Di-n-butyl phthalate
Hazard assessment of Di-n-butyl phthalate
[Di-n-butyl phthalate, CAS No. 84-74-2]
Chemical name:
Synonyms:
Di-n-butyl phthalate
n-Butylphthalate, 1,2-Benzenedicarboxylic acid dibutyl ester, Dibutyl 1,2benzene dicarboxylate, DBP
Molecular formula: C16H22O4
Molecular weight: 278.34
Structural formula:
O
C-O-CH2CH2CH2CH3
C-O-CH2 CH2 CH2CH3
O
Appearance: Colorless to faint yellow viscous liquid1)
Melting point: -35°C1)
Boiling point: 340°C1)
Specific gravity:
d 420 = 1.0465
2.68 x 10-3Pa (25°C)1)
Vapor pressure:
Partition coefficient:
Degradability:
1)
Log Pow = 4.9 (Calculated)1)
Hydrolyzability: No report.
Biodegradability: Easily biodegradable (BOD = 69%, 14 days)3)
Solubility: Water: 11.2 mg/l (20°C) 1), 13 mg/ l (25°C)1)
Organic solvents: Freely soluble in alcohol, ether, benzene, acetone, etc.1).
Amount of production/import:
Usage:
1998: 11.982 t (Production 11.766 t, Import 216 t) 3)
Used as plasticizer for vinyl chloride, vinyl acetate, nitrocellulose and metacryl
resins 1).
Applied laws and regulations:
Law Concerning the Examination and Regulation of
Manufacture, etc. of Chemical Substances, Law on Industrial Safety and Hygiene,
Law Relating to Prevention of Marine Pollution and Maritime Disaster
1)
HSDB 2001; 2) "Tsusansho Koho" (daily), 1975; 3) Ministry of Economy, Trade and Industry, 1999;
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Di-n-butyl phthalate
1. Toxicity Data
1) Information on adverse effects on human health
A 23-year-old male worker who accidentally ingested about 10 g of DBP experienced
vomiting, dizziness, ocular pain, lacrimation and conjunctivitis.
His urine was dark
yellow in color, and the urinary sediment contained numerous erythrocytes and leukocytes.
However, all these symptoms completely disappeared after 1 month (IPCS, 1997).
A 30-year-old woman who used an anti-perspirant containing DBP developed dermatitis,
and the results of patch testing was positive for DBP (IPCS, 1997).
A 32-year-old woman who used a DBP-containing deodorant spray developed itching
and redness, and the results of patch test were positive for DBP (IPCS, 1997).
A 44-year-old man who used a watch strap containing 5% of DBP developed eczema
(IPCS, 1997).
In a survey of 38 workers involved in manufacturing phthalate esters, the frequency of
dysesthesia of four extremities increased with increasing duration of the working hour in
the phthalate ester exposure group.
Some workers complained of excessive perspiration
of feet and hands and vasomotor irregularity indicative of autonomic effects.
Polyneuritis was observed in 57% of the workers, and decreased sensitivity to pain,
decreased senses of hands and feet were noted in some workers.
However, the authors
reported that they could not conclude whether the findings of polyneuropathy described in
this survey report were attributable to DBP or not, because the number of the subjects
included in the survey was small (IPCS, 1997).
Regarding the toxic effects of DBP on reproductive organs, there was a report of a
survey involving 189 female workers who underwent occupational exposure. However,
since the exposure levels were unknown and these workers were also exposed to other
nonspecific substances, the authors could not draw any conclusion (IPCS, 1997).
In Puerto Rican girls, the incidence of premature breast development (thelarche) was
high. In the analysis of serum samples from the girls with premature thelarche (6 months
- 8 years old), phthalate esters mainly in DBP and DEHP (di-(2-ethylhexyl) phthalate)
were detected in 28/41 samples, and, of these 28 samples, DBP and DEHP were detected
in 13 (15-276 µg/l) and 25 (187-2,098 µg/l) samples, respectively. The serum levels of
DBP and DEHP were significantly higher than those in 35 control serum samples from the
age-matched normal girls, suggesting the possible involvement of phthalate esters mainly
consisting of DBP and DEHP in the premature thelarche in these girls. However, the
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Di-n-butyl phthalate
authors concluded that further epidemiological studies and animal experiments would be
needed to substantiate the causal relationship of the endocrine-disrupting effect of
phthalate esters with premature thelarche (Colon et al., 2000).
2) Influence on endocrine system and reproductive system
(1) in vitro results related to receptor binding (Attachment-1)
The relative binding affinity of DBP for estrogen receptor was reported to be
(1/36,000 that of 17 β-estradiol (E2)) in a binding assay using uterine homogenate from
immature female SD rats (Zacharewski et al., 1998) and 1/28,000 that of E2 in a
binding assay using human estrogen receptors expressed in Sf9/Baculovirus (Nakai et
al., 1999).
In an estrogen receptor binding assay using uterine homogenate from
ovariectomized female SD rats, DBP was reported not to bind to estrogen receptors up
to 1 mM (Blair et al., 2000).
In a receptor binding assay, DBP was reported not to
bind to human estrogen receptors up to 10-4 M (CERI, 2001).
In a reporter gene assay using human breast carcinoma cell line MCF-7, 10 µM of
DBP showed 37% of 10 nM E2 taken as 100% (Zacharewski et al., 1998).
In cell
proliferation assay with yeast strain S. cerevisiae PL3 transfected with human estrogen
reporter gene which utilizes cell proliferation in response to ligand binding to human
estrogen receptor, DBP at 10 µM induced weak cell proliferation (Zacharewski et al.,
1998). In the assay with stable transformant of HeLa cells incorporated with the
same genes as those of the above MCF-7 cells and in the yeast two-hybrid assay, DBP
had no estrogen-like activity up to 10 and 0.3 µM, respectively (Nishihara et al., 2000).
In a reporter gene assay with cultured cells, DBP did not activate gene transcription
within the concentration range of 10-11 - 10-5M (CERI, 2001).
Thus, it was suggested that DBP may bind to estrogen receptor and induce
intracellular transcriptional activation, but several reports described that DBP does not
have any affinity for estrogen receptor or induce any gene transcriptional activation
(Zacharewski et al., 1998; Nakai et al., 1999; Blair et al., 2000; Nishihara et al., 2000;
CERI, 2001).
(2)
in vivo results in mammals (Attachments-2)
The effects of DBP and its metabolite mono-butyl phthalate (MBP) on endocrine
and reproductive systems in mammals are shown in Attachments-2.
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Di-n-butyl phthalate
The results of the assays by each screening technique are summarized below.
In an uterotrophic assay, juvenile female SD rats (aged 20 days) were given DBP
subcutaneously at doses of 0, 40, 200 and 1,000 mg/kg/day for 3 days (in accordance
with the OECD draft guidelines), but uterine weight remained unchanged (Yamasaki et
al., 2001).
In ovariectomized SD rats (aged 31-34 days), oral administration of DBP
at doses of 0, 20, 200 and 2,000 mg/kg/day for 4 days had no effect on uterine weight
(Zacharewski et al., 1998).
Further, in ovariectomized SD rats (age, unspecified)
given DBP subcutaneously at 0, 200 and 400 mg/kg/day or orally at 1,000 mg/kg/day
for 2 days and then 0.5 mg/rat of progesterone once subcutaneously on the third day,
uterine weight remained unaffected (Gray et al., 1999).
In the Hershberger assay for detection of anti-androgenic activity (in accordance
with the OECD draft guidelines), castrated male Alpk:Apf SD rats (aged 7 weeks)
were given DBP by oral gavage at 0, 500 and 1,000 mg/kg/day in combination with
subcutaneous administration of testosterone propionate at 0.4 mg/kg/day for 10 days,
and 4 replicate assays were performed. The results suggested the anti-androgenic
activity of DBP, but the positive result was not definitely reproducible (Ashby &
Lefevre, 2000b).
In peripubertal male rat assay, DBP was administered by gavage at
0 and 500 mg/kg/day to male Alpk:Apf SD rats for 14 days immediately after weaning
in accordance with the procedures proposed by EDSTAC (Endocrine Disruptor
Screening and Testing Advisory Committee), causing decrease in epididymal and
seminal vesicle weights.
When administration was continued for 34 days, these
changes plus delayed preputial separation were observed (Ashby & Lefevre, 2000a).
The results of studies on the effects of DBP on male reproductive organs are shown
below.
In a male mouse assay (strain and age, unspecified) in which DBP was administered
orally at 0 and 2,000 mg/kg/day for 10 days, decrease in testis weight and testicular
tissue injuries (details, unknown) were reported in 2,000 mg/kg/day group (Gangolli,
1982).
In a 1-week feeding study in which male Wistar rats (aged 5 weeks) fed the diet
containing 0 and 2% (equivalent to 0 and 1,000 mg/kg/day) of DBP, testis showed
decrease in its weight, decrease in primary spermatocytes, marked increase in
testicular testosterone content and decrease in zinc content in the 1,000 mg/kg/day
group (Oishi & Hiraga, 1980a).
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Di-n-butyl phthalate
In a male Wistar rat (aged 5 weeks) assay in which DBP was administered by
gavage at 0, 250, 500 and 1,000 mg/kg/day for 15 days, the testicular toxicities
included degeneration of seminiferous tubules and biochemical changes such as
decrease in acid phosphatase activity and increase in LDH, γ-GTP, β-glucuronidase (βG) and glucose-6-phosphate dehydrogenase (G6PDH) activities in 250 mg/kg/day or
higher groups and decrease in testicular weight, impaired spermatogenesis and
decrease in sorbitol dehydrogenase (SDH) activity in 500 mg/kg/day or higher groups
(Srivastava et al., 1990).
In a 13-week feeding study in male F344 rats (aged 5-6 weeks) fed the diets
containing DBP at 0, 2,500, 5,000, 10,000, 20,000 and 40,000 ppm (equivalent to 0,
176, 359, 720, 1,540 and 2,964 mg/kg/day), the abnormal changes included focal
atrophy of seminiferous tubules in 720 mg/kg/day or higher groups, decrease in testis
weight and decrease in testicular zinc and serum testosterone levels in the 1,540
mg/kg/day or higher groups and decrease in serum zinc level in 2,964 mg/kg/day
group (CERHR, 2000; Marsman, 1995).
In an inhalation toxicity study in which male Wistar rats (aged 4 weeks) were
exposed to 0, 0.5 and 50 mg/m3 (0, 0.044 and 4.4 ppm) of DBP mist for 6 hours/day
for 3 or 6 months, testis weight remained unaffected (Kawano, 1980).
In a 10-day oral administration study of DBP in male guinea pigs (strain and age,
unspecified) at 0 and 2,000 mg/kg/day, decrease in testis weight and degeneration of
Sertoli cells were observed in 2,000 mg/kg/day group (Gangolli, 1982).
In experiments in which DBP was administered orally at 0 and 2,000 mg/kg/day for
7-9 days, Gray et al. reported decrease in testis weight and toxic effects on
seminiferous tubules in TO mice, SD rats and Dunkin-Hartley guinea pigs receiving
2,000 mg/kg/day, but no testicular toxicities in Syrian hamsters (Gray et al., 1982).
The results of the reproductive/developmental toxicity studies are shown below.
In an NTP protocol study in which male and female CD-1 mice (aged 11 weeks)
were fed the diets containing DBP at 0, 0.03, 0.3 and 1.0% (equivalent to 0, 52.5, 525
and 1,750 mg/kg/day) for 105 days (7 days before and 98 days during co-housing),
there was a significant decrease in the percentage of pregnant rat pairs, the number of
live pups per litter, propotion of pups born alive and live pups in 1,750 mg/kg/day
group. In the cross-over mating, the percentage of pregnant rat, litter size, percentage
of pups born alive and body weight of live fetuses decreased in females in high-dose
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Di-n-butyl phthalate
group mated with the control males (Lamb et al., 1987).
In a teratogenicity study in which female ICR mice (mated at the age of 8-16 weeks)
were fed the diets containing DBP at 0, 0.05, 0.1, 0.2, 0.4 and 1.0% (equivalent to 0,
80, 180, 350, 600 and 2,100 mg/kg/day) on gestation days 0-18, increase in embryonic
death, exencephaly, spina bifida and decrease in maternal body weight were observed
in 2,100 mg/kg/day group (Shiota et al., 1982).
In feeding studies in which female B6C3F1 mice (age, unspecified) were fed the
diets containing DBP at 0 and 20,000 ppm (equivalent to 0 and 2,600 mg/kg/day)
during the gestation period, complete resorption of all embryos occurred in 2,600
mg/kg/day group (ATSDR, 1990; Killinger et al., 1988a).
In a developmental toxicity study in which female Wistar rats (mated at the age of
10-14 weeks) were given DBP by oral gavage at 0, 750, 1,000 and 1,250 mg/kg/day on
gestation days 7-9, 10-12 or 13-15, post-implantation losses increased in all dams
treated on these gestation days in 750 mg/kg/day or higher groups. When DBP was
given on gestation days 7-9, increase in incidence of skeletal malformation and
decreases in number of live fetuses and in fetal body weight were observed in 750
mg/kg/day or higher groups. When given on gestation days 10-12, DBP caused
decrease in number of live fetuses in 750 mg/kg/day or higher groups and decrease in
fetal body weight in 750 and 1,250 mg/kg/day groups, but no fetal malformations were
observed. Administration on gestation days 13-15 resulted in increase in incidences
of cleft palate and abnormal fusion of sternebrae in 750 mg/kg/day or higher groups
and decrease in number of live fetuses in 1,000 mg/kg/day or higher groups (Ema et al.,
1995a). In this experiment, malformation of fetuses were observed when DBP was
given on gestation days 7-9 and 13-15, but not on gestation days 10-12, and the same
results are reported in a similar experiment using 1,500 mg/kg/day as the high dose
(Ema et al., 1994). In another developmental toxicity study in which female Wistar
rats (mated at the age of 14 weeks) were given single oral administration of DBP at 0
and 1,500 mg/kg/day on one day during gestation days 6-16, DBP induced obvious
skeletal malformations (cervical and thoracic vertebrae and ribs) when given on
gestation days 8, 9 or 15, exencephaly and dilatation of renal pelvis when given on
gestation day 9 and cleft palate when given on gestation day 15, and these findings
supported the results in the above study (Ema et al., 1997).
In a feeding study in which female Wistar rats (mated at the age of 14 weeks) were
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Di-n-butyl phthalate
fed the diets containing DBP at 0, 0.5, 1.0 and 2.0% (equivalent to 0, 331, 555 and 661
mg/kg/day) on gestation days 11-21, maternal body weight gain was suppressed in 555
mg/kg/day or higher groups.
Undescended testis and reduced anogenital distance
(AGD) were observed in 555 mg/kg/day or higher groups, and decrease in fetal body
weight, cleft palate and abnormal sternal fusion in 661 mg/kg/day group.
However,
DBP had no effect on development of female reproductive organs (Ema et al., 1998).
Based on the results of oral administration study in female Wistar rats (mated at the
age of 14 weeks) given DBP at 0, 500 (only on gestation days 15-17), 1,000 and
1,500 mg/kg/day on gestation days 12-14, 15-17 and 18-20, the authors concluded that
the critical period for induction of undescended testis and reduced AGD by DBP was
gestation days 15-17 (Ema et al., 2000a).
In an oral administration study in which female SD rats (mated at the age of 8
weeks) were give DBP at 0, 100, 250 and 500 mg/kg/day or at 0, 0.5, 5, 50, 100 and
500 mg/kg/day on gestation days 12-21, abnormal findings in offsprings included
nipple retention in male offsprings in 100 mg/kg/day or higher groups, reduced AGD
in 250 mg/kg/day or higher groups and hypospadias, cryptorchidism, hypoplasia of
prostate, epididymis, seminal vesicle and ductus deferens, epithelial degeneration of
seminiferous tubules, interstitial cell hyperplasia in testis, atrophy of vas deferens and
decreased weights of testis, seminal vesicle, epididymis, prostate and levator anibalbocarvernosus in male offspring in 500 mg/kg/day group. Based on these, the
authors concluded that the NOAEL and LOAEL of DBP for male reproductive
malformations are 50 and 100 mg/kg/day, respectively, under the conditions of 10-day
exposure during pregnancy (Mylchreest et al., 1999; Mylchreest et al., 2000).
In an oral administration study in which female LE rats (age, unspecified) were
given DBP at 0 and 500 mg/kg/day on gestation days 16-19, increase in resorptions
and abnormalities in male offsprings such as reduced AGD, decreased weights of
seminal vesicle, prostate and levator ani-balbocavernosus and retained nipple (areolas)
in 500 mg/kg/day group. In a similar experiment, female SD rats (age, unspecified)
were given DBP by oral gavage at 0 and 500 mg/kg/day from gestation day 14 to day
3 postpartum.
In 500 mg/kg/day group, the number of pups delivered decreased, and
male offsprings showed reduced AGD, hypospadias, atrophy or hypoplasia of the testis
and epididymis, decreased weights of seminal vesicle, prostate, epididymis, testis,
levator ani-balbocavernosus and penis and retained nipple (Gray et al., 1999).
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In another study, male and female LE or SD rat weanlings were given DBP by
gavage at 0, 250, 500 and 1,000 (only to males) mg/kg/day from weaning through the
subsequent periods of growth, mating and lactation of F1 animals, and then the F1
animals from the treated groups were mated with untreated animals.
In F0 animals,
the abnormal findings included delayed sexual maturation in both sexes in 250
mg/kg/day or higher groups, reduced fertility in 500 mg/kg/day group, testicular
atrophy and hypospermatogenesis in males in 500 mg/kg/day or higher groups and
infertility in males in 1,000 mg/kg/day group.
In the F1 animals, malformations,
reduced conception rate and reduced epididymal sperm count were observed in 250
mg/kg/day or higher groups (Gray et al., 1999).
In a continuous breeding study in male and female SD rats (aged 10 weeks), the
diets containing DBP at 0, 0.1, 0.5 and 1.0% (equivalent to 0, 52, 256 and
509 mg/kg/day in males and to 0, 80, 385 and 794 mg/kg/day in females) were
administered to F0 animals.
Dietary administration of DBP caused decreased total
number of live F1 pups in 0.1% (equivalent to 52-80 mg/kg/day) or higher groups,
decreased body weight of live F1 pups in 0.5% (equivalent to 256-385 mg/kg/day) or
higher groups and suppressed maternal body weight gain in 1.0% (equivalent to 509794 mg/kg/day) group. In the cross-over mating trial of parental F0 animals, the body
weight of the pups from the high-dose females paired with the control males decreased.
When the control females were paired with the high-dose males (reversed pairing),
however, the offspring body weight was not changed.
In F0 animals, liver and kidney
weights increased in both sexes in 1.0% (equivalent to 509-794 mg/kg/day) group.
However, male and female reproductive organs were grossly intact, nor were there any
abnormalities in sperm count, sperm motility and estrus cycle. In F1 animals, DBP
caused decreased body weight of live F2 pups in 0.1% (equivalent to 52-80 mg/kg/day)
or higher groups and marked decrease in copulation and pregnancy indexes and
decrease in body weight in male and female F1 parental animals in 1.0% (equivalent to
509-794 mg/kg/day) group.
In F1 generation, increased kidney weight was also
observed in males in 0.5% (equivalent to 256-385 mg/kg/day) or higher groups, and
increased liver weight, decreased weights of prostate, seminal vesicle and testis,
decrease in epididymal sperm count and testicular spermatid head counts, degeneration
of seminiferous tubules, interstitial cell hyperplasia and epididymal hypoplasia in
males in 1.0% (equivalent to 509-794 mg/kg/day) group. The authors therefore
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concluded that reproductive/developmental effects of DBP on offspring generation
was greater than those on the parental generation (Wine et al., 1997).
In male SD rats (aged 4-6 weeks), oral administration of MBP, a metabolite of DBP,
at 0 and 2,000 mg/kg/day resulted in decrease in testis weight and diffuse atrophy of
seminiferous tubules in 2,000 mg/kg/day group (Gray et al., 1982). When MBP was
administered orally to pregnant Wistar rats, the offspring showed skeletal
malformation, cleft palate, dilatation of renal pelvis and undescended testis
(Attachment-3) (CERHR, 2000; Ema et al., 1995b, 1996a, 1996b; Imajima et al.,
1997).
In the NTP-CERHR (Center for Evaluation of Risks to Human Reproduction)
Expert Panel Report on DBP, it was described that various malformations seen in male
F1 animals from the pregnant rats given DBP orally were not mediated by androgen
receptors but were due to inhibition of testosterone biosynthesis. However, the
literature that constitutes the basis of this speculation was not given, and the
mechanistic details remained unknown (CERHR, 2000).
3) Information on general toxicity
(1) Acute toxicity (Table 1)
The LD50 values for each administration route in mice, rats and rabbits are shown in
Table 1 (ACGIH, 1991; ATSDR, 1990; German Chemical Society, 1987). In the
inhalation exposure experiment in mice, the symptoms of acute toxicity such as
labored breathing, motor ataxia, local paralysis, convulsion and coma were observed,
with some deaths due to respiratory failure. In rats, weight loss and decrease in blood
components were reported (ACGIH, 1991). In an inhalation experiment in cats,
salivation, unrest and hypoactivity were reported (German Chemical Society, 1987).
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Table 1
Results of acute toxicity studies
Mouse
Oral LD50
More than 20,000 mg/kg
Rat
Rabbit
3,000 – 8,000 mg/kg*
−
Inhalation LD50
−
−
Percutaneous LD50
−
−
Intraperitoneal LD50
4,000 mg/kg
3,050 mg/kg
−
More than 20,000 mg/kg
−
*: Variable depending on the studies.
(2) Repeated-dose toxicity (Attachment-3)
In a 7-day feeding study in male ICR mice (age, unspecified) fed the diets
containing DBP at 0 and 20,000 ppm (equivalent to 0 and 2,600 mg/kg/day), mice in
2,600 mg/kg/day group showed decrease in body weight, increase in liver weight,
decrease in kidney weight and decreased zinc concentrations in testis and liver
(ATSDR, 1990; Oishi & Hiraga, 1980b). In another feeding study in mice (strain and
age, unspecified) in which DBP was administered in diet at 0, 628 and 1,248
mg/kg/day for 21 days, body weight decreased in 1,248 mg/kg/day group (ATSDR,
1990). In a 13-week feeding study in which B6C3F1 mice (aged 6 weeks) were fed
the diets containing DBP at 0, 1,250, 2,500, 5,000, 10,000 and 20,000 ppm (equivalent
to 163, 353, 812, 1,601 and 3,689 mg/kg/day in males and to 238, 486, 971, 2,137 and
4,278 mg/kg/day in females), the abnormal changes included suppressed body weight
gain and increase in liver weight in males in 812 mg/kg/day or higher groups, increase
in kidney weight in females in 238 mg/kg/day or higher groups and eosinophilic
granules, increased staining intensity of cytoplasm and increased lipofuscin granules
in hepatocytes in males in 1,601 mg/kg/day or higher groups and females in 4,278
mg/kg/day group (CERHR, 2000; Marsman DS, NTP, 1995). In a long-term study in
which CD-1 mice (aged 11 weeks) were fed the diets containing DBP at 0, 0.03, 0.3
and 1.0% (equivalent to 0, 52.5, 525 and 1,750 mg/kg/day) for 126 days, decrease in
body weight and increase in liver weight were observed in 1,750 mg/kg/day group
(CERHR, 2000; Reel et al., 1984).
In a 21-day feeding study in rats (strain and age, unspecified) in which DBP was
administered at doses of 0 and 348 mg/kg/day, decrease in blood cholesterol and
increase in liver weight were observed in 348 mg/kg/day group (ATSDR, 1990; Bell,
1982). In another 21-day feeding study in rats (strain and age, unspecified) in which
DBP was administered at doses of 0, 628 and 1,248 mg/kg/day, liver and kidney
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Di-n-butyl phthalate
weight increased in 628 mg/kg/day or higher groups and in 1,248 mg/kg/day group,
respectively (ATSDR, 1990; BIBRA). In a feeding study in male and female Wistar
rats (age, unspecified) in which DBP was administered in diet at doses equivalent to 0
and 250 mg/kg/day for 34-36 days, decrease in body weight and hepatocellular
necrosis were observed in 250 mg/kg/day group, and inhibition of hepatic
mitochondrial energy metabolism was also reported (ATSDR, 1990; Murakami et al.,
1986a). In another feeding study in which male and female Wistar rats (age,
unspecified) received DBP in diet at doses equivalent to 0 and 2,500 mg/kg/day for
35-45 days, hepatic mitochondrial oxidation decreased, and spleen weight increased
(ATSDR, 1990; Murakami et al., 1986b). When male and female Wistar rats (aged 6
weeks) were fed the diets containing DBP at 0, 400, 2,000 and 10,000 ppm (equivalent
to 0, 27, 142 and 688 mg/kg/day in males and to 0, 33, 161 and 816 mg/kg/day in
females) for 3 months, males in 688 mg/kg/day group showed peroxisome
proliferation and histopathological changes in liver, decrease in thyroid hormone (T3)
levels and anemia, and females in 816 mg/kg/day group showed increases in liver and
kidney weight, decrease in thyroid hormone (T3) levels with no histopathological
changes. And also, to evaluate neurological toxicity neurologic functional (behavior,
reflex, audition, vision, ordor detection sense, nociception, etc.) and histopathological
examinations were conducted and no effects were observed at all dose levels.
Therefore, considering these parameters NOAEL was determined as 142 mg/kg/day in
male and 161 mg/kg/day in female. (CERHR, 2000; BASF, 1992).
In a 13-week
study in which male and female F344 rats (aged 5-6 weeks) were fed the diets
containing DBP at 0, 2,500, 5,000, 10,000, 20,000 and 40,000 ppm (equivalent to 0,
176, 359, 720, 1,540 and 2,964 mg/kg/day in males and to 0, 177, 356, 712, 1,413 and
2,943 mg/kg/day in females), the abnormal changes in males included decrease in
hemoglobin and red blood cell count, increase in platelet count and serum albumin,
increase in hepatic palmitoyl CoA oxidase (PCAO) activity and increase in liver and
kidney weights in 359 mg/kg/day or higher groups, suppressed body weight gain and
histopathological changes in liver in 720 mg/kg/day or higher groups and peroxisome
proliferation in liver in 2,964 mg/kg/day group, and those in females included increase
in hepatic PCAO activity in 356 mg/kg/day or higher groups, increase in liver and
kidney weight in 712 mg/kg/day or higher groups, suppressed body weight gain in the
1,413 mg/kg/day or higher groups and peroxisome proliferation in liver in 2,943
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Di-n-butyl phthalate
mg/kg/day group (CERHR, 2000; Marsman DS, NTP, 1995).
In an inhalation study in which male Wistar rats (aged 4 weeks) were exposed to 0,
0.5 and 50 mg/m3 (0, 0.044 and 4.4 ppm) of DBP mist for 6 hours/day, 5 days/week,
for 3-6 months, decreased body weight and increased relative weight of lung were
observed in 50 mg/m3 (4.4 ppm) group (ATSDR, 1990; Kawano, 1980a; Kawano,
1980b). In another inhalation study in which rats (strain and age, unspecified) were
exposed to DBP at 0 and 2.5 ppm for 6 hours/day for 5 days, pulmonary cytochrome
P-450 content decreased in 2.5 ppm group (ATSDR, 1990; Walseth & Nilsen, 1984).
In rabbits (strain and age, unspecified), the topical application of DBP at 0 and
4,200 mg/kg/day for 90 days caused renal toxicities (details unknown) (ATSDR, 1990;
Lehman, 1955).
4) Information on mutagenicity/genotoxicity and carcinogenicity
(1) Mutagenicity/genotoxicity (Table 2)
DBP was reported to be negative in many of the reverse mutation tests using
Salmonella typhimurium strains, but positive results were also reported in some studies.
The positive results were reported in the absence of metabolic activation, but increase
in the number of revertant colonies was about twice the solvent control value and
showed no dose-response relationship (IPCS, 1997).
Two gene mutation tests with
mouse lymphoma cells were reported, and DBP was positive in one assay without
metabolic activation system but only at the dose which was cytotoxic (IPCS, 1997).
In another assay, DBP was positive in the presence of exogenous metabolic activation
system (Barbar, 2000).
DBP was reported to be negative in all chromosomal
aberration tests (IPCS, 1997).
DBP was also negative in BALB/3T3 cell
transformation assay (Barbar et al., 2000).
In a DNA repair test using human
mucosal cells of upper respiratory tract, however, DBP was positive for DNAdamaging potential (Kleinsasser, 2000).
No reports are available about the in vivo
assays on DBP.
252
Di-n-butyl phthalate
Table 2 Results of mutagenicity/genotoxicity studies
In vitro
Test method
Reverse
mutation test
Chromosomal
aberration test
Sister chromatid
exchange test
Gene mutation
test
Transformation
assay
DNA repair test
Cells/animal species used
S. typhimurium strain TA100, S9(+/-), 13 - 50
µg/m l
(DBP induces significant increases in the
number of revertant colonies under the S9(-)
condition, but the increases are not 2-fold or
greater than that in the control group.)
S. typhimurium strains TA98, TA100, TA1535,
and TA1537, S9(+/-), 100 - 10,000 µg/plate
S. typhimurium strains TA98, TA100, TA1535,
TA1537 and TA2637, S9(+/-), 100 - 2,000
µg/plate
(Positive under the S9(-) condition in TA100
and TA1535.)
S. typhimurim strains TA98, TA100, TA1535
and TA1537, S9(+/-), concentration that
produces precipitation
Chinese hamster Don cells, 0.28 - 27.8 mg/m l
Human lymphocytes, 0.03 mg/m l
CHL cells, 0.03 mg/m l
Chinese hamster Don cells, 0.28 - 27.8 mg/m l
Results*
-
L5178Y mouse lymphoma cells, S9(+/-)
(Positive under the S9(-) condition at the
concentration that produces severe cytotoxicity.)
L5178Y mouse lymphoma cells, S9(+/-)
(Positive under the S9(+) condition at 0.1 µl /m
l.)
BALB/3T3 cells, 0.0034 – 0.082 µl /m l
Human mucosal cells of the upper respiratory
tract, 354 µmol/m l
*-: Negative +: Positive +w: Weakly positive
References
IPCS, 1997
-
IPCS, 1997
+w
IPCS, 1997
-
IPCS, 1997
±
-
IPCS, 1997
IPCS, 1997
IPCS, 1997
IPCS, 1997
+
IPCS, 1997
+
Barbar, 2000
-
Barbar, 2000
+
Kleinsasser, 2000
(2) Carcinogenicity (Table 3)
In a one-year study, Wistar rats given to 0 and 55 mg/kg/day of DBP in diet showed
no DBP-related tumor development.
In rats (strain and age, unspecified) given DBP
at 0 and 100-500 mg/kg/day for 15-21 months and those fed the diet containing DBP
at 2,500 ppm for 18 months or more, no DBP-related tumor development was
observed (ATSDR, 1990; German Chemical Society, 1987).
253
Di-n-butyl phthalate
Table 3
Carcinogenicity assessment by national and international organizations.
Organization
EPA
EU
NTP
IARC
ACGIH
Japan Society for
Occupational Health
Category
Group D
−
−
–
−
−
Significance
Not classifiable as a human carcinogen.
No evaluation.
No evaluation
No evaluation
No evaluation
References
IRIS, 2002
ECB, 2000
NTP, 2000
IARC, 2001
ACGIH, 2001
Japan Society for
Occupational Health,
2001
No evaluation
5) Information on immune system
At present, no reports are available on the effects on the immune system.
6) Fate and Metabolism
DBP was slowly absorbed from skin, but was rapidly absorbed from gastrointestinal
tracts (IPCS, 1997). In a single oral administration study in which rats were given
60 mg/kg of 14C-DBP (the position of radiolabel, unspecified), radioactivity was detected
in liver, kidney, blood, muscle, adipose tissues, stomach and small intestine 24 hours after
administration (IPCS, 1997; Keys et al., 2000). In a 12-week study, however, the
radioactivity was not accumulated in rats fed the 0.1% DBP diet (IPCS, 1997).
In rats
given single oral administration of 7-14C-DBP at 0.27 or 2.31 g/kg, 92% and 83% of the
dose, respectively, were excreted into urine by 48 hours after administration.
In the urine,
phthalic acid (2%), mono-n-butyl phthalate (88%), mono-3-hydroxybutyl phthalate (8%)
and mono-4-hydroxybutyl phthalate (2%) were detected (IPCS, 1997). No report is
available on metabolism of DBP in humans, but after oral administration of DBP to
experimental animals mainly butyl ester linkage is rapidly hydrolyzed in the intestine to
yield mono-butyl phthalate (MBP).
MBP undergoes further ω and ω-1 oxidation to
yield oxidation products of MBP (IPCS, 1997; Keys et al., 2000).
MBP was considered
to be a reproductive and developmental toxicant and reported to cause testicular atrophy in
rats treated orally at 400 mg/kg or above (Keys et al., 2000). MBP and other metabolites
were excreted into urine mainly as the glucuronide conjugates.
The proportion of
unconjugated MBP in urine was higher in rats than in hamsters, and this species difference
was thought to be responsible for severer testicular toxicity in rats than in hamsters (IPCS,
1997; Keys et al., 2000).
254
Di-n-butyl phthalate
(2)
CH3
CH3
CH2OH
(2)
CH2OH
COOCH2 CH2CH2CH3
COOH
COOH
COOCH2 CH2CH2CH3
COOCH 2CH2CH2 CH3
COOH
(4)
MBP(3)
(1)
COOH
COOH
COOCH 2CH2CH(OH)CH3
COOCH2CH2CH2CH2OH
(5)
(6)
(1) Dibutyl phthalate (DBP)
(4) Phthalic acid
(2) Butanol
(5) Mono-2-hydroxy butyl phthalate
(3) Mono-butyl phthalate (MBP)
(6) Mono-1-hydroxy butyl phthalate
Fig. 1
Metabolic pathway of di-n-butyl phthalate
2. Hazard assessment at present
The obvious relationship of endocrine and reproductive toxicities in humans with the
exposure to DBP has not been described in the literature.
In an in vitro assay DBP was reported to have no binding affinity for estrogen receptors
and to induce no gene transcriptional activation in one study, but was also reported to have
a weak binding affinity for estrogen receptors as compared with natural estrogen in
another study;1/28000~1/36000 of E2.
In an in vivo study, administration of DBP at high
dose (2,000 mg/kg/day) had no effect on uterine weight in the uterotrophic assay. These
findings suggest little possibility for DBP to have any estrogenic activity.
In the
Hershberger assay, DBP has been shown to have anti-androgenic activity, but the results
were not clearly reproducible.
In a peripubertal male rat assay, however, the delayed
prepuce separation was observed.
In the study by administration during the pregnancy
and lactation periods, retained nipple and reduced AGD were observed in male F1
255
Di-n-butyl phthalate
offspring. These effects were thought to be possibly due to anti-androgenic effect of this
compound. The testicular toxicities of DBP were reported in many repeatedly dosed
toxicity studies, and the effects on testis and accessory reproductive organs, increased
resorptions, decreased litter size and induction of various malformations were noted in the
reproductive/developmental toxicity studies. Thus, it is evident that DBP affects mainly
male reproductive function.
In the NTP-CERHR (Center for Evaluation of Risks to Human Reproduction) Expert
Panel Report on DBP, it was reported that various malformations seen in F1 males from
dams given DBP orally during gestation period were not mediated by androgen receptors
but due to inhibition of testosterone biosynthesis.
In addition, relating to endocrine effects of DBP decrease of thyroid hormone (T3)
levels was reported in the feeding study, however, this was shown only at the high dose
level (male 688 mg/kg/day, female 816 mg/kg/day). In this study the major effects of DBP
administration were changes in liver (peroxisome proliferation and decrease of lipid
deposition in hepatocytes) and anemia, and no histopathological change in thyroid was
found. Also, no effects on nervous system were found by neurologic functional and
histopathological examinations. Namely, toxicologically specific effects suspected of the
relation to decrease of thyroid hormone were not clearly shown.
As the hazardous effect of DBP in man, a case of nephritis in a worker who accidentally
ingested 10 g of DBP was reported. Skin sensitization after exposure to the products
containing DBP was reported in some cases.
Hepatic and renal toxicities were observed after repeated exposure in laboratory animals.
In mutagenicity studies, DBP was reported to be negative in most of the assays, although
positive in some assays. Carcinogenicity of DEP was negative in rats. No reports are
available about the carcinogenic potential of DBP in humans.
3. Risk assessment and other necessary future measures
DBP is thought to have little potential to induce endocrine disruption mediated by
estrogen receptors, but DBP is evidently a reproductive/developmental toxicant. The
possible involvement of anti-androgenic activity of DBP in its reproductive/developmental
toxicities, particularly its effects on male reproductive system is not yet eliminated, and
CERHR suggests that its androgen receptor-independent anti-androgenic activity
(inhibition of testosterone biosynthesis) is responsible for these toxicities. Considering
256
Di-n-butyl phthalate
these, it will be necessary to investigate the presence or absence of anti-androgenic
activity of DBP and involvement of androgen receptors in this activity based on the ongoing in vitro assay to assess binding affinity for androgen receptors and Hershberger
assay.
Since DBP is a reproductive/developmental toxicant regardless of the presence or
absence of endocrine disrupting effect, it is thought necessary to perform its risk
assessment based on the results of the hazard assessment and exposure assessment and to
explore an appropriate method for risk control.
257
Di-n-butyl phthalate
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262
Di-n-butyl phthalate
263
Di-n-butyl phthalate
Attachment-1 Results of in vitro studies on receptor binding
Item
ER binding
assay
Test methods and conditions
Results
Methods: Competitive binding assay
IC50 value: 5.83 ×10- 5M
3
with [ H]-E2 as a ligand. Receptor:
(E2: 2.09×10-9M)
Human ER expressed in
Relative binding affinity
Sf9/Baculovirus. Temperature: 25°C, (vs E2): (E2=1): 3.6×10 -5
pH: 7.4
Methods: Competitive binding assay
IC50 value: >1×10 - 3M
3
with [ H]-E2 as a ligand. Receptor:
(E2: 8.99×10-10M)
Uterine homogenate from
ovariectomized SD rats.
Temperature: 4 °C, pH: 7.4
Methods: Competitive binding assay
IC50 value: 4.7 ×10- 5M
3
with [ H]-E2 as a ligand. Receptor:
(E2: 1.3×10- 9M)
Uterine homogenate from immature
SD rats. Concentration: 1 – 1000
µM, Temperature: 30°C, pH: 7.6
Methods: Binding assay on human ER
IC50 value: >10 -4M
(E2: 1.2×10-9M)
(recombinant ERα ligand domain)
In the presence of 10 µM
Reporter gene Cells: MCF-7 cells transiently
of DBP,
assay with
transfected with Gal4-human ER gene
the reporter gene
cultured
and Gal4-regulated luciferase reporter
transcription was 37% of
recombinant
gene.
the activity at 10 nM of
cells
Concentration: 0.1, 1, 10µM (DBP), 1
E2.
pM-10 nM (E2)
Negative within a range
Cells: HeLa cells stably transfected
of 0.1 - 10 µM.
with Gal4-human ER gene and Gal4regulated luciferase reporter gene.
Concentration: 0.1, 1, 10µM (DBP), 1
pM-10 nM (E2)
Negative for the agonist
Cells: HeLa cells transfected with
activity within a range of
human ER expression plasmid and ER
10- 11-10-5.
response element.
- 11
-5
(E2: PC50: <10 -11M)
Concentration: 10 -10 M (DBP)
Negative for the agonist
Cells: HeLa cells transfected with rat
activity within a range of
ER expression plasmid and ER
response element.
10- 11-10-5.
- 11
-5
Concentration: 10 -10 M (DBP)
(E2: PC50: <10 -9M)
Cells: S. cerevisiae PL3 strain
Weak cell growth was
Human ERtransfected with human ER.
detected on the 5th day.
dependent
yeast growth
E2 induced obvious cell
Concentration: 10 µM (DBP), 1 nM
growth from the 3rd day.
assay
(E2)
Incubation: 5 days
Yeast twoCells: Yeast transfected with Gal4
REC10: >3×10-4M or
hybrid assay
DNA binding domain/human ER
above (E2: 3×10 -10M)
ligand binding domain genes, Gal4
activation domain/coactivator TIF2
genes and β-galactosidase reporter
gene
ER: Estrogen receptor E2: 17 β-estradiol IC50: 50% inhibition concentration
REC10: Concentration equivalent to 10% of activity at 10-7M of E2
PC50: Concentration equivalent to 50% of the maximal activity of E2.
264
Conclusion
DBP has binding affinity
for ER.
References
Nakai et al.,
1999
(The binding affinity is
1/28,000 that of E2.)
DBP has no binding
affinity for ER.
Blair et al.,
2000
DBP has binding affinity
for ER.
Zacharewski
et al., 1998
(The binding affinity is
1/36,000 that of E2.)
DBP has no binding
affinity for ER.
DBP activates ERmediated gene
transcription.
CERI, 2001
Zacharewski
et al., 1998
DBP does not activate
ER-mediated gene
transcription.
DBP does not activate
ER-mediated gene
transcription.
CERI, 2001
DBP does not activate
ER-mediated gene
transcription.
Yamasaki et
al., 2001
DBP induces cell
proliferation.
Zacharewski
et al., 1998
DBP does not activate
ER-mediated gene
transcription.
Nishihara et
al., 2000
Di-n-butyl phthalate
Attachment-2 Results of studies on mammalian endocrine and reproductive systems
(1) Results of reproduction studies by screening technique
Administration
method
Rat (SD,
s.c. (uterotrophic
female)
assay, juvenile
rat)
Oral gavage
Rat (SD,
(uterotrophic
female)
Ovariectomized assay,
at the age of 19 ovariectomized
rat)
days (n=10)
Rat (SD,
s.c. (uterotrophic
female)
assay,
ovariectomized
rat)
Administration
period
20 days of age
3 days
Rat
(Alpk:ApfSD,
male)
Castrated at the
age of 6 weeks
Oral gavage
(Hershberger
assay)
Started from 8
days after
castration for
10 days
Rat
(Alpk:ApfSD,
male)
Oral gavage
22-23 days of age
14 days
22-23 days of age
14 days of dosing
period + 20 days
of recovery period
35-36 days of age
14 days
22-23 days of age
34 days
Animal species
Dose
Results
References
0, 40, 200, 1,000
mg/kg/day
No effect on uterine weight.
Yamasaki et
al., 2000
31- 34 days of age 0, 20, 200, 2,000
4 days
mg/kg/day
No effect on uterine weight.
Zacharewski
et al., 1998
Age, unspecified
2 days
No effect on uterine weight.
Gray et al.,
1999
DBP at 0, 200 and
400 mg/kg/day, s.c.,
or at 1,000
mg/kg/day by oral
gavage for 2 days,
followed by
progesterone at 0.5
mg/rat, s.c., on the
third day.
DBP at 0, 500 and
1,000 mg/kg/day +
testosterone
propionate (TP) at
0.4 mg/kg/day, s.c.
0, 500 mg/kg/day
0, 500 mg/kg/day
0, 500 mg/kg/day
0, 500 mg/kg/day
265
Four replicate assays were
performed. In the second assay,
weights of bulbospongiosus muscle
+ levator ani muscle, bulbourethral
gland, seminal vesicle and prostate
decreased significantly in 500
mg/kg/day or higher groups as
compared with the control group
(treated with TP). In other 2
assays, however, no significant
decrease was observed in seminal
vesicle and prostate weights in
1,000 mg/kg/day group, and,
moreover, no significant decrease
was observed in bulbourethral gland
weight in one of these two assays.
It is thought that DBP may have an
anti-androgenic effect, but the
results were not definitely
reproducible in four replicate
assays.
Decreased testis and seminal vesicle
weights at 500 mg/kg/day.
Decreased testis and epididymal
weights at 500 mg/kg/day.
No abnormal changes in testis and
accessory reproductive organs.
Decreased testis and seminal vesicle
weights and delay in prepuce
separation at 500 mg/kg/day.
Ashby &
Lefevre,
2000b
Ashby &
Lefevre,
2000a
Di-n-butyl phthalate
(2) Results of reproductive/developmental toxicity studies
Administration
method
Oral gavage
Administration
period
4-6 weeks of age
7-9 days
Mouse, male
Strain,
unspecified
Rat (SD, male)
p.o.
Age, unspecified
10 days
0, 2000 mg/kg/day
Oral gavage
4-6 weeks of age
7-9 days
0, 2000 mg/kg/day
Rat (Wistar,
male)
By feeding
5 weeks of age
1 week
0, 2%
(Corresponding to 0
and 1,000
mg/kg/day)
Rat (Wistar,
male)
Oral gavage
5 weeks of age
15 days
0, 250, 500, 1,000
mg/kg/day
Animal species
Mouse (TO,
male)
Dose
Results
References
0, 2000 mg/kg/day
Decreased testis weight and slight
atrophy of seminiferous tubules at
2,000 mg/kg/day.
Decreased testis weight and
testicular tissue injuries at 2,000
mg/kg/day.
Decreased testis weight and diffuse
atrophy of vas deference at 2,000
mg/kg/day.
Decreased testis weight and primary
spermatocytes and increased
concentration of testosterone and
decreased concentration of zinc in
testis at 1,000 mg/kg/day.
Degeneration of seminiferous
tubules, interstitial edema,
decreased acid phosphatase (AP)
activity and increased activities of
LDH, γ-GTP, β-glucuronidase (β-G)
and glucose-6-phosphate
dehydrogenase (G6PDH) in testis at
250 mg/kg/day or above.
Decreased testis weight, impaired
spermatogenesis and decreased
sorbitol dehydrogenase (SDH)
activity in testis at 500 mg/kg/day
or above.
(The decreased SDH and increased
LDH are thought to indicate
destruction of germinal epithelium.
β-G and γ-GTP are the markers for
Sertoli cells, and the increases in
these marker enzymes are thought
to be associated with decrease in
germinal epithelium. AP exists in
lysosomes of Sertoli cells and
germinal epithelium and is known
to increase during development of
primary spermatocytes and
testicular maturation. The
decrease in AP seems to imply
inhibition of spermatogenic
processes. The G6PDH activity is
reported to increase at the time of
testicular injuries.)
Gray et al.,
1982
266
Gangolli,
1982
Gray et al.,
1982
Oishi &
Hiraga,
1980a
Srivastava
et al., 1990
Di-n-butyl phthalate
Administration
method
By Feeding
Administration
period
5-6 weeks of age
13 weeks
Rat (Wistar,
male)
Inhalation
0, 0.5, 50 mg/m3
(0, 0.044, 4.4 ppm)
Guinea pig
(DunkinHartley, male)
Guinea pig
(male) Strain,
unspecified
Syrian hamster,
male
Mouse (CD-1,
male and
female)
Oral gavage
4 weeks of age
3-6 months
(6 hr/day)
4-6 weeks of age
7 days
p.o.
Age, unspecified
10 days
0, 2,000 mg/kg/day
Oral gavage
4-6 weeks of age
9 days
11 weeks of age
106 days
0, 2,000 mg/kg/day
Mouse (ICR,
female)
By Feeding
Mated at the age
of 8-16 weeks
On gestation days
0-18
Mouse
(B6C3F1,
female)
By Feeding
Age, unspecified
Gestation day 0lactation day 28
(48 days)
0, 0.05, 0.1, 0.2,
0.4, 1.0%
(Corresponding to
0, 80, 180, 350, 660
and 2,100
mg/kg/day)
0, 20,000 ppm
(Corresponding to 0
and 2,600
mg/kg/day)
Animal species
Rat (F344,
male)
By Feeding
Dose
0, 2,500, 5,000,
10,000, 20,000,
40,000 ppm
(Corresponding to
0, 176, 359, 720,
1,540 and 2,964
mg/kg/day)
0, 2,000 mg/kg/day
0, 0.03, 0.3, 1.0 %
(Corresponding to
0, 52.5, 525 and
1,750 mg/kg/day)
267
Results
References
Focal atrophy of seminiferous
tubules in testis at 720 mg/kg/day or
above, decreases in testis weight,
testicular concentration of zinc and
serum concentration of testosterone
at 1,540 mg/kg/day or above and
decrease in serum concentration of
zinc at 2,964 mg/kg/day.
No effect on the relative testis
weight.
CERHR,
2000,
Marsman,
1995
Decreased testis weight and diffuse
atrophy of seminiferous tubules at
2,000 mg/kg/day.
Decreased testis weight and
degeneration of Sertoli cells at
2,000 mg/kg/day.
No abnormal changes in the testis.
Gray et al.,
1982
Decrease in fertility, number of
pups delivered and number of live
pups at 1,750 mg/kg/day.
In cross-over mating, decrease in
fertility, number of pups delivered,
number of live pups and body
weight of live fetuses in females in
the high dose group mated with
control males.
Decreased maternal body weight,
increased embryonic deaths and
exencephaly/spina bifida at
2,100 mg/kg/day.
Complete resorptions of all embryos
at 2,600 mg/kg/day.
Kawano,
1980
Gangolli,
1982
Gray et al.,
1982
Lamb et
al., 1987
Shiota,
1982
ATSDR,
1990,
Killinger et
al., 1988a
Di-n-butyl phthalate
Animal species
Rat (Wistar,
female)
Administration
method
Oral gavage
Administration
period
Mated at the age
of 14 weeks
On one day
during gestation
days 6-16
Dose
Results
References
0, 1,500 mg/kg/day
Suppressed maternal body weight
gain immediately after
administration in all DBP groups.
Increased post-implantation losses
upon exposure upon gestation days
6, 8-10 and 12-16.
A decrease in number of live fetuses
upon exposure on gestation days 9
and 13-15.
Ema et al.,
1997
Rat (SD,
female)
Oral gavage
Age, unspecified
Gestation day 14
0, 500, 1,000, 1,500,
2,000 mg/kg/day
Rat (Wistar,
female)
Oral gavage
Mated at the age
of 10-14 weeks
Gestation days 79
0, 750, 1,000, 1,500
mg/kg/day
Mated at the age
of 10-14 weeks
Gestation days
10-12
0, 750, 1,000, 1,500
mg/kg/day
Mated at the age
of 10-14 weeks
Gestation days
13-15
0, 750, 1,000 ,1,500
mg/kg/day
268
Skeletal malformations upon
exposure on gestation day 8.
Skeletal malformations,
exencephaly, dilatation of renal
pelvis, etc. upon exposure on
gestation day 9.
Skeletal malformations and cleft
palate upon exposure on gestation
day 15.
Increased resorptions, decreased
fetal body weight and skeletal
abnormalities at 1,500 mg/kg/day or
above.
The transplacentral transfer of DBP
into fetuses was 0.12-0.15% or less
of the dose.
DBP is metabolized into MBP and
then transferred to fetal tissues.
Maternal deaths and complete
resorptions of all embryos at 1,500
mg/kg/day.
Skeletal malformations, increased
post-implantation losses, decreased
number of live fetuses and
decreased fetal body weight at 750
and 1,000 mg/kg/day.
Complete resorptions of all embryos
at 1,500 mg/kg/day.
Increased post-implantation losses
and decreased number of live
fetuses.
No malformed fetuses.
Maternal deaths and complete
resorptions of all embryos at 1,500
mg/kg/day. Cleft palate, skeletal
malformations and increased postimplantation losses at 750 and 1,000
mg/kg/day. Decreased number of
live fetuses and decreased fetal
body weight at 1,000 mg/kg/day.
Saillenfait
et al., 1998
Ema et al.,
1994
Di-n-butyl phthalate
Animal species
Rat (Wistar,
female)
Administration
method
Oral gavage
Administration
period
Mated at the age
of 10-14 weeks,
Gestation days 79
Dose
Results
References
0, 750, 1,000 ,1,250
mg/kg/day
Increases in the number of
malformed fetuses (skeletal
malformations) and postimplantation losses and decreases in
the number of live fetuses and fetal
body weight at 750 mg/kg/day or
above.
Increase in post-implantation losses
and decrease in number of live
fetuses at 750 mg/kg/day or above.
decrease in fetal body weight at 750
and 1,250 mg/kg/day.
No increase in the number of
malformed fetuses.
Increases in number of malformed
fetuses (cleft palate, abnormal
sternal fusion) and postimplantation losses at 750
mg/kg/day or above. Decreased
number of live fetuses at 1,000
mg/kg/day or above.
Suppressed maternal body weight
gain at 555 mg/kg/day or above.
Undescended testis and reduced
ano-genital distance (AGD) at 555
mg/kg/day or above. Decreased
fetal body weight, cleft palate and
abnormal sternal fusion at 661
mg/kg/day.
No effect on female reproductive
organs.
Suppressed maternal body weight
gain and decreased fetal body
weight at 1,000 mg/kg/day or
above. Increased resorptions,
decreased number of live fetuses
and undescended testis at 1,500
mg/kg/day
Suppressed maternal body weight
gain at 1,000 mg/kg/day or above.
Undescended testis and reduced
AGD at 500 mg/kg/day or above.
Increased resorptions, decreased
number of live fetuses and
decreased fetal body weight at
1,500 mg/kg/day.
Suppressed maternal body weight
gain and decreased fetal body
weight at 1,000 mg/kg/day or
above.
Ema et al.,
1995a
Mated at the age
of 10-14 weeks,
Gestation days
10-12
0, 750, 1,000 ,1,250
mg/kg/day
Mated at the age
of 10-14 weeks,
Gestation days
13-15
0, 750, 1,000 ,1,250
mg/kg/day
Rat (Wistar,
female)
By feeding
Mated at the age
of 10-14 weeks,
Gestation days
11-21
0, 0.5, 1.0, 2.0%
(Corresponding to
0, 331, 555 and 661
mg/kg/day)
Rat (Wistar,
female)
Oral gavage
Mated at the age
of 10-14 weeks,
Gestation days
12-14
0, 1,000, 1,500
mg/kg/day
Mated at the age
of 10-14 weeks,
Gestation days
15-17
0, 500, 1,000, 1,500
mg/kg/day
Mated at the age
of 10-14 weeks,
Gestation days
18-20
0, 1,000, 1,500
mg/kg/day
269
Ema et al.,
1998
Ema et al.,
2000
Di-n-butyl phthalate
Animal species
Rat (SD,
female)
Rat (SD,
female)
Administration
method
Oral gavage
Administration
period
Age, unspecified
Gestation days 321
Days 1-20 after
birth
Oral gavage
Mated at the age of 0, 100, 250, 500
8 weeks
mg/kg/day
Gestation days 1221
Dose
0, 250, 500, 750
mg/kg/day
270
Results
References
Effects on F1 animals:
Hypospadias, absent or hypoplastic
epididymis, degeneration and
atrophy of seminiferous tubules and
absence of germinal cells in male
offspring at 250 mg/kg/day or
above. Reduced AGD, testicular
atrophy and absence or atrophy of
the prostate and seminal vesicle at
500 mg/kg/day or above.
Decreased offspring viability at
750 mg/kg/day.
No effect on female reproductive
organs.
Effects on dams:
Decreased maternal body weight on
the day of delivery at 500
mg/kg/day.
Effects on F1 animals:
Delayed prepuce separation in male
offspring at 100 mg/kg/day or
above. Nipple retention and
reduced AGD in male offspring at
250 mg/kg/day or above.
Hypospadias, undescended testis,
hypoplasia of the prostate,
epididymis and vas deferens,
epithelial degeneration in
seminiferous tubules and
hyperplasia of interstitial cells in
testis in male offspring at 500
mg/kg/day.
Lowest-observed adverse-effectlevel (LOAEL) for F1 generation =
100 mg/kg/day.
Mylchreest
et al., 1998
Mylchreest
et al., 1999
Di-n-butyl phthalate
Administration
method
Oral gavage
Administration
Dose
period
Mated at the age of 0, 0.5, 5, 50, 100,
500 mg/kg/day
8 weeks
Gestation days 1221
Rat (F344,
female)
By feeding
0, 20,000 ppm
(Corresponding to
1,000 mg/kg/day)
Rat (Wistar,
female)
Oral gavage
Rat (LE, SD,
male and
female)
Oral gavage
Age, unspecified
Gestation day 0 lactation day 28
(48 days)
Age, unspecified
During gestation
period
From weaning
through growth,
mating and
lactation periods
Mating between
DBP-treated
animals and
untreated animals
Rat (LE,
female)
Oral gavage
Age, unspecified
Gestation days 1619
0, 500 mg/kg/day
Rat (SD,
female)
Oral gavage
Age, unspecified
Gestation day 14 day 3 after birth
0, 500 mg/kg/day
Animal species
Rat (SD,
female)
Results
References
No maternal toxicity.
Effects on F1 animals:
Retained nipple in male offspring at
100 mg/kg/day or above.
Hypospadias, absent ventral
prostate, hypoplastic epididymis,
hypoplastic vas deferens,
hyperplasia of testicular interstitial
cells, hypoplastic seminal vesicle,
atrophy of vas deferens, reduced
AGD and decreased weights of the
testis, seminal vesicle, epididymis,
prostate and levator anibalbocarvenosus in male offspring
at 500 mg/kg/day.
No-observed adverse-effect-level
(NOAEL) for F1 generation = 50
mg/kg/day, LOAEL = 100
mg/kg/day.
Complete resorptions of all embryos
at 1,000 mg/kg/day.
Mylchreest
et al., 2000
0, 120, 600
mg/kg/day
Increased resorptions at 600
mg/kg/day.
0, 250, 500 and
1,000 (only in
males) mg/kg/day
F0: Delayed sexual maturation in
both sexes at 250 mg/kg/day or
above. Reduced fertility at 500
mg/kg/day or above (1,000
mg/kg/day: Infertile).
Testicular atrophy and decreased
spermatogenic capacity in males at
500 mg/kg/day or above.
F1: Malformations, decreased
conception rate and decreased
epididymal sperm count at 250
mg/kg/day or above.
LOAEL for F1 generation = 250
mg/kg/day.
Increased resorptions, reduced
AGD, decreased weights of seminal
vesicle, prostate and
bulbospongiosus muscle+levator
ani-balbocarvenosus and retained
nipple at 500 mg/kg/day.
Decreased number of pups
delivered, reduced AGD,
hypospadias, testicular and
epididymal atrophy or hypoplasia,
decreased weights of the seminal
vesicle, prostate, epididymis, testis,
bulbospongiosus muscle+levator
ani-balbocarvenosus and penis and
retained nipple at 500 mg/kg/day.
271
ATSDR,
1990,
Killinger et
al., 1988b
Nikonorow,
1973
Gray et al.,
1999
Di-n-butyl phthalate
Animal species
Rat (F344,
female)
Rat (SD, male
and female)
Administration
method
By feeding
Administration
period
Age, unspecified
Gestation day 0 –
lactation day 28
(48 days)
NTP protocol
10 weeks of age
Continuous breeding protocol study
Dose
Results
0, 2,500 ppm
(Corresponding to 0
and 125 mg/kg/day)
Suppressed offspring body weight
gain at 125 mg/kg/day.
0, 0.1, 0.5 and 1.0%
in diet
(equivalent to 0, 52,
256 and 509
mg/kg/day in males
and to 0, 80, 385
and 794 mg/kg/day
in females)
F0: Suppressed body weight gain
and increased liver and kidney
weights in dams at 1%.
Decreased number of live F1 pups at
0.1% or above. Decreased body
weight of live F1 pups at 0.5% or
above.
F1: Increased kidney weight in
males at 0.5%. Decreased
copulation and pregnancy indexes,
decreased body weight in both
sexes, and increased liver and
kidney weights, decreased prostate,
seminal vesicle and testis weights,
decreases in epididymal sperm
count and testicular spermatid head
count, degeneration of seminiferous
tubules, hyperplasia of testicular
interstitial cells and poorly
developed epididymis in males at
1.0%.
Decreased body weight of live F2
pups at 0.1% or above.
In the cross-over mating, body
weight decreased in offspring from
the pairs between females in highdose group and control males.
272
References
ATSDR,
1990,
Killinger et
al., 1988b
Wine et al.,
1997
Di-n-butyl phthalate
<The results of mono-butyl phthalate as a metabolite.>
Animal species
Rat (SD, male)
Administration
method
Oral gavage
Rat (Wistar,
female)
Oral gavage
Rat (Wistar,
female)
Oral gavage
Administration
period
4-6 weeks of age,
9 days
Age, unspecified
Gestation days 715
Dose
Results
References
0, 2,000 mg/kg/day
Decreased testis weight and diffuse
atrophy of seminiferous tubules.
Increased fetal mortality, decreased
fetal body weight, skeletal
malformations, cleft palate and
dilatation of renal pelvis at 500
mg/kg/day or above.
Suppressed maternal body weight
gain at 625 mg/kg/day or above.
Skeletal malformations and
decreased fetal body weight at 500
mg/kg/day or above. Increased
post-implantation losses and
external malformations at 625
mg/kg/day or above. Decreased
number of live fetuses at 750
mg/kg/day.
Suppressed maternal body weight
gain at 625 mg/kg/day or above.
Increased post-implantation losses
and decreased number of live
fetuses at 625 mg/kg/day or above.
Decreased fetal body weight at 750
mg/kg/day.
No malformed fetuses.
Suppressed maternal body weight
gain at 500 mg/kg/day or above.
Increased post-implantation losses
at 500 mg/kg/day or above.
Decreased number of live fetuses,
cleft palate and abnormal sternal
fusion at 625 mg/kg/day or above.
Undescended testis in male pups
(30-40 days of age) at 300
mg/animal/day.
Gray et al.,
1982
CERHR,
2000, Ema
et al., 1995b
0, 250, 500, 625
mg/kg/day
Mated at the age of 0, 500, 625, 750
12 weeks,
mg/kg/day
Gestation days 7-9
Mated at the age of 0, 500, 625, 750
mg/kg/day
12 weeks,
Gestation days 1012
Mated at the age of 0, 500, 625, 750
mg/kg/day
12 weeks,
Gestation days 1315
Rat (Wistar
p.o.
King A, female)
Age, unspecified
Gestation days 1518
0, 300
mg/animal/day
273
Ema et al.,
1996b
Imajima et
al., 1997
Di-n-butyl phthalate
Attachment-3 Results of repeated-dose toxicity studies
Administration
method
By feeding
Administration
period
Age, unspecified
7 days
Mouse
Strain,
unspecified
Mouse
(B6C3F1, male
and female)
By feeding
Age, unspecified
21 days
By feeding
6 weeks of age
13 weeks
Mouse (CD-1,
male and
female)
By feeding
11 weeks of age
126 days
Rat
Strain,
unspecified
Rat
Strain,
unspecified
By feeding
Age, unspecified
21 days
By feeding
Age, unspecified
21 days
Corresponding to 0,
628 and 1,248
mg/kg/day
Rat (Wistar,
male and
female)
By feeding
Age, unspecified
34-36 days
Corresponding to 0
and 250 mg/kg/day
Rat (Wistar,
male and
female)
By feeding
Age, unspecified
35-45 days
Corresponding to 0
and 2,500
mg/kg/day
Animal species
Mouse (ICR,
male)
Dose
0, 20,000 ppm
(Corresponding to 0
and 2,600
mg/kg/day)
Corresponding to 0,
628 and 1,248
mg/kg/day
0, 1,250, 2,500,
5,000, 10,000,
20,000 ppm (Male:
Corresponding to 0,
163, 353, 812,
1,601 and 3,689
Female:
Corresponding to 0,
238, 486, 971,
2,137 and 4,278
mg/kg/day)
0, 0.03, 0.3, 1.0%
(Corresponding to
0, 52.5, 525 and
1,750 mg/kg/day)
Corresponding to 0
and 348 mg/kg/day
274
Results
References
Decreased body weight, increased
liver weight, decreased kidney
weight and decreased zinc
concentration in testis and liver at
2,600 mg/kg/day.
Decreased body weight at 1,248
mg/kg/day.
ATSDR,
1990, Oishi
& Hiraga,
1980b
Suppressed body weight gain and
increased liver weight in males at
812 mg/kg/day or above.
Increased kidney weight in females
at 238 mg/kg/day or above.
Eosinophilic granules, increased
staining intensity of cytoplasm and
increased lipofuscin granules in
hepatocytes in females at 4,278
mg/kg/day.
NOAEL = 353 mg/kg/day for
males, - for females.
Decreased body weight and
increased liver weight at 1,750
mg/kg/day.
CERHR,
2000,
Marsman
1995
Decreased blood cholesterol level
and increased liver weight at 348
mg/kg/day.
Increased liver weight at 628
mg/kg/day.
Increased kidney weight at 1,248
mg/kg/day.
Decreased body weight,
hepatocellular necrosis and
inhibition of hepatic mitochondrial
energy metabolism at 250
mg/kg/day.
Decreased body weight, increased
spleen weight and decreased
mitochondrial oxidation in liver at
2,500 mg/kg/day.
ASDR,
1990
CERHR,
2000;
Reel et
al., 1984
ATSDR,
1990;
Bell, 1982
ATSDR,
1990
ATSDR,
1990,
Murakami
et al. 1986a
ATSDR,
1990,
Murakami
et al.
1986b
Di-n-butyl phthalate
Animal species
Rat (Wistar,
male and
female)
Administration
method
By feeding
Administration
period
6 weeks of age
3 months
Dose
Results
References
0, 400, 2,000,
10,000 ppm
(Male:
Corresponding to 0,
27, 141 and 688
Female:
Corresponding to 0,
33, 162 and 816
mg/kg/day)
Peroxisome proliferation and
histopathological changes in liver,
decrease thyroid hormone (T3)
levels in serum and anemia in males
at 688 mg/kg/day. Increased liver
and kidney weights and decrease
thyroid hormone (T3) levels in
serum in females at 816 mg/kg/day.
CERHR,
2000,
BASF,
1992
NOAEL = 142 mg/kg/day for
males, 162 mg/kg/day for females.
Males: Decreases in hemoglobin
concentration and erythrocyte
count, increases in platelet count
and serum albumin, increased
palmitoyl CoA oxidase (PCAO)
activity in liver and increased liver
and kidney weights at 359
mg/kg/day or above. Suppressed
body weight gain and
histopathological changes in liver at
720 mg/kg/day or above.
Peroxisome proliferation in liver at
2,964 mg/kg/day.
Females: Increased PCAO activity
in liver at 356 mg/kg/day or above.
Increased liver and kidney weights
at 712 mg/kg/day or above.
Suppressed body weight gain at
1,413 mg/kg/day or above.
Peroxisome proliferation in liver at
2,943 mg/kg/day.
NOAEL = 176 mg/kg/day for
males, 177 mg/kg/day for females.
Decreased cytochrome P-450
content in lung at 2.5 ppm.
Rat (F344, male By feeding
and female)
5-6 weeks of age
13 weeks
0, 2,500, 5,000,
10,000, 20,000,
40,000 ppm (Male:
Corresponding to 0,
176, 359, 720,
1,540 and 2,964
Female:
Corresponding to 0,
177, 356, 712,
1,413 and 2,943
mg/kg/day)
Rat
Strain,
unspecified
Inhalation
Age, unspecified
6 hr/day×5 days
0, 2.5 ppm
Rat (Wistar,
male)
Inhalation
4 weeks of age
6 hr/day×5
days/weeks×3-6
months
0, 0.5, 50 mg/m3
(0, 0.044, 4.4 ppm)
Decreased body weight and
increased relative lung weight at 4.4
ppm.
Rabbit
Strain,
unspecified
Dermal
Age, unspecified
90 days
0, 4,200 mg/kg/day
Renal toxicities (details, unknown)
at 4,200 mg/kg/day.
275
CERHR,
2000,
Marsman
1995
ATSDR,
1990,
Walseth &
Nilsen
1984
ATSDR,
1990,
Kawano
1980a;
1980b
ATSDR,
1990,
Lehman
1955