and then established molecular and cellular key
events of the mixture experimentally, in both in
vitro and in vivo models, making EDC mixtures
experimentally tractable as the“real-life”relevant
unit of human exposure. This finally allowed
us to identify the population at risk from empi-
rically derived EDC mixture concentrations.
Results
An integrated epidemiological-experimental
design assessing the impact of EDC mixtures on
human health and development
To enable risk assessment of real-life exposure
to EDC mixtures, we harnessed (i) a prospec-
tive population-based mother-child pregnancy
cohort (SELMA) to measure prenatal EDC
exposures combined with biostatistical tools
to infer the EDC mixture associated with child
neurodevelopment (Fig. 1A); (ii) complementary
assays in in vitro human cellular systems to
define molecular mechanisms linking EDC-
mixture exposure to adverse outcomes (Fig. 1B);
(iii) in vivo models to determine the physiolog-
ical impact and dose-response of key affected
pathways (Fig. 1C); and (iv) a combination of in
vivo evidence of effects from this EDC mixture
and exposure information from the pregnancy
cohort to assess risks associated with the mix-
ture using a similar mixture approach (SMACH)
(Fig. 1D).Definition and establishment of the EDC mixture
that affects human neurodevelopment
Humans are exposed to several classes of
EDCs, such as phthalates, phenols, and per-
fluorinated alkyl acids ( 12 ). We focused on
prenatal exposures to mixtures of 15 parent
compounds (comprising 20 analytes and
metabolites) measured in the SELMA study, a
population-based pregnancy cohort that recruited
more than 2300 women in the first trimester
from prenatal clinics in Värmland County,
Sweden, from November 2007 to March 2010
( 13 ). Chemicals were selected based on an
evaluation matrix that ranked their (i) known
or suspected endocrine-disrupting properties
based on scientific literature; (ii) inclusion in
the European Chemical Agency (ECHA) list ofvery-high-concern substances ( 14 ); (iii) preva-
lence of empirically measured exposure in
the SELMA pregnancy cohort; (iv) established
knowledge on the most likely routes of ex-
posure; (v) experimental tractability in terms
of multiplex chemical analysis in complex mix-
tures; and (vi) available evidence on health
outcomes of very serious concern. Although
obviously not comprehensive, this served as
a representative selection to spearhead the
evaluation of the effects of mixture exposures.
As a marker of adverse outcomes in child
neurodevelopment, we focused on language
delay at 30 months of age. Delays in language
development in early childhood, as assessed
by validated tests, have been shown to be pre-
dictive of later academic achievement and the
need for special education ( 15 ). SELMA data
confirm that language delay at 30 months of
age is significantly (P= 0.001) associated with
lower cognitive function [Wechsler Intelligence
Scale for Children (WISC-IQ)] at 7 years of age
when adjusted for covariates ( 8 ). Thus, language
delay in early childhood serves as a marker
for more general neurodevelopmental pro-
cesses rather than reflecting language devel-
opment only ( 5 , 6 ).
The current analyses included 1874 pregnant
women, who were assessed for their urinary or
serum EDC concentrations at the 10th week
(median) of gestation (Table 1 and table S1).
Specifically, we profiled urine concentrations
of 10 metabolites of five phthalates, BPA, and
triclosan, as well as serum concentrations of
eight PFAS.
A specific mixture (in terms of both com-
position and concentration) was found to be
associated with language delay in a three-step
procedure (Fig. 1A). First, we identified the
prenatal exposures to EDCs that were asso-
ciated with language delay in 594 children
(prevalence of 10%) by using weighted quan-
tile sum (WQS) regression ( 16 ), a strategy for
estimating empirical weights for a weighted
sum of concentrations that are most associ-
ated with the health outcomes. EDCs included
in these exposures are hereafter referred to as
sEDCs (selected EDCs based on WQS regres-
sion weights). Second, we estimated the
equivalent daily intake of sEDCs measured in
urine (phthalates and alkyl phenols) and esti-
mated serum concentrations from the daily
intake for these urinary measurement–based
compounds. Finally, we used the geometric
means, on a molar basis, for either the mea-
sured or estimated serum concentrations of
the sEDCs in 1874 women to establish mixing
proportions for the reference mixture used
for experimental validation (MIX N, where
N stands for neurodevelopment), (Fig. 1A and
tables S2 and S3). The mixture was tested
across concentrations (0.01X, 0.1X, 1X, 10X,
100X, and 1000X) corresponding to human
exposure, where 1X denotes the geometricCaporaleet al.,Science 375 , eabe8244 (2022) 18 February 2022 3 of 15
Table 1. Single-compound concentrations in prenatal urine and serum of SELMA women.
Distribution of phthalate and phenol metabolites in urine and perfluorinated compounds (PFAS) in
serum analyzed during the first trimester of 1874 pregnant women in the SELMA study. CI, confidence
interval; GM, geometric mean; MEP, mono-ethyl phthalate; MBP, mono-n-butyl phthalate; MBzP,
monobenzyl phthalate; DEHP, di(2-ethylhexyl) phthalate; MEHP, mono-(2-ethylhexyl) phthalate;
MEHHP, mono-(2-ethyl-5-hydroxylhexyl); MEOHP, mono-(2-ethyl-5-oxohexyl) phthalate; MECPP,
mono-(2-ethyl-5-carboxypentyl) phthalate; DiNP, diisononyl phthalate; MHiNP, mono-hydroxy-iso-
nonyl phthalate; MOiNP, mono-, oxo-iso-nonyl phthalate; MCiOP, mono-carboxy-iso-octyl phthalate;
PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PFNA, perfluorononanoic acid; PFDA,
perfluorodecanoic acid; PFUnDA, perfluoroundecanoic acid; PFDoDA, perfluorododecanoic acid;
PFHxS, perfluorohexane sulfonate; PFHpA, perfluoroheptanoic acid.Compound Metabolite Median 95% percentile GM (95% CI).....................................................................................................................................................................................................................Phthalate and metabolites in urine (ng/ml)
DEP.....................................................................................................................................................................................................................MEP 62.6 507.7 68.7 (65.3–72.3)
DBP.....................................................................................................................................................................................................................MBP 71.9 233.1 69.0 (66.5–71.5)
BBzP.....................................................................................................................................................................................................................MBzP 16.8 99.4 16.6 (15.8–17.4)
DEHP .......................................................................................................................................................................MEHP 3.8 15.6 3.8 (3.6–3.9)
.......................................................................................................................................................................MEHHP 16.6 66.6 16.3 (15.7–17.0)
.......................................................................................................................................................................MEOHP 11.2 45.0 11.1 (10.7–11.6)
.....................................................................................................................................................................................................................MECPP 15.7 62.7 15.8 (15.2–16.4)
DiNP .......................................................................................................................................................................MHiNP 5.9 54.6 6.2 (5.9–6.6)
.......................................................................................................................................................................MOiNP 2.7 19.2 2.9 (2.8–3.0)
.....................................................................................................................................................................................................................MCiOP 8.7 74.9 9.8 (9.3–10.2)
BPA.....................................................................................................................................................................................................................1.5 6.2 1.5 (1.4–1.6)
Triclosan.....................................................................................................................................................................................................................0.8 351.4 1.3 (1.2–1.5)
.....................................................................................................................................................................................................................Perfluorinated compounds (PFAS) in serum (ng/ml)
PFOA.....................................................................................................................................................................................................................1.60 3.96 1.60 (1.56–1.64)
PFOS.....................................................................................................................................................................................................................5.35 12.29 5.30 (5.18–5.43)
PFNA.....................................................................................................................................................................................................................0.53 1.29 0.54 (0.53–0.55)
PFDA.....................................................................................................................................................................................................................0.25 0.59 0.26 (0.25–0.27)
PFUnDA.....................................................................................................................................................................................................................0.23 0.54 0.21 (0.21–0.22)
PFDoDA.....................................................................................................................................................................................................................0.03 0.08 0.03 (0.03–0.03)
PFHxS.....................................................................................................................................................................................................................1.23 3.71 1.32 (1.29–1.36)
PFHpA.....................................................................................................................................................................................................................0.02 0.09 0.02 (0.02–0.02)RESEARCH | RESEARCH ARTICLE