hazard assessment of di-n-butyl phthalate

Di-n-butyl phthalate

241

Hazard assessment of Di-n-butyl phthalate

[Di-n-butyl phthalate, CAS No. 84-74-2]

Chemical name: Di-n-butyl phthalate

Synonyms: n-Butylphthalate, 1,2-Benzenedicarboxylic acid dibutyl ester, Dibutyl 1,2-

benzene dicarboxylate, DBP

Molecular formula: C16H22O4

Molecular weight: 278.34

Structural formula:

C-O-CH2CH2CH2CH3

C-O-CH2CH2CH2CH3

O

O

Appearance: Colorless to faint yellow viscous liquid1)

Melting point: -35°C1)

Boiling point: 340°C1)

Specific gravity: 0465.1204 =d

1)

Vapor pressure: 2.68 x 10-3Pa (25°C)1)

Partition coefficient: Log Pow = 4.9 (Calculated)1)

Degradability: 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: 1998: 11.982 t (Production 11.766 t, Import 216 t)3)

Usage: Used as plasticizer for vinyl chloride, vinyl acetate, nitrocellulose and metacryl

resins1).

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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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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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 10-4M (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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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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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 (β-

G) 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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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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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 ani-

balbocarvernosus 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 509-

794 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 Rat Rabbit

Oral LD50 More than 20,000 mg/kg 3,000 – 8,000 mg/kg* －

Inhalation LD50 －－－

Percutaneous LD50 －－ More than 20,000 mg/kg

Intraperitoneal LD50 4,000 mg/kg 3,050 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


251

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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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 DNA-

damaging potential (Kleinsasser, 2000). No reports are available about the in vivo

assays on DBP.


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Table 2 Results of mutagenicity/genotoxicity studies

Test method Cells/animal species used Results* ReferencesS. typhimurium strain TA100, S9(+/-), 13 - 50µg/m l(DBP induces significant increases in thenumber of revertant colonies under the S9(-)condition, but the increases are not 2-fold orgreater than that in the control group.)

- IPCS, 1997

S. typhimurium strains TA98, TA100, TA1535,and TA1537, S9(+/-), 100 - 10,000 µg/plate

- IPCS, 1997

S. typhimurium strains TA98, TA100, TA1535,TA1537 and TA2637, S9(+/-), 100 - 2,000µg/plate(Positive under the S9(-) condition in TA100and TA1535.)

+w IPCS, 1997

Reversemutation test

S. typhimurim strains TA98, TA100, TA1535and TA1537, S9(+/-), concentration thatproduces precipitation

- IPCS, 1997

Chinese hamster Don cells, 0.28 - 27.8 mg/m l - IPCS, 1997Human lymphocytes, 0.03 mg/m l - IPCS, 1997

Chromosomalaberration test

CHL cells, 0.03 mg/m l ± IPCS, 1997Sister chromatidexchange test

Chinese hamster Don cells, 0.28 - 27.8 mg/m l - IPCS, 1997

L5178Y mouse lymphoma cells, S9(+/-)(Positive under the S9(-) condition at theconcentration that produces severe cytotoxicity.)

+ IPCS, 1997Gene mutationtest

L5178Y mouse lymphoma cells, S9(+/-)(Positive under the S9(+) condition at 0.1 µl /ml.)

+ Barbar, 2000

Transformationassay

BALB/3T3 cells, 0.0034 – 0.082 µl /m l - Barbar, 2000

In vitro

DNA repair test Human mucosal cells of the upper respiratorytract, 354 µmol/m l

+ Kleinsasser, 2000

*-: Negative +: Positive +w: Weakly positive

(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).


254

Table 3 Carcinogenicity assessment by national and international organizations.

Organization Category Significance ReferencesEPA Group D Not classifiable as a human carcinogen. IRIS, 2002EU － No evaluation. ECB, 2000NTP － No evaluation NTP, 2000IARC – No evaluation IARC, 2001ACGIH － No evaluation ACGIH, 2001

Japan Society forOccupational Health － No evaluation

Japan Society forOccupational Health,

2001

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).


255

COOH

COOCH2CH2CH(OH)CH3

COOH

COOCH2CH2CH2CH3

COOCH2CH2CH2CH3

COOCH2CH2CH2CH3

(1)MBP(3)

(5)

COOH

COOCH2CH2CH2CH2OH

COOH

COOH

(4)

(6)

CH3CH2OH

(2)CH3

CH2OH

(2)

(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


256

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


257

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 on-

going 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.


258

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263


264

Attachment-1 Results of in vitro studies on receptor binding

Item Test methods and conditions Results Conclusion ReferencesMethods: Competitive binding assaywith [3H]-E2 as a ligand. Receptor:Human ER expressed inSf9/Baculovirus. Temperature: 25°C,pH: 7.4

IC50 value: 5.83 ×10- 5M(E2: 2.09×10-9M)Relative binding affinity(vs E2): (E2=1): 3.6×10-5

DBP has binding affinityfor ER.

(The binding affinity is1/28,000 that of E2.)

Nakai et al.,1999

Methods: Competitive binding assaywith [3H]-E2 as a ligand. Receptor:Uterine homogenate fromovariectomized SD rats.Temperature: 4°C, pH: 7.4

IC50 value: >1×10- 3M(E2: 8.99×10-10M)

DBP has no bindingaffinity for ER.

Blair et al.,2000

Methods: Competitive binding assaywith [3H]-E2 as a ligand. Receptor:Uterine homogenate from immatureSD rats. Concentration: 1 – 1000µM, Temperature: 30°C, pH: 7.6

IC50 value: 4.7 ×10- 5M(E2: 1.3×10- 9M)

DBP has binding affinityfor ER.

(The binding affinity is1/36,000 that of E2.)

Zacharewskiet al., 1998

ER bindingassay

Methods: Binding assay on human ER(recombinant ERα ligand domain)

IC50 value: >10-4M(E2: 1.2×10-9M)

DBP has no bindingaffinity for ER.

CERI, 2001

Cells: MCF-7 cells transientlytransfected with Gal4-human ER geneand Gal4-regulated luciferase reportergene.Concentration: 0.1, 1, 10µM (DBP), 1pM-10 nM (E2)

In the presence of 10 µMof DBP,the reporter genetranscription was 37% ofthe activity at 10 nM ofE2.

DBP activates ER-mediated genetranscription.

Cells: HeLa cells stably transfectedwith Gal4-human ER gene and Gal4-regulated luciferase reporter gene.Concentration: 0.1, 1, 10µM (DBP), 1pM-10 nM (E2)

Negative within a rangeof 0.1 - 10 µM.

DBP does not activateER-mediated genetranscription.


Cells: HeLa cells transfected withhuman ER expression plasmid and ERresponse element.Concentration: 10- 11-10-5M (DBP)

Negative for the agonistactivity within a range of10- 11-10-5.(E2: PC50: <10-11M)


CERI, 2001

Reporter geneassay withculturedrecombinantcells

Cells: HeLa cells transfected with ratER expression plasmid and ERresponse element.Concentration: 10- 11-10-5M (DBP)

Negative for the agonistactivity within a range of10- 11-10-5.(E2: PC50: <10-9M)


Yamasaki etal., 2001

Human ER-dependentyeast growthassay

Cells: S. cerevisiae PL3 straintransfected with human ER.Concentration: 10 µM (DBP), 1 nM(E2)Incubation: 5 days

Weak cell growth wasdetected on the 5th day.E2 induced obvious cellgrowth from the 3rd day.

DBP induces cellproliferation.


Yeast two-hybrid assay

Cells: Yeast transfected with Gal4DNA binding domain/human ERligand binding domain genes, Gal4activation domain/coactivator TIF2genes and β-galactosidase reportergene

REC10: >3×10-4M orabove (E2: 3×10-10M)


Nishihara etal., 2000

ER: Estrogen receptor E2: 17 β-estradiol IC50: 50% inhibition concentrationREC10: Concentration equivalent to 10% of activity at 10-7M of E2

PC50: Concentration equivalent to 50% of the maximal activity of E2.


265

Attachment-2 Results of studies on mammalian endocrine and reproductive systems

(1) Results of reproduction studies by screening technique

Animal species Administrationmethod

Administrationperiod Dose Results References

Rat (SD,female)

s.c. (uterotrophicassay, juvenilerat)

20 days of age3 days

0, 40, 200, 1,000mg/kg/day

No effect on uterine weight. Yamasaki etal., 2000

Rat (SD,female)Ovariectomizedat the age of 19days (n=10)

Oral gavage(uterotrophicassay,ovariectomizedrat)

31- 34 days of age4 days

0, 20, 200, 2,000mg/kg/day

No effect on uterine weight. Zacharewskiet al., 1998

Rat (SD,female)

s.c. (uterotrophicassay,ovariectomizedrat)

Age, unspecified2 days

DBP at 0, 200 and400 mg/kg/day, s.c.,or at 1,000mg/kg/day by oralgavage for 2 days,followed byprogesterone at 0.5mg/rat, s.c., on thethird day.

No effect on uterine weight. Gray et al.,1999

Rat(Alpk:ApfSD,male)Castrated at theage of 6 weeks

Oral gavage(Hershbergerassay)

Started from 8days aftercastration for10 days

DBP at 0, 500 and1,000 mg/kg/day +testosteronepropionate (TP) at0.4 mg/kg/day, s.c.

Four replicate assays wereperformed. In the second assay,weights of bulbospongiosus muscle+ levator ani muscle, bulbourethralgland, seminal vesicle and prostatedecreased significantly in 500mg/kg/day or higher groups ascompared with the control group(treated with TP). In other 2assays, however, no significantdecrease was observed in seminalvesicle and prostate weights in1,000 mg/kg/day group, and,moreover, no significant decreasewas observed in bulbourethral glandweight in one of these two assays.It is thought that DBP may have ananti-androgenic effect, but theresults were not definitelyreproducible in four replicateassays.

Ashby &Lefevre,2000b

22-23 days of age14 days

0, 500 mg/kg/day Decreased testis and seminal vesicleweights at 500 mg/kg/day.

22-23 days of age14 days of dosingperiod + 20 daysof recovery period

0, 500 mg/kg/day Decreased testis and epididymalweights at 500 mg/kg/day.


0, 500 mg/kg/day No abnormal changes in testis andaccessory reproductive organs.

Rat(Alpk:ApfSD,male)

Oral gavage


0, 500 mg/kg/day Decreased testis and seminal vesicleweights and delay in prepuceseparation at 500 mg/kg/day.

Ashby &Lefevre,2000a


266

(2) Results of reproductive/developmental toxicity studies


Administrationperiod

Dose Results References

Mouse (TO,male)

Oral gavage 4-6 weeks of age7-9 days

0, 2000 mg/kg/day Decreased testis weight and slightatrophy of seminiferous tubules at2,000 mg/kg/day.

Gray et al.,1982

Mouse, maleStrain,unspecified

p.o. Age, unspecified10 days

0, 2000 mg/kg/day Decreased testis weight andtesticular tissue injuries at 2,000mg/kg/day.

Gangolli,1982

Rat (SD, male) Oral gavage 4-6 weeks of age7-9 days

0, 2000 mg/kg/day Decreased testis weight and diffuseatrophy of vas deference at 2,000mg/kg/day.

Gray et al.,1982

Rat (Wistar,male)

By feeding 5 weeks of age1 week

0, 2%(Corresponding to 0and 1,000mg/kg/day)

Decreased testis weight and primaryspermatocytes and increasedconcentration of testosterone anddecreased concentration of zinc intestis at 1,000 mg/kg/day.

Oishi &Hiraga,1980a

Rat (Wistar,male)

Oral gavage 5 weeks of age15 days

0, 250, 500, 1,000mg/kg/day

Degeneration of seminiferoustubules, interstitial edema,decreased acid phosphatase (AP)activity and increased activities ofLDH, γ-GTP, β-glucuronidase (β-G)and glucose-6-phosphatedehydrogenase (G6PDH) in testis at250 mg/kg/day or above.Decreased testis weight, impairedspermatogenesis and decreasedsorbitol dehydrogenase (SDH)activity in testis at 500 mg/kg/dayor above.(The decreased SDH and increasedLDH are thought to indicatedestruction of germinal epithelium.β-G and γ-GTP are the markers forSertoli cells, and the increases inthese marker enzymes are thoughtto be associated with decrease ingerminal epithelium. AP exists inlysosomes of Sertoli cells andgerminal epithelium and is knownto increase during development ofprimary spermatocytes andtesticular maturation. Thedecrease in AP seems to implyinhibition of spermatogenicprocesses. The G6PDH activity isreported to increase at the time oftesticular injuries.)

Srivastavaet al., 1990


267




Rat (F344,male)

By Feeding 5-6 weeks of age13 weeks

0, 2,500, 5,000,10,000, 20,000,40,000 ppm(Corresponding to0, 176, 359, 720,1,540 and 2,964mg/kg/day)

Focal atrophy of seminiferoustubules in testis at 720 mg/kg/day orabove, decreases in testis weight,testicular concentration of zinc andserum concentration of testosteroneat 1,540 mg/kg/day or above anddecrease in serum concentration ofzinc at 2,964 mg/kg/day.

CERHR,2000,

Marsman,1995

Rat (Wistar,male)

Inhalation 4 weeks of age3-6 months(6 hr/day)

0, 0.5, 50 mg/m3

(0, 0.044, 4.4 ppm)No effect on the relative testisweight.

Kawano,1980

Guinea pig(Dunkin-Hartley, male)

Oral gavage 4-6 weeks of age7 days

0, 2,000 mg/kg/day Decreased testis weight and diffuseatrophy of seminiferous tubules at2,000 mg/kg/day.

Gray et al.,1982

Guinea pig(male) Strain,unspecified

p.o. Age, unspecified10 days

0, 2,000 mg/kg/day Decreased testis weight anddegeneration of Sertoli cells at2,000 mg/kg/day.

Gangolli,1982

Syrian hamster,male

Oral gavage 4-6 weeks of age9 days

0, 2,000 mg/kg/day No abnormal changes in the testis. Gray et al.,1982

Mouse (CD-1,male andfemale)

By Feeding 11 weeks of age106 days

0, 0.03, 0.3, 1.0 %(Corresponding to0, 52.5, 525 and1,750 mg/kg/day)

Decrease in fertility, number ofpups delivered and number of livepups at 1,750 mg/kg/day.In cross-over mating, decrease infertility, number of pups delivered,number of live pups and bodyweight of live fetuses in females inthe high dose group mated withcontrol males.

Lamb etal., 1987

Mouse (ICR,female)

By Feeding Mated at the ageof 8-16 weeksOn gestation days0-18

0, 0.05, 0.1, 0.2,0.4, 1.0%(Corresponding to0, 80, 180, 350, 660and 2,100mg/kg/day)

Decreased maternal body weight,increased embryonic deaths andexencephaly/spina bifida at2,100 mg/kg/day.

Shiota,1982

Mouse(B6C3F1,female)

By Feeding Age, unspecifiedGestation day 0-lactation day 28(48 days)

0, 20,000 ppm(Corresponding to 0and 2,600mg/kg/day)

Complete resorptions of all embryosat 2,600 mg/kg/day.

ATSDR,1990,

Killinger etal., 1988a


268




Rat (Wistar,female)

Oral gavage Mated at the ageof 14 weeksOn one dayduring gestationdays 6-16

0, 1,500 mg/kg/day Suppressed maternal body weightgain immediately afteradministration in all DBP groups.Increased post-implantation lossesupon exposure upon gestation days6, 8-10 and 12-16.A decrease in number of live fetusesupon exposure on gestation days 9and 13-15.

Skeletal malformations uponexposure on gestation day 8.Skeletal malformations,exencephaly, dilatation of renalpelvis, etc. upon exposure ongestation day 9.Skeletal malformations and cleftpalate upon exposure on gestationday 15.

Ema et al.,1997

Rat (SD,female)

Oral gavage Age, unspecifiedGestation day 14

0, 500, 1,000, 1,500,2,000 mg/kg/day

Increased resorptions, decreasedfetal body weight and skeletalabnormalities at 1,500 mg/kg/day orabove.The transplacentral transfer of DBPinto fetuses was 0.12-0.15% or lessof the dose.DBP is metabolized into MBP andthen transferred to fetal tissues.

Saillenfaitet al., 1998

Mated at the ageof 10-14 weeksGestation days 7-9

0, 750, 1,000, 1,500mg/kg/day

Maternal deaths and completeresorptions of all embryos at 1,500mg/kg/day.Skeletal malformations, increasedpost-implantation losses, decreasednumber of live fetuses anddecreased fetal body weight at 750and 1,000 mg/kg/day.

Mated at the ageof 10-14 weeksGestation days10-12

0, 750, 1,000, 1,500mg/kg/day

Complete resorptions of all embryosat 1,500 mg/kg/day.Increased post-implantation lossesand decreased number of livefetuses.No malformed fetuses.

Rat (Wistar,female)

Oral gavage

Mated at the ageof 10-14 weeksGestation days13-15

0, 750, 1,000 ,1,500mg/kg/day

Maternal deaths and completeresorptions of all embryos at 1,500mg/kg/day. Cleft palate, skeletalmalformations and increased post-implantation losses at 750 and 1,000mg/kg/day. Decreased number oflive fetuses and decreased fetalbody weight at 1,000 mg/kg/day.

Ema et al.,1994


269




Mated at the ageof 10-14 weeks,Gestation days 7-9

0, 750, 1,000 ,1,250mg/kg/day

Increases in the number ofmalformed fetuses (skeletalmalformations) and post-implantation losses and decreases inthe number of live fetuses and fetalbody weight at 750 mg/kg/day orabove.

Mated at the ageof 10-14 weeks,Gestation days10-12

0, 750, 1,000 ,1,250mg/kg/day

Increase in post-implantation lossesand decrease in number of livefetuses at 750 mg/kg/day or above.decrease in fetal body weight at 750and 1,250 mg/kg/day.No increase in the number ofmalformed fetuses.

Rat (Wistar,female)

Oral gavage


0, 750, 1,000 ,1,250mg/kg/day

Increases in number of malformedfetuses (cleft palate, abnormalsternal fusion) and post-implantation losses at 750mg/kg/day or above. Decreasednumber of live fetuses at 1,000mg/kg/day or above.

Ema et al.,1995a

Rat (Wistar,female)

By feeding Mated at the ageof 10-14 weeks,Gestation days11-21

0, 0.5, 1.0, 2.0%(Corresponding to0, 331, 555 and 661mg/kg/day)

Suppressed maternal body weightgain at 555 mg/kg/day or above.Undescended testis and reducedano-genital distance (AGD) at 555mg/kg/day or above. Decreasedfetal body weight, cleft palate andabnormal sternal fusion at 661mg/kg/day.No effect on female reproductiveorgans.

Ema et al.,1998


0, 1,000, 1,500mg/kg/day

Suppressed maternal body weightgain and decreased fetal bodyweight at 1,000 mg/kg/day orabove. Increased resorptions,decreased number of live fetusesand undescended testis at 1,500mg/kg/day


0, 500, 1,000, 1,500mg/kg/day

Suppressed maternal body weightgain at 1,000 mg/kg/day or above.Undescended testis and reducedAGD at 500 mg/kg/day or above.Increased resorptions, decreasednumber of live fetuses anddecreased fetal body weight at1,500 mg/kg/day.

Rat (Wistar,female)

Oral gavage


0, 1,000, 1,500mg/kg/day

Suppressed maternal body weightgain and decreased fetal bodyweight at 1,000 mg/kg/day orabove.

Ema et al.,2000


270




Rat (SD,female)

Oral gavage Age, unspecifiedGestation days 3-21Days 1-20 afterbirth

0, 250, 500, 750mg/kg/day

Effects on F1 animals:Hypospadias, absent or hypoplasticepididymis, degeneration andatrophy of seminiferous tubules andabsence of germinal cells in maleoffspring at 250 mg/kg/day orabove. Reduced AGD, testicularatrophy and absence or atrophy ofthe prostate and seminal vesicle at500 mg/kg/day or above.Decreased offspring viability at750 mg/kg/day.No effect on female reproductiveorgans.

Mylchreestet al., 1998

Rat (SD,female)

Oral gavage Mated at the age of8 weeksGestation days 12-21

0, 100, 250, 500mg/kg/day

Effects on dams:Decreased maternal body weight onthe day of delivery at 500mg/kg/day.Effects on F1 animals:Delayed prepuce separation in maleoffspring at 100 mg/kg/day orabove. Nipple retention andreduced AGD in male offspring at250 mg/kg/day or above.Hypospadias, undescended testis,hypoplasia of the prostate,epididymis and vas deferens,epithelial degeneration inseminiferous tubules andhyperplasia of interstitial cells intestis in male offspring at 500mg/kg/day.Lowest-observed adverse-effect-level (LOAEL) for F1 generation =100 mg/kg/day.



271


Administrationperiod Dose Results References

Rat (SD,female)

Oral gavage Mated at the age of8 weeksGestation days 12-21

0, 0.5, 5, 50, 100,500 mg/kg/day

No maternal toxicity.Effects on F1 animals:Retained nipple in male offspring at100 mg/kg/day or above.Hypospadias, absent ventralprostate, hypoplastic epididymis,hypoplastic vas deferens,hyperplasia of testicular interstitialcells, hypoplastic seminal vesicle,atrophy of vas deferens, reducedAGD and decreased weights of thetestis, seminal vesicle, epididymis,prostate and levator ani-balbocarvenosus in male offspringat 500 mg/kg/day.No-observed adverse-effect-level(NOAEL) for F1 generation = 50mg/kg/day, LOAEL = 100mg/kg/day.


Rat (F344,female)

By feeding Age, unspecifiedGestation day 0 -lactation day 28(48 days)

0, 20,000 ppm(Corresponding to1,000 mg/kg/day)

Complete resorptions of all embryosat 1,000 mg/kg/day.

ATSDR,1990,

Killinger etal., 1988b

Rat (Wistar,female)

Oral gavage Age, unspecifiedDuring gestationperiod

0, 120, 600mg/kg/day

Increased resorptions at 600mg/kg/day.

Nikonorow,1973

Rat (LE, SD,male andfemale)

Oral gavage From weaningthrough growth,mating andlactation periodsMating betweenDBP-treatedanimals anduntreated animals

0, 250, 500 and1,000 (only inmales) mg/kg/day

F0: Delayed sexual maturation inboth sexes at 250 mg/kg/day orabove. Reduced fertility at 500mg/kg/day or above (1,000mg/kg/day: Infertile).Testicular atrophy and decreasedspermatogenic capacity in males at500 mg/kg/day or above.F1: Malformations, decreasedconception rate and decreasedepididymal sperm count at 250mg/kg/day or above.LOAEL for F1 generation = 250mg/kg/day.

Gray et al.,1999

Rat (LE,female)

Oral gavage Age, unspecifiedGestation days 16-19

0, 500 mg/kg/day Increased resorptions, reducedAGD, decreased weights of seminalvesicle, prostate andbulbospongiosus muscle+levatorani-balbocarvenosus and retainednipple at 500 mg/kg/day.

Rat (SD,female)

Oral gavage Age, unspecifiedGestation day 14 -day 3 after birth

0, 500 mg/kg/day Decreased number of pupsdelivered, reduced AGD,hypospadias, testicular andepididymal atrophy or hypoplasia,decreased weights of the seminalvesicle, prostate, epididymis, testis,bulbospongiosus muscle+levatorani-balbocarvenosus and penis andretained nipple at 500 mg/kg/day.


272




Rat (F344,female)

By feeding Age, unspecifiedGestation day 0 –lactation day 28(48 days)

0, 2,500 ppm(Corresponding to 0and 125 mg/kg/day)

Suppressed offspring body weightgain at 125 mg/kg/day.

ATSDR,1990,

Killinger etal., 1988b

Rat (SD, maleand female)

NTP protocol10 weeks of ageContinuous breeding protocol study

0, 0.1, 0.5 and 1.0%in diet(equivalent to 0, 52,256 and 509mg/kg/day in malesand to 0, 80, 385and 794 mg/kg/dayin females)

F0: Suppressed body weight gainand increased liver and kidneyweights in dams at 1%.Decreased number of live F1 pups at0.1% or above. Decreased bodyweight of live F1 pups at 0.5% orabove.

F1: Increased kidney weight inmales at 0.5%. Decreasedcopulation and pregnancy indexes,decreased body weight in bothsexes, and increased liver andkidney weights, decreased prostate,seminal vesicle and testis weights,decreases in epididymal spermcount and testicular spermatid headcount, degeneration of seminiferoustubules, hyperplasia of testicularinterstitial cells and poorlydeveloped epididymis in males at1.0%.Decreased body weight of live F2

pups at 0.1% or above.

In the cross-over mating, bodyweight decreased in offspring fromthe pairs between females in high-dose group and control males.

Wine et al.,1997


273

<The results of mono-butyl phthalate as a metabolite.>




Rat (SD, male) Oral gavage 4-6 weeks of age,9 days

0, 2,000 mg/kg/day Decreased testis weight and diffuseatrophy of seminiferous tubules.

Gray et al.,1982

Rat (Wistar,female)

Oral gavage Age, unspecifiedGestation days 7-15

0, 250, 500, 625mg/kg/day

Increased fetal mortality, decreasedfetal body weight, skeletalmalformations, cleft palate anddilatation of renal pelvis at 500mg/kg/day or above.

CERHR,2000, Ema

et al., 1995b

Mated at the age of12 weeks,Gestation days 7-9

0, 500, 625, 750mg/kg/day

Suppressed maternal body weightgain at 625 mg/kg/day or above.Skeletal malformations anddecreased fetal body weight at 500mg/kg/day or above. Increasedpost-implantation losses andexternal malformations at 625mg/kg/day or above. Decreasednumber of live fetuses at 750mg/kg/day.


0, 500, 625, 750mg/kg/day

Suppressed maternal body weightgain at 625 mg/kg/day or above.Increased post-implantation lossesand decreased number of livefetuses at 625 mg/kg/day or above.Decreased fetal body weight at 750mg/kg/day.No malformed fetuses.

Rat (Wistar,female)

Oral gavage


0, 500, 625, 750mg/kg/day

Suppressed maternal body weightgain at 500 mg/kg/day or above.Increased post-implantation lossesat 500 mg/kg/day or above.Decreased number of live fetuses,cleft palate and abnormal sternalfusion at 625 mg/kg/day or above.

Ema et al.,1996b

Rat (WistarKing A, female)

p.o. Age, unspecifiedGestation days 15-18

0, 300mg/animal/day

Undescended testis in male pups(30-40 days of age) at 300mg/animal/day.

Imajima etal., 1997


274

Attachment-3 Results of repeated-dose toxicity studies




Mouse (ICR,male)

By feeding Age, unspecified7 days

0, 20,000 ppm(Corresponding to 0and 2,600mg/kg/day)

Decreased body weight, increasedliver weight, decreased kidneyweight and decreased zincconcentration in testis and liver at2,600 mg/kg/day.

ATSDR,1990, Oishi& Hiraga,

1980b

MouseStrain,unspecified


Corresponding to 0,628 and 1,248mg/kg/day

Decreased body weight at 1,248mg/kg/day.

ASDR,1990

Mouse(B6C3F1, maleand female)

By feeding 6 weeks of age13 weeks

0, 1,250, 2,500,5,000, 10,000,20,000 ppm (Male:Corresponding to 0,163, 353, 812,1,601 and 3,689Female:Corresponding to 0,238, 486, 971,2,137 and 4,278mg/kg/day)

Suppressed body weight gain andincreased liver weight in males at812 mg/kg/day or above.Increased kidney weight in femalesat 238 mg/kg/day or above.Eosinophilic granules, increasedstaining intensity of cytoplasm andincreased lipofuscin granules inhepatocytes in females at 4,278mg/kg/day.NOAEL = 353 mg/kg/day formales, - for females.

CERHR,2000,

Marsman1995

Mouse (CD-1,male andfemale)

By feeding 11 weeks of age126 days

0, 0.03, 0.3, 1.0%(Corresponding to0, 52.5, 525 and1,750 mg/kg/day)

Decreased body weight andincreased liver weight at 1,750mg/kg/day.

CERHR,2000;

Reel etal., 1984

RatStrain,unspecified


Corresponding to 0and 348 mg/kg/day

Decreased blood cholesterol leveland increased liver weight at 348mg/kg/day.

ATSDR,1990;

Bell, 1982RatStrain,unspecified


Corresponding to 0,628 and 1,248mg/kg/day

Increased liver weight at 628mg/kg/day.Increased kidney weight at 1,248mg/kg/day.

ATSDR,1990

Rat (Wistar,male andfemale)

By feeding Age, unspecified34-36 days

Corresponding to 0and 250 mg/kg/day

Decreased body weight,hepatocellular necrosis andinhibition of hepatic mitochondrialenergy metabolism at 250mg/kg/day.

ATSDR,1990,

Murakamiet al. 1986a


By feeding Age, unspecified35-45 days

Corresponding to 0and 2,500mg/kg/day

Decreased body weight, increasedspleen weight and decreasedmitochondrial oxidation in liver at2,500 mg/kg/day.

ATSDR,1990,

Murakamiet al.1986b


275





By feeding 6 weeks of age3 months

0, 400, 2,000,10,000 ppm(Male:Corresponding to 0,27, 141 and 688Female:Corresponding to 0,33, 162 and 816mg/kg/day)

Peroxisome proliferation andhistopathological changes in liver,decrease thyroid hormone (T3)levels in serum and anemia in malesat 688 mg/kg/day. Increased liverand kidney weights and decreasethyroid hormone (T3) levels inserum in females at 816 mg/kg/day.

NOAEL = 142 mg/kg/day formales, 162 mg/kg/day for females.

CERHR,2000,BASF,1992

Rat (F344, maleand female)

By feeding 5-6 weeks of age13 weeks

0, 2,500, 5,000,10,000, 20,000,40,000 ppm (Male:Corresponding to 0,176, 359, 720,1,540 and 2,964Female:Corresponding to 0,177, 356, 712,1,413 and 2,943mg/kg/day)

Males: Decreases in hemoglobinconcentration and erythrocytecount, increases in platelet countand serum albumin, increasedpalmitoyl CoA oxidase (PCAO)activity in liver and increased liverand kidney weights at 359mg/kg/day or above. Suppressedbody weight gain andhistopathological changes in liver at720 mg/kg/day or above.Peroxisome proliferation in liver at2,964 mg/kg/day.Females: Increased PCAO activityin liver at 356 mg/kg/day or above.Increased liver and kidney weightsat 712 mg/kg/day or above.Suppressed body weight gain at1,413 mg/kg/day or above.Peroxisome proliferation in liver at2,943 mg/kg/day.NOAEL = 176 mg/kg/day formales, 177 mg/kg/day for females.

CERHR,2000,

Marsman1995

RatStrain,unspecified

Inhalation Age, unspecified6 hr/day×5 days

0, 2.5 ppm Decreased cytochrome P-450content in lung at 2.5 ppm.

ATSDR,1990,

Walseth &Nilsen1984

Rat (Wistar,male)

Inhalation 4 weeks of age6 hr/day×5days/weeks×3-6months

0, 0.5, 50 mg/m3

(0, 0.044, 4.4 ppm)Decreased body weight andincreased relative lung weight at 4.4ppm.

ATSDR,1990,

Kawano1980a;1980b

RabbitStrain,unspecified

Dermal Age, unspecified90 days

0, 4,200 mg/kg/day Renal toxicities (details, unknown)at 4,200 mg/kg/day.

ATSDR,1990,

Lehman1955

hazard assessment of di-n-butyl phthalate

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