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Endocrine Active UV Filters: Developmental Toxicity and Exposure Through Breast Milk



Several UV filters exhibit endocrine activity. Evidence for transdermal passage and presence in the food chain (fish) suggests potential exposure of humans during development. Developmental toxicity was studied in rats for the estrogenic UV filters 4-methylbenzylidene camphor (4-MBC, 0.7, 7, 24, 47 mg/kg/day) and 3-benzylidene camphor (3-BC, 0.07, 0.24, 0.7, 2.4, 7 mg/kg/day) administered in chow to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. Neonates exhibited enhanced prostate growth after 4-MBC and altered uterine gene expression after both filters. 4-MBC and 3-BC delayed male puberty and affected reproductive organ weights of adult offspring. Interactions with the thyroid were noted. Expression and estrogen sensitivity of target genes and nuclear receptor coregulators were altered at mRNA and protein levels in adult uterus, prostate and brain. Female sexual behavior was affected by 4-MBC and 3-BC, estrous cycles by 3-BC. Classical endpoints exhibited LOAELs/NOAELs of 7/0.7 mg/kg/day for 4-MBC and 0.24/0.07 mg/kg/day for 3-BC. Molecular endpoints were affected by the lowest doses. In order to obtain information on human exposure, we conducted a monitoring study on human milk with three series of mother-child pairs (2004, 2005, 2006), with focus on cosmetic UV filters in relation to other endocrine disrupters. Methods for UV filter analysis followed the principles of European standardized methods for pesticide residue analysis (EN 15289). In cohorts 2004 and 2005, 78.8% of women reported use of product(s) containing cosmetic UV filters in a questionnaire, and 76.5% of milk samples contained these filters. Use of UV filters and concentration in human milk were significantly correlated. The results agree with the idea of transdermal passage of UV filters. They also indicate that it may be possible to reduce human exposure during critical periods such as pregnancy and lactation by transiently abstaining from use.
CHIMIA 2008,62, No. 5
Chimia 62 (2008) 345–351
© Schweizerische Chemische Gesellschaft
ISSN 0009–4293
Endocrine Active UV Filters: Developmental
Toxicity and Exposure Through Breast Milk
Margret Schlumpf*ab, Karin Kypkec , Claudia C. Vöktd , Monika Birchlerd, Stefan Durrerb, Oliver
Faassb, Colin Ehnesb , Michaela Fuetschb , Catherine Gailleab, Manuel Henselerb , Luke Hofkampe,
Kirsten Maerkelb, Sasha Reolonb , Armin Zenkerf , Barry Timmse, Jesus A. F. Tresguerresg, and
Walter Lichtensteigerab
Abstract: Several UV filters exhibit endocrine activity. Evidence for transdermal passage and presence in the food
chain (fish) suggests potential exposure of humans during development. Developmental toxicity was studied in rats
for the estrogenic UV filters 4-methylbenzylidene camphor (4-MBC, 0.7, 7, 24, 47 mg/kg/day) and 3-benzylidene
camphor (3-BC, 0.07, 0.24, 0.7, 2.4, 7 mg/kg/day) administered in chow to the parent generation before mating,
during pregnancy and lactation, and to the offspring until adulthood. Neonates exhibited enhanced prostate growth
after 4-MBC and altered uterine gene expression after both filters. 4-MBC and 3-BC delayed male puberty and
affected reproductive organ weights of adult offspring. Interactions with the thyroid were noted. Expression and
estrogen sensitivity of target genes and nuclear receptor coregulators were altered at mRNA and protein levels
in adult uterus, prostate and brain. Female sexual behavior was affected by 4-MBC and 3-BC, estrous cycles by
3-BC. Classical endpoints exhibited LOAELs/NOAELs of 7/0.7 mg/kg/day for 4-MBC and 0.24/0.07 mg/kg/day for
3-BC. Molecular endpoints were affected by the lowest doses. In order to obtain information on human exposure,
we conducted a monitoring study on human milk with three series of motherchild pairs (2004, 2005, 2006), with
focus on cosmetic UV filters in relation to other endocrine disrupters. Methods for UV filter analysis followed the
principles of European standardized methods for pesticide residue analysis (EN 15289). In cohorts 2004 and 2005,
78.8% of women reported use of product(s) containing cosmetic UV filters in a questionnaire, and 76.5% of milk
samples contained these filters. Use of UV filters and concentration in human milk were significantly correlated. The
results agree with the idea of transdermal passage of UV filters. They also indicate that it may be possible to reduce
human exposure during critical periods such as pregnancy and lactation by transiently abstaining from use.
Keywords:3-Benzylidene camphor (3-BC) · Developmental toxicity · Human milk ·
4-Methylbenzylidene camphor (4-MBC) · UV filters
1. Endocrine Activity of UV Filters
UV filters are either physical filters like
titanium dioxide and zinc oxide, which
mainly scatter and reflect UV rays, or or-
ganic molecules absorbing light in the UV
range (UVA 400320 nm, UVB 320280
nm). These organic compounds often pos-
sess single or multiple aromatic structures
capable of absorbing energetic solar pho-
tons and returning to the ground state by
thermally emitting the absorbed energy. [1]
Only substances listed in cosmetic direc-
tives like EU Cosmetics Directive, Swiss
Ordinance for Cosmetics, are allowed for
use in sunscreens and as additives in cos-
metics. Currently 27 UV filters are permit-
ted for cosmetic use in Europe. In spite of
considerable structural similarities with au-
thorized cosmetic UV filters, technical UV
filters in plastics and other products need
not be declared.
Since the introduction of cosmetic UV
filters, the main concern regarding their
use was the efficiency to protect human
skin from adverse effects of UV light while
avoiding dermatological side effects. Ob-
servations made in the 1990s on penetration
of human skin by UV filters,[2,3] and on their
presence in fish[4] indicated the possibility
of systemic effects of these chemicals and
prompted us to investigate their endocrine
activity.When tested on MCF-7 cells in
vitro , a number of UV filters used in sun-
screens exhibited estrogenic activity; some
also stimulated growth of the immature rat
uterus in a short-term in vivo test for estro-
genic activity. [5] Estrogenic activity of UV
filters has subsequently been confirmed in
several in vitro and in vivo tests on mammals
and fish.[614] Certain UV filters also dis-
play anti-androgenic activity in vitro,[15,16]
and can affect the thyroid axis.[1719]
Two UV filters with comparatively high
estrogenic activity are 4-methylbenzylidene
camphor (4-MBC) and 3-benzylidene cam-
phor (3-BC).[5,12] Both compounds exhibit
ER beta preference, but they are also active
* Correspondence: Dr. M. Schlumpfab
Tel.: +41 43 233 9517
Fax: +41 43 268 9573
a GREEN Tox GmbH
Langackerstrasse 49
CH-8057 Zürich
b Institute of Anatomy
University of Zurich
Winterthurerstrasse 190, CH-8057 Zurich
c State Institute for Chemical and Veterinary Analysis
of Food
Freiburg, Germany
d University Women’s Hospital
e University of South Dakota
Vermillion, USA
f University of Applied Sciences Northwestern
g Complutense University
Madrid, Spain
CHIMIA 2008,62, No. 5
in ER alpha, typically in in vivo tests such
as the uterotrophic assay, 4-MBC possibly
because of a hydroxylated metabolite.[20]
Whereas a 90-day dermal exposure study
in adult rats reported to the Scientific Com-
mittee on Consumer Products[21] failed to
disclose estrogenic effects of 4-MBC in
this chronic model, significant estrogenic
and antiestrogenic effects were observed
in 90-day studies on adult ovariectomized
rats that addressed this question more spe-
cifically. Typical estrogen targets affected
by 4-MBC include luteinizing hormone,
leptin, fat depots, bone, and genes such
as insulin-like growth factor-1.[22,23] Inter-
actions with estrogenic mechanisms were
also observed after three months exposure
to benzophenone-2.[24,25] Effect patterns
of UV filters do not fully mimic those of
natural estrogens, which may be explained,
i.a., by partial agonist activity, different af-
finities for estrogen receptor (ER) alpha and
ER beta, or interactions with different hor-
mone axes.[5,12,17]
2. Developmental Toxicity of
4-Methylbenzylidene Camphor
(4-MBC) and 3-Benzylidene
Camphor (3-BC)
Development of the sexual pheno-
type depends on the intervention of sex
hormones. In mammals, testosterone (T)
directs development of the male, but this
action is not exclusively mediated by an-
drogen receptors (AR). In brain, T is con-
verted to estradiol (E2)[26,27] to control male
differentiation. Observations in aromatase
knockout mice indicate that development of
the female brain also depends on E2.[28] Cer-
tain peripheral male tissues such as prostate
convert T to dihydrotestosterone acting on
AR as well as to E2 acting on ER.[29,30] With
the exception of tissues with local conver-
sion of T to E2, endogenous estrogen lev-
els are very low in fetuses of both sexes in
rodents[31] and also comparatively low in
humans,[32] thus facilitating competition of
weak estrogenic chemicals for ER.
2.1. Treatment Design
Potential effects of exposure to 4-MBC
and 3-BC during pre- and postnatal de-
velopment were investigated in a mam-
malian model, the rat.[17,19,3335] Male and
female rats of the parent generation were
exposed to 4-MBC (47, 24, 7.0, 0.7 mg/kg/
day) or 3-BC (7.0, 2.4, 0.7, 0.24, 0.07 mg/
kg/day) administered in chow at least ten
weeks before mating, females were further
treated during pregnancy and lactation, and
their F1 offspring until adulthood (age of
three months). 47 mg/kg/day 4-MBC cor-
responds to 40% of uterotrophic LOAEL,
3-BC was dosed according to uterotrophic
potency relative to 4-MBC.[12] Schauer et
al.[36] suggested that toxicity of 4-MBC
should be studied with dermal application.
Yet, certain processes of sexual differentia-
tion like sexual brain differentiation have
long been known to be highly sensitive to
pre- and postnatal stress and handling,[37] so
that results obtained with topical applica-
tion of chemicals to the skin of F0 and F1
rats, each for three months, would be ex-
pected to yield questionable results.
2.2. 4-MBC and 3-BC in Rat Milk
For comparison of experimental data
with internal human exposure, the chemi-
cals were determined in rat milk taken from
the stomach on postnatal day 6. Concentra-
tions (ng/g lipid) were as follows (mean ±
SD, number of samples/litters): 4-MBC, 7
mg/kg/day: 208.6 ±108.7 (5/5), 0.7 mg/kg/
day: 86.3 ±40.5 (4/3). 3-BC, 0.24 mg/kg/
day: 132.1 ±75.2 (3/3), 0.07 mg/kg/day:
not detectable (4/4). 4-MBC and 3-BC were
undetectable in milk of controls.
2.3. Postnatal Development
In the higher dose range, both UV fil-
ters reduced survival rate.[17,38] An impair-
ment of the developing immune system was
indicated by a decrease in thymus weight
at postnatal day (PN) 14. A prominent ef-
fect of both UV filters during early post-
natal development is the significant delay
of puberty in males (preputial separation).
Puberty onset in females remained unaf-
fected (Tables 1, 2 [17,34]). 4-MBC and 3-BC
thus mimic the typical effect of E2 only in
males, but differ from E2 in females, where
E2 advances puberty. [39] Body weight at pu-
berty was slightly reduced in females but
normal in males, indicating that the delay of
male puberty did not result from nutritional
effects. Adult body weights were at control
level after 4-MB[19,33,34] and 3-BC except
for a slight reduction after the highest dose
of 3-BC, possibly as a result of estrogenic
activity. [39]
2.4. Low-dose Effects in Neonatal
Uterus and Prostate
Estrogen target gene mRNA levels in
early postnatal uterus (PN 6), determined
by real-time RT PCR,[33] were affected at
doses as low as 0.07 mg/kg/day 3-BC and
0.7 mg/kg/day 4-MBC (Table 2). The same
doses also affected gene expression in sex-
ually dimorphic brain regions at PN 6 (M.
Fuetsch, C. Gaille, unpublished data). The
changes in mRNAs encoding for vascular
epithelial growth factor (VEGF), induc-
ible nitric oxide synthase (iNOS) and, in
part, endothelial cell nitric oxide synthase
(ecNOS) suggest that angiogenesis and
blood flow in uterus may be influenced.
Effects on male accessory sex glands were
investigated by morphometric analysis in
collaboration with L. Hofkamp and B.
Timms. Maternal exposure to 4-MBC re-
sulted in significant increases in the size of
prostate, seminal vesicles and coagulating
gland at PN 1 (day of birth). In line with
other findings on estrogenic chemicals,
marked differences in growth responses
of specific regions of the prostate were
observed (Table 1, Hofkamp et al.[40] and
unpublished data).
2.5. Reproductive Organs of Adult
Offspring: Regulation of Target
Gene Expression
Exposure to 4-MBC and 3-BC af-
fected reproductive organ weights (Tables
1, 2[17,33,34]). Testes of 4-MBC-exposed
offspring showed decreased weight at
PN14[38] and increased relative weight at
the highest dose in adulthood. The adult
finding is reminiscent of neonatal admin-
istration of weak estrogens.[41,42] In con-
trast, the decrease in prostate weight of
4-MBC-exposed offspring resembles the
effect of perinatal administration of the
potent ER agonist diethylstilbestrol.[41,43]
This suggests a differential sensitivity of
male target organs.
The same estrogen target genes were
studied in reproductive organs and brain of
male and female offspring in order to com-
pare effects on gene regulation. In ventral
and dorsolateral prostate and uterus, gene
expression was affected at mRNA and pro-
tein levels in a tissue-specific manner. [33,34]
The 4-MBC-induced decrease in prostate
weight was accompanied by a decrease in
AR, ER alpha, and insulin-like growth fac-
tor-I (IGF-I) (Table 1). In 4-MBC-exposed
uterus, affected target genes include ER al-
pha and progesterone receptor (PR) (Table
2). Effect patterns differed between the two
camphor derivatives also at the molecular
level, in spite of close structural relation-
ship and similar actions in acute assays for
Malfunctions may also be caused by
changes in the sensitivity of tissues to natu-
ral estrogens. In order to assess such chang-
es, 4-MBC-exposed offspring were gonad-
ectomized in adulthood, injected two weeks
later with a single dose of E2 (10 or 50 µg/
kg s.c.), and investigated 6 h after the in-
jection. 4-MBC exposure reduced the acute
up-regulation of PR and IGF-I and down-
regulation of ER alpha and AR mRNA
by E2 in uterus, and the down-regulation
of AR and IGF-I mRNA by E2 in ventral
prostate (Table 1[33,34]). The reduced up-
regulation of estrogen target genes in uterus
was accompanied by decreased steroid re-
ceptor coactivator-1 (SRC-1) protein levels,
while reduced down-regulation of genes in
prostate was paralleled by reduced nuclear
receptor corepressor (N-CoR) protein.[33,34]
This identifies nuclear receptor coregula-
tors as targets of endocrine receptors, and
suggests that they are involved in changes
in estrogen sensitivity.
CHIMIA 2008,62, No. 5
Table 1. Effect of 4-MBC and 3-BC on selected endpoints in male rat offspring
4-Methylbenzylidene camphor
[mg/kg/day p.o.]
3-Benzylidene camphor
[mg/kg/day p.o.]
0.7 724470.07 0.24 0.7 2.4 7
Puberty (preputial separation)
Adult body weight Ø
Postnatal Day 14, Testis relative weight
Adult F1, Testis relative weight Ø
Postnatal Day 1, Duct number (dorsal) and duct volume (ventral)
Adult F1, Ventral lobe relative weight
Gene expression, adult F1 prostate, mRNA/protein
Androgen receptor (AR) dorsolateral prostate (DP)
Androgen receptor (AR) ventral prostate (VP)
AR mRNA down-regulation by estradiol in VP
N-CoR protein, DP
N-CoR protein, VP
( )
( )
Central nervous system, adult F1, mRNA
Gene expression in Ventromedial Hypothalamic Nucleus
Estrogen Receptor-alpha
Progesterone Receptor
,:significant increase or decrease versus control for p < 0.05 or better. Ø: no statistically significant change. blank or −: not analyzed.
PN 1 = day of birth. Adult F1 offspring: 12 weeks of age, studied under baseline conditions.
Data from Schlumpf et al. [17, 38], Durrer et al. [34], Maerkel et al. [19], Hofkamp et al. [40], O. Faass, M. Fuetsch, C. Ehnes, C. Gaille, unpublished data.
Table 2. Effect of 4-MBC and 3-BC on selected endpoints in female rat offspring
4-Methylbenzylidene camphor
[mg/kg/day p.o.]
3-Benzylidene camphor
[mg/kg/day p.o.]
0.7 724470.07 0.24 0.7 2.4 7
Puberty (vaginal opening)
Adult body weight Ø
Adult F1, Ovary relative weight ØØ
Postnatal Day 6, Uterus relative weight
Adult F1, Uterus relative weight
Postnatal Day 6 Uterus, Gene expression, mRNA
Estrogen receptor-alpha
( )
Adult F1 Uterus, Gene expression, mRNA/protein
Progesterone Receptor (PR-A protein)
PR mRNA up-regulation by estradiol
SRC-1 protein
( )
Ø/ Ø
( )
Ø/ Ø
Central nervous system, adult F1, mRNA
Gene expression in Ventromedial Hypothalamic Nucleus
Estrogen Receptor-alpha
Progesterone Receptor
PR mRNA up-regulation by estradiol
Female sexual behavior (proceptive and lordosis
Estrous cycle
,:significant increase or decrease versus control for p < 0.05 or better. Ø: no statistically significant change. blank: not analyzed.
PN 1 = day of birth. Adult F1 offspring: 12 weeks of age, studied under baseline conditions, females in diestrus.
Data from Schlumpf et al. [17, 38], Durrer et al. [33], Maerkel et al. [19], O. Faass, M. Fuetsch, C. Ehnes, C. Gaille, unpublished data.
CHIMIA 2008,62, No. 5
2.6. Sexually Dimorphic Gene
Expression in Brain and Female
Sexual Behavior
In consideration of the data on brain
differentiation and fetal estrogen levels as
outlined above, we hypothesized that the fe-
male brain should be sensitive to estrogenic
chemicals. Female sexual behavior was re-
corded in adult female offspring exposed to
one of two doses of 4-MBC (7, 24 mg/kg/
day) or 3-BC (2.4, 7 mg/kg/day). Exposed
females were mated with normal experi-
enced males in the evening of proestrus, at
the onset of the dark phase (16.00), when
behavioral receptivity of gonadally intact
rats is high.[44] Behavior was recorded on
coded videotapes in a room illuminated
by an infrared light source. Va ginal smears
were recorded for at least 10–14 days be-
fore behavioral testing. All four treatments
strongly suppressed female sexual behavior
(Table 2, Faass et al., unpublished data).
The treatments affected proceptive behav-
ior (jumping and ear wiggling, displayed to
attract the male), as well as receptive (lor-
dosis) behavior (decreased lordosis quotient
LQ = number of lordosis responses/number
of mounts x 100). At the same time, the male
attempting to mount was rejected more fre-
quently by the female (rejection behavior).
Detailed analyses of estrous cycles were
performed on additional groups of animals
for 21 and 16 days in 4-MBC- and 3-BC-
exposed offspring, respectively. In 4-MBC-
exposed offspring, female sexual behavior
was disturbed in the presence of normal es-
trous cycles, whereas 3-BC exposure caused
irregular cycles. The two functions thus are
differentially affected.
Gene expression was analyzed by real-
time RT PCR in adult male and female off-
spring in two brain regions involved in the
control of gonadal function and sexual be-
havior, medial preoptic region (MPO) and
ventromedial hypothalamic nucleus (VMH)
(Tables 1, 2, 4-MBC:[19,35] 3-BC: Faass et
al., unpublished data). Both compounds
caused sex- and region-specific changes in
ER, in nuclear receptor coactivator SRC-1,
and in target gene mRNA levels. A drop of
PR mRNA in female VMH down to male
levels emerged as a common feature ob-
served after all doses of 4-MBC and after
the higher two doses of 3-BC (tested for
behavioral effects). Reduced PR mRNA in
female VMH was correlated with impaired
female sexual behavior. A similar relation-
ship had been observed with a polybromi-
nated flame retardant (PBDE 99) and with
a PCB mixture.[45] Lordosis behavior is di-
rectly correlated with the expression of PR
mRNA in VMH of female rats.[46,47] Loss of
sexual dimorphism of PR in female VMH
thus appears to represent a signal of altered
regulation of PR that is linked with behav-
ioral impairment across different endocrine
3. UV Filters in Environment and
Food Chain
There is good evidence that pharmaceu-
ticals and ingredients of personal care prod-
ucts (PPCPs) can spread to the biosphere
and reach the food chain. UV filters may
be directly introduced into surface waters
during swimming or may enter wastewa-
ter from households or industry at several
levels of industrial production or commer-
cial use.[1] Cosmetic compounds, synthetic
perfumes and UV filters, were detected in
high amounts in Swiss sewage sludge.[48]
UV filters and synthetic musks are pres-
ent in surface waters and in biota at various
trophic levels, in particular in fish.[4,4952]
UV filter levels in fish from rivers receiv-
ing inputs from wastewater treatment plants
(WWTPs) had considerably higher chemi-
cal loads than fish from Swiss lakes with in-
puts from WWTPs,[50] suggesting increased
availability of these contaminants for fish in
rivers. These studies identified WWTPs as
a major source for UV filters in the aquatic
environment and demonstrate the presence
of UV filters in the food chain.
4. Human Exposure: Monitoring
of Human Milk
Assessment of chemical risks requires
information on quality and quantity of
chemicals present in human body during
critical and sensitive life stages such as
pre- and postnatal development. Acute and
short-term (4 d) experiments with percu-
taneous application of 4-MBC to human
volunteers indicated transdermal passage
of the compound,[36,53] but such studies do
not yield information on internal exposure
of the human population under realistic
patterns of cosmetic use. This informa-
tion can be provided by analysis of human
milk, which informs on internal exposure
of mother and fetus and on contamination
of the food provided to the nursing infant.
Most of the existing data relate to organo-
chlorine compounds. Unfortunately, their
trend to decrease in human milk has been
considered as a success in the campaign
against chemical exposure of babies, with-
out asking for possible exposures to other
chemicals from the food web, like cosmet-
ics, pharmaceuticals, industrial or house-
hold products. Thus, phthalate exposure
has recently been linked with alterations
in male genital development and hormone
5. The Swiss Cohort
Since there was no information on inter-
nal exposure of human populations to UV
filters, and very limited information on ad-
ditional cosmetic ingredients, we started a
monitoring study of human milk at the Uni-
versity Hospital Basel with the approval of
the Basel University Ethics Committee. The
study focused primarily on chemical analy-
sis of UV filters in relation to several other
groups of endocrine-disrupting chemicals
(EDCs and EDC candidates) and consisted
of three different cohorts over three years
(2004, 2005, 2006). So far, the first two co-
horts have been evaluated. For the first time
the questionnaire given to the mothers con-
tained very detailed questions on the use of
cosmetics in pregnancy and lactation. The
aim was to detect a possible correlation
between exposure to certain UV filters and
their presence in human milk.
5.1. Questionnaires
All mothers had to fill out a question-
naire and to give written consent for par-
ticipation in the study. The questionnaires
asked for mother and child data on birth
date, sex, height, weight, sisters and broth-
ers, education, professional career, living
area (urban, suburban, rural), nutritional
and smoking habits of mothers. The ques-
tionnaire then asked for detailed qualitative
and semiquantitative (daily, weekly, month-
ly or less) use of different types and brands
of cosmetic products during pregnancy and
lactation, including sunscreens, lipsticks,
perfumes, deodorants, skin care creams,
body lotions, shower lotions, bubble baths,
hair dyes, make-ups etc.
5.2. Sampling of Human Milk
Sampling was supervised by Claudia
Vökt with the assistance of the study nurse
Monika Birchler. Care was taken to avoid
contamination. The mothers were instruct-
ed to clean breast and nipples thoroughly
with warm tap water before milk sampling.
The milk was obtained using a freshly hot
water-rinsed milk pump (Type Harmony,
Medela AG, Baar). The milk servings were
collected in a clean sterilized bottle (Schott
Duran ISO 4796) stored in the freezer at
20 °C. Milk sampling mainly included the
transitory phase of lactation (day 6 to 14 af-
ter birth), occasionally also the first days of
the mature phase of lactation (from 14 days
after birth on), rarely the colostral phase
(first 6 days after birth) (Wünschmann et
al.[56]). The numbers of milk servings per
bottle (around 100 ml) representing the in-
dividual milk sample of each mother taken
for chemical analysis, varied between 4 and
5.3. Chemicals Analyzed
Our intention was to simultaneously
analyze different groups of EDCs in order
to obtain information on their relative im-
portance. Together with Karin Kypke from
the Community Reference Laboratory for
Pesticides in Food of Animal Origin at the
CHIMIA 2008,62, No. 5
State Institute for Chemical and Veterinary
Analysis of Food in Freiburg/Germany, we
analyzed eight of a total of 27 authorized
cosmetic UV filters (Tables 3, 4) in the same
human milk sample, as we wanted to know
whether these cosmetic UV filters concen-
trate in human milk, and whether there is a
correlation between use of cosmetics con-
taining these chemicals and their presence
in human milk. The same milk samples
were also analyzed for synthetic fragrances
such as nitro musks (musk xylene, musk ke-
tone), polycyclic musks including HHCB
(Galaxolide) and AHTN (Tonalide), mac-
rocyclic musks, several polybrominated
diphenylethers (BDE28, 47, 99, 100, 153,
154), organochlorine pesticides (including
DDT/DDE, methoxychlor, hexachloro-
cyclohexane (HCH), hexachlorobenzene,
toxaphene), seven indicator PCB conge-
ners, and cyclodiene insecticides (aldrin,
dieldrin, chordane, endrine, endosulfan,
heptachlor bromocyclene) (Schlumpf et al.,
in preparation).
5.4. Extraction of UV Filters
The amount of human milk used as a
sample depends on the lipid content of
the milk. Routinely a sample amount of
0.250.5 g lipid per sample was analyzed.
Human milk samples were centrifuged and
the UV filters of interest (Table 3) were
extracted from the cream together with
lipid, using sodium sulfate and the solvent
n-hexane/acetone (1:1) at first, followed
by dichlormethane/acetone (1:1). Follow-
ing evaporation of the solvent in a rotary
evaporator, the extract containing the UV
filters was re-dissolved in cyclohexane/
ethyl acetate (1:1), centrifuged and three
internal standards for the eight UV filters
were added. To remove lipid, gel perme-
ation chromatography was performed on
Bio-Beads S-X3 with cyclohexane/ethyl
acetate as eluting solvent. The eluate was
concentrated to a defined volume. Analo-
gous procedures were used for additional
groups of lipophilic xenobiotic substances
analyzed in the same milk sample, like
persistent organochlorine compounds, syn-
thetic musks and PBDEs. These data will
be presented in the final report on all three
cohorts (Schlumpf et al., in preparation).
In rats, the whole stomach of the pup
was homogenized and extracted using ac-
etone and n-heptane in a Dispomix homog-
enization system (Medic Tools). After add-
ing the internal standards, the sample was
shaken 15 min at 0 °C. and centrifuged, the
supernatant collected and dried. The extrac-
tion step was repeated and the UV filters
were separated from lipids by RP-HPLC
using a octadecylsilyl column (Zenker et
al., in preparation).The fraction containing
4-MBC and 3-BC was dried in a vacuum
centrifuge, re-dissolved in ethanol and de-
termined by GC-MS (see below).
5.5. Determination of UV Filters
The method for analysis of UV filters
in human milk samples followed the prin-
ciples of the European standardized meth-
ods for pesticide residue analysis.[57] Deter-
mination of all UV filters except Bp-2 was
done by GC-LRMS (GC: HP 6890; MS: HP
5973; 30 m HP5-MS, 0.25 mm i.d., 0.25
µ mfilm thickness + 2.5 m pre-column)
with MSD-EI detection mode, using se-
lected ion monitoring (SIM mode) and se-
lecting one target and three qualifier ions
as characteristic mass ions. To compensate
for matrix effects matrix-matched calibra-
tion was used. For determination of Bp-2
LC-MSD (LC: HP 1100; MS: Quattro LC,
50 ×2 mm Luna C18 (2), 5 µm Phenom-
enex) with the detection mode ESI pos was
applied, using multiple reaction monitoring
(MRM), eluent: A being 1 mM ammonium
acetate, pH 4.75 and eluent B being metha-
nol, using matrix-matched calibration. The
concentrations of the substances are report-
ed as ng per g of milk lipid (ng/g lipid). The
limit of quantification (LOQ) and the limit
of determination (LOD) for the UV filters:
HMS, 3-BC, BP-3, 4-MBC and OC were
4.0 (ng/g lipid) (for LOQ) and 2.0 (ng/g
lipid) for LOD. For OD-PABA, EHMC and
BP-2, LOQ was 2.0 ng/g lipid and LOD 1.0
ng/g lipid. The mean level for each residue
was calculated with the assumption of zero
level for undetected value and half LOQ for
levels determined between LOD and LOQ.
The levelwas stated as ‘nd’, i.e.undetected,
if it was below LOD.
5.6. Biostatistics
Possible relationships between use of
UV filters and chemical-analytical data
in human milk were analyzed by Va lentin
Rousson, Biostatistics Unit, University
of Zurich, using Pearson Chi-Square and
Fisher’s exact test.
5.7. Use of UV Filters in Cosmetics
and Presence in Human Milk
An analysis of the first two cohorts from
the Basel cohort study, pilot study (2004)
and Study 1 (2005), revealed that during the
periods of pregnancy and lactation, 78.8%
of the women used some cosmetic product
containing UV filters. In 76.5% of human
milk samples, UV filters were detected
(Table 4). Ethylhexyl-methoxicinnamate
(EHMC, previously known as octyl-meth-
oxycinnamate (OMC)) and octocrylene
(OC) were the UV filters most frequently
used according to the questionnaire and
most frequently detected in milk samples.
For these two filters, a significant correla-
tion between use and presence in human
milk could be demonstrated for the indi-
vidual chemical (p = 0.031 for EHMC, p
= 0.046 for OC, Fisher’s exact test). The
correlation was also significant across all
UV filters for use and presence in the cor-
responding milk sample (p = 0.009). Inter-
estingly, only 45.5% of women reported
use of sunscreens with UV filters, whereas
60.6% of the women used other cosmetics
containing UV filters.
These data demonstrate concentration
of UV filters in a relevant proportion of
human milk samples. Except for lipsticks
where oral uptake is probably important,
these results agree with the idea of transder-
mal passage of UV filters from cosmetics,
as proposed from animal and human stud-
ies.[5,36,53,58] However, it should be kept in
mind that there are also other sources for
Table 3. UV lters analyzed in human milk
Chemical INCIa) NomenclaturePurity of
Bp-2 2,2`4,4`- Tetrahydroxi-benzophenone Benzophenone-2 97%
Bp-3 2-Hydroxi-4-methoxi-benzophenone Benzophenone-3 98%
3-BC 3-Benzylidene-bornane-2-on 3-Benzylidene Camphor > 97%
4-MBC 3-(4`-Methyl)benzylidene bornane-2-on 4-Methylbenzylidene Camphor > 99.7%
2-Ethylhexyl-4-methoxicinnamate Ethyl-hexylcinnamate
HMS 3,3,5-Trimethyl-cyclohexyl-salicylate,
Homosalate > 98%
OC 2-Cyano-3,3`-diphenyl-acrylic acid
Octocrylene 98%
4-Dimethylamino-benzoic acid-2 ethyl-
Octyl-dimethyl PABA > 98.5 %
a)INCI: International Nomenclature of Cosmetic Ingredients
CHIMIA 2008,62, No. 5
these compounds, since they are present in
the ecosphere and food chain (see above).
The Basel cohort study shows multiple
chemical exposures of neonates to groups
of identified and candidate EDCs. Accord-
ing to these results, it is evident that expo-
sure (use) of cosmetics containing UV fil-
ters will produce UV filter-positive human
milk samples. Abstinence from use of or-
ganic UV filter containing sunscreens and
cosmetics could therefore be considered as
an important step to diminish the total load
of chemicals in human milk, in order to re-
duce exposure during particularly sensitive
life stages.
6. Comparison of Experimental Rat
Data with Human Exposure
Our data indicate that pre- and postna-
tal exposure to 4-MBC and 3-BC can in-
terfere with sexual development at brain
and reproductive organ levels. Classical
toxicological endpoints such as puberty
and reproductive organ weights exhib-
ited lowest observed adverse effect lev-
els (LOAEL) and no observed adverse
effect levels (NOAEL) of 7 and 0.7 mg/
kg/day for 4-MBC and of 0.24 and 0.07
mg/kg/day for 3-BC, respectively. Mo-
lecular endpoints were affected by the
lowest doses studied. At the LOAEL of
7 mg/kg/day, 4-MBC concentration in rat
milk (208.6 ng/g lipid) was eleven times
the highest value so far found in human
milk (19 ng/g lipid, Table 4). The ratio
at the classical NOAEL and molecular
LOAEL of 0.7 mg/kg/day 4-MBC is 4.5.
This comparatively small ratio indicates
that the potential risk posed by UV filters
warrants further considerations.
The investigations were supported by Swiss
NationalResearchProgram 50, EU 5th Framework
Program (EURISKED), Swiss Federal Office for
the Environment, Hartmann-Müller Stiftung, and
the Olga Mayenfisch Stiftung.
Received: April 2, 2008
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n = 34
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b Abbreviations: EHMC = ethylhexylmethoxy cinnamate = OMC = octylmethoxy cinnamate, 4-MBC = 4-methylbenzylidene camphor, 3-BC = 3
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... Moreover, some of the organic UV filters are capable of disrupting endocrine systems. Published reports indicate their contribution to the reduction of fertility and reproduction [55][56][57][58] and prove to have estrogenic activity [45,[58][59][60][61]. ...
... Moreover, some of the organic UV filters are capable of disrupting endocrine systems. Published reports indicate their contribution to the reduction of fertility and reproduction [55][56][57][58] and prove to have estrogenic activity [45,[58][59][60][61]. ...
Full-text available
UV filters in environmental compartments are a source of concern related to their ecotoxicological effects. However, little is known about UV filters’ toxicity, particularly those released into the environment as mixtures. Acute toxicity of nine organic UV filters benzophenone-1, benzophenone-2, benzophenone-3, 4-methoxy benzylidene camphor, octocrylene, ethylhexyl methoxycinnamate, 2-ethylhexyl salicylate, homosalate, and butyl methoxydibenzoylmethane was determined. UV filter solutions were tested as single, binary, and ternary mixtures of various compositions. Single solutions were tested using a set of bio tests, including tests on saline crustaceans ( Artemia franciscana ), freshwater crustaceans ( Daphnia magna ), marine bacteria ( Aliivibrio fischeri ), and freshwater plants ( Lemna minor ). The tests represent different stages of the trophic chain, and hence their overall results could be used to risk assessment concerning various water reservoirs. The toxicity of binary and ternary mixtures was analyzed using the standardized Microtox ® method. Generally, organic UV filters were classified as acutely toxic. Octocrylene was the most toxic for Arthemia franciscana (LC 50 = 0.55 mg L –1 ) and Daphnia magna (EC 50 = 2.66–3.67 mg L –1 ). The most toxic against freshwater plants were homosalate (IC 50 = 1.46 mg L –1 ) and octocrylene (IC 50 = 1.95 mg L –1 ). Ethylhexyl methoxycinnamate (EC 50 = 1.38–2.16 mg L –1 ) was the most toxic for marine bacteria. The least toxic for crustaceans and plants were benzophenone-1 (EC 50 = 6.15–46.78 mg L –1 ) and benzophenone-2 (EC 50 = 14.15–54.30 mg L –1 ), while 4-methoxy benzylidene camphor was the least toxic for marine bacteria (EC 50 = 12.97–15.44 mg L –1 ). Individual species differ in their sensitivity to the tested organic UV filters. An assessment of the toxicity of mixtures indicates high and acute toxicity to marine bacteria after exposition to a binary mixture of benzophenone-2 with octocrylene, 2-ethylhexyl salicylate, or homosalate. The toxicity of mixtures was lower than single solutions predicting antagonistic interaction between chemicals. Graphical abstract
... Degradation patterns may have a significant impact on the occurrence of BP-2 in the marine environment. Negligible-to-moderate detectability of some UV filters, mainly 4-MBC (negligible) and BP-3 (moderate) may be additionally related to the restriction of their use in cosmetic products due to possible negative impacts on consumers [63,64] and the environment [65][66][67][68]. In 2017 and 2022, the European Commission introduced limitations on the maximum allowed concentration of BP-3 in cosmetics. ...
Full-text available
Spatiotemporal changes in the concentration of UV filters were investigated along the shore according to increasing distance from breakwaters, from the shoreline, as well as according to seasonality in three locations of different anthropogenic pressures, involving those from cosmetic products being released during touristic activity. Nine organic UV filters (benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), octocrylene (OCR), 4-methoxy benzylidene camphor (4-MBC), ethylhexyl methoxycinnamate (EHMC), ethylhexyl salicylate (EHS), homosalate (HMS), and butyl methoxydibenzoylmethane (BMDM)) were determined in core sediments, and the range of determined concentrations above the limit of quantification was between 19.2 ng·kg −1 d.w. (HMS) and 539.5 µg·kg −1 d.w. (4-MBC). Unexpectedly, contrary to the level of anthropogenic pressure, the concentrations of four (BP-1, BP-2, BP-3, OCR) UV filters decreased in the following order: Darłówko > Ustka > Rowy. Higher concentrations of BP-1, BP-2, BP-3, and OCR were determined in spring than in summer and autumn. The maximal concentration of HMS and EHMC/EHS was found in the summer and in the autumn, respectively. BMDM was determined occasionally only in two samples collected in Ustka. The higher maximal concentration range of all UV filters was determined in core sediments taken from the eastern (539.5 µg·kg −1 d.w.) rather than from the western (11.3 µg·kg −1 d.w.) parts of the beaches. According to increasing distance from the breakwaters, higher concentrations of UV filters were determined in sites located up to 100 m away in all locations and seasons. Spatial variation in the concentration of UV filters was observed in profiles perpendicular to the water line. Typically, higher concentrations were determined at sites having contact with water, although incidentally, high concentrations were also noticed at sites located further into the beach. The Polish coast of the Baltic Sea is not free from organic UV filters, and expectations concerning the abundance of UV filters in a given location are far from recorded data due to the impact of hydro-technical treatments (i.e., stony and wooden breakwaters, artificial reefs, nourishment) and coastal littoral drift.
... Specifically, some widely used UVFs are known to have toxic effects on protozoa, microalgae, and crustaceans [15]. Also, identification of harmful side-effects of UV-filters, such as endocrine disruption and breast cancer risk, has increased the interest to controlling and monitoring the dose of organic UVFs in PCPs and their environmental fate and transport [16,17]. Therefore, use of the advanced technologies to remove or reduce PCPs released into the environment and their subsequent impacts has become a necessity. ...
Full-text available
In this study, simultaneous photocatalytic degradation of different parabens (methyl-, ethyl-, propyl-, and butyl paraben) and UV filters (benzophenone-3, 4-methylbenzylidene camphor, 2-ethylhexyl 4-(dimethylamino) benzoate, ethylhexyl methoxycinnamate and octocrylene) in water matrices was performed under visible light irradiation using novel double plasmonic Ag@Ag3PO4/Ag@AgCl nanophotocatalyst, synthesized by an easy and fast photochemical conversion and photo-reduction. It was found that the nanophotocatalyst with appropriate mole ratio of Ag@Ag3PO4/Ag@AgCl (1:3) showed superior photocatalytic activity than individual plasmonic nanoparticles. This is because there are two simultaneous surface plasmon resonances (SPR) generated by the metallic Ag nanoparticles, in addition to the hetero-junction structure formed at the interface between Ag@Ag3PO4 and Ag@AgCl. The structures of the synthesized photocatalysts were characterized, and the principal reactive oxygen species in the photocatalytic process were identified via a trapping experiment, confirming superoxide radicals (∙O2-) as the key reactive species of the photocatalytic system. The process of photodegradation of the target pollutants was monitored using an optimized method that incorporated solid-phase extraction in combination with gas chromatography-mass spectrometry. The simultaneous photodegradation process was modeled and optimized using central composite design. The kinetic study revealed that the degradation process over Ag@Ag3PO4 (30%)/Ag@AgCl (70%) under visible light followed a pseudo-first-order kinetic model. The simultaneous degradation of target compounds was further investigated in sewage treatment plant effluent as well as tap water. It was found that the matrix constituents can reduce the photodegradation efficiency, especially in the case of highly contaminated samples.
Cardiovascular diseases (CVD) represent the number one cause of death worldwide. The vascular endothelium may play a role in the pathophysiology of CVD diseases. Octylmethoxycinnamate (OMC) is a UV-B filter (CAS number: 5466-77-3) widely used worldwide in numerous personal care products, including sunscreens, daily creams, and makeup. This UV-B filter is considered an endocrine disruptor. Therefore, this investigation aimed to evaluate the direct effects of OMC in human umbilical arteries (HUAs) with endothelium and the possible mechanisms involved in the response. The results demonstrated that OMC exerts a rapid (non-genomic) and endothelium-dependent arterial relaxant effect on HUAs previously contracted with serotonin (5-HT) and Histamine (His). On the other hand, when HUAs were contracted with potassium chloride (KCl), the relaxing effect was only observed in HUAs without endothelium, and it appeared to be inhibited in HUAs with endothelium. Thus, the vasorelaxant effect of OMC depends on the endothelium and depends on the contractile agent used, suggesting that OMC may act through different signaling pathways. Furthermore, computational modulation studies, corroborated the binding of OMC to all the proteins under investigation (eNOS, COX-2, ET-1, and TxA2), with higher affinity for COX-2. In summary, the vascular effect of OMC may involve activating different pathways, i.e., acting through the NO pathway, COX pathway, or activating the endothelin-1 pathway.
Hundreds of xenobiotics, with very diverse origins, have been detected in human milk, including contaminants of emerging concern, personal care products and other current-use substances reflecting lifestyle. The routes of exposure to these chemicals include dermal absorption, ingestion and inhalation. Specific families of chemicals are dominant among human milk monitoring studies (e.g., organochlorine pesticides, bisphenol A, dioxins), even though other understudied families may be equally toxicologically relevant (e.g., food-processing chemicals, current-use plasticizers and flame retardants, mycotoxins). Importantly, the lack of reliable human milk monitoring data for some individual chemicals and, especially, for complex mixtures, is a major factor hindering risk assessment. Non-targeted screening can be used as an effective tool to identify unknown contaminants of concern in human milk. This approach, in combination with novel methods to conduct risk assessments on the chemical mixtures detected in human milk, will assist in elucidating exposures that may have adverse effects on the development of breastfeeding infants.
Ultraviolet (UV) filters are of great concern due to their wide occurrence, bioaccumulation, and toxicity. Little is known about human exposure to UV filters. A total of 3467 individual human milk samples from 24 Chinese provinces were collected during 2017-2019. The concentrations of 12 UV filters in 100 pooled milk samples were determined. The total UV filter concentration was 78-846 (mean 235 ± 120) ng/g lipid weight. The highest and lowest total mean concentrations were for samples from Qinghai and Sichuan provinces, respectively. A significant positive correlation was found between UV radiation levels and UV concentrations in the samples. The dominant UV filters were 2-(2-hydroxy-5-methylphenyl) benzotriazole (UV-P) and ethylhexyl methoxycinnamate (EHMC), which contributed means of 32 and 22%, respectively, to the total concentrations. Plastic products and sunscreens were probably the sources of UV-P and EHMC in the human milk from China, respectively. The mean 2-(3,5-di-tert-amyl-2-hydroxyphenyl) benzotriazole (UV-328) concentration was 2.6 ± 2.6 ng/g lipid weight. The UV filter profiles were similar to profiles for samples from Japan, the Philippines, and Switzerland but not for samples from Korea and Vietnam. The estimated daily UV filter intake for breastfed infants was below the corresponding reference dose. This was the first large-scale study of UV filters in human milk and will help assess the risks posed.
Résumé Les produits de protection solaire (PPS) composés de filtres chimiques et/ou de filtres minéraux occupent aujourd’hui une place importante en photoprotection. Ils ont été à l’origine de larges polémiques, dont certaines ne sont pas éteintes, sur leur efficacité réelle en prévention primaire des cancers cutanés (CC), ainsi que sur leurs risques sanitaire et écologique potentiels. Les filtres chimiques agissent en absorbant le rayonnement lumineux ; on distingue les filtres UVB purs et les filtres larges dont le spectre d’absorption s’étend à l’UVA. Les filtres minéraux diffractent ou réfléchissent le rayonnement ; les deux principaux sont le dioxyde de titane (TiO2) et l’oxyde de zinc (ZnO) ; pour des contingences cosmétiques la taille de leurs particules a été réduite aux nanoparticules. Un PPS doit répondre à des qualités de rémanence, de substantivité et de photostabilité. Deux types de méthodes sont proposés pour l’évaluation des coefficients de protection (CP) des PPS : des méthodes in vivo sur volontaires sains, et des méthodes in vitro utilisant des substrats variés et basés sur la spectrométrie. Le sun protective factor (SPF) in vivo est le CP UVB universellement admis ; pour le CP UVA, la méthode in vivo de la persistent pigment darkening (PPD), bien que non consensuelle, reste à ce jour la plus utilisée en Europe. La mesure de la longueur d’onde critique (Lc) a un intérêt majeur pour définir l’étendue de la protection dans les UVA longs les plus délétères. Les filtres chimiques sont connus pour être à l’origine de réactions locales ; un risque particulier pour l’octocrylène a été évoqué ces dernières années. Récemment, le débat s’est surtout centré sur le risque de perturbations endocriniennes (effet mimant l’estradiol après administration orale à très forte dose chez le rat) pouvant être induites par certains (mais pas les autres !) filtres chimiques, essentiellement le 4-méthylbenzylidène camphre et les benzophénones. Le risque humain paraît peu pertinent dans des conditions normales d’usage de PPS ; le risque environnemental de certains filtres, s’il était avéré, relèverait probablement de leur présence dans bien d’autres produits que les PPS. Les filtres minéraux, souvent prônés chez l’enfant, soulèvent la question du risque des nanoparticules. En tout état, dans le cadre du « Plan d’action national sur la fertilité » et d’une évaluation du risque des nanoparticules, l’Afssaps/ANSM a donné les recommandations adéquates concernant l’incorporation de ces filtres, chimiques et minéraux, dans les produits cosmétiques et le bon usage des PPS. Enfin, les données actuelles ne laissent pas à penser que les PPS puissent avoir un effet délétère en inhibant les effets bénéfiques du soleil, en particulier la synthèse de vitamine D. Les PPS ont démontré, sous couvert d’une utilisation correcte, une efficacité protectrice contre la plupart des effets délétères du soleil à condition d’être conçus en respectant le cahier des charges parfaitement identifié dans les recommandations européennes de 2006. En particulier, les études épidémiologiques démontrent leur efficacité dans la prévention primaire de tous les CC qu’il s’agisse des expositions environnementales ou intentionnelles, et des PPS de protection moyenne suffisent pour cela. Les risques plus que spéculatifs ne doivent certainement pas conduire à dissuader de les utiliser.
Throughout human life, an extensive and varied range of emerging environmental contaminants, called endocrine disruptors (EDCs), cause adverse health effects, including in the cardiovascular (CV) system. Cardiovascular diseases (CVD) are worryingly one of the leading causes of all mortality and mobility worldwide. The UV-B filter octylmethoxycinnamate (also designated octinoxate, or ethylhexyl methoxycinnamate (CAS number: 5466-77-3)) is an EDC widely present in all personal care products. However, to date, there are no studies evaluating the OMC-induced effects on vasculature using animal models to improve human cardiovascular health. This work analysed the effects of OMC on rat aorta vasculature and explored the modes of action implicated in these effects. Our results indicated that OMC relaxes the rat aorta by endothelium-dependent mechanisms through the signaling pathways of cyclic nucleotides and by endothelium-independent mechanisms involving inhibition of L-Type voltage-operated Ca²⁺ channels (L-Type VOCC). Overall, OMC toxicity on rat aorta may produce hypotension via vasodilation due to excessive NO release and blockade of L-Type VOCC. Moreover, the OMC-induced endothelial dysfunction may also occur by promoting the endothelial release of endothelin-1. Therefore, our findings demonstrate that exposure to OMC alters the reactivity of the rat aorta and highlight that long-term OMC exposure may increase the risk of human CV diseases.
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Zur Erfassung der UV-Filtersubstanzen wird eine Methode vorgestellt, die die simultane Bestimmung dieser Verbindungen und von Organochlorverbindungen wie PCB und DDT in unterschiedlichen Fischkompartimenten bis in den ng/kg-Bereich ermöglicht. Die Nachweisgrenzen liegen zwischen 40 und 90 ng/kg Filet und die Wiederfindungsraten bewegen sich zwischen 78 und 104%. Die Belastung von Wasser und Fischen des Meerfelder Maares, Eifel, mit UV-Filtersubstanzen wurde 1991 und 1993 exemplarisch untersucht. Insgesamt konnten in den Fischen des Meerfelder Maares 6 UV-Filtersubstanzen identifiziert und quantifiziert werden. Die im Sommer 1991 gefangenen Barsche waren mit 2,0 mg/kg Fett (Summe nachgewiesener UV-Filtersubstanzen) und die Rotaugen von 1993 mit 0,50 mg/kg Fett belastet. In beiden Fischarten lag die Belastung in der gleichen Größenordnung wie die der Organochlorverbindungen PCB und DDT. In den Rotaugen aus drei weiteren deutschen Seen konnte im Filet Methylbenzylidencampher, eine lipophile UV-Filtersubstanz, nachgewiesen werden. Diese Befunde zeigen, daß UV-Filtersubstanzen in deutschen Seen weir verbreitet und vermutlich als relevante Umweltchemikalien zu betrachten sind. Da im Wasser der Seen die Konzentrationen der UV-Filter substanzen meistens unter der Bestimmungsgrenze lagen, können Fische für diese lipophilen Verbindungen als Expositionsmonitor verwendet werden. Eine ökotoxikologische Bewertung ist zum jetzigen Zeitpunkt nicht möglich, da die Datenlage völlig unzureichend ist.
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Over the past several years, there has been increasing concern that chemicals and pesticides found in the environment may mimic endogenous estrogens, potentially producing adverse effects in wildlife and human populations. Because estrogenicity is one of the primary concerns, a 90-day&sol;one-generation reproduction study with 17β-estradiol was designed to set dose levels for future multigenerational reproduction and combined chronic tox-icity&sol;oncogenicity studies. The purpose of these studies is to evaluate the significance of a range of responses as well as to provide benchmark data for a risk assessment for chemicals with estrogen-like activities. This 90-day&sol;one-generation reproduction study was conducted in male and female Crl: CD BR rats using dietary concentrations of 0, 0.05, 2.5, 10, and 50 ppm 17β-estradiol. Endpoints were chosen in order to evaluate both subchronic and reproductive toxicity. In addition, several mechanistic&sol;biochemical endpoints were evaluated for their usefulness in follow-up studies. In the P 1 generation, dietary administration of 2.5, 10, and 50 ppm 17β-estradiol produced dose-dependent decreases in body weight, body weight gain, food consumption, and food efficiency. At 10 and 50 ppm 17β-estradiol, minimal to mild nonregenerative anemia, lymphopenia, decreased serum cholesterol (50 ppm only), and altered splenic lymphocyte subtypes were also observed in the P 1 generation. Additionally, at these concentrations, there were changes in the weights of several organs. Evidence of ovarian malfunction, characterized by reduced numbers of corpora lutea and large antral follicles, was observed at 2.5 ppm 17β-estradiol and above. Other pathologic changes in males and females fed 10 and 50 ppm 17β-estradiol included centrilobular hepatocellular hypertrophy; diffuse hyperplasia of the pituitary gland; feminiza-tion of the male mammary glands; mammary gland hyperplasia in females; increased number of cystic follicles in the ovary; hypertrophy of the endometrium and endometrial glands in the uterus; degeneration of seminiferous epithelium; and atrophy of the testes and the accessory sex glands. In the reproduction portion of this study, rats fed 10 or 50 ppm 17β-estradiol did not produce litters. While there was no evidence that the 50 ppm treated rats mated, 33.3&percnt; of the rats fed 10 ppm mated but did not produce litters. No effects on mating and fertility indices were observed in rats fed 0.05 and 2.5 ppm 17β-estradiol. Pup weights at birth were statistically decreased relative to control in the groups fed 0.05 and 2.5 ppm 17β-estradiol. Weights of the rats in the 0.05 ppm group recovered by postnatal day 4 and remained similar to control throughout the remainder of the study. The mean gestation length of the 0.05 ppm group was slightly, albeit not statistically significantly, shorter (0.5 days) than that of the control group, which may have contributed to the decrease in birth weight of the 0.05 ppm group. In contrast, the weights of the F 1 generation rats fed 2.5 ppm 17β-estradiol remained decreased relative to the control group throughout the study. Parental administration of 17β-estradiol did not alter anogenital distance in male or female pups. The onset of sexual maturation, as measured by day of preputial separation in males and day of vaginal opening in females, was delayed in male rats fed 2.5 ppm (by 8.2 days) and was hastened in female rats fed 0.05 and 2.5 ppm (by 1.6 and 8.8 days, respectively). The age at vaginal opening ranged from 26 to 37, 26 to 35, and 21 to 25 days for rats fed 0, 0.05, and 2.5 ppm 17β-estradiol, respectively. Hence, the range of age at vaginal opening was similar between the control and 0.05 ppm group. The organ weight and pathologic alterations observed in the adult F 1 generation rats were similar to those observed in the P 1 generation rats. However, in some instances the observed effects, such as histopathological findings in the female reproductive organs, were more severe in the F 1 generation than in the P 1 generation. Apparent differences in sensitivity between the P 1 and F 1 generations may be explained by the increased daily intake of 17β-estradiol by the young F 1 generation rats, in utero exposure, or a combination of both. Dietary administration of 10 and 50 ppm 17β-estradiol clearly exceeded a maximum tolerated dose. Future studies are needed to define the dose-response curve at dietary concentrations below 10 ppm.
A central step in the development and sexual differentiation of the brain is the intraneuronal conversion of testosterone to estrogen. This conversion is catalyzed by an enzyme complex comprised of cytochrome P450 aromatase and the ubiquitous NADPH-dependent cytochrome P450 reductase. Brain aromatase activity is developmentally regulated and expressed only in specific hypothalamic and limbic structures. To determine whether developmental change in aromatase activity are the result of differential regulation of aromatase gene expression, we designed two specific oligonucleotides complementary to a 5'-region or to the heme-binding region of the rat aromatase complementary DNA. Relative amounts of messenger RNA (mRNA) encoding aromatase cytochrome P450 were examined on cryostat sections from male fetuses and offspring of time-pregnant Long-Evans rats using the in situ hybridization technique. On gestational day (GD) 16 (GD 1 = 24 h after mating), aromatase-specific mRNA was detected in the preoptic/hypothala...
: Chemical UV-filters are used in sun protection products and various kinds of cosmetics. The lipophilic chemical UV-filter 3-benzylidene camphor was investigated for its capability to cause vitellogenin induction, possibly via oestrogen receptor binding, in a well-established in vivo fish assay (juvenile rainbow trout, Oncorhynchus mykiss, vitellogenin ELISA). A clear relationship was demonstrated between the dose of injected 3-benzylidene camphor and the concentration of plasma vitellogenin with a 105-times induction from 68 mg 3-benzylidene camphor /kg/injection and above compared to the control vitellogenin level. The relationship between the injected dose of 3-benzylidene camphor and the percent of responding fish (vitellogenin) was evaluated by logistic regression analysis and effective dose-values (ED-values) were determined. ED10, ED50 and ED90 of 3-benzylidene camphor after 6 days (2 injections) were 6.4, 16 and 26 mg/kg/injection, respectively. These ED-values place 3-benzylidene camphor among the more potent xenooestrogens discovered to date and necessitates investigations on the distribution, concentration, persistence and bioaccumulation of 3-benzylidene camphor and other UV-filters in nature.
4-Methylbenzylidene-camphor (4-MBC) is an organic sunscreen that protects against UV radiation and may therefore help in the prevention of skin cancer. Recent results on the estrogenicity of 4-MBC have raised concerns about a potential of 4-MBC to act as an endocrine disruptor. Here, we investigated the direct interaction of 4-MBC with estrogen receptor (ER) α and ERβ in a series of studies including receptor binding, ER transactivation and functional tests in human and rat cells. 4-MBC induced alkaline phosphatase activity, a surrogate marker for estrogenic activity, in human endometrial Ishikawa cells. Interestingly, 4-MBC induced weakly ERα and with a higher potency ERβ mediated transactivation in Ishikawa cells at doses more than 1 μM, but showed no distinct binding affinity to ERα or ERβ. In addition, 4-MBC was an effective antagonist for ERα and ERβ. In an attempt to put 4-MBC's estrogenic activity into perspective we compared binding affinity and potency to activate ER with phyto- and xenoestrogens. 4-MBC showed lower estrogenic potency than genistein, coumestrol, resveratrol, bisphenol A and also camphor. Analysis of a potential metabolic activation of 4-MBC that could account for 4-MBC's more distinct estrogenic effects observed in vivo revealed that no estrogenic metabolites of 4-MBC are formed in primary rat or human hepatocytes. In conclusion, we were able to show that 4-MBC is able to induce ERα and ERβ activity. However, for a hazard assessment of 4-MBC's estrogenic effects, the very high doses of 4-MBC required to elicit the reported effects, its anti-estrogenic properties as well as its low estrogenic potency compared to phytoestrogens and camphor has to be taken into account.
Recognition of the harmful effects of ultraviolet (UV) radiation on the skin has triggered development of organic chemicals (commonly referred as UV filters) that can absorb UV radiation and attenuate the negative effects of sunlight exposure. Depending on the properties and the intended degree of protection, a wide array of combinations is being marketed as delivering protection against most kinds of UV-induced skin damage. However, some UV filters have dermatological implications, so maximum applicable concentrations have been established. To monitor to what extent commercial products comply with the mandatory limits, several analytical methods have been used for their determination in cosmetics and related products.
In this paper, a novel technique has been developed for the determination of benzophenone-3 and its metabolites in urine using solid-phase microextraction (SPME) combined with gas chromatography–quadrupole ion trap mass spectrometry (GC–MS). Benzophenone-3 is a common ingredient found in sunscreens and other products. Because benzophenone-3 and other sunscreening compounds may be absorbed into the body, methods for monitoring the accumulation, metabolism, and excretion of these compounds need to be developed. Three kinds of SPME fibers were used and compared for this study. Parameters for the detection were examined in an aqueous medium and are listed as follows: equilibrium time, multiple extractions from the same vial vs. different vials, the maximum desorption temperature and time, and the effects of both salt and solvent on the extraction efficiency. Human urine samples were then spiked with standard benzophenone solutions and analyzed with the SPME method to determine linear range, limit of detection, and precision. Linear range determinations fell within 10–1000 ng/ml with precisions averaging 7% RSD. Ultimately a urine specimen taken after a human subject had applied a commercially available sunscreen product was tested for benzophenone and its metabolites. Benzophenone-3 and 2,4-dihydroxybenzophenone were detected and the former was subjected to both SPME and SPE quantitation methods with comparable results of 260 and 200 ng/ml, respectively.
Recently, we reported on in vitro and in vivo estrogenic activity of UV filters and on developmental toxicity of 4-methylbenzylidene (4-MBC) camphor [Schlumpf, M., Cotton, B., Conscience, M., Haller, V., Steinmann, B., Lichtensteiger, W., 2001a. In vitro and in vivo estrogenicity of UV screens. Environ. Health Perspect. 109, 239; Schlumpf, M., Berger, L., Cotton, B., Conscience-Egli, M., Durrer, S., Fleischmann, I., Haller, V., Maerkel, K., Lichtensteiger, W., 2001b. Estrogen active UV screens. SÖFW-J. 7, 10]. 4-MBC (7, 24, 47mg/(kgday)) was administered in chow to long Evans rats from 10 weeks before mating of the parent (F0) generation until adulthood of the F1 generation. Peripheral reproductive organs and central nervous system were studied in adult offspring. mRNA expression of progesterone receptor (PR), an estrogen-regulated gene, was investigated in medial preoptic area (MPO) and ventromedial hypothalamic nucleus (VMH) by real-time RT-PCR. We analyzed intact 12-week-old male and female offspring under steady state conditions and adult gonadectomized offspring 6h after a single s.c. injection of estradiol-17β (E2) (10 or 50μg/kg) in order to assess estrogen sensitivity. At steady state conditions we observed significantly higher PR mRNA expression in VMH of control females versus control males. 4-MBC exposed females exhibited a decrease in PR mRNA to levels of control males. The increase in PR mRNA in response to E2 was higher in VMH of males of both 4-MBC groups as compared to control males. PR mRNA levels were similar in MPO of control males and females. Developmental 4-MBC exposure increased PR mRNA levels in male MPO, but did not significantly change female levels. The acute response to the lower E2 dose was decreased in MPO of 4-MBC-exposed males, whereas females of the 7mg/kg dose group exhibited an increased reaction to 50μg/kg of E2. Our data indicate that developmental exposure to endocrine active chemicals such as the UV filter 4-MBC can interfere with sexually dimorphic gene expression in brain in a sex- and region-specific manner.