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Toxicological information

Epidemiological data

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Administrative data

Endpoint:
epidemiological data
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline, non-GLP study using a prospective cohort study.

Data source

Reference
Reference Type:
publication
Title:
Prenatal Phthalate Exposures and Anogenital Distance in Swedish Boys
Author:
Bornehag, Carl-Gustaf 1 ; Carlstedt, Fredrik; Jönsson, Bo Ag; Lindh, Christian H; Jensen, Tina K; Bodin, Anna; Jonsson, Carin; Janson, Staffan; Swan, Shanna H
Year:
2015
Bibliographic source:
Environmental health perspectives

Materials and methods

Study type:
cohort study (prospective)
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich
EC Number:
271-090-9
EC Name:
1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich
Cas Number:
68515-48-0
Molecular formula:
C26 H42 O4
IUPAC Name:
1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich
Constituent 2
Reference substance name:
936021-98-6
Cas Number:
936021-98-6
IUPAC Name:
936021-98-6
Constituent 3
Reference substance name:
936022-00-3
Cas Number:
936022-00-3
IUPAC Name:
936022-00-3
Constituent 4
Reference substance name:
936022-02-5
Cas Number:
936022-02-5
IUPAC Name:
936022-02-5
Details on test material:
Diisononyl phthalate (DiNP) 68515-48-0
Mono-(4-methyl-7-hydroxyloctyl) phthalate oh-MMeOP936021-98-6 0.02
Mono-(4-methyl-7-oxo octyl) phthalate oxo-MMeOP 936022-00-3 0.01
Mono-(4-methyl-7-carboxyheptyl) phthalate cx-MMeHP936022-02-5 0.02

Method

Type of population:
general
Details on study design:
Biological samples (blood and urine) were collected from the 2,582 pregnant women and their children participating in The Swedish Environmental Longitudinal, Mother and child, Asthma and allergy (SELMA) study, a prospective birth cohort study in Sweden. The SELMA study recruited women in the 10th week of pregnancy in the county of Värmland, Sweden, between September 2007 and March 2010. Of 8,394 reported pregnant women, 6,658 were invited to participate. Among the invited women, 2,582 agreed to participate, corresponding to a participating rate of 39%.

Boys born between September 2009 and November 2010 (i.e., those who were < 18 months of age in the SELMA study) (n = 325) and their parents were invited to participate in the study; of these, 228 participated (70%). AGD measurements were obtained in 225 boys, and of these, 196 children are included in the present analyses. Mean age of the 196 boys was 20.8 months. Information related to lifestyles, socioeconomic status, living conditions, diet, and medical history were also collected using annual questionnaires.
Exposure assessment:
measured
Details on exposure:
Of the 2,582 pregnant women participating in the SELMA study, a first morning urine sample was obtained from 2,356 women (91%) during weeks 9–11 of the pregnancy. These samples were analyzed for the MBP, MEP, MBzP, MEHP, oh-MEHP, oxo-MEHP, cx-MEPP, sum DEHP, oh-MMeOP, oxo-MMeOP, cx-MMeHP, sum DiNP . Briefly, 0.2 mL of urine were added with 0.1 mL of ammonium acetate (pH 6.5) and 0.01 mL glucoronidase (Escherichia coli) and thereafter incubated at 37°C for 30 min. Then 0.05 mL was added of a 50:50 (vol:vol) water and acetonitrile solution of labeled (3H or 13C) internal standards of all analyzed compounds. A C18 column (2.1 mm i.d. × 50 mm; Genesis Lightn; Grace, Deerfield, IL, USA) was used before the injector to reduce interference. The phthalate metabolites in the samples were separated on a C18 column (1.5 μm, 2.0 mm i.d. × 30 mm VisionHT; Grace). The mobile phases were water and acetonitrile with 0.08% formic acid. The samples were analyzed on a Shimadzu UFLC system (Shimadzu Corporation, Kyoto, Japan) coupled to a QTRAP5500 triple quadrupole linear ion trap mass spectrometer equipped with a TurboIon Spray source (AB Sciex, Foster City, CA, USA). The samples were analyzed in triplicate, and the mean of the two closest were reported. All samples were analyzed in a randomized order. For quality control of the analyses, chemical blanks and two different in-house prepared quality control samples were analyzed in all sample batches. The limit of detection (LOD) was defined as the concentration corresponding to a peak area ratio of three times the standard deviation of the chemical blanks. The creatinine concentrations were analyzed.
Statistical methods:
Urinary levels of phthalate metabolites were log-transformed to normalize distributions and geometric means (GMs) with 95% confidence interval (CIs). Associations between log-transformed phthalate metabolite concentrations and AGD were estimated using a general linear model. Covariate- adjusted AGD (AGDas and AGDap) was grouped into “short” (< 25th percentile), “medium” (25th–75th percentile), with the category “long” (≥ 75th percentile) as the referent. Logistic regression models were used to estimate the odds ratio (OR) of having a boy with a short (adjusted) AGD compared with a long AGD as a function of log-transformed concentrations of phthalate metabolites in prenatal urine. ORs for having a short AGD compared with a long AGD were computed as a function of quartiles of phthalate metabolite concentration in the pregnant women’s urine, with the lowest quartile as a reference.

Results and discussion

Results:
The median AGDas was 40.7 mm [interquartile range (IQR) = 37.8–45.0 mm] while the median AGDap was 82.6 mm (IQR = 78.0–87.4 mm). The mean CV was 2.3% for AGDas and 1.6% for AGDap. Most of the phthalate metabolites were negatively associated with AGD both before and after adjustment for covariates in multiple linear regression models; however, most of the associations did not reach significance. Strongest and most significant inverse associations were found between AGDas and DINP metabolites and most strongly for oh-MMeOP [mono-(4-methyl-7-hydroxyl-octyl) phthalate] and oxo-MMeOP [mono- (2-ethyl-5-oxohexyl) phthalate] and the sum of DINP metabolites. The ORs for having an AGDas in the 2nd–3rd quartile when compared with the 4th quartile as a function of (log-transformed) DINP metabolite concentrations were not significant, whereas the ORs for having an AGDas in the 1st quartile when compared with the 4th quartile were in the range of 2.6–3.1 and significant. Weaker inverse associations were observed between AGDap and DINP metabolites, which did not reach statistical significance. Associations between prenatal DEHP metabolite exposure and shorter AGDas were also seen, though these associations did not reach statistical significance. Finally, the odds of having a short AGDas (in the 1st quartile) when compared with a long AGDas (4th quartile) as a function of quartile of phthalate metabolite concentration showed a linear dose–response relationship for prenatal DINP metabolite exposure. Similar but somewhat weaker associations were seen between AGDas and DEHP metabolites, as well as well as MBzP metabolites, whereas no associations were seen with MEP and MBP metabolites. No consistent patterns were seen between AGDap and any of the phthalate metabolites.
Confounding factors:
All models were adjusted for weight for age, boys age at time of examination, gestational week of urine sampling, and urinary creatinine concentration.
Strengths and weaknesses:
This study benefitted from a large base cohort of pregnant women who are followed prospectively after obtaining a first morning void specimen. However, there are several limitations of this study. First, there are inherent problems with the AGD measurement that persist at this time. These limitations include: (1) no standardized method of measurement, (2) limited normative data, (3) no established method for statistical adjustment of age, height and length/weight, and (4) lack of agreement/understanding as to the clinical significance of this outcome.
In addition, investigators reported a self-selection bias in the SELMA cohort when compared with the nonparticipant group—participating families smoked less (14% vs. 19%), had more frequent asthma and allergy symptoms in the family (58% vs. 38%), had higher education (university level) among the mothers (51% vs. 36%), and more often lived in single-family houses (67% vs. 60%). Moreover, 30% of those eligible did not participate in this secondary study.
Additional limitations relate to the single urine sample and correction of urine concentration with specific gravity. As there is no standard method of correction for urinary concentration, reported phthalate concentrations may not be accurate and, in addition, there is limited comparability across studies with different methods of adjustment.

Applicant's summary and conclusion

Conclusions:
AGD was measured in a cohort of 196 boys at 21 months of age and maternal first-trimester urine was analyzed for 10 phthalate metabolites of DEP (diethyl phthalate), DBP (dibutyl phthalate), DEHP, BBzP (benzylbutyl phthalate) and DiNP. Data on covariates were collected by questionnaires. Significant associations were found between the shorter of two AGD measures (anoscrotal distance; AGDas) and DiNP metabolites and strongest for oh-MMeOP and oxo-MMeOP. However, interpretation must be viewed in light of the methodological limitations described earlier, the lack of consistency within this study (e.g. no effects observed for DEHP, which is considered a stronger reproductive toxicant) and across similar studies, and the finding for only one of the two AGD measurements. In addition, AGDas reduction was small (4%) in relation to more than an interquartile range increase in DiNP exposure.
Executive summary:

According to the authors, concentrations of most of the phthalate metabolites were negatively associated with the measures of AGD, both before and after adjustment for covariantes. However, most of the associations did not reach statistical significance. The strongest and most significant inverse associations were found between AGDs and some individual DINP metabolites and the sum of DINP metabolites excreted in maternal urine. However, extend of AGD reduction remained small.

• A single urine sample does not provide a measure of exposure over a longer time period (such as the potentially sensitive period of pregnancy), specifically for longer side chain phthalates (see Report to the U.S. Consumer Product Safety Commission by the CHRONIC HAZARD ADVISORY PANEL ON PHTHALATES AND PHTHALATE ALTERNATIVES). This is due to the very rapid metabolism and excretion of phthalates and considerable variation in phthalate exposure patterns (see Koch et al., Tox. Letters 231, 261-­‐269 (2104); Aylward et al., J Toxicol Env Health 17, 45-­‐61 (2014)). Therefore, a single urine sample can only provide an indication of individual exposure over a very short time-­‐period (< 24 hours) before sampling. Due to potential differences in phthalate exposure sources and events, exposure assessment based on single urine samples are inadequate for characterization of exposures over a longer time period.

•Bornehag et al. state that "The smaples were analyzed in triplicate, and the mean of the two closest were reported." This introduces bias in exposure data and results from all quantitations should be used for exposure assessment unless specific criteria for exclusion of samples have been developed.

• Urinary concentrations of phthalate metabolites are not directly related to the extent of maternal (or fetal) exposures to the different phthalates due to the differences in bioavailability, biotransformation and toxicokinetics within the phthalates. Correction for the relative importance of the different metabolic pathway and the overall recovery of an oral dose of a phthalate as metabolites excreted with urine are required for a transformation of the urinary metabolite concentrations to phthalate exposure.

• Bornehag et al. do not report an association between AGD and DEHP exposures or exposures to the other phthalates with high potency for reproductive toxicity in rats. This is biologically implausible since DEHP, di-­‐butyl phthalate and benzylbutyl phthalate are at least one order of magnitude more potent as compared to DINP regarding reproductive effects in rats. Excretion of DEHP metabolites reported by Bornehag et al. is app. 2 fold higher as compared to DINP metabolites. Therefore, an association between AGDs and DEHP is expected to be much more likely as compared to an association between AGDs and DINP.

• Margins-­‐of-­‐exposures for DINP in the general population even when using subtle reproductive changes in rats after in utero exposure to DINP are well above 1,000 indicating minimal concern regarding health risks in humans at best. Rats are more sensitive as compared to mice and non-­‐ human primates to phthalate-­‐induced reproductive toxicity. Therefore, risk assessments based on rat data are conservative and very high margins-­‐of-­‐exposures for DINP in humans can be derived.

• Absence of an association between AGD and DEHP exposures reported by Bornehag et al. analyzed is inconsistent with the previous work by the senior author of the Bornehag et al. (for overview, see Kay et al., Crit. Rev. Toxicol. 44, 467-­‐498 (2014) and with some other papers. Conclusions The publication adds to the inconsistent database on potential associations between exposures to phthalates (or other chemicals) and human health outcomes. The data are inconclusive due to the reasons listed above.