Phthalates are used industrially as plasticizers
and solvents and as stabilizers for colors and fragrances.
They are found in personal care products, medications,
paints, adhesives, and medical equipment made with
polyvinyl chloride plastics [Agency for Toxic Substances
and Disease Registry (ATSDR) 1995, 2001, 2003]. Diethyl
phthalate (DEP), di(2-ethylhexyl) phthalate (DEHP),
butylbenzyl phthalate (BBzP), and di-n-butyl
phthalate (DBP) are used in personal care products
(Houlihan et al. 2002; Koo and Lee 2004). The potential
effects of phthalates on human health are not well
characterized. There is a paucity of existing data
describing phthalate-associated human health outcomes,
although animal studies have found testicular toxicity
associated with phthalate exposure (Li et al. 1998;
Parks et al. 2000).
Two studies provide preliminary evidence of associations
between urinary concentrations of monoethyl phthalate
(MEP), a metabolite of DEP, and DNA damage in human
sperm (Duty et al. 2003a), as well as relationships
of monobutyl phthalate (MBP) and monobenzyl phthalate
(MBzP) phthalate, metabolites of DBP and BBzP, respectively,
with decreased sperm motility (Duty et al. 2003b).
In a recent epidemiologic study prenatal exposure
to MEP, MBP, MBzP, and monoisobutyl phthalate was
associated with shortened anogenital distance (AGD)
in male infants (Swan et al. 2005). In rodent studies
AGD is a sensitive measure of prenatal antiandrogen
exposure.
Despite the recent public and scientific interest
on the potential human health effects of phthalates,
routes of human exposure to phthalates have not been
adequately characterized. Potential routes include
dietary ingestion of phthalate-containing foods,
inhalation of indoor and outdoor air, and dermal
exposure through the use of personal care products
that contain phthalates. As far as we know, the proportional
contribution of phthalate-containing personal care
products to total body burden has not been studied.
Houlihan et al. (2002) quantified phthalate levels
in 72 personal care products obtained at a supermarket
in the United States, including hair gel/hair spray,
body lotion, fragrances, and deodorant. DEP was detected
in 71% of these products, DBP in 8%, BBzP in 6%,
and DEHP in 4% of the products tested (Houlihan et
al. 2002). In a recent study (Koo and Lee 2004),
high-performance liquid chromatography (HPLC) was
used to quantify the levels of the same four phthalates
in 102 hair sprays, perfumes, deodorants, and nail
polishes purchased at retail stores in Seoul, Korea.
DBP was detected in 19 of the 21 nail polishes and
in 11 of the 42 perfumes; DEP was detected in 24
of the 42 perfumes and 2 of the 8 deodorants.
The assertion that phthalates are absorbed into
the circulation through human skin is physiologically
plausible and is supported by a limited number of
human and animal studies (ATSDR 1995, 2001, 2003).
The stratum corneum of the epidermis regulates transdermal
absorption, and uptake is achieved through passive
diffusion (Howard et al. 2001). Water-soluble substances
penetrate hydrolyzed keratin, whereas lipid-soluble
substances such as phthalates, especially DEP and
other low-molecular-weight phthalates, can dissolve
into lipid materials between keratin filaments. After
penetration of the epidermis, diffusion into the
dermal and subcutaneous layers is generally uninhibited
because of the nonselective and porous aqueous mediums
in these layers. Substances can then enter the systemic
circulation through venous and lymphatic capillaries.
With increased hydration, rates of absorption of
more hydrophilic compounds can be increased 3-5
times more than usual. Epidermal permeability also
varies greatly between species (Howard et al. 2001).
In one study dermal doses of DEHP were administered
to human volunteers over a 24-hr period, and approximately
1.8% of the total dose was absorbed (Wester et al.
1998). Another experiment involved the topical application
of DBP to human volunteers. The authors determined
that 68 mg would be absorbed in 1 hr if the skin
surface of the whole body were saturated with the
chemical (Hagedorn-Leweke and Lippold 1995). In another
study human breast skin was exposed in vitro to 14C-DEP,
and average absorption under conditions of occlusion
was 3.9% compared with 4.8% without occlusion at
72 hr. However, this was much slower and less complete
compared with absorption through rat skin (Mint et
al. 1994).
In vitro and animal experiments have also
indicated that phthalates are absorbed percutaneously
(Barber et al. 1992; Deisinger et al. 1998; Elsisi
et al. 1989; Melnick et al. 1987; Mint et al. 1994;
Ng et al. 1992; Scott et al. 1987). However, the
mechanism explaining differential rates of uptake
is not agreed upon. Scott et al. (1987) attributed
the phthalate-specific rates of absorption to varying
degrees of lipophilicity. Elsisi et al. (1989) observed
that the lengths of the alkyl chains were inversely
associated with the relative rates of absorption;
except for dimethyl phthalate, DEP has the shortest
alkyl chain (ATSDR 1995).
Although diester and monoester phthalates have
short biologic half-lives of approximately 6-12
hr and do not accumulate (ATSDR 1995, 2001, 2003),
the frequent application of personal care products
may result in semi-steady-state levels, making it
possible to estimate typical phthalate body burden
from a single urine sample (Hauser et al. 2004; Hoppin
et al. 2002). After exposure, diester phthalates,
which may be found in personal care products, are
metabolized to monoester metabolites, the suspected
toxic agents (Li et al. 1998). For this reason and
to avoid contamination, monoester phthalate metabolites
rather than the parent diesters are commonly measured
(Blount et al. 2000).
Our objective in the present study was to determine
whether the use of personal care products predicted
urinary levels of phthalate monoesters, and to identify
subject characteristics that predicted phthalate
levels.
Design and setting. This study was
approved by the Human Subject Committees at
the Harvard School of Public Health, Massachusetts General Hospital (MGH),
and Simmons College. All subjects signed an informed consent. Subjects were
participants in an ongoing study on phthalates and male reproductive health.
They were recruited between January 2000 and February 2003 from the Andrology
Laboratory at MGH. Males between 20 and 54 years of age who were partners of
subfertile couples were eligible; those who have had a vasectomy were excluded.
Approximately 65% of eligible men agreed to participate. The most frequently
cited reason for not participating was lack of time. A total of 406 men were
recruited.
Personal care product use assessment. A
trained research nurse administered a brief questionnaire
to each subject at the time of his visit to the MGH
andrology clinic for semen and urine sample collection.
Information was obtained on personal care product
use, smoking status, age, height, weight, race, and
use of medications. Participants were specifically
asked whether they had used hair gel/hair spray,
lotion, aftershave, cologne, or deodorant in the
48 hr before the collection of the urine sample.
They were also asked to record the time they last
used the products within the 48-hr period.
Urinary phthalate monoester measurement. A
single spot urine sample was collected from each
participant in a sterile plastic specimen cup (which
was prescreened for phthalates) on the same day that
the questionnaire was administered. The analytical
approach has been described in detail (Blount et
al. 2000) and adapted to both enable the detection
of additional monoesters and improve efficiency of
the analysis (Silva et al. 2003). Briefly, measurement
of monoester metabolites, namely, MEP, MBP, mono(2-ethylhexyl)
phthalate (MEHP), MBzP, and monomethyl phthalate
(MMP), entailed enzymatic deconjugation of the phthalates
from their glucuronidated form, solid-phase extraction,
HPLC separation, and tandem mass spectrometry detection.
The limits of detection (LODs) were approximately
1 ng/mL. One method blank, two quality control samples
(human urine spiked with phthalate monoesters), and
two sets of standards were analyzed along with every
21 unknown urine samples. Analysts at the Centers
for Disease Control and Prevention (CDC) were blind
to all information concerning subjects. To control
for urinary dilution, urinary concentrations were
adjusted according to specific gravity. Specific
gravity was measured using a handheld refractometer
(National Instrument Company Inc., Baltimore, MD).
The following formula was used to adjust phthalate
concentrations by specific gravity: Pc = P[(1.024 - 1)/SG - 1],
where Pc represents specific gravity-corrected
phthalate concentration (ng/mL), P is
the measured phthalate concentration (ng/mL), and
SG is the specific gravity of the sample. Specific
gravity-adjusted monoester phthalate levels
were used as continuous outcome variables in statistical
models.
Statistical methods. All analyses
were performed using SAS software (version 8.1; SAS
Institute Inc., Cary, NC). The use of each personal
care product was categorized into a dichotomous variable
(yes/no use in the 48 hr before the urine sample
collection).
Because the phthalate monoester levels were not
normally distributed, nonparametric tests were used
to assess univariate associations between personal
care product use and urinary phthalate levels. Multiple
linear regression was used to explore the relationship
between each of the five personal care products and
each of the five log-transformed monoester phthalate
concentrations. In addition, a six-level sum variable
was created, representing the number of different
types of products used by a participant in the past
48 hr; possible values for this variable were 0,
1, 2, 3, 4, or 5. To determine if a dose-response
relationship existed between urinary phthalate levels
and the number of types of personal care products
used, a trend test was performed using sum variable
as an ordinal variable. For urinary phthalate concentrations
that were below the LOD, a value equal to half the
LOD was imputed (except when quantification was given)
as follows: MEP, 0.605 ng/mL; MBzP, 0.235 ng/mL;
MBP, 0.47 ng/mL; MEHP, 0.435 ng/mL; and MMP, 0.355
ng/mL.
After evaluating appropriateness using quadratic
terms, we modeled age and body mass index (BMI; kilogram
per square meter) as continuous independent variables.
Smoking status was categorized as current smoker
and current nonsmoker (includes ex-smokers and never
smokers). Race was coded as African American, Hispanic,
and other race, with Caucasian as the reference group.
On the basis of biologic plausibility and statistical
factors (i.e., change in parameter estimate), we
included age, BMI, race, and smoking variables in
all models as potential confounders.
To explore the relationship between time of product
use and urinary levels of the phthalates, we regressed
log-phthalate levels on the time between product
use and urine sample collection (referred to as TIMEDIF).
TIMEDIF was categorized into four intervals: product
use 0-3 hr before urine sample collection (TIME0-3); > 3
but ≤ 6
hr (TIME3-6); > 6 but ≤ 8
hr (TIME6-8), and > 8 hr (TIME9). Approximately
75-85% of subject’s product use was within
8 hr of urine collection, and therefore we used TIMEDIF > 8
hr as the reference category.
Subject demographics. Of the 406
men recruited for an ongoing semen quality study,
37 did not provide urine samples. Of the remaining
369, specific-gravity analyses were not available
for 32, leaving 338 for primary analysis. Additionally,
one urine sample was missing MMP concentrations.
The study population was composed largely of white
(
n = 275, 82%), nonsmoking men (
n =
304, 91%) (Table 1). There were 19 African-American
men, 18 Hispanic men, and 24 men of other race/ethnicity.
Personal care product use. Eleven
men (3%) did not provide complete product use information
(Table 1). Most men reported use of deodorant (89%),
whereas fewer men reported using hair gel (37%),
lotion (34%), cologne (29%), and aftershave (13%).
Nine men (2.7%) did not use any of the personal care
products listed on the questionnaire, 114 (33.7%)
used only one type of product, 119 (35.3%) used two
types of products, 71 (21%) used three types of products,
22 (6.5%) used four different types of products,
and only 3 (0.9%) of the men used five or more different
types of products within 48 hr of urine collection.
The percentage of African-American (59%) and Hispanic
(53%) men who reported using cologne within 48 hr
of urine collection was higher the percentage of
Caucasian men (25%) or men of other races (25%).
Additionally, African-American men (65%) were more
likely than Hispanic (44%), Caucasian (30%), or men
of other races (43%) to use lotion. No other associations
were seen between any other personal care products
and race. Interestingly, men who used aftershave
were almost twice as likely (18.5%) to also use cologne
as non-aftershave users (9.8%) (chi squared p =
0.03). There were no consistent relationships among
any of the other products used.
Urinary phthalate monoesters. There
was a wide distribution of both specific gravity- adjusted (Table 2) and -unadjusted phthalate monoester levels (Table 3). Five
phthalate monoesters were detected in 75-100% of subjects. MEP was the
most prevalent (100%), followed by MBP (95%) and MBzP (90%). MEHP and MMP were
both found in about 75% of subjects. Phthalate metabolite concentrations are
presented both adjusted for specific gravity and unadjusted for comparison
with other studies. The highest geometric mean levels were found for MEP (179
ng/mL), followed by MBP (16.6 ng/mL), MBzP (7.1 ng/mL), MEHP (6.6 ng/mL), and
last, MMP (4.5 ng/mL).
Covariate relationships. Race and
cigarette smoking status were predictors of MEP and
MBP levels (Table 4). We found significantly higher
median MEP levels among African-American men (506
ng/mL) and Hispanic men (395 ng/mL) compared with
Caucasian men (140 ng/mL) and those men categorized
as other race (125 ng/mL). Median MBP levels
in Caucasian men (15.3 ng/mL) were also lower than among African-American men
(32.7 ng/mL) and Hispanic men (29.1 ng/mL), and among men identified as other
race (26.5 ng/mL). Median MEP levels in current smokers (250 ng/mL) were significantly
higher than among nonsmokers (143 ng/mL) (Table 4). BMI was weakly, although
positively, correlated with MEP (Spearman correlation coefficient of 0.1, p < 0.05).
Age was not associated with any of the five phthalate concentrations. Wilcoxon
rank-sum tests showed positive associations between the sum variable for product
use and African-American men and men of other races compared with Caucasians.
BMI was positively associated with those identified as other race.
Product use and urinary phthalate relationship. In
the univariate analyses, median MEP levels were higher
among cologne users (265 ng/mL) compared with those
who did not use cologne (108 ng/mL). Likewise, men
who used aftershave had higher median MEP levels
(266 ng/mL) than men who did not (133 ng/mL). Fragranced
products such as cologne and aftershave contain relatively
higher DEP levels than other personal care products.
Figure 1, created on a subset of men who used cologne
plus additional products, depicts the rise in MEP
levels with specific combinations of personal care
product use.
Median MBP was lower among men who had used deodorant
(16.3 ng/mL) compared with those who did not use
deodorant (22.5 ng/mL). The use of lotion was associated
with lower median levels of MBP (14.9 ng/mL), MBzP
(6.1 ng/mL), and MEHP (4.4 ng/mL) compared with men
who did not use lotion (MBP, 16.8 ng/mL; MBzP, 8.6
ng/mL; MEHP, 7.2 ng/mL) (Table 5).
Men of Hispanic, Caucasian, and other races who
used cologne had considerably higher median MEP levels
(981, 444, and 178 ng/mL, respectively) than non-cologne
users of similar race (138, 102, and 116 ng/mL; p =
0.09, < 0.001, and 0.13, respectively). Interestingly,
African-American men who used cologne had 30% lower
median MEP levels compared with non-cologne users
(371 ng/mL vs. 508 ng/mL), although the differences
were not statistically significant. Hispanic and
Caucasian men had substantially higher MEP levels
if they used aftershave (1076 and 220 ng/mL) than
if they did not (138 and 126 ng/mL; p = 0.08
and 0.03, respectively). African-American men who
used aftershave had 33% lower MEP levels compared
with non-aftershave users (340 ng/mL vs. 508 ng/mL),
although the differences were not statistically significant.
No other race/product associations were observed.
Multiple linear regression. In multiple
linear regression models, after adjusting for race,
smoking status, BMI, and age, urinary levels of MEP
were 2.57 times higher among men who had used cologne
and 1.71 times higher among aftershave users compared
with men who did not report the use of these products
(p < 0.0001 and 0.02, respectively) (Table
6). There was also a dose-response relationship
between urinary phthalate MEP levels and the number
of types of personal care products used. For every
additional type of product used, MEP concentrations
increased 33% (95% confidence interval, 14-53%;
trend test p = 0.0002) (Figure 2). The use
of deodorant was associated with 30% lower MBP levels
(p = 0.08). MBP, MBzP, and MEHP levels were
31% (p = 0.004), 34% (p = 0.003), and
34% (p = 0.003) lower, respectively, among
men who had used lotion within the past 48 hr before
urine collection compared with men who had not.
Time of product use. In secondary
analyses, we explored the relationship between time
of product use and urinary levels of phthalate monoesters.
Statistical power was limited in these secondary
analyses as a result of small sample sizes, generally
fewer than 15 subjects for each of the four TIME
strata. The analyses were performed only among users
of each specific product. Cologne use at TIME0-3,
TIME3-6, and TIME6-8 compared with cologne
use at TIME9 was associated with an increase in MEP
of 1.7-fold (p = 0.17), 2.8-fold (p < 0.01),
and 1.1-fold (p = 0.75), respectively. No
consistent time trends were observed for the other
phthalates and cologne use. Aftershave was inconsistently
associated with a 2- to 3-fold increase in MEP levels--3.0-fold
increase at TIME0-3 (p = 0.15), 2.0-fold
increase at TIME3-6 (p = 0.25), and
2.6-fold increase at TIME6-8 (p = 0.11)--compared
with aftershave use at TIME9. No time trends were
observed for the other phthalates and aftershave
use. For lotion use at TIME0-3, TIME3-6,
and TIME6-8, MBP concentration increased 1.9-fold
(p = 0.03), 1.2-fold (p = 0.55), and
1.2-fold (p = 0.52) compared with lotion use
at TIME9. No significant time relationships were
found between lotion use and any other phthalate
or between deodorant or hair gel use and any of the
phthalates.
In the present study, men who used cologne and/or
aftershave within the 48-hr period before the collection
of the urine sample had higher urinary levels of
MEP. This is not unexpected because previous studies
have demonstrated that DEP, the parent compound of
MEP, is an ingredient in many colognes, deodorants,
and fragranced products (Houlihan et al. 2002; Koo
and Lee 2004) and that percutaneous absorption of
DEP occurs (Api 2001; ATSDR 1995; Mint et al. 1994;
Scott et al. 1987). More striking is the steepness
of the dose-response relationship between the
number of product types used in the 48 hr before
urine collection and urinary MEP levels. DEP was
found in 71% of the personal care products tested
in one study (Houlihan et al. 2002), whereas DEHP,
DBP, and BBzP were found in fewer than 10% of products.
In another study, DEP was found in 57% of the perfumes
and 25% of the deodorants surveyed; DBP, DEHP, and
BBzP were not detected in any of the deodorants and
in fewer than 27% of the perfumes (Koo and Lee 2004).
Therefore, it is plausible that MEP would have a
strong relationship with multiple product use, whereas
the other phthalate monoesters would not.
Interestingly, the use of body lotion was associated
with lower levels of MBP, MBzP, and MEHP. The reason
for this relationship is not known, although several
hypotheses are plausible. It is possible that other
ingredients in body lotion may act as a barrier to
the absorption of DBP, BBzP, and DEHP. It is also
feasible that men who use lotion use fewer other
personal care products. However, chi-squared tests
did not show significant inverse relationships between
the use of body lotion and other products (data not
shown). An alternative explanation is that the urinary
levels of these monoesters reflect exposure to their
parent phthalates other than by use of personal care
products. Percutaneous absorption after dermal exposure
is expected to be lower for DBP, BBzP, and DEHP than
for DEP.
The quantities of phthalates present in different
brands of deodorant, aftershave, hair gel/hair spray,
lotion, and cologne are known to be quite variable
(Houlihan et al. 2002; Koo and Lee 2004). In
the present study, because information on the use
of specific brand name products was not gathered,
the analysis was performed by category of product.
This approach is likely to introduce bias toward
the null because not all products within a given
category contain phthalates and those that do contain
phthalates do so at variable concentrations. Because
the participants in this study are all male, it is
unclear whether the findings of this study may be
generalizable to women, who may use different types
and combinations of personal care products.
It is unclear why current smokers had higher levels
of MEP. The results, however, were unstable because
the sample size was small: only 31 men (9%) were
current smokers. One potential explanation is that
smoking may alter the toxicokinetics of DEP. Although
DBP, unlike DEP, is listed as an ingredient in the
filters of Phillip Morris cigarettes (Phillip Morris
2004), MBP was not found to be related to current
smoking status.
Racial differences in urinary levels of MEP and
MBP were consistent with previous data from the National
Health and Nutrition Examination Survey (NHANES)
1999-2000 that have shown African Americans
and Hispanics have higher urinary levels of MEP and
MBP than do Caucasians (CDC 2003; Silva et al. 2004).
In our study we explored the MEP and race associations
for use of specific personal care products. The higher
MEP levels for Hispanic than for Caucasian men appeared
related to differentially higher cologne and aftershave
use. Interestingly, the higher urinary MEP levels
in African-American than in Caucasian men did not
appear to be related to higher cologne and/or aftershave
use. Therefore, the use of other products not identified
in this study, different sources of exposure to DEP,
or differential toxicokinetics may be driving the
high MEP levels among African-American men. After
accounting for race, age, and smoking status in the
statistical models, MEP levels were still significantly
higher among cologne and aftershave users; African-American
race remained an independent predictor of MEP levels.
However, it is important to note that only 18 African
Americans participated in the study, and these findings
may be related to chance because of the small numbers.
Further study on racial/ethnic differences is warranted.
In an earlier study on the relationship between
demographic characteristics and urinary phthalate
levels among a nonrepresentative subset of 289 participants
of NHANES III, MBP, MBzP, and MEHP were higher in
individuals of low socioeconomic status (Koo et al.
2002). Urban residence was also significantly associated
with higher MEHP and MBP levels. Socioeconomic status
and area of residence were not controlled for in
the present study, and these factors could potentially
account for some of the differences measured between
the racial groups. Finally, it is also possible that
higher personal care product use or the selection
of certain types of products among racial groups
may contribute to differences in urinary phthalate
levels.
The time elapsed between product use and urine
sample collection influenced the relationship between
cologne use and MEP concentrations. MEP was 2.7-fold
higher when cologne was used between 3 and 6 hr before
urine collection compared with when it was used 8
hr or more before urine collection. Therefore, to
best assess the relationship of cologne use on urinary
MEP levels, we suggest that urine collection should
occur 3-6 hr after cologne use.
When time of lotion use was not accounted for in
the analysis, there was an inverse association between
urinary levels of MBP and lotion use. However, in
analyses in which time of use was explored, MBP concentrations
were significantly higher within the first 3 hr after
lotion use compared with lotion use 8 hr or more
before. The lotion use MBP relationship may require
a larger data set to determine how use correlates
with MBP levels in urine samples collected at variable
times after applying lotion.
Although aftershave use between 0 and 8 hr before
urine collection was associated with 2- to 3-fold
higher MEP concentration compared with aftershave
use more than 8 hr before urine collection, each
strata had fewer than 10 subjects, and the reference
group had only 15. This could explain why the aftershave-time
of use relationships did not reach statistical significance.
To put these findings into perspective, a comparison
with previous work is offered. The interquartile
difference (443 ng/mL) in MEP, associated with increased
DNA damage in sperm (Duty et al. 2003b), was approximately
2- to 3-fold higher than the difference in levels
of MEP observed between men who did versus those
who did not use cologne (312 ng/mL) or aftershave
(131 ng/mL), respectively. MBP and MBzP, found in
our previous study to be associated with decreased
sperm motility and concentration (Duty et al. 2003b),
were not found to be associated with aftershave or
cologne use.
Cologne and aftershave use were associated with
significantly higher urinary MEP levels after controlling
for age, BMI, smoking, and race. Additionally, a
dose-response relationship was found between
the number of different types of personal care products
used and MEP urinary concentrations. Interestingly,
lotion was inversely associated with most phthalate
levels. Secondary analysis revealed that, for cologne,
product use 3 to 6 hr before urine collection was
most predictive of urinary MEP concentration. However,
for lotion, product use in the 3 hr before urine
collection was most predictive for MBP concentration.
The identification of personal care products as
contributors to phthalate body burden is an important
step in exposure characterization. Additionally,
the results of this study suggest that the time that
products are used in relation to the time that the
urinary samples are collected should be documented.
This will help reduce random measurement error in
statistical analysis. Further work is needed to identify
additional predictors of phthalate exposure.