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epidemiological data
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)

Data source

Reference Type:
Phthalate excretion pattern and testicular function: A study of 881 healthy Danish men
Joensen, Ulla Nordström 1 ; Frederiksen, Hanne 1 ; Jensen, Martin Blomberg 1 ; Lauritsen, Mette Petri 2 ; Olesen, Inge Ahlmann 1 ; Lassen, Tina Harmer 1 ; Andersson, Anna-Maria 1 ; Jørgensen, Niels
Bibliographic source:
Environ Health Perspect. 2012 Oct;120(10):1397-403

Materials and methods

Study type:
cross sectional study
Endpoint addressed:
toxicity to reproduction / fertility
Test guideline
no guideline available
Principles of method if other than guideline:
Cross sectional study measuring phthalate metabolites, hormone levels and sperm quality parameteris in 888 healthy, young Danish men recruited from 2007 to 2009.
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
Constituent 2
Reference substance name:
Constituent 3
Reference substance name:
Constituent 4
Reference substance name:
Details on test material:
No direct testing was conducted. Study subjects provided urine samples which were used for analytical assessment of DINP metabolites.


Type of population:
Ethical approval:
confirmed, but no further information available
Details on study design:
Between 2007 and 2009, 881 healthy, young Danish men were recruited during compulsory examination assessing their fitness for military service. A fraction of the recruited population (19 of 900) was deemed ineligible (e.g. self-reported or suspected use of anabolic steroids, history of testicular cancer, missing blood or semen samples) and excluded from the study sample. Neither the population denominator nor the final response proportion was reported in this paper, but the response proportion in a previous semen quality study of Danish conscripts was 24% (Ravnborg et al., 2011). At the time of examination, spot urine, blood and semen samples were collected from each man who also completed a self-administered questionnaire.
Exposure assessment:
Details on exposure:
Exposure was estimated based on urinary metabolite concentrations of fourteen phthalates: MEP, MnBP, MiBP, MBzP, MEHP, MEHHP, MEOHP, MECPP, MOP, MCPP, MiNP, MHiNP, MOiNP and MCiOP. In addition to examining individual metabolite concentration, several exposure constructs were also examined: (1) Molar sums of low-molecular-weight phthalate metabolites (MEP, MiBP, MnBP), high-molecular-weight phthalate metabolites (MBzP, MCPP, DEHP, DiNP) and total metabolites, (2) molar sums of DEHP and DiNP metabolites, (3) percentage of total DEHP metabolites expressed as MEHP and (4) percentage of total DiNP expressed as MiNP.
Statistical methods:
Exposure was modeled both as a continuous variable and also in quartiles. Outcomes were normalized using transformation methods appropriate to the dependent measure. For example, hormone levels and semen volume were logged transformed; square root was used to transform percent morphologically normal sperm. Multiple linear regression models, adjusted for potential confounders (e.g. age, smoking) were used to conduct statistical analyses.

Results and discussion

Results indicated few associations between any measure of phthalate exposure (either single metabolites or composite/constructed variables) and alterations in reproductive hormone levels, hormone ratios or sperm quality indicators (data not shown in paper). In general, exceptions were observed only when phthalate exposure was specified as %MiNP in the regression models. For hormone levels, %MINP was associated with a 15% lower free andogen index [95% confidence limit (CL) -23% to -8%], 9% lower total testosterone/luteinizing hormone ratio [CL: -18% to -0.4%] and 19% lower free androgen/luteinizing hormone ratio [CL: -30% to -8%] among men in the highest v. the lowest quartiles. MiNP percentile was negatively associated with a 13% lower follicle-stimulating hormone level [CL: -25% to -1%] comparing the highest v. lowest quartiles. %MiNP was associated with relatively higher values of the sperm quality indicators among men in the highest quartile v the lowest quartile; other sperm quality results were not statistically significant.
Confounding factors:
age, BMI, smoking, alcohol intake, time of day of blood samples
Strengths and weaknesses:
Joensen, et al. improves upon earlier work with its unselected (i.e. not from infertility clinics) and relatively healthy sample of young Danish men. It is notable for being among the first epidemiologic studies of male endocrine/reproductive functioning to incorporate markers of biologic “susceptibility” to high molecular weight phthalates. The sole finding from Joensen et al comes from the novel and largely un-validated measures of susceptibility to DEHP and DiNP exposure.
There are limitations to these computed susceptibility measures. First, primary metabolites, MEHP and MiNP are non-specific biomarkers of exposure to their respective phthalate diester (i.e. DEPH and DiNP) because they share the same metabolic pathway as other chemicals that require cytochrome p450 Phase I enzymes for detoxification. As such, MEHP, MiNP and any measure on which they are computationally based may be confounded by other chemical exposures. Secondly, MiNP may be formed from DINP in the environment. It is an “insensitive” biomarker of DiNP exposure.

Another significant limitation relates to the appropriateness of examining hormonal levels (or sperm quality), using a cross-sectional study design, in light of the exceedingly short biological half-lives of the exposure markers. Beyond questions of validity, the clinical significance of the results seems, as suggested by the authors, is negligible.

Applicant's summary and conclusion

Executive summary:

This is a cross-sectional study of 7 phthalate diesters and reproductive hormones and testicular function among 881 healthy, young Danish men recruited during evaluation for military fitness for duty. Few alterations were reported for any of the individual phthalate metabolites or their molecular sums and the reproductive or testicular function outcomes. Modest decrements observed in free androgen index, T/LH ratio and T/FSH ratio were associated with relatively higher levels of %MiNP, which were not observed to be in conjunction with any of the sperm quality parameters. To the contrary, positive associations were reported between levels of %MiNP and semen volume and progressively motile sperm.