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Diethyl phthalate
140
Hazard assessment of Diethyl phthalate
[Diethyl phthalate, CAS No. 84-66-2]
Chemical name : Diethyl phthalate
Synonyms : 1,2-Benzenedicarboxylic diethylester, DEP
Molecular formula : C12H14O4
Molecular weight : 222.24
Structural formula :
C-OCH2CH3
C-OCH2CH3
O
O
Appearance : Colorless or white, oily liquid1)
Melting point : -40.5°C1)
Boiling point : 298°C1),
Specific gravity : 254d = 1.1201)
Vapor pressure : 0.28 Pa (20°C)1)
Partition coefficient : Log Pow = 2.47 (calculated value) 1)
Degradability : Hydrolyzability: No report.
Biodegradability: Easily biodegradable (BOD = 88%, 4 weeks)2)
Solubility : Water 1,000 mg/l (25°C)1)
Organic solvent Soluble in ketones, esters, alcohol, ether,
benzene, acetone and aromatic
hydrocarbons1)
Amount of production/import : 394 t in 1998 (manufactured: 371 t, imported: 23 t)3)
Usage : Plasticizer for cellulose acetate, methacrylate, vinyl acetate,
polystyrene resin, etc., perfume reservant1)
Applied laws andregulations : Industrial Safety and Health Law, Marine Pollution Prevention
Diethyl phthalate
141
Law
1) HSDB, 2001; 2) "Tsusansho Koho" (daily), 1975; 3) Ministry of International Trade and Industry,
1999
1. Toxicity Data
1) Information on adverse effects on human health
It was reported that exposure to diethyl phthalate (DEP) in combination with alcohol
resulted in tentative and moderate irritation of the eye and oral mucosa in the workers
engaged in manufacture of DEP, even though no irritation occurred in the surface of hands
and body contacting with oily DEP several times (Smith, 1924).
When a patch test was conducted in 30 people with contact dermatitis working in a
plant producing shoes from polyvinyl chloride (PVC) pellets containing dioctyl phthalate,
one of them was positive to DEP (none in the control group not engaging in the work),
suggesting a cross-sensitization. One of 30 workers without any dermatitis symptoms
was sensitive to DEP (Vidovic & Kansky, 1985).
Among 26 dialysis patients with renal disorder using dialyzers equipped with PVC
tubes, 2 hepatitis cases occurred. One was diagnosed as non-specific hepatitis while the
other was diagnosed as drug-induced hepatitis. When the polyvinyl chloride tubes were
perfused with saline, 10 - 20 mg (determined by UV) and 20 - 50 mg (determined by IR)
of DEP per 1 liter of purfusate was detected, suggesting the influence of DEP (Neergaard
et al., 1971).
2) Information on endocrine system and reproductive system
(1) in vitro results related to receptor binding (Attachment-1)
In an estrogen receptor binding assay using human estrogen receptor (ER) expressed
in Sf9/Baculovirus, homogenate of female Sprague Dawlay (SD) rat uterus, or
homogenate of human uterus and prostate, the affinity of DEP to ER was not observed up
to the concentration of 0.1 - 1 mM (Nakai et al., 1999; Blair et al., 2000; Paganetto et al.,
2000; CERI, 2001a).
No estrogenic activity of DEP was observed up to 1 mM in a reporter gene assay
conducted by yeast two-hybrid method (Nishihara et al., 2000). No estrogen responsive
Diethyl phthalate
142
sequence (ERE)-dependent gene transcription activity was noted in a reporter gene assay
using HeLa cell in which human estrogen receptor gene was introduced (CERI, 2001a).
Weak activity, however, was observed in a reporter gene assay using yeast in which human
estrogen gene was introduced (1/2,000,000 of E2)(Harris et al., 1997).
Proliferation of estrogen-responsive human breast cancer cells (MCF-7, ZR-75-1)
was not stimulated by DEP (Harris et al., 1997).
(2) in vivo results in mammals (Attachment-2)
In a uterotrophic assay, DEP at 7 daily subcutaneous doses of 0, 200, 600 or 2,000
mg/rat, exerted no effect on uterus weight in ovariectomized female SD rats (8 weeks old)
indicating no estrogenic activity. In addition, when DEP was similarly administered
together with 17 α-ethynylestradiol at 7 daily subcutaneous doses of 0.5 µg/kg body
weight to detect anti-estrogenic activity, no effect was observed on uterus weight (CERI,
2001b).
In Hershberger assay no effect was noted on weights of accessory reproductive
organs in castrated male SD rats (8 weeks old) by DEP at 10 daily gavage dose of 0, 200,
600 or 2,000 mg/rat, indicating no androgenic activity. Furthermore, when DEP was
similarly administered to rats together with testosterone propionate at 10 daily
subcutaneous dose of 0.4 mg/kg BW, no effect was again observed on weights of
accessory reproductive organs, indicating no anti-androgenic activity (CERI, 2001b).
When DEP at 0 and 1,600 mg/kg BW/day was administered by oral gavage to male
SD rats (5 weeks old) for 4 days, no effect was noted on progesterone binding to
microsomal membrane to induce testicular toxicity, or on enzymes activity (17-α-
hydroxylase, 17-20-lyase, 17-β-dehydrogenase) involved in progesterone-testosterone
metabolism (Foster et al., 1980; 1983). On the other hand, the testosterone levels in
serum and testes decreased by 7-day dietary administration of DEP at the concentrations
of 2% (corresponding to 2,000 mg/kg /day) in male Wistar rats (5 weeks old), whereas
testis weights or serum dihydrotestosterone levels were not altered(Oishi & Hiraga, 1980a;
1980b).
DEP at 10 daily gavage of 0 or 1,596 mg/kg neither induced testicular atrophy nor
affected accessory reproductive organ weights in Wistar male rats (4 weeks old) (Gray &
Butterworth, 1980).
Diethyl phthalate
143
In a fertility test (F0 generation) by continuous mating between CD-1 male and
female mice (7 days old) (20/sex/group for the dose groups and 40/sex for control group)
under dietary administration of DEP at 0, 0.25, 1.25 and 2.5% (corresponding to 0, 370,
1,942 and 3,742 mg/kg /day, respectively), the fertility was 100% in each group without
any effect by DEP on dams delivery, pups body weight at the time of birth or their sex
ratio (Lamb et al., 1987). When the final delivered pups (F1 generation) of 0 or 2.5%
group (no DEP administration during nursing period) were mated within the group
(20/group/sex) from day 74 (± 10) of birth onward, the number of surviving offspring at
birth decreased in F2 generation despite no difference in dam fertility. Body weights of F1
females in the dosing groups significantly decreased at the time of autopsy. In addition,
increased prostate weights and decreased sperm concentrations were noted in F1 males,
while increased liver weight and decreased pituitary weight were noted in F1 females
(Lamb et al., 1987).
In a teratogenicity test using female SD rats administered DEP at the dietary
concentrations of 0, 0.25, 2.5 and 5% (corresponding to 0, 198, 1,909 and
3,215 mg/kg/day) from days 6 to 15 of gestation, no effect was observed on uterus weights
as well as the numbers of corpus luteum, implantations, fetal death or surviving offspring
per dam , or fetal body weights or sex ratio. The incidence of supernumerary ribs in 5%
group was 21%, significantly higher than 8.8% in the control group (Field, et al., 1993).
When DEP was subcutaneously administered to female ICR mice at the doses of 0,
500, 1,650 and 5,600 mg/kg /day from day 0 to 17 of gestation, thymus and spleen
weights decreased in dams of all dosed groups. Also pituitary weight decreased, while
adrenal and spleen weights increased in 5,600 mg/kg /day group. On the other hand,
body weight decreased, while the incidences of cervical and lumbar ribs increased in
fetuses of the 5,600 mg/kg /day group(Tanaka et al., 1987).
When DEP at the doses of 0, 570, 1,130 and 1,890 mg/kg was intraperitoneally
administered to female SD rats on days 5, 10 and 15 of gestation, there was no difference
in fertility among groups, but fetal body weights decreased in all dosed groups with
increase of incidence of skeletal variation and delayed ossification (Singh et al., 1972).
3) Information on general toxicity
(1) Acute toxicity (Table 1) (German Chemical Society, 1994; AGGIH, 1991)
Diethyl phthalate
144
Table 1 shows the LD50 and LC50 values of DEP in mice, rats and rabbits in various
administration routes. The acute symptoms observed in rats, rabbits, dogs and chicks
after oral or intravenous administration were increased respiration rate, dysequilibrium,
convulsion, lethargy and respiratory arrest. After intraperitoneal administration to mice,
pulmonary congestion, edema and petechial bleeding as well as degeneration of renal
tubule were observed. After inhalation of DEP vapor at 511 ppm for 6 hours (prepared
by passing DEP heated at 150°C through the air), no death occurred in rats despite
transient vascular dilation in ears and feet, disappearing after 14 days. After inhalation of
DEP vapor at 1,100 ppm for 5 hours, irritation in the nose was noted in cats.
Table 1 Acute toxicity test results
Mice Rats RabbitsOral LD50 6,178-8,600 mg/kg BW* 9,168 – 31,000 mg/kg BW * 1,000 mg/kg BW **Inhalation LC50 － － －Percutaneous LD50 － － －Intraperitoneal LD50 2,749-3,220 mg/kg* 5,675 mg/kg －
*: Some differences between the reports ; **: with relatively low reliability on data
(2) Repeated-dose toxicity (Attachment-3)
When DEP at concentrations of 0 and 2% (corresponding to 0 and 2,000 mg/kg/day,
respectively) was dietarily administered for 3 weeks to male Fischer 344 (F344) rats (age
unknown), increased liver weight, decreased serum triglyceride level, increased hepatic
catalase and carnitin acetyltransferase activities and increased ratio of peroxisome to
mitochondria were observed in the dosed group (Moody & Reddy, 1978; 1982).
When DEP at concentration 0, 0.2, 1.0 and 5.0% (corresponding to 0, 150, 770 and
3,160 mg/kg /day in males, 0, 150, 750 and 3,710 mg/kg /day in females) was dietarily
administered to SD rats (age unknown), relative weights of liver, stomach, small intestine
and cecum increased in female 0.2% group, while suppression of body weight increase,
decreased food consumption (day 1 only) and increased relative weights of liver and small
intestine were noted in 1.0% female group. Relative weights of thyroid, adrenal and,
pituitary as well as heart increased in the male group. In both males and females, relative
weight of stomach increased in 1% group, suppression of body weight increase and
increased relative weights of brain, liver, stomach, kidney, small intestine and cecum were
noted in 5% group (Brown et al., 1978).
When DEP at concentration 0, 0.5, 2.5 and 5.0% in diet was administered to male
Diethyl phthalate
145
and female rats (age unknowm) for 2 years, body weight increase was inhibited in 5%
group (German Chemical Society, 1994).
When DEP at doses of 0, 12.5, 25, 50 and 100 µl/day (corresponding to 0, 468, 935,
1,870 and 3,740 mg/kg/day, respectively) was subcutaneously administered to male and
female B6C3F1 mice (6 weeks old) for 4 weeks, absolute and relative liver weights
increased in 25 and 100 µl/day female groups (NTP, 1993).
When DEP at doses of 0, 7.5, 15 and 30 µl/day/ (corresponding to 0, 193, 386 and
772 mg/kg /day, respectively) was subcutaneously administered to male and female
B6C3F1 mice (6 weeks old) for 103 weeks, kidney weights increased in female 15 and 30
µl/day groups (NTP, 1993).
When DEP at doses of 0, 37.5, 75, 150 and 300 µl/day (corresponding to 0, 214, 429,
858 and 1,715 mg/kg/day, respectively) was subcutaneously administered to male and
female F344 rats (6 weeks old) for 4 weeks, liver weights increased in female 150 and 300
and male 300 µl/day groups. The kidney weight increased in female 150 and male 150 and
300 µl/day groups (NTP, 1993).
When DEP at doses of 0, 100 and 300 µl/day (corresponding to 0, 285 and
855 mg/kg /day, respectively) was subcutaneously administered to male and female F344
rats (6 weeks old) for 104 weeks, mortality increased in all dosed males. Hematocrit value
(Ht), hemoglobin concentration (Hb) and number of erythrocytes (RBC) increased in
female 300 µl/day group. Mean body weight slightly decreased in male 300 µl/day group.
Degree of fatty liver dcreased by DEP in both males and females in a dose dependent
manner(NTP, 1993).
By inhalation of DEP to cats (strain, age and sex unknown) at concentration of 356
ppm for 6 hours/day (corresponding to 3,289 mg/kg/day) for 7 days, decreased activity,
vomiting, suppression of central nerve system, thirst and decreased appetite were observed
(BIBRA, 1994).
No macroscopic or histopathological abnormality was observed in liver of rats
(strain, age and sex unknown), intravenously administered DEP at the total amount of 40
mg (20 mg twice with an interval of 2 or 3 days, 10 mg x 4 times or 5 mg x 8 times)
(Neegaard et al., 1975).
Diethyl phthalate
146
4) Information on mutagenicity/genotoxicity and carcinogenicity
(1) Mutagenicity/genotoxicity (Table 2)
Weakly positive results were reported in a reverse mutation assay using Salmonella
typhimurium (TA100, TA1535) without metabolic activation (Agarwal et al., 1985;
Kozumbo et al., 1982; Rubin et al., 1979). However, negative result was obtained using
high purity DEP (99.7%) (German Chemical Society, 1998). Chromosome aberration
assay also gave negative results (Ishidate & Odashima, 1977; Omori, 1976; Tsuchiya &
Hattori, 1976). No reports have been available for in vivo mutation study for DEP.
Table 2 Results of mutagenicity/genotoxicity assays
Test method Cells and animal species used Result* References
Salmonella typhimurium TA98, TA100, TA1535,TA1539, S9(+/-), 10-10,000 µg/ml
－ Zeiger et al.,1982; 1985
Salmonella typhimurium TA98, TA1538, S9(+/-), 100 -2,000 µg/plate －
Agarwal et al.,1985
Salmonella typhimurium TA100, TA1535, S9(-), 100-2,000 µg/plate (TA100, negative with S9(+) to TA100and TA1535)
＋wAgarwal et al.,
1985
Salmonella typhimurium TA98, S9(+/-), 100-1,000µg/plate －
Kozumbo et al.,1982;
Rubin et al., 1979Salmonella typhimurium TA100, S9(-), 100-1,000µg/plate (negative with S9(+)) ＋w
Kozumbo et al.,1982;
Rubin et al., 1979
Reverse mutation test
Salmonella typhimurium TA98, TA100, TA1535,TA1537, S9(+/-), up to 5,000 µg/ml (minimumconcentration unknown)
－German
ChemicalSociety, 1998
Bacillus subtilis recA(-S9), H17(+S9), M45(-S9), 10mg/plate －
Omori, 1976DNA repair test
Escherichia coli uvrA(-S9), PolA(-S9), recA(-S9),10 mg/plate －
Omori, 1976;Sato et al., 1975
CHL cell (-S9), 0.125-0.5 mg/ml－
Ishidate &Odashima, 1977;
Omori, 1976
In vitro
Reverse mutation test
Human leukocyte, 0.03 mg/ml－
Tsuchiya &Hattori, 1976
*－: negative ＋: positive ＋w: weak positive
(2) Carcinogenicity (Table 3 and Attachment-4)
When DEP at the doses of 0, 7.5, 15 and 30 µl/day (corresponding to 0, 193, 386
and 772 mg/kg/day, respectively) was subcutaneously administered to male and female
B6C3F1 mice (6 weeks old) for 103 weeks, the incidence of hepatocellular adenoma as
well as total hepatocellular tumors (adenoma and cercinoma) increased in 7.5 and
15 µl/day groups but without relation to dose (7/50, 16/50, 19/50 and 12/50 in the 0, 7.5,
15 and 30 µl/day groups, respectively). On the other hand, the incidence of basophilic
Diethyl phthalate
147
foci, increased in the male 15 µl/day group but again no relation to dose. Although in
males incidence of total hepatocellular tumors (adenoma and carcinoma) increased in a
dose-dependent manner with statistical significace in 30 µl/day group, the relation with
doseages was questionable, because the control value was too low (9/50, 14/50, 14/50 and
18/50 in the 0, 7.5, 15 and 30 µl/day groups, respectively) (NTP, 1993).
When DEP at doses of 0, 100 and 300 µl/day (corresponding to 0, 285 and
855 mg/kg BW/day, respectively) was subcutaneously administered to male and female
F344 rats (6 weeks old) for 104 weeks, incidence of mammary fibroma decreased in a
dose-dependent fashion in female 100 and 300 µl/day groups, while acanthosis occurred in
the administration site of skin in female 100 and male and female 300 µl/day groups (NTP,
1993).
No information is available for the carcinogenicity in human.
Table 3 Carcinogenicity assessment by national and international organizations
Organizations Classifica-tion
Significance References
EPA Group D Not classifiable as a human carcinogen IRIS, 2002EU － No evaluation. ECB, 2000NTP － No evaluation. NTP, 2000IARC － No evaluation. IARC, 2001ACGIH A4 Not classifiable as a human carcinogen ACGIH, 2001Japan Society forOccupational Health － No evaluation.
Japan Society forOccupational Health, 2001
5) Information on immune system
No information is available for the effect of this substance on the immune system.
6) Fate and Metabolism
The absorption of DEP is relatively quick, from the skin and digestive tract.
When 157 µmole (34.9 mg/kg BW) of DEP labeled with 14C in benzene ring was
subcutaneously applied to in the shaved back (1.3 cm in diameter) of male F344 rats, 24,
35 and 50% were recovered in urine and feces 24, 48 and 168 hours thereafter,
respectively (Elsisi et al., 1989). When DEP at 100 mg/rat was orally administered,
about 60% was recovered in urine within 24 hours (Kawano, 1980).
When 14C-labeled DEP at benzene ring was percutaneously applied to the back skin
Diethyl phthalate
148
of F344 male rats, radioactivity was detected in fatty tissue (0.03%), muscle (0.14%) and
skin (0.06%) as well as other organs and tissues (<0.5%), including brain, lung, liver,
spleen, small intestine, kidney, testis, spinal cord and blood, 168 hours after the
administration (Elsisi et al., 1989). DEP was distributed in lung, heart, liver, kidney,
gonad, spleen and brain also after gavage of 100 mg/rat (Autian, 1973).
In Wistar rats given DEP at gavage of 100 mg/rat, phthalic acid (49.95%) and
monoethyl phthalate (7.95%) were detected in urine (Kawano, 1980) (Fig. 1). It was
reported in an in vitro metabolism study that DEP was hydrolyzed by carboxyesterase in
human liver and in liver and small intestine of rat, ferret and baboon to generate
monoethyl phthalate (Mentlin & Butte, 1989; Lake et al., 1977).
(1) (2) (3)
COOH
COOC2H5
COOC2H5
COOC2H5
COOH
COOH
(1) Diethyl phthalate (DEP) (2) Monoethyl phthalate (3) Phthalic acid
Fig. 1 Metabolic pathway of diethyl phthalate
2. Hazard Assessment at present
No information is avaliable for the effect of DEP on human endocrine system and
reproductive system.
Most of in vitro experiments conducted to investigate the effect of DEP on endocrine
system indicated negative binding capability to ER. No estrogenic or androgenic actions
were detected in in vivo uterotrphic and Hershberger assays. It will thus be unlikely that
DEP exerts endocrine disrupting activity.
As for the effect on reproductive system, there are reports denying the effect of DEP
on testis and accessory reproductive organs. DEP did not demonstrate any reproductive or
developmental toxicity. On the other hand, DEP has been reported to exert harmful
effects on body weight, prostate weight, pituitary weight and sperm concentration in the
offspring, at such high dose as 3,742 mg/kg/day. However, it is difficult to conclude that
there are sufficient scientific findings to evaluate effects of DEP on fertility and
development of offspring in multi-generations.
As information on hazardous properties, DEP vapor was reported to irritate human
Diethyl phthalate
149
eye, skin and respiration system and to suggest sensitization. Its acute toxicity in
experimental animals was weak, but orally and subcutaneously DEP demonstrated effects
mainly on liver and kidney in repeated toxicity tests. Results of reverse mutation, DNA
repair and chromosome aberration assays indicate the negative mutagenicity for DEP. As
for carcinogenicity, hepatocellular adenoma and carcinoma was reported to be induced in
mice without dose-dependency. No information is available for carcinogenicity of DEP in
humans.
3. Risk assessment and other necessary future measures
On DEP 2-generation reproductive toxicity test is being conducted. DEP is
unlikely to have endocrine disruptive activity mediated by sex hormone receptors. But
there are not sufficient findings concerning effects on fertility and development of
offspring in multi-generations. The endocrine disrupting activity and related toxic effects
of DEP will be comprehensively evaluated by combining the results of the above test.
Diethyl phthalate
150
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phthalate esters. Hokkaido-ritsu Eiseikenkyushohou, 25, 146-147.
Singh, A.R., Lawrence, W.H., and Autian, J. (1972) Teratogenicity of phthalate Esters in Rats. J.
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Singh, A.R., Lawrence, W.H., and Autian, J. (1975) Maternal-fatal transfer of 14C-di-2-ethylhexyl
phthalate and 14C-diethyl phthalate in rats. J. Pharmaceut. Sci., 64, 1347-1350.
Smith, O. M. (1924) Toxic properties of diethylphthalate. J. Am. Pharm. Assoc., 13, 812.
Tanaka, C., Siratori, K., Ikegami, K., and Wakisaka, Y. (1987) A teratological evaluation following
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Tran, D.Q., Klotz, D.M., Ladlie, B.L., Ide, C.F., McLachlan, J.A., and Arnold, S.F. (1996)
Inhibition of progesterone receptor activity in yeast by synthetic chemicals. Biochem.
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Tsuchiya, K. and Hattori, K. (1976) Chromosomal study on human leucocytes cultures treated with
phthalate acid ester. Rep. Hokkaido Inst. Public Health, 26, 114.
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Zeiger, E., Haworth, S., Speck, W., and Mortelmas, K. (1982) Phthalate ester testing in the national
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ethylhexyl) phthalate and related chemicals in salmonella. Environ. Mutagen., 7, 213-232.
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investigation/research on environment-compatible technology development on behalf of the
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Diethyl phthalate
154
Attachment-1 Results of in vitro assays relating to receptor binding
Item Test methods and conditions Result Conclusion ReferencesMethod: Competitive binding testusing [3H]-E2 as ligand, Receptor:Human ER expressed withSf9/Baculovirus, Temperature:25°C, pH: 7.4Concentration: unknown
IC50: Unknown(E2: 2.09 × 10-9 M)
No binding Nakai et al.,1999
Method: Competitive binding testusing [3H]-E2 as ligand, Receptor:Uterus homogenate ofovariectomized SD rat,Temperature: 4℃ , pH: 7.4Concentration: 10-3 M (DEP)
IC50: >10-3 M(E2: 8.99 × 10-10 M)
No binding Blair et al.,2000
Method: Competitive binding testusing [3H]-E2 as ligand, Receptor:Cytoplasm fractionation obtainedby crushing frozen normal humanuterus, Temperature: 5℃, pH: 7.4,Concentration: 10-4 M (DEP)
Ligand bindinginhibition rate: <10%KD and Bmax of [3H]-E2
KD: 2.4 × 10-8 MBmax: 34.5 fmol/mgproteinDEP: >10-4 M
No binding Paganetto etal., 2000
ER bindingtest
Method:Human ER binding test(recombinant ER α ligand domain)
IC50: ＞10-4 M(E2: 1.3 × 10-9 M)
No binding CERI, 2001a
Yeast two-hybrid assay
Cells: Yeast transfected with Gal4DNA binding domain/human ERligand binding domain gene, Gal4activation domain/coactivator TIF2gene and β-galactosidase reportergene
REC10: >10-3 M(E2: 3 × 10-10 M)
No genetranscriptionactivity
Nishihara etal., 2000
Reportergene assayusingrecombinantyeast
Cells: Yeast in which human ERgene and lac-Z reporter gene werestably transfected.Concentration: 4.8 × 10-7 - 10-3 M(DEP), 4.8 × 10-12 - 10-8 M (E2)incubation: 4 - 6 days
Weak activity dependenton the conclusion wasdetected within the rangeof 10-4 - 10-3 M. Therelative maximumresponse (E2=100%) toE2 at 10-3 M DEP was30%.
Presence of genetranscriptionactivity(1/2,000,000 of E2)
Harris et al.,1997
Reportergene assayusingrecombinantcultured cell
Cell: HeLa cell transfected withhuman ER expression gene and ERresponse sequenceConcentration: 10-11 -10-5 MIncubation: 20 – 24 hr
Agonist activity wasnegative within the rangeof 10-11 - 10-5 M(E2: PC50: <10－11 M)
No genetranscriptionactivity
CERI, 2001a
Human breastcancer cellgrowth assay
Cells: Human breast cancer cells(MCF-7 and ZR-75 cells)MCF-7 cellConcentration: 10-5 M(DEP), 10-8
M(E2),Incubation: 11 daysZR-75-1 cellConcentration:10-5 M, 10-6 M, 10-7 M(DEP),10-8 M, 10-10 M, 10-12 M(E2)Incubation: 10 days
MCF-7 cell assay: Nosignificant growth wasobserved atconcentrations of 10-5 M.ZR-75 cell assay: Nosignificant growth wasobserved at 10-7 - 10-5 M.(Growth correspondingto E2 exposure wasobserved within therange of 10-12 - 10-8 M)
No cellproliferatingactivity
Harris et al.,1997
ER: Estrogen receptor; E2: 17β-estradiolREC10: Concentration corresponding to 10% of 10-7 M E2 activity level; PC50: Concentration
Diethyl phthalate
155
corresponding to 50% of the maximum activity by E2; IC50: Concentration to 50% inhibition by E2
Diethyl phthalate
156
Attachment-2 Results of in vivo assays for effects on endocrine and reproductive systems of
mammals
Animalspecies
Adminis-trationmethod
Administrati-on period
Dose Result References
0, 200, 600, 2,000mg/kg
No influence on weight of uterusRat (SD,female)6 rats/group;
Ovariectomized rats,
ovariec-tomy whenthey were6 weeks
old
s.c.(utero-trophicassay)
Administrat-ion for 7days whenthey were 8weeks old.The uteruswas extractedafter 24hours andweighed.
0, 200, 600, 2,000mg/kg+
17 α-ethynylestradiolat 0.5 µg/kg/day wassubcutaneouslyadministered
No influence on weight of uterus
CERI, 2001b
0, 200, 600, 2,000mg/kg/day
No influence on weights ofaccessory reproductive organs
Rat(SD, male)castrated
when theywere 6
weeks old
Oralgavage
(Hershb-ergerassay)
Administrat-ion for 10days whenthey were 8weeks old,autopsy after24 hours
0, 200, 600, 2,000mg/kg/day
+Testosteronepropionate (TP)0.4 mg/kg/day wassubcutaneouslyadministered
No influence on weights ofaccessory reproductive organs
CERI, 2001b
Rat(SD, male,
5 weeksold)
Oralgavage
4 days(sacrificed
afteradministrati-
on)
0, 1,600 mg/kg/day No influence on testicular tissue (noeffect on binding of microsomemembrane to progesterone).No influence on activity of enzymes(17-α-hydroxylase, 17-20-lyase, 17-β-dihydrogenase) that are involvedin progesterone-testosteronemetabolism
Foster et al.,1980; 1983
Rat(Wistar,5 weeks,
male)
Byfeeding
7 days 0, 2％(corresponding to 0,2,000 mg/kg/day)
No effect on testis weight or serumdihydrotestosteroneDecreased testosterone levels inserum and testis
Oishi &Hiraga,
1980a; 1980b
Rat(Wistaralbino,4 weeks
old, male)
Oralgavage
10 days 0, 1,596 mg/kg/day No testicular atrophy. No influenceon weights of accessory reproductiveorgans
Gray &Butterworth,
1980
Mouse(ICR,
female)
p.c. Day 0 - 17 ofgestation,sacrificed onday 18
0, 500, 1,650, 5,600 mg/kg/day
Thymus and spleen weightsdecreased in dams at≥ 500 mg/kg/dayPituitary weight decreased, andadrenal and spleen weightsincreased, without differences infertility, numbers of corpus luteum,implantations or surviving fetuses, orfatal male/female ratio as to thedams at 5,600 mg/kg/dayDecreased fetal body weight andincreased incidence of cervical ribsand lumbar ribs at 5,600 mg/kg/day
Tanaka et al.,1987
Diethyl phthalate
157
Animalspecies
Adminis-trationmethod
Administrati-on period
Dose Result References
Death occurred in 1 male in 1.25%group and in 2 males and 1 female in2.5% groupNo influence on fertility, deliveryfrequency, body weight of F1 at thetime of birth or sex ratio
By feedingContinuous mating test(F0 generation); Theadministration duringnursing period wasdiscontinued
Mouse(CD-1,
female andmale, 20
rat/group)(7 weeks
old)By feedingFertility test (F1
generation)
0, 0.25, 1.25, 2.5％(corresponding to 0,370, 1,942, 3,742 mg/kg/day)
No difference in fertilityDecreased number of survivingoffspring (F2 generation) at the timeof birthF1 males: Increased prostate weightand decreased sperm concentrationF1 females: Increased liver weightand decreased pituitary weight
Lamb et al.,1987
Rat(SD,
female)
Byfeeding
Day 6 - 15 ofgestation,sacrificed onday 20
0, 0.25, 2.5, 5％(corresponding to 0,198, 1,909, 3,215 mg/kg/day)
0.25%: Increased body weight ofdams (at the time of autopsy)2.5%: Decreased body weight ofdams (day 9 of gestation)5%: Decreased body weight of dams(day 9 - 18 of gestation and at thetime of autopsy)No difference in uterus weight,numbers of corpus luteum,implantations, resorption, deadfetuses or surviving fetuses, fetalbody weight or fetal sex ratio perdam5%: Increase in the incidence ofsupernumerary ribs of fetuses(21% against 8.8% in the control )
Field et al.,1993
Rat(SD,
female)
i.p. Day 5, 10, 15of gestation,sacrificed onday 20
0, 570, 1,130, 1,890mg/kg
No difference in fertility between thegroups.Decreased fetal body weight, andincreased incidence of skeletalvariation and delayed ossification at≥ 570 mg/kg/day
Singh et al.,1972
Diethyl phthalate
158
Attachment-3 Results of repeated dosing toxicity test
Animalspecies
Adminis-trationmethod
Administrati-on period
Dose Test method and result References
Rat(F344,male)(ageunknown)
Byfeeding
3 weeks 0, 2％(corresponding to0, 2,000mg/kg/day)
Increased liver weight, decreasedserum triglyceride, increasedhepatic catalase activity andcarnitin acetyltransferase activityand increased ratio of peroxisometo mitochondria
Moody &Reddy,
1978, 1982
Rat (SD,femaleandmale)(ageunknown)
Byfeeding
16 weeks 0, 0.2, 1.0, 5.0％(male:corresponding to0, 150, 770 3,160mg/kg/dayfemale:corresponding to0, 150, 750, 3,710mg/kg/day)
0.2%: Increased relative weights ofliver, stomach, small intestine andcecum in the females1%: suppression of body weightincrease, decreased foodconsumption (day 1 only) andincreased relative weights of liverand small intestine in females5%: Increased relative weights ofthyroid gland, adrenal, pituitaryand heart in males1%: Increased relative weight ofstomach in males and females5%: Inhibition on body weightincrease and increased relativeweights of brain, liver, stomach,kidney, small intestine and cecumin males and females (the increasein relative weight of cecum inmales was noted only when therewas some content)
NOEL: 150 mg/kg/day
Brown et al.,1978
Rat(femaleandmale)(strainand ageunknown)
Byfeeding
2 years 0, 0.5, 2.5, 5.0％ Suppression of on body weightincrease at 5.0%
GermanChemicalSociety,
1994
Dog(strain,age andsexunknown)
Byfeeding
1 years 0, 0.5, 1.5, 2.0,2.5％(equivalent to0, 114, 343, 500,629 mg/kg/day)
No influence GermanChemicalSociety,
1994
MouseB6C3F1(female
andmale)
(6 weeksold)
p.c. 4 weeks(5 day/week)
0, 12.5, 25, 50, 100µl/day/rat(equivalent to 0,468, 935, 1,870,3,740 mg/kg/day)
Increased absolute and relativeweights of liver in females at 25and 100 µl/day
NTP, 1993
Diethyl phthalate
159
Animalspecies
Adminis-trationmethod
Administrati-on period
Dose Test method and result References
MouseB6C3F1(female
andmale)
(6 weeksold)
p.c. 103 weeks(5 day/week)
0, 7.5, 15, 30µl/day/rat(equivalent to 0,193, 386, 772 mg/kg/day)
A slight increase in kidney weightin females at≥ 15 µl/day
NTP, 1993
RatF344
(femaleand
male)(6 weeks
old)
p.c. 4 weeks(5 day/week)
0, 37.5, 75, 150,300 µl/day/rat(equivalent to 0,214, 429, 858,1,715 mg/kg/day)
Increased liver weight in females at≥ 150 µl/day and in males at 300µl/dayIncreased kidney weight in thefemales at 150 µl/day and in themales at ≥ 150 µl/day
NTP, 1993
RatF344
(femaleand
male)(6 weeks
old)
p.c. 104 weeks(5 day/week)
0, 100, 300µl/day/rat(equivalent to 0,285, 855mg/kg/day)
Decreased mean body weight inmales at 300 µl/dayIncreased Ht, Hb and RBC in thefemales at 300 µl/dayIncreased mortality in the males at≥ 100 µl/dayA decrease in fatty liver occurredin males and females in correlationto the dose
NTP, 1993
Rat(strain,age and
sexunknown
)
i.v., 1%DEP/salinecontaining20%alcohol
Theadministration wasconductedfor thenumber oftimesindicated onthe right forabout 4 to 20days with aninterval of 2or 3 days
40 mg/rat5 mg × 8 times10 mg × 4 times20 mg × 2 times
No abnormality was noted bymacroscopic or histopathologicalexamination in livers of any group
Neergaard etal., 1975
Diethyl phthalate
160
Animalspecies
Adminis-trationmethod
Administrati-on period
Dose Test method and result References
Mouse(SwissWebster)(age andsexunknown)
i.p. 6 weeks 0, 125 mg/kg/day No macroscopic orhistopathological abnormality
Calley et al.,1966
Cat(strain,age and
sexunknown
)
Inhalat-ion
6 hours/days× 7 days
356 ppm(corresponding to3,289 mg/kg/day)
Decreased activity, vomiting,inhibition on central nerve system,thirst, decreased appetite
BIBRA,1994
Diethyl phthalate
161
Attachment-4 Result of carcinogenicity tests
Animalspecies
Adminis-trationmethod
Administrat-ion period Dose Result References
MouseB6C3F1
(femaleand
male)(6 weeks
old)
s.c. 103 weeks(5 days/weeks)
0, 7.5, 15, 30 µl/day(corresponding to 0,193, 386, 772 mg/kg/day)
Females:Increased incidence ofhepatocellular adenoma as well astotal incidence of totalhepatocellular tumors (carcinomaand adenoma) at 7.5 and 15 µl/day(7/50, 16/50, 19/50 and 12/50 inthe control group, 7.5, 15 and 30µl/day groups, respectively) (nodose correlation).Males:Increased number of basophilicfoci at 15 µl/day (no dosecorrelation).Increased incidence of totalhepatocellular tumors(carcinomaand adenoma) at 30 µl/day (9/50,14/50, 14/50 and 18/50 in thecontrol group, 7.5, 15 and 30µl/day groups, respectively) (Datawith low reliabilityl)
NTP, 1993
RatF344
(femaleand
male)(6 weeks
old)
s.c. 104 weeks(5 day/week)
0, 100, 300 µl/day(corresponding to 0,300, 1,000 mg/kg/day)
Decreased incidence of mammaryfibroma in females at 100 and 300µl/day (dose correlation)Onset of acanthosis inadministration site in the skin offemales at 100 µl/day and in themales and females at 300 µl/day
NTP, 1993
