National Health and Nutrition Examination Survey

Phthalates are a group of chemicals used to make plastics more flexible and harder to break. They are often called plasticizers. Some phthalates are used as solvents (dissolving agents) for other materials. They are used in many products, such as vinyl flooring, adhesives, detergents, lubricating oils, automotive plastics, plastic clothes (raincoats), and personal-care products (soaps, shampoos, hair sprays, and nail polishes). Phthalates are used widely in polyvinyl chloride plastics, which are used to make products such as plastic packaging film and sheets, garden hoses, inflatable toys, blood-storage containers, and medical tubing. 1,2-Cyclohexane dicarboxylic acid, diisononyl ester (DINCH) is used as a replacement of some high molecular weight phthalates. Once phthalates or DINCH enter a person's body, they are converted into breakdown products (metabolites) that pass out quickly in urine. Biomeasures of phthalates and phthalates alternatives in humans are necessary to evaluate potential human health effects from exposure to these chemicals.

Examined participants aged 6 years and older from a one-third sample.

The test principle utilizes high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) for the quantitative detection in urine of the following phthalate metabolites: monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobutyl phthalate (MBP), mono-isobutyl phthalate (MIBP), mono (3-carboxypropyl) phthalate (MCPP), mono(2-ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), monoisononyl phthalate (MNP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), monocarboxyoctyl phthalate (MCOP), monocarboxynonyl phthalate (MCNP)), and cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester (MHNCH)  (Silva, et al., 2007). Urine samples are processed using enzymatic deconjugation of the glucuronidated metabolites followed by on-line solid phase extraction (SPE) coupled with reversed phase HPLC-ESI-MS/MS. Assay precision is improved by incorporating isotopically-labeled internal standards of the phthalate metabolites and MHNCH. In addition, 4-methyl umbelliferone glucuronide is used to monitor deconjugation efficiency. This selective method allows for rapid detection of monoester metabolites of commonly used phthalate diesters and DINCH in human urine with limits of detection in the low ng/mL range.

Refer to NHANES 2011-2012 Lab Methods for Metabolites of Phthalates and Phthalate Alternatives for detailed description of the laboratory method used.

Urine specimens are processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention for analysis.

Detailed specimen collection and processing instructions are discussed in the NHANES Laboratory Procedures Manual (LPM). Vials are stored under appropriate frozen (–20°C) conditions until they are shipped to National Center for Environmental Health for testing.

Mobile Examination Centers (MECs)

Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured quality assurance evaluation during unscheduled visits to evaluate both the quality of the laboratory work and the quality-control procedures. Each laboratory staff person is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff. Formal retraining sessions are conducted annually to ensure that required skill levels are maintained.

The NHANES QA/QC protocols meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed QA/QC instructions are discussed in the NHANES LPM.

Analytical Laboratories

NHANES uses several methods to monitor the quality of the analyses performed by the laboratories. In the MEC, these methods include performing blind split samples collected on “dry run” sessions. In addition, contract laboratories randomly perform repeat testing on 2.0% of all specimens.

NCEH developed and distributed a quality control protocol for all the laboratories which outlined the Westgard rules (Westgard, et al., 1981) used when running NHANES specimens. Any problems encountered during shipping or receipt of specimens, instrument calibration, reagents, and any special considerations are submitted to NCHS. Summary statistics for each control pool, QC graphs, are reviewed by NCHS for trends or shifts in the data. The laboratories are required to explain any identified areas of concern.

All QC procedures recommended by the manufacturers were followed. Reported results for all assays meet the Division of Laboratory Sciences’ quality control and quality assurance performance criteria for accuracy and precision, similar to the Westgard rules (Westgard, et al., 1981).

The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.

Refer to the 2011-2012 Laboratory Data Overview for general information on NHANES laboratory data.

Subsample Weights

Urinary phthalates and plasticizers metabolites were measured in a one third subsample of persons 6 years and older. Special sample weights are required to analyze these data properly. Specific sample weights for this subsample are included in this data file and should be used when analyzing these data.

Variance Estimation

The analysis of NHANES laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES Demographic Data File contains demographic and sample design variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.

Links to NHANES Data Files

This laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier SEQN.

Detection Limits

The detection limits were constant for all of the analytes in the data set.  Two variables are provided for each of these analytes.  The variable named URD___LC indicates whether the result was below the limit of detection.  There are two values: “0” and “1”.  “0” means that the result was at or above the limit of detection.   “1” indicates that the result was below the limit of detection. The other variable named URX___ provides the analytic result for that analyte. For analytes with analytic results below the lower limit of detection (i.e., URD___LC=1), an imputed fill value was placed in the analyte results field. This value is the lower limit of detection divided by square root of 2 (LLOD/sqrt(2)).

The lower limit of detections (LLOD, in ng/mL) for the phthalate metabolites and MHNCH are:

Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues.