Identification of Phthalate Esters in the Serum of Young Puerto Rican Girls with Premature Breast Development 

Environmental Health Perspectives v.108, n.9, Sep00

Ivelisse Colón,¹ Doris Caro,¹ Carlos J. Bourdony,^2,3 and Osvaldo Rosario¹

¹Department of Chemistry, University of Puerto Rico, San Juan, Puerto Rico
²Pediatric Endocrinology and Diabetes Division, San Juan City Hospital, San Juan, Puerto Rico
³Department of Pediatrics, University of Puerto Rico, School of Medicine, San Juan, Puerto Rico

• Introduction
• Methods
• Results
• Discussion

http://ehpnet1.niehs.nih.gov/docs/2000/108p895-900colon/abstract.html

Address correspondence to O. Rosario, Department of Chemistry, University of Puerto Rico, P.O. Box 23346, San Juan, Puerto Rico 00931-3346. Telephone: (787) 764-0000, ext. 7367. Fax: (787) 763-6899. E-mail: rosario129@hotmail.com

We acknowledge the Environmental Protection Agency-Minority Academic Institutions Traineeship Program, Research Centers at Minority Institutions Program (grant RR03641); the Pediatric Endocrinology Division, San Juan City Hospital; the Clinical Laboratory, San Juan City Hospital; and the Premature Thelarche and Early Sexual Development Registry, Puerto Rico Department of Health.

Received 18 May 1999; accepted 9 May 2000.

Introduction

Hyperplasia of breast tissue in neonates is a normal physical finding that may be present during the first 6 months of life as a result of placental passage of maternal estrogenic hormones. When this condition persists beyond this period or occurs before 8 years of age in females, it is known as premature or precocious thelarche (Figure 1). The majority of cases occur before 2 years of age. Girls diagnosed with premature thelarche have one or both breasts enlarged without any other physical signs of the onset of puberty (3).

Figure 1. Twenty-three-month-old Puerto Rican girl with premature breast development (thelarche).

Since 1979, pediatric endocrinologists in Puerto Rico have detected an alarming increase in the number of patients with premature thelarche (4,5). Among the hypotheses proposed to explain the observed premature sexual development in this U.S. Caribbean Island territory, the most controversial theory associated thelarche with the subject's diet. Sáenz et al. (6) suggested that dairy and meat products were contaminated with anabolic estrogenic chemicals, which are used for increasing muscle mass in cattle and poultry. In 1985, studies conducted by the U.S. Department of Agriculture in conjunction with a scientific commission from the Puerto Rico Department of Health led to the conclusion that no abnormal levels of the suspected chemicals were present in the approximately 800 samples of meat and dairy products that were analyzed (7). Other theories are still under consideration, such as the association with ovarian cysts, premature endogenous production of sexual hormones, and environmental contamination by pharmaceutical waste products. These theories do not establish a strong association with the majority of the cases reported (8). Also, a genetic predisposition of Puerto Rican girls for developing premature thelarche is unlikely. Investigation among this ethnic group in the Philadelphia, Pennsylvania, area did not reveal a similar pattern of early sexual development (8). Moreover, other ethnic groups living in Puerto Rico are also affected by the condition (8).

In 1987, the Puerto Rico Department of Health created by law the Premature Thelarche and Early Sexual Development (PTESD) Registry in response to the observed increase in cases (9). This is the only world registry created for the study of premature sexual development in a human population. The objectives of this epidemiologic surveillance system are to define the epidemiologic, clinical, and etiologic aspects of the different manifestations of premature sexual development on the island. Although the registry was established in 1988, retrospective data to 1969 and prospective data to 1998 have been collected. In this time period, 6,580 cases of premature sexual development have been registered, of which 4,674 (71%) are premature thelarche cases. Based on the data accumulated by the registry, the estimated annual average incidence rate of premature thelarche in Puerto Rican girls 6-24 months of age is 8 cases per 1,000 live female births from 1984 to 1993 (10). This incidence is, to our knowledge, the highest ever reported. Compared to a study conducted in Minnesota (11), the incidence of premature thelarche in the Puerto Rican female population is 18.5 times higher.

In the last decade, there has been a growing interest and concern for the study of the impact of man-made chemicals on wildlife and humans. These studies have suggested that synthetic and naturally occurring substances in the environment may affect the normal function of the endocrine system. These substances are also referred to as endocrine-disrupting chemicals (EDCs). In wildlife, alterations in sexual reproductive behavior have been observed in areas of contamination with EDCs. For example, malformations in the sexual organs of alligators have been reported in Lake Apopka, Florida, where high concentrations of DDT and its degradation products have been detected (12,13), and feminization of trout in the Great Lakes has been associated with the high levels of polychlorinated biphenyls (PCBs) in water samples (14). Other studies have indicated that many chemicals, including phthalate esters, may affect development and reproduction, including germ cells, sperm motility, chryptorchidism, and hypospadias, in laboratory animals (15-19). The specific mechanisms by which these chemicals may affect human health are unknown. Extrapolations to humans of the effect of these substances on wildlife are difficult. A limited number of reports (20-22) in the scientific literature describe the accidental exposure of humans to chemicals such as lindane and other organochlorinated pesticides, dioxins, and PCBs with known endocrine-disrupting properties. The study of these exposures has led to the conclusion that these compounds can alter the female-to-male ratio in offspring (20) and cause learning disabilities, behavioral problems, suppression of the immune system, and gynecomastia in the exposed subjects (21-22).

At present, the cause of the observed high incidence of premature sexual development in Puerto Rico is unknown, and the long-term consequences of the aberrant premature sexual development in this population are also unknown. Many of the chemicals that are classified as EDCs have been imported or produced in high quantities in Puerto Rico. Until 1988, a total of 450 million pounds of chlorinated pesticides were imported to the island (23). Although many of these pesticides are currently banned for use in U.S. territories, these pesticides are known to bioaccummulate and to have a long persistence in the environment. Also, many of these substances such as phthalate esters, alkyl phenols, and surfactants are present in commercial products commonly used for packaging, storing, and preserving food (24). The environmental load of plasticizers in Puerto Rico is unknown, but it is assumed to be significant because of the high level of consumption of dietary products in plastic containers imported to the island. Based on the high exposure to these substances in the general population in Puerto Rico and the fact that exposure of human fetuses, newborns, and young girls to exogenous estrogenic chemicals may lead to adverse effects in their sexual development, we designed this study to search for known EDCs in the serum of Puerto Rican girls with premature thelarche. We focused specifically on girls with premature thelarche because this condition represents the majority (71%) of the total cases of premature sexual development reported on the island.

Methods

This research protocol was approved by the San Juan City Hospital's Institutional Review Board before the initiation of the study.

Case and control subjects. Study subjects were females from 6 months to 8 years of age (mean age 31 months; median age 20 months), all diagnosed with premature thelarche. We analyzed all samples from thelarche patients taken from January 1994 to April 1998. Control subjects were females from 6 months to 10 years of age (mean age 70 months; median age 46 months). We obtained control serum samples from the San Juan City Hospital general clinical laboratory. Control individuals did not have evidence of premature sexual development or any other endocrine disease (they were seen in the institution for general pediatric care).

Serum samples were provided by the Pediatric Endocrinology Section of the San Juan City Hospital. This hospital is the only municipal tertiary care public health institution for the city of San Juan and primarily serves an indigent population, including the subjects and controls for this study (25).

We used a number coding system for the handling of samples during the analysis. To assure confidentiality, subject's names and clinical data were not available to the University of Puerto Rico's Environmental Analytical Chemistry Laboratory personnel during the study.

Moni-Trol samples. Moni-Trol (Dade, EF Baragaño, Puerto Rico), a lyophilized product derived from human serum, is available commercially for optimization and calibration of instrumentation in clinical laboratories. We reconstituted the lyophilized samples as needed with 5.00 mL of a carbonate diluent supplied by the manufacturer. Samples were stored at 4°C and protected from light.

Serum samples. Whole blood samples were collected by venipuncture into glass tubes with no chemical additives. After collection, the samples were allowed to coagulate and were then centrifuged. Serum samples were stored at -23°C in glass tubes and protected from light until analysis.

Optimization of the extraction procedure. Moni-Trol samples were prepared, and 1 mL samples were spiked with standard solutions of diethyl phthalate (DEP) and dibutyl phthalate (DBP) as model compounds of the phthalate ester family. The spiked concentrations were between 50 ppb and 25 ppm. The samples were stored at 4°C protected from light for 18 hr. After reaching room temperature, the samples were extracted with hexane/dichloromethane (8:1). The extracts were concentrated to 1.0 mL with a flow of nitrogen and 1 µL was injected into the gas chromatography/mass spectrometry (GC/MS) system (HP 5890/5971; Hewlett Packard, Wilmington, DE). The samples were analyzed by selected ion monitoring (SIM) using a SPB-1 column (100% polydimethyl siloxane; Supelco, Bellefonte, PA). Samples were heated to an initial temperature of 70°C for 4 min and then heated to 250°C at 10°C/min. We compared the relative area of the peaks for each standard to the area of standards in a calibration curve generated under the same conditions and calculated the percent of recovery for each concentration. All methodology was tested using serum samples as well. Serum samples were spiked with the model compounds in concentrations ranging from 20 to 80 ppb.

Extraction of organic compounds from serum samples. Serum samples were transported to the analytical laboratory in glass tubes stored at 0°C. Precipitation of serum and plasma proteins was accomplished by adding 1 mL acetonitrile to 1.0 mL sample. Five milliliters of the extraction mixture, which consisted of an 8:1 solution of hexane and dichloromethane, was added to each sample. Samples were then submitted to ultrasound extraction for 5 min in a sonicator bath (Fisher Scientific, Cayey, Puerto Rico). The phases were allowed to separate, and the extract was transferred to a centrifuge tube. The extraction procedure was performed twice and the extracts were combined.

Concentration and analysis. The combined extracts were concentrated to 0.5 mL with a flow of nitrogen. The concentrated extract (1 µL) was injected into the GC/MS system. The samples were heated to an initial temperature of 70°C for 4 min and then raised to 130°C at 5°C/min and then to 250°C at a rate of 10°C/min.

Special precautions. We applied essential quality control standards to avoid incorrect interpretation of results. This is particularly important when the expected concentrations of the analytes occur at trace levels. Phthalate esters have been detected as interferences in many chromatographic analyses (26,27). We analyzed adequate sets of analytical blanks before sample analysis; these included system and solvent blanks as well as blanks for the sampling device, pipettes, and storage tubes.

Results

Optimization of the extraction and analysis procedures. For the optimization of the extraction procedure, Moni-Trol samples spiked with DEP and DBP (concentrations ranging from 50 ppb to 25 ppm) were extracted and analyzed by GC/MS. We compared the relative area for each compound in the extract to the relative area for standards in a calibration curve. The calibration curves had R² values > 0.99 for the model phthalates. We calculated the amount extracted (in parts per million) for each concentration and generated recovery curves for each compound by plotting the amount extracted as a function of the amount of standard added to the sample. The slope of the regression line gives the average percent of recovery for each compound for the concentration range. In Moni-Trol samples, the average percent recovery (± SD) for DEP was 79.9 ± 0.4%, whereas the average percent recovery for DBP was 88.2 ± 0.2%. The method for extraction and analysis showed excellent linear correlation (R² values > 0.99) for the concentration range tested.

When we tested the optimized method using real blood serum instead of Moni-Trol for the sample preparation and recovery curves, we used concentrations ranging from 20 to 80 ppb; the recovery curves are presented in Figure 2. Samples were analyzed by GC/MS in the SIM mode. As calculated from the recovery curves, the average percent of recovery (± SD) was 118 ± 13% for DBP and 88 ± 3% for DEP.

Figure 2. Recovery curve for real serum samples spiked with (A) DEP (y = 0.8334x - 8.2971; R² = 0.9939) and (B) dibutyl phthalate (y = 1.18x - 17; R² = 0.9504).

Serum samples from thelarche patients and control samples. We analyzed 41 samples from patients diagnosed with premature thelarche at the San Juan City Hospital's Pediatric Endocrinology Division and included in the Premature Thelarche and Early Sexual Development Registry of the Puerto Rico Department of Health. We used a GC/MS instrument operating in the scan mode to analyze the samples. Figure 3 shows a representative total ion chromatogram for serum samples from study subjects. Figure 4 shows the extracted ion chromatogram for m/z = 149 for the same chromatogram. The m/z = 149 is one of the characteristic ions for phthalate ester detection. As confirmed by mass spectral data, four of the peaks in the extracted ion chromatogram correspond to compounds of the phthalate ester family. Phthalate esters were consistently detected at significant concentration levels (ranging from tens of parts per billion to units of parts per million) in 28 of 41 (68%) serum samples obtained from the thelarche patients. The concentration of phthalate esters was calculated by means of calibration curves (R² > 0.99) constructed with standards. As shown in Table 1, the concentration for phthalate esters is in the parts per billion range. The concentration values of phthalate esters shown in this table have been corrected for the presence of phthalates in analytical blanks. DEP was detected in samples in concentrations of tens of parts per billion, whereas the phthalate esters with the most common commercial uses, di-(2-ethylhexyl) phthalate (DEHP) and DBP, were detected in much higher concentrations. For those samples with high concentrations of DEHP, one of the major metabolites, mono(2-ethylhexyl) phthalate (MEHP), was also detected. Benzyl butyl phthalate was detected in two samples (5 and 6), whereas dioctyl phthalate (DOP) was detected in only one (sample #15).

Figure 3. Total ion chromatogram representative of those obtained for samples from thelarche patients.

Figure 4. Extracted ion chromatogram for the total ion chromatogram in Figure 2. The peaks identified as a, b, c, and d correspond to compounds of the phthalate ester family.

To determine if the detection of these compounds has a strong correlation with premature thelarche patients, control samples were also analyzed. These samples were collected, stored, and analyzed in the same manner and by the same nurses and laboratory technicians as described for the study samples. From the 35 samples analyzed from girls with no signs of thelarche or other conditions characteristic of premature sexual development, DOP (562 mg/L or ppb) was detected in only one sample (control #2). DEHP was detected in only 5 (14%) of the control samples in concentrations ranging from 276 ppb to 719 ppb. Table 2 shows the individual data for control samples.

For purposes of comparison between thelarche and control subjects, we calculated the average concentration for each of the phthalate esters detected. The results are shown in Figure 5. The most dramatic difference is in the case of DEHP, where the ratio of average concentrations between control:case samples was 70:450 ppb. This difference proved to be statistically significant to the 95% confidence interval. Comparison of the levels of DEHP and DBP in the individual patients did not show any pattern or correlation. When these were compared to the age of the patient, no correlation was evident.

Figure 5. Average concentrations (in ppb) of DBP, DEP, benzylbutyl phthalate (BBP), DOP, DEHP, and MEHP in thelarche patients and control samples. Data correspond to 41 case samples and 35 control samples.

Eight subject samples (numbered 34-41) were also analyzed using the GC/MS instrument in the SIM mode for ions characteristic of the phthalate esters family. This MS modality offers a higher degree of selectivity as well as lower detection and quantification limits. DBP and DEHP were detected by SIM in all eight samples from the thelarche patients. Dimethyl phthalate (DMP) was detected in two samples (36 and 39) and DEP in samples 36, 37, and 38. As summarized in Table 3, DMP and DEP are present at low concentration levels (tens of milligrams per liter) that could not necessarily be detected by the scan modality of the MS system.

Analytical blanks. Because of their common use as plasticizers and their ubiquitous dispersal in the environment, phthalate esters have been detected as impurities in solvents, water, glassware, and many items of clinical and analytical laboratory equipment (26,27). We tested several analytical blanks to establish if contamination from the sampling, storage, extraction, and analysis of samples could interfer with the real samples. These blanks included solvent, butterfly sampling devices, distilled water, and storage tubes with Teflon caps. We also analyzed plastic tubes and pipettes blanks, although these were not used for sample handling or storage. The concentration of the compounds studied in the blanks was subtracted from the average concentration determined from the repetitions of each case and control sample. Background correction is essential when analysis at trace levels is required. DEHP was not detected in significant levels (> 10 ppb) in any of the blanks analyzed by GC/MS in the scan mode. Table 4 summarizes the average concentration of the phthalate esters detected in blanks as calculated from calibration curves. The levels shown in Table 4 are the average values for the repetition of at least three blanks of the same kind. Each subject and control sample was corrected with the set of blanks tested the same day of the analysis.

Discussion

In this study we optimized and validated a method for the analysis of phthalate esters in blood serum using DEP and DBP as model compounds.

Phthalate esters are chemicals with known endocrine-disrupting properties (15-19). There is significant concern for their ubiquitous presence in the environment, and scientists, clinicians, and regulatory agencies currently debate their potential for adverse health effects in humans (28-30). Nevertheless, no studies have been reported on concentration levels of phthalate esters in the general population. The analysis of phthalate esters in human serum has been limited to samples from hemodialysis patients, which are in continuous exposure to these substances due to blood transfusions (31). The methodology used by Malik et al. (31) includes HPLC analysis. The optimized methodology for the present study provides a simpler approach for the analysis of phthalate esters in blood samples. This methodology, consisting of a liquid-liquid extraction followed by concentration of the extracts with a flow of nitrogen and analysis by GC/MS in the scan mode, proved to be linear over a wide concentration range (tens of parts per billion to parts per million), efficient, and reproducible as demonstrated by the recovery curves. The extraction efficiency was > 80% for model phthalate esters in samples prepared in Moni-Trol and in real serum. The method resulted in extraction efficiencies > 80% for chlorinated pesticides.

As demonstrated by the eight samples analyzed by SIM, improvements in the detection limits of the methodology is necessary to detect some phthalate esters such as DEP and DMP, which are present at much lower concentrations. This is also important for the successful detection of phthalate ester metabolite residues in samples. This optimized method permits the use of the SIM modality when lower detection limits are required. This methodology may allow the development of other analytical research protocols that could result in enhanced detection limits, thus offering investigators the possibility of assessing levels of phthalate esters and other known pollutants in blood samples from the general population.

We detected high levels of phthalate esters in 68% of the samples from thelarche patients. DEP, DBP, and DEHP were detected in levels ranging from tens of parts per billion (nanograms per milliter) to units of parts per million (milligrams per milliliter) in case samples. MEHP, one of the major metabolites of DEHP, was detected in five case samples (Table 1). The presence of this phthalate ester was not caused by sample contamination, because contamination by a metabolite rarely occurs. We detected DEHP in 14% of the control samples. The concentration of this phthalate in control samples was significantly lower than the levels in study samples (Table 2).

Many of the phthalate esters detected in study samples have been shown to be estrogenic when assayed by the recombinant yeast screen test (32). Although phthalates have low estrogenic activity compared to estradiol and other potent estrogenic substances (33), these chemicals may cause endocrine disruption through several mechanisms. Chemicals with weak estrogenic activity may cause disruption in biologic systems if they act at critical periods of development (34). The intrauterine period of human embrionic differentiation and development is particularly sensitive to weak estrogens. Besides their estrogenic activity, phthalates such as DBP have blocking antiandrogen action (35). If these properties of phthalate esters play a role in the cause of premature breast development in Puerto Rican females, further research should address this possibility.

The detection of high levels of phthalate esters in this group of Puerto Rican girls could reflect an elevation in the exposure of the general Puerto Rican population to these substances. Further research will address this issue. The total daily consumption of DEHP from all sources of exposure has been estimated at 5.8 mg in the United States and 2.1 mg in Japan (36). The most important sources of exposure for children are ingestion of contaminated formulas, food, and water from contact with plastic wrappings and containers and mouthing of plastic toys and pacifiers (36-38). There is mounting concern for exposure to phthalate esters (specifically DEHP), especially through polyvinyl chloride plastic medical equipment.

The present study provides the first analytical evidence of the presence of plastic additives with known estrogenic activity in girls with premature thelarche. In a study conducted in 1997 that included 17,077 girls, Herman-Giddens et al. (39) reported that girls in the United States are developing pubertal characteristics at younger ages than previously reported. These authors concluded that

the possibility that the increasing use of certain plastics and insecticides that degrade into substances that have estrogen-related physiological effects on living things should be investigated in relation to the earliest onset of puberty.

The findings of this study cannot be interpreted as the cause of premature thelarche in Puerto Rican girls at present. It may well be that the etiology of the various manifestations of premature sexual development (including thelarche) on this island is multifactorial. Further research should be performed to clarify if phthalate esters by themselves, or in association with other endogenous or exogenous estrogenic compounds, are capable of inducing precocious sexual development in animals and humans. Other possible environmental cofactors related to exposure should also be considered, especially those unique to the Puerto Rican environment. The following have already been associated with premature sexual development in Puerto Rico: the presence of anabolic steroids in poultry (6) and consumption of soy-based formula with a high phytoestrogen content by Puerto Rican infants (8). The higher exposure to phthalate esters in the Puerto Rican infant population is supported by the high importation of plastic packaged foods and because Puerto Rico is a tropical island with year-round high temperature and humidity, which promotes closed environments with the frequent use of air conditioners in homes and public buildings (36).

The issue of endocrine disruptors causing adverse health effects to humans and wildlife is not free of controversy (40,41). Other studies should address this issue, particularly that of critical stage susceptibility to endocrine-disrupting chemicals and alterations in sexual development of humans and other animal species. If the hypothesis holds true, premature sexual development in Puerto Rico may prove to be an unfortunate example of the impact of endocrine-disrupting environmental chemicals at a critical stage of human development.

References and Notes

1. Liwnicz BH, Liwnicz RG. On endocrine function. In: Clinical Chemistry: Theory, Analysis and Correlation, 2nd ed (Kaplan LA, ed). St. Lewis, MO:CV Mosby Company, 1989;607-619.
2. Sippell WG. Diagnosis and treatment of central precocious puberty--can final height be improved? Horm Res 41(suppl 2):14-15 (1994).
3. Kulin HE, Müller J. The biological aspects of puberty. Pediatr Rev 17:75-86 (1996).
4. Pérez A. Precocious sexual development in Puerto Rico. Lancet 1:1299-1300 (1982).
5. Bongiovanni AM. An epidemic of premature thelarche in Puerto Rico. J Pediatr 103:245-246 (1983).
6. Sáenz CA, Bondiovanni AM, Conde L. An epidemic of precocious development in Puerto Rican children. J Pedriatr 107:393-396 (1985).
7. Montgomery EE. Unpublished data.
8. Freni-Titulaer LW, Cordero JF, Haddock L, Lebrón G, Martínez R, Mills JL. Premature thelarche in Puerto Rico: a search for environmental factors. Am J Dis Child 140:1263-1267 (1986).
9. Commonwealth of Puerto Rico. Regulation of the Secretary of Health No. 64, 28 September 1989.
10. Bourdony CJ. Premature Thelarche and Early Sexual Development Registry. Annual report. San Juan, Puerto Rico:Department of Health, 1998.
11. Van Winter JT, Noller KL, Zimmerman D, Melton LJ. Natural history of premature thelarche in Olmstead County, Minesota, 1940 to 1984. J Pediatr 116:278-279 (1990).
12. Guillette LJ Jr, Gross TS, Masson GR, Matter JM, Percival HF, Woodward AR. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ Health Perspect 102:680-688 (1994).
13. Vonier PM, Crain DA, McLachlan JA, Guillette LJ Jr, Arnold SF. Interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the American alligator. Environ Health Perspect 104:1318-1322 (1996).
14. Colborn T, vom Saal FS, Soto AM. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ Health Perspect 101:378-384 (1993).
15. Siddiqui A, Srivastava SP. Effect of di(2-ethylhexyl) phthalate administration on rat sperm count and on sperm metabolic enzymes. Bull Environ Contam Toxicol 48:115-119 (1992).
16. Fredricsson B, Müller L, Pousette A, Westerholm R. Human sperm motility is affected by plasticizers and diesel particle extracts. Pharmacol Toxicol 72:128-133 (1993).
17. Imajima T, Shono T, Zakaria O, Suita S. Prenatal phthalate causes cryptorchidism postnatally by inducing transabdominal ascent of the testis in fetal rats. J Pediatr Surg 32:18-21 (1997).
18. Poon R, Lecavaliert P, Mueller R, Valli V, Procter B, Chu I. Subchronic oral toxicity of di-n-octyl phthalate and di(2-ethylhexyl) phthalate in the rat. Food Chem Toxicol 35:225-239 (1997).
19. Arcadi R, Costa C, Imperatore C. Oral toxicity of DEHP during pregnacy and suckling in the Long-Evans rat. Food Chem Toxicol 36:963-974 (1998).
20. Mocarelli P, Brambilla P, Gerthous PM, Patterson DG Jr, Needham LI. Change in sex ratio with exposure to dioxin. Lancet 348:409 (1996).
21. Weisglas-Kuperus N, Sas TCJ, Koopman-Esseboom C, van der Zuan CW, de Ridder MAJ, Keishuizen A, Hooijkaas H, Sauer PJJ. Immunological effect of background prenatal and postnatal exposure to dioxins and polychlorinated biphenyls in Dutch infants. Pediatr Res 38:404-410 (1995).
22. Sattin RW, Roisin A, Kafrissen ME, Dugan JB, Farer LS. Epidemic of gynecomastia among illegal Haitian entrants. Public Health Rep 99:504-510 (1984).
23. Quiñones A. Importación de plaguicidas a Puerto Rico [M.S. Thesis]. San Juan, Puerto Rico:University of Puerto Rico, 1992.
24. Ross G. 1,001 Chemicals in Everyday Products. New York:Van Nostrand Reinhold, 1994.
25. Puerto Rico Planning Board. Unpublished data.
26. Leung S, Giang BY. Phthalate esters as potential contaminants during GC analysis of environmental samples using electron capture and mass spectrometric detectors. Bull Environ Contam Toxicol 50:528-532 (1993).
27. López-Avila V, Milanés J, Constantine F, Beckert WF. Typical phthalate ester contamination incurred using EPA method 8060. J Assoc Off Anal Chem 73:709-720 (1990).
28. Kavlock RJ, Daston GP, DeRosa C, Fenner-Crisp P, Gray LE, Kaattari S, Lucier G, Luster M, Mac MJ, Maczka C, et al. Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the U.S. EPA-sponsored workshop. Environ Health Perspect 104(suppl 4):715-740 (1996).
29. Hileman B. Environmental hormone disrupters focus of major research initiatives. Chem Eng News 74:28-35 (1996).
30. Patlak M. A testing deadline for endocrine disrupters. Environ Sci Technol 30:540A-544A (1996).
31. Malik S, Kenny M, Ahmad S. A convenient method to measure di-2-ethylhexyl phthalate from the serum of hemodialysis patients. Toxicol Environ Chem 37:333-337 (1993).
32. Harris CA, Henttu P, Parker MG, Sumpter JP. The estrogenic activity of phthalate esters in vitro. Environ Health Perspect 105:802-811 (1997).
33. Jobling S, Reynolds T, White R, Parker MG, Sumpter JP. A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ Health Perspect 103:582-587 (1995).
34. Colborn T, Clement C. Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection. Princeton, NJ:Princeton Scientific Publishing Co., 1992.
35. Mylchreest E, Cattley R, Foster P. Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl)phthalate: an antiandrogenic mechanism? Toxicol Sci 43:47 (1998).
36. Giust JA, Seipelt T, Anderson BK, Deis DA, Hinders JD. Determination of bis(2-ethylhexyl) phthalate in cow's milk and infant formula by high performance liquid chromatography. J Agric Food Chem 38:415-418 (1990).
37. Rastogi SC. Gas chromatographic analysis of phthalate esters in plastic toys. Chromatographia 47:724-726 (1998).
38. Marín ML, López J, Sánchez A, Vilaplana J, Jiménez A. Analysis of potentially toxic phthalate plasticizers used in toy manufacturing. Bull Environ Contam Toxicol 60:68-73 (1998).
39. Herman-Giddens ME, Slora EJ, Wasserman RC, Bourdony CJ, Bhapkar MV, Koch GG, Hasemeier CM. Secondary sexual characteristics and menses in young girls seen in office practice: a study from the Pediatric Research in Office Settings network. Pediatrics 99:505-512 (1997).
40. Safe S. Environmental and dietary estrogens and human health: is there a problem? Environ Health Perspect 103:346-351 (1995).
41. Zacharewski T, Meek M, Clemons J, Wu Z, Fielden, Mathews J. Examination of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters. Toxicol Sci 46:282-293 (1998).

Last Updated: August 9, 2000