The Association between Biomarker-Based Exposure Estimates for
Phthalates and Demographic Factors in a
Human Reference Population

Environmental Health Perspectives v.110, n.4, Apr02

[Excerpts only-not complete paper]

Jung-Wan Koo,1 Frederick Parham,1 Michael C. Kohn,1 Scott A. Masten,1 John W. Brock,2 Larry L. Needham,2 and Christopher J. Portier1
1 Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA;
2 National Center for Environmental Health, Center for Disease Control and Prevention, Atlanta, Georgia, USA

Abstract
Population-based estimates of environmental exposures using biomarkers can be difficult to obtain for a variety of reasons, including problems with limits of detection, undersampling of key strata, time between exposure and sampling, variation across individuals, variation within individuals, and the ability to find and interpret a given biomarker. In this article, we apply statistical likelihoods, weighted sampling, and regression methods for censored data to the analysis of biomarker data. Urinary metabolites for seven phthalates, reported by Blount et al., are analyzed using these methods. In the case of the phthalates data, we assumed the underlying model to be a log-normal distribution with the mean of the distribution defined as a function of a number of demographic variables that might affect phthalate levels in individuals. Included as demographic variables were age, sex, ethnicity, residency, family income, and education level. We conducted two analyses: an unweighted analysis where phthalate distributions were estimated with changes in the means of these distributions as a function of demographic variables, and a weighted prediction for the general population in which weights were assigned for a subset of the population depending on the frequency of their demographic variables in the general U.S. population. We used statistical tests to determine whether any of the demographic variables affected mean phthalate levels. Individuals with only a high school education had higher levels of di-n-butyl phthalate than individuals with education beyond high school. Subjects who had family income less than $1,500 in the month before sampling and/or only high school education had higher levels of nbutyl benzyl phthalate levels than other groupings. Di(2-ethylhexyl) phthalate was higher in males and/or in urban populations and/or in people who had family income less than $1,500 per month. Our findings suggest that there may be significant demographic variations in exposure and/or metabolism of phthalates and that health-risk assessments for phthalate exposure in humans should consider different potential risk groups. Key words: demographic factors, phthalates, risk assessment. Environ Health Perspect 110:405–410 (2002). [Online 11 March 2002] http://ehpnet1.niehs.nih.gov/docs/2002/110p405-410koo/abstract.html

Phthalates are important industrial chemicals used in the manufacture of a wide range of plastic and nonplastic products and can be divided into two basic groups: those used as plasticizers for synthetic polymers that are incorporated into food wrap, medical tubing, and molded toys, and those used primarily in consumers products such as varnishes, perfumes, nail polishes, and insect repellents. It is conceivable that the route of exposure of an organism to phthalates is an important parameter when considering metabolism of these chemicals in vivo. Phthalates are readily metabolized in the gut, such that oral exposure would not lead to accumulation of high concentrations of these chemicals (1). However, few data are available on the metabolism of this group of chemicals after inhalation or dermal exposure. The primary route of phthalate exposure to the general human population has been presumed to be ingestion. Lower molecular-weight phthalates such as diethyl phthalate (DEP) and di-nbutyl phthalate (DBP) can be absorbed percutaneously, and the more volatile congeners can be inhaled. Dermal absorption is important for products applied to skin.

Blount et al. (2) reported the concentrations of seven phthalate monoesters [monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP), monocyclohexyl phthalate (MCHP), mono-2-ethylhexyl phthalate (MEHP), monooctyl phthalate (MOP), monoisononyl phthalate (MINP)] in the urine of 289 people, providing the first systematic compilation of data that address phthalate exposures to the general population from commercially important phthalate diesters. Kohn et al. (3) applied a simple pharmacokinetic model to estimate the total daily intake of phthalates that would result in the reported urinary concentrations of monoester metabolites. These intake estimates were used as a measure of total exposure to diethyl phthalate (DEP), di-n-butyl phthalate (DBP), n-butyl benzyl phthalate (BBP), dicyclohexyl phthalate (DCHP), di-(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DOP), di-inonyl phthalate (DINP).

Blount et al. (2) reported a considerable number of observations in which the analyte levels in urine were below the limit of detection (LOD) for the procedure being used.

This analysis excluded analytes for which more than 25% of the studied individuals were below the LOD and discarded individuals below the LOD for analytes they did analyze. This represents a substantial loss of information. Maximum likelihood methods for censored observations (4–7) have been used for many years to analyze survival data and data for which some observations cannot be seen, but it is known that the observation is beyond some critical point. For urinary metabolite data, an observation below the LOD can be assumed to have a metabolite concentration less than the LOD. Methods have been developed for analyzing biomarkers of exposure—including observations below the LOD—by using statistical likelihoods and regression methods for censored data (8). Using a likelihood for censored data, these fractional pieces of information contribute to the overall interpretation of the data and can be used in a natural framework to estimate parameters and test for population differences. To account for strata differences of demographic factors, we estimated population-based exposures to phthalates using a weighted analysis in which weights were assigned for each individual group depending on the frequency of their demographic variables in the general U.S. population.

The aim of this study was to present methods for the analysis of exposure estimates based on urinary biomarker data accounting for strata differences and problems with LOD and to investigate the association between biomarker-based exposure estimates for phthalates and demographic factors in a human reference population.

p.408

Phthalates are used in the manufacture of a wide range of plastic and nonplastic products. Most of a phthalate dose is cleared in 24 hr and completely eliminated in 3–5 days (13,19–22). Because phthalates are lipophilic (23), it might be predicted that these compounds would accumulate in fat. However, with other lipophilic compounds, such as polychlorinated biphenyls, deposition of the compound into fat may not occur until several hours or several months after dosing (24–26). Because of the rapid metabolism of phthalates to more polar metabolites, these compounds are not sequestered in fat. Phthalates are widely distributed in the body, with the liver being the major, initial repository organ. Clearance from the body is rapid, and there is only a slight cumulative potential (16). Even though there is only a slight cumulative potential, phthalates are found in a wide variety of extensively used products, have been identified in all environmental compartments, and are a serious concern for the possibility of adverse effects. The acute toxicity of phthalates is low, with LD50 values ranging from 0.7 to > 20 g/kg (27); however, changes in lipid metabolism (28–30), testicular atrophy (31,32), alterations in xenobiotic metabolism (33,34), liver peroxisome proliferation (35), and carcinogenicity (36,37) have been observed. Regarding reproductive and developmental effects, phthalates vary in potency, with DEHP being the most potent and DBP and BBP roughly an order of magnitude less potent (38–45).

Another difficulty in estimating the environmental hazard posed by phthalates is the lack of sufficient data documenting the human and wildlife exposure. Furtmann (46) has suggested that the main source of phthalates is consumer products, and that as a result of disposal of these products, there are considerable phthalate emissions into the

especially with respect to potentially susceptible populations. Our analysis suggests that people with a high school education or less have higher urinary output of DBP and BBP metabolites; individuals with a family income less than $1,500 in the month before sampling have higher urinary output of BBP and DEHP metabolites; and males and urban populations have higher urinary output of DEHP metabolites. The analysis used assumed that the pharmacokinetics of these compounds is the same in all individuals; this may not be true because genetic polymorphisms in the genes controlling the metabolism and elimination of phthalates may exist and could have an impact on levels of these metabolites in the urine. Hence, our findings may derive from differences in actual exposures, differences in metabolism, or a combination of these. Further study is needed to determine which of these may drive the observed differences.

Table 3. Estimated phthalates exposure (µg/kg/day) weighted using demographic characteristics in the general US population and using regression parameters which are significant (p < 0.15) from the LIFEREG procedure.^a

Phthalates 	Variableb 	     Mean 	Median 	   percentile 	percentile
DEP 		Ethnicity (black)    10.1 	10.2 	   0.43 	229
DBP 		Education, ethnicity 1.66	1.66 	   0.31 	8.78
		  (Mexican)
BBP 		Family income, 	     0.84 	0.85 	   0.19 	3.65
		  education
DCHP 		Family income 	     1.26×10–5  1.30×10–5  1.18×10–9 	0.14
DEHP 		Sex, residence,
		  family income      0.41	0.41	   0.015	11.3
DOP 		Residence, education 6.16×10–5 	6.26×10–5  2.19×10–9 	1.56
DINP 		None 		     8.99×10–7 	9.28×10–7  4.25×10–13 	1.67

a Data from U.S. Census Bureau (18).
b Below 0.15 significant level.

REFERENCES AND NOTES

1. Harris CA, Henttu P, Parker MG, Sumpter JP. The estrogenic activity of phthalate esters in vitro. Environ Health Perspect 105:802–811 (1997).

2. Blount BC, Silva MJ, Caudill SP, Needham LL, Pirkle JL, Sampson EJ, Lucier GW, Jackson RJ, Brock JW. Levels of seven urinary phthalate metabolites in a human reference population. Environ Health Perspect 108:979–982 (2000).

3. Kohn MC, Parham F, Masten SA, Portier CJ, Shelby MD, Brock JW, Needham LL. Human exposure estimates for phthalates. Environ Health Perspect 108:A440–442 (2000).

4. Tsay JY, Chen IW, Maxon HR, Heminger L. A statistical method for determining normal ranges from laboratory data including values below the minimum detectable value. Clin Chem 25:2011–2014 (1979).

5. Vlachonikolis IG, Marriott FH. Evaluation of censored contanimation data. Food Addit Contam 12:637–644 (1995).

6. Lindsey JK, Byrom WD, Wang J, Jarvis P, Jones B. Generalized nonlinear models for pharmacokinetic data. Biometrics 56:81–88 (2000).

7. Burmaster DE, Wilson AM. Fitting second-order finite mixture models to data with many censored values using maximum likelihood estimation. Risk Anal 20:261–271 (2000).

8. Tobin J. Estimation of relationships for limited dependent variables. Econometrica 26:24–36 (1958).

9. Department of Health and Human Services. National Center for Health Statistics. Third National Health and Nutrition Examination Survey, 1988–1994, NHANES III Laboratory Data File. Public Use Data File Documentation No. 76200. Hyattsville, MD:Centers for Disease Control and Prevention, 1996.

10. Tanaka A, Matsumoto A, Yamaha T. Biochemical studies on phthalic esters. III. Metabolism of dibutyl phthalate (DBP) in animals. Toxicology 9:109–123 (1978).

11. Foster PMD, Cook MW, Thomas LV, Walters DG, Gangolli SD. Differences in urinary metabolic profile from di-nbutyl phthalate-treated rats and hamsters. A possible explanation for species differences in susceptibility to testicular atrophy. Drug Metab Dispos 11:59–61 (1983).

12. Nativelle C, Picard K, Valentin I, Lhugenot JC, Chagnon MC. Metabolism of n-butyl benzyl phthalate in the female Wistar rat. Identification of new metabolites. Food Chem Toxicol 37:905–917 (1999).

13. Eigenberg DA, Bozigian HP, Carter DE. Distribution, excretion, and metabolism of butylbenzyl phthalate in the rat. J Toxicol Environ Health 17:445–456 (1986).

14. Castle L. Personal communication.

15. Peck CC, Albro PW. Toxic potential of the plasticizer di(2-ethylhexyl) phthalate in the context of its disposition and metabolism in primates and man. Environ Health Perspect 45:11–17 (1982).

16. Kluwe WM. Overview of phthalate ester pharmacokinetics in mammalian species. Environ Health Perspect 45:3–9 (1982).

17. Albro PW, Moore B. Identification of the metabolites of simple phthalate diesters in rat urine. J Chromatogr 94:209–218 (1974).

18. U.S. Census Bureau. Statistical Abstract of the United States: 2000. Available: http://www.census.gov/statab/www/ [cited 1 August 2001].

19. Williams DT, Blanchfield BJ. The retention, distribution, excretion and metabolism of dibutyl-7-14C in the rat. J Agric Food Chem 23:854–858 (1975).

20. Tanaka A, Adachi T, Takahaski T, Yamaha T. Biochemical studies on phthalic esters. I. Elimination, distribution and metabolism of di-(2-ethylhexyl) phthalate in rats. Toxicology 4:253–264 (1975).

21. Daniel JW, Bratt H. The absoption, metabolism and tissue distribution of di(2-ethylhexyl) phthalate in rats. Toxicology 2:51–65 (1974).

22. Williams DT, Blanchfield BJ. The retention, excretion and metabolism of di(2-ethylhexyl) phthalate administered orally to the rat. Bull Environ Contam Toxicol 11:371–378 (1974).

23. Leyder F, Boulanger P. Ultraviolet absorption, aqueous solubility and octanol-water partition for several phthalates. Bull Environ Contam Toxicol 30:152–157 (1984).

24. Matthews HB, Anderson MW. The distribution and excretion of 2,4,5,2’,5’-pentachlorobiphenyl in the rat. Drug Metab Dispos 3:211–219 (1975).

25. Pittman KA, Wiener M, Treble DH. Mirex kinetics in the rhesus monkey. II. Pharmacokinetic model. Drug Metab Dispos 4:288–295 (1976).

26. Rozman T, Rozman K, Williams J, Greim H. Enhanced fecal excretion of mirex in rhesus monkeys by 5% mineral oil in the diet. Drug Chem Toxicol 4:251–262 (1981).

27. Autian J. Toxicity and health threats of phthalate esters: review of the literature. Environ Health Perspect 4:3–26 (1973).

28. Reddy JK, Moody DE, Azarnoff DL, Rao MS. Di-(2-ethyl hexyl) phthalate: an industrial plasticizer induces hypolipidemia and enhances hepatic catalase and carnitine acetyltransferase activities in rats and mice. Life Sci 18:941–946 (1976).

29. Bell FP, Patt CS, Brundage B, Gillies PJ, Phillips WA. Studies on lipid biosynthesis and cholesterol content of liver and serum lipoproteins in rats fed various phthalate esters. Lipids 13:66–74 (1978).

30. Oishi S, Hiraga K. Effects of monoesters of o-phthalic acid on serum lipid composition of rats. Toxicol Lett 14:79–84 (1982).

31. Creasy DM, Foster JR, Foster PMD. The morphological development of di-n-pentyl phthalate induced testicular atrophy in the rat. J Pathol 139:309–321 (1983).

32. Oishi S, Hiraga K. Testicular atrophy induced by di-2-ethylhexyl phthalate: effect of zinc supplement. Toxicol Appl Phamacol 70:43–48 (1983).

33. Aitio A, Parkki M, Effect of phthalate esters on drug metabolizing enzyme activities in rat liver. Arch Int Pharmacodyn Ther 235:187–195 (1978).

34. Walseth F, Toftgard R, Nilsen OG. Phthalate esters I: Effects on cytochrome P-450 mediated metabolism in rat liver and lung, serum enzymatic activities and serum protein levels. Arch Toxicol 50:1–10 (1982).

35. Moody DE, Reddy JK. Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol Appl Pharmacol 45:497–504 (1978).

36. Reddy JK, Azarnoff DL, Hignite CE. Hypolipidaemic hepatic peroxisome proliferators form a novel class of chemical carcinogens. Nature 283:397–398 (1980).

37. Kluwe WM, McConnell EE, Huff JE, Haseman JK, Douglas JF, Hartwell VW. Carcinogenicity testing of phthalate esters and related compounds by the National Toxicological Program and the National Cancer Institute. Environ Health Perspect 45:129–133 (1982).

38. Ema M, Itami T, Kawasaki H. Teratogenic phase specificity of butyl benzyl phthalate in rats. Toxicology 79:11–19 (1993).

39. Shiota K, Chou MJ, Nishimura H. Embryotoxic effects of di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) in mice. Environ Res 22:245–253 (1980).

40. Foster PM, Thomas LV, Cook MW, Gangolli SD. Study of the testicular effects and changes in zinc excretion produced by some n-alkyl phthalates in the rat. Toxicol Appl Pharmacol 54:392–398 (1980).

41. Heindel JJ, Powell CJ. Phthalate ester effects on rat Sertoli cell function in vitro: effects of phthalate side chain and age of animal. Toxicol Appl Pharmacol 115:116–123 (1992).

42. Gray TJ, Beamand JA. Effect of some phthalate esters and other testicular toxins on primary cultures of testicular cells. Food Chem Toxicol 22:123–131 (1984).

43. Ema M, Harazono A, Miyawaki E, Ogawa Y. Developmental toxicity of mono-n-benzyl phthalate, one of the major metabolites of the plasticizer n-butyl benzyl phthalate, in rats. Toxicol Lett 86:19–25 (1996).

44. Gray LE Jr, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby J. Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p´-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health 15:94–118 (1999).

45. Pirkle JL, Sampson EJ, Needham LL, Patterson DG, Ashley DL. Using biological monitoring to assess human exposure to priority toxicants. Environ Health Perspect 103(suppl 3):45–48 (1995).

46. Furtmann RNK. Phthalates in the aquatic environment. Report no. 6/93. Brussels:European Chemical Industry Council (European Council for Plasticisers and Intermediates), 1996.

47. ECPI. Phthalate Esters Used in PVC. Assessment of the Release, Occurrence and Possible Effects of Plasticizers in the Environment [Partial Copy]. Brussels:European Chemical Industry Council (European Council for Plasticisers and Intermediates), 1996.

48. The National Toxicology Program (NTP) Center for the Evaluation of Risk to Human Reproduction (CERHR). Available: http://cerhr.niehs.nih.gov [cited 1 August 2001].

49. CDC. National Report on Human Exposure to Environmental Chemicals. Available: http://www.cdc.gov/nceh/dls/ report/default.htm [cited 1 October 2001].