PHTHALATES AND HUMAN HEALTH

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1 PHTHALATES AND HUMAN HEALTH RHauser,AMCalafat 806 ROUTES See end of artile for authors affiliations Correspondene to: Prof. Assoiate R Hauser, Oupational Health Program, Harvard Shool of Publi Health, Building 1, Room 1405, 665 Huntington Avenue, Boston, MA 02115, USA; rhauser@hohp.harvard. edu T Oup Environ Med 2005; 62: doi: /oem he diesters of 1,2-benzenediarboxyli aid (phthali aid), ommonly known as phthalates, are a group of man-made hemials with a wide spetrum of industrial appliations (fig 1, table 1). High moleular weight phthalates (for example, di(2-ethylhexyl) phthalate [DEHP], di-isononyl phthalate [DiNP], di-n-otyl phthalate [DnOP]), are primarily used as plastiizers in the manufature of flexible vinyl whih, in turn, is used in onsumer produts, flooring and wall overings, food ontat appliations, and medial devies. 1 3 Manufaturers use low moleular weight phthalates (for example, diethyl phthalate [DEP] and dibutyl phthalate [DBP]) in personal-are produts (for example, perfumes, lotions, osmetis), as solvents and plastiizers for ellulose aetate, and in making laquers, varnishes, and oatings, inluding those used to provide timed releases in some pharmaeutials. 3 5 In this paper, we review the uses and metabolism of phthalates, and the studies on health effets of phthalates in human populations published between 1973 and June The referenes inluded in this review were searhed using the Web of Siene database whih provides interative itation and literature searhing of the Institute for Sientifi Information s Siene Citation Index Expanded. The database ontains data from more than 5000 sientifi journals and overs the period from 1980 to present. We also searhed the bibliography ited in the seleted referenes for additional relevant itations. OF EXPOSURE AND METABOLISM Beause phthalates are widely used in many personal are and onsumer produts, the opportunity is high for non-oupational human exposure (table 1). However, to date, the proportional ontribution from the various soures and routes of exposure to phthalates is unknown. Traditionally, ingestion has been onsidered an important route of exposure. Although phthalates have low volatility, they off-gas and are present in residential indoor air. 6 7 Dermal ontat and parenteral exposure from medial devies ontaining phthalates may also ontribute to exposure. 2 8 On exposure, phthalates are rapidly metabolised and exreted in urine and faees During a phase I biotransformation, the relatively polar and low moleular weight phthalates (for example, DEP) primarily metabolise into their hydrolyti monoesters by hydrolysis of one of the ester bonds (fig 1). 4 9 In ontrast, the high moleular weight phthalates are first metabolised to their respetive hydrolyti monoesters, and then, after enzymati oxidation of the alkyl hain, to more hydrophili, oxidative metabolites (fig 2) Monoesters and the oxidative metabolites of phthalates an be exreted in the urine and faees unhanged or they an undergo phase II biotransformation to produe gluuronide onjugates with inreased water solubility and therefore inreased urinary exretion. 2 4 Gluuronidation not only failitates urinary exretion of phthalate metabolites, but also may redue their potential for biologial ativity if the putative biologially ative speies is the free metabolite. The perentage of free monoester exretion in humans varies depending on the aqueous solubility of the phthalate metabolites Mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), two oxidative metabolites of DEHP, are mostly exreted gluuronidated in humans. 16 Most of mono(2-ethylhexyl) phthalate (MEHP), the hydrolyti monoester metabolite of DEHP, is also exreted as a gluuronide, while monoethyl phthalate (MEP), the hydrolyti monoester of DEP, is mostly exreted in its free form. 17 For other phthalates, the perentage of free monoester exretion varies between that of MEP and MEHP. Although the metabolism of the relatively low moleular weight phthalates (for example, DEP) ends with the hydrolyti monoester, the metabolism of the higher moleular weight phthalates (for example, DEHP, DiNP, di-isodeyl phthalate [DiDP]) ontinues with transformation of the hydrolyti monoester to oxidative produts (fig 2) Therefore, the use of the hydrolyti monoesters as sole biomarkers for omparing relative exposures to various phthalates in a given study ould be misleading. This is espeially true when omparing onentrations of the

2 Figure 1 Generi hemial struture of phthalates diesters and phthalate monoester metabolites. R is an alkyl and/or aryl group: CH 3 (dimethyl phthalate), CH 2 CH 3 (diethyl phthalate), CH 2 CH 2 CH 2 CH 3 (dibutyl phthalate), CH 2 CH(CH 2 CH 3 )CH 2 CH 2 CH 2 CH 3 (di[2-ethylhexyl) phthalate), CH 2 CH 2 CH 2 CH 3 and CH 2 C 6 H 5 (butylbenzyl phthalate). high moleular weight monoester phthalates, suh as MEHP, with the lower moleular weight phthalate monoesters, suh as MEP, beause the metabolism of the higher moleular weight phthalates is more omplex and results in more metabolites, thus dereasing the relative amounts of their monoester metabolite. On the other hand, hydrolyti monoesters of high moleular weight phthalates (for example, MEHP) may be used as biomarkers for omparing exposure to their orresponding phthalate diesters (for example, DEHP) among studies, assuming that the hydrolyti monoester is present in detetable onentrations. However, using the hydrolyti phthalate monoesters as the sole biomarkers of exposure in an epidemiologial study ould introdue exposure mislassifiation beause the other metabolites are not aounted for. This is partiularly important for the hydrolyti monoesters of high moleular weight phthalates, inluding isomeri phthalates suh as DiNP, whih represent only a small perentage of all of the urinary metabolites; oxidative phthalate metabolites predominate in urine. Most of the researh on oxidative metabolism of phthalates in humans has been onduted on DEHP The urinary onentrations of two DEHP oxidative metabolites MEOHP and MEHHP have been found to be higher than MEHP, the hydrolyti metabolite of DEHP A third oxidative metabolite of DEHP, mono(2-ethyl-5-arboxypentyl) phthalate has also been found at higher onentrations in urine than MEHP These findings suggest that the relatively low urinary onentrations of MEHP ompared to the onentrations of the hydrolyti metabolites of other phthalates (for example, DEP, DBP) may result, at least in part, from alternative metaboli pathways leading to the formation of oxidative monoester metabolites whih are more amenable than MEHP to urinary exretion. In rodents, DiNP and DnOP, like DEHP, are also initially metabolised into their hydrolyti monoesters, whih then form oxidative phthalate monoesters whih are the major metabolites deteted in the urine Although there may be some differenes in the metabolism among speies, it is likely that all large moleular weight phthalates undergo similar metaboli pathways in humans. MEASURES OF INTERNAL DOSE OF PHTHALATES Phthalates are widely used in laboratory and medial produts and sample ontamination is therefore diffiult to avoid. Beause environmental exposure levels are normally muh lower than those resulting from sampling ontamination, human studies using the phthalate diesters as biomarkers of exposure were limited to highly exposed populations A different approah uses urinary phthalate monoester metabolites as biomarkers of exposure. By measuring the phthalate monoesters, ontamination from the ubiquitous parent ompounds is minimised, thus allowing for the study of environmentally exposed populations. Another advantage of using the phthalate monoesters as biomarkers is that the monoesters are generally onsidered the biologially ative moleules. Phthalate monoesters an be measured in biologial matries (for example, urine, serum, breast milk, saliva, ovarian folliular fluid, seminal plasma, and amnioti fluid) by using hromatography oupled with mass spetrometri tehniques. High performane liquid hromatography (HPLC) is most ommon Gas hromatography an also be used to measure phthalate monoesters after onversion to volatile derivatives. 38 Analytial methods using isotope dilution HPLC oupled with tandem mass spetrometry (MS/MS) for measuring trae levels of seleted phthalate monoesters in biologial matries have been reported These methods are highly speifi, sensitive (detetion limits are in the low nanogram per millilitre range), aurate (relative reoveries are,100%), and preise (inter-day relative standard deviations are generally,15%). In isotope dilution mass spetrometry, a stable isotope of the analyte(s) of interest is added to the matrix before sample preparation; the quantifiation of the analyte(s) is based on a ratio of the signal from the analyte relative to the signal from the known amount of its stable isotope. Isotope dilution mass spetrometry is onsidered the most aurate and preise method for trae analysis. Briefly, the determination of phthalate monoesters involves the enzymati deonjugation of these metabolites 807 Table 1 Potential soures of exposure and health effets of seleted phthalates Phthalate diester Potential soures of exposure* Potential health effetsà Diethyl phthalate Di-n-butyl phthalate Butylbenzyl phthalate Di(2-ethylhexyl) phthalate Personal are produts (e.g. fragranes), oatings (e.g. pharmaeutials), dyes, insetiides Cellulose aetate plastis, personal are produts (e.g. nail polish, osmetis), laquers, varnishes, oatings (e.g. pharmaeutials) Vinyl flooring, adhesives and sealants, ar-are produts, toys, food pakaging, syntheti leather, industrial solvents, personal are produts PVC plastis used in household produts (e.g. toys, floor tiles and furniture upholstery, wall overings), food pakaging, blood storage bags and medial devies Redued growth rate, food onsumption and inreased organ weights Hepati and renal effets, developmental and reprodutive effets, redued fetal weight, ryptorhidism, hypospadias, redued anogenital distane in males. Testiular toxiity, ryptorhidism, redued anogenital distane, teratogeni, modulates steroid hormone levels Hepatoellular arinoma, testiular toxiity, anovulation, teratogeni at high doses, affets fetal growth *List is not inlusive. ÀHealth effets are in animals, frequently at high dose levels; human health effets are reviewed in the text.

3 808 Figure 2 Seleted metabolites of di[2-ethylhexyl) phthalate) (DEHP) in humans: mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5- hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethyl-5-arboxypentyl) phthalate (MECPP). from their gluuronidated form, followed by solid phase extration, separation with HPLC, and detetion by MS/MS. Analytial methods whih do not inlude mass spetrometri (MS) detetion are generally less seletive and sensitive than those based on MS detetion. HUMAN EXPOSURE DATA (POPULATION LEVELS) Beause of the potential human toxiities of phthalates, internal dose measurements of phthalates are neessary for exposure and risk assessment. Measuring human exposure to phthalates requires information on their onentrations and/ or their metabolites in biologial media as well as on the pharmaokinetis of the phthalates. Indiret measures of exposure inluding environmental monitoring and exposure history/questionnaire data an be used to assess human exposure to phthalates. Advanes in analytial hemistry have made possible measuring trae levels of phthalates in biologial tissues (that is, biomonitoring) and have ontributed to inreased reent use of biomonitoring in exposure assessment. The most ommon biomonitoring approah for investigating human exposure to phthalates is the measurement of urinary onentrations of phthalate metabolites. Biomonitoring data ombined with indiret measures of exposure are the most appropriate tools for assessing exposure. Variability in an individual s exposure to phthalates an result from hanges in the use of personal are produts, diet, or daily ativities, and exposure may vary over time. Although phthalate metabolites in urine an be used to aurately measure a person s exposure at a single point in time, determining exposure over weeks or months may require multiple measurements. Therefore, information on the temporal variability of urinary levels of phthalate metabolites is needed to optimise the design of exposure assessment in epidemiologial studies. Two studies have addressed this issue. The first study doumented relatively good reproduibility of urinary phthalate monoester levels in two first-morning urine speimens olleted for two onseutive days from 46 Afrian-Amerian women; day-to-day intra-lass orrelation oeffiients ranged from 0.5 to Reently, the temporal variability in urinary phthalate metabolite levels among 11 men who olleted up to nine urine samples eah during a three month period was evaluated. 44 Although substantial day-to-day and monthto-month variability in eah individual s urinary phthalate metabolite levels existed, a single urine sample was moderately preditive of eah subjet s exposure over three months. The sensitivities ranged from 0.56 to Both between- and within-subjet varianes and the preditive ability of a single urine sample differed among phthalate metabolites, suggesting that the most effiient exposure assessment strategy for a partiular epidemiologial study may depend on the phthalates of interest. Furthermore, a ross-setional study of more than 2540 people in the United States showed variations in the population distributions of several phthalate metabolites depending on the time of day of olletion of the urine samples. 26 These observations, along with the non-persistent nature of phthalates, may reflet differenes in the timing of exposure to phthalates during the day. Beause olleting 24-hour urine samples is not pratial for epidemiologial studies, onsideration should be given to standardising the time of sample olletion. When the goal is to ompare relative exposures to various phthalates aross populations or among different studies, olleting first-morning urine samples is preferred. However, studies designed to explore potential health risks of phthalates should not restrit sample olletion to first-morning urine samples beause the data above suggest that relevant exposure opportunities in the ourse of a day may be missed and thus exposure ould be mislassified. At the very least, timing of the urine olletion should always be reorded. In the United States, the National Health Nutrition and Examination Survey (NHANES) is an ongoing survey, onduted by the National Center for Health Statistis at the Centers for Disease Control and Prevention (CDC), designed to ollet data on the health and nutritional status of the ivilian, non-institutionalised US population. The data estimates from NHANES, presented by age group, gender, and rae/ethniity, are probability based, and hene, are representative of the US population. Although the biologial speimens olleted from NHANES partiipants are used mostly for linial and nutritional testing, some of them are

4 used for assessing exposure to environmental hemials, inluding phthalates. In Germany, German Environmental Surveys (GerESs), large sale representative population studies for assessing the exposure of the general population to environmental ontaminants, have been onduted sine the mid-1980s. Although phthalates were not inluded in early surveys, DEHP metabolites were measured reently in a subset of samples olleted for the 2001/2002 pilot study for the GerES on hildren. 45 In the United States, CDC reported the levels of seven urinary phthalate monoester metabolites in two subsets of NHANES partiipants The first study inluded the analysis of a non-representative all-bak ohort of 289 urine samples, olleted from adults during for NHANES III. 24 NHANES provided the first nationally representative, population based, phthalate monoester onentrations in urine for seleted demographi groups in the United States. Although the two data sets are not diretly omparable, the frequenies of detetion of the phthalate monoesters were similar. The high moleular weight phthalate monoesters (for example, MEHP, mono-n-otyl phthalate, monoisononyl phthalate) were deteted less frequently and at lower levels than the low moleular weight phthalates (for example, MEP, monobutyl phthalate [MBP], monobenzyl phthalate [MBzP]). However, the mean and median onentrations of MEP, MBP, and MBzP were lower in the NHANES than in the NHANES III subset population while the MEHP onentrations remained essentially unhanged. These findings may be related to the small size and non-representative nature of the sampling for the NHANES III all-bak ohort or to redued exposure to some phthalates for the NHANES population. The relatively low frequeny of detetion of the high moleular weight phthalate monoesters may have been due, at least in part, to the fat that these hydrolyti metabolites may not be the most sensitive urinary biomarkers; the oxidative phthalate monoester metabolites are (vide infra). Nonetheless, these two investigations onfirmed that human exposure to seleted phthalates is widespread. Future NHANES will be useful for monitoring temporal hanges in phthalate exposures. However, even a omprehensive programme suh as NHANES has limitations: urine samples from young (that is,,6 years of age) individuals are not olleted in the population sampled, the design is ross-setional, and no data on fetal exposures are olleted. Therefore, there is a pressing need for assessing exposure during ritial periods of development, periods of inreased suseptibility to the potential adverse effets of phthalates. Few studies have evaluated exposure to phthalates in pregnant women and young hildren, despite their potential sensitivity to adverse effets of phthalates. MEP, MBP, MBzP, and MEHP were frequently deteted in 25 urine samples olleted from pregnant Afrian-Amerian and Dominian women living in New York City. 6 The median onentrations of MEP and MEHP were somewhat higher and the median MBzP onentration was similar to that measured in NHANES for several demographi groups, inluding adults, women, non-hispani blaks, and Mexian Amerians. The median onentration of MBP was times higher than the NHANES levels, both olletively or when ompared within the demographi groups. There are only four published studies on the levels of phthalate metabolites in hildren environmentally exposed to phthalates. Phthalate monoester onentrations in urine were measured in 19 toddlers aged months in Imperial Valley, California. 46 Twelve toddlers provided two urine samples within one month period. Similar to other studies, the metabolites predominantly deteted were MEP, MBP, MBzP, and MEHP. The unadjusted mean onentration of MEP and MBP were approximately two and three times higher, respetively, than the geometri mean onentrations in hildren aged 6 11 years in NHANES , whereas the mean onentration of MBzP and MEHP were similar. The onentration of DBP, butylbenzyl phthalate (BBzP), and DEHP metabolites in two groups of German hildren have been reported reently. The median urinary onentrations of MBP and MBzP in these German hildren 21 were about three times higher and two times lower, respetively, than in US hildren 6 11 years of age. 26 The urinary onentrations of MBP, MBzP, and MEHHP and MEOHP, two oxidative metabolites of DEHP, were signifiantly higher in 36 hildren (2 6 years of age) than in 19 adults (inluding four of their teahers and parents). In ontrast, the urinary levels of MEHP were signifiantly higher in the adults than in the hildren. More importantly, urinary onentrations of the two oxidative metabolites MEHHP and MEOHP were higher than MEHP for both hildren and adults. The higher onentrations of oxidative metabolites in the hildren than in the adults might indiate an enhaned oxidative metabolism in hildren. The higher urinary levels of DEHP oxidative metabolites than of MEHP were onfirmed in a larger study of 254 hildren 3 14 years old. 45 Boys showed higher urinary onentrations than girls for all three DEHP metabolites. The oldest hildren (13 and 14 years old) had the lowest mean urinary onentrations of DEHP oxidative metabolites. The median urinary onentrations of MEHP in these two groups of German hildren were about 1.5 times higher than in US hildren 6 11 years of age The urinary onentrations of DEHP oxidative metabolites were not measured in NHANES , but they will be in future NHANES. The urinary levels of several phthalate monoesters in various populations in Germany have been reported In eight non-oupationally exposed people, the mean urinary levels of MEP, MEHP, and MBP were onsiderably higher than the levels found in the NHANES population, but the MBzP levels were about half The same group of investigators also measured the onentrations of these phthalate metabolites in the firstmorning voids from 53 females and 32 males, aged 7 64 years, who live in southern Germany. 20 Similar to the NHANES population, females had higher reatinine adjusted urinary levels of MEP, MBP, MBzP, and MEHP than males. However, the median levels of MBP were eightfold higher and the median levels of MEHP were threefold higher in the German study than in the NHANES population. Conentrations of MEP were about half those found in NHANES , but levels of MBzP were similar. These data suggest similar sex related differenes in exposure to phthalates, both in the United States and in Germany, although differenes in the exposure to phthalates may exist geographially. Furthermore, the varying amounts of phthalates used within eah ountry and varying sampling olletion times (for example, first-morning voids versus non first-morning voids, seasonal versus throughout-the-year olletion) may aount for the 809

5 810 observed differenes when omparing the levels of urinary phthalates found in Germany and in the United States. Another limitation of NHANES is that the survey by its design evaluates the general population, but not speifi populations who might be highly exposed These speifi populations need to be examined by other studies designed to determine possible assoiations between high exposures and adverse health effets. Human populations at risk of high exposure to phthalates inlude individuals reeiving medial treatments with polyvinyl hloride (PVC) plasti devies made flexible by the addition of large amounts (up to 40% by weight) of plastiizers, suh as DEHP. Speifially, infants born prematurely who often spend their first days or weeks in neonatal intensive are units (NICU) are at espeially high risk of exposure to high levels of DEHP. 51 In the first study to evaluate exposure to DEHP in infants undergoing intensive therapeuti interventions, the median urinary onentrations of MEHP in six premature newborns were found to be about 50 times higher than in hildren between 6 and 11 years of age from the general US population. 49 A seond study on urinary levels of MEHP in NICU infants onfirmed the higher levels in NICU patients ompared to levels in hildren from the general US population. 52 The researhers assessed the use of DEHP ontaining produts in the are of 54 infants admitted to one of two NICUs, and examined the intensity of use of these produts in relation to levels of MEHP in the infants urine. Before data olletion, three DEHP exposure ategories (low, medium, and high) were defined based on a review of medial produts typially used in both NICUs and information provided by their manufaturers with respet to DEHP ontent. There was a diret relation between urinary MEHP levels and the extent of medial produt use. Urinary MEHP levels among infants onsidered to be in the highest DEHP exposure ategory based on the use of high DEHP ontaining produts were five times as high as levels among infants in the lowest DEHP exposure group; urinary MEHP levels among infants in the medium DEHP exposure group were twie as high. Conerns exist regarding the overall benefits of medial proedures using PVC devies and the potential risks assoiated with exposure to DEHP. 51 Additional researh is needed to establish whether hildren undergoing intensive therapeuti interventions using DEHP ontaining devies are at higher risk for altered health outomes than hildren undergoing similar treatments but not potentially exposed to DEHP. HUMAN HEALTH ENDPOINTS Overview Although the review on the potential human health effets of phthalates fouses on epidemiologial studies, in vitro and experimental animal studies are briefly disussed at the beginning of eah health setion to provide both bakground information and guidane in identifying relevant human health endpoints that may be assoiated with phthalates. Laboratory studies have shown that phthalates exhibit marked differenes in toxiity in relation to their struture (table 1). The most well studied struture-ativity relationships are for the indution of testiular toxiity in rats. The length of the alkyl side-hain predited testiular toxiity, with speifi phthalates being toxi while others were not. Furthermore, the branhing of the alkyl side-hain suh as that observed within strutural isomers (for example, butyl phthalates) resulted in differenes in toxiity Laboratory studies have also shown that the phthalate monoester metabolites are assoiated with testiular toxiity while the parent diesters are generally not. 57 In addition to the struture of the phthalate ester prediting toxiity, there is also evidene that there is an age dependeny in the testiular toxiity. Adult rats were generally less sensitive than young pubertal rats 58 or rats exposed in utero. Administration of some phthalates (for example, DEHP and DiNP) to rodents resulted in adverse liver effets inluding inreased liver weights, elevated liver enzyme levels, histologial hanges, and, in some ases tumours These effets were assoiated with peroxisomal proliferation, 59 a proess related to metabolism of holesterol and fatty aids. However, the relevane of these effets to humans is debatable due in part to metaboli differenes among speies Developmental outomes In experimental animal studies, primarily in rodents, some phthalates indued reprodutive trat developmental anomalies that onsisted of epididymal malformations or absene of the epididymis, inreased inidene of hypospadias, ryptorhidism, dereased anogenital distane (AGD), delayed preputial separation (pubertal milestone), retention of thorai nipples, and testiular lesions. The testiular lesions were haraterised by seminiferous tubule atrophy and Leydig ell hyperplasia. Reprodutive trat developmental anomalies were seen in rats dosed during gestation and/or latation with DBP, DEHP, 54 BBzP, and DINP. 54 Gray and oworkers failed to find similar effets for DEP or dimethyl phthalate (DMP). 54 Although the effets of DBP, BBzP, DEHP, and DINP were onsistent with an antiandrogeni mode of ation, in human androgen reeptor (AR) transriptional ativation assays, DBP and DEHP or their metabolites did not interat with the AR in vitro. 69 Later evidene suggests that the mehanism may be through redued fetal testosterone synthesis during sexual differentiation in the male. 70 Developmental anomalies Developmental endpoints of interest in humans inlude hypospadias, ryptorhidism, and AGD. Although there are many published human studies on potential risk fators for hypospadias and ryporhidism, few have explored their relationships with exposure to phthalates. There is urrently limited evidene to onlude whether an assoiation between phthalates and male developmental anomalies exists. Van Tongeren and oworkers in 2002 developed a jobexposure matrix for investigating the assoiation between maternal oupational exposure to potential endorine disrupting ompounds, inluding phthalates, and hypospadias. 71 Vrijheid and oworkers applied this job exposure matrix in a large study on 3471 ases of hypospadias reorded from 1980 to 1996 by the National Congenital Anomaly System in England and Wales. 72 Overall there was no assoiation of hypospadias with maternal oupational exposure to phthalates. However, for the period there was an inreased odds ratio (OR) for probable versus unlikely exposure to phthalates before adjustment for soial lass (1.52, 95% CI 1.05 to 2.20) and an OR of 1.26 (95% CI 0.81 to 1.97) after soial lass adjustment. Hairdressers made up the majority of ases in the probable exposure ategory. However, in another analysis in whih births, rather than all ongenital anomalies, were used as the denominator, hairdressers did not show an inreased risk of

6 hypospadias (observed to expeted ratio was 0.95, 95% CI 0.66 to 1.32). The authors stated that the assoiation between phthalates and hypospadias should be interpreted autiously sine assoiations were only seen in one time period and adjustment for soial lass weakened the assoiation. Furthermore, in addition to potential exposure to phthalates, hairdressers are exposed to several lasses of hemials, inluding solvents and dye formulations, making it diffiult to attribute potential health risks to phthalates in this oupational group. The job-exposure matrix developed by Van Tongeren and oworkers was also applied by Pierik and oworkers in 2004 in the Netherlands to assess maternal and paternal oupational exposure to endorine disruptors in relation to ryptorhidism and hypospadias. 73 In this nested ase-ontrol study within a ohort of 8695 male newborns in the ity of Rotterdam, there were 78 ases of ryptorhidism and 56 ases of hypospadias. Although not reported in the manusript, maternal and paternal oupational exposures to phthalates were not assoiated with either abnormality (Pierik, personal ommuniation). In a reent study, Swan and olleagues explored the relationship between prenatal exposure to phthalates and shortened AGD in 85 male infants from a multientre study in Los Angeles, CA, Minneapolis, MN, and Columbia, MO. 74 The anogenital index (AGI), defined as AGD (millimetres) divided by weight (kg) at examination, was obtained in 134 boys 2 30 months of age, and the age adjusted AGI was alulated by regression analysis. For 85 of these male infants, a maternal urine sample olleted during pregnany was available. The mean length of gestation at the time of maternal urine sample olletion was 28.3 weeks and the mean age at examination of the infants was 12.6 months (interquartile range was 5 16 months). In regression analyses, maternal prenatal urinary onentrations of MEP, MBP, MBzP, and mono-isobutyl phthalate (MiBP) were inversely related to age adjusted AGI. MEHP was unrelated to AGI; however, the DEHP oxidative metabolites, MEOHP and MEHHP, were of borderline signifiane with AGI. Monomethyl phthalate and mono-3-arboxypropyl phthalate were not assoiated with AGI. Covariates onsidered for inlusion in the regression models inluded mother s ethniity and smoking status, time of day and season in whih the urine sample were olleted, and gestational age at urine olletion. None of the ovariates were retained in the models sine their inlusion did not alter the regression oeffiients by more than 15%. The authors also performed ategorial analyses in whih boys with AGI below the 25th entile were lassified as having short AGI. The OR for short AGI were 10.2 (95% CI 2.5 to 42.2) for high ompared to low MBP onentration, while for medium MBP onentration ompared to low the OR was 3.8 (95% CI 1.2 to 12.3). The OR for high ompared to low MEP, MBzP, and MiBP onentration were 4.7, 3.8, and 7.3, respetively (all p values,0.05). Interestingly, shorter AGI was assoiated with an inreased proportion of boys with inomplete testiular desent. It is important to note that among the 134 boys in the study for whom genital measurements were available, no frank genital malformations or disease were deteted, and 86.6% of them had both testes lassified as normal or normal-retratile. The results of this study are noteworthy beause they represent the first human data showing an inverse relationship between prenatal phthalate exposure and shortened AGD. These data are generally onsistent with evidene from experimental animal studies on phthalates and shortened male AGD, a sensitive measure of prenatal anti-androgen exposure Several study limitations inlude the olletion of only a single urine sample late in pregnany to measure prenatal phthalate exposure, the lak of adjustment for urinary dilution, the relatively small number of boys, and that the reliability of AGD measurements in humans has not been established beause the use of AGD in humans is new. However, these limitations would likely introdue random measurement error and thus bias effet estimates to the null. Repliation of the AGD results in other study populations is strongly reommended. In addition, the potential reprodutive impliations of shortened AGD in males are unlear and require further investigation. Pubertal development Colon et al in 2000 onduted a study on the relationship between serum levels of phthalates and premature breast development (thelarhe) in young girls in Puerto Rio. 77 Forty one patients with thelarhe from the San Juan City Hospital s Pediatri Endorinology Division (median age 20 months, range 6 months to 8 years) and 35 ontrol subjets (median age 46 months, range 6 months to 10 years) were studied. The ontrols were seen in the San Juan City Hospital for general paediatri are and did not have thelarhe or premature sexual development. High levels of DMP, DEP, DBP, DEHP, and MEHP in serum were deteted in 28 thelarhe patients (68%) ompared to 1 and 5 ontrol subjets who had detetable levels of di-isootyl phthalate and DEHP, respetively. The largest differenes between ases and ontrols were for DEHP where the average onentrations were 450 and 70 ng/ml (ppb), respetively (p, 0.05). Beause of onern with potential sample ontamination when measuring the parent diester, eah ase and ontrol sample was orreted with the set of blanks tested the same day of the analysis. Although this study is noteworthy sine the relationship between environmental hemials and pubertal development is an understudied area, diffiulties surrounding the analytial methods used in this study to analyse serum for phthalate diesters have been raised. MKee expliitly desribed several inonsistenies between the diester serum levels reported in the Colon study and those found in other studies onduted in both experimental animals and humans. 78 Furthermore, phthalate esters are not oestrogeni in vivo in animal studies, 79 so it is unlikely that phthalate oestrogeniity is a relevant mehanism in humans. Other limitations with the design of the study inlude the large differenes in ages between ases and ontrols, and assessing phthalate levels at the same time as the assessment of outome (a ross-setional design). Despite these limitations, Colon and olleagues study has generated interest and researh on the potential assoiation of phthalates with pubertal development. A novel, though very small, study explored whether pubertal development was altered in 19 adolesents (13 boys and 6 girls) age years who had undergone extraorporeal membrane oxygenation (ECMO) as newborns. 80 ECMO treatments are known to result in high exposure to DEHP. 81 The growth entiles for the ECMO boys and girls were normal for age and sex, and the levels of LH, FSH, testosterone (in boys), and oestradiol (in girls) were within 811

7 812 the normal referene ranges for stage of pubertal development. In ECMO boys in Tanner stage 4 5, the mean phalli length was slightly longer than the referene range (mean for ECMO boys was 11.2 m, referene range m). In ECMO boys in Tanner stage 2 3, the mean testiular volume was slightly larger than the referene range (11 ml, range 5 10 ml). The authors onluded that adolesents exposed to signifiant quantities of DEHP as neonates showed no effets on physial growth and pubertal maturity. However, it is diffiult to make definitive onlusions based on this study beause it was very small and normal referene ranges for reprodutive hormones, physial growth, and sexual maturation are quite wide. Furthermore, the postnatal levels of DEHP or its metabolites in these hildren were not measured. A larger study would be neessary to detet subtle hanges, if they exist. Male reprodutive health Experimental animal studies have reported assoiations between pubertal and adult exposure to DBP, BBzP, DEHP, and testiular toxiity Pubertal animals were found to be more sensitive than sexually mature animals to the testiular toxiity of phthalates Testiular toxiity with germ ell loss was primarily indued through effets on Sertoli ells, the ell type responsible for initiation and maintenane of spermatogenesis It is important to note that speies differenes in sensitivity to DEHP toxiity have been observed, with markedly lower responses in marmosets than in rodents. 91 Semen quality The human studies desribed below provide limited evidene of an assoiation between some phthalates, speifially MBP and MBzP, and poor semen quality. Duty and olleagues have published four manusripts exploring the relationships between environmental exposure to phthalates and semen harateristis, sperm DNA damage, and serum reprodutive hormones Study subjets onsisted of male partners of subfertile ouples that presented to an infertility lini in Massahusetts, USA. At the time of the lini visit, one semen sample, and blood and urine samples were olleted. Computer aided sperm analysis (CASA) was used to measure sperm onentration and motility, as well as motion parameters. Strit riteria were used to assess sperm morphology. Sperm DNA damage was assessed with the neutral omet assay. Beause the Duty et al study was ongoing, the number of subjets in eah publiation varies; however, there is overlap of subjets among the publiations. Among 168 men, they found dose-response relationships (after adjusting for age, abstinene time, and smoking status) between MBP and sperm motility (OR per tertile: 1.0, 1.8, 3.0; p for trend = 0.02) and sperm onentration (OR per tertile: 1.0, 1.4, 3.3; p for trend = 0.07). 93 They also found a doseresponse relationship between MBzP and sperm onentration (OR per tertile: 1.0, 1.4, 5.5; p for trend = 0.02). There was weak evidene of an assoiation between MBP and sperm morphology, MBzP and sperm motility, and MMP and sperm morphology. Among 220 men, MBP, MBzP, and MEHP had inverse assoiations, although not signifiant, with VSL (straight line veloity), VCL (urvilinear veloity), and LIN (linearity = VSL/VCL6100) measured by CASA. 94 Unexpetedly, positive relationships were found between MEP and both VSL and VCL. To quantify sperm DNA damage in samples analysed using the neutral omet assay, Duty and oworkers used VisComet image analysis software to measure omet extent, a measure of total omet length (mm), perent DNA in tail (tail %), a measure of the proportion of total DNA present in the omet tail, and tail distributed moment (TDM), an integrated measure of length and intensity (mm). 92 In multiple regression models, after adjusting for age and smoking status, for an interquartile range (IQR) inrease in MEP onentration the omet extent inreased by 3.6 mm (95% CI 0.74 to 6.47) and TDM inreased by 1.2 mm (95% CI to 2.38). There were no relationships between MBP, MBzP, MEHP, and MMP and any omet assay parameters. Duty et al reently published data from 295 men on the relationships between urinary levels of phthalate metabolites and serum levels of FSH, LH, SHBG, testosterone, and inhibin-b. 95 After adjusting for age, body mass index, and time of day that the blood sample was drawn, there was an inverse assoiation between MBzP and FSH, and a positive assoiation between MBP and inhibin B. An IQR inrease in MBzP was assoiated with a 10% derease (95% CI 216 to 24) in FSH. For an IQR inrease in MBP, inhibin B inreased 4.8% (95% CI 0 to 10). MEHP was inversely assoiated with testosterone (20.47 nmol/ml; 95% CI to 0.10; p = 0.1). In summary, Duty and oworkers found evidene of assoiations between MBP and MBzP and altered semen quality, 93 MEP and sperm DNA damage, 92 and MBP and MBzP and reprodutive hormones. 95 Although there is some onsisteny of their results with experimental animal studies, there are also inonsistenies. For instane, in studies on laboratory animals, MEHP, MBP, and MBzP have been shown to adversely effet semen prodution and quality. In the Duty et al study, assoiations were found with MBP and MBzP, but no assoiations were found with MEHP. 93 In addition, there is no experimental evidene showing that MEP damages DNA. The hanges in the serum levels of inhibin B and FSH in assoiation with MBP and MBzP, respetively, did not hange in the expeted patterns. Therefore, it is unlear whether these assoiations represented physiologial relevant alterations in these hormones, or whether they represented assoiations found as a result of onduting multiple omparisons. However, despite these inonsistenies with animal data, this work begins to provide us with human data neessary to assess potential risk and the publi health impliations related to ubiquitous human exposure to phthalates. Murature and oworkers reruited 21 university students to explore the relationship between sperm onentration and DBP onentrations in the ellular frations of ejaulates. 96 The geographial loation from whih the subjets were reruited was not indiated. Furthermore, the statistial analyses performed were not traditional that is, they did not treat the subjets as a single population. Instead, the authors assumed that there were two populations that differed in their ability to metabolise DBP. It is not entirely lear, but it seems that the two populations were defined by a visual inspetion of DBP onentrations. Based on DBP onentrations, the subpopulations were defined as those with a lower ability to metabolise and those with a greater ability to metabolise DBP. Within eah subpopulation, they explored the relationship between DBP and sperm onentration. In the subpopulation with a lower ability to metabolise DBP, there was an inverse relationship between sperm onentration and DBP (r = 20.4; slope of regression 20.7). In the subpopulation with a greater ability to

8 metabolise DBP, there was also an inverse orrelation of 20.4 (slope of regression 20.6) between DBP and sperm onentration. The study did not measure or adjust for potential onfounders. In India, Rozati and oworkers studied 53 men: 21 infertile men with poor semen quality and 32 ontrol men with normal semen parameters. 97 Phthalate esters were measured in seminal plasma and results were reported as the sum of a mixture of DMP, DEP, DBP, BBzP, DEHP, and DnOP. The onentration of phthalates was inversely orrelated with sperm morphology (r = 20.77, p, 0.001) and positively orrelated with the perentage of single stranded DNA in sperm (r = 0.86, p, 0.001) assessed with the sperm nulear hromatin ondensation test. The onentration of phthalates was not orrelated with ejaulate volume, sperm onentration, or motility. The authors measured total phthalate diesters and did not report results for individual phthalates. The results are noteworthy beause they demonstrate the presene of phthalates in seminal plasma. However, beause diesters were measured, sample ontamination is a potential onern. Time to pregnany In Sweden, Modigh and olleagues onduted a well designed retrospetive study on the relationship between time to pregnany among partners of men oupationally exposed to DEHP. 98 They reruited ouples in whih the male partner was employed at plants produing or using DEHP (that is, plants proessing PVC plasti); 227 ouples partiipated and ontributed 397 pregnanies. Exposure was estimated using air sampling measurements ombined with questionnaires assessing work loation and work tasks. Men were assigned to three exposure groups (none, low [,0.1 mg/ m 3 ], and high [.0.1 mg/m 3 ]. No assoiations were found between exposure to DEHP and time to pregnany. Statistial methods were used in whih they also onsidered the time lag for sperm prodution and maturation, as well as restriting the analyses to the first reorded pregnany. The results remained unhanged. Although maximum exposure was 2.1 mg/m 3, paternal exposure exeeded 0.5 mg/m 3 for only four pregnanies. Testiular aner Although exposure to phthalates was not diretly measured, the relationship between testiular aner and oupational exposure to PVC plastis has been explored in two studies. Hardell et al used the Swedish Caner Registry to identify both ases of testiular aner (n = 148) and ontrols (n = 315). 99 A self-administered questionnaire was used to assess oupational histories. Potential oupational exposure to PVC plastis were onfirmed by the employers. Six ases with seminoma and two ontrols reported exposure to PVC (OR = 5.6, 95% CI 1.1 to 196). Exposures to other types of plastis were not assoiated with inreased risk. In a subsequent larger study, Hansen in 1999 did not find an assoiation (OR,1.0) between oupational exposure to plastis, mainly PVC, and testiular aner. 100 Hansen s study was a nationwide ase-ontrol study (3745/7212) with objetive assessment of work histories using the national Supplementary Pension Fund in Denmark. In onlusion, based on these two studies there is inadequate evidene of an assoiation between oupational exposure to PVC plastis and testiular aner. Female reprodutive health Reent studies in experimental animals have found assoiations between DEHP and its metabolite MEHP and ovarian toxiity in sexually mature rats Effets of DEHP inlude prolonged oestrous yles, suppressed or delayed ovulation, smaller preovulatory folliles due to redued granulosa ell size, and dereased irulating serum oestradiol. Suppression of oestradiol prodution by granulosa ells seondarily resulted in inreased serum FSH levels and an absene of the LH surge neessary for ovulation. DEHP also suppressed serum progesterone levels. Thus, DEHP and MEHP resulted in hypoestrogeni, hypoprogestini, anovulatory yles in adult female rats. MMP, MEP, monopropyl phthalate, MBP, and monopentyl phthalate had no effets on oestradiol prodution. 103 Studies in pregnant and pseudopregnant rats have also found assoiations between DBP and BBzP and impaired implantation in mated females and dereased deidualisation in the pseudopregnant animals. Based on the experimental studies, endpoints of interest in humans should inlude ovulatory funtion, whih may inlude redued fertility or prolonged time to pregnany, and serum levels of oestradiol, progesterone, LH, and FSH. Endometriosis Cobellis and olleagues in Italy published in 2003 a aseontrol study on the relationship between plasma and peritoneal fluid levels of DEHP and endometriosis. 106 Study subjets were fertile women that underwent diagnosti laparosopy for ovarian ysts or hroni pelvi pain and dysmenorrhoea. Patients with histologial onfirmation of endometriosis were enrolled as ases (n = 35). Controls (n = 24) were healthy age mathed subjets without known infertility or reprodutive diseases. Blood samples were obtained the day before the surgial proedure or immediately before anaesthesia for laparosopy. Peritoneal fluid was obtained by uldoentesis during laparosopy. Women with endometriosis had higher plasma DEHP onentrations (median 0.57 mg/ml, IQR ) than ontrol women (0.18 mg/ml, IQR ) (p = ). Plasma MEHP onentrations did not differ between groups and were generally very low exept for two ases. For ases, the median MEHP was 0.38 mg/ml (IQR ) and for ontrols the median was 0.58 mg/ml (IQR ). Cases and ontrols had similar levels of peritoneal DEHP and MEHP onentrations. The orrelation between serum DEHP and MEHP onentrations was weak (r = 0.16, p > 0.3). The weak relationship between serum levels of DEHP and MEHP raises questions regarding the utility of the DEHP measurements. Beause peritoneal fluid may ontain esterases apable of hydrolysing DEHP to MEHP, further exploration into why the relationships between DEHP and MEHP with endometriosis differed is warranted. Pregnany outomes (gestational age, ompliations of pregnany) Latini and oworkers measured serum DEHP and MEHP in the ord blood of 84 newborns born at the general pratie Brindisi Hospital, Brindisi, Italy in The mean gestational age was 38.4 weeks (SD 2.2, range weeks) and the mean birth weight was 3220 g (SD 680, range g). Eleven of the newborns were preterm births and three were very low birth weight babies. All ord blood speimens were olleted and handled with glass devies to avoid ontamination with DEHP. DEHP and MEHP were 813

9 814 eah present in 65 of the samples (77%). MEHP positive newborns (n = 65) had a younger gestational age than MEHP negative newborns (n = 19) (38.16 weeks (SD 2.34) ompared to weeks (SD = 1.35), respetively, p = 0.033). In logisti regression analyses on gestational age, the OR for absene of MEHP was 1.50 (95% CI to 2.21). Gestational age was not assoiated with DEHP positive newborns ompared to DEHP negative newborns. There were no differenes in birth weight when DEHP or MEHP positive newborns were ompared to DEHP or MEHP negative newborns, respetively. In addition, DEHP or MEHP did not predit sex of the infant, delivery mode, maternal smoking, premature rupture of the membranes, presene of ord loops, neonatal jaundie, or small size for gestational age. Potential study limitations inlude the lak of information on whether all the deliveries were unompliated vaginal births, whether any pregnanies required medial interventions during delivery that may have lead to exposure to DEHP, and the relatively low seletivity and sensitivity of the analytial method used for measuring DEHP and MEHP. The authors hypothesise that the shorter pregnany duration may be due to phthalates playing a role in induing an intrauterine inflammatory proess, a risk fator for preterm birth. Tabaova and oworkers in 1999 published an abstrat desribing the results of a ross-setional study in Bulgaria on 93 pregnant women residing in proximity to a plastis prodution plant. 108 Urinary levels of non-speified phthalates were measured and ompared in women with normal pregnanies to those ompliated by anaemia, toxaemia (inludes onditions suh as preelampsia, hyperemesis), and spontaneous abortion. They ontrolled for demographi harateristis, reprodutive history, and lifestyle fators. The overall mean phthalate level for women with any ompliations was 217 mg/100 ml urine ompared to 81 mg/ 100 ml for women with normal pregnanies (p = 0.02). Although these results are intriguing, beause only an abstrat was published, the potential strengths and limitations of the methods used in the study are unknown. Therefore, this study is only presented for ompleteness of the review. Respiratory health Interior materials used in the home environment inlude plastiised PVC produts used as wall and floor overing materials. These PVC produts are potential emission soures of hemials inluding, among others, DEHP widely used as a PVC plastiizer. Other soures of emissions inlude visosity modifiers and stabilisers used in the PVC produts. Phthalates have been measured in residential indoor environments in both house dust and indoor air. 109 The emission hemials from these PVC produts may ause airway inflammation and inrease the risk of bronhial obstrution and asthma. MEHP has been shown to indue bronhial hypersensitivity in rats, 110 and preterm infants exposed to respiratory PVC tubing materials had a higher risk of bronhial asthma. 81 MEHP may also have properties similar to prostaglandins, potent mediators of inflammation. 111 Airway obstrution and lung funtion Several epidemiologial studies have explored the relationship between interior materials in the indoor environment and airway obstrution and asthma. These studies used questionnaires or experts observation to identify and ategorise residential indoor surfae materials In another study, researhers explored the relationship between wheeze in hildhood and syntheti bedding. 114 Jaakkola and oworkers onduted a mathed pair aseontrol study (251 ases and ontrols) within a birth ohort of 3754 hildren born in Oslo, Norway from 1992 to Information on the hildren s health and environment was obtained at 6, 12, 18, and 24 months of follow up. The interior surfaes were ategorised by trained experts and exposure was ategorised based on the materials used; in addition, a plastiizer exposure index was alulated. Physiian or medial reords or information form questionnaires was used to identify bronhial obstrution, defined as two or more episodes with symptoms and signs of bronhial obstrution or one episode lasting more than one month. In both the rude analysis and in the adjusted analysis, the risk of bronhial obstrution was greater in the presene of PVC in the floors (adjusted OR = 1.89; 95% CI 1.14 to 3.14) and of textile wallpaper in one or more rooms (adjusted OR = 1.58; 95% CI 0.98 to 2.54) ompared with the homes with wood or parquet flooring and painted walls and eilings. Bronhial obstrution was also assoiated with the plastiizer exposure index (adjusted OR = 2.72; 95% CI 1.50 to 4.91, omparing the highest quartile of exposure to the referene ategory). The study was well designed and a variety of potential onfounders were olleted and adjusted for. Among the same ohort of hildren, Oie and oworkers in 1999 found that the relation of bronhial obstrution to plastiizer exposure index was stronger in homes in the low air hange ategory than in the high air hange ategory. 115 Jaakkola and oworkers in 2000 onduted a population based ross-setional study on 2568 Finnish hildren aged 1 7 years to assess the relationship between the presene of plasti wall materials in the home and respiratory health. 113 A questionnaire was used to assess the hild s health and home environment, speifially fousing on the presene of plasti wall materials. The outomes of interest were dotor diagnosed asthma, allergi rhinitis, persistent wheezing, persistent ough, persistent phlegm, weekly nasal ongestion/exretion, and respiratory infetions. There were assoiations between the presene of plasti wall materials and lower respiratory trat symptoms: persistent wheeze (OR = 3.42, 95% CI 1.13 to 10.36), ough (OR = 2.41, 95% CI 1.04 to 5.63), and phlegm (OR = 2.76, 95% CI 1.03 to 7.41). Upper respiratory symptoms were not assoiated with plasti wall materials. Although there was an assoiation with asthma, the onfidene interval was wide (OR = 1.52, 95% CI 0.35 to 6.71). Ponsonby and oworkers in 2003 published the results of a prospetive study on a birth ohort of infants in Tasmania. 114 Parental report of bedding data at 1 month of age was available for 863 infants (of 6378 hildren) who were partiipating in a seven year follow up survey. Syntheti bedding inluded syntheti overlying quilts or syntheti pillows whih inluded foam, sponge, tontine, polyester, and Daron. One outome measure was frequeny of wheeze defined as more than 12 episodes in the past year. Syntheti pillow use at 1 month of age was assoiated with frequent wheeze at 7 years of age (adjusted relative risk (arr) = 2.5, 95% CI 1.2 to 5.5) independent of later hildhood exposure to syntheti bedding. In addition, urrent syntheti pillow and quilt use was assoiated with frequent wheeze (arr = 6.4, 95% CI 1.2 to 35). Among hildren with asthma, the age of onset was earlier if syntheti bedding was used in infany.

10 Although this study was well designed and the assoiations between syntheti bedding and wheeze were onsistent and strong, it is diffiult to determine the potential role, if any, of phthalates emitted from the syntheti bedding. There are many other potential explanations for the observed assoiations beause syntheti bedding may lead to inreased exposure to house dust mite, volatile organi ompounds, and redued exposure to endotoxin, all potential risk fators for hildhood asthma and wheeze. Hoppin and oworkers in 2004 explored the assoiation between urinary levels of phthalate monoesters and pulmonary funtion parameters (fored vital apaity (FVC), fored expiratory volume in 1 seond (FEV 1 ), peak expiratory flow (PEF), and maximum mid-expiratory flow (MMEF)) among 240 adults from NHANES III all-bak ohort. 116 After ontrolling for rae, age, height, body mass index, smoking history, and urrent smoking, there was an assoiation between MBP urinary levels and three measures of pulmonary funtion (FVC, FEV 1, PEF) in males but not females. Among men there were large derements in lung funtion; for an IQR hange (25th to 75th entile) in urinary MBP levels, FEV 1 dereased 112 ml (SE = 51, p = 0.04), FVC dereased 131 ml (SE = 63, p = 0.04), and PEF dereased 367 ml (SE = 181, p = 0.05). MEP was assoiated with lower FVC and FEV 1 in men. MEHP was not assoiated with any of the lung funtion measures. In analyses limited to nonsmoking men, point estimates remained essentially unhanged. Among non-smoking women, MEHP was positively assoiated with FEV 1 and MMEF. In this rosssetional study, it is unlear why assoiations were only found in men and why MEHP, a metabolite of DEHP whih is found in PVC plastis and impliated in the studies on residential use of PVC plasti interior materials, was not assoiated with lung funtion derements. Beause the Hoppin et al study was ross-setional it was also not possible to determine whether the derements in lung funtion were assoiated with long term phthalate exposure or assoiated with reent exposure and thus represent short term derements that may be reversible. However, despite these unertainties, the study had several strengths inluding objetive measures of lung funtion and the use of biologial measures of phthalate exposure. Bornehag and olleagues in 2004 explored the relationship between allergi symptoms in hildren and the onentration of phthalates in house dust. 117 A nested ase ontrol study was onduted within a ohort of hildren 1 6 years of age in Varmland, Sweden. The ases onsisted of 198 hildren with persistent allergi symptoms and 202 ontrols without symptoms. Persistent symptoms were defined as ezema, wheezing, or rhinitis without a old during the preeding 12 months on the initial questionnaire and at least two of three symptoms reported 18 months later on the follow up questionnaire. Controls did not report symptoms on either questionnaire. In addition to questionnaire assessment of symptoms, physiians performed examinations and obtained a medial history to assess asthma, rhinitis, and ezema. Apart from 23 hildren, ase-ontrol assignment based on the questionnaire agreed with the physiian diagnosis. Separate statistial analyses were presented for ase ontrol status based on the questionnaire and for physiian diagnosis of disease. PVC flooring materials in the hild s bedroom were assoiated with questionnaire ase status (OR = 1.59, 95% CI 1.05 to 2.41). Children with physiian diagnosed asthma, rhinitis, or ezema had higher BBzP onentrations in bedroom dust than hildren without disease. Higher DEHP dust onentrations were also found in the bedrooms of hildren with physiian diagnosed asthma. The BBzP relationships remained after restriting the analysis to homes with PVC flooring in the hild s bedroom. In dose-response analyses in whih phthalates were ategorised into quartiles, BBzP was assoiated with ase status in the rude and adjusted analyses. Adjustments were made for the following ovariates: environmental tobao smoke, gender and age of hild, type of building, onstrution period of building, self reported water leakage, and exposure to other phthalates. In addition, assoiations between BBzP and dotor diagnosed rhinitis and ezema, and between DEHP and dotor diagnosed asthma were found. The authors hypothesised that in addition to the different toxiologial and pharmaokineti properties of the phthalates, the physial and hemial properties of the different phthalates may partially aount for the differenes noted between BBzP and DEHP with regard to the type of symptom. BBzP would likely have higher onentrations in the gas phase while DEHP would likely have higher onentrations in the ondensed phase (that is, dust and airborne partiles). This may aount for the assoiation of DEHP with lower airway symptoms ompared to the assoiation of BBzP primarily with skin and muosal irritation. Potential onfounding by soioeonomi status (SES) was onsidered unlikely in the study beause of a more homogeneous distribution of SES in Sweden. Furthermore, when the analyses were restrited to single family houses the results were unhanged. In onlusion, there is limited epidemiologial evidene of an assoiation between some phthalate esters and allergi and airway symptoms. A ross-setional study found assoiations between urinary levels of MBP and lower pulmonary funtion in adults, 116 while a ase-ontrol study of hildren found assoiations between allergi symptoms and house dust levels of BBzP and DEHP. 117 Several studies have also reported relationships between bedding materials and PVC building materials in the indoor environment and airway obstrution or asthma OCCUPATIONAL HEALTH STUDIES: GENERAL HEALTH EFFECTS Nielsen and oworkers ondued a small study on 44 workers at a PVC proessing plant in whih DiDP, DEHP, and BBzP were used as plastiizers. 118 Workers were employed for an average of 8 years (range 1 21). The ontrols were five healthy men who were members of the laboratory staff. Exposure was assesses by phthalate diester measurements in air and phthalate monoester metabolites in urine. Air exposures varied and in speifi job titles exeeded 2 mg/ m 3. In workers, they did not find an inreased prevalene of peripheral neuropathy, obstrutive lung disease, or dereased lung funtion measures. However, the study was small and underpowered to detet hanges in the prevalene of these outomes. Among 147 workers exposed to phthalate plastiizers in the manufature of artifiial leather and films based on PVC resins, Milkov and oworkers in 1973 reported a moderately inreased prevalene of toxi polyneuritis. 119 It was diffiult to relate findings to speifi phthalates or phthalates in general as there were other workplae hemial (for instane, vinyl 815

11 816 hloride, arbon monoxide, and hydrogen hloride) and physial exposures. Oupational studies on potential assoiations with health risks are very limited. Further researh among oupationally exposed workers is needed to better define potential risk. Oupations with potential exposure inlude, among others, produers of phthalates, PVC plasti manufaturers, fragrane industry, and workers in beauty are, suh as nail tehniians and hairdressers. CONCLUSIONS Depending on the health endpoint of interest, there is urrently only limited or inadequate human data on the relationships between exposure to phthalates and human health effets. Although phthalates have been in ommerial use for over 50 years, human studies using biologial measures of exposure (that is, monoester) have generally been onduted within the last five years. Further epidemiologial studies are needed to advane our understanding of potential human health risks of phthalates. The following are the most ommon limitations of the published studies on assoiations between exposure to phthalates and health outomes: (1) small human populations; (2) sensitivity, seletivity, or ontamination issues related to some analytial methods; and (3) lak of either Key points Phthalates are a family of ompounds that exhibit marked differenes in toxiity in relation to their struture. In laboratory animal studies, some phthalates have been assoiated with developmental and reprodutive toxiity. These studies show that the most sensitive life stages are: fetal. peri-pubertal. adult (mature). Most of the US population is exposed to phthalates. Data suggest that phthalate exposure is also prevalent in other ountries (e.g. Germany and Denmark). Exposure is believed to be primarily through ingestion and inhalation, although dermal exposure may be important for some phthalates (e.g. diethyl phthalate). Speial populations, suh as neonates in intensive are units, may be highly exposed to di(2-ethylhexyl phthalate, DEHP) through the use of medial devies. Phthalates have short biologial half-lives, metabolise quikly, do not aumulate, and are primarily exreted in the urine. Therefore, the urinary onentrations of phthalate metabolites provide an exellent biomarker of exposure. The metabolism and exretion of phthalates varies based on their hemial struture. Oxidative metabolism is prevalent for high moleular weight phthalates (e.g. DEHP, di-isononyl phthalate). Therefore, biomonitoring strategies should be designed with this onsideration in mind. Exposure to high doses of some phthalates auses reprodutive and developmental toxiities in both male and female animals. Although several human studies have explored possible assoiations between phthalate esters and altered semen quality, shortened gestation, redued anogenital distane in baby boys, and premature breast development in young girls, data are limited and further study is reommended. There is limited epidemiologial evidene of an assoiation between some phthalate esters, speifially DEHP, dibutyl phthalate, and butylbenzyl phthalate, and allergi and airway symptoms. environmental or biologial measures of phthalates. In ontrast, toxiologial studies provide suffiient evidene that some phthalate esters and their metabolites, speifially MBP, MBzP, and MEHP, are reprodutive and developmental toxiants in rats. Based on our review of the literature and our understanding of the toxiity of phthalates from experimental studies, the following reommendations for future researh are put forward. Studies need to be onduted to identify the phthalate metabolites relevant to human health and the relative ontribution of the pathways through whih humans are exposed to phthalates. A few outstanding questions and issues related to these needs inlude the following. What oxidative metabolites of high moleular weight phthalates (for example, DEHP, DiNP, DiDP) are the best biomarkers of exposure to these phthalates? To assess DEHP exposure, is it suffiient to measure only urinary levels of MEHP or should the oxidative metabolites also be measured? If MEHP and oxidative metabolites are measured, how do we use these measures in epidemiologial studies? What is the proportional ontribution of ingestion, inhalation, and dermal exposure to phthalate body burden? Human health effets studies are needed to explore: (1) the relationship of gestational exposure to phthalates with male reprodutive trat development (AGD, hypospadias, ryptorhidism); (2) the relationship of both early life (that is, gestational and pubertal) and adult exposure with male and female reprodutive funtion (that is, menstrual yle and semen quality assessments, as well as measures of fertility and feundity); and (3) the relationship of early life and adult exposure with asthma and obstrutive airway disease. Another area of interest is the relationship, if any, between early life exposure and testiular aner, part of the triad of the testiular dysgenesis syndrome Authors affiliations R Hauser, Department of Environmental Health, Oupational Health Program, Harvard Shool of Publi Health, Boston, MA 02115, USA A M Calafat, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA Funding: this work was supported in part by grants ES09718 and ES00002 from the National Institute of Environmental Health Sienes, NIH Competing interests: none REFERENCES 1 ATSDR. Toxiologial profile for di-n-otyl phthalate (DNOP). Atlanta, GA: Ageny for Toxi Substanes and Disease Registry, ATSDR. Toxiologial profile for di(2-ethylhexyl)phthalate (DEHP). Atlanta, GA: Ageny for Toxi Substanes and Disease Registry, David RM, MKee RH, Butala JH, et al. Esters of aromati mono-, di-, and triarboxyli aids, aromati diaids, and di-, tri-, or polyalohols. 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