Review

Toxicological prole 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, carcinogenicity, in vitro toxicity, ecotoxicity, environmental fate and potential
human exposure. No toxicological endpoints of concern have been identied. Comparison of estimated exposure (0.73 mg/kg/day)
from dermal applications of fragrances and cosmetic products with other accepted industrial (5 mg/m
3
in air) and consumer expo-
sures (350 mg/l in water; 0.75 mg/kg/day oral exposure) indicates no signicant 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.
PI I : S0278- 6915( 00) 00124- 1
Food and Chemical Toxicology 39 (2001) 97108
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 dermal exposure...................................................................................101
10.2. Subchronic toxicity in animals 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
1. 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 298

C, a specic gravity
of 1.12 (20

C) and a vapor pressure of <0.001 torr

(20

C). 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 signicant technical advantage as a
vehicle for fragrance and cosmetic products. DEP has
many industrial uses, but this toxicological prole will
emphasize its safety in use as a solvent and vehicle for
fragrance and cosmetic ingredients.
Important reviews of the toxicological prole 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 131 g/kg by the oral route and
15 g/kg by the intraperitoneal route in mice, rats, guinea
pigs, rabbits and chickens. Intravenous doses in the
range of 0.070.3 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 signicantly 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
98 A.M. Api / Food and Chemical Toxicology 39 (2001) 97108
DEP. 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 (20 times) 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/cm
2
of undiluted DEP once daily
for 10 days in an occluded patch test on 10 healthy
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, Skin
2
(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-specic 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
A.M. Api / Food and Chemical Toxicology 39 (2001) 97108 99
recorded. 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 20 perfume-sensitive patients and
50 control 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 (30 with skin lesions and 30
without skin lesions) in the shoe factory and 30 normal
unexposed subjects were patch tested with several dif-
ferent phthalates. No eects due to DEP were found in
normal subjects, but 1/30 workers with dermatitis and
1/30 workers 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 1/30 workers with derma-
titis and 4/30 workers without dermatitis (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 2448-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 70 dermatitis 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/cm
2
of UVA irradiation within 10 min
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 10 min 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/cm
2
of 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
Signicant percutaneous absorption has been reported
for DEP. A single dermal application of
14
C-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, 00.5% of the dose
(Elsisi et al., 1989). Application of
14
C-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 of
14
C-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%
100 A.M. Api / Food and Chemical Toxicology 39 (2001) 97108
without 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/cm
2
/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 0.0020.006%) of the applied dose was
found in the kidneys and 0.004% (range 0.0010.006%)
in the liver (RIFM, 1973b). Oral administration of
14
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 20 min, 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 90 and 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 specic 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 puried 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 clobrate-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 SpragueDawley
males treated with phenobarbital intraperitoneally for 3
days, had no eect on cytochrome P450 or 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 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 15 ml/kg. In a
2-week dermal study, undiluted DEP was applied to
male and female SpragueDawley 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
B6C3F
1
mice received dermal applications of undiluted
DEP, 12.5, 25, 50 or 100 ml (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, 150 or 300 ml (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 150 and 300 ml females. Increased
relative kidney weights were seen in 150 and 300 ml
males and in 150 ml females. There were no clinical signs
of toxicity and no dermatotoxicity (NTP, 1995).
10.2. Subchronic toxicity in animals oral exposure
Toxic signs after 16 weeks of exposure to DEP in the
diet consisted of an increase in liver weight (without sig-
nicant abnormal histological ndings) in female rats at
A.M. Api / Food and Chemical Toxicology 39 (2001) 97108 101
doses 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 denite histopathologic
damage in the liver and kidneys at the highest dose and
questionable changes at 250 and 500 mg/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 signicant 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,
750 or 3710 mg/kg/day for females) DEP to male and
female SpragueDawley 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 signicant eects on water intake, or on the
results of the hematological examinations, serumenzyme
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-
nicant 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 showa 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 615; the rats were sacriced 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 fromrats 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 0 to 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
LD
10
dose of 4500 mg/kg/day by gavage, once daily on
gestation days 613 and were allowed normal delivery.
Two of the 50 pregnant 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
102 A.M. Api / Food and Chemical Toxicology 39 (2001) 97108
cohabitation 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 F
1
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 signicant 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-450 or 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 signicance 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 (018
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
(4550% of control eggs injected with sesame or
Crisco
1
oil, respectively died). One of the 10 eggs 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 10 immature female Wistar [Crl(WI)BR]
rats received a single oral dose of 0 (vehicle control), 50,
150 or 500 mg/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 sacriced, 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
signicant 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-
nicant 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 displace
3
H-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
5
m DEP with the estrogen-responsive
human breast cancer cell line MCF-7. They also reported
A.M. Api / Food and Chemical Toxicology 39 (2001) 97108 103
an extremely weak estrogenic activity using yeast cells
with the human estrogen receptor. Activity was
observed only at concentrations greater than 10
4
m (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 50 mg/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 prole 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
B6C3F
1
mice (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 signicant 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 signicant 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 1068%), 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 initiationpromotion 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 sacriced rats revealed no
unusual pathology, either gross or microscopic, which
appeared to bear any relation to the DEP in the diet
(RIFM, 1955).
104 A.M. Api / Food and Chemical Toxicology 39 (2001) 97108
15. 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 prole 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:
EPAhas 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 (LC
50
), 12 to 100
milligrams/liter (mg/l); daphnid 48 hour LC
50
, 50
to 90 mg/l; and algae 96 hour median eective
concentration (EC
50
), 30 to 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/m
3
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 10 m
3
of air during an 8-h workday for his
working lifetime, this would represent acceptable expo-
sure to inhalation of 50 mg DEP each workday. The
ambient water quality criteria for protection of human
health established by the US Environmental Protection
Agency (EPA) is 350 mg/l, or a dose of 350 mg 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 19951996 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 prole 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 di(2-ethyl-
A.M. Api / Food and Chemical Toxicology 39 (2001) 97108 105
hexyl) phthalate (DEHP), mono(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 signicant 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 signicant 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 signicant 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
Ptzer and Ms Jennifer Cocchiara for their assistance in
developing this manuscript.
References
Adams, W.J., Biddinger, G.R., Robillard, K.A., Gorsuch, J.W., 1995.
A summary of the acute toxicity of 14 phthalate esters to repre-
sentative aquatic organisms. Environmental Toxicology and Chem-
istry 14, 15691574.
Api, A.M., 1997. In vitro assessment of phototoxicity. In Vitro Tox-
icology 10, 339350.
Ashour, M-B.A., Moody, D.E., Hammock, B.D., 1987. Apparent
induction of microsomal carboxylesterase activities in tissues of
clobrate-fed mice and rats. Toxicology and Applied Pharmacology
89, 361369.
ATSDR, 1995. Toxicological Prole for Diethyl Phthalate. Agency for
Toxic Substances and Disease Registry, Division of Toxicology/
Toxicology Information Branch. 1600 Clifton Road NE, E-29,
Atlanta, GA 30333, USA.
Augustinsson, K.-B., Ekedahl, G., 1962. On the specicity of aryles-
terases. Acta Chemica Scandinavica 16, 240241.
Balaguer, P., Gillesby, B.E., Wu, Z.F., Meek, M.D., Annick, J.,
Zacharewski, T., 1996. Assessment of chemicals alleged to possess
estrogen receptor-mediated activities using in vitro recombinant
receptor/reporter gene assays. Fundamental and Applied Toxicol-
ogy 30 (Suppl.), 143.
Benson, W.H., Stackhouse, R.A., 1986. Evaluation of a new approach
to the safety assessment of biomaterials. Drug and Chemical Tox-
icology 9, 275283.
Blickensdorfer, P., Templeton, L., 1930. A study of the toxic proper-
ties of diethylphthalate. Journal of the American Pharmaceutical
Association 19, 11791181.
Bower, R.K., Haberman, S., Minton, P.D., 1970. Teratogenic eects
in the chick embryo caused by esters of phthalic acid. Journal of
Pharmacology and Experimental Therapeutics 171, 314324.
Brown, D., Butterworth, K.R., Gaunt, I.F., Grasso, P., Gangolli,
S.D., 1978. Short-term oral toxicity study of diethyl phthalate in the
rat. Food and Cosmetics Toxicology 16, 415422.
Buehler, E.V., 1996. Nonspecic hypersensitivity: false-positive
responses with the use of Freund's complete adjuvant. Contact
Dermatitis 34, 111114.
Chambon, P., Riotte, M., Daudon, M., Chambon-Mougenot, R.,
Bringuier, J., 1971. Etude du me tabolisme des phtalates de dibutyle
et du die thyle chez le rat. Comptes Rendus des Se ances de L'Aca-
de mie des Sciences 273, 21652168.
CIR, 1985. Cosmetic Ingredient Review. Final report on the safety
assessment of dibutyl phthalate, dimethyl phthalate, and diethyl
phthalate. Journal of the American College of Toxicology 4, 267303.
de Groot, A.C., Liem, D.H., 1983. Facial psoriasis caused by contact
allergy to linalool and hydroxycitronellal in an after-shave. Contact
Dermatitis 9, 230232.
de Groot, A.C., Weyland, J.W., 1992. Systemic contact dermatitis
from tea tree oil. Contact Dermatitis 27, 279280.
de Leeuw, J., den Hollander, P., 1987. A patient with a contact allergy
to jogging cream. Contact Dermatitis 17, 260261.
Draize, J.H., Woodard, G., Calvery, H.O., 1944. Methods for the
study of irritation and toxicity of substances applied topically to the
skin and mucous membranes. Journal of Pharmacology and
Experimental Therapeutics 82, 377390.
Elsisi, A.E., Carter, D.E., Sipes, I.G., 1989. Dermal absorption of
phthalate diesters in rats. Fundamental and Applied Toxicology 12,
7077.
EPA, 1978. Chemical Hazard Information Prole; Draft Report;
Alkyl Phthalates. US Environmental Protection Agency, Oce of
Toxic Substances, Oce of Pesticide and Toxic Substances,
Washington, DC.
EPA, 1981. An Exposure and Risk Assessment for Phthalate Esters. US
Environmental Protection Agency, Oce of Water Regulations and
Standards, WH-553, Washington, DC. EPA/440/4-81-020.
EPA, 1987. Health and Environmental Eects Prole for Phthalic acid
Esters. US Environmental Protection Agency NTIS Oce of Toxic
Substances, Oce of Pesticide and Toxic Substances, Washington,
DC.
EPA, 1996. Diethyl phthalate; toxic chemical release reporting; Com-
munity right-to-know. Federal Register 61, 3935639359 (40 CFR
Part 372, 29 July).
Farli, M., Gasperini, M., Francalanci, S., Gola, M., Sertoli, A., 1990.
Occupational contact dermatitis in 2 dental technicians. Contact
Dermatitis 22, 282287.
Field, E.A., Price, C.J., Sleet, R.B., George, J.D., Marr, M.C., Myers,
C.B., Schwetz, B.A., Morrissey, R.E., 1993. Developmental toxicity
evaluation of diethyl and dimethyl phthalate in rats. Teratology 48,
3344.
Ford, R.A., Domeyer, B., Easterday, O., Maier, K., Middleton, J.,
2000. Criteria for development of a database for safety evaluation of
fragrance ingredients. Regulatory Toxicology and Pharmacology
31, 166181.
Foster, P.M.D., Thomas, L.V., Cook, M.W., Gangolli, S.D., 1980.
Study of the testicular eects and changes in zinc excretion pro-
duced by some n-alkyl phthalates in the rat. Toxicology and
Applied Pharmacology 54, 392398.
Foster, P.M.D., Thomas, L.V., Cook, M.W., Walters, D.G., 1983.
Eect of di-n-pentyl phthalate treatment on testicular steroidogenic
106 A.M. Api / Food and Chemical Toxicology 39 (2001) 97108
enzymes and cytochrome P-450 in the rat. Toxicology Letters 15,
265271.
Fredricsson, B., Mo ller, L., Pousette, A

., Westerholm, R., 1993.

Human sperm motility is aected by plasticizers and diesel particle
extracts. Pharmacology and Toxicology 72, 128133.
Gollamudi, R., Lawrence, W.H., Rao, R.H., Autian, J., 1985. Eects
of phthalic acid esters on drug metabolizing enzymes of rat liver.
Journal of Applied Toxicology 5, 368371.
Gray, T.J.B., Butterworth, K.R., 1980. Testicular atrophy produced
by phthalate esters. Archives of Toxicology 4 ( Suppl), 452455.
Gray, T.J.B., Gangolli, S.D., 1986. Aspects of the testicular toxicity of
phthalate esters. Environmental Health Perspectives 65, 229235.
Gray, T.J.B., Beamand, J.A., Gangolli, S.D., 1982. Eects of phtha-
late esters on rat testicular cell cultures and on Sertoli cell function
in the intact testis. The Toxicologist 2, 78.
Gray, T.J.B., Lake, B.G., Beamand, J.A., Foster, J.R., Gangolli, S.D.,
1983. Peroxisomal eects of phthalate esters in primary cultures of
rat hepatocytes. Toxicology 28, 167179.
Greif, N., 1967. Cutaneous safety of fragrance material as measured by
the maximization test. American Perfumer and Cosmetics 82, 5457.
Guerra, L., Vincenzi, C., Peluso, A.M., Tosti, A., 1993. Prevalence
and sources of occupational contact sensitization to acrylates in
Italy. Contact Dermatitis 28, 101103.
Hardin, B.D., Schuler, R.L., Burg, J.R., Booth, G.M., Hazelden,
K.P., MacKenzie, K.M., Piccirillo, V.J., Smith, K.N., 1987. Eva-
luation of 60 chemicals in a preliminary developmental toxicity test.
Teratogenesis, Carcinogenesis, and Mutagenesis 7, 2948.
Harris, C.A., Henttu, P., Parker, M.G., Sumpter, J.P., 1997. The
estrogenic activity of phthalate esters in vitro. Environmental Health
Perspectives 105, 802811.
Hayakawa, R., Matsunaga, K., Arima, Y., 1987. Airborne pigmented
contact dermatitis due to musk ambrette in incense. Contact Der-
matitis 16, 9698.
Heindel, J.J., Powell, C.J., 1992. Phthalate ester eects on rat Sertoli
cell function in vitro: Eects of phthalate side chain and age of ani-
mal. Toxicology and Applied Pharmacology 115, 116123.
Holness, D.L., Nethercott, J.R., 1997. Results of patch testing with a
specialized collection of plastic and glue allergens. American Jour-
nal of Contact Dermatitis 8, 121124.
Hotchkiss, S.A.M., 1998. Absorption of fragrance ingredients using in
vitro models with human skin. In: Frosch, P.J., Johansen, J.D.,
White, I.R. (Eds.), Fragrances. Benecial and Adverse Eects.
Springer-Verlag, Berlin, pp. 125135.
Hotchkiss, S.A.M., Mint, A., 1994. Metabolism of phthalic acid esters
during percutaneous absorption through rat and human skin in
vitro. Journal of Investigative Dermatology 102, 647.
Iijo, C., 1975. U

ber die beeinussing von sulfathiazol, phtha-

lylsulfathiazol und phthalsa ureestern auf die entwicklung des hu h-
nerembryos. Showa Igakkai Zasshi 35, 187201.
Ioku, T., Mukaide, A., Kitanaka, H., Sakagami, Y., Kamevama, T.,
1976. In vivo distribution of drugs. Labeled compounds. Yakuri To
Chiryo 4, 510514.
Ishihara, M., 1977. Problems of closed patch tests with ingredients of
cosmetic products. Journal of Japanese Cosmetic Science Society 1,
87102.
Jones, H.B., Garside, D.A., Liu, R., Roberts, J.C., 1993. The inuence
of phthalate esters on Leydig cell structure and function in vitro and
in vivo. Experimental and Molecular Pathology 58, 179193.
Jordan, W.P., Dahl, M.V., 1971. Contact dermatitis to a plastic sol-
vent in eyeglasses. Archives of Dermatology 104, 524528.
Kamrin, M.A., Mayor, G.H., 1991. Diethyl phthalate: A perspective.
Journal of Clinical Pharmacology 31, 484489.
Kawano, M., 1980. Toxicological studies on phthalate esters. 2.
Metabolism, accumulation and excretion of phthalate esters in rats.
Japanese Journal for Hygiene 35, 693701.
Kayano, Y., Watanabe, K., Matsunaga, T., Yamamoto, I., Yoshi-
mura, H., 1997. Involvement of a novel mouse hepatic microsomal
esterase, ES46.5K, in the hydrolysis of phthalate esters. Biological
and Pharmaceutical Bulletin 20, 749751.
Klecak, G., 1979. The open epicutaneous test (OET), a predictive test
procedure in the guinea pig for estimation of allergenic properties of
simple chemical compounds, their mixtures and of nished cosmetic
preparations. International Federation Societies Cosmetic Chemists,
18 September.
Klecak, G., Geleick, H., Frey, J.R., 1977. Screening of fragrance mate-
rials for allergenicity in the guinea pig: Comparison of four testing
methods. Journal of the Society of Cosmetic Chemists 28, 5364.
Lake, B.G., Phillips, J.C., Hodgson, R.A., Severn, B.J., Gangolli,
S.D., Lloyd, A.G., 1976. Studies on the hydrolysis in vitro of
phthalate esters by hepatic and intestinal mucosal preparations from
various species. Biochemical Society Transactions 4, 654655.
Lake, B.G., Phillips, J.C., Linnell, J.C., Gangolli, S.D., 1977. The in
vitro hydrolysis of some phthalate diesters by hepatic and intestinal
preparations from various species. Toxicology and Applied Phar-
macology 39, 239248.
Lamb IV, J.C., Chapin, R.E., Teague, J., Lawton, A.D., Reel, J.R.,
1987. Reproductive eects of four phthalic acid esters in the mouse.
Toxicology and Applied Pharmacology 88, 255269.
Larsen, W.G., 1975. Cosmetic dermatitis due to a perfume. Contact
Dermatitis 1, 142145.
Larsen, W.G., 1977. Perfume dermatitis: a study of 20 patients.
Archives of Dermatology 113, 623626.
Lawrence, W.H., Malik, M., Turner, J.E., Singh, A.R., Autian, J.,
1975. A toxicological investigation of some acute, short-term, and
chronic eects of administering di-2-ethylhexyl phthalate (DEHP)
and other phthalate esters. Environmental Research 9, 111.
Marsman, D.S., Herbert, R.A., Haseman, J.K., 1994. Dermal carci-
nogenesis studies of diethylphthalate (DEP) and dimethylphthalate
(DMP) in F344/N rats and B6C3F
1
mice, with initiation/promotion
studies in male CD-1 mice. Toxicologist 14, 302.
Mentlein, R., Butte, W., 1989. Hydrolysis of phthalate esters by pur-
ied rat and human liver carboxylesterases. Biochemical Pharma-
cology 38, 31263128.
Meynadier, J.-M., Meynadier, J., Peyron, J.-L., Peyron, L., 1986.
Formes cliniques des manifestations cutane es d'allergie au parfums.
Annales de Dermatologie et du Ve ne re ologie 113, 3139.
Mint, A., Hotchkiss, S.A.M., Caldwell, J., 1994. Percutaneous
absorption of diethyl phthalate through rat and human skin in vitro.
Toxicology in Vitro 8, 251256.
Moody, D.E., Reddy, J.K., 1978. Hepatic peroxisome (microbody)
proliferation in rats fed plasticizers and related compounds. Tox-
icology and Applied Toxicology 45, 497504.
Moody, D.E., Reddy, J.K., 1982. Serum triglyceride and cholesterol
contents in male rats receiving diets containing plasticizers and
analogues of the ester 2-ethylhexanol. Toxicology Letters 10, 379
383.
Morrissey, R.E., Lamb IV, J.C., Morris, R.W., Chapin, R.E., Gulati,
D.K., Heindel, J.J., 1989. Results and evaluations of 48 continuous
breeding reproduction studies conducted in mice. Fundamental and
Applied Toxicology 13, 747777.
Nethercott, J.R., Nield, G., Holness, D.L., 1989. A review of 79 cases
of eyelid dermatitis. Journal of the American Academy of Derma-
tology 21, 223230.
Neuhauser, E.F., Loehr, R.C., Malecki, M.R., Milligan, D.L., Dur-
kin, P.R., 1985. The toxicity of selected organic chemicals to the
earthworm Eisenia fetida. Journal of Environmental Quality 14,
383388.
NTP, 1984. Diethyl Phthalate: Reproduction and Fertility Assessment
in CD-1 Mice when Administered in the Feed. NTP-84-262.
National Toxicology Program, National Institute of Environmental
Health Sciences, Research Triangle Park, NC 27709, USA.
NTP, 1988. Developmental Toxicity Evaluation of Diethyl Phthalate
(CAS No. 84-66-2) Administered to CD Rats on gestational Days 6
through 15. NTP-88-336. National Toxicology Program, National
A.M. Api / Food and Chemical Toxicology 39 (2001) 97108 107
Institute of Environmental Health Sciences, Research Triangle
Park, NC 27709, USA.
NTP, 1995. Toxicology and Carcinogenesis Studies of Diethylphtha-
late in F344/N Rats and B6C3F
1
Mice (dermal studies) with Dermal
Initiation/Promotion Study of Diethylphthalate and Dimethylph-
thalate in Male Swiss (CD-1
1
) Mice. Technical Report Series No.
429. National Toxicology Program, PO Box 12233, Research Tri-
angle Park, NC 27709, USA.
O'Grady, D.P., Howard, P.H., Werner, A.F., 1985. Activated sludge
biodegradation of 12 commercial phthalate esters. Applied and
Environmental Microbiology 49, 443445.
Oishi, S., Hiraga, K., 1980. Testicular atrophy induced by phthalic
acid esters: eect on testosterone and zinc concentrations. Toxicol-
ogy and Applied Pharmacology 53, 3541.
Oliwiecki, S., Beck, M.H., Chalmers, R.J.G., 1991. Contact dermatitis
from spectacle frames and hearing aid containing diethyl phthalate.
Contact Dermatitis 25, 254265.
Peakall, D.B., 1975. Phthalate esters: Occurrence and biological
eects. Residue Reviews 54, 141.
Price, C.J., Sleet, R.B., George, J.D., Marr, M.C., Schwetz, B.A.,
Morrissey, R.E., 1989. Developmental toxicity evaluation of diethyl
phthalate (DEP) in CD
1
rats. Teratology 39, 473474.
Remaut, K., 1992. Contact dermatitis due to cosmetic ingredients.
Journal of Applied Cosmetology 10, 7380.
RIFM 1955. Toxicological Studies of Diethyl Phthalate. Submission
to FDA by Celanese Corporation of America, 23 December. Docu-
ment number 23199.
RIFM 1963. Eye Irritation Study of Diethyl Phthalate in Rabbits.
Unpublished report from International Flavors & Fragrances, Inc.,
23 September. Report number 14297.
RIFM 1964. Repeated Insult Patch Test of Diethyl Phthalate in
Humans Subjects. Unpublished report from International Flavors &
Fragrances, Inc., 3 April. Report number 14572.
RIFM 1968. Primary Irritation Patch Test on Diethyl Phthalate in
Human Subjects. Unpublished report from international Flavors &
Fragrances, Inc., 26 January. Report number 14298.
RIFM 1971. Repeated Insult Patch Test of Diethyl Phthalate in
Human Subjects. Unpublished report from International Flavors &
Fragrances, Inc., 29 July. Report number 14299.
RIFM 1973a. Report on the Primary Irritation Potential of DEP on
Human Volunteers. RIFM report number 1802, June 11A.
RIFM 1973b. Tissue Distribution and Excretion of Diethyl Phthalate
Following Percutaneous Administration to Female Albino Rabbits.
RIFM report number 9984, January 12.
RIFM 1974. Primary Skin Irritation Tests with Diethyl Phthalate in
Rabbits. Unpublished report from International Flavors & Fra-
grances, Inc., 10 December. Report number 14300.
RIFM 1978a. Acute Oral Toxicity (LD
50
) of Diethyl Phthalate in
Rats. Unpublished report from International Flavors & Fragrances,
Inc., 31 March. Report number 14303.
RIFM 1978b. Acute Dermal Toxicity (LD
50
) Study of Diethyl Phtha-
late in Albino Rats. Unpublished report from International Flavors
& Fragrances, Inc., 31 March. Report number 14302.
RIFM 1978c. Primary Eye Irritation Study in the Albino Rabbit.
Submission to EPA, Anonymous, 3 December. Document number
12327.
RIFM 1978d. Report on Human Maximization Studies. RIFM report
number 1698, June 5.
RIFM 1978e. Guinea Pig Sensitization (Buehler). Unpublished report
from International Flavors & Fragrances, Inc., 25 April. Report
number 14304.
RIFM 1983a. Investigation of Toxicity of Certain Plasticizers. Report 1.
Acute Toxicity to Small Animals. Submission to EPAby E.I. DuPont
de Nemours & Co., Inc., 4 February. Document number 12315.
RIFM 1983b. Investigation of Toxicity of Certain Plasticizers. Report
3. Chronic Toxicity to Small Animals. Submission to EPA by E. I.
DuPont de Nemours & Co., Inc., 4 February. Document number
12324.
RIFM 1984a. Acute Dermal Irritation Study. RIFM report number
1795, June 1.
RIFM 1984b. Results of Skin Irritation Tests on Diethyl Phthalate.
Submission to EPA by The Dow Chemical Company, 29 June.
Document number 12320.
RIFM 1985. Acute Dermal Irritation Study. RIFM report number
3099, June 1.
RIFM 1994. 2-Week Dermal Dose Range Finding Study in Rats.
RIFM report number 23238, February 24.
RIFM 1997a. Evaluation of Human Photoallergy by Repeated Insult
Patch Test. RIFM report number 30623, July 2.
RIFM 1997b. Evaluation of Human Photoallergy by Repeated Insult
Patch Test. RIFM report number 30624, July 2.
RIFM 1998. Evaluation of Phototoxicity in Humans. RIFM report
number 34768, December 8.
RIFM 1999a. Evaluation of Phototoxicity in Humans. RIFM report
number 34769, July 20.
RIFM 1999b. Diethyl Phthalate: Assessment of Oestrogenic Potential.
Unpublished report from Jones, P., Baker, V., 19 August. Report
No. 34981.
Rowland, I.R., Cottrell, R.C., Phillips, J.C., 1977. Hydrolysis of
phthalate esters by the gastro-intestinal contents of the rat. Food
and Cosmetics Toxicology 15, 1721.
Schulsinger, C., Mollgaard, K., 1980. Polyvinyl chloride dermatitis not
caused by phthalates. Contact Dermatitis 6, 477480.
Scott, R.C., Dugard, P.H., Ramsey, J.D., Rhodes, C., 1987. In vitro
absorption of some o-phthalate diesters through human and rat
skin. Environmental Health Perspectives 74, 223227.
Shibko, S.I., Blumenthal, H., 1973. Toxicology of phthalic acid esters
used in food-packaging material. Environmental Health Perspec-
tives 3, 131137.
Singh, A.R., Lawrence, W.H., Autian, J., 1972. Teratogenicity of
phthalate esters in rats. Journal of Pharmaceutical Sciences 61, 51
55.
Smith, E.L., Calnan, C.D., 1966. Studies in contact dermatitis. XVII.
Spectacle frames. Transactions of the St. John's Hospital Dermato-
logical Society 52, 1034.
Tabak, H.H., Quave, S.A., Mashni, C.I., Barth, E.F., 1981. Biode-
gradability studies with organic priority pollutant compounds.
Journal Water Pollution Control Federation 53, 15031518.
Takenaka, T., Hasegawa, E., Takenaka, U., Saito, F., Odaka, T. 1986.
Fundamental studies of safe compound perfumes for cosmetics.
Part 1. The primary irritation of compound materials to the skin.
Unknown Source, pp. 313329.
Tanaka, C., Siratori, K., Ikegami, K., Wakisaka, Y., 1987. A ter-
atological evaluation following dermal application of diethyl
phthalate to pregnant mice. Oyo Yakuri 33, 387392.
van Ketel, W.G., 1983. Sensitization to cis-3-hexenyl salicylate. Con-
tact Dermatitis 9, 154.
Vidovic, R., Kansky, A., 1985. Contact dermatitis in workers proces-
sing polyvinyl chloride plastics. Dermatosen in Beruf und Umwelt
33, 104105.
Yoshikawa, K., Tanaka, A., Yamaha, T., Kurata, H., 1983. Muta-
genicity study of nine monoalkyl phthalates and a dialkyl phthalate
using Salmonella typhimurium and Escherichia coli. Food and Che-
mical Toxicology 21, 221223.
Zou, E., Fingerman, M., 1997. Eects of estrogenic xenobiotics on
molting of the water ea. Daphnia magna. Ecotoxicology and
Environmental Safety 38, 281285.
Zou, E., Fingerman, M., 1999. Eects of exposure to diethyl phthalate,
4-(tert)-octylphenol, and 2,4,5-trichlorobiphenyl on activity of chit-
obiase in the epidermis and hepatopancreas of the ddler crab, Uca
pugilator. Comparative Biochemistry and Physiology Part C 122,
115120.
108 A.M. Api / Food and Chemical Toxicology 39 (2001) 97108