOTAL AGE <40 years >40 years TOTAL 31 (75.6%) 6(14.6%) 4 (9.8%) 41 (100%) 31 (75.6%) 10 (24.4%) 41 (100%) 43 (75.4%) 11 (19.3%) 3 (5.3%) 57 (100%) 28 (49.1%) 29 (50.9%) 57 (100%) 13 (76.4%) 3 (17.7%) 1 (5.9%) 17 (100%) 6(352% )* 11 (64.7%) 17 (100%) *t test p=.005 71 3M MN03112263 TABLE 4.1.9 TOTAL SERUM FLUORIDE DISTRIBUTION 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLUORINE (P P M ) NUMBER PERCENT <1 1-3 >3-10 >10-15 >15-26 TOTAL MEAN TF SO MEDIAN TF RANGE 23 65 16 6 5 115 3.3 4.7 2 0-26 20.0 56.5 13.9 52 4.4 100.0 72 3M MN03112264 TABLE 4.1.10 TOTAL SERUM FLUORIDE BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA ---------------------------------------------- FLUORINE (ppm) N (% ) MEAN SD MEDIAN RANGE TEST# BMI <25 41(35.7) 2.8 3.74 2 0-19 F1.47# 25-30 57(49.6) 4.0 5.47 2 0-26 Pb54 >30 17(14.8) 2.1 351 1 0-14 AGE <31 21(18.3) 3.7 455 2 0-20 F-.10# 31-40 48(41.7) 35 4.08 2 0-14 P-.96 41-50 27(235) 35 456 2 0-19 51-60 19(165) 3.0 6.42 1 0-26 Alcohol <ioz/d 87(75.6) 3.4 5.15 2 0-26 p*53* 1-3oz/d 20(17.4) 35 257 2 0-12 missing 8(7.0) 2.1 253 1 0-6 Tobacco smoker 28(245) 3.6 456 2 0-20 p*>.66* nonsmoker 85(755) 35 4.13 2 0-26 missing 2(1.7) 3.0 454 3 0-6 TOTAL 115 35 4.67 2 0-26 #univariate Anova Student!test, Prob>T 73 3M MN03112265 TABLE 4.1.11 AGE DISTRIBUTION BY TOTAL SERUM FLUORINE CATEGORY. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA AGE 20-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 TOTAL TOTAL SERUM FLUORINE (ppm) <1 1-3 >3-10 >10-15 NUMBER (PERCENT) 1 (4.4) 3(13.0) 6(26.1) 4(17.4) 2 (8.7) 0 (0) 6(26.1) 1 (4-3) 23(100) 1 (15) 10 (15.4) 13 (20.0) 12 (18.5) 13 (20.0) 7(10.7) 6 (9.3) 3 (4.6) 65(100) 0 (0) 4(25.0) 4(25.0) 5(31.2) 2(125) 0 (0) 0 (0) 1 (65) 16 (100) 1 (16.7) 0 (0) 2(335) 0 (0) 1 (16.7) 1 (16.7) 1 (16.7) 0 (0) 6 (100) >15-26 0 (0) 1(20.0) 1 (20.0) 1 (20.0) 0 (0) 1(20.0) 0 (0) 1 (20.0) 5 (100) MEANAGE SO MEDIAN AGE AGE RANGE 39.9 10.2 37 25-59 39.6 85 38 24-56 36.0 75 355 27-57 395 11.1 375 25-54 41.6 105 40 30-57 74 3M MN03112266 TABLE 4.1.12 DISTRIBUTION OF TOBACCO USE BY TOTAL SERUM FLUORIDE CATEGORY. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA _______________ TOTAL SERUM FLUORINE (ppm)_______________ <1 1-3 >3-10 >10-15 >15-26 TOTAL Tobacco use Smoker Nonsmoker Missing Total NUMBER(%) 3 (13.0) 19(82.7) 1(45) 23(100) 16 (24.6) 49(75.4) 0(0) 65(100) 6 (37.5) 9(565) 1 (65) 16 (100) 2(33.3) 4(66.7) 0(0) 6(100) 1 (205) 4 (8 0 5 ) 0(0) 5(100) 28(245) 85(73.9) 2(1.7) 115(100) Cigarettes/day (among smokers) MEAN 165 SD 14.0 MEDIAN 17 RANGE 2-30 24J* 85 20 7-40 18.0 95 20 3-30 20 0 20 20 ` significantly different from <1 ppm mean (pc.005) 20 215 - 10.1 20 20 20 2-40 75 3M MN03112267 TABLE 4.1.13 DISTRIBUTION O F ALCOHOL USE BY TOTAL SERUM FLUORIDE CATEGORY. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA ALCOHOL USE <1 oz/day 1-3 oz/day MISSING TOTAL SERUM FLUORINE (ppm ) <1 1-3 >3-10 >10-15 NUMBER (PERCENT) 17 (73.9) 2 (8.7) 4 (17.4) 51 (78.5) 13(20.0) 1 (1 5 ) 9 (56.3) 4 (25.0) 3(18.7) 5 (83.3) 1 (16.7) 0(0) >15-26 5(100) 0 (0) 0 (0) TOTAL 23(100) 65 (100) 16(100) 6(100) 5(100) 76 3M MN03112268 TABLE 4.1.14 BODY MASS INDEX DISTRIBUTION BY TOTAL SERUM FLUORINE CATEGORY. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL SERUM FLUORINE (ppm) <1 1-3 >3-10 >10-15 >15-26 BMIfkg/m2) >15-20 >20-25 >25-30 >30-35 >35-40 >40-45 TOTAL MEAN BM 8D MEDIAN BMI RANGE 1 (4.4) 9(39.1) 5(21.7) 7 (30.4) 0 (0) 1 (4.4) 23(100) 27.6 55 27 18.8-40.5 NUMBER PERCENT) 0(0) 0(0) 21 (32.3) 8(50.0) 39 (60.0) 5(31.2) 5(7.7) 3 (18.8) 0(0) 0(0) 0(0) 0(0) 65(100) 16 (100) 26.6 2.6 26.8 225-33.7 26.3 35 25.7 21.4-325 0(0) 1 (16.7) 4 (66.6) 0(0) 1 (16.7) 0(0) 6(100) 29.4 3.7 295 245-355 0(0) 1 (20.0) 4 (80.0) 0(0) 0(0) 0(0) 5(100) 26.0 1.4 25.6 24.1-27.6 3M MN03112269 TABLE 4.1.15 COEFFICIENT OF VARIATION FOR SEVEN HORMONE ASSAYS. 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA HORMONE BOUND TESTOSTERONE FREE TESTOSTERONE ESTRADIOL TSH LH PROLACTIN FSH cv 10.6% 12.1% 18.3% 10.0% 8.6% 3.1% 5.6% 78 3M MN03112270 TABLE 4.1.16 THE OBSERVED VERSUS EXPECTED NUMBER O F WORKERS WITH HORMONE ASSAYS OUTSIDE THE ASSAY REFERENCE RANGE 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA OBSERVED EXPECTED Estradiol >=44 pg/ml Testosterone bound <=300 ng/dl Testosterone free <=9 ng/dl Prolactin >=15 ng/ml LH 2-12m U/m l FSH 1-12m U/m I TSH >=4.6 mU/ml 17 13 11 10 3 1 1 2.8 2.8 2.8 2.8 2.8 2.8 2.8 O /E * 6.0 4.5 3.9 3.5 1.1 .4 .4 95% c r (3.6,9.8) (2.6,8.1) (2.0,7.1) (1.8,6.7) (0.3,3.3) (0.1,2 .0) (0.1,2.0) *0/E - OBSERVED TO EXPECTED RATIO "C l -95% CONFIDENCE INTERVAL 79 3M MN03112271 TABLE 4.1.17 PEARSON CORRELATION COEFFICIENTS BETWEEN SERUM HORMONES. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA. ESTRADIOL ESTRADIOL 1 FREE TEST. .40 P-.Q0Q1 BOUND .M TESTri .32 P-.0006 PROLACTIN .16 p-,08 LnLH++ .06 FSH+ -.14 P-.15 LnTSH* .05 FREE TESTOSTERONE* BOUND TESTOSTERONE* PROLACTIN# LnLH*+ FSH* ' 1 .74 .13 P-.0001 .10 -.05 1 .21 .28 .16 P-.03 P-.003 P-.04 m 1 .15 .004 .07 -.02 .11 1 .63 -.15 P-.0001 p.11 m -.23 P-.01 *ng/dl #ng/ml ++LOG LUTENIZING HORMONE (mll/ml) * FOLLICLE STIMULATING HORMONE (mU/ml) LOG THYROID STIOMULATING HORMONE (mU/ml) 3M M N03112272 TABLE 4.1.18 PEARSON CORRELATION COEFFICIENTS BETWEEN TOTAL SERUM FLUORIDE, AGE, BODY MASS INDEX (BMI), DAILY ALCOHOL USE, DAILY TOBACCO CONSUMPTION, AND SERUM HORMONES. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL AGE (years) FLUORINE (ppm) BMI (kg/m2 ) ALCOHOL TOBACCO (oz/day) (dgs/day) ESTRADIOL FREE TESTOSTERONE* BOUND TESTOSTERONE* PROLACTIN# LnLH++ .13 P-.16 .03 .08 .19 P-.045 .04 -2 5 P-.01 -.45 D..0001 -24 D-.01 -.19 D-.01 .11 -.01 -2 6 Om.005 -.36 D-.0001 -.06 20 P-.03 .05 -.08 -.16 D=.11 .03 -.14 .12 P -2 .05 .11 -.16 P-.09 .18 P<b.06 FSH* -.03 .33 -.08 P-.0003 LnTSH# 26 .09 P-.005 @pg/ml *ng/dl #ng/ml +LOG LUTEN1ZING HORMONE (mll/ml) FOLLICLE STIMULATING HORMONE (mU/ml) #LOG THYROID STIMULATING HORMONE (mU/ml) .04 2 4 P-.01 .15 P-.15 .17 p>.06 -.03 81 3M MN03112273 TABLE 4.1.19 BOUND TESTOSTERONE (TB) BY BODY MASS INDEX, AGE, SMOKING, DRINKING STATUS AND TOTAL SERUM FLUORIDE 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA. . N(%) MEAN TB(ng/dl) SD MEDIAN RANGE TEST# BMI (kg/m2) <25 25-30 >30 Age <31 31-40 41-50 51-60 Alcohol <1oz/d 1-3oz/d missing Tobacco smoker nonsmoker musing Total n u o rin e <1 ppm 1-3 >3-10 >10-15 >15-26 Total 40(35.4) 56(49.6) 17(15.0) 20(17.7) 48(42.5) 26(23.0) 19(16.8) 86(76.1) 19(16.8) 8(7.1) 27(23.9) 84(74.3) 2(1.8) 23(20.4) 64(56.6) 15(133) 6(5.3) 5(4.4) 113(100) 641 565 436 598 634 512 470 581 484 690 622 559 432 584 567 530 600 6% 572 242.9 196.8 172.7 232.8 214.1 185.6 226.1 2123 215.1 2723 177.7 233.0 97.6 295.4 202.9 1893 234.6 149.8 220.7 592 275-1192 F-5.64 560 141-954 P-.005 438 210-803 673 278-1192 F-3.60 605 275-1189 P-.016 498 141-947 409 210-954 574 210-1192 F-133 417 141-1039 P-37 602 409-1101 617 379-1039 F-1.69 556 141-1192 p-3 0 432 363-501 438 275-1192 F-039 572 141-1039 P-.82 574 210-819 563 244-947 659 517-880 561 141-1192 #univariate Anova 82 3M MN03112274 TABLE 4.1.20 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE BOUND TESTOSTERONE (ng/dl) AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA variable 8 sm -- p-value Intercept Total Fluorine (ppm)* 1027 -148 190.7 67.2 .0001 .05 Age (years) Age X Total Fluoride* BMI (kg/m2) -9 3 -16 3.3 .009 1.6 .04 5.4 .003 Smoker** 74 45.0 .28 Alcohol (<1oz/day)# Estradiol (pg/ml) 89 47.5 .11 2 1.0 .02 LH (mU/mi) Prolactin (no/ml) 116 6.1 .004 8 4.1 .04 R2= .39 . ` Square root transformation of total serum fluoride measured in ppm. " Reference category is nonsmokers. #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. 83 3M MN03112275 TABLE 4.1.21 FREE TESTOSTERONE (TF) BY BODY MASS INDEX AGE, SMOKING AND DRINKING STATUS AND TOTAL SERUM FLUORIDE 3M CHEMOLFTE PLANT. COTTAGE GROVE, MINNESOTA N f% ) MEAN TF(ng/dl) SD MEDIAN RANGE TEST# BMI kg/m2 <25 40(35.4) 17.4 6.22 16.7 7.4-452 F-3.58 25-30 56(49.6) 15.1 4.13 152 32-232 p.03 >30 17(15.0) 13.7 6.08 13.5 5.6-302 Age years <30 31-40 20(17.7) 18.7 7.64 16.7 92-452 F-9.14 48(42.5) 17.0 3.75 17.1 7.4-2937 p.,0001 41-50 26(23.0) 14.1 4.73 14.3 32-232 51-60 19(16.8) 11.5 3.78 112 5.6-19.0 Alcohol <1oz/d 86(76.1) 15.8 526 15.8 5.6-452 F-1.45 1-3 oz/d 19(16.8) 14.2 4.79 152 32-232 p-23 missing 8(7.1) 18.1 6.40 172 11.0-29.7 Tobacco smoker 27(23.9) 16.6 3.71 17.1 8.4-242 F-.95 nonsmoker 84(74.3) 15.4 5.84 152 32-452 p-23 missing 2(1.8) 15.9 4.45 15.9 12.7-19.0 Total Fluorine <1 ppm 23(20.4) 16.4 8.4 13.9 6.4-452 F-0.13 1-3 64(56.6) 15.6 42 152 32-302 P-.97 >3-10 15(132) 152 3.8 152 7.1-19.7 >10-15 6(52) 15.9 52 17.6 5.6-19.9 >15-26 5(4.4) 152 22 14.1 132-182 Total 113(100) 15.7 5.4 16 32-452 #univariate Anova 84 3M MN03112276 TABLE 4.1.22 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING TH E FREE TESTOSTERONE VALUE (ng/dl) AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B $ E (B ) D-vaiue Intercept Total Fluorine (ppm)* Age (years) Age X Total Fluoride* BMi (kg/m2) Smoker** Alcohol (<1oz/day)# Estradiol (pg/ml) LH (mU/ml) 29.72 -3.56 -.34 .07 -21 1.46 1.65 .10 .18 4.57 1.62 .08 .04 .13 1.03 1.14 .03 .15 .0001 .03 .0001 .05 .11 .16 .15 .003 20 R2= .39 `Square root transformation of total serum fluoride measured in ppm. " Reference category is nonsmokers. #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. 85 3M MN03112277 TABLE 4.1.23 PARTICIPANT ESTRADIOL BY BODY MASS INDEX, AGE, SMOKING DRINKING STATUS AND TOTAL SERUM FLUORIDE 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA N (% ) MEAN ESTRADIOL (M/m l) SD MEDIAN RANGE TEST# BMI (kgffli*) <25 40(35.4) 34.1 12.91 40 8-69 F-.13 25-30 56(49.6) 332 13.89 33 8-83 p-28 >30 17(15.0) 322 1226 27 18-57 AGE <30 31-40 41-50 51-60 20(17.7) 34.4 10.15 34 19-58 F-3S0 48(42.5) 36.8 11.54 38 12-69 P-.018 26(23.0) 31.6 18.48 28 8-83 19(16.8) 25.9 7.93 24 15-47 Alcohol <1 oz/d 86(76.1) 33.0 11.78 33 8-66 F-.14 1-3 oz/d 19(16.8) 31.8 16.61 30 8-69 P-.71 missing 8(7.1) 41.1 1820 40 23-83 Tobacco smoker 27(23.9) 36.3 17.40 34 14-83 F-.13 nonsmoker 84(74.3) 32 11.63 32 8-66 P-.88 missing 2(1 A) 30 13.44 30 21-40 Total Fluorine <1 ppm 23(20.4) 362 13.1 34 14-60 F-127 *-1 -3 64(56.6} 31.4 13.6 30 8-83 p--29 >3-10 15(132) 32.8 10.6 34 10-58 >10-15 6(52) 382 152 35.5 22-66 >15-26 5(4.4) 412 11.4 42 26-56 Total 113(100) 33.4 132 33 8-83 86 3M MN03112278 TABLE 4.1.24 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE ESTRADIOL VALUE (pg/dl) AMONG 113 MALE WORKERS. 3M CHEMOLITE PLANT. COTTAGE GROVE, MINNESOTA Variable 0 SE(ft) p-value Intercept Total Fluorine (ppm)* Age (years) BMI (kg/tn2) Cigarettes/day Alcohol (<1oz/day)# Free Testosterone (ng/dl) 12.89 .03 -J22 .51 .16 .09 .85 12.13 .01 .15 .34 .11 .11 24 29 .03 .14 .14 .15 .98 .0007 R2= 2 4 ` Square transformation of total serum fluoride measured in ppm. #Reference category Is moderate drinkers who consume 1-3 oz ethanol/day. 87 3M MN03112279 TABLE 4.25 LUTENIZING HORMONE (LH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS, AND TOTAL SERUM FLUORINE 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA N<%) MEAN LH (mU/ml) SD MEDIAN RANGE TEST# BMI mg/kg2 <25 40(35.4) 5.49 3.08 4.60 2.6-21.7 F-6.19 25-30 >30 56(49.6) 5.84 325 5.15 1.7-23.0 P-.003 17(15.0) 3.72 121 3.60 2.0-72 Age years <30 31-40 20(17.7) 48(42.5) 4.81 5.49 226 3.14 4.45 1.7-10.1 F-.69 4.75 2.4-21.7 p-28 41-50 51-60 26(23.0) 19(16.8} 5.33 5.90 1.64 4.73 5.15 22-9.8 4.10 2.0-23.0 Alcohol <1oz/d 1-3oz/d 86(76.1) 19(16.8) 5.60 4.69 324 120 4.70 1.7-232 F-124 421 22-10.1 p-27 missing 8(7.1) 4.86 1.00 4.05 3.4-62 Tobacco smoker 27(23.9) 6.30 3.78 5.30 2.6-21.7 F-5.16 nonsmoker 84(742) 5.05 2.71 422 1.7-23.0 p.025 missing 2(1.8) 7.45 2.47 7.45 Total <1 ppm 23(20.4) 52 2.1 4.4 2 2 -9 2 F-0.16 . >-1-3 64(56.6) 5.6 3.6 42 1.7-232 P -.98 >3-10 15(13.3) 5.1 2.7 4.9 2.0-13.9 >10-15 6(5.3) 5.4 02 4.9 3.7-72 >15-26 5(4.4) 52 12 52 3.7-72 Total 113(100) 5.4 32 4.7 1.7-23.0 #univariate A nova 88 3M MN03112280 TABLE 4.1.26 LINEAR MULTIVARIATE REGRESSION MODEL #1 OF FACTORS PREDICTING THE LUTENIZING HORMONE* VALUE (mU/ml) AMONG 113 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B SE?B) p-value Intercept Total Fluorine (ppm)* Age (years) BMI (kg/m2) Smokers** Alcohol (<1oz/day)# Bound Testosterone (ng/dl) 1.26 .001 .01 -.02 24 .06 .001 .40 .008 .005 .01 23 .10 .0002 .002 .93 .03 .15 .29 .60 .008 R2= .28 'logarithmic transformation of lutenizing hormone (LH). ** Reference category is nonsmokers. #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. 89 3M MN03112281 TABLE 4 1 .2 7 FOLLICLE STIMULATING HORMONE (FSH) BY BODY MASS INDEX,' AGE, SMOKING AND DRINKING STATUS, AND TOTAL SERUM FLUORINE 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA N (% ) F S H (m U /m i) mean" Sd MEDIAN------R A N 6E TEST#" BMlmg/kg2 <25 25-30 >30 Age yam <30 31-40 41-50 51-60 40(35.4) 56(49.6) 17(15.0) 20(17.7) 48(42.5) 26(23.0) 19(165) Alcohol <1oz/d 1-3oz/d missing 86(76.1) 19(165) 8(7.1) Tobacco smoker nonsmoker missing Total Fluorine <1 ppm 1-3 >3-10 >10-15 >15-26 Total 27(23.9) 84(743) 2(13) 23(20.4) 64(56.6) 15(13.3) 6(53 ) 5(4.4) 113(100) #univariate Anova 5.02 559 451 338 438 5.65 6.22 537 4.18 458 5.77 4^5 6.10 4.4 5.4 4.8 5.4 4.9 5.1 239 2.71 1.75 1.86 224 255 3.01 2.62 1.92 1.49 2.46 4.49 0.42 1.95 2.75 223 2.14 256 2.49 4.6 15-105 F-157 43 1.4-14.8 p -59 33 1.6-8.3 3.6 1.4-95 F-3.72 4.6 1.6-105 p-,014 4.6 2.1-14.8 5.0 2.7-14.8 4.6 1.4-145 F-3.47 3.9 2.0-95 p-,065 43 25-6.4 4.9 2.6-11.9 F-2.80 42 1.4-145 p-,09 6.1 55-6.4 4.4 1.6-105 F-0.75 4.8 1.4-145 P-.56 4.9 2.1-9.7 4.4 35-85 3.7 25-7.7 45 1.4-145 90 3M MN03112282 TABLE 4.1.28 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE FOLLICLE STIMULATING HORMONE VALUE (mU/ml) AMONG 113 MALE WORKERS. 13M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B SE(B) p-vaiue Intercept Total Fluorine(ppm)* Age (years) BMI (kg/m2) Cigarettes/day Alcohol(<1oz/day)# TSH (mU/ml)@ LH (m U /m lp 1.20 .004 .08 -.04 .02 .45 -.43 .44 1.62 .04 .02 .05 .02 .48 22 .06 .46 .91 .0006 .41 .29 .34 .05 .0001 R2= 48 logarithmic transformation of follicle stimulating hormone (FSH). #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. Thyroid Stimulating Hormone MLutienizing Hormone 91 3M MN03112283 TABLE 4.1 J29 THYROID STIMULATING HORMONE (TSH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS, AND TOTAL SERUM FLUORINE. 3M CHEM OUTE PLANT, COTTAGE GROVE, MINNESOTA TSH(mU/ml) N (% )_ _ MEAN SD MEDIAN RANGE TEST# BMI mofluj2 <25 40(35.4) 135 0.66 1.04 037-3.14 F -35 25-30 >30 56(49.6) 1.64 1.01 1038 0.45-630 P-.70 17(15.0) 1.72 0.71 135 0.62-333 Age years <30 31-40 20(17.7) 1.43 036 1.42 038-233 r.47 48(42.5) 136 1.04 1.46 037-630 p**.70 41-50 26(23.0) 1.64 0.75 134 0.75-336 51-80 19(16.8) 1.70 0.74 133 0.62-3.09 Alcohol <1oz/d 86(76.1) 137 0.70 1.40 038-336 F-133 1-3oz/d 19(163) 133 139 135 0.60-630 pa>37 missing 8(7.1) 1.49 0.63 1.61 037-232 Tobacco smoker 27(23.9) 133 0.61 137 0.61-3.03 F-.09 nonsmoker 84(74.3) 1.66 0.92 1.49 037-630 P-.76 missing 2(ie> 138 0.42 138 038-137 Total fluorine it r 23(20.4) 13 0.64 13 03-33 F -2 3 0 64(56.6) 1.6 034 13 0.4-63 P-.08 >3-10 15(133) 1.6 0.67 1.4 03-3.0 >10-15 6(53) 2.4 037 23 033-33 >15-26 5(4.4) 23 1.66 2.1 1.7-33 Total 113(100) 1.8 0.85 1.4 03-63 #univariate Anova 92 3M MN03112284 TABLE 4.1.30 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE THYROID STIMULATING HORMONE* VALUE (mU/ml) AMONG 113 MALE WORKERS. 3M CHEMOLFTE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le B p-value Intercept Total Fluorine (ppm)* Age (years) BM1 (kg/m2) Cigarettes/day Alcohol (<3oz/day)# Free Testosterone** FSH## -.190 .027 .006 -.002 -.001 -.140 .020 .060 .465 .009 .005 .013 .004 .194 .009 .019 .68 .004 .29 .89 .74 .26 .04 .003 R2= .30 logarithmic transformation of thyroid stimulating hormone (TSH). #Reference category is moderate drinkers who consume 3 oz ethanol/day. ** ng/dl M Foltide stimulating hormone mU/ml 93 3M MN03112285 TABLE 4.1.31 PROLACTIN BY BODY MASS INDEX, AGE, SMOKING, DRINKING STATUS, AND TOTAL SERUM FLUORINE 3M CHEMOLiTE PLANT, COTTAGE GROVE, MINNESOTA N (% ) PROLACTIN (ng/ml) MEAN SD MEDIAN RANGE TEST# BMI (kg/m2) <25 25-30 >30 40(35.4) 56(49.6) 17(15.0) Age <30 31-40 41-50 51-60 20(17.7) 48(42.5) 26(23.0) 19(16.8) Alcohol <1oz/d 1-3oz/d missing 86(76.1) 19(16.8) 8(7.1) Tobacco smoker nonsmoker missing Total Fluorine <1 ppm 1 -3 >3-10 >10-15 >15-26 T o ta l 27(23.9) 84(74.3) 2(1.8) 23(20.4) 64(56.6) 15(133) 6(53) 5(4.4) 113(100) #univariate Anova 9.10 8.71 7.45 9.63 9.01 838 7.16 8.61 9.46 725 6.97 9.13 11.65 73 83 4.9 15.1 83 8.7 5.13 5.18 3.08 430 530 532 337 437 8.87 233 3.14 5.18 9.40 3.19 434 1.15 11.01 4.16 4.90 8.4 2.7-243 F-.69 73 13-33.7 p-31 72 23-13.6 83 3.9-183 F-.96 8.7 12-33.7 p-31 63 23-233 63 23-15.1 73 13-243 F-.44 8.7 2.9-33.7 p -3 0 6.9 43.-103 6.6 13-123 F-4.18 83 23-33.7 P-.043 11.7 5.0-183 73 23-183 F-3.02 8.1 13-243 P-.02 6.6 1.4-18.1 9.4 6.8-33.7 7.7 33-15.1 7.7 13-33.7 94 3M MN03112286 TABLE 4.1.32 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE PROLACTIN VALUE (ng/ml) AMONG 113 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B p-value Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day# Estradiol (pg/ml) 7.41 1.43 -.04 -.08 -.08 .06 4.14 .36 .05 .13 .04 .03 .07 .0002 .41 .53 .08 .07 Alcohol Use## Light (<1oz/day) Nonresponse (NR) Light X total fluoride NR X total fluoride 3.21 2.14 -1.67 -1.34 1.65 2.69 .77 .37 .05 .43 .03 .0006 R2= .22 ##Reference category is moderate drinkers who consume 1-3 oz ethanol/day. Nonrespondants (NR) failed to complete the alcohol use questionnaire items. Light X total fluoride and NR X total fluoride are interaction terms for alcohol categories and total serum fluoride. 95 3M MN03112287 TABLE 4.1.33 PEARSON CORRELATION COEFFICIENTS BETWEEN HORMONE RATIOS AND TOTAL FLUORIDE, AGE, BODY MASS INDEX, ALCOHOL AND TOBACCO CONSUMPTION 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL AGE (years) BMI (kg/m2) ALCOHOL TOBACCO FLUORINE (oz/day) (clgs/day) ETB@ E/TF* E/LHTB/LH+ TF/LH++ TB/TF" -.01 .11 .001 .002 -.09 .16 D.09 .004 .15 D>.15 -J26 OmJQQB -3 2 P-.001 -.40 P-.0001 34 D-.01 32 P-.001 37 Db.004 .18 D-.06 -.14 P-.13 -.02 -.16 Ds.08 .05 .01 .05 -.01 .03 -.12 .05 .01 .04 -.01 .03 .09 @ESTRADIOL TO BOUNDTESTOSTERONE RATIO ESTRADIOL TO FREE TESTOSTERONE -ESTRADIOL TO LUTENIZING HORMONE RATIO +BOUNDTESTOSTERONE TO LUTENIZING HORMONE RATIO FREE TESTOSTERONE TO LUTENIZING HORMONE RATIO "BOUNDTESTOSTERONE TO FREE TESTOSTERONE RATIO 96 3M MN03112288 TABLE 4.1.34 PEARSON CORRELATION COEFFICIENTS BETWEEN PROLACTIN HORMONE RATIOS AND TOTAL FLUORIDE, AGE, BODY MASS INDEX, ALCOHOL AND TOBACCO CONSUMPTION 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TEW* TF/P* EIP*FSH/P" P/LH" P/TSH++ TOTAL FLUORINE (oom) -.05 -.08 -.03 -.09 .11 .07 AGE (years) BMI (kg/m*) ALCOHOL TOBACCO (oz/day) (clgs/day) -.04 -.11 -.05 J37 Ds.0001 -2 4 O-.003 .17 P-.07 -.13 -.08 .03 .004 .09 .07 -.03 .06 .007 -.13 D-.18 .15 Ds.11 .17 P-.07 24 p=.01 22 D-.02 25 P-.008 21 O-.02 -2 2 Db.02 .09 @Free testosterone to prolactin ratio *Free testosterone to prolactin ratio Estradiolto prolactin ratio "Follicle stimulating hormone to prolactin ratio "Prolactin to lutenizing hormone ratio Prolactin to thyroid stimulating hormone ratio 97 3M MN03112289 TABLE 4.1.35 PEARSON CORRELATION COEFFICIENTS BETWEEN THYROID STIMULATING HORMONE RATIOS AND TOTAL FLUORIDE, AGE, BODY MASS INDEX, ALCOHOL AND TOBACCO CONSUMPTION 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL AGE (years) BMl(kfl/m2)ALCOHOL TOBACCO FLUORINE (oz/day) (cigs/day) TB/TSH# TF/TSH* E/TSH** -.13 -.18 O-.05 -.13 -.23 P-.01 d-,0002 -.24 P-.01 -24 P-.01 -2 3 D-.01 -.05 -.16 p>.09 -.13 -.05 .03 .01 .04 #Bound testosterone to thyroidstimulating hormone ratio Free testosterone to thyroidstimulating hormone ratio Estradiolto thyroid stimulating hormone ratio TABLE 4.1.36 PEARSON CORRELATION COEFFICIENTS BETWEEN FOLLICLE STIMULATING HORMONE RATIOS AND TOTAL FLUORIDE.AGE, BODY MASS INDEX, ALCOHOL AND TOBACCO CONSUMPTION 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL AGE (years) Blfl(ka/m 2) ALCOHOL TOBACCO FLUORINE ' (oz/day) (cigs/day) TB/FSH# TF/FSH* E/FSH** .07 -.43 -.16 P-.0001 P-.08 -.01 -.47 D-.0001 .04 -.36 p=.0001 .04 .07 .06 .08 .04 -.08 -.12 -.02 #Bound testosterone to follicle stimulating hormone ratio Free testosteroneto follicle stimulating hormone ratio Estradiol to follicle stimulating hormone ratio 98 3M MN03112290 TABLE 4.1.37 PEARSON CORRELATION COEFFICIENTS BETWEEN PITUITARY GLYCOPROTIEN HORMONE RATIOS AND TOTAL FLUORIDE, AGE, BODY MASS INDEX, ALCOHOL AND TOBACCO CONSUMPTION 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL AGE (years) BMI (kg/m2) ALCOHOL TOBACCO FLUORINE (oz/day) (clgs/day) TSH/FSH TSH/LH* F/LH+ .12 .09 -.05 -.16 p -,0 8 -.02 .28 P-.003 .04 .15 .13 M P-.01 21 Om.03 -.14 -.14 -.14 .05 Thyroid stimulating hormone to follide stimulating hormone ratio "Thyroid stimulating hormone to lutenizing hormone ratio Follicle stimulating hormone to lutenizing hormone ratio 99 3M MN03112291 TABLE 4.1.38 LINEAR MULTIVARIATE REGRESSION MODEL1 O F FACTORS PREDICTING THE BOUND-FREE TESTOSTERONE RATIO AMONG 112 MALE WORKERS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le ft SlflS ) p-vaiue Intercept Toted Fluorine (ppm)* Age (years) BMI (kg/m2) 36.60 .02 .19 -.48 6.87 .008 .101 .244 .0001 .02 .07 .05 LH+ .12 .337 .73 FSH@ .92 .440 .04 R2= .21 `square transformation of toted serum fluoride flutienizing hormone mU/ml @ follicle stimulating hormone mU/ml 100 3M MN03112292 TABLE 4.1.39 LINEAR MULTIVARIATE REGRESSION MODEL2 OF FACTORS PREDICTING THE BOUND-FREE TESTOSTERONE RATIO AMONG 112 MALE WORKERS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable S mm p-value Intercept Total Fluorine (ppm)* Age (years) BMI (kg/m2) 37.3 .02 .25 -.52 6.97 .009 .097 J250 .0001 .03 .009 .03 LH+ .55 271 .05 R2= .17 square transformation of total serum fluoride +luteinizing hormone mU/ml @ follicle stimulating hormone mU/ml 101 3M MN03112293 TABLE 4.1.40 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE ESTRADIOL-BOUND TESTOSTERONE RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B E (B ) p-value o cn Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Free Testosterone* LH+ .05 .00001 -.0004 .002 -.00001 .003 -.001 .0001 .027 .00001 .0004 .0007 .00002 .007 .0006 .0006 CO CO .74 .29 .006 .96 .008 .94 FSH@ -.002 .001 .12 TSH++ Prolactin** -.003 .0001 .003 .0005 .30 .78 R2 .2 1 #Reference category Is moderate drinkers who consume 1-3 oz ethanol/day. *ng/dl -flutelnizing hormone mU/ml @ follicle stimulating hormone mU/ml ++ Thyroid stimulating hormone (mU/ml) ** prolactin ng/ml 102 3M MN03112294 TABLE 4.1.41 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE ESTRADIOL-FREE TESTOSTERONE RATIO AMONG 112 |u a | p WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le B p-value Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Bound Testosterone* LH+ FSH TSH++ 1.31 .002 .012 .048 .005 .090 -.001 .012 -.059 -2 0 4 .880 .001 .011 .026 .008 .730 .0004 .035 .046 .110 .15 .03 .34 .07 51 .70 .01 .73 21 .05 Prolactin** .027 .018 .15 R 2 -2 2 #Reference category is moderate drinkers who consume 1-3 oz ethanoi/day. *ng/dl +lutienizing hormone mUAnl @ follicle stimulating hormone mU/ml ++ Thyroid stimulating hormone (mU/ml) ** prolactin ng/ml 103 3M MN03112295 TABLE 4.1.42 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING TH E ESTRAD10L-LH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B SI?S5 p-value Intercept Total Fluorine (ppm) 3.07 .02 3.58 .07 .39 .80 Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Free Testosterone* Bound Testosterone* FSH@ -.03 Z1 .009 .37 .13 .001 -.7 5 .05 .10 .03 .90 .10 .002 .15 .39 .008 .77 .68 2A .71 .0001 t s h -h Prolactin** -.39 .42 .35 -.03 .07 .72 R2= .34 +estradiol to iutenizing hormone (mU/ml) ratio #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * ng/dl follicle stimulating hormone mU/ml ++ Thyroid stimulating hormone (mU/ml) ** prolactin ng/ml 104 3M MN03112296 TABLE 4.1.43 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE BOUND TESTOSTERONE-LH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable 6 sew o-value Intercept 74.46 48.53 .13 Total Fluorine (ppm) 2 7 .98 .79 Age (years) 2 9 .62 .64 BMI (kg/m2) -.43 1.35 .75 Cigarettes/day -.15 .44 .73 Alcohol (<1oz/day)# 7.55 12.1 .54 Free Testosterone* 5.96 1.01 .0001 estradiol -2 8 .38 .45 f s h @@ -8.65 2.03 .0001 CO TSH++ 5.69 .97 Prolactin** .11 .95 .90 R2= .43 fbound testosterone to lutenlzing hormone (mUArrtl) ratio #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. *ng/dl @ pg/ml @@foDide stimulating hormone mU/mi ++ Thyroid stimulating hormone (mU/ml) ** prolactin ng/ml 105 3M MN03112297 TABLE 4.1.44 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE FREE TESTOSTERONE-LH+ RATIO AMONG 112 MALE WORKERS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B p-value Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Bound Testosterone* Estradiol f s h @@ TSH++ Prolactin** 3.00 -.05 .001 .07 -.007 .30 .003 .001 -.33 .18 -.05 1.38 .03 .01 .04 .01 .36 .0007 .01 .06 .17 .03 .03 .09 .91 .08 .58 .41 .0001 .91 .0001 .30 .08 R2= .46 +free testosterone to lutenizing hormone (mU/ml) ratio #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. @ pg/ml @@foliicle stimulating hormone mU/ml h-4- Thyroid Emulating hormone (mU/ml) ** prolactin ng/ml 106 3M MN03112298 TABLE 4.1.45 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE BOUND TESTOSTERONE-PROLACTIN RATIO AMONG 111 MALE WORKERS. 13M CHEMOLITE PLANT, COTTAGE GROVE. MINNESOTA Variable B SE(B) p-value Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Estradiol++ Free Testosterone * LH** FSH@ 60.05 -.15 .84 -1.54 1.49 13.9 -2 2 3.93 -2J23 -2.55 68.04 1.38 .88 1.92 .62 17.2 .53 1.45 2.63 3.50 .38 .91 .34 .42 .02 .42 .68 .008 .40 .47 o00 TSH+ -.95 .24 R2= .17 ++pg/ml #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * ng/dl ......... ** lutenizing hormone mU/ml @ follicle stimulating hormone mU/ml + Thyroid stimulating hormone mU/ml 107 3M MN03112299 TABLE 4.1.46 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE FREE TESTOSTERONE-PROLACTIN RATIO AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B E(B) p-value Intercept 2.41 1.76 .17 Total Fluorine (ppm) -.03 .04 .35 (OlO Age (years) -.004 .02 BMI (kg/m2) -.004 .05 .93 Cigarettes/day .04 .02 .03 Alcohol (<1 oz/day)# -.03 .76 .97 Estradiol4-*- -.0001 .01 .99 Bound Testosterone * .002 .0001 .03 LH** -.08 .07 .24 FSH@ -.12 .09 2 \ TSH+ -.18 .21 .40 R2= .15 -H-pg/ml #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * ng/dl ** lutenizing hormone mU/ml @ follicle stimulating hormone m ll/ml + Thyroid stimulating hormone mU/ml 108 3M MN03112300 TABLE 4.1.47 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE ESTRADIOL-PROLACTIN RATIO AMONG 111 MALE WORKERS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B SE(B) p-value intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Bound Testosterone* Free Testosterone * LH** FSH@ 2.65 .005 .01 .07 .10 .86 -.001 .12 -.13 -.29 4.01 .081 .05 .116 .036 1.01 .003 .12 .15 2i .51 .95 .80 .53 .005 .40 .95 .31 .39 .17 TSH+ -.67 .47 .16 R2= .16 #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * ng/di ** lutenizlng hormone mU/ml @ follicle stimulating hormone mU/ml + Thyroid stimulating hormone mU/ml 109 3M MN03112301 TABLE 4.1.48 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE PROLACTlN-FSH@ RATIO AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le 0 1 (1 ) p-value Intercept Total Fluorine (ppm) Alcohol # low (<1 oz/day) nonresponse (NR) low X Fluoride 2.56 .31 .81 .19 -.31 1.52 .11 .52 .85 .12 .09 .008 .13 .82 .01 NR X Fluoride Age (years) BMI (kg/m*) Cigarettes/day Estradiol++ -.08 .01 -.03 .02 too 29 .02 .04 .01 .01 .78 .01 .86 .03 .06 Bound Testosterone* Free Testosterone * LH** TSH+ -.001 .01 -.0 7 .31 .001 .04 .05 .17 .92 .75 .15 .07 r2 .31 ++pg/m l #Reference category Is moderate drinkers who consume 1-3 oz ethanol/day. ** lutenizing hormone m ll/m l @ follicle stimulating hormone m ll/m l + Thyroid stimulating hormone m ll/m l 110 3M MN03112302 TABLE 4.1.49 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE PROLACTIN-LH** RATIO AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT. COTTAGE GROVE, MINNESOTA Variable B E (B ) p-value Intercept Total Fluorine (ppm) Alcohol # low (<1oz/day) nonresponse (NR) low X Fluoride NR X Fluoride Age (years) BMI (kg/m2) Cigarettes/day Estradiol** Bound Testosterone* Free Testosterone * FSH@ 1.07 .34 .68 .41 -.35 -.39 -.02 .05 -.02 .003 .001 -.04 -.11 1.27 .09 .43 .69 .09 2.0 .01 .04 .01 .009 .0008 .03 .05 .38 .0003 .11 .55 .0004 .05 .12 .17 .09 .76 .17 .30 .03 TSH+ .15 .14 29 " fiO i **lutenizing hormone *+pg/m l ^Reference category is moderate drinkers who consume 1-3 oz ethanol/day. no/dl @ tolBde stimulating hormone m ll/m l + Thyroid stimulating hormone mU/ml 111 3M MN03112303 TABLE 4.1.50 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE PROLACTIN-TSH+ RATIO AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA variable ft ------ SE(B)-------- p-value Intercept Total Fluorine (ppm) Alcohol # low(<1oz/day) nonresponse (NR) low X Fluoride NR X Fluoride Age (years) BMI (kg/m2) Cigarettes/day Estradiol-*-*Bound Testosterone* Free Testosterone' FSH@ 5.06 1.61 4.16 3.85 -1.76 -2.11 -.19 .11 -.06 .03 .008 -.35 .51 4.83 .37 1.67 2.72 .38 .77 .06 .14 .04 .04 .003 .14 .20 .30 .0001 .01 .16 .0001 .008 .003 .43 .14 .46 .02 .01 .01 ++pg/m l # Reference category is moderate drinkers @ follicle stimulating hormone m ll/m l + Thyroid stimulating hormone m ll/m l consume 1-3 oz ethanol/day, 112 3M MN03112304 TABLE 4.1.51 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE BOUND TESTOSTERONE-TSH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B E<B) p-value Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Free Testosterone* Estradiol f s h @ LH++ Prolactin** 559.5 37.7 -1.1 -9.8 -.66 74.9 12.5 -1.6 47.1 5.7 -1.1 360.7 109.8 6.2 8.9 2.9 78.3 6.6 2.5 15.9 12.2 6.2 iono .12 .73 Z? .82 .34 .06 .51 .004 .64 .85 R2= .29 +bound testosterone to thyroid stimulating hormone (mU/ml) ratio #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * ng/dl @ pg/ml @@follicle stimulating hormone mU/ml ++ Thyroid stimulating hormone (mU/ml) ** prolactin ng/ml 113 3M MN03112305 TABLE 4.1.52 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE FREE TESTOSTERONE-TSH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le ft SE(B) D-value Intercept Total Ruorlne (ppm) Age (years) BMI (kg/m2) 15.65 -.28 -.29 -.01 6.34 .13 .08 .19 .02 .03 .003 .94 Cigarettes/day Alcohol (<1oz/day)# Bound Testosterone* Estradiol FSH@@ -.03 1.50 .01 -.01 .68 .06 1.64 .003 .05 .33 .65 .36 .006 .80 .04 LH++ Prolactin** -.001 -.18 .25 .99 .13 .17 R2= .37 +free testosterone to thyroid stimulating hormone (mU/ml) ratio #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * ng/di @ pg/m l @@foliide stimulating hormone mU/m++ Thyroid stimulating hormone (mU/ml) ** prolactin ng/ml 114 3M MN03112306 TABLE 4.1.53 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE ESTRADIOL-TSH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA V a ria b le ------------- S lB ) p-value Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Free Testosterone* Bound Testosterone* FSH@ LH++ Prolactin** 30.80 -.425 -.53 .32 .06 2.36 -.28 .009 .81 J20 -.0 7 16.10 .31 20 .46 .14 4.00 .46 .01 .81 .62 .32 .06 .18 .01 .50 .70 .55 .55 .42 .31 .75 .83 R2= .15 +estradiol to thyroid stimulating hormone (mU/mi) ratio #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. *ng/dl follicle stimulating hormone mU/ml ++ thyroid stimulating hormone (mU/ml) ** prolactin ng/ml 115 3M MN03112307 TABLE 4.1.54 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE BOUND TESTOSTERONE-FSH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le B 5BB5 p-value Intercept 101.89 61.25 .10 Total Fluorine (ppm) .66 124 .60 Age (years) -.13 .75 .14 BMI (kg/m2) -1.08 1.70 .53 Cigarettes/day Alcohol (<1oz/day)# -.37 .55 .29 15.30 .50 .98 Free Testosterone* 6.87 1 2 8 .0001 LH@@ -7.61 1.93 .0002 Estradiol .77 .47 .11 TSH++ 8.90 7.03 21 Prolactin** -.03 1 2 0 .97 R2= .50 -abound testosterone to follicle stimulating hormone (mU/ml) ratio #Referenc8 category Is moderate drinkers who consume 1-3 oz ethanol/day. *ng/dl @luteinizing hormone mU/ml @@estradiol pg/ml ++ Thyroid stimulating hormone (mU/ml) ** prolactin ng/ml TABLE 4.1.55 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE FREE TESTOSTERONE-FSH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable 0 SE(B) p-vaiue Intercept 4.31 2.01 .03 Total Ruorine (ppm) -.04 .04 2 7 Age (years) -.10 .02 .0001 BMI (kg/m2) .06 .06 2 8 Cigarettes/day -.02 .02 .31 Alcohol (<1oz/day)# .18 .52 .74 Bound Testosterone* .003 .001 .02 LH@@ -.25 .07 .0003 Estradiol .03 .04 2 7 TSH++ .49 2A .04 Prolactin** -.05 .04 2 3 R2= .43 +free testosterone to follicle stimulating hormone (mU/ml) ratio #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * ng/dl luteinizing hormone mU/ml @@estradiol pg/ml ++ Thyroid stimulating hormone (mU/ml) " prolactin ng/ml 117 3M MN03112309 TABLE 4.1.56 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE ESTRADIOL-FSH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le B SE(B) p-value Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Free Testosterone* Bound Testosterone* LH@ 6.91 .006 -.19 27 .03 .52 .26 -.002 -.49 5.69 .006 .07 .16 .05 1.42 .16 .004 .18 23 .34 .008 .10 .57 .71 .11 .62 .009 TSH++ .08 .65 .90 Prolactin** .04 .11 .70 R2= 2S +estradiol to follicle stimulating hormone (mU/ml) ratio #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * ng/dl luteinizing hormone mU/ml ++ Thyroid stimulating hormone (mU/ml) ** prolactin ng/ml 118 J 3M MN03112310 TABLE 4.1.57 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE BOUND TSH-FSH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA I Variable B Sfc(B) Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1oz/day)# Estradiol** Bound Testosterone* Free Testosterone* Prolactin** LH@ .72 .01 .002 .00007 -.003 -.16 -.001 -.0002 -.01 .002 -.03 .33 .006 .004 .01 .003 .08 .003 .0002 .009 .007 .01 .03 .14 .59 .94 .37 .05 .56 .28 .15 .73 .005 R2= .26 H-pg/ml #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. *ng/dl " prolactin ng/ml @ lutenlzing hormone mU/ml + thyroid stimulating hormone (mU/ml) to follicle stimulating hormone (mU/ml) ratio 119 3M MN03112311 TABLE 4.1.58 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE TSH-LH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE. MINNESOTA v a ria b le 5 SE(B) p-vaiue Intercept Total Fluorine (ppm) Age (years) BMI (kg/m2) Cigarettes/day Alcohol (<1 oz/day)# Estradiol** Bound Testosterone* Free Testosterone* Prolactin** FSH@ .32 .006 .004 .008 -.001 -.07 -.004 -.0001 .007 .001 -.05 25 .005 .003 .007 .002 .06 .002 .001 .007 .005 .01 21 21 26 27 .53 26 .07 .84 .32 .91 .0001 R2= 2 6 ++pg/ml #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. ng/dl ** prolactin ng/ml @ follicle stimulating hormone mU/mi + Thyroid stimulating hormone (mU/ml) to lutenizing hormone (mU/ml) ratio I 3M M N03112312 TABLE 4.1.59 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE BOUND LH-FSH+ RATIO AMONG 112 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B SE(B) p-value Intercept Total Fluorine (ppm) Age (years) BMI (kgAn2) Cigarettes/day Alcohol (<1oz/day)# Estradiol++ Bound Testosterone* Free Testosterone* Prolactin** TSH@ .60 -.0001 .009 .01 .0001 .04 .004 .0001 -.004 -.005 -.05 .43 .009 .005 .01 .004 .11 .003 .0002 .01 . .009 .05 .17 .98 .09 .40 .82 .71 .18 .18 .78 .57 .29 R2= .12 H-pg/ml #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. *ng/dl ** prolactin ng/ml @ thyroid stimulating hormone mU/ml + lutenizing hormone (mU/ml) to follide stimulating hormone (mU/ml) ratio 121 3M MN03112313 TABLE 4.1.60 PEARSON CORRELATION COEFFICIENTS BETWEEN TOTAL SERUM FLUORIDE, AGE, BODY MASS INDEX (BMI), DAILY ALCOHOL USE, DAILY TOBACCO CONSUMPTION, AND LIPOPROTEINS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLU O R ID E _________________________ iPPTM )___ AG E (years) CHOLESTEROL* .07 2S P -.008 BM I (kg/m z ) .19 D -.0S LDL** .02 .13 .0 6 HDLi -.01 .03 -.1 3 TRIG LYCERIDES* .09 mg/dl **tow density lipoprotein #high density lipoprotein .19 o .0 4 27 D -.004 ALCOHOL TOBACCO (o z/d ay) (ciga/d ay) .0 9 -.0 0 8 .1 8 P -.0 8 .0 7 .3 5 D -.0001 28 D -.002 -.0 9 .1 9 ____ P - 04 l 122 3M MN03112314 TABLE 4.1.61 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE CHOLESTEROL AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable 6 1(55 p-value Intercept Total Fluoride (ppm) Cigarettes/day BMI (kg/m2) Age (years) Alcohol # 107.30 .52 1.12 1.44 .77 33.00 .67 .31 1.01 .38 .002 .44 .0005 .16 .05 low (<1oz/day) -5.50 8.71 .53 nonresponse (NR) -13.53 14.75 .35 GOT (IU/dl)* .41 .12 .001 Bound Testosterone** .03 .02 R2=* 2.9 # Reference category is moderate drinkers who consume 1-3 oz ethanol/day. gamma glutamyl transferase .07 123 3M MN03112315 TABLE 4.1.62 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE LOW DENSITY LIPOPROTIEN AMONG 111 MALE WORKERS. 3M CHEMOLITE PUNT, COTTAGE GROVE, MINNESOTA v a ria b le B s e (B) p-value Intercept Total Fluoride (ppm) Cigarettes/day BMI (kg/m2) Age (years) Alcohol# low (<1 oz/day) nonresponse (NR) Prolactin (ng/ml) Bound Testosterone(ng/dl) 73.93 22 .69 1.26 .37 -3.02 -10.85 -1.59 .04 32.00 .65 .30 .95 .37 8.33 13.93 .66 .02 .03 .73 .02 .19 .32 .71 .43 .02 .0071 R *-1 9 ' ^Reference category is moderate drinkers who consume 1-3 oz ethanol/day. 124 3M MN03112316 TABLE 4.1.63 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE HIGH DENSITY LIPOPROTIEN (HDL) AMONG 111 MALE WORKERS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B----------- SEfBJ p-value intercept 65.00 10.07 .0001 Total Fluoride (ppm) -1.61 .77 .04 Alcohol # low (<1 oz/day) -9.92 3.51 .006 nonresponse (NR) -6.77 5.73 2A low X Fluoride 1.62 .80 .04 NR X Fluoride* 2.05 1.63 .21 Age (years) -.004 .12 .97 BMI (kg/m2) -.31 .29 .28 Cigarettes/day -.12 .09 .18 Bound Testosterone** .018 .007 .009 Free Testosterone** -.77 .28 R 2-.17 ^Reference category is moderate drinkers who consume 1-3 oz ethanol/day. interaction terms between total fluoride and alcohol category **n g /d l .008 125 3M MN03112317 TABLE 4.1.64 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE TRIGLYCERIDES AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE MINNESOTA variable ------------ g ------- S e <b> p-value Intercept -114.50 117.20 .33 Total Fluoride (ppm) 2.38 2.31 .15 Cigarettes/day 2.28 1.05 .03 BMI (kg/m2) 6.07 3.39 .08 Age (years) 2.32 1.44 .11 Alcohol # low (<1oz/day) -11.48 29.4 .70 nonresponse (NR) -19.94 49.03 .69 Free Testosterone* 7.34 3.37 .03 Bound Testosterone* -.21 .08 R2-.19 Reference category is moderate drinkers who consume 1-3 oz ethanol/day. ng/dl .009 126 3M MN03112318 TABLE 4.1.65 PEARSON CORRELATION COEFFICIENTS BETWEEN TOTAL SERUM FLUORIDE, AGE, BODY MASS INDEX (BMI), DAILY ALCOHOL USE, DAILY TOBACCO CONSUMPTION, AND HEPATIC PARAMETERS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA sG or TOTAL F L U O R IN E (ppm) .01 A G E (years) BM I (kg/m 2) -.10 .09 ALCOHOL TOBACCO (o z/d ay) (d g s /d a y ) .12 -.11 SGPT** .01 .01 20 D-.0 2 GGT* -.04 .12 27 Pb .004 AKPH** -.03 .27 .19 D s.004 P -.0 4 SERUM GLUTAMIC OXALOACETIC TRANSAMINASE lU/dl " SERUM GLUTAMIC PYRUVIC TRANSAMINASE lU/dl #GAMMA GLUTAMYL TRANSFERASE lU/dl ##ALKALINE PHOSPHATASE lU/dl .03 .15 -.19 Pb .05 -.11 .03 2B Dai.006 127 3M MN03112319 TABLE 4.1.66 PEARSON CORRELATION COEFFICIENTS BETWEEN HEPATIC ENZYMES, SERUM HORMONES, AND LIPOPROTEINS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA SgOY sgpt Gfff EKPIT CHOLESTEROL* LDL- .0 7 25 .1 9 D- . 0 0 8 Db .0 5 .0 9 .0 2 1 3 .0 6 -.008 HDL# TRIG LYCERIDES* E S T R A D IO L + FREE TESTOSTERONE" BOUND TESTOSTERONE" PRO LACTIN* *mg/dl **Iow density lipoprotein #high density l^oprotein pg/ml "ng/dl -.0 1 .0 9 -.1 6 D .0 9 -.1 2 -.1 6 D=>.09 20 P-.0 3 .0 3 .1 9 D- . 0 4 -.0 4 -.1 4 -.1 0 -.1 5 -.1 3 27 D- .0 0 4 JO3 -2 3 Do.0 1 -.1 2 -.1 6 P-.0 9 .1 8 D -.O S .0 7 -.0 0 3 -.0 3 -.1 2 -.2 0 p .0 3 128 3M MN03112320 TABLE 4.1.67 PEARSON CORRELATION COEFFICIENTS BETWEEN W PPATIf! PA RA M PTPQ C 3M CHEMOLITE PLANT. COTTAGE GROVE, MINNESOTA sGor SGPT** "ggP AKPH** SGOT 1 * SSPT .68 P -.0001 1 GGT .43 P -.0001 .6 0 P -.0001 1 m AKPH .0 4 .09 21 P -.0 2 1 SERUM GLUTAMIC OXALOACETIC TRANSAMINASE lU/dl -S E R U M GLUTAMIC PYRUVIC TRANSAMINASE lU /dl GAMMA GLUTAMYL TRANSFERASE lU/dl ##ALKAUNE PHOSPHATASE lU/dl 129 3M MN03112321 TABLE 4.1.68 SERUM GLUTAMIC OXALOACETIC TRANSAMINASE (S G O T), GLUTAMIC PYRUVIC TRANSAMINASE (SGPT),GAMMA GLUTAMYL TRANSFERASE (GGT), AND ALKALINE PHOSPHATASE (AKPH) BY TOTAL SERUM FLUORINE 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL flu o r in e <1 ppm >1-3 >3-10 >10-15 >15-26 TOTAL N 23 65 16 6 5 115 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 65 16 6 5 115 <1 ppm >=1-3 >3-10 >10-15 >15-26 TOTAL 23 65 16 6 5 115 <1 ppm >=1-3 >3-10 >10-15 >15-26 TOTAL 23 65 16 6 5 115 #univariate Anova MEAN SD MEDIAN RANGE 22.5 24.1 25.8 25.7 222. 24.0 SGOT (IU/dl) 4.1 8.6 14.5 11.3 5.1 8.9 22 23 222 222 22 23 13-29 10-74 17-77 17-47 14-27 10-77 SGPT (IU/dl) 47.7 10.7 46 30-69 51.3 3 02 45 4-263 53.0 14.0 5 02 29-40 73.2 532 5 22 38-177 44.6 8.6 42 34-54 51.7 26.8 47 4-263 Alkaline Phosphatase (IU/dl) 86.1 25.6 85 85.9 19.9 80 77.9 202 7 1 2 872 34.0 752 89.0 42.1 84 83.3 2 22 80 43-153 38-137 54-123 61-153 41-153 38-153 GGT (IU /d l) 372 29.4 27 6-117 32.4 26.7 25 5-174 35.4 35.4 26 10-158 38.3 16.7 362 19-80 22.2 112 20 11-37 33.7 27.6 26 5-174 TEST# P-0.41 P -.80 F -1 .1 9 p -2 2 F -0 .4 3 P -.78 P -0 .3 9 P-.81 130 3M MN03112322 TABLE 4.1.69 SERUM GLUTAMIC OXALOACETIC TRANSAMINASE (SGOT) BY BODY MASS INDEX AGE, SMOKING AND DRINKING STATUS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA N (% ) SGOT (lU/dl) MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 4 1 (3 5 .7 ) 5 7 (4 9 .6 ) 17(14.8) AGE <30 31-40 41-50 51-60 2 1 (1 8 .3 ) 4 8 (4 1 .7 ) 2 7 (2 3 5 ) 1 9 (1 6 5 ) Alcohol <1oz/d 1-3oz/d missing 8 7 (8 1 5 ) 2 0 (1 8 .7 ) 8 Tobacco smoker nonsmoker missing 2 8 (2 4 .8 ) 8 5 (7 5 5 ) 2 TOTAL 115 #univariate Anova 24 23 27 25 24 22 26 26 24 23 24 24 20 12.4 55 8.1 12.7 9.1 5 .4 75 135 8.0 45 8 .4 115 35 22 13-77 F -.S 2 23 10-42 P -.40 S3 17-47 23 17-77 F -.7 8 23 10-74 P-.51 23 13-40 23 14-47 22 16-77 F -5 1 23 10-74 P -.44 21 19-31 23 13-77 F -.0 2 22 1 0 -4 2 p59 20 17-47 131 3M MN03112323 TABLE 4.1.70 SERUM GLUTAMIC PYRUVIC TRANSAMINASE (SGPT) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA N (% ) SGPT(lU/dl) MEAN SD MEDIAN RANGE TEST BMI <25 25-30 >30 AGE <30 31-40 41-50 51-60 A lc o h o l <1oz/d 1-3oz/d missing 41(35.7) 57(49.6) 17(14.8) 21(18.3) 48(41.7) 27(23.5) 19(165) 87(813) 20(18.7) 8 49 50 64 49 53 47 57 53 47 51 35.4 143 32.8 113 33.6 153 32.0 2935 16.9 10.9 41 49 55 45 47 46 50 47 46 52 29-263 4-95 38-177 31-80 29-263 4-99 34-177 29-263 4-99 35-67 F-2.1 P-.12 F-.61 P-..61 F-.68 P-..41 Tobacco smoker nonsmoker missing 28(24.8) 85(753) 2 TOTAL 115 48 53 49 153 47 29.6 48 253 49 4-90 30-263 31-67 F-.78 P -.3 9 iunivariate Anova 132 3M MN03112324 TABLE 4.1.71 GAMMA GLUTAMYL TRANSFERASE (GGT) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA GGT (lU /dl) N(% ) _ MEAN SD M EDIAN RANG E TEST# BMI <25 25-30 >30 AGE <30 31-40 41-50 51-50 A lc o h o l <1oz/d 1-3oz/d missing Tobacco smoker nonsmoker missing TOTAL 41(35.7) 57(49.6) 17(14.8) 21(18.3) 48(41.7) 27(233) 19(16.5) 87(81.3) 20(18.7) 8 28(24.8) 85(75.2) 2 115 28 34 48 32 31 33 44 40 32 41 36 32 85 31.1 23.1 28.6 23.4 32.7 17.2 233 253 253 50.4 213 263 1033 17 5-174 F-3.54 19 8-158 P-.03 44 19-117 25 11-111 - F-158 22 5-174 P-.36 29 8-72 35 11-117 35 8-89 F-1.64 26 6-174 P-.36 23 12-158 33 5-89 F-.55 25 6-174 pa.46 85 12-158 #univariate Anova 133 3M MN03112325 TABLE 4.1.72 ALKALINE PHOSPHATASE (AKPH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 3M CHEMOUTE P U N T , COTTAGE GROVE, MINNESOTA N(% ) a Kp H (lu /d iT MEAN SD M EDIAN RANG E TEST# BMI <25 25-30 >30 AGE <30 31-40 41-50 51-60 A lc o h o l <1oz/d 1-3oz/d missing Tobacco smoker nortsmoker missing TOTAL 41(35.7) 57(49.6) 17(145) 21(185) 48(41.7) 27(23.5) 19(16.5) 87(815) 20(18.7) 8 28(24.8) 85(755) 2 115 79 84 90 78 80 86 95 85 77 82 85 77 86 22.1 21.9 27.1 225 205 24.1 24.1 24.0 16.9 22.5 23.8 22.0 24.8 75 38-153 F-153 81 41-153 p*.22 90 43-153 76 38-153 F-2.78 76 50-15. P-.45 83 43-153 94 41-130 2 38-153 F-2.05 75 51-124 P-.16 70 60-115 85 61-153 F-6.48 77 38-153 P-.012 86 68-103 #univariate Anova 134 3M MN03112326 TABLE 4 .1 .73A UNEAR M ULTIVARIATE REG RESSIO N M ODEL 1 O F FACTORS PREDICTING TH E SERUM GLUTAM IC OXALOACETIC TRANSAM INASE (SG O T) AMONG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, COTTAG E G RO VE, M INNESO TA V a ria b le B p -v a lu e Intercept Total Fluorine (ppm) BMI (kg/m2) 26.71 -3 .2 3 -.0 0 0 4 7.1 1.31 .23 .0 0 0 3 .02 .99 BMI X T . Fluorine* Age (years) Alcohol # low (<1oz/day) nonresponse (NR) Cigarettes/day Prolactin (ng/ml) .1 2 -.0 0 3 .7 0 -1 .1 0 -.0 9 -.3 7 .05 .0 8 1.85 3 .1 0 .0 7 .1 5 .0 1 5 .9 7 .71 .7 2 .16 .01 f 1* * f ^Reference category tomoderatedrinkem whoconsume 1-3 oz ethanol/day. j * interaction term between total serumfluoride and BMI. 135 3M MN03112327 TABLE 4 .1 .73B LINEAR MULTIVARIATE REG RESSIO N M O DEL 2 O F FACTORS PREDICTING TH E SERUM GLUTAMIC OXALOACETIC TRANSAM INASE (SG O T) AMONG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, CO TTAG E G R O VE, M INNESO TA V a ria b le B s E fln p-value Intercept Total Fluorine (ppm) 27.71 -2 .7 0 6 .2 2 1.23 .0001 .02 BMI (kg/m2) -.09 .06 .11 BMI X T . Fluorine* .10 .04 .02 Age (years) Cigarettes/day Alcohol # -.02 .07 .74 -.11 .06 .11 low (<1oz/day) 1.84 1.61 28 nonresponse (NR) -1 .3 2.7 .64 Prolactin (ng/ml) -2 7 .13 .04 GGT (IU/dl)** .13 .02 .0001 R2- .35 #Referenoe category tomoderate drinkerswhoconsume 1-3 oz ethanol/day. * Interaction term between totalserum fluoride and BMI ** Gammaglutamyl transferase 136 3M MN03112328 TABLE 4 .1 .73C LINEAR M ULTIVARIATE REG RESSIO N M O DEL 3 O F FACTORS PREDICTING TH E SERUM GLUTAM IC OXALOACETIC TRANSAM INASE (SG O T) AMONG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, COTTAG E G R O VE, M INNESO TA v a ria b le s -------- H 5) p -v a lu e Intercept Total Fluorine (ppm) BMI (kg/m2) BMI X T . Fluorine* Age (years) Cigarettes/day 120.60 .6 3 -.0 7 -.0 3 .0 6 -.0 2 4.0 .002 .78 .42 .13 .58 .03 .34 .05 .23 .04 .45 Alcohol # low (<1 oz/day) nonresponse (NR) Prolactin (ng/ml) SGPT (IU /dl)** -.6 5 -1.40 -.0 9 2A 1.03 1-72 .0 8 .01 .53 .42 .29 .0001 R2- .74 #Referencecategory is moderatedrinkers whoconsume 1-3 oz ethanoi/day. * Interaction term between total serum fluoride and BMI ** Serum glutamicpyruvictransaminase 137 3M MN03112329 TABLE 4.1.74A LINEAR M ULTIVARIATE REG RESSIO N M ODEL 1 O F FACTORS PREDICTING TH E SERUM GLUTAM IC PYRUVIC TRANSAM INASE (SG PT) AMONG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, COTTAG E G R O VE, M INNESO TA variab le B S E (B ) p -v a lu e Intercept 58.13 2 4 .2 6 .0 2 Total Fluorine (ppm) -1 5 .8 0 4 .5 8 .0 0 0 8 BMI (kg/im2) .30 .82 .72 BMI X T . Fluorine* Age (years) .62 .17 .0004 -24 28 .39 Alcohol # low (<1oz/day) 5 .5 4 6.36 .3 9 nonresponse (NR) Cigarettes/day 1.31 -2 7 10.63 .2 3 .90 24 Prolactin (ng/hnl) -g -_ -1 .1 8 .51 .02 #Reference category is moderate drinkerswhoconsume 1*3 oz ethanol/day. * interaction term between total serum fluoride and BMI. 138 3M MN03112330 TABLE 4.1.74B LINEAR MULTIVARIATE REGRESSION MODEL 2 OF FACTORS PREDICTING THE SERUM GLUTAMIC PYRUVIC TRANSAMINASE (SGPT) AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA V a ria b le B S e (B) p -v a lu e Intercept Total Fluoride (ppm) BMI (kg/m2) BMI X T . Fluorine* Age (years) Cigarettes/day A lc o h o l# 62.09 -1 3 .7 0 -.7 0 .5 4 -.3 3 -.0 2 7 19.63 3 .6 4 .6 6 .1 4 22 .1 8 .0 0 2 .0 0 0 3 .3 0 .0001 .14 .1 4 low (<1oz/day) 10.02 5 .0 9 .0 5 nonresponse (NR) .48 8 .4 4 .9 5 Prolactin (ng/ml) -.7 4 .41 .07 GGT (IU/dl)** .56 .07 .0001 R2-51 #Referenoe category is moderatedrinkers whoconsume 1-3oz ethanol/day. * Interaction term between total serum fluoride arte BMI ** Gammaglutamyl transferase 139 3M MN03112331 TABLE 4 .1 .74C LINEAR M ULTIVARIATE REG RESSIO N M O DEL 3 O F FACTORS PREDICTING TH E SERUM GLUTAM IC PYRUVIC TRANSAM INASE (SG PT) AM O NG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, CO TTAG E G RO VE, M INNESO TA v a ria b le ft S B fft) o -v a lu e Intercept Total Fluorine (ppm) -1 8 .2 5 -6 .6 5 14.36 2.61 2\ .01 BMI (kg/m2) BMI X T . Fluorine* Age (years) Cigarettes/day Alcohol # .30 27 -2 3 -.001 .45 .51 .10 .007 .16 .14 .13 .99 low (<1oz/day) 3 .5 5 3 .5 3 .3 2 nonresponse (NR) 4 .3 9 5.91 .4 6 Prolactin (ng/ml) -.11 2 9 .72 S G O T (IU /d ir 2 .8 5 .19 .0001 R2- .75 " fReference category is moderate drinkers whoconsume 1*3 oz ethanol/day. * Interaction term between total serum fluoride and BMI ** serum glutamic oxaloacetictransaminase 140 i 3M MN03112332 TABLE 4 .1 .75A UNEAR MULTIVARIATE REG RESSIO N M ODEL 1 O F FACTORS PREDICTING TH E GAMMA GLUTAMYL TRANSFERASE (G G T) AM ONG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, COTTAG E G RO VE, M INNESO TA V a ria b le 6 p -v a lu e Intercept -12.59 2 2 .6 2 .5 8 Total Fluorine (ppm) -1.93 2.11 .36 Alcohol# low (<1oz/day) -1 2 .3 7 9 .5 0 .20 nonresponse (NR) -2 8 .1 3 15.46 .07 low X Fluorine* 1.59 2 .1 8 .47 NR X Fluorine* 13.90 4 .4 8 .0 0 3 o CO CO Age (years) .29 .33 BMI (kg/m2) 1.71 .03 Cigarettes/day .09 2 4 .72 F&..18 #Referencecategory is moderate drinkerswhoconsume 1-3 oz ethanol/day. Interaction terms between total fluoride end alcohol category 141 3M MN03112333 TABLE 4.1.75B LINEAR MULTIVARIATE REGRESSION MODEL 2 OF FACTORS PREDICTING THE GAMMA GLUTAMYL TRANSFERASE (GGT) AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT. COTTAGE GROVE. MINNESOTA V a ria b le 6 SE iB i D -v a lu e Intercept n Total Fluoride (ppm) -5 8 .7 8 -1 .7 9 2 1 .5 5 1.83 .0 0 8 .3 3 Alcohol # low (<1 oz/day) -9 .0 4 8 .2 5 .2 8 nonresponse (NR) -2 0 .0 8 13.49 .1 4 low X Fluorine* 1.39 1.90 .4 7 NR X Fluorine* 12.18 3.91 .0 0 2 Age (years) .15 .26 .57 BMI (kg/m2) 1.30 .66 .05 Cigarettes/day .01 .23 .96 Cholesterol (mg/dl) SGOT (lU/dl) .1 5 1.18 .06 .02 2 4 .0001 R2=.38 ^Reference category is moderate drinkBrswhoconsume 1-3oz ethanol/day. interaction terms between total fluoride and alcoholcategory 142 3M MN03112334 TABLE 4 .1 .75C UNEAR M ULTIVARIATE REG RESSIO N M O DEL 3 O F FACTORS PREDICTING TH E GAMMA GLUTAMYL TRANSFERASE (GG T) AMONG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, COTTAG E G R O VE, M INNESO TA v a ria b le ft -- 5 H B )-- p -v a lu e Intercept -32.39 18.47 .0 8 Total Fluorine (ppm) -1 .6 3 1.60 .31 Alcohol # low (<1 oz/day) -13.58 7 .1 7 .0 6 nonresponse (NR) -26.68 11.75 .025 low X Fluorine* .92 1.66 .58 NR X Fluorine* 1 2 .0 4 3.41 .0 0 0 6 Age (years) 25 23 27 BMI (kg/m2) .51 .59 .38 Cigarettes/day .09 2 0 .65 Cholesterol (mg/dl) .12 .06 .04 SGPT (IU/cB)** .59 .07 .0001 R2 .53 ^Reference category is moderatedrinkers whoconsume 1-3 oz ethanol/day. interaction terms between total fluoride ami alcohol category ** serumglutamic pyruvictransaminase 143 3M MN03112335 TABLE 4.1.76 LINEAR MULTIVARIATE REGRESSION MODEL 1 OF FACTORS PREDICTING THE ALKALINE PHOSPHATASE (AKPH) AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le B -- sm -- p -v a lu e Intercept Total Fluorine (ppm) Cigarettes/day Cigarettes/day X Fluorine* BMI (kg/m2) Age (years) Alcohol # low (<1 oz/day) nonresponse (NR) 2 4 .5 0 -1 .0 3 -.0 6 22 1.10 .54 5 .7 8 8 .1 2 15.69 .4 3 22 .0 5 JS5 22 4 .9 0 8 .1 3 .0 9 .0 2 .7 9 .0001 .0 5 .0 2 24 .32 R2- .31 dReferencecategory is moderatedrinkerswhoconsume 1-3 oz ethanol/day. * interactionterm between total serum fluoride and dgarettes/day. 144 3M MN03112336 TABLE 4 .1 .7 7 PEARSON CORRELATION C O EFFIC IEN TS BETW EEN TOTAL SERUM FLUO RIDE, AGE, BODY M ASS INDEX (BM I), DAILY ALCOHOL USE, DAILY TO BACCO CO NSUM PTIO N, AND HEM ATOLOGY PARAM ETERS 3M CHEM O LITE PLANT, COTTAG E G RO VE, M INNESO TA HEMAGLOBIN* WBC** PMN COUNT4EOSINOPHILS LYMPHOCYTES MONOCYTES PLATLETS BASOPHILS TOTAL FLUORINE tow ") -.07 .10 .05 -.10 .19 D.04 .05 .10 .04 AGE (years) BMI fka/m2} -.03 .04 .07 .07 .08 .09 .13 .05 -.05 .04 .04 -22 D=.02 -.13 -.11 -.08 -.02 ALCOHOL TOBACCO (oz/day) (dgs/day) 20 D-.04 -.07 -.10 .02 .15 -21 Da.03 .05 20 D-.008 .70 D-.0001 .64 D-.0001 23 Db.003 28 D-.002 J32 D=>.0004 29 P-.002 -.14 -.05 BANDS *g/dl white bloodceil count polymorphonuclearleukocyte count 26 C-.005 -.14 145 3M MN03112337 TABLE 4.1.78 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE HEMAGLOBIN AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA v a ria b le B S E (fi) . p -v a lu e intercept Total Fluorine (ppm)* 14.51 -.0 0 2 .6 7 .0 0 0 9 .0001 .0 2 Alcohol# low (<1 oz/day) 22 20 27 nonresponse (NR) Age (years) BMI (kg/m2) Cigarettes/day .56 .001 .01 .01 .3 3 .009 .02 .0 0 7 .0 9 .8 8 .6 5 .2 0 Clgs/day X Fluorine2** .0 0 0 3 .0001 .0 0 0 5 Estradiol (pg/ml) .01 .006 .0 7 R^-23 'square transformation oftotal fluoride iReference category is moderatedrinkerswhoconsume 1-3 oz ethanol/day. ** interactionterm between cigarettes per day and square transformation of total fluoride 146 3M MN03112338 TABLE 4.1.79 LINEAR M ULTIVARIATE REGRESSIO N M ODEL O F FACTORS PREDICTING TH E MEAN CORPUSCULAR HEMOBLOBIN (M CH) AM ONG 111 MALE WORKERS 3M CHEM O LITE PLANT, COTTAG E G R O VE, M INNESO TA v a ria b le 8 p -v a lu e Intercept 31.65 .95 .0001 Total Fluorine (ppm) .15 .09 .10 Alcohol # low (<1oz/day) -2.9 .65 .65 nonresponse (NR) .0 3 .01 .02 low X Fluorine -.1 6 .09 .08 NR X Fluorine -.0 4 .19 .80 Age (years) .03 .01 .02 BMI (kg/m2) -.07 .03 .01 Cigarettes/day .02 .01 .13 Cigs/day X Fluorine* .006 .003 .03 R2-J24 #Reference category is moderatedrinkers whoconsume 1-8 oz ethanol/day. ` interactionterms; alcoholcategory by total fluoride,cigarettes perday by total fluoride 147 3M MN03112339 TABLE 4.1.1.80 LINEAR MULTIVARIATE REGRESSION MODEL O F FACTORS PREDICTING THE MEAN CORPUSCULAR VOLUME (MCV) AMONG 111 MALE WORKERS. 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA Variable B sa M B S B a B a aB a B m n m&i p-vaiue E S 9B B IB H H II^^R 9B S B S I^E & S S S S S S R H E M Intercept 8 .7 4 2 .5 0 .0001 Total Fluorine (ppm) -.0 4 .07 .52 Alcohol # low (<1oz/day) -.61 .78 .43 nonresponse (NR) -.9 5 1.27 .46 Age (years) .11 .03 .002 BMI (kg/tn2) -.06 .08 .05 Cigarettes/day .04 .03 .21 Cigs/day X Fluorine* .0 2 .007 .0 0 4 TSH (mU/ml) .38 .35 2 9 R2- 2B #Raferencecategory is moderate drinkers who consume 1-3 oz ethanol/day. * interaction term; cigarettes perday bytotal fluoride 148 3M MN03112340 TABLE 4.1.81 LINEAR MULTIVARIATE REG RESSIO N M O DEL O F FACTORS PREDICTING TH E W H ITE BLOOD CELL C O U N T (W BC)* AMONG 111 MALE WORKERS 3M CHEM O LITE PLANT, COTTAG E G RO VE, M INNESO TA variable 5 5Efl3l p-value Intercept Total Ruorine (ppm) Alcohol# low (<1 oz/day) 2 .8 7 .07 .4 4 1.32 .10 .46 .03 .4 9 .3 3 nonresponse (NR) low X Ruorine NR X Ruorine Age (years) -1 .0 8 -.0 4 39 -.0 0 7 .7 4 .1 0 .21 .02 .1 5 .68 .0 0 6 .6 4 BMI (kg/m2) .07 .04 .05 Cigarettes/day Free Testosterone(ng/dl) LH@ .1 3 .0 4 .1 0 .01 .0001 .03 .13 .04 .02 R2- .67 WBC/1000 #Referencecategory is moderatedrinkers whoconsume 1-3 oz ethanol/day. @ lutenizing hormone mli/rnl 149 3M MN03112341 TABLE 4.1.82 LINEAR M ULTIVARIATE REG RESSIO N M O DEL O F FACTORS PREDICTING T H E POLYMORPHONUCLEAR LEUKOCUTE C O U N T (POLY) AM ONG 111 MALE W ORKERS. 3M CHEM O LITE PLANT, CO TTAG E G R O VE, M INNESO TA Intercept 368 1151 .75 Total Fluorine (ppm) 165 88 .06 Alcohol # low (<1oz/day) 746 399 .06 nonresponse (NR) 49 651 .94 low X Fluorine -161 90 .08 NR X Fluorine 370 185 .05 Age (years) 6 14 .66 BMI (kg/m2) 45 33 .17 Cigarettes/day 95 10 .0001 LH (mU/ml)++ 79 36 .03 Bound Testosterone* -1 .6 2 .8 .04 Free Testosterone * 84 3 2 .01 T t t ------------------------------------------------------------------------------------- + Lutanizing hormond Reference category is moderate drinkers whoconsume 1-3 oz ethanol/day. *ng/dl 150 3M MN03112342 TABLE 4.1.83 LINEAR MULTIVARIATE REGRESSION M ODEL O F FACTORS PREDICTING TH E BAND C O U N T (BAND) AM ONG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, COTTAGE G R O VE, M INNESO TA V a ria b le B p -v a lu e Intercept Total Fluorine (ppm) Alcohol# low (<1 oz/day) nonresponse (NR) Age (years) BMI (kg/m2) -1 1 .4 -3 .4 7 8 .2 14.9 1.0 2 .2 129.6 3 .2 4 0 .3 6 7 .8 1 .8 4 .6 .9 3 .30 .0 5 .83 .56 .63 Cigarettes/day 4.2 1.5 .005 R2* .12 Reference category is moderate drinkers whoconsume 1-3 oz ethanol/day. 151 3M MN03112343 TABLE 4.1 .8 4 LINEAR M ULTIVARIATE REGRESSIO N M O DEL O F FACTORS PREDICTING TH E LYM PHO CYTE C O U N T (LYM PH) AM ONG 111 MALE WORKERS. 3M CHEM O LITE PLANT, CO TTAG E G RO VE, M IN N ESO TA Intercept Total Fluorine (ppm) 2205.6 -3 4 2 .7 611.1 125.3 .0 0 0 5 .007 Alcohol # low (<1 oz/day) -5 2 6 .6 2 2 2 .7 .02 nonresponse (NR) -977.1 355.7 .0 0 7 low X Fluorine 189.0 5 2 .3 .0 0 0 5 NR X Fluorine 247.9 103.9 .0 2 Cigarettes/day 3 4 .0 6.9 .0001 Cigs/day X Fluorine* -3 .3 1.45 .0 2 BMI (kg/m2) 1.58 19.6 .94 BMI X Fluorine* 7 .1 5 4.1 .08 CoO e Age (years) -16.1 8 .6 Prolactin (ng/ml) TSH (mU/ml)+ 3 8 .5 170.4 14.2 772 .008 .0 3 ^Reference category is moderatedrinkerswhoconsume 1-3 oz ethanal/day. interaction terms alcoholcategory by total fluoride; cigarettes/day by total fluoride, BMI bytotal fluoride. thyroidstimulating hormone mUftnl 152 3M MN03112344 TABLE 4.1.85 LINEAR MULTIVARIATE REGRESSIO N M ODEL O F FACTORS PREDICTING TH E M O NO CYTE C O U N T (M O NO ) AM ONG 111 MALE WORKERS. 3M CHEM O LITE PLANT, COTTAG E G R O VE, M INNESO TA variable 5 m n) p-value Intercept Total Fluorine (ppm) Alcohol # low (<1oz/day) 397.4 110.4 132.1 198.9 3 8 .6 5 3 .8 .0 5 .005 .0 2 nonresponse (NR) Age (years) BMI (kg/m2) BMI X Fluorine* Cigarettes/day LH@ 40.1 -.3 7 -2.66 -4 .0 7 .0 13.9 89.1 2 .4 7 .0 1.42 1.9 6.8 .6 6 .88 .70 .0 0 6 .0 0 0 4 .04 R 2.30 #Reference category is moderatedrinkers who consume 1-3 oz ethanol/day. interaction term, BMI bytotal fluoride. @lutenizing hormone mU/ml 153 3M _ M N 0 3 112345 TABLE 4.1.86 LINEAR M ULTIVARIATE REGRESSIO N M O DEL O F FACTORS PREDICTING TH E EO SINO PHIL C O U N T (EO S) AMONG 111 MALE W ORKERS. 3M CHEM O LITE PLANT, CO TTAG E G R O VE, M INNESO TA V ariable B S E (B ) p -v a lu e intercept Total Fluorine (ppm) A lc o h o l# low (<1 oz/day) nonresponse (NR) Age (years) BMI (kg/m2) Clgarettes/day Cigs/day X Fluorine* TSH@ 50.45 -7.31 -1 2 .1 0 2 1 .7 9 1.56 2 .1 0 3 .0 4 .62 30.1 122.30 3.35 * 37.91 6 2 .2 5 1.67 4 .1 3 1.69 .35 17.1 .68 .03 .7 5 .73 .35 .61 .0 8 .0 8 .0 8 #Reference category is moderate drinkers who consume 1-3 oz ethanol/day. * interaction term, cigarettes per dayby total fluoride @Thyroidstimulating hormone mU/mi 154 3M MN03112346 TABLE 4.1.87 LINEAR MULTIVARIATE REGRESSIO N M ODEL O F FACTORS PREDICTING TH E PLATELET C O U N T (PLATE) AM ONG 111 MALE WORKERS. 3M CHEM O LITE PLANT, COTTAG E G R O VE, M INNESO TA variable B S E (B ) D-value Intercept Total Fluorine (ppm) Alcohol # low (<1oz/day) nonresponse (NR) Age (years) BMI (kg/m2) B M jX Fluorine* Cigarettes/day Cigs/day X Fluorine* Prolactin (ng/mi) Bound Testosterone** 264.7 29.8 8.2 .9 -1 .3 1.1 -1 .0 2 .7 -.3 2 .6 -.0 4 54.8 9 .5 13.3 2 2 .5 .6 1.7 .4 .6 .1 .0 3 .0 3 .0001 .0 0 2 .5 4 .9 7 .04 .5 3 .0 0 4 .0001 .0 4 .0 9 .1 0 R2,j28 #Reference category is moderate drinkers whoconsume 1-3 oz ethanol/day. Interaction terms, BMI by total fluoride, cigarettes perday by total fluoride, "ng/dl 155 3 M _ M N 0 3 1 12347 TABLE 4.1 .8 8 LINEAR MULTIVARIATE REG RESSIO N M ODEL O F FACTORS PREDICTING TH E BASOPHIL C O U N T (BASO) AM ONG 111 MALE W O RKERS. 3M CHEM O LITE PLANT, COTTAG E G R O VE, M INNESO TA Intercept 4 4 .3 5 54.19 .4 2 Total Ruorlne (ppm)* -.0 3 .06 .61 Alcohol # low (<1oz/day) nonresponse (NR) -1 .7 3 -.5 6 13.61 2 2 .5 4 .9 0 .81 Age (years) BMI (kg/fn2) Cigarettes/day Clgs/day X Ruorine2** Bound Testosterone## -.0 7 -.61 .0 2 -.0 7 0o0 .6 8 1.58 .52 .007 .0 4 .9 2 .70 .12 .0 0 7 .06 Free Testosterone## LH@ 2 .5 5.5 1.6 .11 1.8 .002 R^ .17 `squaretransformation oftotal serum fluoride #Reference category is moderate drinkerswho consume 1-8 oz ethanol/day. Interaction term, cigarettes per day bytotal fluoride. ##ng/dl @ lutenizing hormone mU/ml 156 3M MN03112348 t 4 Mortality Tables TABLE 4.2.1 CHARACTERISTICS O F 749 FEMALE EM PLO YEES, 1947-1989. Chemical Division Non chemical Division Total number of workers 245 504 749 person years of observation mean follow-up (years) mean age at employment (years) mean year of employment (years) mean year of death (years) mean age at death (years) 6029.0 2 4 .6 28.8 1965.0 1981.3 5 8 .7 13280.4 2 6 .4 2 6 .9 1962.8 1979.2 5 4 .4 1 9 3 0 9 .4 2 5 .8 2 7 .6 1963.5 1979.6 5 5 .4 157 3M MN03112349 TABLE 4 .2.2 CHARACTERISTICS O F 2788 MALE EM PLO YEES, 1947-1990. C hem ical Division Non chemical Division Total number of workers 1339 1449 2788 person years of observation mean follow-up (years) mean age at employment (years) mean year of employment (years) mean year of death (years) mean age at death (years) 33385.3 2 4 .8 2 5 .6 1963.8 1978.3 54.2 3 7 7 3 2 .4 2 6 .0 2 8 .9 1962.3 1978.1 58.1 7 1 1 1 7 .7 2 5 .5 2 7 .3 1963.0 1978.2 5 6 .4 158 3M MN03112350 TABLE 4 .2.3 VITAL STATUS AND CAUSE O F DEATH ASCERTAINM ENT AMONG 749 FEMALE EMPLOYEES, 1947-1990. Vital status Chemical Division No. % Non chemical Division No. % Total No. % Alive Dead* 234 95.3 11 4 .7 465 91.6 39 8.4 699 93.3 50 6.7 Total 245 100.0 504 100.0 749 100 tw o deaths occurred outside the U.S. with cause of death ascertained from sources other than death certificates. TABLE 4 .2 .4 VITAL STATUS AND CAUSE O F DEATH ASCERTAINM ENT AMONG 2788 MALE EMPLOYEES, 1947-1989. Vital status Alive Dead* Chemical Division No. % 1191 88.9 148 11.1 Non chemical Division No. % 1249 86.2 200 13.8 Total No. 2440 348 % 8 7 .5 12.5 Total 1339 100.0 1449 100.0 2788 100.0 *two deaths occurred outside the U.S. with cause of death ascertained from sources other than death certificates. 159 3M MN03112351 TABLE 4.2.5 NUM BERS O F DEATHS AND STANDARDIZED M O RTALITY RATIOS (SM Rs) AMONG 749 FEMALE EM PLO YEES, 1947-1989. Cause of Death -------5 5 1 -- --------Ex p ---------" g m -- 95% Cl All causes 50 66.74 Cancer 17 23.04 Gastrointestinal 2 4 .5 4 Respiratory 4 4.72 Breast 3 5.87 Genital 2 3.37 Lymphopoietic 3 2.04 He'art'disease 10 12.39 Cerebrovascular 3 3.51 Gastrointestinal 3 3.41 Injuries 4 6.23 Suicide 1 1.78 .75 .71 .44 .95 .51 .59 1.47 .81 .86 .88 .64 .56 .5 6 -.9 9 .4 2 -1 .1 4 .0 5 -1 .5 9 .2 6 -2 .4 3 .1 0 -1 .4 9 .0 7 -2 .1 4 .3 0 -4 .2 9 .4 9 -1 2 9 .0 1 -4 .8 0 .1 8 -2 .5 7 .1 7 -1 .6 4 .0 1 -3 .1 3 160 3M MN03112352 TABLE 4.2.6 NUMBERS O F DEATHS AND STANDARDIZED M O RTALITY RATIOS (SM Rs) BY DURATION O F EM PLO YM ENT AM ONG FEMALE EMPLOYEES, 1947-1989. Cause of Death Obs Sm r 95% Cl Duration 10 years All causes Cancer Cardiovascular 50 66.74 .75 .5S-.99 17 23.04 .71 .42-1.14 18 22.00 .82 .48-1.29 Duration >10 years All causes Cancer Cardiovascular 20 26.62 .75 .46-1.16 6 9 .4 2 .64 .23-1.39 8 10.27 .78 .34-1.54 Abbreviations used are: Obs, observed; Exp, expected; C l, confidence interval. 161 3M MN03112353 TABLE 4.2,7 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY LATENCY AMONG FEMALE EMPLOYEES, 1947-1989. Cause of Death Obs X0 M R 95% Cl Latency>10 years All causes Cancer Cardiovascular Latency>15 years 41 16 13 5 6 .9 4 .7 2 .S 2 -.9 8 20.93 .76 .44-1.24 19.86 .65 .35-1.12 Ail causes Cancer Cardiovascular Latency>20 years 37 14 13 4 9 .3 7 18.25 17.79 .75 .53-1.03 .77 .4 2 -1 2 9 .73 .3 9 -1 2 5 All causes Cancer Cardiovascular 29 39.20 .74 .49-1.06 11 14.47 .76 .38-1.36 10 14.67 .68 .3 3 -1 2 5 Abbreviations used are: Obs, observed; Exp, expected; C l, confidence interval. 162 3M MN03112354 TABLE 4.2.8 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY ANY EMPLOYMENT IN THE CHEMICAL DIVISION AMONG FEMALE EMPLOYEES, 1947-1989. Cause ot Death Obs Exp -- sm -- 95% Cl Not employed in CD All causes Cancer Cardiovascular Heart disease All Gl All respiratory Injuries 39 14 13 8 2 2 3 4 3 .0 5 15.46 13.82 7.69 Z23 2 .2 3 1.48 .91 .91 .94 1.04 .9 0 .9 0 2 .0 2 .6 4 -1 .2 4 .4 9 -1 .5 2 .50-1.61 .4 5 -2 .0 5 .1 0 -3 .2 3 .1 0 -3 .2 3 .4 1 -5 .9 0 employed in CD All causes 11 23.69 Cancer 3 8.38 Cardiovascular 5 8.19 Heart disease 2 4 .6 9 All Gl 1 1.18 All respiratory 1 1.28 Injuries 1 1.98 .46 .23-.83 .36 .07-1.05 .61 .20-1.43 .43 .05-1.54 .85 .01-4.73 .78 .01-4.81 .51 .51-2.81 Abbreviations used are: Obs, observed; Exp, ex__p__e__c_tJe. dA; Ci "l, confidence interval; CD, Chemical Division. 163 3M MN03112355 TABLE 4.2.9 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs), BASED ON U.S. WHITE MALE RATES, AMONG 2788 MALE EMPLOYEES, 1947-1989. Cause of Death Obs Exp 95% Cl All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Testis Bladder Lymphopoietic Cardiovascular CHD Cerebrovascular All Gastrointestinal All respiratory Diabetes Injuries Suicide 347 103 24 9 8 31 29 6 1 3 13 145 110 10 12 13 8 38 12 473.56 107.80 25.94 9.11 5.33 40.53 38.72 5.10 .82 2.20 11.42 203.31 147.04 19.92 23.99 25.89 6.53 46.56 17.10 .73 .95 .93 .99 1.50 .76 .75 1.18 1.22 1.36 1.14 .71 .75 .50 .50 .50 1.23 .82 .70 .66- .81 .77-1.15 .59-1.38 .45-1.88 .65-2.96 .52-1.09 .50-1.08 .43-2.56 .02-6.80 .27-3.98 .54-1.84 .60-.84 .61-.90 .24-.92 26-.87 .27-.86 .53-2.42 .58-1.12 .32-12 3 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 164 3M MN03112356 TABLE 4.2.10 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs), BASED ON MINNESOTA WHITE MALE RATES, AMONG 2788 MALE EMPLOYEES, 1947-1989. Cause of Death 55s Exp SMR 95% Cl All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Testis Bladder Lymphopoietic Cardiovascular CHD Cerebrovascular All Gastrointestinal All respiratory Diabetes Injuries Suicide 347 103 24 9 8 31 29 6 1 3 13 145 110 10 12 13 8 38 12 450.79 97.29 26.78 9.42 5.58 30.42 28.94 6.07 .92 2.18 12.07 212.19 159.09 24.66 21.13 21.75 6.52 47.74 15.09 77 1.05 .90 .96 1.43 1.02 1.00 .99 1.09 1.37 1.09 .68 .69 .60 .57 .60 1.23 .80 .79 .69-.86 .86-12 7 .57-1.33 .44-1.81 .62-2.83 .69-1.45 .67-1.44 .36-2.15 .01-6.05 .28-4.01 .57-1.84 .58-.80 .57-.83 .32-1.02 29-.99 .32-1.06 .53-2.42 .56-1.08 .41-1.39 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 165 3M MN03112357 TABLE 4.2.11 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY LATENCY, BASED ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES, 1947-1989. Cause of Death .LATENCY* 10 YEARS Obs EXP SMR 95% Cl All causes Cancer Gastrointestinal Pancreas Respiratory Lung Skin Prostate Bladder Lymphopoietic Cardiovascular All Gastrointestinal All respiratory Diabetes Injuries Suicide 299 98 24 8 29 27 3 6 3 11 130 8 11 8 21 11 398.27 88.71 24.78 5.20 28.81 27.44 1.53 5.94 1.75 10.03 195.91 18.58 20.16 5.37 27.61 10.19 .77 1.10 .97 1.54 1.01 .98 1.96 1.01 1.72 1.10 .66 .43 .55 1.49 .76 1.08 .68-.86 .90-1.35 .62-1.44 .66-3.03 .67-1.45 .65-1.43 .39-5.73 .37-2.20 .34-5.01 .55-1.96 .55-.79 .19-.86 -27-.98 .64-2.94 .47-1.16 .54-1.93 Abbreviations used are: 6bs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 166 3M MN03112358 TABLE 4.2.12 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY LATENCY, BASED ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES, 1947-1989. Cause of Death LATENCY* 15 YEARS "5b S -- Exp SMR 95% Cl Ait causes Cancer Gastrointestinal Pancreas Respiratory Lung Skin Prostate Bladder Lymphopoietic Cardiovascular All Gastrointestinal All respiratory Diabetes injuries Suicide 266 90 24 8 27 25 3 5 3 9 119 8 9 7 23 9 344 80.64 22.63 4.72 26.71 25.45 1.29 5.73 1.96 8.68 178.25 16.17 18.60 4.54 29.21 7.47 .77 1.12 1.06 1.69 1.01 .98 2.33 .87 1.53 1.04 .67 .49 .48 1.54 .79 1.21 .68-87 .90-1.37 .68-1.51 .73-3.32 .67-1.47 .64-1.45 .47-6.80 .28-2.04 .37-4.47 .47-1.97 .55-.80 .21-.97 .22-.92 .62-3.18 .50-1.16 .55-2.29 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 167 3M MN03112359 TABLE 4.2.13 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY LATENCY, BASED ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES, 1947-1989. Cause of Death m r~LATENCY 20 YEARS Obs' " EXP -- 95% Cl All causes Cancer Gastrointestinal Pancreas Respiratory Lung Skin Prostate Bladder Lymphopoietic Cardiovascular All Gastrointestinal All respiratory Diabetes Injuries Suicide 216 73 15 4 25 23 2 5 3 7 99 8 9 7 13 7 286.9 68.74 19.33 4.06 23.06 21.06 .98 5.29 1.75 7.01 151.80 12.90 16.3 3.65 19.47 5.01 .75 1.06 .77 .99 1.08 1.05 2.02 .95 1.72 .99 1.06 .62 .55 1.92 .67 1.40 .66-.86 .83-1.34 .43-1.28 .27-2.52 .70-1.60 .66-1.57 .23-7.34 .30-2.21 .34-5.01 .39-2.03 .83-1.34 .27-1.21 .25-1.05 .77-3.95 .36-1.14 .56-2.80 Abbreviations used are: Obs, observed; Exp, expected; Ct, confidence Interval; CHD, coronary and atherosclerotic heart disease. 168 3M MN03112360 TABLE 4.2.14 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY DURATION OF EMPLOYMENT, BASED ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES, 1947-1989. Cause ot Death DURATIONS 5 YEARS Obs Exp 95% Cl All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Bladder Brain Lymphopoietic Cardiovascular CHD Cerebrovascular All Gastrointestinal All respiratory Diabetes Injuries Suicide 256 80 22 8 7 25 23 4 2 3 6 114 90 6 7 9 8 29 9 321.20 721 20.21 7.10 4.22 23.72 22.10 4.47 1.68 2.51 8.41 159.50 120.20 18.44 15.20 16.30 4.53 36.60 8.81 .80 1.11 1.09 1.13 1.66 1.08 1.04 .84 1.19 1.20 .71 .71 .75 .33 .46 .55 1.77 .79 1.02 .70-.90 .88-1.38 .68-1.65 .49-2.22 .66-3.42 .70-1.59 .66-1.56 .23-2.15 .13-4.29 .24-1.50 .26-1.55 .59-.86 .60-.92 .12-.71 .18-.95 .25-1.05 .76-3.48 .53-1.14 .47-1.94 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 169 3M MN03112361 TABLE 4.2.15 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY DURATION OF EMPLOYMENT, BASED ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES, 1947-1989. Cause of Death DURATION 10 YEARS "Obs Exp SMR 95% Cl All causes 203 257.30 Cancer 67 59.36 Gastrointestinal 20 16.75 Colon 7 5.92 Pancreas 6 3.50 Respiratory 22 19.38 Lung 20 18.47 Prostate 4 4.20 Bladder 1 1.44 Brain 3 1.89 Lymphopoietic 5 6.58 Cardiovascular 92 132.13 CHD 75 99.75 Cerebrovascular 5 15.49 All Gastrointestinal 4 11.96 All respiratory 7 13.80 Diabetes 8 3.49 Injuries 19 23.46 Suicide 8 5.88 .79 1.13 1.19 1.18 1.71 1.13 1.08 .95 .69 1.59 .76 .70 .73 .32 .33 .51 2.29 .68 1.36 .68-.91 .87-1.43 .73-1.84 .47-2.44 .63-3.71 .71-1.72 .66-1.67 .26-2.44 .01-3.85 .32-4.64 .24-1.77 .56-.85 .57-.92 .10-.75 .09-.86 .20-1.05 .99-4.51 .34-1.22 .59-2.68 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 170 3M MN03112362 TABLE 4.2.16 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY DURATION OF EMPLOYMENT, BASED ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES, 1947-1989. ________________________ DURATION 20 YEARS________________ All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Bladder Brain Lymphopoietic Cardiovascular CHD Cerebrovascular All Gastrointestinal All respiratory Diabetes Injuries Suicide 104 35 10 5 1 11 10 2 1 1 4 48 39 1 2 5 5 2 3 152.36 37.31 10.52 3.77 21 12.69 12.10 2.83 .94 1.03 3.82 80.6 61.25 9.13 6.87 8.61 1.94 6.61 2.54 .68 .94 .95 1.33 .45 .87 .83 .71 1.06 .97 1.05 .58 .64 .11 29 .58 2.58 .30 1.18 .56-.83 .65-1.30 .46-1.75 .43-3.09 .01-2.52 .43-1.55 .40-1.52 .08-2.55 .01-5.91 .01-5.40 .28-6.02 .19-1.36 .4S-.87 00-.61 .03-1.05 .19-1.36 .83-6.02 .03-1.09 .24-3.45 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 171 3M MN03112363 TABLE 4.2.17 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs), BASED ON MINNESOTA W HITE MALE RATES, AMONG 1339 MALE EMPLOYEES EVER EMPLOYED IN THE CHEMICAL DIVISION, 1947-1989. Cause of beath ok-- S'MR 95% Cl All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Testis Bladder Lymphopoietic Cardiovascular CHD Cerebrovascular All Gastrointestinal All respiratory Diabetes Injuries Suicide 148 40 9 4 4 12 11 4 1 1 5 54 43 4 8 7 3 31 10 172.96 36.31 9.77 3.46 2.04 11.26 10.70 1.97 .44 .75 4.76 76.65 57.74 8.53 8.27 7.770 2.55 31.72 6.99 .86 1.10 .92 1.15 1.96 1.07 1.03 2.03 2.28 1.33 1.05 .70 .74 .47 .97 .91 1.18 .98 1.43 .72-1.01 .79-1.50 .42-1.75 .31 -4.01 .53-5.01 .55-1.86 .51-1.84 .55-4.59 .03-12.66 .02-7.40 .34-2.45 .53-.92 .54-1.00 .13-1.20 .42-1.91 .36-1.87 .24-3.44 .66-1.39 .68-2.63 Abbreviations used are: Obs, observed; xp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 172 3M MN03112364 TABLE 4.2.18 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs). BASED ON MINNESOTA W HITE MALE RATES, AMONG 1449 MALE EMPLOYEES NEVER EMPLOYED IN TH E CHEMICAL DIVISION, 1 9 4 7 -1 9 8 9 . Cause of Death dbs MR 95% t \ All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Testis Bladder Lymphopoietic Cardiovascular CHD Cerebrovascular All Gastrointestinal All respiratory Diabetes Injuries Suicide 200 63 15 5 4 19 18 2 0 2 8 91 67 6 4 6 5 23 2 291.25 67.56 16.46 5.79 3.37 25.58 24.44 3.45 .43 1.45 6.89 129.77 93.84 12.93 14.56 16.77 4.05 38.28 9.26 .69 .93 .91 .89 1.19 .74 .74 .58 - .00 1.38 1.16 .70 .71 .46 JZ7 .36 1.24 .60 .60 .59-.79 .72-1.19 .51-1.50 .28-2.01 .32-3.04 .45-1.16 .44-1.16 .07-2.09 .00-8.45 .16-4.99 .50-2.29 .5B-.86 .55-.91 .17-1.01 .07-.70 .13-.78 .40-2.88 .38-.S8 02-.78 Abbreviations used are: Obs, observed; Exp, e_x__p_e__ctAedI"; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease. 173 3M MN03112365 TABLE 4.2.19 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY LATENCY, BASED ON MINNESOTA W HITE MALE RATES, AMONG MALE EMPLOYEES NEVER EMPLOYED IN THE CHEMICAL DIVISION, 1947-1989. Cause of Death LATENCY 15 YEARS 6bs EXP Sm r 95% Cl All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Lymphopoietic Cardiovascular All Gastrointestinal All respiratory Diabetes Injuries 161 56 15 5 4 17 16 2 5 75 4 4 5 7 216.10 50.70 14.37 5.13 2.99 16.73 15.94 3.86 5.40 113.60 9.80 12.14 2.82 11.17 .75 1.10 1.05 .98 1.34 1.02 1.00 .52 .93 .66 .41 .33 1.77 .63 .63-.B7 .83-1.43 .59-1.73 .31-2.28 .36-3.43 .59-1.67 .57-1.63 .06-1.87 .30-2.16 .52-.83 .11-1.05 09-.84 .57-4.14 .25-1.29 Abbreviations used are: Obs, observed; Exp, expected; til, confidence interval; CHD, coronary and atherosclerotic heart cBsease; CD, Chemical Division. 174 3M MN03112366 TABLE 4.2.20 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY LATENCY, BASED ON MINNESOTA W HITE MALE RATES, AMONG MALE EMPLOYEES EVER EMPLOYED IN THE CHEMICAL DIVISION, 1947-1989. Cause of Death LATENCY 2 1 5 YEARS Obs EXP SFffH 45% Cl All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Lymphopoietic Cardiovascular All Gastrointestinal All respiratory Diabetes Injuries 105 34 9 7 4 10 9 3 4 44 4 5 2 6 128.4 29.95 8.30 3.00 1.75 9.98 9.50 1.87 3.28 64.67 6.37 6.49 1.72 8.54 .82 1.14 1.08 1.33 2.28 1.00 .95 1.61 1.72 .68 .63 .77 1.17 .70 67-.99 .79-1.59 .49-2.06 .36-3.42 .61-5.85 .48-1.94 .43-1.80 .32-4.70 .33-3.12 .49-.91 .17-1.61 .25-1.80 .43-4.21 .26-1.53 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease; CD, Chemical Division. 175 3M MN03112367 TABLE 4.2.21 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY DURATION O F EMPLOYMENT, BASED ON MINNESOTA WHITE MALE RATES. AMONG MALE EMPLOYEES EVER EMPLOYED IN THE CHEMICAL DIVISION, 1947-1989. Cause of Death DURATION 2 1 0YEARS OBs EXP M R 95% til All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Lymphopoietic Cardiovascular All respiratory Diabetes Injuries 90 27 6 3 2 8 7 3 4 38 3 3 7 108.7 24.4 6.92 2.47 1.46 8.16 7.78 1.55 2.84 54.60 5.42 1.51 8.11 < CD .83 1.08 .87 1.22 1.37 .90 1.94 1.41 .70 .55 1.99 .86 .67-1.02 .71-1.58 .22-1.89 .24-3.55 .75-4.86 .42-1.93 .36-1.86 .39-5.66 .38-3.61 .50-.97 .11-1.62 .40-5.80 .35-1.78 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease; CD, Chemical Division. 176 3M MN03112368 TABLE 4.2.22 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY ^ RATIOS (SMRs) BY DURATION OF EMPLOYMENT. BASE& ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES EVER EMPLOYED IN THE r.HFMinAL DIVISION. 1947-1989. Cause ot Death DURATION 20YEARS Qbs Exp SMR 95% CT All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Lymphopoietic Cardiovascular Ail respiratory Diabetes Injuries 45 16 3 3 0 5 4 2 3 18 2 2 2 66.29 16.21 4.53 1.61 . .96 5.57 5.31 1.10 1.67 34.48 3.52 .84 327 .68 .99 .66 1.84 .00 .90 .75 1.82 1.79 .52 .57 2.37 .61 .50-.91 .56-12 0 .13-1.94 .37-5.38 0-3.84 .29-2.09 .20-1.93 .20-6.58 .36-5.24 .31-.83 .06-2.52 .27-8.56 .07-2.21 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease; CD, Chemical Division. 177 3M MN03112369 TABLE 4.2.23 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY DURATION O F EMPLOYMENT, BASED ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES NEVER EMPLOYED IN THE CHEMICAL DIVISION, 1947-1989. Cause of Death DURATION S 10YEARS 555" Exp SMR 95% U i All causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Lymphopoietic Cardiovascular All respiratory Diabetes Injuries 113 40 14 4 4 14 13 1 1 54 4 5 4 148.60 34.43 9.82 3.45 2.04 11.22 10.69 2.65 3.47 78.31 8.35 1.98 7.96 .76 1.16 1.43 1.16 1.96 1.25 1.22 .38 Zl .69 .48 2.52 .50 .63-.91 .83-1.58 .78-2.39 .31-2.97 .53-5.01 .68-2.09 .65-2.08 .01-2.10 .01-1.49 .52-.90 .13-1.27 .81-3.87 0.14-1.29 Abbreviations used are: Obs, observed; Exp, ex_p_e_cted; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease; CD, Chemical Division. 178 3M MN03112370 TABLE 4.2.24 NUMBERS OF DEATHS AND STANDARDIZED MORTALITY RATIOS (SMRs) BY DURATION OF EMPLOYMENT, BASED ON MINNESOTA WHITE MALE RATES, AMONG MALE EMPLOYEES NEVER EMPLOYED IN THE CHEMICAL DIVISION, 1947-1989. Cause of Death DURATION 20YEARS Obs E p M R 95% Cl Ail causes Cancer Gastrointestinal Colon Pancreas Respiratory Lung Prostate Lymphopoietic Cardiovascular All respiratory Diabetes Injuries 59 19 7 2 1 6 6 0 1 30 3 3 0 86.1 21.09 5.99 2.15 1.25 7.12 6.79 1.73 2.15 46.14 5.43 1.10 3.34 .69 .90 1.17 .93 .80 .84 .88 .00 .46 .65 .59 2.74 .00 .52-.88 .54-1.41 .47-2.41 .10-3.32 .01-4.45 .31-1.83 .32-1.92 .0-2.12 .01-2.58 .44-.93 .12-1.72 .55-8.00 .00-1.14 Abbreviations used are: Obs, observed; Exp, expected; Cl, confidence interval; CHD, coronary and atherosclerotic heart disease; CD, Chemical Division. TABLE 4 .2 2 5 AGE ADJUSTED STANDARDIZED RATE RATIOS (SRRs) FOR ALL CAUSE, CANCER, AND CARDIOVASCULAR MORTALITY BY DURATION OF EMPLOYMENT. AMONG MALE EMPLOYEES, 1947-1989. Cause of death all causes all cancers all cardiovascular SRR* .81 1.04 .91 95%CI .63-1.03 .67-1.61 .62-1.34 Abbreviations used are: SRR, standardized rate ratio; Cl, confidence interval. * less than 10 years of employment as referent category I 180 3M MN03112372 TABLE 4.2.26 AGE ADJUSTED STANDARDIZED RATE RATIOS (SRRs) FOR ALL CAUSE, CANCER* LUNG CANCER, Gl CANCER, AND CARDIOVASCULAR MORTALITY BY EVER/NEVER EMPLOYED IN THE CHEMICAL DIVISION, AMONG MALE EMPLOYEES, 1947-1989. Cause of death all causes all cancers lung cancer Gl cancer all cardiovascular SRR* 1.18 1.10 1.09 1.16 1.05 95%CI (.95,1.47) (.74,1.65) (.67,2.31) (.50,2.69) (.76,1.48) Abbreviations used are: SRR, standardized rate ratio; Cl, confidence interval; Gl, gastrointestinal. * Never employed in the Chemical Division as referent category 181 3M MN03112373 TABLE 4.2.27 AGE STRATIFIED, YEARS OF FOLLOW-UP ADJUSTED RATE RATIOS (RRmh) FOR ALL CAUSE, CANCER, AND CARDIOVASCULAR MORTALITY BY EVER/NEVER EMPLOYED IN THE CHEMICAL DIVISION, AMONG MALE EMPLOYEES, 1947-1989. Aae at employment RRmh* 95%CI All causes 15-19 years 20-29 years 30-39 years 40-65 yeans All cancers 15-19 years 20-29 years 30-39 years 40-65 years All cardiovascular 15-19 years 20-29 years 30-39 years 40-65 years 1.22 .95 .95 1.02 .95 .72 1.10 .66 1.40 .86 .78 1.11 (.6 2 ,2 .4 0 ) (.6 8 ,1 .3 2 ) (.61 1.50) (.72-1.44) (.2 1 ,4 .3 4 ) (.3 8 ,1 .3 5 ) (.6 2 ,1 .9 0 ) (.2 7 ,1 .6 0 ) (.3 9 ,5 .0 3 ) (.4 4 ,1 .6 7 ) (.4 4 .1 .2 9 ) (.7 3 ,1 .8 2 ) Abbreviations used are: RRmh, Mantel-Haenszel age adjusted rate ratio; Cl, confidence interval. * Adjusted for years of follow-up and stratified by four age categories. Never employed in the Chemical Division as referent category 182 3M MN03112374 TABLE 4.2.28 AGE STRATIFIED, YEARS O F FOLLOW-UP ADJUSTED RATE RATIOS (RRmh) FOR ALL CAUSE, CANCER, AND CARDIOVASCULAR MORTALITY BY DURATION OF EMPLOYMENT IN THE CHEMICAL DIVISION, AMONG MALE EMPLOYEES, 1947-1989. Aae at employment RRmh* 95%CI All causes 15-19 years 20-29 yearn 30-39 years 40-65 years All cancers 15-19years 20-29 years 30-39 years 40-65 years All cardiovascular 15-19 years 20-29 years 30-39 years 40-65 years 1.30 1.16 2.16 1.69 2.17 .84 1.75 2.67 .88 1.38 3.53 1.50 (.5 8 ,3 .2 8 ) (.8 1 ,1 .6 5 ) (1 .5 2 ,2 .7 0 ) (1 .0 7 ,2 .6 0 ) (.4 0 ,1 1 .6 1 ) (.4 4 ,1 .5 1 ) (.9 5 ,3 .2 1 ) (.995,7.14) (.25, 3.33) (.7 3 ,2 .6 0 ) (1 .6 8 ,6 .2 1 ) (.8 1 ,2 .7 9 ) Abbreviations used are: RRm h >Mantel-Haenszel age adjusted rate ratio ; Cl, confidence interval. * Adjusted for years of follow-up and stratified by four age categories, less than 10 years employment as referent category 183 3M MN03112375 TABLE 4.2.29 PROPORTIONAL HAZARD REGRESSION MODEL OF FACTORS PREDICTING THE ALL CAUSE MORTALITY AMONG 2788 MALE WORKERS. v a ria b le 8 SW ) p-value RR* Year of first employment Age at first employment* Duration of employment* -.55 .079 -.34 .009 .006 .001 .0001 .0001 .0001 .946 1.082 .967 Months in chemical division .001 .001 .24 1.001 Abbreviations used are: 8, regression parameter; SE(B), standard error of the slope parameter; RR, relative risk. # relative risk for one unit change in independent variable ` years TABLE 4.2.30 PROPORTIONAL HAZARD REGRESSION MODEL O F FACTORS PREDICTING THE CARDIOVASCULAR MORTALITY AMONG 2788 MALE WORKERS. Variable B SE(B) p-value RR* Year of first employment Age at first employment* Duration of employment* Months in chemical cfivision -.075 .119 .230 .0002 .016 .009 .294 .001 .001 .0001 .45 .85 .928 1.126 .852 1.00 slope parameter; RR, relative risk. # relative risk for one unit change in independent variable years 184 3M MN03112376 TABLE 4.2.31 PROPORTIONAL HAZARD REGRESSION MODEL OF FACTORS PREDICTING THE CANCER MORTALITY AMONG 2788 MALE WORKERS. Variable B p-value RR* Year of first employment -.031 .019 .11 .969 Age at first employment* .078 .011 .0001 1.081 Duration of employment* -.028 .009 .002 .972 Months in chemical division .002 .001 2 0 1.002 Abbreviations used are: B, regression parameter; SE(B), standard error of the slope parameter; RR, relative risk. # relative risk for one unit change in independent variable years TABLE 4.2.32 PROPORTIONAL HAZARD REGRESSION MODEL OF FACTORS PREDICTING THE LUNG CANCER MORTALITY AMONG 2788 MALE WORKERS. Variable B SE(B) p-value RR* Year of first employment Age at first employment* Duration of employment* Months in chemical division -.019 .070 -.062 -.026 .042 .021 .133 .016 .65 .981 .001 1.072 .64 .940 .11 .975 slope parameter; RR, relative risk. # relative risk for one unit change in independent variable years 185 3M MN03112377 TABLE 4.2.33 PROPORTIONAL HAZARD REGRESSION MODEL OF FACTORS PREDICTING THE Gl CANCER MORTALITY AMONG 2788 MALE WORKERS. Variable B SE(B) p-value RR* Year of first employment .015 .038 .71 1.015 Age at first employment* .130 .021 .001 1.139 Duration of employment* .005 .020 .82 1.005 Months in chemical division .001 .002 .56 1.001 Abbreviations used are: Gl, Gastrointestinal; B, regression parameter; SE(B), standard error of the slope parameter; RR, relative risk. # relative risk for one unit change in independent variable * years TABLE 4.2.34 PROPORTIONAL HAZARD REGRESSION MODEL OF FACTORS PREDICTING THE PROSTATE CANCER MORTALITY AMONG 2788 MALE WORKERS. Variable B SE(B) p-vaiue RR# Year of first employment .010 .081 .90 1.011 Age at first employment* .082 .045 .06 1.085 Duration of employment* -.070 .052 .18 .932 Months in chemical division .010 .005 .03 1.010 Abbreviations used are: 3, regression parameter; SE(B), standard error of the slope parameter; RR, relative risk. # relative risk for one unit change in independent variable years 186 3M MN03112378 TABLE 4.2.35 PROPORTIONAL HAZARO REGRESSION MODEL OF FACTORS PREDICTING THE PANCREATIC CANCER MORTALITY AMONG 2788 MALE WORKERS. v a ria b le 6 s m -- p-value RR* Year of first employment Age at first employment* .046 .136 .066 .034 .48 .0001 1.047 1.146 Duration of employment* Months in chemical division -.012 -.002 .035 .006 .73 .73 .988 .998 Abbreviations used are: 8, regression parameter; SE(0), standard error of the slope parameter, RR, relative risk. # relative risk for one unit change in independent variable y e a s TABLE 4.2.36 PROPORTIONAL HAZARD REGRESSION MODEL O F FACTORS PREDICTING TH E DIABETES MELLITUS MORTALITY AMONG 2788 MALE WORKERS. Variable B SE(B) p-value RR* Year of first employment -.405 .221 .06 .667 Age at first employment* .092 .044 .04 1.096 Duration of employment* .009 .030 .75 1.009 Months in chemical division -.001 .004 .76 .999 slope parameter; RR, relative risk. # relative risk for one unit change in independent variable years 187 3M MN03112379 TABLE 4.2.37 PROPORTIONAL HAZARD REGRESSION MODEL OF FACTORS PREDICTING THE ALL CAUSE MORTALITY AMONG 749 FEMALE WORKERS. Variable B 1 (5 ) p-value RR* Year of first employment -.02 .03 .41 .977 Age at first employment* .08 .02 .0001 1.08 Duration of employment* 2-10 years >10 years 1.31 .54 .85 .57 .01 3.72 .14 2.33 Months in chemical division -.003 .004 .48 .997 Abbreviations used are: B, regression parameter; SE(B), standard error of the slope parameter; RR, relative risk. # relative risk for one unit change in independent variable years TABLE 4.2.38 PROPORTIONAL HAZARD REGRESSION MODEL OF FACTORS PREDICTING THE CARDIOVASCULAR MORTALITY AMONG 749 FEMALE WORKERS. Variable B SE(B) p-value RR* Year of first employment Age at first employment* Duration of employment* Months in chemical division -.034 .119 -.011 -.015 .048 .024 .025 .017 .48 .0001 .67 .37 .966 1.126 .986 .985 slope parameter; RR, relative risk. # relative risk for one unit change in independent variable * years 188 3M MN03112380 TABLE 4.2.39 PROPORTIONAL HAZARD REGRESSION MODEL OF FACTORS PREDICTING THE CANCER MORTALITY AMONG 749 FEMALE WORKERS. variable B S e TbT " p-value RR* Year of first employment Age at first employment* Duration of employment* Months in chemical division -.043 .085 -.021 .001 .053 .025 .025 .005 .42 .958 .001 1.089 .65 .980 .87 1.001 slope parameter; RR, relative risk. # relative risk for one unit change in independent variable * years 189 3M MN03112381 FREE TESTOSTERONE (ng/dl) FIGURE 1. Free testosterone and total serum fluorine 1990 3M Chemoilte study a ------ AGE-30 BMI-25 TOTAL FLUORINE (ppm) 190 3M MN03112382 Figure 2. Bound testosterone and total serum fluorine 1990 3M Chemollte study AGE-30 BMI-2S AGE-30 BMI-3S AGE-SO BMI-25 AGE-80 BMI-3S TOTAL FLUORINE (ppm) 191 3M MN03112383 Figure 3. Estradiol and total serum fluorine 1990 3M Chemolite study TOTAL FLUORINE (ppm) 192 3M MN03112384 LH(pg/m l) Figure 4. Lutenizing hormone and total serum fluorine 1990 3M Chemollte study ion 9 8765432* 1 0 1 ........ . l ~ 'l 1l o 10 20 30 TOTAL FLUORINE (ppm) 193 3M MN03112385 FSH (pg/m l) Figure 5. Follicle stimulating hormone and total serum fluorine 1990 3M Chemollte study TO TA L FLU O R IN E (ppm ) 194 3M MN03112386 Figure 6. Prolactin and total serum fluorine 1990 3M Chemollte study Moderate drinkers Light drinkers Nonrespondents 195 3M MN03112387 TSH (pg/ml) Figure 7. Thyroid stimulating hormone ami total serum fluorine 1990 3M Chemolfte study TOTAL FLUORINE (ppm) 196 3M MN03112388 BOUND /FREE TESTOSTERONE RATIO Figure 8. Bound to free testosterone ratio ami total serum fluorine 1990 3M Chemoltte study TOTAL FLUORINE (ppm) 197 3M MN03112389 5. DISCUSSION s 1 Physiologic Effects Study 5-1-1 Introduction This was a cross-sectional study of the relationship between selected physiologic parameters and PFOA exposure which was assessed using total serum fluorine. Participants were recruited from workers employed during November, 1990 in the Chemical Division of the 3M Chemolite Plant in Cottage Grove, M innesota All current workers who had worked in high exposure Jobs at any tim e in the five previous years were invited to participate. A sample of workers employed in low exposure jobs was frequency matched to the age distribution of workers in high exposrue jobs. Participants completed a corporate medical history questionnaire and had vital parameters measured by an occupational health nurse. Blood was drawn for assays of total serum fluorine, seven hormones involved in the hypothalamicpituitary-gonadal axis, serum lipids, lipoproteins, hepatic function parameters, and hematology indices. Blood was drawn in the morning after workers were assigned to the day shift for at least three days. In 93% of participants serum fluorine levels were at least 10 tim es the background levels in the general population and in 3M workers not employed at Chemolite. Many workers who lacked PFOA exposure by job history had elevated PFOA levels. The sources of the unexpected PFOA exposure are unknown. The findings from this study are consistent with the hypothesis that perfluorcoctanolcadd (PFOA) affects the human hypothalamic-pituitary-gonadal axis. This study showed that relatively low levels of serum PFOA (20pM ) depressed free testosterone and elevated estradiol but did no affect LH or FSH levels. The association between free testosterone and PFOA was different in 198 3M MN03112390 older men than in younger men. in older men, free testosterone (FT) was depressed below 10 ng/mt at serum fluoride levels below one part per million (estimated PFOA levels below 1 pM ). In younger men, FT decreased toward 10 ng/ml at serum fluoride levels above 15 ppm (estimated PFOA levels below 15 liM). Increasing age may increase men's susceptibility to the testosterone lowering effects of PFOA. The associations between PFOA and the hormone levels may reflect a true causal relationship, or may be a result of chance, bias, or uncontrolled confounding. There are no human studies of PFOA associated reproductive toxicity available for comparison. Studies of the effects of PFOA in rodents have demonstrated a similar decrease in testosterone, increase in estradiol, and little change in L H 19. The association between PFOA and free testosterone may have been mediated by elevated estradiol and prolactin. Elevated estradiol decreases testosterone and other steroid hormone synthesis in Leydig cells. LH response to low testosterone is attenuated by estradiol through negative feedback mechanisms at the pituitary and hypothalamic levels112,113. Elevated prolactin sensitizes the hypothalamus and pituitary to estrogens feedback. The combined effect of elevated estradiol and prolactin could have reduced the secretion of LH and the subsequent Leydig ceil response. Estradiol has direct effects on Leydig cell testosterone synthesis. In rats, PFOA decreased androstenedione and testosterone, but not 17 alpha-hydroxyprogesterone19. The metabolism of 17 alpha-hydroxyprogesterone to androstenedione was inhibited at the step of the C-17,20 lyase. The activity of the rate limiting C -17,20 lyase has been reported to be under estradiol regulation in rat Leydig c e lls 114> 115. Thus, in PFOA treated rats, elevated levels of estradiol may inhibit the C -17,20 lyase and thereby reduce testosterone synthesis. The increase in the estradiol-testosterone ratio observed in workers is compatible with this mechanism for decreased free testosterone. The primary source of estradiol in males is the P450 (P450 19) mediated aromatization of testosterone116- 117. Additional estradiol is secreted directly from Leydig cells. The observed increase in estradiol may be the result of increased production from one of these two, sources or may be the result of inhibition of P450 mediated estradiol m etabolism 118. Perfluorooctanoic acid, a 199 3M MN03112391 prototype peroxisome proliferator, may regulate steroidogenesis by binding to a member of a new family of cytosolic receptors (PPAR) belonging to the nuclear hormone receptor superfamily and transactivating the transcription of genes involved in steroid synthesis 119-121. PFOA was positively associated with the TB /TF and E /TF ratios. PFO A binding to sex hormone bindinf globulin (SHBG) may have produced changes in the bound to free testosterone ratio. However, this would result in a change in the TB/TF that is In the opposite direction to the observed association between PFOA and TB /TF. The associations of PFOA with these ratios are consistent with a mechanism that involves decreased production of testosterone and increased production of estradiol. The HPG axis of older men appeared to be more susceptible to PFOA compared to that of younger men. No animal data has been reported concerning age related sensitivity to the effects of PFOA. However, the onset of Leydig cell tumors has been reported to occur late in two year rat feeding studies122. This finding may represent increased susceptibility for hormonal alterations in aged rats. Further animal research is needed to define any age related susceptibility factors. Prolactin levels w ere positively associated with total serum fluoride in participants who reported moderate drinking (1-3 drinks/day). Since the function of prolactin in men is uncertain, the clinical significance of such an association is undear. Alcohol ingestion is a stimulus for prolactin secretion. The mechanism of this effect appears to be mediated by alterations in caldum m ediated signal transduction pathw ays123. This suggests that the elevation of prolactin associated with PFOA and alcohol may be mediated by alterations in caldum mediated events such as transmembrane signal transduction pathways. Thyroid stimulating hormone was positively assodated with total serum fluoride. Animal studies have shown that perfluorodecanoic ad d depressed peripheral thyroid hormone levels without produdng a hypothyroid response 75*781101. In the present study, peripheral thyroid hormone levels were not assayed. Therefore, it is not possible to assess whether the observed assodation between 200 3M MN03112392 PFOA and TSH could be a direct hypothalamic effect, a pituitary regulatory effect, or an effect mediated by changes in peripheral thyroid hormone levels. in summary, this is the first report of hormonal changes associated with PFOA in humans. The present findings in humans are consistent with those previously reported in animal studies19.. The consistent findings indude low free testosterone, increased estradiol, and unchanged LH. Rodent and human reproductive endocrine systems cfiffer greatly, yet the suggested effects of PFOA are similar. In light of the observed similarities in effect, it is tempting to speculate that PFOA may effect the humans and rodents reproductive endocrine system through the sam e mechanism. A hypothesis that PFOA alters a calcium mediated cellular signal transduction pathway, such as the cAMP or inositol triphosphate mediated second messenger response, may provide a unified mechanism for the multiple lod of putative effects. No adverse health effects have been observed in exposed 8. The present study did not examine adverse health effects, although several adverse outcomes associated with hormonal alterations are possible. The etiology of a number of cancers including adenocarcinomas of the prostate, endometrium, colon, rectum, pancreas and breast, have been linked to changes in endogenous horm ones124. Cancers in this etlologic category indude. Perfluorooctanoic ad d is not a genotoxic carcinogen in standard assays 9. However, PFOA is a nongenotoxic rodent carcinogen. In rate exposured to PFOA over a two year period, there was assodated increase in Leydig cell tum ors12s. Leydig cell tumors have te e n observed in assodation with other peroxisome proliferators in ra te 122. It has te e n hypothesized that chronically elevated LH produced testicular neoplasm s19>122 However, in PFOA treated rats, LH was not elevated. This may be due to estrogens feedback inhibition as discussed previously, or due to insuffident experimental induction tim e 19. Alternatively, another mechanism may have been operative in produdng Leydig cell tumors. Exogenous estradiol produces Leydig cell tumors In m ic e 126. High estradiol levels are assodated with Leydig cell tumors in both rate and humans 127' 129 The tissue surrounding the Leydig cell adenomas also produces increased estrogens127. High estradiol may be a stimulus for Leydig cell 201 3M MN03112393 proliferation and tum or formation. This hypothesis is supported by the observation that estradiol stimulates TQ F-a secretion in Leydig cells TG F -a binds to EG F receptors expressed on Leydig c e ils 129 and stimulates cell proliferation. The hormonal changes associated with PFOA may be a mechanism for nongenotoxic carcinogenesis. The role of PFOA in human nongenotoxic carcinogenesis needs to be clarified. Adequate androgen levels are necessary for maintenance of potency, spermatogenesis, libido and male reproductive organs. Low testosterone amt high estrogens may decrease libido, and fertility in males 13. Decreased male fertility may be one potential adverse outcome of PFOA. The reproductive toxicity of PFO A has not been extensively studied. No studies have te e n conducted in humans. PFOA was not teratogenic in rats 9*131 132. No adverse effects on fertility were noted for fem ale rats in a teratogenesis study 9. M ale rats were not studied. No other reproductive studies in animals have been reported. Studies of human reproductive function are needed since human reproductive processes are thought to be more sensitive to xenobiotic insults compared to other animal species133. 5.1.3 Cholesterol. Triglycerides, and Lipoproteins Cholesterol, triglycerides, and LDL were not significantly associated with PFOA. The lack of association of PFOA with cholesterol or triglycerides is consistent with observations in experimental animal models. No animal stucBes of PFOA's effect on LDL are available for comparison. The are no studies in humans concerning the relationship of PFOA with LDL, cholesterol, or triglycerides. In light drinkers, PFOA had little effect on HDL levels. In moderate drinkers, increasing PFOA reduced H D L The putative effects of PFOA and alcohol may be mediated by alteration of a common HDL regulatory process. The findings are limited by the small number of exposed workers, the limited range of total fluoride values, and the limitations of the study design. The conclusion and suggested mechanism must be considered preliminary. 202 3M MN03112394 The mechanism by which PFOA modifies the alcohol-HDL relationship could be mediated by alterations in fatty acid metabolism or fatty ad d binding. Alcohol intake induces spedfic P450 metabolic enzymes induding 2E1 and alters lipid metabolism 134. PFOA induces a spedfic P450 A 1 fam ily of metabolic enzymes and alters lipid metabolism in rodents. The joint effect of alcohol and PFOA on P450 mediated lipid metabolism could alter HDL dynamics. The primary structure of PFOA suggests that PFOA could affect the ligand binding of tatty add in hepatocytes and HDLs. The competition for NEFA binding sites could reduce the effect of alcohol on HDL levels. Studies of the joint effect of PFOA and alcohol on HDL may clarify the regulatory mechanisms for H D L The decrease in HDL assodated with increasing PFOA levels m ay be dinically significant. In a meta-analysis of 12 prospective studies of the relationship between HDL levels and coronary heart disease (CHD ), Gordon estimated that the change in CHD risk assodated with a one mg/dl change in HDL level is approximately the sam e as the change in risk assodated with a 2-4 mg/dl change in LDL le v e l13s. The predicted drop in HDL for a moderate drinking partidpant with a total fluoride of 20 ppm is 30 mg/dl. A change of this order of magnitude may have a measurable impact on the occurrence of cardiovascular disease. In the retrospective mortality study, there was no increase in mortality from cardiovascular disease. However, there are a limited number of workers with total serum fluorine levels of 20 ppm of more. Any increase in risk for cardiovascular diseases among a small group of highly exposed workers may not be readily apparent in a study of all Chem dite or CD employees. Further research is needed to confirm and darify the assodation between PFOA and HDL level. Future studies could test the hypothesis that PFOA and alcohol jointly alter NEFA metabolism resulting in a decrease in HDL and an increase in cardiovascular morbidity and mortality risks for exposed workers who drink alcohol. 5 J .4 Hepatic Parameters Changes in SG O T (AST) and SG PT (ALT) appear to be assodated with total serum fluoride through an interaction with adiposity, in obese partidpants, both SGOT and SG PT increased with increasing PFOA. However, there did not 203 3M MN03112395 appear to be an independent effect of PFOA on SG O T after adjusting for SG PT. The findings are limited by the small number of exposed workers, the limited range of total fluoride values, and the previously discussed limitations of the study design. The conclusion and suggested mechanisms must be considered preliminary. Compared to SG O T, SG P T is a relatively specific m arker for hepatocyte disruption13S. The lade of assodation of SG O T with PFO A after adjusting for SGPT suggests that the liver is the primary source for the small PFOA associated changes in transaminases. Since S G P T is a enzyme assodated with the ER membrane, the increase in SG PT m ay have been the result of PFOA assodated ER proliferation, it may indicate a disruption in the integrity of hepatocyte membranes which allows increased release of cytosolic hepatic enzymes. The tissue spedfic effect suggested for hepatocyte membranes could be due to a higher hepatic concentration of PFOA. Liver injury is generally considered to be a multifactorial process. There is evidence that interactions between endogenous and exogenous factors play a role in hepatotoxicity observed in w orkers137. The modification of the adipositySGPT assodation by PFOA suggests that the mechanisms of transaminase elevation may be linked. Obesity has been assodated with elevation of transaminases as well as dinically important hepatitis138>139. The observation that some obese individuals evidence little adiposity effect while other obese individuals develop hepatic fibrosis has not been explained. It has been hypothesized that metabolic polymorphisms or other hepatotoxin exposure may play a role 14. Animal studies and limited human data suggest that xenobiotics, such as certain solvents and alcohol, may potentiate the effects of other hepatotoxins141> 142. Following this model, PFOA may directly or indirectly potentiate the hepatotoxic effect of obesity. A mitochondrial site of PFOA action may occur. The mitochondria plays an essential role in fat metabolism. Disruption of mitochondrial function can produce impairment of mitochondrial oxidation of long chain and medium chain fatty adds. Studies of fatty ad d metabolism in PFOA exposed humans have not been carried out Valproic add , an eight carbon branched chain fatty ad d (2 propyl-pentanoic 204 3M MN03112396 add) that impairs mitochondrial function and fatty ad d metabolism, is an example of a hepatotoxic xenobiotic of similar carbon structure to P F O A 143. Commercial grade PFOA contains isomers with carbon backbones identical to valproic adds structure 39. The valproate-like isomers of PFOA could produce toxicity similar to that of valproate. The modification of the assodation between PFOA and the transaminases by adiposity could be mediated by disturbances of mitochondrial fatty add metabolism in humans. GGT increased as alcohol use increased. The increase in G G T was sm aller as PFOA increased. This assodation was independent of changes in SG O T, SGPT, and AKPH. Perfiuorooctanoic add may inhibit the hepatotoxic effects of alcohol. The GGT-alcohol dose response relationship is thought to be secondary to the induction and increased release of G G T. Increased serum G G T levels indicate proliferation of the endoplasmic reticulum and induction of cytochrome P450 system, leakage from hepatocytes, or injury to other tissues144"147. Perfiuorooctanoic ad d may decrease serum G G T by altering cell membrane permeability, by redudng the alcohd mediated induction of G G T, or by changing alcohol oxidation pathways and redudng the production of toxic intermediates such as acetaldehyde. Perfiuorooctanoic ad d was negatively associated with AKPH in non-smokers. In workers who smoke greater than five dgarettes per day, PFOA was positively assodated with AKPH. The assodation of AKPH with PFOA was independent of GGT, transaminases, and hormones. Smoking has been reported to elevate A K PH 148. The mechanism of this effect is thought to be the result of AKPH induction by compounds in dgarette smoke. The joint effect of smoking and PFOA could increase the induction of AKPH. In summary, the assodations between PFOA and hepatic enzymes are w eak and are not dinicaliy significant In the retrospective mortality study, there was no increased in mortaiityassocaited with liver disease. Future studies of the effects of PFOA may eiuddate possible mechanisms of action of nongenotoxic hepatic carcinogens. The hepatic enzyme results are illustrative of the problem of extrapolating findings observed in rodent animal models to other species, induding hum ans149. In humans, PFOA does not cause the dramatic hepatic 205 3M MN03112397 effects observed in rodents. Instead, the observed associations may result from PFOA modification of the hepatic effects of obesity, alcohol consumption, and smoking. Each of these factors are independently associated with hepatotoxidty. Further studies of the joint effects of PFOA and BMI, alcohol, and smoking on hepatic enzymes are needed. 5.1.5 Hematology Counts and Parameters PFOA was weakly, but significantly associated with hemoglobin levels, M CV, and MCH. The associations between PFOA and erythrocyte indices appeared to be mediated through interactions with smoking, and perhaps alcohol consumption. The findings in animal studies * 150 are consistent with a decrease in red cell volume and a larger decrease in red cell number. Together, these changes produce an increase in cellular hemoglobin concentration. The estimated changes in erythrocytes indices are not of clinical significance over the range of total serum fluoride. However, these findings suggest that further studies of the effect of PFOA on red cell regulation and function are needed. The fincfings are limited by the small number of exposed workers, the limited range of total fluoride values, and the previously discussed limitations of the study design. Pharmacological doses of androgens increase erythrocyte number and mass but produce little change in M CV or M C H 1S1*152. The mechanisms by which androgens increase hemoglobin appear to mediated by modulating the erythropoietin responsiveness of multi-potential stem cells and by stimulating erythropoietin production151 1S3*155. in physiologic doses, the effect of testosterone bn erythrocyte indices is controversial. Palacios et al. and Cunningham et al. reported that testosterone is associated with a small increase in hemoglobin, but no change in M CV or MCH 156>157. Mauss et al. reported no change in red cell indices for physiologic levels of testosterone158. In the present study, the testosterone level was not strongly or significantly related to the red ceil indices. Estradiol was weakly association with HGB but not M C V or MCH. The effect of physiologic estradiol levels on the m ale hematological system is poorly understood. Tell et al. reported that the effect of smoking on red cell indices was different in male than in fem ale adolescents159. This suggests that estrogen levels m ay play a role in the effect of xenobiotics on red cell 206 3M MN03112398 indices. Taken together, the evidence suggests that the association between PFOA and erythrocyte indices was not mediated by the PFOA associated changes in testosterone, but may have been mediated in part by changes in estradiol. Thyroid hormone was associated with changes in HGB and M CV. A decreased availability of thyroxin (T4 ) to myxedma levels produces a mild macrocytic anemia in humans. The increased cell volume is due to alterations in lipid deposition in erythrocyte membranes that occurs during ineffective erythropoiesis 16. TSH confounded the association between PFOA and M CV. Decrease in T 4 could explain some of the increase in M CV and TSH . However, PFOA appeared to have an independent and opposite effect on M CV. Therefore, the association between PFOA and changes in red ceil indices was probably not related to changes in thyroid function. The immune system effects associated with PFOA present a complex picture. As expected, smoking had a strong effect on leukocyte counts. Smoking modified the association between cell count and PFOA for total lymphocytes, eosinophils, platelets and basophils. However, smoking did not modify the estimated PFOA effect on W BC, PM N, band count, or monocyte count Alcohol modified the association between PFOA and cell count for W BC, PM N, and lymphocyte count Adiposity modified the association between PFOA and lymphocyte count, monocyte count and platelet count Taken together, this preliminary data suggests that PFOA is associated with changes in peripheraly leukocyte counts. The negative association with lymphocyte count is consistent with the lymphocytes effects observed in primate studies. PFOA could modulate cell counts by altering the effects of smoking, alcohol consumption, and adiposity on peripheral leukocyte counts. The magnitude of the W BC and PMN associations were not clinically significant from an infectious disease perspective. Increased W BC is positively associated with mortality from ail causes, cardiovascular diseases, cancer and myocardial infarction161' 168. It is unclear if the alteration in W BC is a consequence of, or the cause of, ongoing pathological processes. Judgment as to the clinical relevance of fite PFOA associated changes in W BC must await further study. 207 t 3M MN03112399 Adiposity modified the association between ceil count and PFOA for monocytes. Alcohol and cigarette consumption were independent determinants in the present study. Monocyte counts have been reported to be low in massively obese individuals169. The biological basis for these effects are not d ear. The univariate and joint effects of adiposity and PFOA on monocyte count m ay a fruitful area for future research. In the present study, the complex relationships between lymphocyte count, PFOA, alcohol use, dgarette use, and body mass may have been the result of the differential effect on T ceil subsets. In order to darify these assodations, specific subsets need to be measured. The assodation of lymphocyte subsets with disease endpoints have yet to be darified. The interpretation of the observed assodation requires further research. Smoking was negatively assodated with basophil count As PFOA level increased, the smoking effect was diminished. Taylor et al. reported an increase in blood basophils in smokers compared to nonsm okers17D. W aiter et al. studied smokers am i nonsmokers and found that acute smoking causes degranulation and loss of basophils. However, chronic smoking is assodated with an elevated basophil count. 171*174. No attempt was made to prohibit subjects from smoking prior to the time of blood sampling. The negative assodation observed in this study may reflect recent smoking by partidpants prior to blood drawing. The apparent reduction in the degranulating effect of smoking suggests that PFOA may interact with the basophil degranuiaition process. Exposure to PFOA may be assodated with changes in immune function beyond simple changes in cell number. The avid oxygen binding by PFCs may alter the effectiveness of peroxidatic killing by PMNs. Cytokine signaling is important in immune function and could be altered by PFOA exposure175. The response to antigen binding depends upon rearrangement of membrane proteins. Changes in the membrane physical characteristics produced by the potent surfactant action of PFOA could alter immune responses. More research is needed in the area of PFOA immunotoxidty. The findings of the present study new t to be confirmed. Lymphocyte could be immunophenotyped using well established flow 208 3M MN03112400 cytometry m ethods176> 177. H ie standard immunotoxicologic assessment defined by the National Toxicology Program 178 should be carried out for PFOA. Smoking has been observed to increase platelet number, survival, adhesiveness, activation, and aggregation when exposed to A D P 179"184. Adhesiveness may change as a result of the effects of smoking on nonesterified fatty adds (NEFA). Smoking increases NEFA which may compete with PFOA for platelet membrane binding sites. Such competition could alter the smoking associated increase in platelet count. This hypothesized mechanism could be tested by in vitro modeling of platelet function in the presence of NEFA and PFOA. The relationship between obesity and platelet count has not been well studied. BMI has been reported to be negatively assodated with platelet countm . The mechanism for this effect is not d ear, but may be related to changes in NEFA assodated with obesity. Thus, the effect modification of the PFOA effect by smoking and obesity may have resulted from a common effect on NEFA. Changes in platelet count have been assodated with risk for cardiovascular disease 186>187 Direct and indirect mechanisms have been hypothesized fOrthe observed increase in disease occurrence. Thus, PFOA assodated changes in platelet count m ay be a marker for increased cardiovascular disease risk. Further study of potential effects of PFOA on platelet count and function are needed. 5J.6 Tstal Fluorine Smoking and total serum fluorine were weakly assodated in partidpants. The adjusted estimate fo rthe difference in mean fluorine between smokers and nonsmokers was small (0.1 ppm) and probably not of biological significance. Smoking intensity was not significantly correlated with total serum fluorine levels. It is unlikely that smoking affects the pharmacokinetics of serum fluorine or PFOA. It is unlikely that smoking was a primary route for absorption of PFOA. Exposure reduction does not need to await the results of future studies. In rodents, removal from exposure results in the reversal of the marked hepatic responses to P F O A 188. Intervention to reduce the PFOA body burdens of employees would prevent any potential adverse effects in the future. The reduction of exposure is espedally important since PFOA has an unusually long 209 3M MN03112401 biological haif-life. A significant reduction in body burden will require years of reduced exposure. 5-1-7 Methodological Considerations fi 1.7.1 Selection Bias Given the occupational study setting, the voluntary participation, and the requirements for blood sample collection, the overall participation was unexpectedly high. Past medical screening programs at Chemolite had participation rates of 60% to 70% . The present study's participation rate exceeded 80% . Given the high participation, non-response bias is likely to be small. Selection Mas is an important validity issue for cross sectional studies189. Only active Chemical Division workers were included in this study. Workers not included m ay have had a different response pattern than those who were included, if continued employment depended on response to exposure and the exposure was associated with the endpoint of interest, then selection bias m a y . have occurred. A finding of the present study w as that PFO A was associated with decreased free testosterone and increased estradiol. If workers who had high susceptibility to the effect of PFOA changed jobs, then the overall slope of the dose response curve could be underestimated. Conversely, if workers with low testosterone associated with PFOA changed jobs less often, then the overall dose response curve may be overestimated. Migration out of the high exposure jobs is unlikely to be the result of subdinical changes in hormone levels. All current Chemical Division employees who worked in high exposure jobs over the last five years were included in the sample. M any workers who had been employed in the high exposure jobs, but who changed jobs were included as participants. The vast majority of workers who had significant exposure over the previous five years would be included in the study sam ple as the turn-over rate in Chemolite employees was low (three percent per year} and the study included all current employees with appropriate job histories. Selection bias Is not a likely explanation for the findings in this study. 210 3M MN03112402 i Bias No worker was unexposed. The lowest potential exposure group had significantly elevated levels of total serum fluorine. In view of this, the observed effects may represent an underestimate of the true effect Total serum fluorine was used as a surrogate variable for PFOA exposure. The use of total serum fluorine has been validated in past biological monitoring in the Chemolite Plant and other plants using PFOA 8. Direct measurement of PFOA using gas chromatographic techniques have been highly correlated with total serum fluorine in Chemolite workers. Approximately 90% of total serum fluorine in Chemolite workers was reported to be in the form of PFO A 8>1Z. The valicfity of using this surrogate measure was not directly assessed in the current study due to cost. Small amounts of PFCs other than PFOA may have been present in serum. The half-life of PFC compounds is cfirectiy related to molecular weight. Compounds with six or less carbon backbones are likely to be rapidly excreted by exhalation 19. Short chain PFCs are unlikely to contribute appreciably to total serum fluorine. Longer chain PFC, such as perfluorodecanoic ad d (PFDA), are not produced at Chemolite. The high toxicity of PFDA exdudes it from commercial applications 88177'79,101< 102. Longer chain PFC are unlikely to be a . significant component of total serum fluorine. O ther organic fluorine containing compounds exist in biological systems and the environm ent However, the small amounts absorbed from the environment in the form of drugs or plant products are rapidly metabolized and excreted191. Inorganic fluorine was not a large constituent of the total fluorine levels. Serum ionic fluorine levels in the 1-5 ppm range are assodated with death in unintentional occupational exposures182. Total serum fluorine is a good surrogate measure for PFOA in this cohort The coeffldent of variation for total serum fluorine was 66% . The repeatability of the assay was better at total serum fluorine levels above five ppm. At the low end of the spectrum (< 1 ppm), where the assay is limited by sensitivity, the total serum fluorine values may overestimate the true value. These measurement errors are likely to lead to an underestimate of the effect of PFO A on the physiologic endpoints. 211 3M MN03112403 Commercial PFOA is a complex mixture of isomers and related compounds 3 9 . It is d ear that structurally related compounds, such as valproic ad d , exhibit toxicity for certain isomeric forms, but not others143,193 It is widely recognized that different drug enantomers have different pharmacokinetic and pharmacodynamic properties191. Thus, different isomers of PFOA m ay have different toxidties. If one isomer of PFO A is assodated with toxidty, then the use of total serum fluorine or total PFO A levels could have produced an under estimate of the true strength of assodation. However, in animal studies using straight chain PFOA, the spectrum of toxidties is sim ilar to those observed in studies using mixed isomer of PFOA 37*** 88>194. Further research is needed to darify the role of PFOA Isomers. The toxicokinetics of PFOA in humans are Afferent from those observed in rodents. Extrapolating the tissue distribution of PFOA from animals to humans may not be valid. No data exist on the relationship between serum and tissue PFOA distribution or body burden in humans. The use of serum levels to extrapolate to the concentrations at the site of PFOA action m ay have been inappropriate. Obtaining pharmacokinetic data in humans or appropriate animal models is an important area for future research efforts. The temporal variability of physiologic parameters is recognized. The ultraridian, arcadian, and drcannual variability of the study endpoints was not assessed directly. Instead, blood samples were drawn at the sam e time of day, on the same shift for ail participants. One sample was drawn to estimate mean parameter values. Considerable measurement error is inherent in this procedure for hormones with short pulsatile intervals such as LH, FSH, and testosterone. However, studies have shown that one sample is as good as three samples in estimating mean v a lu e s 19s. The use of a single sample to estimate mean hormone level produced random measurement error and would be expected to attenuate the observed relationships. Mean serum values of the assayed hormones m ay not represent the biologically important quantities at the site of action. Validation studies of self-reported smoking status, using biochemical markers such as exhaled carbon monoxide, serum and urine thiocyanate, and serum 212 3M MN03112404 thiocyanate, have shown that smokers underreport their sm oking19S. Smoking is associated with changes in physiologic parameters such as hematological counts i 97'199, cholesterol 20, lipoproteins 2011202, and hepatic enzym es148. The strength and direction of the association between self-reported smoking information and these parameters can be used to indirectly assess the validity of the smoking information m . in this study, smoking status and intensity was strongly and significantly associated with leukocyte count, band count, eosinophil count, platelet count, and monocyte count. As expected, smoking intensity was negatively associated with basophil count. No participant reported drinking more than 3 ounces of alcohol per day. This may reflect the company's success in cBscouraging heavy alcohol consumption in employees, a reporting bias, or the fact that heavy drinkers may not be able to continue employment due to the demands of Chemical Division jobs. Alcohol consumption is associated with changes in physiologic parameters such as hepatic enzymes 2M, erythrocyte mean corpuscular volume, triglycerides, and high density lipoprotein144. As with smoking, the strength and direction of the association between self-reported alcohol consumption information and these parameters can be used to indirectly assess the validity of the alcohol information. Increased serum HDL is associated with moderate alcohol intake 144,205,206 j h e expected relationship between alcohol intake and HDL was observed in this study in individuals with low PFOA levels. As expected, there was a positive association between alcohol intake and triglycerides. Alcohol has a direct toxic effect upon erythrocyte size, maturation, am i num ber2071208. The specificity of M CV is 90% in identifying alcoholics from social drinkers with a positive predictive value of 96% 209. In the present study alcohol consumption of 1 to 3 drinks per day was associated with an increase in M CV of the sam e order as reported previously 2oa. Alcohol induces GGT. The sensitivity of G G T in detecting alcohol use varies from 52% to 94% . G G T is highly non-specific for alcohol consumption or for hepatic abnorm alities14*210. Heavy drinking of two to five drinks per day over one week or more are necessary to induce G G T 145> 208. G G T may be the only commonly assayed hepatic enzyme to increase with 213 3M MN03112405 heavy drinking. A significant positive association between G G T and self-reported alcohol use was observed. The presence of these associations indicates that the misdassification of alcohol use w as unlikely to produce a d a s large enough to explain the observed associations. Alcohol use was weakly assodated with hepatic transaminases. SG O T and SGPT are less sensitive indicators of alcohol use than G G T. In alcohol induced liver disease, SG O T may be slightly elevated and SG P T little changed. SG P T has been shown to decrease in some cases of alcohol induced liver disease 211. Considering the relationship known to exist between alcohol use, S G O T and SGPT, little alcohol assodated change in transaminases would be expected. The observed w eak assodation is not an unexpected finding and therefore probably does not reflect misdassification of alcohol use. Nonrespondents to the alcohol item were different than respondents. They were treated as a separate group in the analysis since the difference could not be explained by measured covariates. Although the power of the study is diminished by treating alcohol information as a nominal categorical variable, the potential for bias was reduced. 5J.7.3 .Confounding.,Bias Information on the duration of employment in exposed jobs was not collected. Plant records did not contain suffident information to reconstruct exposures more than five years in the p a s t The duration of exposure may be an important determinant of PFOA effect Duration of employment m ay be related to PFOA level since PFOA has the potential for bioaccumulation. The duration of exposure may have been a confounder for peptide hormonal endpoints in this study. In rodents, steroid hormonal and hepatic enzyme effects of PFOA exposure occur after two weeks of exposure, whereas peptide hormonal effects may require longer exposures19. Leydig cell tumors m ay require a considerable length of exposure or latency to develop122. There are many compounds in complex androgen-estrogen system. The present study measured only a few of them. Other biologically Important steroid 214 3M MN03112406 hormones include cortisol, androstenedione, dihydroepiandrostenedione sulfate (DHEAS), estrone, estriol, estrogenic catechols, and dihydrotestosterone (DHT). A total estrogen index or estrogen to testosterone ratio (E /T) m ay be more important than assays of individual compounds 212 Sex hormone binding globulin (SHBG), a major determinant of the estrogen to testosterone balance at the tissue le v e l213, was not assayed. More research is needed to clarify the potential role of these hormones as confounders of the observed associations. The relationship for bound testosterone may have been confounded by steroid hormone binding globulin (SHBG). Sex hormone binding globulin is an important determinant of testosterone and estradiol levels in different tissues as well as their metabolism 21S. Plasma SHGB levels are positively associated with estrogens and negatively associated with androgens 214. Thyroid hormone levels affect SHBG 21s. The ratios of estradiol to testosterone and testosterone to DHT may be regulated by SHBG levels 213. The association between PFOA and bound testosterone may have been, in part, related to estradiol and thyroid hormone changes in SHBG levels. Adult rats do not express SHBG 216. The decline in total testosterone observed in rats is not the result of changes in the amount or binding characteristic of SHBG. The observed depression of free testosterone in men is analogous to changes in total testosterone in rats and is probably not significantly related to changes in SHGB binding. Major stresses, such as surgical procedures, have been shown to markedly affect hormones in men 217. It is unlikely that major physical stresses were associated with PFOA. Therefore, stress was not a significant confounder In the present study. Shiftwork has been shown to affect a variety of physiologic endpoints including biochemical parameters, hematologic indices, and hormones 218. Study participants rotated weekly through three shifts. All samples w ere collected on the day shift at least three days post shift change. Given the rotating shifts and standard day shift sampling, It is unlikely that shiftwork and PFOA were associated. Shiftwork did not appear to be a significant confounder of the estimated dose response relationships. 215 3M MN03112407 Several dietary factors are determinants of the endpoints considered in this study. The effects of dietary fat and cholesterol on serum lipoproteins and lipids is widely appreciated 200. Dietary calories, fat, and carbohydrates affect steroid hormones 219>^ . Diet can also affect the metabolism of steroid hormones 221 222 Since it is unlikely that diet is associated with PFOA, it is probably not a confounding covariate in this study. Physical activity affects many physiologic parameters including hormones 223, enzymes 22A, lipoproteins 20, and hematologic indices. For hormones, only maximal exercise produced an effect No effect was noted for submaximal physical activity. It is unlikely that many participants engaged in maximal physical activity. Therefore, in this group, it is unlikely that physical activity is a determinant of the hormonal endpoints under study. Physical activity may effect HDL levels, but it is unlikely that physical activity was associated with PFOA. Therefore, physical activity was unlikely to be a significant confounder in this study. Medication usage and diseases such as diabetes mellitus are important determinants for some of the physiologic parameters measured in this study 193> 19S. Questionnaire items concerning medication use and medical history were incomplete and were not validated. PFOA exposure has not been associated with any medical conditions . If the use of medication or the diagnosis of a medical condition that affects one of the physiologic endpoints is associated with PFOA exposure, then confounding may occur. However, no such relationships have been described. Inflammatory processes, which are major determinants of W BC, w ere not assessed in this study. There is no evidence that inflammatory processes are related to total serum fluorine or serum PFOA. Therefore, these determinants of leukocyte count are unlikely to confound the estimated relationships. & 1-7.4 Analytic Model Specification Bias The analytic multivariate approach used in this study assumed that a linear model with additive effects was an adequate model with which to summarize the 216 3M MN03112408 data. A normal error term was used. Similar models of physiologic variables have been extensively used in the past and their assumptions tested 225. The model form was partially defined a prion* based on a biological hypothesis. The choice of a final model was based on biological knowledge plus best predictive power. The variable transformations used were not based on a specific biological mechanism, but instead reflect the basic form of dose response relationships observed in nature. 5 .2199 0 Chemolite Mortality Study 5.2.1 introduction This was a retrospective cohort study of mortality in workers employed in a PFOA production plant for greater than six months during the period from January 1 ,1 9 4 7 to Decem ber 3 1 ,1 9 8 9 . Completeness of the cohort was assessed from independent sources. Demographic and work history data were collected from plant records and verified from independent sources where possible. Cohort members were not individually contacted for additional information on confounding variables such as smoking. Vital status was confirmed for 100% of the cohort. Cause of death was obtained from death certificates for 99.6% of deaths and other sources for 0.4% of deaths. Cause of death was coded by IC D -8 categories by a nosologist. Reliability of death certificate coding was assessed by random resubmission of death certificates for recoding. The concordance was 100% for three digit IC D -8 codes. 5.2.2,Participant-Characteristics The 749 women were observed for 19,309 person-years, had a mean age at first employment of 27 years and mean follow-up of 26 years. H ie number of expected events given the age and size of the cohort was small. The study had limited power to detect moderate increases in cause-specific mortaltiy. The 2788 men were observed for over 70,000 person years. The mean age at first employment was 27 years,the mean length of follow-up was 25 years and a the mean age of death was 56 years. Non-CD men were older on average than 217 3M MN03112409 CD men and had more person-years in the older age groups where mortality was the highest internal comparisons were confounded by age as well as other tim e correlated factors such as length of follow-up. 5-2.3 Mortality Results In females, 6.7% were deceased compared to 12.5% in the males. Given that the mean age at first employment amt mean length of follow-up was sim ilar tor males and fem ales, this reflects the expected survival advantage of women. Fbr both males and females the proportion of deaths was sm aller in the C D cohort Employment in the Chemical Division did not produce a large survival disadvantage. The all causes, all cancer, and all cardiovascular mortality among women was less than expected in the overall cohort The SMRs were remarkably stable when stratified on ten year exposure groups, and ten, fifteen, and twenty year latency periods. The all causes SM R was .75 in the total cohort .75 in those employed for at least ten years or for those employed longer than ten years, and .75 in all three latency periods. Cardiovascular diseases and cancer mortality followed a sim ilar pattern. In males, the all causes, cardiovascular diseases, all gastrointestinal, and all respiratory diseases SM Rs were significantly less than one. The all causes SM R was .77 using Minnesota mortality rates and .73 using national rates. The low SMRs are most likely a result of the healthy worker effect (HW E). As expected, the cancer SM R is less affected by the HW E. The all causes SM Rs were .75 for all three latency groups. Latency did not have a strong relationship with the HWE. The all causes SM R was .80 in the greater than five year employment duration group and .68 in the greater than 20 year employment group. The low all causes SM R in the greater than 20 year duration group suggests that working for 20 or more years was associated with continued selection based on good health. The all causes SM R decreased with duration of employment in one meta-analysis of retrospective cohort studies 226 .but increased in the meta analysis by Fox and C o llier227. 218 3M MN03112410 The SRRs for all causes, all cancer, and all cardiovascular diseases for less that ten years employment to more than ten years employment w ere not significantly different from one. Because the rates were based on small numbers of events, the 95% Cl were wide. Due to the small number of events in the fem ales, SRRs were not calculated. The SRRs are similar to the SM Rs for the less than ten year employment and greater than ten year employment groups. The SRRs for CD versus non-CD male workers for all causes, all cancer, and all cardiovascular diseases were not significant and were sim ilar to the SMRs. Working in the CD did not substantially alter the rates of death. The small number of events observed for rare causes of death or specific causes of death make it unlikely that moderate increases in rates could be detected in this cohort for the follow-up period through 1989. More follow-up tim e will be needed to allow sufficient power to detect moderate increases in rates for specific causes of death. The results from the adjusted R R m h contrasting the mortality rates for ail causes, all cancer, and all cardiovascular diseases between C D and non-CD male workers were sim ilar to those for the SRRs and SM Rs. None of R R m h point estimates were statistically different from one. The contrast of rates between less than ten years of employment and greater than ten years of employment presented a different picture. All cause RRm h were significantly elevated in the oldest two age groups, while the RRm h for cardiovascular diseases was significantly elevated in the 30 to less than 40 year age group. The all cancer RRm h displayed a trend toward a statistically significant elevation in the oldest two groups. The RRm h were not adjusted for year of first employment. They may have been substantially confounded by changes of exposure over time since year of first employment. As seen in several PH regression models, year of first employment was significantly associated with the mortatlity. After age and length of follow-up, calendar tim e is the strongest tim e factor associated with m ortality189. Hence, it is likely that the elevated RRs for composite categories of cause of death in the oldest groups were a result of uncontrolled confounding by calendar period. Given the small number of events in strata, it was not feasible to further stratify the data on year of first em ploym ent 219 3M MN03112411 In the PH regression analysis, prostate cancer mortality w as positively and significantly related to time in the Chemical Division. Ten years of employment in the CD was associated with a 3 fold increase in prostate cancer mortality compared to men never employed in the CD. This trend was evident in the SM R analysis stratified by CD and non-CD em ploym ent This association was independent of duration of employment and year of first em ploym ent As expected, age at first employment was positively related to prostate cancer mortality rate. The interpretation of this estimated relative rate is tem pered by a number of factors. The estimates were based on six prostate cancer deaths, four in the CD cohort and two in the non-CD cohort A change of one case could significantly alter the estimates. Ascertainment of ail prostate cancer deaths may have been incomplete. Diagnosis m ay have been more complete in the CD cohort. Given that death certificate cause of death information is known to be imperfect, misdassification of one or more deaths could occur. The use of mortality as the event of interest for etiologic studies of prostate cancer is not the best study endpoint because of the long natural history and low mortality of prostate cancer. The majority of incident prostate cancers do not progress and cause death 2281228 For localized disease, an 80% ten year survival in untreated patients have been reported 23. Studies of prostate cancer incidence in this workforce are needed to clarify the suggested increase in prostate cancer risk. The findings of hormonal alterations in PFOA exposed men suggests a possible biologic mechanism for the increase in prostate cancer mortality 231. Incidence studies of other diseases that are hormonally mediated m ay be indicated if the PFOA associated hormonal changes are confirmed. 5.2.4 Methodological Considerations nMiiWiiMi! The use of death certificates to categorize cause of death im perfect232-235. The size of the potential bias depends on the cause of death. In one study cancer as a cause of death was under-reported by 13% 232 Leukemias and lymphomas were underreported in 19% of autopsy confirmed cases. Colorectal cancers were underreported in 12% of cases. Therefore, it appears that cancer deaths were not severely misdassified. All cardiovascular diseases as a group may 220 I * 3M MN03112412 have been inaccurate. Individual disease with the whole may be severely misclassificated and may produce large biases. For example, specific causes of death in the cardiovascular group, such as cerebrovascular disease, are inaccurately designated on death certificates. Three measures of PFOA exposure based on job history w ere used in this study. First, the cohort was dichotomized into those who ever worked in the CD and those who never worked in the CD . Second, the number of months worked in the CD until 1985 was used as a continuous param eter for PFOA exposure. Third, the total duration of Chemolite employment was used as a continuous param eter for the effect of work in a plant producing PFOA among a large number of products. Each of these surrogate variables may produce a different spectrum of misclassification. Categorization of workers into ever versus never employed in the CD may not reflect the biological effective dose of PFOA. Many C D jobs do not entail PFOA exposure. A number of workers were employed in the CD for short periods in the distant p as t Their exposure may not have been significant This categorization may misclassify unexposed workers as exposed. Conversely, PFOA exposure was widespread among Chemical Division (CD) employees working in jobs with no exposure to PFOA. No exposure measurements have been done in non-CD employees. It is possible that non-CD employees had significant body burdens of PFOA. if this was the case, exposed workers would have been classified as unexposed. Such misclassification would be expected to bias the effect estimates toward the null. The months of employment in the CD was the best available estimate of PFOA dose. Not ail CD jobs have PFOA exposure. The misclassification produced by classifying unexposed workers as exposed could have biased the estimate toward the null. The use of duration of employment at Chemolite as a continuous exposure parameter is less specific for PFOA than tim e in the CD. If another xenobiotic exposure in the plant has modulated disease occurrence rates, the use of duration may produce less misclassification than use of duration in the CD . 5.2.4.2 Confounding and Selection Bias The healthy worker effect strongly affects the validity of many occupational studies189>23S. It is a complex bias that results, In part, from the selection of 221 3M MN03112413 individuals for employment who are healthier than those in the comparison population. The HW E is usually stronger for cardiovascular diseases and respiratory diseases. Because cardiovascular diseases mortality accounts for a significant portion of ail causes mortality, the HW E usually reduces the all causes SMR. The age at first employment, age at risk, length of follow-up, and duration of employment are four tim e factors that are associated with changes in the HW E 189. Generally, the HW E diminishes with age and tim e. Collection of confounder information for individuals is difficult in retrospective cohort mortality studies. The present study included workers followed for more than 40 years. It was not feasible to colled: individual information on such covariates as smoking, health status, medical history, or dietary habits. The proportion of workers at Chemolite who smoke has been lower than in other facilities owned by the same corporation. In recent health maintenance studies, the self-reported smoking prevalence (25% ) is lower than the statewide smoking prevalence. The observation that all respiratory diseases and lung cancer rates are lower than expected may be the result of historically low smoking prevalence. The low smoking prevalence may depress the ail causes SM R, ail cancer SM R, and all respiratory disease SM R. The use of Internal comparison groups may reduce this smoking related bias 23,1. Time fadors such as age at risk, age at first employment, year of first employment, and duration of employment are associated with the occurrence of many diseases189. The use of an internal comparison group may reduce certain selection effects, but may n d control confounding if the exposure defined internal comparison groups have different distributions of these tim e fadors. Although the mean age at first employment and mean year of first employment are similar in the CD and non-CD cohorts of men and women, the comparisons of the rates of disease are confounded by differences in the distribution of age at risk. These time fadors are strongly correlated, with some being e x a d linear combinations of others. The relationship between measures of exposure and disease occurrence may be complex functions of these inter-related time fadors. Adjustment for time fadors may reduce the effects of confounding, but may not control confounding 238. if the disease occurrence relationship is defined in terms of cumulative 222 3M MN03112414 exposure, the true effect of exposure may be biased toward the null by uncontrolled confounding due to the complex tim e factors189. Some workers were exposed to many other potentially disease causing xenobiotics, such as benzene and asbestos, during their employment at Chemolite. Adjustment for their effects was not possible in this study. Even if information was available, exposures are often highly correlated making the separation of individual effects impossible. S.2.4.4 Analytic Model Specification Bias Comparison of SM Rs and RRm h between exposure groups may not be strictly valid. However, if the distribution of the person tim e in the comparison groups is not strongly discordant, then such a comparison m ay be useful. In the current study, the person-time distributions are different in the exposed groups. However, the differences appear to be of a magnitude that makes useful comparisons of SMRs possible. Although the proportional hazard (PH) model has been used frequently for cohort studies and clinical trials, it has not been widely used in occupational studies. In the past, it has been suggested that Poisson regression was the analytic strategy of choice because computational costs were less and the conceptualization of the model straight forw ard189. However, PH models are now easily run with standard computer packages109 Their wide application in clinical trials and cohort studies has fostered the understanding of the PH models. Poisson models appear less frequently in the literature and may not be as well understood. Poison regression and PH models have theoretical links and have been shown to give similar results when used to analyze the sam e data s e t189. Cox PH regression was chosen as the multivariate model to employ in this study. The validity of the proportional hazards assumptions was examined using the two standard techniques. The assumptions did not appear to be grossly violated. However, in analyses involving a small number of events, the assessment of the validity of assumptions may be limited. The use of the factors as continuous variables was based on lade of statistical evidence for a significant nonlinear 223 3M MN03112415 effect Although this strategy has been widely used for control of confounding, it has not been extensively validated in simulation studies. 224 3M MN03112416 6. SUMMARY. CO NCLUSIONS AND RECO M M ENDATIO NS 6.1 Cross-Sectional Study of the Physiologic Effects of PFQA This was a cross-sectional study of selected physiologic effects of PFOA, as quantified by total serum fluorine. Participants were recruited from workers employed during November 1990 in the Chemical Division of the 3M Chemolite Plant in Cottage Grove, Minnesota. All current workers who were employed in high exposure jobs at any time during the previous five years and an age matched sample of workers employed in low exposure jobs were invited to participate. Participants completed a corporate medical history questionnaire and had vital parameters measured by an occupational health nurse. Blood was drawn for assays of total serum fluorine, seven hormones involved in the hypothalamicpituitary-gonadal axis, serum lipids, lipoproteins, hepatic function parameters, and hematology indices. Blood was drawn in the morning after workers were assigned to the day shift for at least three days. In past studies, the majority of total serum fluorine found in Chemolite workers was in the form of PFOA. T h is , total serum fluorine is a valid surrogate measure of PFOA in Chemolite employees. For 93% of workers, total serum fluorine levels were 20 times greater than community and corporate background levels. Findings in the current study are consistent with other data suggesting that PFOA has a long biological half-life in both men and women. The long half-life of PFOA may result in significant bioaccumulation from small frequent doses or large, infrequent doses. The hormonal findings from this study are consistent with the hypothesis that PFOA depresses the human hypothalamic-pituitary-gonadal axis. The results show that low levels of serum PFOA (20pM ) depressed free testosterone and elevated estradiol with little observed change in LH levels. In older men, flee testosterone was depressed below 9 ng/dl at serum fluorine levels below one ppm (estimated PFOA levels below 1 pM ). 225 3M MN03112417 Mean prolactin levels w ere positively associated with PFOA in moderate drinkers, but not in light drinkers. Since the function of prolactin in men is uncertain, the clinical significance of this finding is unclear. Mean thyroid stimulating hormone was positively associated with PFOA. Since peripheral thyroid hormone leveis were not assayed, it was not possible to assess whether the observed association between PFOA and TS H was the result of a direct effect on the hypothalamus, pituitary, thyroid gland, or peripheral thyroid hormone metabolism. Cholesterol, triglycerides, and LDL were not significantly associated with PFOA. PFOA was negatively associated with HDL in moderate drinkers. PFOA w as not associated with the marked hepatic changes in humans that have been observed in rodents. PFOA appeared to alter the hepatic response to endogenous factors and xenobiotics. PFOA w as significantly associated with hemoglobin levels, M CV, and M CH. The estimated changes in erythrocytes are not of dinical significance over the range of observed total serum fluorine. The changes in leukocyte counts associated with PFO A exposure presented a complex picture. For example, the negative assodation between PFOA and lymphocytes was increased by smoking more than 10 dgarettes per day and decreased by alcohol use and adiposity. The magnitude of these associations are not dinically significant from an infectious disease perspective. However, elevated W BC has been assodated with increased all causes, cardiovascular diseases, and cancer mortality as well as increased inddence of myocardial infarction. 6 .2 Retrospective Cohort Mortality Study O f The Chemolite Workforce. 19471990 This was a retrospective cohort study of mortality in workers employed in a PFOA production plant All causes mortality in both male and fem ale Chemolite employees were significantly less than expected based on comparisons to the 226 3M MN03112418 mortality experience of the Minnesota and United States population. The SM Rs for several other causes of death including all respiratory diseases w ere less than expected. Since the healthy worker effect was apparently strong in the Chemoiite cohort, internal comparisons of SMRs were made between Chemical Division (C D ) and non-Chemical Division (non-CD) employees. These comparisons did not suggest any significant excesses in mortality in CD or nonCD employees. Generally, the findings from this study provide no evidence that employment at Chemoiite results in elevated mortality rates from any cause. However, prostate cancer mortality may be associated with length of employment in the Chemical Division. Ten years of employment in the CD was associated with a significant three fold increase in prostate cancer mortality. There was no association between prostate cancer mortality and employment (ever/hever) in the Chemical Division. Given the small number of deaths from prostate cancer in this study and the natural history of the disease, the association between employment in the CD and prostate cancer must be viewed as hypothesis generating and should not be over interpreted. However, the biological plausibility for any association between CD employment and prostate cancer is increased by animal and human toxicological data suggesting an association between PFOA and steroid sex hormone changes. 6.3 Conclusion Perfluorooctanoic ad d was assodated with reproductive hormonal changes in exposed workers. The dinical significance of these findings are unknown. The assodafions of PFOA with hormones, HDL, hematology parameters, prostate cancer mortality in men indicates the need for further research. ^Recommendations Research is needed in five areas. 227 3M MN03112419 1. An assessm ent of the horm onal effect of P FO A in w om en is needed. A crosssectional study should be conducted using specific assays fo r P FO A and accounting for tem poral horm onal variations. 2 . The clinical significance of the associations of P FO A with the physiologic param eters need clarification. Since morbidity from diseases such as prostate cancer is reflected in m ortality, an update of th e retrospective m ortality study is needed in five years. M orbidity studies should be conducted of endpoints that m ay be produced by hormonal c h a n g e s . Since exposed w orkers are relatively young and are lim ited in num ber, the feasible endpoints fo r a short term m orbidity study are lim ited. Pooling of workers from a num ber of plants could increase the num ber of exposed workers and allow endpoints with low er incidence to be studied. Th e morbidity study should be a long term which would allow the study of endpoints that occur at higher frequency in older ag e groups. In m en, endpoints should include the incidence of benign prostatic hypertrophy and prostate cancer. T h e feasibility of including inflam m atory bowel disease and colorectal cancer as endpoints should also be evaluated. In w om en, endpoints should indude the age of m enopause, the inddence of osteoporosis and related fractures, uterine fibroids, and cholelithiasis. If there are a suffident num ber of events, endom etrial cancer and inflam m atory bowel disease should be evaluated. If the cross-sectional hormonal study in w om en finds no assodation between P FO A and horm ones, then the morbidity study can be lim ited to m en. 3. Studies of reproductive outcom es in both m en and w om en are needed. Libido, potency, and fertility are cBrectly assodated with steroid horm ones levels. T h e feasibility of a retrospective study of reproductive endpoints o r a prospective study of tim e-to-pregnancy needs to be explored. 4. Th e m echanism s of action of P FO A need to be studied concurrently with morbidity. M echanistic studies are needed to define the relevance o f anim al studies for hum ans and provide a firm biological basis fo r the fincflngs of the mortality, morbidity, and reproduction studies. In vhm and cell line studies could clarify the m echanism s of action of P FO A on the pituitary secretion of LH, FS H , T S H , and prolactin. Pituicyte cultures m ay be 228 3M MN03112420 helpful in evaluating the direct effect of P FO A pituitary function. T h e effect of PFO A on other autocrine o r paracrine fe d o ra such as T G F -a , TG F-B , FG F, and T N F could also be evaluated. Hum an adiptocyte cultures could be used to study the effect of P FO A on arom atase activity. Additionally, studies are needed to clarify the relationship betw een P FO A and the tem poral variability of reproductive horm ones. 5. 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M ath M odeling 1 9 8 6 ;7 :1 3 93 -1 5 12 . 254 3M MN03112446 AEPENDIX1 P H Y S IO LO G IC E FFE C TS S TU D Y Q U E S TIO N N A IR E 255 3M MN03112447 Medical History Questionnaire 256 3M MN03112448 M.....e d ic a l H is to rym Q u e s tio n n a ire 257 mJl 3M MN03112449 47. Neuropathy (nerve aonormnlity m arms or legs. 46. Sem ite disofdar .6. MuOple aO eress SO. Other nervous system disease 81. YPP NO 52. Carpal Tunnelsydreme 63. Chrome low Peek pain 94. Herniated or raptured disc in the lew Peek SS. Hamiaiatf driupturaPdMeintfM m ck 56. Any cancar 87. mmmm Tobacco Smoking Have you ever smoked cigarettes? (Ns Y et amt Itt* then 20 packa el eiganttpe D-r 12 ox. of toPacoo in a lifetime or less than 1 cigarette day to rt year.) 58. # y tst (Compote the following tuaslienp) Do yeu now amokoefgerottoo (aa el ene month age?) 66. NC Howeld were you when you fi* narted regular operetta smoking? Ityou have stopped amoking egarattet eompletely. haw old were you when yeu stepped? Age so p p e d * 80. 61. Hew many operettas de you smoke par day now? Per day 82. On the average of the entire time you smoked, how many Ogarettea 00 you smoke par day'' Per day > 83 ii j Door did yeu inhale the egarette smeke? 6 4 .0 Does net appiy 1 O Not at a t O Sightly O Modern O Deeply Have you ever smoked a pipe mragaurle12rtye'z.(oYtatosbmacecaontvmt aore Hernia.) yM IS. L J Ryaet Howold were you when you started to amok* som e regularly? A ge* 66. I you have stopped smoking a pipe completely. hew OHware you whan you sopped? Ags stopped 67. hq 1--1 On the average ewarthe antife lima you wnekad a pipe, hew much pipe Mfaaacadid you imaha par week? * 66. (Standard pouch of wbanco contains 1 1/2ez. per weak.) Howmuch pipe tobacco era you smoking new? Oz. per weak? *" Do you cr did yeu MiaM P it pip* sm ote? Not smoking i epiae | ii 70. Never smoked D Not at M U Sightly Deeply Medatataiy Have you ever smoked cigars regularly? (Yes means m art than 1 egar a weak lor a year) ' If y a s: Yet 71. NC Hew oMsmis you when you firs started smokingagars regularly? Again y e a r* '* ! II I you have stopped smoking cigars opmpletaly, hew eld were yeu when you stopped? Ags to p p e d * 73. On the average ever the entire time you smoked cigars, how many cigars did you smoke a weak? Cigars per weak y* Hew many ogars are you smoking p v iw k iiD w t CiptfS per w ttk ^ 75. Ncftvnotane < Do or did you inhale the Ogar smoke? 78. Never smoked Nat S an Slightly Deeply Mooerateiy Cheek! yeu use snu or chawing loPacco. 77 Q I yet. hew many years naua you used S? Years used * 78. What is the hew description el the number ot alcoholic beverages you consume (1 dnnk * 1 120tXr*..b-ee*r, 1glass o! wine or 1 sc. of nilS HQUBrj 7*- None O Leas than f ennk per day OO 1 to 3 drinks a day 4 or mere draws per day M Opam nw t M Cttm r SMB 229-BW41 St *ui. MX at44-IOOO 3M 258 3M MN03112450 APPENDIX 2 TA B LES O F H O R M O N E R A TIO S BY B O D Y M ASS IN D E X , A G E , SM O K IN G S TA TU S , A ND A LC O H O L C O N S U M P TIO N 259 3M MN03112451 TABLE A4.1.1 BOUND TESTOSTERONE TO FREE TESTOSTERONE RATIO (TB/TF) BY BODY MASS INDEX, AGE. SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TB/TF N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 37.2 9.06 37.1 22.3-622 F-1.47 56 37.6 9.31 37.1 19.3-62.4 p-23 17 33.3 9.18 31.2 19.7-52.4 AGE <31 31-40 41-50 51-60 20 32.4 6.92 312 20.0-43.6 F-2.39 48 37.3 9.37 37.1 192-62.6 P-.07 26 37.1 9.30 38.8 19.7-58.8 19 39.9 9.90 39.9 22.3-62.4 Alcohol < lo z/d 86 37.4 9.90 372 19.3-62.6 F-2.06 1-3oz/d 19 33.9 6.70 322 22.7-44.1 P-.15 m issing 8 38.0 5.70 38.6 26.4-432 Tobacco sm oker nonsm oker m isting 27 84 2 37.9 7.96 372 25.0-58.8 F-.32 36.7 9.64 36.3 19.3-62.6 P-.57 27.5 1.57 272 26.4-28.6 TOTAL.., 113 #univariate Anova 260 3M MN03112452 TABLE A4.1.2 ESTRADIOL TO FREE TESTOSTERONE RATIO (E/TF) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA fc/tF N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 AGE <31 31-40 41-50 51-60 40 2.07 0.88 1.94 0.73-5.0 56 228 0.92 217 0.75229 >30vs-30 17 256 0.98 242 1.44221 T-2.35 P-.02 20 1.94 026 121 1.44-327 F-1.19 48 2.25 0.81 217 0.77-4.18 p-22 26 2.31 120 204 0.77229 19 2.48 1.05 242 1.07-521 Alcohol <10z/d 86 223 0.92 210 0.74221 F.01 1-3oz/d 19 221 0.76 221 0.73-4.18 pa.92 missing 8 2.46 1.96 209 1.41229 Tobacco smoker nonsmoker missing 27 84 2 2.19 0.98 208 0.74229 F..15 227 0.92 219 0.73221 P-.70 1.88 022 128 1.65-211 TOTAL 113 #univariate Anova ` Student t test, Prob>T 261 3M MN03112453 TABLE A4.1.3 ESTRADIOL TO BOUND TESTOSTERONE RATIO (E/TB) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA E/TB X100 N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 5.8 255 5.7 11.8-135 F-3.70 56 6.3 2.73 55 22-13.4 P-.03 17 8.0 251 7.8 3.7-14.4 AGE <31 31-40 41-50 51-60 20 6.1 1.79 5.9 3.0-95 F-.07 48 6.4 2.78 5.6 11.8-135 P-.98 26 65 353 5.0 1.7-14.4 19 6.4 76 6.0 25-11.6 Alcohol <10Z/d 1-3oz/d missing 86 19 8 6.3 2.90 5.7 15-14.4 F-.08 65 1.98 6.8 3.0-10.5 P-.98 6.6 3.45 5.4 3.9-135 Tobacco smoker 27 5.9 2.73 5.1 11.8-135 F-1.01 nonsmoker 84 65 254 6.0 25-13.4 p-52 missing 2 6.9 155 6.9 3.7-14.4 TOTAL 113 #univariate Anova 262 3M MN03112454 TABLE A4.1.4 ESTRADIOL TO LUTENIZING HORMONE RATIO (E/LH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA E/LH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 7.0 3.11 75 2.0-16.4 F-2.59 56 75 429 65 1.0-20.6 P-.08 17 95 3.92 8.8 35-18.4 AGE <31 31-40 41-50 51-60 20 8.7 458 7.6 2550.6 F-2.51 48 7.8 355 7.6 15-16.4 P-.06 26 6.4 4.58 5.1 1.6-18.8 19 5.8 2.89 65 15-115 Alcohol <10Z/d 86 75 3.95 7.0 1.0-20.6 F-.04 1-3oz/d 19 7A 4.16 6.7 15-16.4 p-56 missing 8 85 3.19 8.7 45-15.4 Tobacco smoker nonsmoker missing 27 84 2 7.0 4.42 65 15-16.4 F-54 7J5 3.77 7.1 1.0-20.6 p-56 4.7 355 4.7 25-7.0 TOTAL 113 #univariate Anova 263 3M MN03112455 TABLE A4.1.5 FREE TESTOSTERONE TO LUTENIZING HORMONE RATIO (TF/LH) BY BODY MASS INDEX, AGE. SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TF/LH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 3.6 1.74 as 1.1-1121 F-1.47 56 3.2 1.74 2.9 0.7-9.1 p-a4 17 34 1.75 a4 1.4-7.1 AGE <31 31-40 41-50 51-60 20 44 2JSS 3.9 a9 F-7ai 48 3.6 1.43 as a3 P -.0 0 0 2 26 22 1.30 a7 a7 19 22 1.14 2JS 2JS Alcohol <1oz/d 86 3.4 1.85 a2 0.7-11a F-.06 1-3oz/d 19 22 1.44 a2 0.7-64 P -.8 1 missing 8 3.8 1.43 3.6 ai-6 2 Tobacco smoker 27 32 1.34 22 1.1-6.9 F-iao nonsmoker 84 3.6 1.87 22 0.7-11a P-.28 missing 2 2.4 1.38 22 1.4-3.3 TOTAL 113 #univariate Anova 264 3M MN03112456 TABLE A4.1.6 BOUND TESTOSTERONE TO LUTENIZING HORMONE RATIO (TB/LH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA tB/LH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 131 57.9 125 39*298 F-.79 56 116 60.6 107 24-288 P-.46 17 122 43.4 121 55-199 AGE <31 31*40 41*50 51-60 20 147 69.7 136 39-298 F-4.72 48 133 58.0 131 36-288 p-,004 26 101 36.7 93 29-163 19 96 47.8 95 24-208 Alcohol <loz/d 86 122 57.1 121 24-298 F-32 1-3oz/d 19 114 56.1 92 29-202 p-57 missing 8 147 63.2 142 77-234 Tobacco smoker nonsmoker missing 27 84 2 116 52.4 114 41-288 F-.54 125 58.8 121 24-298 p-,46 64 34.3 64 39-88 TOTAL 113 #univariate Anova 265 3M MN03112457 TABLE A4.1.7 THYROID STIMULATING HORMONE TO LUTENIZING HORMONE RATIO (TSH/LH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TSH/LH x10 N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 3.2 1.78 35 0.6-85 F-3.40 56 35 2.80 3.0 0.4-17.0 P-.Q2 17 55 354 4.4 1.7-135 AGE <31 31-40 41-50 51-60 20 3.7 2.45 3.1 1.0-9.9 F-.14 48 3.8 3.16 35 0.4-17.0 p-53 26 3.4 1.89 2.9 0.8-85 19 3.8 2.48 3.5 0.4-11.0 Alcohol <1oz/d 1-3oz/d m issing 86 19 8 35 259 3.1 0.4-11.0 F-2.92 4.7 4.05 35 1.0-17.0 P-.09 35 1.74 3.6 0.8-5.8 Tobacco sm oker 27 3.0 1.68 2.8 0.4-7.6 F-2.89 nonsm oker 84 4.0 2.89 3.3 0.4-17.0 P-.09 m issing 2 1.7 0.01 1.7 1.7-1.7 TOTAL 113 #univariate Anova 266 3M MN03112458 TABLE A4.1.8 FOLLICLE STIMULATING HORMONE TO LUTENIZING HORMONE RATIO (FSH/LH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA FSH/LH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 30 40 14) 0.42 0.8 0.4-24 F-2.S4 56 1.0 0.37 0.9 0.4-1.9 P-.08 17 12 0.46 1.1 0.4-24 AGE <31 31-40 41-50 51-60 20 0.9 0.4 02 04-14 F-3.06 48 04 0.46 02 0.4-14 P-.03 28 1.1 0.46 1.0 0.4-14 19 12 0.49 1.1 04-24 Alcohol < lo z/d 1-3oz/d m issing 86 19 8 1.0 0.42 0.9 0.4-24 0.9 0.41 0.9 0.4-1.7 F-.48 0.9 0.24 0.9 04-14 P-.49 Tobacco sm oker nonsm oker m issing 27 84 2 1.0 0.40 0.9 .04-14 1.0 0.42 0.9 0.4-24 F-0.0 0.9 0.43 0.9 0.7-1.0 P-.98 TOTAL 113 #univariate Anova 267 3M MN03112459 TABLE A4.1.9 PROLACTIN TO LUTENIZING HORMONE RATIO (P/LH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA . pTlh N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 1.84 1.14 123 020229 F-.73 56 1.78 1.40 1.46 0.39-9.11 P-.49 17 221 120 1.83 1.18-4.91 AGE <31 31-40 41-50 51-60 20 229 1.70 123 1.18-4.91 F-125 48 1.95 126 1.67 029-9.11 P-21 26 1.61 0.93 1.18 026-3.70 19 1.54 0.87 129 025-327 Alcohol <1oz/d 86 1.78 1.04 1.61 0.35-629 F-3.19 1-3oz/d 19 2.37 214 1.70 026-9.11 P-.08 m issing 8 1.57 0.71 1.47 0.88-3.00 Tobacco sm oker nonsm oker m issing 27 84 2 1.27 0.73 1.12 0.39-2.74 F-8.25 2.07 1.38 1.72 025-9.11 p-,005 1.43 0.78 1.43 0.88-2.00 TOTAL 113 #univariat0 Anova 268 3M MN03112460 TABLE A4.1.10 BOUND TESTOSTERONE TO THYROID STIMULATING HORMONE RATIO (TB/TSH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TB/TSH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 500 331 413 170-1682 F-2.42 56 461 364 329 51-2102 P-.09 17 296 152 297 87-589 AGE <31 31-40 41-50 51-60 20 522 367 416 122-1682 F-2.64 48 521 388 421 51-2102 P-.05 26 359 203 345 131-1035 19 328 231 286 87-1122 Alcohol < lo z/d 86 468 352 353 87-2102 F-2.74 1-3oz/d 19 329 210 278 51-900 P-.10 m issing 8 563 326 456 184-1154 Tobacco sm oker 27 468 232 403 184-1185 F-.07 nonsm oker 84 448 364 321 5141102 p.80 m isting 2 371 198 371 231-511 TOTAL 113 #univariate Anova 269 3M MN03112461 TABLE A4.1.11 FREE TESTOSTERONE TO THYROID STIMULATING HORMONE RATIO (TF/TSH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA ............ TF/TSH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 56 17 AGE <31 31-40 41-50 51-60 Alcohol <1oz/d 1-3oz/d missing 20 48 26 19 86 19 8 Tobacco smoker nonsmoker missing TOTAL 27 84 2 113 #univariate Anova 13.8 8.62 11.7 7.01 9.3 5.05 15J 9.87 13.4 7.62 9.7 4.58 8.1 4.46 12.4 1.65 9.5 5.10 15.3 9.35 12J5 11.9 13.7 -- 5.53 8.06 7.99 10.8 3.8-43.7 F-2.43 9.7 1.7-35.8 P-.09 8.0 2.0-19.7 10J 6.1-43.7 F-5.36 11.9 1.7-35.8 P-.002 8.7 3.7-22.0 7.8 2.0-21.3 10.0 2.0-43.7 F-2.48 8.7 1.7-20.7 P-.12 12.3 5.0-33.5 11.3 5.0-27.2 F-.12 9.8 1.7-43.7 p.73 13.7 8.1-19.4 270 3M MN03112462 TABLE A4.1.12 ESTRADIOL TO THYROID STIMULATING HORMONE RATIO (E/TSH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA E/TSH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 AGE <31 31-40 41-50 51-60 Alcohol <1oz/d 1-3oz/d missing Tobacco smoker nonsmoker missing TOTAL 40 56 17 20 48 26 19 86 19 8 27 84 2 113 26.7 17.73 23.1 3.3-108.1 F-.27 25.4 14.04 21.3 15-59.0 P-.76 23.4 16.79 18.4 7.7-54.8 27.8 12.00 24.6 9.7-50.0 F-3.21 29.5 18.40 223 1.8-108.1 P-.03 21.8 13.06 19.0 35-525 18.2 10.80 16.8 7.8-54.8 25.1 13.08 21 3.3-59.0 F-.57 22.4 15.30 18.0 15-545 P-.45 37.8 31.81 285 10.4-108.1 26.7 15.13 23.4 10.3-59.0 F-.20 25.1 15.85 20.7 1.8-108.1 P-.65 27.1 19.40 27.1 13.4-40.8 #univariate Anova 271 3M MN03112463 TABLE A4.1.13 THYROID STIMULATING HORMONE TO FOLLICLE STIMULATING HORMONE RATIO (TSH/FSH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TSH/FSH N MEAN SD MEDIAN RANGE TEST# BMI <25 25*30 >30 40 0.39 0.25 0.34 0.08-1.09 F-.39 56 0.41 0.39 0.31 0.06-224 P-.6 8 17 0.48 129 0.43 0.15-126 AGE <31 31*40 41*50 51*60 20 0.46 0.31 0.44 0.12-126 F-.93 48 0.46 0.40 0.35 0.06-224 P-.43 26 0.37 028 0.30 0.09-121 19 0.33 0.18 0.31 0.06-0.75 Alcohol < lo z/d 1*3oz/d 86 0.38 0.26 0.32 .06-126 F-5.36 19 0.58 0.54 0.39 0.15-224 P-.02 m issing 8 0.40 0.24 0.39 0.08-0.76 Tobacco smoker . nonsm oker m issing 27 84 2 0.34 1.04 026 0.06-0.92 F-2.39 0.45 228 0.40 0.06-224 P-.12 0.21 0.05 021 0.17-025 TOTAL 113 #univariate Anova 272 I l 3M MN03112464 TABLE A4.1.14 FREE TESTOSTERONE TO FOLLICLE STIMULATING HORMONE RATIO (TF/FSH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TF/FSH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 4.3 2.66 3.6 15-15.6 F-1.03 56 3.6 2.18 3.0 0.7-11.1 p-56 17 4.0 2.86 3.1 05-115 AGE <31 31-40 41-50 51-60 20 5.8 354 5.0 1.7-15.6 F-1055 48 42. 2.16 3.7 1.7-11.3 P-.0001 26 3.0 1.62 2.5 0.7-6.6 19 22 157 2.0 0.7-65 Alcohol <1oz/d 86 2.8 2.60 3.1 0.7-16.6 F-.01 1-3oz/d 19 3.9 2.18 3.7 15-10.1 P-51 m issing 8 4.4 157 4.7 25-75 Tobacco sm oker 27 35 1.76 3.0 0.7-75 F-1.14 nonsm oker 84 4.1 2.67 3.5 0.7*15.6 p-58 m issing 2 2.6 0.91 z e 25-75 TOTAL 113 #univariato Anova 273 3M MN03112465 TABLE A4.1.15 BOUND TESTOSTERONE TO FOLLICLE STIMULATING HORMONE RATIO (TB/FSH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TF/FSH N MEAN SD MEDIAN RANGE TEST BMI <25 25-30 >30 40 155 87.3 138 39-411 F-2.00 56 126 67.9 113 23-297 P-.14 17 122 75.7 115 34-306 AGE <31 31-40 41-50 51-60 Alcohol <10z/d 1-3oz/d m issing 20 48 26 19 86 19 8 182 S0.3 184 39-411 F-8.75 154 74.5 141 45-348 P-.0001 101 52.9 91 39-227 87 53.2 78 23-264 133 76.5 120 23-411 F-0.0 133 78.6 112 39-325 pa.98 173 80.6 190 82-303 Tobacco sm oker nonsm okar m issing 27 84 2 130 74.4 106 39-325 F-.28 139 78.5 131 23-411 p-,60 72 70.9 72 57-86 TOTAL 113 #univariate Anova 274 3M MN03112466 TABLE A4.1.16 ESTRADIOL TO FOLLICLE STIMULATING HORMONE RATIO (E/FSH) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA E/FSH N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 8.7 5.51 6.8 1.3-23.3 F-.50 56 7.8 5.76 6.6 1.4-29.6 P-.61 17 9.3 7.04 7.6 2.65-33.1 AGE <31 31-40 41-50 51-60 20 10.8 5.80 9.6 3.1-25.0 F-5.00 46 9 6.01 72 1.4-33.1 P-.003 26 6.9 6.13 4.8 1.3-29.6 18 4.9 227 4.6 1.5-9.3 Alcohol <ioz/d 86 8.1 5.83 6.6 1.3-33.1 F-.01 1-302/d 19 8.3 4.99 7.1 3.1-19.1 P-.91 missing 8 10.9 7.94 83 4.6-29.6 Tobacco smoker nonsmoker missing 27 84 2 8.1 6.82 4.7 1.4-29.6 F-.13 8.5 5.59 7.0 1.3-33.1 P-.72 5.1 2JS6 5.1 3J2-6.9 TOTAL 113 #univariate Anova 275 3M MN03112467 TABLE A4.1.17 THYROID STIMULATING HORMONE TO PROLACTIN RATIO (TSH/P) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT. COTTAGE GROVE, MINNESOTA TSH/P N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 0.22 0.17 0.18 0.05-0.83 F-.71 56 0.25 0.22 ai9 0.02-121 P-.49 17 0.28 0.19 026 0.07-021 AGE <31 31-40 41-50 51-60 20 0.17 0.09 0.15 0.05-029 F-1.38 48 0.25 023 0.17 0.02-121 p-25 26 0.26 0.18 022 0.06-0.83 18 0.29 020 020 0.07-0.81 Alcohol <1oz/d 86 0.24 0.19 0.19 0.04-121 F-2.15 1-3oz/d 19 0.29 024 0.17 0.02-1.00 P-.15 m issing 8 0J21 0.08 020 0.09-020 Tobacco sm oker nonsm oker m issing 27 84 2 0.29 025 021 0.09-121 F-1.00 0.23 0.18 0.18 0.02-1.00 p-22 0.14 0.08 0.14 0.09-020 TOTAL 113 #univariate Anova 276 3M MN03112468 TABLE A4.1.18 FREE TESTOSTERONE TO PROLACTIN RATIO (TF/P) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TF/P N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 2JS 1.47 22 0.6-7.8 F-.19 56 23 Z13 1.9 05-15.0 P-.83 17 2.1 1.12 2.0 05-4.6 AGE <31 31-40 41-50 51-60 20 2.4 1.67 Z0 0.7-7.8 F-.72 48 2.6 227 Z1 05-15.0 p-54 26 Z1 1.05 ZO 0.7-42 19 2.0 1.19 1.6 0.8-5.6 Alcohol <ioz/d 1-3oz/d missing 86 19 6 2.4 1.94 ZO 0.6-15.0 F-.19 2JO 1.20 1.5 0.5-5.1 p-57 2.6 0.75 Z7 1.5-3.8 Tobacco smoker 27 32 2.81 Z4 1.1-15.0 F-9.58 nonsmoker 84 Z1 1.18 1.9 0.5-7.8 P-.003 missing 2 22 220 22 0.7-35 TOTAL 113 #univariate Anova 277 3M MN03112469 TABLE A4.1.19 BOUND TESTOSTERONE TO PROLACTIN RATIO (TB/P) BY BODY MASS INDEX AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PUNT, COTTAGE GROVE, MINNESOTA tB /P .. N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 AGE <31 31-40 41-50 51-60 Alcohol < io z/d 1-3oz/d m issing Tobacco sm oker nonsmoker m issing TOTAL 40 56 17 20 48 26 19 86 19 8 27 84 2 113 87 46.4 82 20-221 F-.60 87 85.0 64 22-624 pmJBS 68 36.1 67 28-158 75 47.1 68 20-206 F-.B3 96 91.0 79 22-624 P-.48 78 41.6 72 23-163 73 34.4 63 34-158 87 73.0 72 20-624 F-1.23 68 49.9 49 22-221 P-.27 95 20.8 100 55-117 121 122.8 94 27-624 F-11.31 73 38.7 66 22-206 P-.001 60 56.8 60 20-100 #univariate Anova 278 3M MN03112470 TABLE A4.1.20 ESTRADIOL TO PROLACTIN RATIO (E/P) BY BODY MASS INDEX, AGE. SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA E/P N MEAN SD MEDIAN RANGE TEST# BMI <25 25-30 >30 40 4.7 2.80 4.4 1.1-13.8 F-.19 56 5.1 4.88 3.9 1.1-325 P-.82 17 5.1 2.68 42 1.9-9.7 AGE <31 31-40 41-50 51-60 20 4.2 250 4.1 1.1-90 F-1.09 48 5.7 5.16 4.6 1.1-325 p-56 26 4.7 322 4.0 1.1-15.1 19 4.3 2.00 3.7 25-9.6 Alcohol <1oz/d 86 5.0 4.11 4.1 1.1-325 F-59 1-3oz/d 19 4.2 2.94 3.1 1.1-13.0 P-.45 m issing 8 6^ 4.15 4.6 3.0-15.1 Tobacco sm oker nonsm oker m issing 27 84 2 75 6.67 52 1.1-32.5 F-1251 42 2.16 4.0 1.1-105 P-.001 4.6 4.84 4.6 1.1-8.0 TOTAL 113 funivariate Anova 279 3M MN03112471 TABLE A4.1.21 FOLLICLE STIMULATING HORMONE TO PROLACTIN RATIO (FSH/P) BY BODY MASS INDEX, AGE, SMOKING AND DRINKING STATUS 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA f s h Tp " N MEAN SD MEDIAN RANGE TEST# BMI <25 25*30 >30 40 0.72 0.51 057 05-2.1 F-52 56 0.79 0.52 0.65 0.1-2.6 50 17 0.74 058 057 05-2.6 AGE <31 31-40 41-50 51-60 20 0.46 053 0.45 05-1.0 F-5.41 48 0.71 051 054 0.1-25 P-.002 26 0.88 050 0.73 05-2.1 19 1.05 0.62 051 05-2.6 Alcohol <loz/d 1-3oz/d 86 0.81 057 0.66 05-2.1 F-3.18 19 0.57 052 050 0.1-25 P-.08 missing 8 0.66 051 0.60 05-25 Tobacco smoker nonsmoker missing 27 84 2 1.00 057 0.79 0.3-25 F-7.90 0.68 0.49 051 0.1-25 P-.006 0.75 057 0.62 0.3-15 TOTAL 113 #umvariate Anova 280 3M MN03112472 APPENDIX 3 TABLES OF HORMONE RATIOS BY TOTAL SERUM FLUORIDE 281 3M MN03112473 TABLE A4.2.1 HORMONE RATIOS BY TOTAL SERUM FLUORIDE: ESTRADIOUFREE TESTOSTERONE (E/TF) ESTRADIOL/BOUND TESTOSTRONE (E/TB) ESTRADIOUTHYROID STIMULATING HORMONE (E/TSH) 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLUORIDE ppm <1 >=1-3 >3-10 >10-15 >15-26 TOTAL N 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 #univariate Anova MEAN 2.5 2.1 23 2.6 2.7 235 73 5.9 6.7 6.9 63 6.4 293 24.9 26.7 193 19.8 253 SD MEDIAN RANGE E/TF TEST# 13 23 0.7-53 F-1.65 03 1.9 03-5.4 P-.16 03 23 0.8-33 1.1 2.1 1.64.0 0.7 3 1.933 .92 2.1 0.7-5.4 E/TB (X100) 33 23 23 23 13 23 6.3 5.5 5.9 5.6 53 53 1.1-14.4 1.7-13.5 2.9-12.3 43-11.7 4.8-8.4 13-14.4 F-1.17 P-.33 E/TSH 21.8 133 16.7 1.9 6.8 153 21.0 10.4-108.1 F-0.75 23.6 1.8523 p=36 21.1 3.859.0 15.8 7.8453 16.9 153-313 213 1.76-108.1 282 3M MN03112474 TABLE A4.2.2 HORMONE RATIOS BY TOTAL SERUM FLUORIDE: BOUND TESTOSTERONE/FREE TESTOSTRONE (TB/TF) BOUND TESTOSTRONE/FOLLICLE STIMULATING HORMONE (TB/FSH) BOUND TESTOSTERONE/PROLACTIN (TB/P) FREE TESTOSTERONE/PROLACTIN (TF/P) 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLUORIDE ppm <1 >=1*3 >3-10 >10-15 >15-26 TOTAL N 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 funivariate Anova MEAN 36.7 36.8 34.5 38.3 43.6 36.8 148.3 130.9 135.3 122.7 162.9 136.1 82.1 87.5 86.4 51.5 90.3 84.5 2.34 2.38 2.64 1.40 2.20 2.35 SD MEDIAN RANGE TEST# TB/TF 9.6 9.6 7.0 7.6 10.6 92 TB/FSH 85.6 76.3 81.4 48.3 76.4 77.1 TB/P 432 812 51.0 27.9 30.6 67.3 TF/P 1.46 1.97 1.84 0.82 0.91 1.78 37.1 35.6 33.3 38.9 39.9 37.0 120.4 114.2 86.9 135.8 143.5 120.0 67.5 63.3 78.0 52.1 83.4 70.7 2.01 1.88 2.40 1.35 2.16 1.95 16.7-62.6 19.2-58.8 25.0-43.6 29.3-47.6 36.9-62.4 16.7-62.6 F-34 P-45 <--10vs>10 T-2.10 P-.15 56.7-411.0 23.1-347.7 49.0-303.3 34.4-169.8 67.1-253.5 23.1-411.0 F-.40 P-.81 19.8-2053 23.0-6242 28.1-2242 222-89.3 58.3-129.7 19.8-6242 F-.40 P-.81 .7-7.8 .6-15.0 .8-8.1 3-2.3 .9-3.5 3-15.0 F-32 P-.72 283 3M MN03112475 TABLE A4.2.3 HORMONE RATIOS BY TOTAL SERUM FLUORIDE: ESTRADIOL/PROLACTIN (E/P) THYROID STIMULATING HORMONE/PROLACTIN (TSH/P) FOLLICLLE STIMULATING HORMONE/PROLACTIN (FSH/P) PR0LACTIN/LUTENI2ING HORMONE (P/LH) 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLUORIDE ppm <1 >=1-3 >3-10 >10-15 >15-26 TOTAL N 23 84 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-% TOTAL 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL. 23 63 15 6 5 112 #univaiiate Anova MEAN 5.34 4.75 5.87 3.13 5.55 4.97 0.22 0.24 0.29 0.24 0.29 0.24 0.65 0.80 0.87 0.52 0.66 0.76 1.71 1.85 1.79 3.14 1.5 1.87 SD E/P 2.77 4.38 4.65 1.08 1.69 3.94 TSH/P 0.11 0.21 0.76 0.14 0.09 0.20 FSH/P 0.47 0.52 0.68 0% 036 0X2 P/LH 0.65 1.75 1.20 3X0 0.44 1% MEDIAN RANGE TEST# 4X1 1X5-10X2 F-.64 3.77 1.09-32X0 P-.63 4.76 1.87-17.89 3.45 1.13-4.07 6.17 3.11-7.09 4.08 1.09-32X0 0X1 0.07-0.48 F-.40 0.17 0.04-1X0 p--.81 0X0 0.07-0.85 0X5 0.02-0.41 0X7 0X0-0.44 0.19 0.02-1X0 0.60 0.18-2X6 F-.89 0.66 0.15-2.17 P-.47 0X7 0.15-2X8 0.47 0.13-1.04 0.47 0X3-1.13 0.60 0.13-2X8 1.64 . 0.35-3.02 1X7 0X1-6X9 1X5 0.39-4.00 1.88 1.10-9.11 1X3 1.03-2.10 1.62 0X5-9.11 F-1.72 P-.15 284 3M MN03112476 TABLE A4.2.4 HORMONE RATIOS BY TOTAL SERUM FLUORIDE: ESTRADIOL/LUTENIZING HORMONE (E/LH) ESTRADIOL/FOLLICLLE STIMULATING HORMONE(E/FSH) FOLLICLLE STIMULATING HORMONE/LUTENIZING HORMONE (FSH/LH) 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLUORIDE ppm <1 >=1-3 >3-10 >10-15 >15-26 TOTAL N 23 63 15 6 5 112 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 63 15 6 5 112 #univariate Anova MEAN B.64 6.85 724 7.21 7.99 7.34 10.27 7.71 7.74 8.04 1022 6.37 0.91 1.04 0.99 1.08 0.91 1.00 SD MEDIAN RANGE TEST# E/LH 4.73 4.01 2.74 2.15 2.71 3.91 E/FSH 6.85 5.98 326 424 6.60 521 FSH/LH 020 0.44 0.43 020 025 0.41 828 6.19 725 7.09 7.03 7.01 8.69 6.10 629 827 7.03 6.85 0.89 0.940.91 1.03 0.99 0.94 121-18.81 0.95-20.59 1.96-1122 429-1027 6.03-12.70 0.95-20.59 126-33.12 120-29.60 3.71-12.42 3.09-15.30 5.45-2120 120-33.12 0.42-1.48 0.37-225 0.41-1.% 0.53-1.78 025-1.40 027-2% F-.92 P-.45 F-1.00 p-041 F-.41 P-.73 285 3M MN03112477 TABLE A4.2.5 HORMONE RATIOS BY TOTAL SERUM FLUORIDE: FREE TESTOSTERONE/THYROID STIMULATING HORMONE (TF/TSH) BOUND TESTOSTRONE/THYROID STIMULATING HORMONE (TB/TSH) FREE TESTOSTERONE/LUTENIZING HORMONE (TF/LH) BOUND TESTOSTERONE/LUTENIZING HORMONE (TB/LH) 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLUORIDE ppm <1 >=1-3 >3-10 >10-15 >15-26 TOTAL N 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 <1 >=1-3 >3-10 >10-15 >15-26. TOTAL 23 64 15 6 5 113 #univariate Anova MEAN SD MEDIAN RANGE TEST# TF/TSH 12.6 8.5 9.5 4.5-35.1 F-.93 12.7 7.5 11.1 1.7-43.7 p-,45 11.9 6.8 10.4 3.2-27.2 8.5 1.7 7.5 12.0-20.7 7.5 1.9 7.9 4.6-9.6 12.1 7.5 9.9 1.7-43.7 TB/TSH 456 330 320 170-1367 Fat.54 479 363 370 51-2102 p-,70 416 270 401 95-1185 334 296 226 87-900 314 49 317 247-370 451 333 353 51-2102 TF/LH 3.7 2.3 3.3 1.2-11.3 F-.28 3.5 1.8 3.2 0.6-9.1 p-,89 3.3 1.0 3.3 1.2-5.6 3.1 1.33 2.89 1.4-4.6 3.0 0.8 3.4 1.9-3.9 3.4 1.7 3.2 0.6-11.3 TB/LH 127 66 125 39-298 F-.13 121 58 114 24-288 P-.93 115 51 105 52-234 118 56 105 61-201 127 21 125 122-149 122.1 57.3 118 24.3-298 286 3M MN03112478 TABLE A4.2.6 HORMONE RATIOS BY TOTAL SERUM FLUORIDE: THYROID STIMULATING HORMONE/FOLLICLE STIMULATING HORMONE (TSH/FSH-. THYROID STIMULATING HORMONE/LUTENIZING HORMONE (TSH/LH) 1990 PERFLUOROCHEMICAL EFFECTS STUDY, 3M CHEMOLITE PLANT, COTTAGE GROVE, MINNESOTA TOTAL FLUORIDE ppm <1 >=1-3 >3-10 >10-15 >15-26 TOTAL N 23 64 15 6 5 113 <1 >*1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 <1 >*1-3 >3-10 >10-15 >15-26 TOTAL 23 64 15 6 5 113 #univariate Anova MEAN 0.42 0.40 0.44 0.49 0.49 0.42 0.36 0.36 0.38 0.45 0.40 0.37 4.40 3.77 3.78 3.42 3.93 SD MEDIAN RANGE TSH/FSH 0.24 0.38 0.33 0.25 0.17 0.33 TSH/LH 0.22 0.30 0.27 0.19 0.37 0.26 TF/FSH 3.27 Z38 1.84 1.63 Z15 0.40 0.29 0.35 0.49 0.45 0.35 0.33 0.28 0.31 0.43 0.40 0.31 3.7 3.1 3.2 3.9 3.6 0.08-0.89 0.06-2.34 0.06-1.20 0.19-0.80 0.27-0.68 0.06-2.34 0.08-1.00 0.04-1.70 0.04-1.1 0.22-0.70 0.36-0 45 0.04-1.70 1.5-15.6 .7-11.1 1.7-7.3 .8-5.3 1.8-6.6 TEST# F-0.23 P-.92 F-0.23 p-,92 F-.34 p-,85 287 3M MN03112479 TABLE 4.1.78 LINEAR MULTIVARIATE REGRESSION MODEL OF FACTORS PREDICTING THE HEMAGLOBIN AMONG 111 MALE WORKERS. 3M CHEMOUTE PLANT, COTTAGE GROVE, MINNESOTA Variable B SE(B) p-value Intercept Total Fluorine (ppm)* Alcohol# low(<1oz/day) nonresponse (NR) Age (years) BMI (kg/m2) Cigarettes/day Cigs/day X Fluorine2** Estradiol (pg/ml) 14.51 -.002 .22 .56 .001 .01 .01 .0003 .01 .67 .0009 .20 .33 .009 .02 .007 .0001 .006 .0001 .02 .27 .09 .88 .65 .20 .0005 .07 R2-.23 1 ` square transformation of total fluoride #Reference category is moderate drinkerswhoconsume 1-3oz ethanol/day. interactiontermbetweencigarettes perdayandsquaretransformationoftotal fluoride 3M MN03112480