Document jmjbQN2yXXBXQdO4JB85Dj5MN

P-1 "McCrea, Deborah" <mccrea@taftlaw.com> 01/21/2010 02:58 PM To NCIC OPPT@EPA cc "Bilott, Robert A." <bilott@taftlaw.com> bcc Subject 01/21/2010 Letter To EPA Docket Center Taft/ Deborah McCrea / Legal Assistant Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1800 Cincinnati, Ohio 45202-3957 Tel: 513.381.2838 Fax: 513.381.0205 www.taftlaw.com / mccrea@taftlaw.com Internal Revenue Service Circular 230 Disclosure: As provided for in Treasury regulations, advice (if any) relating to federal taxes that is contained in this communication (including attachments) is not intended or written to be used, and cannot be used, for the purpose of (1) avoiding penalties under the Internal Revenue Code or (2) promoting, marketing or recommending to another party any transaction or matter addressed herein. This message may contain information that is attorney-client privileged, attorney work product or otherwise confidential. If you are not an intended recipient, use and disclosure of this message are prohibited. If you received this transmission in error, please notify the sender by reply e-mail and delete the message and any attachments. CONTAINS NO CBI ft)R 3 & W S P-2 Taft/ Taft Stettinius & HoHister LlP 4 2 5 W alnut Street, Suite 1 8 0 0 /C incinnati. OH 4 5 2 02 -3 9 5 7 /Tel: 5 1 3 .3 8 1 .2 8 3 8 /F ax: 513.3 8 1 .0 2 0 5 / w w w .taftlaw .com Cincinnati / Cleveland / Columbus / Dayton / Indianapolis / Northern Kentucky / Phoenix / Beijing ROBERT A . BtLOTT 5 1 3 -3 5 7 -9 6 3 8 January 21, 2010 FEDERALEXPRESS EPA Docket Center, MC 2822T U.S. Environmental Protection Agency EPA W est, Room 3334 1301 Constitution Avenue, NW Washington, D.C. 20004 r- Re: Submission to IRIS and AR-226 Database For PFOA/PFOS: EPA-HQO R D -2003-0016 To IR IS Database for PFOA/PFOS: In response to the Notice issued by USEPA on February 2 3 ,2 0 0 6 , regarding USEPA's efforts to consider perfluorooctanoic acid ("PFOA") and perfluorooctane sulfonate ("PFOS") within the Integrated Risk Information System ("IRIS"), 71 Fed. Reg. 9333-9336 (Feb. 23, 2006), we are submitting the following additional information to USEPA for inclusion in that review, and for inclusion in the AR-226 database: 1. Melzer, D., ef a/., "Association Between Serum Perfluorooctanoic Acid (PFOA) and Thyroid Disease in the NHANES Study," Environ. Health . Persp. (online doi: 10.1289/ehp.0901584) (Jan. 2 0 ,2 0 1 0 )." RABrmdm Enclosure cc: Gloria Post (NJDEPXw/ end.) (via U.S. Mail) Helen Goeden (M DH)(w/ end.) (via U.S. Mail) Lora W erner (ATSDR)(w/ end.) (via U.S. Mail) 11617362.1 M R r 32-4-2/S ehponline.org ENVIRONMENTAL HEALTH PERSPECTIVES P-3 Association Between Serum Perfluorooctanoic Acid (PFOA) and Thyroid Disease in the NHANES Study David Melzer, Neil Rice, Michael H Depledge, William E Henley, Tamara S Galloway doi: 10.1289/ehp.0901584 (available at http://dx.doi.org/) Online 20 January 2010 National Institute of ` Environmental Health Sciences Page 1 of 35 P-4 Association Between Serum Perfiuorooctanoic Acid (PFOA) and Thyroid Disease in the NHANES Study David Melzer1, Neil Rice1, Michael H Depledge2, William E Henley3, Tam ara S Galloway4. From the Epidemiology and Public Health Group Peninsula Medical School1, Exeter, UK, Environment and Human Health Group, Peninsula Medical School, Exeter, UK2, School of Mathematics and Statistics, University of Plymouth3, School of Biosciences, University of Exeter, UK4; Corresponding author: Tamara Galloway, Professor of Ecotoxicology, School of Biosciences, Prince of Wales Road, Exeter EX4 4PS, United Kingdom email t.s.gallowav@exeter.ac.uk Tel. +44 (0)1392 263436, Fax. +44 (0)1392 263700 1 P-5 Page 2 of 35 Acknowledgments We thank everyone involved in NHANES especially those who carried out the assays of PFOA concentrations at the Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention. Funding was from Peninsula Medical School and University of Exeter internal support. No funding organization or sponsor played any part in the design or conduct of the study; in the analysis or interpretation of the data; or preparation, review, or approval of the manuscript. The authors declare no competing interests. Short running head: PFOA and thyroid disease Key words: C8, human population, PFOA, PFOS, thyroid disease Abbreviations C8 alternative brief name for Perfluorooctanoic acid PFOA Perfluorooctanoic acid PFOS Periluorooctane sulfonate PFC Perfluorinated chemicals 2 Outline of manuscript section headers Abstract Introduction Aim Methods Results Discussion Conclusions P-7 Page 4 of 35 Abstract: BACKGROUND: Perfluorooctanoic acid (PFOA, `C8') and perfluoroctane sulphonate (PFOS) are stable compounds with many industrial and consumer uses. Their persistence in the environment plus toxicity in animal models has raised concern over low-level chronic exposure effects to human health. OBJECTIVES: To estimate associations between serum PFOA and PFOS concentrations and thyroid disease prevalence in representative samples of the United States general population. METHODS: Analyses of PFOA/PFOS against disease status in the Health and Nutrition Examination Survey (NHANES) 1999-2000, 2003-2004 and 2005-2006. Included were 3974 adults with measured for PFC concentrations. Regression models were adjusted for age, sex, race/ethnicity, education, smoking status, body mass index, and alcohol intake. RESULTS: In women, the NHANES weighted prevalence of reporting any thyroid disease was 16.18% (n=292), for men 3.06% (n=69): prevalence of current thyroid disease taking related medication was 9.89% (n=163) for women and 1.88% (n=46) for men. In fully adjusted logistic models, women with PFOA >5.7ng/ml (top population quartile `4') were more likely to report current treated thyroid disease: Odds Ratio = 2.24 (95%CI: 1.38 to 3.65, p=0.002) compared to PFOA <4.0ng/ml (quartiles 1&2). In men, there was a near significant similar trend OR=2.12 (Cl 0.93 to 4.82, p=0.073). 4 Page 5 of 35 p.8 For PFOS in men, a similar association was present comparing those with PFOS>36.8 ng/ml (Q4) to those with PFOS concentrations <25.5 ng/ml (Q1&Q2): OR for treated disease 2.68 Cl: 1.03 to 6.98, p=0.043. In women this association was not significant. CONCLUSIONS: Higher concentrations of serum PFOA and PFOS are associated with current thyroid disease in the US general adult population. More work is needed to establish the mechanisms involved and to exclude confounding and pharmacokinetic explanations. 5 P-9 Page 6 of 35 Introduction The perfluoroalkyl acids (PFAAs) are a family of synthetic, highly stable perfluorinated compounds with a wide range of uses in industrial and consumer products, from stainand water-resistant coatings for carpets and fabrics to fast-food contact materials, fireresistant foams, paints and hydraulic fluids (OECD 2005). The carbon-fluoride bonds that characterize PFAAs and make them useful as surfactants are highly stable and recent reports indicate the widespread persistence of certain PFAAs in the environment and in wildlife and human populations globally (Fromme 2009; Giesy and Kannan 2001; Lau 2007; Saito et al. 2004). Two of the PFAAs of most concern are the eight carbon chained perfluoroctane sulphonate (PFOS) and perfliiorooctanoic acid (PFOA, `C8')- Most persistent organic pollutants are lipophilic and accumulate in fatty tissues, but PFOS and PFOA are both lipo- and hydrophobic, and following absorption will bind to proteins in serum rather than accumulating in lipid (Hundley 2006; Jones et al. 2003). The renal clearance of PFOA and PFOS is negligible in humans, leading to reported half lives in blood serum of 3.8 and 5.4 years for PFOA and PFOS respectively (Olsen 2007). Human biomonitoring of the general population in various countries has shown that in addition to the near ubiquitous presence of PFOS and PFOA in blood, they may also be present in breast milk, liver, seminal fluid and umbilical cord blood (Lau 2007). Extensive laboratory studies of die toxicology of PFOA and PFOS have reported enlargement of the liver, modulation of sex hormone homeostasis, developmental and immune system toxicity, hypolipidemia and reduced body weight in rodent and non Page 7 of 35 p. 10 human primate models (reviewed in Lau 2004;Lau 2007). Research interest has focused on the ability of these compounds to bind to nuclear receptors including the peroxisome proliferator activating receptor (PPARa), and to disrupt serum protein ligand binding (Luebker 2002), highlighting PFOA and PFOS as potential endocrine disruptors (Jensen 2008). Endocrine systems that may be targets of endocrine disrupting chemicals include the hypothalamus-pituitary-thyroid axis (HPT) (Boas 2006). Thyroid hormone is essential for the normal physiological function of nearly all mammalian tissues. Thyroid hormone status is controlled by a well-established feedback mechanism, in which thyroidstimulating hormone (TSH) stimulates the thyroid to synthesize T4. which is then converted to the biologically active T3. The rate of release of TSH is regulated by the hypothalamus as well as by the circulating levels of T3 and T4. Therefore, multiple physiological steps including hormone biosynthesis, transport, metabolism or action on target cells are required for thyroid hormone homeostasis. Numerous studies have now shown PFAAs to impair thyroid hormone homeostasis in animal studies. Depression of serum T4 and T3has been reported by several authors in PFOS-exposed rats (Lau et al. 2003;Luebker 2005; Seacat 2003), without the concomitant increase in TSH that would be expected through feedback stimulation. Earlier mechanistic studies of the structurally related compound perfluorodecanoic acid (PFDA) showed that it could reduce serum thyroid hormone levels by apparently reducing the responsiveness of the HPT axis and by displacing circulating thyroid hormones from their plasma protein binding sites (Gutshall 1989). Whilst circulating hormone levels were depressed, the activities of thyroid hormone sensitive liver enzymes were elevated, suggesting that functional hypothyroidism was not occurring. A similar 7 p. 11 Page 8 of 35 mechanism for PFOS has been hypothesized (Chang 2007). A recent study of the mechanisms involved in PFOS-induced hypothyroxinemia in rats has indicated that increased conjugation of T4 in the liver, catalysed by the hepatic enzyme uridine dihosphoglucuronosyl transferase (UGT1A1), and increased thyroidal conversion of T4 to T3 by type 1 deiodinase may be partly responsible for the effects seen (Yu 2009). Taken together, these findings suggest that the effects of PFAAs on thyroid hormone physiology are multiple and complex. Extrapolations from animal laboratory studies such as these to an estimation of the risks posed by PFOA and PFOS to thyroid function in humans are complicated by the extreme variations reported in their toxicokinetic profile between species (Johnson 1979; Olsen 2007). The extremely long half lives of PFOA and PFOS in humans are in contrast with the relatively rapid elimination seen in animal models: the serum half life of PFOS in rats is around 100 days (Hundley et al. 2006), drawing attention to the potential risks to human health. Disruption to thyroid hormone balance was not found in previous studies of community exposure to PFOA (Emmett 2006; Olsen et al. 2003a) or PFOS (Inoue 2004). Modest associations between PFOA and thyroid hormones (negative for free T4 and positive for T3) were reported in 506 PFOA production workers across three production facilities (Olsen and Zobel 2007). There were no associations between TSH or T4 and PFOA and the free hormone levels were within the normal reference range. Given the.evidence from animal studies of thyroid hormone imbalance and the varied epidemiological results from community and occupational exposures, we aimed to explore the hypothesis that higher serum PFOA and PFOS concentrations would be 8 p. 12 Page 9 of 35 associated with thyroid disease in the general adult population. The U.S. Centers for Disease Control and Prevention (CDC) environmental chemical biomonitonng program, using samples from the US National Health and Nutrition Examination Survey (NHANES), provides large scale data on serum PFAA concentrations in population representative samples. Here, we use these data to estimate associations between PFOA/S concentrations and thyroid disease in representative samples of the general population of the USA. Methods Study population. Data were from three independent cross-sectional waves of the US National Health and Nutrition Examination Survey (NHANES) 1999-2000,2003-2004 and 2005-2006. NHANES surveys assess the health and diet of the non-institutionalized civilian population of the United States and are administered by the National Center for Health Statistics. The study protocol for NHANES was approved by the National Centers for Health Statistics Institutional Review Board. Assessment o f PFOA/S concentrations. Solid phase extraction coupled to High Performance Liquid Chromatography-Turbo Ion Spray ionization-tandem Mass Spectrometry with isotope labeled internal standards was used for the detection of PFOA and PFOS, with a limit of detection of 0.2 ng/ml. 9 p. 13 Page 10 of 35 (Kuklenyik 2005). The laboratory methods and comprehensive quality control system were consistent in each NHANES wave, and documentation for each wave are available (1999-2000: http.7ywww.cdc.gov/nchs/data/nhanes/freQuencv/ssDfc a.ndf ; 2003-2004: http://www.cdc.gov/nchs/data/nhanes/nhanes 03 04/124pfc c.pdf ; and 2005-2006: http://www.cdc.gov/nchs/nhanes/nhanes2005-2006/lab methods 05 06.htm - accessed 2nd October 2009). Serum polyfluorinated chemicals (PFCs) were measured in a one third representative random subset of persons 12 years and over in each NHANES wave. Data from individuals undergo years old were excluded, as questions relating to disease prevalence were only asked of adults. Disease outcomes. In all NHANES waves, adult respondents were asked about physician diagnosed diseases. Associations were examined between PFOA and PFOS concentrations and thyroid disease outcomes. Individuals were asked whether they had ever been told by a doctor or health professional that they had a thyroid problem (in the 1999-2000 survey the questions related to goiter and other thyroid conditions), and whether they still had the condition. We further defined thyroid disease by considering those people who said they currently had thyroid disease and were taking any thyroid related medication, including lvothyroxine; liothyronine; `thyroid desiccated' and `thyroid drugs unspecified' for hypothyroidism and propylthiouracil and methimazole for hyperthyroidism. No details were available on specific thyroid disease diagnosis and the PFC samples did not overlap with the thyroid hormone measurement sub-samples in NHANES. 10 p. 14 Page 11 of 35 To assess disease specificity, associations were examined between PFOA and the other NHANES disease categories elicited: Ischemic heart disease (combining any diagnoses of coronary heart disease, angina, and/or heart attack), diabetes, arthritis, current asthma, Chronic Obstructive Pulmonary Disease (COPD: bronchitis or emphysema) and current liver disease. Statistical analysis. NHANES uses a complex cluster sample design with some demographic groups (including less privileged socioeconomic groups and Mexican Americans) over-sampled to ensure adequate representation. Prevalence estimates and models were therefore survey weighted using the NHANES primary sampling unit, strata and population weights, unless otherwise stated. Multivariate logistic regression modeling was used to estimate odds ratios of thyroid disease outcomes by quartile of PFOA and PFOS concentrations, and associations of other physician-diagnosed diseases. As thyroid disease prevalence is markedly higher in women, we used sex specific models. Because the distribution of PFC concentrations is skewed (with most people having relatively low-exposures and with considerably more variance at the higher exposure end), all available data were pooled and PFOA and PFOS concentrations were divided into population weighted quartiles. Using the Hsieh method (Hsieh 1998), our estimated power to detect an association of OR>1.8 with current treated disease comparing the top PFOA quartile to bottom quartile is 67% in women. Combining the lowest two quartiles into a larger control group provides 80% power. The corresponding minimum detectable effect size in men is an OR>2.9. Assumptions for the 11 p. 15 Page 12 of 35 power calculations include a significance level of 5%, and a multiple correlation coefficient of 0.2 relating PFOA exposure to potential confounders. Models were adjusted for the following potential confounding factors: year of NHANES study; age; gender, race/ethnicity, from self-description and categorized into: Mexican American, other Hispanic, non-Hispanic White, non-Hispanic Black, and other race (including multi-racial); education, categorized into: less than high school, high school diploma (including GED), more than high school, and unknown education; smoking (from self-reported status asked in those aged 20 and over), categorized into: never smoked, former smoker, smoking some days, smoking every day, and unknown smoking status; Body Mass Index (`BMP, measured weight in kilograms divided by the square of measured height in meters), categorized into: underweight (BMI <18.5), recommended weight (BMI 18.5 to 24.9), overweight (BMI 25.0 to 29.9), obese I (BMI 30.0 to 34.9), obese II (BMI 35.0 or above), and unknown BMI; alcohol consumption (in adults aged 20 and over - based on responses to the question "In the past 12 months, on those days that you drank alcoholic beverages, on the average day, how many drinks did you have?"), categorized into: 0,1, 2, 3, 4,5 or more drinks per day, and unknown alcohol consumption. Regression analyses were conducted using STATA SE 10.1. Results Serum concentrations of PFOA were available for n=3974 individuals aged 20 years and older from NHANES waves 1999-2000 (n=1040), 2003-2004 (n=1454) and 2005-2006 (n=1480). In age, gender, NHANES wave and ethnicity adjusted analyses, mean levels of PFOA were higher in men than women (by 0.76ng/ml (95% Cl: 0.73 to 0.80) p<0.0001), 12 Page 13 o f 35 p. 16 and there were significant differences between ethnic groups (Table 1). Individuals with more education had high: PFOA levels (highest vs. lowest education: l.lng/ml (95% Cl: 1.03 to 1.19, p=0.008). Increased alcohol consumption levels were also associated with higher PFOA concentrations (e.g. those drinking 5 or more drinks per day had mean PFOA levels 1.24ng/ml higher than non-drinkers (95% Cl: 1.14 to 1.37, p<0.0001)). In age, gender, NHANES wave and ethnicity adjusted analyses of die full sample (men and women), mean levels of PFOA were higher in men than women (p<0.0001), and there were significant differences between ethnic groups (Table 1). Individuals with more education had higher PFOA levels (p=0.008). Increased alcohol consumption levels were also associated with higher PFOA concentrations (p<0.0001)). Similar differences were found in PFOS concentrations. Mean levels of PFOS were higher in men (p<0.0001), with significant differences in levels between ethnic groups, and individuals with more education had higher PFOS levels (p=0.008). Eight individuals did not answer questions about thyroid disease, so the included sample size was n=3966, with 1900 men and 2066 women (Table 2). In women, the overall (unweighted) numbers reporting any thyroid disease was n=292 and the NHANES weighted but unadjusted prevalence was 16.18%: for men. n=69, weighted prevalence 3.06%. The study weighted prevalence of current thyroid disease taking medication was necessarily lower (n=163,9.89% for women; n=46,1.18% for men). Population weighted quartiles of PFOA and PFOS concentrations were computed for men and women separately (Table 2): the highest quartile (Q4) of PFOA for women 13 p. 17 Page 14 of 35 ranged from 5.7 to 123.0 ng/ml and for men 7.3 ng/ml to 45.9 ng/ml. Study weighted but unadjusted prevalences of current thyroid disease taking related medication in women varied across die quartiles, but with wide confidence intervals: Q l=8.14% (95%CI 5.75 to 10.53), Q4=16.19% (Cl 11.74 to 20.62). In men, unadjusted prevalence rates were far lower throughout (prevalence = 2.27% in Q1 and Q4). For PFOS the prevalence of treated thyroid disease ranged from 8.14% (Ql) to 12.55% (Q4) in women and for men 1.85% to 3.89%. In logistic regression models adjusting for age, ethnicity and study year (Table 3), there were associations between PFOA quartiles and both definitions of thyroid disease in women. For logistic models additionally adjusted for educational status, Body Mass Index, smoking status and alcohol consumption, these associations remained significant: e.g. comparing those with PFOA concentrations >5.7ng/ml (Q4) to PFOA <2.6ng/ml (Q l) the odds ratio for current thyroid disease on medication was OR= 1.86 (95%CI: 1.12 to 3.09), p=0.018. Comparing Q4 to the larger control group of PFOA<4.0ng/ml (Ql & Q2) the estimated odds ratio for treated thyroid disease was OR=2.24 (Cl 1.38 to 3.65), p=0.002. In men, there was a similar suggestive trend, but it narrowly missed significance: comparing PFOA >7.3ng/ml (Q4) to those with PFOA concentrations <5.2ng/ml (Q1&Q2) the odds ratio for treated disease was OR=2.12 (0.93 to 4.82), p=0.073. For PFOS concentrations, in women odds ratios for disease trended in a similar direction, but were far from significance. However, in men as association was present comparing 14 those with PFOS>36.8 ng/ml (Q4) to those with PFOS concentrations <25.5 ng/ml (Q1&Q2): odds ratio for treated disease was OR=2.68 (1.03 to 6.98), p=0.043. Sensitivity analyses. As a sensitivity analysis we computed a logistic regression model including both men and women, testing an interaction term between gender and PFOA levels for treated thyroid disease risk: the interaction term was not significant (p-value for interaction = 0.152. As a post-hoc analysis we examined associations between chemical concentration quartile and any of the other major disease categories covered in NHANES: arthritis, asthma, Chronic Obstructive Pulmonary Disease (COPD), diabetes, Heart disease, or liver disease. Combining men and women (to reduce multiple testing and because these diseases are less gender related) we found no significant associations for PFOA (Table 4) except for comparisons of the intermediate quartiles to Q 1 for arthritis: this did not reach significance in the top quartile. For PFOS, there were no `positive' associations between higher serum concentrations and higher prevalence of disease. There was one statistically significant `negative' association suggesting that people reporting having Chronic Obstructive Pulmonary Disease (COPD) may be less likely to be in the highest PFOA concentration quartile (OR=0.58 Cl 0.43 to 0.76, p=0.0003). p. 19 Page 16 of 35 Discussion This study aimed to determine whether increased serum PFOA or PFOS concentrations were associated with thyroid disease in a general adult US population sample. The prevalence of thyroid disease is markedly higher in women than in men, and therefore we have estimated sex specific associations. We have shown that across all the available data from NHANES, thyroid disease associations with serum PFOA concentrations are present in women and are strongest for those currently being treated for thyroid disease. For men, a near significant association between PFOA and treated thyroid disease was also present: an interaction term analysis suggests that the PFOA trends in men and women are not significantly different, despite the relative rarity of thyroid disease in men. In addition, a nominally significant association was present between PFOS concentrations and treated thyroid disease in men, but not in women. The presence of associations with both PFOA and PFOS raises the issue of how best to perform risk assessments for combinations of perfluorochemicals. The somewhat divergent risk patterns for the two compounds supports their separate risk assessment (Scialli et al. 2007), given that current legislative advice (US EPA 2000; MDH 2008) is to consider the combined effects of chemicals only when two or more chemicals in a mixture affect the same tissue, organ or organ system. Our results are important because PFAAs are detectable in virtually everyone in society (Kannan et al. 2004) with ubiquitous presence across global populations (Calafat et al. 2006). Occupational exposure to PFOA reported in 2003 showed mean serum values of 16 Page 17 of 35 1,780 ng/ml (range 40-10,060 ng/ml (Olsen et al. 2003b) and 899 ng/ml (range 722-1,120 ng/ml, (Olsen et al. 2003c)) Production of PFOS was halted in 2002 in the USA by its principal producer, due largely to concerns over bioaccumulation and toxicity. Since then, voluntary industry reductions in production and usage of other perfluorinated compounds, such as the US EPA initiated PFOA Stewardship Programme (US EPA 2006) have contributed to a decreasing trend in human exposure for all perfluorinated compounds (with the notable exception of perfluorononanoic acid, PFNA) ( Calafat et al. 2007; Olsen et al. 2007). In May 2009, PFOS was listed under the Stockholm Convention on persistent organic pollutants (http://chm.pops.int/). Our results can be compared with previous studies of human populations and also of non human primates. A six month study of cynomolgus monkeys chronically exposed to PFOA showed no associations between PFOA and thyroid parameters, at mean serum PFOA concentrations higher than those reported in NHANES, although only male monkeys were involved (Butenhoff et al. 2002). The largest human study of PFOA is centered on an industrial facility in Washington, West Virginia, horn which PFOA spread to the population through air, water, occupational and domestic exposure in a `point source' contamination. The C8 Health Project (Steenland et al. 2009) has measured PFOA concentrations in over 69,000 residents. Markedly high concentrations were found, with an arithmetic mean of 83 ng/ml and a median concentration in serum of 28 ng/ml (http://c8sciencepanel.org/pdfs/Status Report factors associated with C8 levels Qct2008.pdf). far higher than the NHANES concentrations in the general population. Preliminary analyses report associations between PFOA and total cholesterol, LDL and triglyceride concentrations in multivariate 17 p. 21 Page 18 of 35 models adjusting for age, body mass index, sex, education, smoking, alcohol and regular exercise. Comprehensive cross-sectional and follow-on analyses of associations with thyroid disease have not yet been reported but are expected to be released in 2010-2011 (http://www.C8sciencepanel.0rg/studies.html#21. Importantly, disruption to thyroid hormone balance was not found in other studies of populations exposed to PFOA, despite the considerably higher levels reported in some studies (Emmet et al. 2006; Olsen et al. 2003a). Emmet et al. (2006) studied 371 residents of a community with long standing environmental exposure to PFOA. They found a median serum PFOA concentration of 181-571 ng/ml, but there was no association between serum PFOA and a history of thyroid disease. In a study which included thyroid hormone levels, a positive association was reported between serum PFOA concentration and T3 levels in occupationally exposed workers, although there were no changes in other thyroid hormones (Olsen et al. 2001). Modest associations between PFOA and thyroid hormones (negative for free T4 and positive for T3) were reported in 506 PFOA production workers across three production facilities (Olsen and Zobel 2007). There were no associations between TSH or T4 and PFOA and the free hormone levels were within the normal reference range. A linear extrapolation of the findings reported here would be expected to lead to associations being more evident at higher exposure levels, yet this is not supported by the literature. Non-linearity of response is not uncommon for receptor mediated systems such as endocrine-signalling pathways that act to amplify the original signal. Large changes in cell function can occur in response to extremely low concentrations, but which may 18 become saturated and hence unresponsive at higher concentrations (vom Saal and Hughes 2005; Welshons et al. 2003). The mechanisms involved in thyroid homeostasis are numerous and complex and there are multiple potential targets for disruption of thyroid hormone homeostasis (Schmutzler et al. 2007). These include thyrotropin receptor (Santini et al. 2003), iodine uptake by the sodium iodide transporter (Schroder van der Elst et al. 2004), type 1 5'-deiodinase (Ferreira et al. 2002), transthyretin (Kohrle et al. 1988), thyroid hormone receptor (Moriyama et al. 2002) and the thyroid hormone dependent growth of pituitary cells (Ghisari and Bonefeld-Jorgensen 2005). Depression of serum T4 and T3has been reported by several authors in PFOS-exposed rats (Lau et al. 2003; Luebker et al. 2005; Seacat et al. 2003). One mechanism by which PFAAs may deplete T4 is through induction of the hepatic uridine diphosphoglucuronosyl transferase (UGT) system, which is involved in hepatic metabolism of thyroid hormone and biliary clearance of T4as T4glucuronide (Barter and Klaassenl994). Since PFOA is an agonist for PPARa, it is plausible that induction of hepatic UGT in PFAA-exposed rats (Yu et al. 2009) could represent a PPARa mediated response. The involvement of another PPARa agononist, WY 14643, in enhancing the hepatic degradation of thyroid hormone has recently been shown (Weineke et al. 2009). A growing body of data describes the in vitro binding affinity of PFOA to human serum binding proteins (Chen and Guo 2009), PPAR a , p and y, and other nuclear receptors (Vanden Huevel et al. 2006), but the contribution of these mechanisms to PFOA's p. 23 Page 20 of 35 thyroid-mediating effects in humans remains to be established. Many cellular and metabolic processes including lipid metabolism, energy homeostasis and cell differentiation are controlled by PPARa. Early studies of the effects of PFAAs in rodents showed that a single dose lowered heart rate and body temperature and depressed T4and T3. Replacement of T4did not reverse the clinical symptoms of hypothermia (Gutshall et al. 1988; Langley and Pilcher 1985). Although circulating thyroid hormone levels were low, liver enzymes responsive to thyroid hormone levels were elevated, suggesting that thyroidal homeostasis was not functionally compromised. Chang et al. (2007) found that exposure to PFOS for up to 3 weeks did not affect functional thyroid status, as free T4, TSH and various thyroid-responsive liver enzymes were all unaffected. These findings, and later results have led to proposals that displacement of circulating thyroid hormones from plasma protein binding sites and a reduced responsiveness of the HPT axis contribute significantly towards PFOA's hypothyroid-inducing effects (Lau et al. 2007). Whatever the mechanisms involved, it is clear that more research is merited to clarify the pathways involved. The feedback mechanism by which the rate of release of TSH and the circulating levels of T3 and T4are regulated tends to show a low level of individual variation (FeltRasmussen et al. 1980). Therefore subtle disruption of the thyroid axis within normal reference ranges may have negative health consequences for the individual, whilst remaining within normal reference values, highlighting the importance of including both clinical and laboratory endpoints in such studies. The NHANES data do not allow specification of precise type of thyroid disease present, since it does not report on 20 p. 24 Page 21 of 35 individual hormone levels. PFOA concentration was positively associated with free T4 and negatively associated with T3 levels in a cohort of 506 exposed workers, with a near significant association with TSH levels (Olsen et al. 2007b), although all effects were regarded as modest. The limitations of these analyses should be noted. The PFOA and PFOS measures were based on a single serum sample. Although PFOA has a half life of four years {Olsen 2007) and therefore a single sample is likely to represent medium term internal dose, samples taken at several time points might be more accurate in classifying exposure. Any misclassification from single measures would tend to decrease power and under estimate the real strengths of association. Secondly, the PFOA concentrations were measured at the same time as disease status, making attribution of causal direction difficult. This raises the possibility of reverse causation. One might hypothesize that following onset of thyroid disease, changes in the nature of exposure or in the pharmacokinetics of PFOA might occur (including patterns of absorption, distribution (including protein binding) or excretion). As the associations we report were present in people who were on thyroid hormone replacements, which effectively mimic normal thyroid function, a mechanism for reverse causation through changes in pharmacokinetics is difficult to imagine. Confounding by unmeasured factors is also possible, but it is unlikely that confounding could explain similar findings reported from some of the diverse experimental and observational studies discussed above. Post-hoc association testing with other common diseases (necessarily involving multiple statistical testing) did not identify other robust associations of higher PFC concentration 21 p. 25 Page 22 of 35 with increased disease prevalence, suggesting specificity of our findings for thyroid disease. An apparent association between higher PFOS concentrations and lower prevalence of COPD requires replication, to exclude a false positive result from multiple testing. In addition to the limitations of our analyses, the strengths should also be noted: this is the first large-scale nationally representative general adult population analysis of directly measured serum concentrations of PFOA and PFOS. In addition, the associations present are strongest for the most specific identification of thyroid disease, based on reported diagnosis with current use of thyroid specific medication. The NHANES study also supported adjustment of models for a range of potential confounding factors, which in fact made relatively minor differences to the key estimates, suggesting that the associations are robust. Further work is clearly needed to characterize the PFOA and PFOS associations with specific thyroid diagnoses and thyroid hormone levels in the general population, and clarify whether the associations reflect pathology, changes in exposure or altered pharmacokinetics. Longitudinal analyses are also needed to establish whether high exposures predict future onsets of thyroid disease, although concurrent alteration of thyroid functioning would still be a cause for concern. Conclusions Higher PFOA and PFOS concentrations are associated with thyroid disease (and being on thyroid related medication) in the NHANES US general adult population representative 22 Page 23 o f 35 study samples. More work is needed to establish the mechanisms underlying this association and to exclude confounding and pharmacokinetic explanations. References Barter R, Klaassen C. 1994. Reduction of thyroid hormone levels and lateration of thyroid function by four representative UDP-glucuronosyl transferase inducers in rats. Toxicol. Appl. Pharmacol. 128: 9-17. Butenhoff J, Costa G, Elcombe C, Farrar D, Hansen K, Iwai H. et al. 2002. Toxicity of ammonium perfluorooctanoate in male cynomolgus monkeys after oral dosing for 6 months. Toxicol. Sci 69: 244-257. 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Environ Toxicol Chem 28(5): 990-996. 30 Page 31 of 35 Table 1: Survey weighted characteristics of sample with survey weighted back-transformed geometric mean concentrations of PFOA and PFOS. n (% within group) 1900 MEN Mean PFOA (96% Cl) 4.91 (4.64 to 5.2) Mean PFOS (95% Cl) 25.08 (23.63 to 26.62) n (% within group) 2066 WOMEN Mean PFOA (95% Cl) 3.77 (3.52 to 4.04) Mean PFOS (95% Cl) 19.14 (17.8 to 20.58) Age 20 to 49 50 to 69 70 + Ethnicity n (survey weighted %) Mexican American Other Hispanic Non-Hispanic White Non-Hispanic Black Other Race Education n (survey weighted %) Less titan high school High school graduate More than high school Unknown BMI Categories n (survey weighted %) Underweight (BMI 0-18.5) Normal (BM118.5-25) Overweight (BMI 25-30) Obese (BMI 30+) Unknown Smoking status n (survey weighted %) Smoked <100 cigarettes in lifetime Former smoker 928 (62.6%) 553 (27.0%) 419 (10.4%) 432 (8.3%) 62 (4.2%) 969 (73.5%) 382 (9.9%) 55 (4.1%) 637 (20.9%) 478 (29.3%) 782 (49.7%) 3 (0.0%) 23 (1.2%) 525 (28.8%) 778 (39.8%) 545 (29.0%) 29(1.2% ) 761 (41.1%) 664 (30.0%) 5.30 (5.02 to 5.59) 4.46 (4.12 to 4.82) 3.99 (3.53 to 4.52) 24.2 (22.66 to 25.84) 26.97 (24.87 to 29.25) 25.8 (23.22 to 28.67) 3.73 (3.48 to 4) 5.63 (4.77 to 6.65) 5.13 (4.83 to 5.45) 4.43 (3.92 to 5) 4.32 (3.47 to 5.38) 18.44 (16.79 to 20.25) 27.73 (21.89 to 35.14) 25.7 (24.04 to 27.47) 27 (24.3 to 30) 22.77 (17.3 to 29.97) 4.14 (3.82 to 4.49) 4.94 (4.56 to 5.35) 5.26 (4.93 to 5.62) 2.22 (1.31 to 3.74) 22.08 (20.47 to 23.82) 25.22 (23.56 to 27) 26.38 (24.54 to 28.36) 17.59 (8.5 to 36.41) 4.42 (2.78 to 7.01) 4.89 (4.44 to 5.39} 5.01 (4.67 to 5.38) 4.9 (4.54 to 5.29) 3.1 (2.23 to 4.3) 17.95 (10.79 to 29.87) 24.36 (21.99 to 26.98) 25.28 (23.55 to 27.13) 26.28 (24.42 to 28.28) 17.51 (10.34 to 29.66) 5.01 (4.68 to 5.37) 4.67 (4.28 to 5.09) 26.16 (24.19 to 28.29) 25.66 (23.7 to 27.79) 1134(59.8%) 545 (28.7%) 387 (13.4%) 481 (7.3%) 92 (5.9%) 1008(71.8%) 415(10.8%) 70 (4.3%) 628 (19.6%) 498 (25.9%) 937 (54.6%) 3 (0.0%) 35 (2.4%) 594 (32.6%) 615(27.8%) 784 (38.0%) 3 8 (1 .2 % ) 1300(58.6%) 417(21.3%) 3.38(3.1310 3.64) 4.62(4.1510 5.14) 4.13 (3.78 to 4.51) 16.72 (15.37 to 18.19) 22.92 (20.81 to 25.23) 24.39 (22.19 to 26.81) 2.44 (2.21 to 2.7) 3.6 (3.17 to 4.09) 4.13 (3.84 to 4.45) 2.98 (2.66 to 3.34) 3.3 (2.61 to 4.17) 12.04 (10.91 to 13,29} 16.28 (14.29 to 18.56) 20.08 (18.57 to 21.72) 20.52 (17.97 to 23.42) 19.64 (15.04 to 25.64) 3.53(3.11 to 4) 4 (3.69 to 4.34) 3.76 (3.49 to 4.04) 1.84 (1.32 to 2.56) 19.26 (17.1 to 21.69) 19.9 (18.15 to 21.83) 18.74 (17.32 to 20.28) 12.64 (7.25 to 22.02) 3.78 (3.07 to 4.65) 3.84 (3.52 to 4.19) 3.64 (3.3 to 4.01) 3.83 (3.49 to 4.2) 3.36 (2.78 to 4.05) 18.39 (14.05 to 24.08) 18.97 (17.53 to 20.52) 19.48 (17.86 to 21.24) 19 (17 to 21.22) 21.81 (17.13 to 27.78) 3.63 (3.36 to 3.93) 3.81 (3.46 to 4.19) 18.91 (17.43 to 20.52) 19.22 (17.33 to 21.32) 31 "O CJ P * Table 1 continued Some days Every day Unknown Drinking {average # drinks per day In past 12 months) n (survey weighted %) Non-drinker 1drink /day 2drinks / day 3 drinks / day 4 drinks/day 5+ drinks / day Unknown 90 (4.8%) 385 (24.1%) 5.12 (4.23 to 6.19) 5.01 (4.7 to 5.33) 334 (15.8%) 298 (15.7%) 282 (17.5%) 181 (11.1%) 97 (6.0%) 283 (16.4%) 425 (17.6%) 4.44 (3.89 to 5.06) 4.67 (4.22 to 5.17) 5.4 (4.86 to 6.01) 5.2 (4.79 to 5.65) 5.58 (4.94 to 6.31) 5.25 (4.88 to 5.65) 4.44 (4.07 to 4.84) Page 32 of 35 21.33(17.97to25.31) 23.43 (21.45 to 25.59) 25.38 (22.78 to 28.28) 26.39 (23.19 to 30.02) 27.7 (24.76 to 30.99) 23.73 (21.2 to 26.56) 25.05 (21.27 to 29.5) 23.09 (20.92 to 25.49) 24.04 (21.44 to 26.96) 46 (2.4%) 302 (17.8%) 1 (0.0%) 4.05 (3.36 to 4.89) 4.17 (3.73 to 4.67) 18.29 (15.06 to 22.22) 19.9 (17.76 to 22.29) 857(35.1%) 316 (17.9%) 291 (18.0%) 124(6.6%) 64 (3.3%) 80 (4.8%) 334 (14.4%) 3.5 (3.22 to 3.8) 3.87 (3.47 to 4.32) 4.15 (3.79 to 4.55) 3.82 (3.4 to 4.29) 3.96 (3.19 to 4.92) 4.63 (3.62 to 5.91) 3.58 (3.22 to 3.98) 18.84 (16.81 to 2 1 .11) 20.37 (18.12 to 22.9) 19.82(17.9510 21.87) 18.07(15.81 to 20.65) 15.71 (12.89 to 19.14) 18.13 (14.76 to 22.27) 19.26 (17.29 to 21.46) p. 35 32 Page 33 of 35 Table 2- n> f a specific summary statistics with thyroid disease prevalence by weighted quartile* of PFOA md ------------- ;--------------- r - 1---------------- Summary statistics by quartile ' Unweighted Weighted Mean (sd) Mean (95% Cl) ng/ml N (case/total) TTkhuyrrjosiMd disease aeuvearr Unweighted Prevalence (%) Weighted prevalence (%) (95% Cl) Cuurrent thyroid idisease with thyroid medication N ,(caseAo.t.a.l) Unweighted Prevalence (%) Weighted prevalence (%) (95% Cl) WOMEN PFO A -A ll and population based quartiles All 2066 0.1 to 123.0 4.25 (4.92) 4.84 (4.35 to 5.33) Q1 689 0.1 to 2.6 1.71 (0.66) 1.79 (1.72 to 1.85) Q2 550 2.7 to 4.0 3.32 (0.40) 3.33 (3.28 to 3.38) 03 441 4.1 to 5.7 4.79 (0.48) 4.78 (4.72 to 4.85) 04 386 5.7 to 123.0 9.47 (9.38) 9.7 (8.43 to 10.98) WOMEN PFO S - All and population based quartiles All 2066 0.14 to 406.0 23.24 (23.13) 24.78 (22.6 to 26.9) Q1 616 0.14 to 12.4 8.13(2.82) 8.49 (8.06 to 8.93) Q2 523 12.5 to 19.4 15.75 (2.02) 15.92 (15.71 to 16.13) Q3 466 19.5 to 29.8 24.21 (2.89) 24.49 (24.03 to 24.94) Q4 461 29.9 to 406.0 50.96 (35.15) 50.48(45.81 to 55.16) 292/2066 65/689 71/550 72/441 84/386 14.13 9.43 12.91 16.33 21.76 292/2066 68/616 71/523 62/466 91/461 14.13 11.04 13.58 13.30 19.74 16.18 (14.44 to 18.09) 12.62 (9.66 to 15.57) 13.87 (9.66 to 18.08) 15.98(11.81 to 20.15) 22.57 (17.44 to 27.71) 16.18 (14.44 to 18.09) 15.14 (10.82 to 19.46) 16.23 (12.58 to 19.89) 12.69 (8.59 to 16.78) 20.66 (16.46 to 24.87) 163/2066 34/689 35/550 39/441 55/386 163/2066 32/616 40/523 39/466 52/461 7.89 4.93 6.36 8.84 14.25 7.89 5.19 7.65 8.37 11.28 9.89 (8.32 to 11.72) 8.14 (5.75 to 10.53) 7.27 (4.37 to 10.16) 8.25 <4.97 to 11.52) 16.18 (11.74 0 20.62) 9.89 (8.32 to 11.72) 8.7(5.4510 11.96) 9.85 (6.58 to 13.13) 8.47 (4.98 to 11.97) 12.55 (8.86 to 16.23) MEN PFOA- All andI population based quartiles All 1900 0.1 to 45.9 5.23 (3.41) Q1 643 0.1 to 3.6 2.47 (0.85) 02 517 3.7 to 5.2 4.42 (0.45) 03 381 5.3 to 7.2 6.12(0.55) Q4 359 7.3 to 45.9 10.39 (4.20) 5.79 (5.41 to 6.18) 2.51 (2.43 to 2.58) 4.44 (4.39 to 4.50) 6.19 (6.12 to 6.26) 10.3 (9.72 to 10.89) 69/1900 24/643 20/517 11/381 14/359 3.63 3.73 3.87 2.89 3.90 MEN PFO S- All andI population based quartiles All 1900 0.3 to 435.0 29.57(22.11) 30.36 (28.2 to 32.5) 69/1900 3.63 Q1 529 0.3 to 18.0 12.29 (4.30) 12.35 (11.94 to 12.76) 18/529 3.40 02 480 18.2 to 25.5 21.82(2.13) 21.83 (21.63 to 22.03) 13/480 2.71 03 454 25.6 to 36.7 30.81 (3.18) 30.93 (30.57 to 31.29) 15/454 3.30 04 437 36.8 to 435.0 57.73 (29.4) 56.45 (52.85 to 60.04) 23/437 5.26 Note: ` quartiles defined to reflect theI US population, accounting for population weighting in NHANES 3.06 (2.40 to 3.88) 3.49(2.01 to 4.97) 3.44 (1.48 to 5.41) 1.51 (0.38 to 2.63) 3.71 (1.67 to 5.75) 3.06 (2.40 to 3.88) 3.22 (1.86 to 4.57) 1.64(0.4010 2.87) 2.68 (1.26 to 4.10) 4.69 (2.44 to 6.95) 46/1900 16/643 13/517 7/381 10/359 46/1900 10/529 8/480 11/464 17/437 2.42 2.49 2.51 1.84 2.79 2.42 1.89 1.67 2.42 3.89 1.88 (1.30 to 2.69) 2.27 (1.25 to 3.30) 2.14 (0.79 to 3.49) 0.77 (0.17 to 1.37) 2.27 (0.22 to 4.33) 1.88 (1.30 to 2.69) 1.85 (0.82 to 2.89) 0.80 (0.12 to 1.48) 1.62 (0.55 to 2.69) 3.24 (1.07 to 5.40) 33 p. 36 Page 34 of 35 Table 3: Gender specific, survey weighted associations between PFOA and PFOS concentrations and thyroid disease. PFOA PFOS Models adjusting for age, ethnicity & study year: OR (95% Cl), p-value Fully adjusted models *: OR (95% Cl) p-value Models adjusting for age, sex, ethnicity & study year: OR (95% Cl), p-value Fully adjusted models *: OR (95% Cl) p-value Women - Thyroid disease ever Q1 1 Quartiles of PFC ^2 Q3 0.98(0.65 to 1.50), p<0.936 1.09 (0.66 to 1.81), p0.729 Q4 1.63 (1.07 to 2.47), p-0.024 ** Top quartile (Q4) vs Q 1&2 1.64 (1.12 to 2.41), p -0.013** Women - Thyroid disease current with medication Q1 1 Quartiles of PFC Q3 0.77 (0.45 to 1.32), p-0.334 0.86 (0.47 to 1.57), p=0.607 0 4 1.83 (1.13 to 2.95), p-0.015' * Top quartile (04) vs Q1&2 2.09 (1.34 to 3.26), p=0.002 ** 1 0.95(0.62 to 1.47), ps0.825 1.11 (0.6710 1.83), p=0.679 1.64 (1.09 to 2.46), p -0 .0 1 9** 1.68 (1.14 to 2.49), p-0.011 ** 1 0.7 (0.41 to 1.22), p-0.205 0.89 (0.4910 1.59), p0.676 1.86 (1.12 to 3.09), p-0.018** 2.24 (1.38 to 3.65), p=0.002 " 1 1.04 (0.63 to 1.71), p=0.875 0.68 (0.4 to 1.17), p-0.155 1.11 (0.66 to 1.86), p<=0.69 1.08(0.7310 1.61), p=0.681 1 1.11 (0.5810 2.14), p=0.747 0.85 (0.46 to 1.59), p-0.609 1.27 (0.69 to 2.32), p=0.435 1.19(0.7710 1.85), p=0.417 1 1.01 (0.63tO l.6),p=0.972 0.64 (0.39 to 1.05), p0.078 1.15 (0.7 to 1.91), p=0.568 1.15 (0.78 to 1.7), p=0.48 1 1.05 (0.55 to 2), p=0.89 0.81 (0.44 to 1.51), p=0.496 1.31 (0.72 to 2.36), p0.369 1.27 (0.82 to 1.97), p=0.269 Men Thyroid disease ever Ql 1 1 1 Quartiles of PFC 2 Q3 1.17 (0.64 to 2.15), p<=0.600 0.58 (0.21 to 1.59), p=0.283 1.11 (0.62 to 1.99), p-0.729 0.57 (0.19 to 1.66),p=0.291 0.50 (0.22 to 1.17), p-0.107 0.81 (0.40 to 1.61), p-0.536 Q4 1.58 (0.79 to 3.16), pe0.191 1.58 (0.74 to 3.39), p0.233 1.51 (0.70 to 3.22), p0.284 Top quartile (Q4) vs Q1&2 1.45 (0.68 to 3.09), p-0.323 Men - Thyroid disease current with medication 1.5 (0.66 to 3.39), p=0.324 1.6 (0.57 to 4.46), p=0.360 Q1 1 1 1 Quartiles of PFC Q2 Q3 1.18 (0.55 to 2.54), p-0.668 0.51 (0.20 to 1.32), p=0.162 1.12 (0.52 to 2.39), p=0.767 0.49 (0.18 to 1.38), p0.171 0.42 (0.16 to 1,10), p-0.077 0.82 (0.29 to 2.27), p=0.694 Q4 1.74 (0.63 to 4.78), p-0.275 1.89(0.60 io 5.90), p=0.268 1.72 (0.73 to 4.05), p0.211 Top quartile (Q4) vs Q1&2 2.02 (0.89 to 4.58), p0.092 2.12(0.93 to 4.82), p0.073 2.44 (1.04 to 5.74), p=0.041 ** Notes: * Models adjusted forage, ethnicity, education, BMI, smoking status, alcohol consumption; ** Significant association with 95% confidence 1 0.51 (0.23 to 1.14), p=0.097 0.88 (0.43 to 1.84), p0.736 1.58 (0.72 to 3.47), p=0.251 1.78 (0.58 to 5.52), p-0.309 1 0.43 (0.17 to 1.08), p0.073 0.95 (0.34 to 2.70), p0.926 1.89 (0.72 to 4.93), p=0.190 2.68 (1.03 to 6.98), p=0.043 *' 34 p. 37 Page 35 of 35 p. 38 Table 4: Associations between PFOA & PFOS concentrations (population based quartiles) and other diseases in fully adjusted logistic regression models (by self reported disease status). PFOA PFOS n (survey weighted %) OR (95% Cl) p-value n (survey weighted %) OR (95% Cl) p-vatue Arthritis ever 1006/3960 (22.8%) 1006/3960 (22.8%) Q1 287/1310(19.2%) 1 1 219/1132(19.0%) Q2 298/1036 (27.6%) 1.63 (1.24 to 2.14) 0.001 267/1009 (23.5%) 03 231/857 (22.7%) 1.31 (1.03 to 1.66) 0.029 260/916 (26.8%) 04 190/757 (21.8%) 1.28 (0.97 to 1.68) 0.082 260/903 (22.0%) Asthma ever 471/3961 (13.2%) 471/3961 (13.2%) Q1 138/1313(11.9%) 1 1 139/1133 (14.0%) Q2 128/1036 (14.2%) 1.25 (0.92 to 1.70) 0.154 140/1013 (15.6%) Q3 122/856(15.8%) 1.44(1.01 to 2.05) 0.045 111/914(13.1%) 04 83/756(11.2%) 0.93 (0.64 to 1.36) 0.716 81/901 (10.3%) COPD ever 302/3953(8.2%) 302/3953 (8.2%) Qt 81/1310 (7.7%) 1 1 83/1131 (8.8%) 02 93/1033(8.8%) 0.91 (0.58 to 1.43) 0.677 85/1008 (8.5%) 03 66/853 (8.3%) 0.88 (0.54 to 1.43) 0.593 67/914 (7.7%) 04 62/757 (8.2%) 0.85 (0.54 to 1.34) 0.473 67/900 (7.9%) Diabetes ever 459/3964 (8.7%) 459/3964 (8.7%) Q1 186/1314(10.9%) 1 1 122/1133(8.6%) Q2 127/1035 (9.2%) 0.80 (0.55 to 1.17) 0.242 119/1012(9.3%) 03 83/857 (7.7%) 0.74 (0.48 to 1.15) 0.177 103/916(7.7%) 04 63/758 (7.0%) 0.69(0.41 to 1 16) 0.158 115/903 (9.4%) Heart disease ever* 321/3966 (5.8%) 321/3966 (5.8%) Q1 93/1314(5.7%) 1 1 69/1134(4.8%) 02 93/1037(6.1%) 0.95 (0.59 to 1.51) 0.816 85/1013(5.1% ) Q3 78/857 (5.9%) 1.02 (0.65 to 1.61) 0.917 80/916 (5.7%) 04 57/758 (5.4%) 1.08 (0.70 to 1.69) 0.715 87/903 (7.4%) Liver Disease current 57/3942(1.4%) 57/3942 (1.4%) Q1 24/1307 (1.4%) 1 1 22/1127 (1.7%) 02 11/1028(1.0%) 0.66 (0.25 to 1.74) 0.391 10/1007(0.9%) Q3 17/855(2.5%) 1.93 (0.96 to 3.88) 0.065 13/910(1.6%) 04 5/752 (0.8%) 0.61 (0.21 to 1.78) 0.355 12/898(1.5%) a any report of coronary heart disease, and/or angina, and/or heart attack 1 1.19(0.91 to 1.54) 1.29 (1.00 to 1.66) 0.74 (0.53 to 1.04) 1 1.16 (0.80 to 1.68) 0.97 (0.65 to 1.43) 0.79 (0.50 to 1.26) 1 0.84 (0.56 to 1.25) 0.67 (0.41 to 1.09) 0.58 (0.43 to 0.76) 1 1.02 (0.70 to 1.47) 0.76 (0.50 to 1.18) 0.87 (0.57 to 1.31) 1 0.77 (0.49 to 1.23) 0.83 (0.46 to 1.51) 0.91 (0.50 to 1.64) 1 0.49 (0.18 to 1.32) 0.94(0.41 to 2.16) 0.95 (0.39 to 2.29) 1 0.193 0.054 0.085 1 0.427 0.867 0.320 1 0.384 0.103 0.0003 1 0.928 0.218 0.491 1 0.270 0.540 0.745 1 0.154 0.880 0.907 35