Available data are reviewed for acute toxicity, eye irritation, dermal irritation, dermal sensitization, phototoxicity, photoallergenicity, percutaneous absorption, kinetics, metabolism,
Trang 1Toxicological pro®le of diethyl phthalate:
a vehicle for fragrance and cosmetic ingredients
A.M Api *
Research Institute for Fragrance Materials Inc., Two University Plaza, Suite 406, Hackensack, NJ 07601, USA
Accepted 1 August 2000
Summary
Diethyl phthalate (DEP; CAS No 84-66-2) has many industrial uses, as a solvent and vehicle for fragrance and cosmetic ingre-dients and subsequent skin contact This review focuses on its safety in use as a solvent and vehicle for fragrance and cosmetic ingredients Available data are reviewed for acute toxicity, eye irritation, dermal irritation, dermal sensitization, phototoxicity, photoallergenicity, percutaneous absorption, kinetics, metabolism, subchronic toxicity, teratogenicity, reproductive toxicity, estro-genic potential, genetic toxicity, chronic toxicity, carcinoestro-genicity, in vitro toxicity, ecotoxicity, environmental fate and potential human exposure No toxicological endpoints of concern have been identi®ed Comparison of estimated exposure (0.73 mg/kg/day) from dermal applications of fragrances and cosmetic products with other accepted industrial (5 mg/m3in air) and consumer expo-sures (350mg/l in water; 0.75 mg/kg/day oral exposure) indicates no signi®cant toxic liability for the use of DEP in fragrances and cosmetic products # 2001 Elsevier Science Ltd All rights reserved
Keywords: Review; Diethyl phthalate; Phthalate ester; Fragrance; Cosmetic
0278-6915/01/$ - see front matter # 2001 Elsevier Science Ltd All rights reserved.
PII: S0278-6915(00 )0 0124-1
www.elsevier.com/locate/foodchemtox
Contents
1 Introduction 98
2 Acute toxicity 98
3 Eye irritation in rabbits 98
4 Dermal irritation in animals 99
5 Dermal irritation in humans 99
6 Dermal sensitization in animals 99
7 Dermal sensitization in humans 100
8 Potential phototoxicity in humans 100
9 Percutaneous absorption 101
10 Kinetics and metabolism 101
10.1 Subchronic toxicity in animals Ð dermal exposure 101
10.2 Subchronic toxicity in animals Ð oral exposure 102
11 Teratogenicity and reproductive toxicity 102
12 Estrogenic potential 103
Abbreviations: DEP, diethyl phthalate; FCA, Freund's complete adjuvant; MED, minimum erythema dose
* Tel.: +1-201-488-5527; fax: +1-201-488-5594.
E-mail address: api@rifm.org
Trang 21 Introduction
The diethyl ester of phthalic acid (DEP; CAS No
84-66-2) is a clear, colorless, practically odorless liquid (see
Fig 1) It has a boiling point of 298C, a speci®c gravity
of 1.12 (20C) and a vapor pressure of <0.001 torr
(20C) Being an oily liquid with slight water solubility,
having an octanol/water partition coecient of log
K=2.47 and being soluble in or partially miscible with
many of the organic molecules with fragrance properties,
provides DEP with a signi®cant technical advantage as a
vehicle for fragrance and cosmetic products DEP has
many industrial uses, but this toxicological pro®le will
emphasize its safety in use as a solvent and vehicle for
fragrance and cosmetic ingredients
Important reviews of the toxicological pro®le of DEP
include those by the Agency for Toxic Substances and
Disease Registry (ATSDR, 1995), Kamrin and Mayor
(1991), the Cosmetic Ingredient Review (CIR, 1985); the
US Environmental Protection Agency (EPA, 1978,
1981, 1987); and Peakall (1975) These reviews recognize
concerns because of the widespread use of DEP, but in
general ®nd no major concerns for toxicity under
cur-rent conditions of use, especially when compared with
other alkyl phthalate esters DEP is sometimes confused
with DEHP (the di-2-ethylhexyl, CAS No 117-81-7)
because of their similar uses and the single letter dierence
in the abbreviated forms of their chemical names This
report will not duplicate information from many of the
articles referenced in these reviews, but will provide
brief summaries from the reviews as well as more recent
publications and previously unpublished data from The
Research Institute for Fragrance Materials, Inc
(RIFM)
2 Acute toxicity
DEP has a low order of acute toxicity Lethal doses are
reported in the range of 1±31 g/kg by the oral route and
1±5 g/kg by the intraperitoneal route in mice, rats, guinea
pigs, rabbits and chickens Intravenous doses in the
range of 0.07±0.3 g/kg caused deaths in mice and rabbits
No deaths were reported with a dose of 11 g/kg by the dermal route in rats Clinical signs have included CNS depression and respiratory paralysis prior to death (Blickensdorfer and Templeton, 1930; Shibko and Blu-menthal, 1973; Lawrence et al., 1975; Peakall, 1975; RIFM, 1978a,b, 1983a,b; Benson and Stackhouse, 1986)
3 Eye irritation in rabbits Minimal irritation of the eye has been reported follow-ing exposure to undiluted DEP Undiluted DEP (0.1 ml) was instilled into the conjunctival sac of the rabbit eye and reactions were scored at 1, 24, 48 and 96 h Irritation was observed at 1 hr that decreased signi®cantly by 24 h (Draize et al., 1944) Lawrence et al (1975) reported no signs of irritation in rabbit eyes using undiluted DEP Minimal irritation was observed following instillation of 0.1 ml DEP to the unwashed eyes of New Zealand rabbits (ATSDR, 1995) Undiluted DEP (0.1 ml) was tested for rabbit eye irritation with or without washing Slight redness of the conjunctivae that did not persist was observed in the unwashed or the washed eye (RIFM, 1978c) Diethyl phthalate (12.5% prepared in 98% ethyl alcohol) was used in a rabbit eye irritation test where 0.1
ml was instilled into the right eye of each rabbit Eyes were examined every 24 h for 4 days and again on day 7 There was no corneal opacity or iris congestion, but a severe conjunctival irritation was observed including che-mosis and discharge On day 7, irritation disappeared, but slight vessel injection was still present The ethyl alcohol alone caused mild conjunctival irritation, although historic control data for ethyl alcohol showed irritation similar to that of the DEP solution in this study (RIFM, 1963)
4 Dermal irritation in animals Slight to moderate irritation has been reported when the skin of rabbits, rats or guinea pigs was treated with undiluted
13 Genetic toxicology 104
14 Chronic toxicity and carcinogenicity 104
15 In vitro toxicity 105
16 Ecotoxicology and environmental fate 105
17 Potential human exposure 105
18 Discussion and conclusions 106
Acknowledgements 106
References 106
Trang 3DEP In a 24-h or 21-day open epicutaneous test on
Himalayan White-Spotted guinea pigs, application of
undiluted DEP caused irritation eects (Klecak et al.,
1977) Application of undiluted DEP on the intact and
abraded skin of six albino rabbits in a closed patch test
caused slight to moderate irritation at both sites at the
24-h evaluation A slight (40%) reduction in irritation
was noticed at the 72-h evaluation (RIFM, 1974) In a
4-h semi-occlusive patch test using rabbits, 0.5 ml of the
undiluted DEP was applied on clipped or intact dorsal
skin Reactions were assessed at 1, 24, 48, 72 and 168 h
after patch removal No irritant eects were noted
(RIFM, 1984a)
DEP was applied (19 times) to intact and (four times)
to abraded abdominal skin of one group of rabbits In a
second group of rabbits, a protective cream `ply 9' was
applied to the abraded skin before exposure to DEP
(four times) In the third group, intact ear skin of rabbits
was exposed (20times) to heated or unheated DEP
Very slight to slight irritation was noted for all
treat-ments regardless of the site or skin condition (RIFM,
1984b) In a 4-h semi-occlusive patch test using New
Zealand White rabbits, 0.5 ml of the undiluted DEP was
applied to a 2.5-cm square lint which was then placed
on clipped dorsal skin of the rabbits The skin was
cleansed after the patch removal, and reactions were
assessed at 1, 24, 48, 72 and 168 h later No irritation
due to DEP was observed (RIFM, 1985)
Undiluted DEP at a dose of 2 ml/kg/day was applied
for 2 weeks to a 4-cm circle of gauze for 6 h/day and
placed on clipped rat skin protected by a semi-occlusive
dressing Irritant eects at the test site were observed as
evidenced by erythema and/or slight desquamation
Histological examination showed very mild epidermal
thickening and slight hyperkeratosis (RIFM, 1994)
5 Dermal irritation in humans
Primary dermal irritation with undiluted DEP has not
been reported in humans No primary irritation due to
DEP was observed in 45 adult human subjects in a
closed patch test using 0.5 ml of the undiluted test
material (RIFM, 1968) No irritation was observed after
application of 0.05 ml/cm2of undiluted DEP once daily
for 10days in an occluded patch test on 10healthy
volunteers (RIFM, 1973a) In a 48-h closed patch test
conducted on the backs of 26 healthy volunteers, DEP
in petrolatum caused no irritation (RIFM, 1978d) In
total, the RIFM database contains reports of 576
human volunteers exposed to undiluted DEP with no
adverse dermal reactions (Api, 1997) However, Api
(1997) reported that in vitro exposure of a human skin
model, Skin2(Advanced Tissue Sciences, Inc.), to DEP
caused marked cytotoxicity to the skin cells This model
is a living, human three-dimensional tissue substrate
with ®broblasts derived from neonatal foreskin seeded and grown onto nylon mesh The reason for this in vitro observation with DEP when no irritation is observed on intact human skin in vivo is unknown
6 Dermal sensitization in animals Undiluted DEP has not been reported to be a sensitizer when tested on the skin of guinea pigs Undiluted DEP was tested for its potential for sensitization on the skin
of male and female Himalayan white-spotted guinea pigs using four dierent testing methods These included
an open epicutaneous test, the Draize intradermal test, the guinea pig maximization test and the Freund's complete adjuvant (FCA) test No dermal sensitization due to DEP was observed with any of the procedures (Klecak et al., 1977; Klecak, 1979) An aqueous 50% solution of DEP was prepared and 0.5 ml was applied
on 3-in square pads The pads were placed on the shaved backs of 12 white male guinea pigs, occluded with an adhesive tape and removed after 6 h Applications were made three times weekly until nine applications had been made Two weeks after the ninth application challenge applications to the same area and a ventral untreated area were made No dermal irritation or sen-sitization was observed (RIFM, 1978e)
Buehler (1996) has observed that the guinea pig max-imization test using FCA as a non-speci®c enhancement
of the immune system will, at times, give false positive responses He reported a group of guinea pigs that had shown a highly reactive response to the vehicle, acetone
On rechallenge these guinea pigs were also hyperreactive
to DEP, although naive guinea pigs were not responsive
to DEP
7 Dermal sensitization in humans DEP has not been reported to be a dermal sensitizer in normal human volunteers, although positive ®ndings have been reported in some of the studies with patients In a Kligman maximization test in 25 human volunteers, 10% DEP was reported to be a non-sensitizer (Greif, 1967) In another maximization test, DEP was tested in
26 normal healthy volunteers No irritation or dermal sensitization reactions were observed (RIFM, 1978d) Irritation and the potential for sensitization were tested
in 42 healthy normal volunteers with undiluted DEP and in an additional 37 normal volunteers using 50% DEP in ethyl alcohol SDA 40 In both studies, 0.5 ml of the test material was added to a 1-in square patch axed
to 13 in adhesive elastic bandage and then applied to the upper arm of subjects Patches were removed 24 h later Patches were applied 3 days a week on alternate days for 3 successive weeks with reaction to each exposure
Trang 4recorded A challenge patch was applied on week 6 at a
previously unexposed site and removed after 24 h
Reactions to challenge were scored at 24 and 72 h after
the patch removal DEP caused little or no primary
irritation and no dermal sensitization (RIFM, 1964, 1971)
No eects were reported from patch tests using 5%
DEP in petrolatum in 20perfume-sensitive patients and
50control patients (Larsen, 1977) Workers in a factory
producing shoes from a polyvinyl chloride granulate
containing dioctyl phthalate (DOP) and coal tar were
examined Sixty workers (30with skin lesions and 30
without skin lesions) in the shoe factory and 30normal
unexposed subjects were patch tested with several
dif-ferent phthalates No eects due to DEP were found in
normal subjects, but 1/30workers with dermatitis and
1/30workers without dermatitis showed positive reactions
to DEP The authors considered it probable that these
eects were due to cross-sensitization to DOP, which
showed positive reactions in 1/30workers with
derma-titis and 4/30workers without dermaderma-titis (Vidovic and
Kansky, 1985) Positive patch test reactions to DEP in
patients with contact dermatitis from eyeglasses frames
and hearing aids have been reported (Smith and Calnan,
1966; Oliwiecki et al., 1991) A 24±48-h occluded patch
test was conducted using 0.5% DEP in 99% ethanol or
in a cream base Of 231 patients, four showed marked
erythema and two showed slight erythema (Takenaka et
al., 1986) No eects due to patch-testing with DEP
were found in a 58-year-old woman who developed
contact dermatitis from the nose pads of her eyeglasses
(Jordan and Dahl, 1971), in a 62-year-old woman with
perfume-associated dermatitis (Larsen, 1975), in 38
contact dermatitis patients (Ishihara, 1977), in nine
children with dry plantar dermatosis (Schulsinger and
Mollgaard, 1980), in a patient with psoriasis (de Groot
and Liem, 1983), in a patient with itchy dermatitis on
her trunk after applying a perfumed toilet lotion (van
Ketel, 1983), in 21 patients allergic to perfumes
(Mey-nadier et al., 1986), in a 35-year-old teacher with a
his-tory of dermatitis on face, hands and feet (de Leeuw and
den Hollander, 1987), in a 73-year-old woman with
itchy pigmentation of her face (Hayakawa et al., 1987),
in 70dermatitis patients (Nethercott et al., 1989), in two
patients with chronic hand eczema (Farli et al., 1990), in
115 patients with cosmetic-related contact allergy
(Remaut, 1992), in a patient with skin sensitivity to tea tree
oil (de Groot and Weyland, 1992), in 82 patients thought
to have occupational acrylic sensitization (Guerra et al.,
1993), and in 51 patients with a variety of allergies in a
patch test clinic (Holness and Nethercott, 1997)
8 Potential phototoxicity in humans
No phototoxicity or photoallergenicity was observed
with 25% DEP in ethanol applied to the skin The minimum
erythema dose (MED) for each volunteer was deter-mined using a 1000 watt Xenon Arc Solar Simulator by exposing unprotected, naive skin to a series of ®ve UVB/UVA exposures each 25% greater than the pre-vious dose The MED was the smallest dose that pro-duced redness at evaluation 24 h after the irradiation Photoirritation was evaluated after application of a sin-gle patch containing 25% DEP in ethanol, using a 25
mm Hill Top Chamber, on the paraspinal region After
24 h, the patch was removed and the site was irradiated with 16 joules/cm2 of UVA irradiation within 10min followed by UVB irradiation at 0.75 MED Reactions at the site were evaluated 1, 24, 48 and 144 h after irra-diation and compared with control sites In 35 female volunteers, mild toxicity was observed in one subject (RIFM, 1999a) Using the identical procedure in a group of 29 volunteers (24 female, ®ve male), no pho-toirritation was observed (RIFM, 1998)
Potential photoallergenicity was evaluated by an induction phase involving application of 25% DEP in ethanol to skin sites twice a week for 3 weeks The induction patch remained in contact with the skin for approximately 24 h, at which time it was removed, and within 10min the site was irradiated with UVA/UVB at 2 MED After the six induction applications and irra-diations, the volunteers had a 2-weeks rest period with-out any application or irradiation The challenge phase involved application of 25% DEP in ethanol to naive skin sites under occluded patches for approximately 24
h, followed by irradiation with 16 joules/cm2of UVA and then 0.75 MED UVB within 10 min after removal
of the patch Reactions at the sites were evaluated 1, 24,
48 and 72 h after challenge irradiation There was no evidence for photoallergy in 29 volunteers (26 female, three male) (RIFM, 1997a) or in another group of 23 volunteers (15 female, eight male) (RIFM, 1997b)
9 Percutaneous absorption Signi®cant percutaneous absorption has been reported for DEP A single dermal application of14C-DEP to the clipped skin of male F-344 rats resulted in a 24% excretion in urine and feces in the ®rst 24 h and a cumulative excretion of 50% in 7 days 34% of the dose remained at the area of application after 7 days Amounts in tissues were minor, 0±0.5% of the dose (Elsisi et al., 1989) Application of 14C-DEP on the shaved backs of rabbits resulted in about 27% excretion
in urine in the ®rst 24 h and a cumulative excretion in urine of 49% and feces of about 1% in 4 days (RIFM, 1973b)
Percutaneous absorption of14C-DEP in vitro through rat dorsal skin was 33% with occlusion and 37% with-out occlusion, whereas average absorption in human breast skin in vitro was 3.5% under occlusion and 4.7%
Trang 5without occlusion at 72 h (Hotchkiss and Mint, 1994;
Mint et al., 1994; Hotchkiss, 1998) Scott et al (1987)
observed that in vitro absorption of DEP through
human skin was slow (steady-state absorption
rate=1.270.11 mg/cm2/h), and that absorption
through rat skin in vitro was higher than through
human skin by a factor of about 30
10 Kinetics and metabolism
Absorbed DEP is distributed throughout body tissues
with the greatest accumulation of the dose in the kidney
and liver Major metabolism is by partial hydrolysis to
ethanol and the monoester, monoethylphthalate, which is
fairly rapidly excreted in the urine Application of 14
C-DEP (ring labelled) on the shaved backs of three female
albino rabbits resulted in about 27% excretion in urine
in the ®rst 24 h and a cumulative excretion in urine of
about 49% and in feces of about 1% in 4 days Blood
levels accounted for about 7% of the dose after 1 h of
application and less than 1% of the dose after 4 days
Tissue distributions showed the greatest accumulation
of the applied dose in kidneys and liver After 4 days
0.003% (range 0.002±0.006%) of the applied dose was
found in the kidneys and 0.004% (range 0.001±0.006%)
in the liver (RIFM, 1973b) Oral administration of14
C-DEP to rats and mice resulted in maximum
concentra-tions of radioactivity in kidney and liver, followed by
blood, spleen and fat Highest levels were observed
within 20min, followed by fairly rapid decrease to only
trace amounts at 24 h Excretion occurred primarily in
urine Cumulative urinary and fecal excretion,
respec-tively, was 47 and 0.7% within 12 h, 82 and 2.5% within
24 h and 90and 2.7% within 48 h after the dose (Ioku et
al., 1976)
Metabolism after oral administration of DEP to rats
results in hydrolysis, with the principal urinary
meta-bolite being monoethyl phthalate and with phthalic acid
as the minor secondary urinary metabolite (Chambon et
al., 1971; Kawano, 1980) Hydrolysis to the monoester
can occur in the lumen of the gastrointestinal tract or in
intestinal mucosal cells after oral administration as well
as in organs such as the liver, kidney and lung after
systemic absorption (Lake et al., 1976, 1977; Rowland
et al., 1977; Kayano et al., 1997) Hydrolysis to the
monoester by skin has been demonstrated using in vitro
percutaneous absorption through rat skin and adult
human skin (Hotchkiss and Mint, 1994; Hotchkiss,
1998)
The speci®c enzymes for hydrolysis of DEP to the
monoester are not well characterized for various species
Human plasma-derived arylesterase did not hydrolyze
DEP (Augustinsson and Ekedahl, 1962) DEP was
hydrolyzed to its monoester by puri®ed carboxylesterase
from human liver and rat liver (Mentlein and Butte,
1989) Microsomal carboxylesterase activity towards DEP was induced in mouse liver and rat kidney, but not
in rat or mouse testes, in clo®brate-fed animals (Ashour
et al., 1987) Kayano et al (1997) isolated a novel esterase from mouse hepatic microsomes that had high catalytic activity compared with the mouse hepatic microsomes DEP was hydrolyzed to the monoester, but the monoester was not hydrolyzed even after prolonged incubation periods
Limited evidence for induction of enzymes by DEP has been reported Preincubation of DEP in microsomal pellets and supernatant isolated from Sprague±Dawley males treated with phenobarbital intraperitoneally for 3 days, had no eect on cytochrome P450or on N-acetyl transferase activity in rat liver microsomal suspensions, but the activity of UDP glucuronyl transferase was reduced (Gollamudi et al., 1985) Increased activity of peroxisomal enzyme carnitine acetyl transferase was observed in rat primary hepatocyte cultures in the pre-sence of DEP (Gray et al., 1983) Male rats fed 2% DEP
in their diet for 3 wk showed marginal hepatic peroxi-some proliferation (Moody and Reddy, 1978, 1982) 10.1 Subchronic toxicity in animals Ð dermal exposure Signs of toxicity in rats and mice after 4 weeks of der-mal exposure to undiluted DEP were limited to increases
in weights of liver and kidneys at doses of 1±5 ml/kg In a 2-week dermal study, undiluted DEP was applied to male and female Sprague±Dawley rats at the dose of 2 ml/kg/day under a 6-h semi-occlusive patch No changes were observed in body weight gain, clinical chemistry, hematology, or by histological examination (RIFM, 1994) In a 4-week study, groups of male and female B6C3F1mice received dermal applications of undiluted DEP, 12.5, 25, 50or 100ml (approx 560, 1090, 2100 or
4300 mg/kg for males and 630, 1250, 2500 or 5000 mg/
kg for females), 5 days/week Increased absolute and relative liver weights were observed only in females receiving 25 and 100 ml DEP No other adverse clinical signs of toxicity or dermatotoxicity were observed (NTP, 1995) Male and female F344/N rats given dermal applications of 37.5, 75, 150or 300ml (approx 200, 400,
800 or 1600 mg/kg for males and 300, 600, 1225 or 2500 mg/kg for females) of undiluted DEP for 5 days/week for 4 weeks showed increased relative liver weights in
300 ml males and in 150and 300ml females Increased relative kidney weights were seen in 150and 300ml males and in 150 ml females There were no clinical signs
of toxicity and no dermatotoxicity (NTP, 1995)
10.2 Subchronic toxicity in animals Ð oral exposure Toxic signs after 16 weeks of exposure to DEP in the diet consisted of an increase in liver weight (without sig-ni®cant abnormal histological ®ndings) in female rats at
Trang 6doses as low as 150 mg/kg/day and increased weights of
other organs in male and female rats at higher doses of
750 to 3710 mg/kg/day Guinea pigs were administered
125, 250, 500 or 1000 mg/kg DEP by mouth, 5 or 6 days
per week for up to 12 doses At necropsy, evidence of
toxicity was limited to slight but de®nite histopathologic
damage in the liver and kidneys at the highest dose and
questionable changes at 250and 500mg/kg (RIFM,
1983b) Oishi and Hiraga (1980) fed DEP to young male
Wistar rats at 2% (approx 1000 mg/kg/day) in the diet
for 1 week They reported an increased liver weight and
decreased concentrations of testosterone in testes and
serum There were no eects on body weight, kidney
weight, testes weight, zinc concentration in testes, liver
kidney or serum, or dihydrotestosterone concentration
in serum NTP (1984) reported no deaths, signs of
toxi-city or signi®cant eects on body weight compared with
controls when diets containing DEP at levels of 0, 0.25,
0.50, 1.0, 2.5 or 5.0% (approx 500, 1000, 2000, 5000 or
10000 mg/kg/day) were fed to male and female CD-1
mice (8 weeks of age) for 14 days
Brown et al (1978) fed diets containing 0.2, 1 or 5%
(approx 150, 770 or 3160 mg/kg/day for males and 150,
750or 3710mg/kg/day for females) DEP to male and
female Sprague±Dawley rats for 16 weeks Reduced
food intake and body weight gain were noted in females
fed 1 and 5% DEP and in males fed 5% DEP No
sta-tistically signi®cant eects on water intake, or on the
results of the hematological examinations, serum enzyme
levels, urinary cell-excretion rate, renal concentration
tests or histological examination were observed There
were increases in weights of several organs in males and
females, primarily at the highest dose (brain, liver, stomach,
small intestine and full caecum); the most consistent
sig-ni®cant ®nding was increased relative liver weight in
females at all treatment levels The authors considered it
likely that the increased organ weights relative to body
weight were a result of the reduced body weight gain
and that the increased liver weight, in particular, the
absence of abnormal histological ®ndings, might be due
to work hypertrophy In the case of work hypertrophy,
there is a stimulation of processing-enzyme activity and an
increase in the amount of smooth endoplasmic reticulum,
whereas damaged livers show a reduction in the activities of
aniline hydroxylase and some other processing enzymes
and in glucose-6-phosphatase activity DEP shows no
marked loss of either aniline-hydroxylase or
glucose-6-phosphatase activity, no increase in smooth endoplasmic
reticulum, and a decrease in alcohol dehydrogenase
Brown et al (1978) conclude that ``overall, this pattern
of change is most consistent with a functional
hyper-trophy of the liver and, on this basis, it is likely that the
liver enlargement reported in this paper was the result of
such hypertrophy On this evidence there is no reason to
assume that the enlarged liver represents an adverse
response to DEP.''
11 Teratogenicity and reproductive toxicity DEP administered to pregnant rats during the period of major organogenesis had no adverse eect on embryo/ fetal development, except for an increased incidence of extra rib (an anatomical variation) at a maternally toxic exposure level Dietary concentrations of DEP at 0.25, 2.5 or 5% were administered to timed-pregnant CD rats
on gestation days 6±15; the rats were sacri®ced on gestation day 20 The average nominal doses based on food consumption of controls were 200, 2000 or 4000 mg/kg/day The actual average doses because of decreased food consumption were approximately 200,
1900 or 3300 mg/kg/day Maternal toxicity was shown
by decreased food consumption, decreased body weight gain and decreased water consumption, particularly at the highest dose Gravid uterine weight, absolute and relative maternal liver and kidney weights were unaected by DEP treatment No adverse eect on embryo/fetal growth, viability or incidence of external, visceral or ske-letal malformations was observed An increased incidence
of one extra rib in the ospring from rats in the maternally toxic high dose group was regarded as a variation (NTP, 1988; Price et al., 1989; Field et al., 1993) DEP was administered percutaneously to pregnant Jcl:ICR mice in daily doses of 500, 1600 or 5600 mg/kg/ day from day 0to day 17 of gestation and fetuses were removed by caesarean section on day 18 Maternal toxicity was indicated at all doses by reduced thymus and spleen weights and at the high dose by increased adrenal weight Fetal body weight was reduced at the high dose and skeletal examinations showed a higher incidence of cervical and lumbar ribs at the high dose However, no external, visceral or skeletal anomalies in the fetuses were attributable to DEP treatment The authors concluded that DEP had no potential to pro-duce teratogenic eects on fetuses under those conditions (Tanaka et al., 1987)
Pregnant CD-1 mice received DEP at an estimated
LD10dose of 4500 mg/kg/day by gavage, once daily on gestation days 6±13 and were allowed normal delivery Two of the 50pregnant mice died; there were no eects
on maternal body weight gain, viable litters, neonate survival or neonatal weight gain (Hardin et al., 1987) A teratogenicity study (Singh et al., 1972) in rats was con-sidered irrelevant for this review because of the inap-propriate intraperitoneal route of exposure
Eects on general reproductive performance with DEP were limited to slight changes at doses causing decreased body weight gain Male and female CD-1 mice were given DEP at concentrations of 0.25, 1.25 or 2.5% in diet, before, during and after cohabitation in a con-tinuous breeding protocol The approximate doses were
460, 2440 or 4400 mg/kg body weight Continuous exposure of mice (11 weeks of age at outset) to these dose levels of DEP during the 7-day premating, 98-day
Trang 7cohabitation and 21-day segregation periods had no
eect on the number of pairs able to produce at least
one litter There was no eect on the number of litters
produced per pair, proportion of pups born alive, sex of
pups born alive and live pup weight The low- and
mid-dose groups actually showed more live pups per litter
compared with the control and high dose group The
fertility and reproductive performance of the ospring
were further assessed for the control and 2.5% groups
The high dose group in the F1 generation showed
reduction in body weight gain, decreased number of live
pups per litter (when sexes were combined, but not
when analyzed by males and females separately),
decreased sperm concentration (no change in sperm
motility or percentage of abnormal sperm), increased
prostate weight in males, increased liver weight in
females, and reduced uterus and pituitary weight in
females There were no statistically signi®cant eects on
mating behavior, proportion of pups born alive, live
pup weight or sex of pups born alive (NTP, 1984; Lamb
et al., 1987; Morrissey et al., 1989)
Special attention has been paid to the male reproductive
system DEP failed to produce any eect on testicular
Ser-toli cell function or on testicular cell cultures contrary to
other phthalate esters tested (Gray et al., 1982; Gray and
Gangolli, 1986; Heindel and Powell, 1992) Testosterone
levels in serum and testes were decreased in rats fed 2%
DEP (approx 1000 mg/kg/day) in their diet for 1 week, but
no testicular damage occurred as evidenced by testes weight
or testes zinc content (Foster et al., 1980; Gray and
Butterworth, 1980; Oishi and Hiraga, 1980) Treatment of
5-week-old male rats by oral intubation with DEP
dis-solved in corn oil (1600 mg/kg) did not cause testicular
atrophy or aect testicular cytochrome P-450or
steroido-genic enzymes following a single dose or up to 4 days of
dosing (Foster et al., 1983) Rats receiving 2000 mg/
kg/day DEP by oral gavage for 2 days showed no eect on
seminiferous tubular structure or Leydig cell
morphol-ogy by light microscopy Ultrastructural examination
of Leydig cells showed mitochondrial swelling and focal
dilatation of smooth endoplasmic reticulum
LH-stimulated testo-sterone secretion from Leydig cells
incubated with the monoester of DEP was not aected
(Jones et al., 1993)
A variety of toxic eects of DEP observed by in vitro
techniques are reported, but their signi®cance is questionable
in view of the high doses and the in vivo results presented
above Human sperm exposed in vitro to DEP showed
decreased motility with prolonging exposure time (0±18
min) Other qualities of motility such as velocity,
line-arity, and amplitude of the track were also aected
(Fredricsson et al., 1993) Eect of DEP on developing
chick embryo was determined DEP (0.025 ml) was
injected into the yolk sac of fertilized eggs before day
3 of embryonic life 69% of the chick embryos died
(45±50% of control eggs injected with sesame or
Crisco1oil, respectively died) One of the 10eggs hatched showed marked malrotation of the left leg (Bower et al., 1970) Iijo (1975) injected 0.025 ml DEP into the yolk sac
of fertilized chicken eggs and found 65% lethality in DEP treated eggs as compared to 21% lethality in control eggs In addition, 1/28 embryos showed malformations
12 Estrogenic potential DEP has not been reported to cause estrogenic activity
in vertebrates, although weak activity has been reported in some, but not all, in vitro studies EPA (1996) determined that there was insucient evidence, at that time, to demonstrate that DEP causes hormonal disruption Groups of 10immature female Wistar [Crl(WI)BR] rats received a single oral dose of 0(vehicle control), 50, 150or 500mg/kg body weight of DEP once a day for 3 consecutive days As a positive control, one group of rats received a single oral dose of 0.4-mg b-estradiol/kg body weight once a day for 3 consecutive days The vehicle used for DEP and b-estradiol was peanut oil Approximately 24 hr after administration of the ®nal dose the rats were sacri®ced, the uterus removed and weighed There were no treatment-related eects of DEP
on clinical observations or on body weights throughout the study The uterus weights were unaected by treat-ment with DEP, while the positive control produced a signi®cant eect on uterus weight (RIFM, 1999b) Using an in vitro recombinant/receptor gene bioassay with HeLa cells stably transfected with the Gal4-human estrogen receptor chimeric construct, Gal4-HEGO and the Gal4-regulated reporter gene, 17m5-G-Luc, no sig-ni®cant induction in luciferase activity was observed with DEP (Balaguer et al., 1996)
Using a recombinant yeast strain (Saccharomyces cerivisiae) containing hER (the human estrogen receptor) and the reporter gene, lac-Z, DEP did not demonstrate estrogenic potential over the range of concentrations (10 8±10 4 m) tested The results were compared against the positive control, b-estradiol, and the negative control, testosterone (RIFM, 1999b)
An in vitro estrogen receptor-binding assay using rat uterine cytosol from the uteri of 10-week-old Wistar rats was conducted The assay measures the potential binding
of DEP to the estrogen receptor by testing its ability
to compete with and displace3H-17b-estradiol bound to the receptors in the cytosol The results indicated that DEP did not bind to the estrogen receptor (RIFM, 1999b)
Harris et al (1997) reported no estrogenic activity using the estrogen-responsive human breast cancer cell line ZR-75, but did observe a slight increase in cell proliferation at day 8, but not at day 5 or 12 using the a concentration of 10 5m DEP with the estrogen-responsive human breast cancer cell line MCF-7 They also reported
Trang 8an extremely weak estrogenic activity using yeast cells
with the human estrogen receptor Activity was
observed only at concentrations greater than 10 4m (a
potency only 0.0000005 that of 17b-estradiol)
Some estrogen-mimicking xenobiotics in vertebrates can
also aect the hormonally regulated molting process in
arthropods by binding and blocking the ecdysteroid
recep-tors Zou and Fingerman (1997) reported that DEP
delayed the molting in the water ¯ea, Daphnia magna, at a
concentration of 22.4 mg/l (over 100 times the
con-centration causing inhibition by diethylstilbestrol) These
authors also reported that DEP at 50mg/l inhibited
the chitobiase activity involved in the premolt stage of
the ®ddler crab, Uca pugilator (Zou and Fingerman,
1999)
13 Genetic toxicology
The weight of evidence from mutagenicity tests supports
the view that DEP is non-genotoxic NTP (1995)
reviewed the published data (seven studies) and reported
that DEP may be weakly mutagenic in Salmonella
strains TA100 and/or TA1535, which mutate via base
substitution However, because the in vitro data were
sparse and no in vivo data were available for analysis,
they considered the mutagenic pro®le to be incomplete
NTP (1995) then proceeded to conduct additional tests
They reported no mutagenic response with DEP in
Sal-monella typhimurium strains TA98, TA100, TA1535 or
TA1537 either with or without rat or hamster liver S9
They also reported no chromosomal aberrations with
DEP in Chinese hamster ovary cells with or without rat
liver S9 However, DEP induced sister chromatid
exchanges at concentrations of 167 to 750 mg/ml with,
but not without, rat liver S9 NTP (1995) noted that,
although the positive sister chromatid exchange test
might indicate a potential for in vivo DNA damage, this
endpoint is highly sensitive and does not correlate well
with carcinogenic eects in rodents
Because DEP is readily hydrolyzed to the monoester
it is relevant to note that monoethyl phthalate showed
no mutagenic eect when tested with S typhimurium
strains TA100 and TA98 and Escherichia coli WP2
strains uvrA+ and uvrA with or without rat liver S9
(Yoshikawa et al., 1983)
14 Chronic toxicity and carcinogenicity
There is no unequivocal evidence for serious toxicity or
carcinogenicity in rats or mice after long-term
adminis-tration of DEP by the oral or dermal route of exposure
Carcinogenic eects of DEP were evaluated in a 2-year
dermal study in male and female F344/N rats and
B6C3F1mice (Marsman et al., 1994; NTP, 1995) Rats
were treated with undiluted DEP at doses of 0, 100 or
300 ml/day (approx 0, 320 or 1015 mg/kg/day for males and 0, 520 or 1600 mg/kg/day for females) dermally to clipped interscapular skin ®ve times/week for 104 weeks
A treatment-related increased incidence of minimal to mild epidermal acanthosis at the site of application was observed in dosed males and females The incidence of fatty degeneration of the liver was decreased in both male and female treated rats Decreased incidence of
®broadenomas of mammary gland occurred in female treated rats No evidence of skin neoplasia was found in male or female rats
Studies conducted in mice with dermal DEP doses of
0, 7.5, 15 or 30 ml (approx 0, 260, 520 or 1050 mg/kg/ day in males and 0, 290, 550 or 1100 mg/kg/day in females) in acetone ®ve times/week for up to 103 weeks showed no signi®cant evidence of toxicity or neoplasia
at the site of application An increased incidence of basophilic foci in the liver was noted in mid-dose male mice However, no dose-related trend was apparent, and
no statistically signi®cant increased incidence was observed in female mice A marginal increased incidence (36%) of combined hepatocellular adenoma or carcinoma was observed in high-dose male mice (the Fisher Exact Test had a P value of 0.035 and the dose-related trend
by the logistic regression test had a P value of 0.034)
In females, the incidence of combined hepatocellular adenoma or carcinoma was higher in low- and mid-dose mice than in high-dose mice or controls Because the incidence of hepatocellular neoplasms in the high-dose male mice was similar to the historical control mean (36%, range 10±68%), and because there was no dose response for liver neoplasms in female mice, these mar-ginal increases were considered to be uncertain ®ndings, providing only equivocal evidence of carcinogenic activity (Marsman et al., 1994; NTP, 1995)
One-year initiation±promotion studies in male mice were conducted to evaluate the potential of dermally applied DEP to initiate tumorigenesis when followed by a strong promoter (TPA: 12-O-tetradecanoylphorbol-13-acetate) or to promote tumorigenesis following admin-istration of a known initiator (DMBA: 7,12-dimethyl-benz[a]anthracene) No initiator or promoter activity of DEP was demonstrated (Marsman et al., 1994; NTP, 1995)
Rats given 0.5, 2.5 or 5% DEP (approx 250, 1250 or
2500 mg/kg/day) in diet for 2 years showed slightly decreased body weight gain throughout the study and diminished eciency of food utilization at the highest dose compared with the control rats No treatment-related eects on hemocytology, blood sugar, non-pro-tein nitrogen levels or urinalyses were observed Post-mortem examination of dead or sacri®ced rats revealed no unusual pathology, either gross or microscopic, which appeared to bear any relation to the DEP in the diet (RIFM, 1955)
Trang 915 In vitro toxicity
A number of in vitro toxicity tests have been reported
using various cell culture systems While some evidence
of inhibition of biochemical and physiological cellular
functions and some cell death have been observed with
DEP, the ®ndings are not considered particularly useful
for this toxicological pro®le and are not reviewed in this
report For further detail, see CIR (1985)
16 Ecotoxicology and environmental fate
Studies have been conducted to determine acute and
chronic toxicity of DEP for many aquatic species (EPA,
1987; Adams et al., 1995) Neuhauser et al (1985) has
determined the acute toxicity for DEP in earthworms
In the decision to remove DEP from the toxic chemical
list requiring reporting, EPA (1996) stated:
EPA has also concluded that DEP does not meet the
toxicity criterion of EPCRA section 313(d)(2)(C)
because it cannot reasonably be anticipated to
cause adverse eects on the environment of sucient
seriousness to warrant continued reporting DEP
exhibits low toxicity to aquatic organisms (®sh 96
hour median lethal concentration (LC50), 12 to 100
milligrams/liter (mg/l); daphnid 48 hour LC50, 50
to 90mg/l; and algae 96 hour median eective
concentration (EC50), 30to 86 mg/l, and is not
likely to bioconcentrate
DEP undergoes rapid degradation by bacteria
com-monly found in the environment as evidenced in studies
by the static-¯ask screening method (Tabak et al., 1981)
and activated sludge tests (O'Grady et al., 1985) For
further details, see the reviews by ATSDR (1995) and
EPA (1987)
17 Potential human exposure
Because of extensive industrial uses, DEP is ubiquitous
in the environment and has been measured in air, water,
soil, ®sh, human adipose tissue and foods wrapped in
cellulose acetate ATSDR (1995) has presented a broad
review of the potential for human exposure to DEP
Acceptable levels of exposure of 5 mg/m3 of air have
been estimated for workers exposed to DEP in the
workplace by the American Conference of
Govern-mental Industrial Hygienists, the Occupational Safety
and Health Administration and the National Institute
for Occupational Safety and Health For a worker
breathing 10m3 of air during an 8-h workday for his
working lifetime, this would represent acceptable
expo-sure to inhalation of 50mg DEP each workday The
ambient water quality criteria for protection of human health established by the US Environmental Protection Agency (EPA) is 350mg/l, or a dose of 350mg DEP in the daily consumption of 1 l of water The oral RfD (the daily exposure to the human population, including sensitive subgroups, that is likely to be without an appreciable risk of deleterious non-cancer eects during a lifetime) was set by EPA at 0.75 mg/kg/day (52.5 mg/day for a 70-kg human)
DEP is an important solvent and vehicle for fragrance and cosmetic ingredients Thus, there is potential exposure
to humans by the intentional application of such pro-ducts to the skin A survey of fragrance manufacturers conducted in 1995±1996 by RIFM reported an annual use of approximately 4000 metric tons of DEP in the preparation of fragrance mixtures A conservative method for estimating dermal exposure assumes that an individual would repeatedly use all types of cosmetic products, each product containing the chemical of concern
at the 97.5 percentile of use (Ford et al., 2000) A survey
of over 2000 perfume compounds intended for hydro-alcoholic cosmetic products reported a 97.5 percentile of use for DEP of 28.6% (International Fragrance Asso-ciation, Geneva, 1999, pers commun.) This use level applied to the conservative method estimates a potential exposure of approximately 44 mg/day or 0.73 mg/kg/ day DEP can also be found in cosmetic products that contain ethanol denatured with DEP It is usually used
at a concentration of 0.5% to denature ethanol used for cosmetic products; in rare cases, it can be used up to 1% This use level applied to the conservative method estimates
a potential exposure of approximately 6 mg per day or 0.1 mg/kg/day (The European Cosmetic, Toiletry & Perfumery Association, COLIPA, pers commun., 2000)
18 Discussion and conclusions The popular use of DEP as a vehicle for fragrances is due not only to its favorable physicochemical properties but also because of its favorable toxicological pro®le as described in this review A particular advantage of DEP
is the safety when applied to the skin as is done inten-tionally with fragrances and cosmetic products Testing for dermal irritation and sensitization in both animals and humans, and for phototoxicity and photo-allergenicity in human volunteers has established the safe concentrations for use However even undiluted DEP has caused only slight to moderate irritation when tested on the skin of animals
Eects related to reproductive and developmental toxicity do not appear to be present with current expo-sures to DEP as evidenced in this report Concern for this type of toxic eect has been due to the embryotoxic and teratogenic eects of some members of the phthalic acid ester class of plasticizers, such as
Trang 10di(2-ethyl-hexyl) phthalate (DEHP), mono(2-ethyl-di(2-ethyl-hexyl) phthalate
(MEHP) and butylbenzyl phthalate (EPA, 1987; Field
et al., 1993)
The more complex phthalic acid esters such as DEHP
and butylbenzylphthalate have also been reported to
produce positive evidence for carcinogenicity in rats
and/or mice (NTP, 1995) The dermal carcinogenicity
studies conducted by NTP with DEP in rats and mice
are considered to be particularly relevant to safety
because of the signi®cant percutaneous absorption
through the skin of experimental animals (Elsisi et al.,
1989; RIFM, 1973b) The marginal increase of combined
hepatocellular adenomas and carcinomas in high-dose
male mice was considered to be an uncertain ®nding by
NTP due to the lack of signi®cant ®ndings in female
mice and the unusually low incidence of hepatocellular
adenomas in the control group of male mice It is
con-sidered reasonable that the weight of evidence from
both carcinogenicity and genotoxicity studies supports a
low level of concern for carcinogenic hazard from DEP
under conditions of use
It is concluded that the potential for dermal exposure to
DEP, based on use data with conservative assumptions
about maximal use patterns, is within the levels of
exposure deemed safe by other routes of exposure and is
not considered to present any signi®cant toxic liability
for its current uses as a solvent and vehicle in cosmetic
products
Acknowledgements
The author is grateful to Drs Arvind Agarwal and Emil
P®tzer and Ms Jennifer Cocchiara for their assistance in
developing this manuscript
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