Food and Chemical Toxicology 39 (2001) 97108 www.elsevier.

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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 ingredients 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, estrogenic 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/m3 in air) and consumer exposures (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

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. Introduction ..........................................................................................................................................................98 Acute toxicity ........................................................................................................................................................98 Eye irritation in rabbits .........................................................................................................................................98 Dermal irritation in animals..................................................................................................................................99 Dermal irritation in humans..................................................................................................................................99 Dermal sensitization in animals ............................................................................................................................99 Dermal sensitization in humans ..........................................................................................................................100 Potential phototoxicity in humans ......................................................................................................................100 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 0278-6915/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0278-6915(00)00124-1

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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

1. Introduction The diethyl ester of phthalic acid (DEP; CAS No. 8466-2) 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 current 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).

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 Blumenthal, 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 following 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 chemosis 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).

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 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

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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 treatments 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).

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 sensitization was observed (RIFM, 1978e). Buehler (1996) has observed that the guinea pig maximization 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.

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/cm2 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, Skin2 (Advanced Tissue Sciences, Inc.), to DEP caused marked cytotoxicity to the skin cells. This model is a living, human three-dimensional tissue substrate

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

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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 different 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 dermatitis 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 (Meynadier et al., 1986), in a 35-year-old teacher with a history 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).

erythema dose (MED) for each volunteer was determined 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 previous dose. The MED was the smallest dose that produced redness at evaluation 24 h after the irradiation. Photoirritation was evaluated after application of a single 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 10 min followed by UVB irradiation at 0.75 MED. Reactions at the site were evaluated 1, 24, 48 and 144 h after irradiation 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 photoirritation 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 irradiations, the volunteers had a 2-weeks rest period without 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/cm2 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 14C-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 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 of 14C-DEP in vitro through rat dorsal skin was 33% with occlusion and 37% without occlusion, whereas average absorption in human breast skin in vitro was 3.5% under occlusion and 4.7%

8. Potential phototoxicity in humans No phototoxicity or photoallergenicity was observed with 25% DEP in ethanol applied to the skin. The minimum

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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/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 14CDEP (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 14CDEP to rats and mice resulted in maximum concentrations 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, respectively, 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 metabolite 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 presence of DEP (Gray et al., 1983). Male rats fed 2% DEP in their diet for 3 wk showed marginal hepatic peroxisome 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 dermal 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 B6C3F1 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 signicant abnormal histological ndings) in female rats at

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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 toxicity 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 statistically signicant 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 signicant 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-6phosphatase 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 hypertrophy 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 skeletal 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 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 produce teratogenic eects on fetuses under those conditions (Tanaka et al., 1987). Pregnant CD-1 mice received DEP at an estimated LD10 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 considered irrelevant for this review because of the inappropriate 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 continuous 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

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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 middose 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 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 Sertoli 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 dissolved in corn oil (1600 mg/kg) did not cause testicular atrophy or aect testicular cytochrome P-450 or steroidogenic 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 morphology by light microscopy. Ultrastructural examination of Leydig cells showed mitochondrial swelling and focal dilatation of smooth endoplasmic reticulum. LHstimulated 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, linearity, 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

Crisco1 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 treatment 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 signicant 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 (108104 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 3H-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 105 m DEP with the estrogen-responsive human breast cancer cell line MCF-7. They also reported

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an extremely weak estrogenic activity using yeast cells with the human estrogen receptor. Activity was observed only at concentrations greater than 104 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 receptors. 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 concentration 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 Salmonella 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 administration 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 B6C3F1 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 marginal 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-13acetate) or to promote tumorigenesis following administration of a known initiator (DMBA: 7,12-dimethylbenz[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 treatmentrelated eects on hemocytology, blood sugar, non-protein nitrogen levels or urinalyses were observed. Postmortem 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).

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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: 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 90 mg/l; and algae 96 hour median eective concentration (EC50), 30 to 86 mg/l, and is not likely to bioconcentrate. DEP undergoes rapid degradation by bacteria commonly 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).

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 products 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 hydroalcoholic cosmetic products reported a 97.5 percentile of use for DEP of 28.6% (International Fragrance Association, 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 intentionally with fragrances and cosmetic products. Testing for dermal irritation and sensitization in both animals and humans, and for phototoxicity and photoallergenicity 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 exposures 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-

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 Governmental Industrial Hygienists, the Occupational Safety and Health Administration and the National Institute for Occupational Safety and Health. For a worker breathing 10 m3 of air during an 8-h workday for his working lifetime, this would represent acceptable exposure to inhalation of 50 mg DEP each workday. The

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A.M. Api / Food and Chemical Toxicology 39 (2001) 97108 Benson, W.H., Stackhouse, R.A., 1986. Evaluation of a new approach to the safety assessment of biomaterials. Drug and Chemical Toxicology 9, 275283. Blickensdorfer, P., Templeton, L., 1930. A study of the toxic properties 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., tabolisme des phtalates de dibutyle Bringuier, J., 1971. Etude du me thyle chez le rat. Comptes Rendus des Se ances de L'Acaet du die mie des Sciences 273, 21652168. de 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; Community 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 produced 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

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 considered 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
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