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    BIS (2-ETHYLHEXYL)PHTHALATE

    EXPLANATION

         Bis (2-ethylhexyl)phthalate (DEHP) was evaluated previously at
    the twenty-eighth meeting of the Joint FAO/WHO Expert Committee on
    Food Additives (Annex 1, reference 66). The information evaluated
    included results of pharmacokinetic and metabolism studies of DEHP,
    data from reproduction, teratogenicity, mutagenicity, short- and
    long-term toxicity, and carcinogenicity studies, and results of
    special studies on testicular atrophy and on the relationship of
    DEHP administration to peroxisome proliferation in the liver. At
    that time it was concluded that DEHP is a hepatocarcinogen in both
    rats and mice. The Committee recommended that human exposure to
    DEHP as a result of its migration from food-contact materials be
    reduced to the lowest level technologically attainable. The
    Committee provisionally accepted the use of food-contact materials
    that contain bis (2-ethylhexyl)phthalate as a potential migrant
    into food, subject to the conditions outlined in Section 2.2 and
    the first paragraph of Section 3.2 of that report.

         Since the previous evaluation, additional data have become
    available on mechanisms of testicular atrophy and DEHP-induced
    hepatocarcinogenicity. Additional data relevant to these two
    mechanisms are summarized and discussed in the following monograph
    addendum.

    BIOLOGICAL DATA

    Special studies on testicular effects, fertility, and teratogenicity

         Rats, mice, guinea pigs, and ferrets have been shown to be
    susceptible to the induction of testicular injury by DEHP and other
    phthalate esters, but Syrian hamsters are comparatively resistant
    (Creasey et al., 1983; Gray et al., 1982b). In rats, repeated
    administration of DEHP results in seminiferous tubular atrophy that
    is characterized by a loss of the meiotic and post-meiotic germ
    cell populations from the seminiferous epithelium (Creasey et
    al., 1983), accessory sex gland atrophy, reduced testicular and
    anterior prostate zinc concentrations (Curto & Thomas, 1982), and
    increased testicular testosterone concentration (Oishi & Hiraga,
    1980). Characteristic testicular changes are similar in all
    susceptible species, involving early detachment of spermatocytes
    and spermatids from the seminiferous epithelium (Gray & Gangolli,
    1986). Morphological changes are evident in Sertoli cells within 3
    to 6 hours following a single dose of di-n-pentyl phthalate, at
    which time the germ cell population appeared unaffected (Creasey
    et al., 1983).

         Oishi & Hiraga (1983) demonstrated that testicular atrophy
    induced by DEHP could not be prevented by co-administration of
    zinc. The ability of dietary zinc and co-administered testosterone
    to inhibit DEHP-induced testicular atrophy in rats also has been
    studied. Agarwal et al. (1986) reported that adult male F344 rats
    on a zinc-deficient diet showed an enhanced susceptibility to the
    gonadotoxic effects of DEHP and argued that this finding supports
    the hypothesis that testicular zinc depletion is casually related
    to the ensuing testicular and accessory sex organ atrophies
    produced by DEHP. Parmar et al. (1987) reported that co-
    administration of testosterone and DEHP to adult male albino Wistar
    rats appeared to prevent testicular injury induced by DEHP
    administration alone. The authors suggested that these results
    argue for the involvement of testosterone in DEHP-induced
    testicular atrophy.

         Effects of age and hormones on induction of testicular atrophy
    in rats were investigated by Gray & Gangolli (1986). DEHP (0 and
    2800 mg/kg/day) was administered orally as corn oil solutions to
    six groups of eight male Sprague-Dawley rats (one group of four-,
    10-, and 15-week old rats per dose). DEHP was administered for 10
    days, after which the rats were killed. Administration of DEHP to
    four-week old rats produced a marked depression in the weight of
    the testes, seminal vesicle, and prostate. In 10-week old rats,
    DEHP administration produced only a slight reduction in testis
    weight but the weights of the seminal vesicle and prostate were
    significantly reduced. In 15-week old rats, DEHP had no effect on
    any of these organ weights. Histopathologically, the testes of the
    treated four-week old rats showed severe atrophy affecting
    virtually all tubules; tubules were populated only by Sertoli    

    cells, spermatogonia, and occasional primary spermatocytes. In the
    10-week old treated rats, these histological changes were present in
    from 5 to 50% of tubules; non-affected tubules appeared normal. No
    histological abnormalities were seen in testes from treated 15-week
    old rats.

         The age-dependent response of the rat testes to DEHP was also
    studied by Sjoberg et al. (1986b). Groups of male Sprague-Dawley
    rats (25, 40, and 60 days old at the beginning of the experiment)
    were given DEHP in the diet (dose adjusted to give a daily intake
    of 0, 1.0, or 1.7 g/kg bw) for 14 days; rats were then killed.
    Testicular weight was markedly reduced in the 25- and 40-day old
    rats given 1.7 g/kg DEHP. All tubules were affected with severe
    testicular damage, in the 25-day old rats. When the mean daily
    intake of DEHP was 1.0 g/kg, however, only a few tubules in each of
    the 25- and 40-day old animals was affected.

         The possibility that DEHP induces testicular atrophy by
    interfering with the production of testosterone or the pituitary
    gonadotrophins was examined in studies involving co-administration
    of these hormones with di-n-butyl phthalate (DBP). Six groups of
    six male Sprague-Dawley rats (4 to 5 weeks old) were given DBP
    orally as corn oil solutions (0 and 2000 mg/kg/day) for five days,
    after which they received 50 units of pregnant mares' serum
    gonadotrophin (PMSG) in corn oil by subcutaneous injection on the
    first two days of DBP treatment, and one group of rats from each
    dosage level received an aqueous solution of testosterone
    propionate (200 痢/kg/day) by subcutaneous injection daily during
    treatment with DBP. Rats treated with DBP alone showed a
    significant reduction in testis and seminal vesicle weight and
    severe testicular atrophy. Administration of PMSG or testosterone
    propionate did not markedly influence the effects of DBP on the
    testis. The authors concluded that these results suggest testicular
    lesions caused by DBP and other phthalate esters are not primarily
    due to lack of availability of pituitary hormones or testosterone,
    thus pointing to a site of action in the seminiferous tubules (Gray
    & Gangolli, 1986).

         Gray & Gangolli (1986) also studied the effects of some
    phthalate esters on two specific markers of Sertoli cell function,
    the secretion of seminiferous tubule fluid and of androgen binding
    protein (ABP), in 4- to 50 week old male phthalate in corn oil
    (2200 mg/kg), a production of fluid and ABP was almost completely
    suppressed. This effect was still marked at a dose of 440 mg/kg,
    but was not evident at 220 mg/kg. After three daily doses of DBP at
    220 mg/kg, however, one out of five rats was partially affected. A
    single oral dose of mono-2-ethylhexyl phthalate in corn oil (MEHP;
    1000 mg/kg), the principal metabolite of orally administered DEHP,
    reduced fluid and ABP production to approximately 50% of control
    levels. After three daily doses of MEHP (1000 mg/kg), fluid and ABP
    production was approximately 25% of control levels. However,
    
    diethyl phthalate, an ester that does not cause testicular atrophy
    (Foster et al., 1980), had no effect on these criteria of Sertoli
    cell function after tree daily oral doses of 1600 mg/kg in corn oil
    (a dose level equimolar with 220 mg/kg DBP).

         In contrast to the preceding results, when 10-week old male
    Sprague-Dawley rats were given a single oral dose of DBP in corn
    oil (2200 mg/kg), seminiferous tubule fluid and androgen binding
    protein production were only reduced to approximately 60% of
    control levels; oral administration of MEHP (1000 mg/kg in corn
    oil) produced no effect. MEHP was still without effect after three
    daily doses (Gray & Gangolli, 1986).

         Several researchers have attempted to identify the active
    metabolite of DEHP that affects the rat testis  in vivo and
     in vitro. Sjoberg et al. (1986a) reported that no testicular
    damage was observed in young male Sprague-Dawley rats receiving
    oral doses of DEHP or 2-EH (2.7 mmol/kg bw) daily for five days.
    In animals receiving corresponding doses of MEHP, however, the
    number of degenerated spermatocytes and spermatids was increased,
    whereas no such effects were seen in animals given MEHP-derived
    metabolites. MEHP (at concentrations as low as 10 Molar) was the
    only compound that enhanced germ cell detachment from mixed primary
    cultures of Sertoli and germ cells. The authors suggested that
    these results indicate the probability that effects on the testes
    observed after administration of DEHP are exerted by its metabolite
    MEHP.

         Because of the effects of phthalate esters on Sertoli cells
    and the early separation of germ cells from Sertoli cells observed
     in vivo, Gray & Beamand (1984) examined the use of primary
    cultures of Sertoli and germ cells as an  in vitro model for
    phthalate-induced testicular toxicity. Addition of 100 然 MEHP to
    the culture medium for 24 hours resulted in a pronounced detachment
    of germ cells from the Sertoli cell monolayer and a change in
    Sertoli cell morphology to a more elongated shape. The effect of
    MEHP was shown to be concentration dependent over the range of 1 to
    100 然. No such changes were produced by DEHP (up to 100 然) or by
    its other primary metabolite, 2-ethylhexanol (EH). Three
    metabolites of MEHP (compounds V, VI, and IX described by Albro et
    al. in 1973) were also tested: compounds V and VI had no effect at
    100 然, but compound IX did produce a slight increase in germ cell
    detachment. In studies with a range of phthalate monoesters, it was
    found that only those causing testicular damage  in vivo produced
    an increase in germ cell detachment at low concentrations (1 to 100 然)
    in culture; of the phthalate monoesters tested, MEHP produced the most
    marked response (Gray & Gangolli, 1986). Finally, because the germ
    cells detaching from the monolayer cultures were viable and
    morphologically normal, and because morphological changes were seen
    in the still-attached Sertoli cells, Gray & Gangolli (1986) suggested
    that these results indicate that the action of phthalates may be
    mediated via a primary effect on Sertoli cells.

         Saxena et al. (1985) investigated the role of several
    enzymes in DEHP-induced testicular injury in rats. Two groups of
    six male Wistar albino rats (13 weeks old) were given either saline
    or DEHP (2000 mg/kg bw) orally for 7 consecutive days. All animals
    were sacrificed on day 8 of the experiment. Following treatment
    with DEHP, relative organ weight of the testis was not
    significantly different from that of controls. Histopathology of
    DEHP-treated testes, however, revealed focal interstitial edema and
    degenerative changes in seminiferous tubules. The following enzyme
    changes were observed following DEHP treatment: succinic
    dehydrogenase activity, distributed throughout the seminiferous
    tubules and interstitial tissue in control rats, was markedly
    reduced; glucose-6-phosphate dehydrogenase activity, demonstrated
    in interstitial cells and tubular epithelium of control animals,
    was increased in interstitial tissue of the testes; NADH-diaphorase
    activity, localized in seminiferous tubules and interstitial tissue
    in control rats, demonstrated decreased activity in the
    interstitial tissue where edema was not noted; ATPase activity,
    observed in the basement membrane and interstitial tissue of
    control rats, was enhanced; alkaline phosphatase activity,
    localized in basement membrane and interstitial tissue of control
    animals, was slightly enhanced; and acid phosphatase activity,
    observed in the form of granules throughout the cross section of
    the control rat, was reduced. The authors suggested that these
    alterations indicate that disruption of cellular energetics in the
    testes may be responsible for DEHP-associated infertility in male
    rats.

         Changes in cell-specific enzyme activities during DEHP-induced
    testicular atrophy were also investigated by Oishi (1986). Ten
    groups of seven male Wistar rats (30 days old) were administered
    saline (2 ml/kg/day) or DEHP (2 g/kg/day, without vehicle) by
    gavage daily. One pair each of the control and DEHP-treated groups
    was sacrificed after 0, 1, 3, 6, or 10 days. Specific activities of
    testicular enzymes associated with postmeiotic spermatogenic cells,
    such as lactate dehydrogenase isozyme-X, hyaluronidase, and
    sorbitol dehydrogenase, were lower than those of control animals by
    day 10, which was coincident with degeneration of spermatogenic
    cells in this experiment. The specific activities of enzymes
    associated with premeiotic spermatogenic cells, Sertoli cells, or
    interstitial cells were higher than those of control animals by day
    10. The specific activities of alcohol dehydrogenase and aldolase,
    zinc-containing enzymes, increased after DEHP treatment in spite of
    the decrease of zinc concentration in the testis. The authors
    noted, however, that all of these changes occurred after or
    simultaneous with massive histological or morphological changes
    rather than prior to such changes.

         Parmar et al. (1986) observed increases in the activities of
    gamma-glutamyl transpeptidase and lactate dehydrogenase in adult
    male albino rats receiving oral doses of 250, 500, 1000 and 2000
    mg/kg DEHP in groundnut oil per day for 15 days. An increase in the
    activity of beta-glucuronidase and decrease in the activity of acid

    phosphatase was observed at the highest dose of DEHP. The authors
    concluded that these results suggest that DEHP can affect
    spermatogenesis by altering the activities of enzymes responsible
    for the maturation of sperm.

         Agarwal et al. (1986) investigated the recovery from
    DEHP-induced testicular toxicity produced upon discontinuance of
    exposure in sexually mature rats. Five groups of 24 male F344 rats
    (15-16 weeks old) were administered DEHP in the diet (0, 320, 1250,
    5000, or 20,000 mg/kg diet; ppm) for 60 consecutive days. Eight
    rats from each group were sacrificed after having been maintained
    on a normal (not containing DEHP) diet for five days following the
    60-day treatment period; the remaining sixteen rats per group were
    placed on a normal diet for 70 days, than killed. Dietary
    administration of DEHP produced toxicity evidenced by reduced
    testicular and accessory organ weights, loss of testicular zinc,
    and induction of seminiferous tubular atrophy. The toxic response
    was dose-dependent and statistically significant with varying
    severity depending upon the target tissue: Histopathological
    evidence of tissue injury to the testis was characterized by severe
    atrophy of the seminiferous tubules and loss of spermatogenesis in
    rats fed a diet containing 20,000 ppm DEHP. Cessation of exposure
    to DEHP initiated partial to complete recovery from toxicity in
    most cases; magnitudes of recovery were available, with the gonads
    being slower that other systems (such as liver).

         Oishi (1985), however, found limited reversibility of
    testicular atrophy induced by DEHP in young rats. Two groups of 20
    male Wistar rats (95-112 g) were administered either a saline
    solution (2 ml/kg/day) or DEHP (2.0 g/kg/day, without vehicle) by
    gavage for 14 days. Ten rats from each group were killed one day after
    treatment. The remaining ten animals per group were killed 45 days
    after cessation of administration of saline or DEHP. For rats
    killed on day 15 of the experiment, testicular and accessory sex
    organ weights for rats administered DEHP were significantly less
    than those of control animals. Histological changes in the testes
    of DEHP-treated rats were characterized by a marked shrinkage of
    seminiferous tubules: The germinal epithelium consisted of only
    Sertoli cells, very few spermatogonia, and several multinucleated
    cells. At 45 days after termination of DEHP administration, the
    majority of tubules showed little more than a lining of Sertoli
    cells, although a small number showed a partially intact germinal
    epithelium. Spermatocytes, spermatids, and spermatozoa were seen in
    the few tubules in which spermatogenesis was regenerated. The
    percentage of spermatogenic tubules in a representative cross
    section was 0 and 12.8%, respectively, at termination of the 2-week
    DEHP treatment and following the 45-day recovery period.

         Douglas et al. (1986) studied mutagenic and other genotoxic
    effects of phthalate esters in adult mice and rats and in Chinese
    hamster ovary cells  in vitro. When 6-8 week old B6C3F1 mice (five
    mice per group) were given intraperitoneal injections of DEHP in


    olive oil (0, 0.6, 3.0, or 6.0 g/kg bw/day) for five consecutive days,
    the numbers of morphologically abnormal sperm in treated groups did
    not differ from controls in the 12 weeks following treatment. When
    6-8 week old Sprague-Dawley rats (three rats per group) were given
    intraperitoneal injections of DEHP in olive oil (0, 0.52, 2.6, or
    5.2 g/kg bw/day) for five consecutive days, the numbers of
    morphologically abnormal sperm were also unaffected by DEHP
    treatment. In addition, DEHP treatment of Chinese hamster ovary
    cultures at concentrations up to 10 mM for one hour induced neither
    sister chromatid exchange nor DNA damage.

         The antifertility and mutagenic effects of parenteral
    administration of DEHP to mice was investigate by Agarwal et al.
    (1985) using a modified dominant lethal test emphasizing the
    repeated administration of small doses of the test compound. Adult
    male and female ICR mice (8-10 weeks old) were used in the
    experiment. Groups of eight male mice were administered DEHP
    subcutaneously (0.99, 1.97, 4.93, or 9.86 gm/kg bw) on days 1, 5,
    and 10; two groups of eight control mice each were given
    subcutaneous injections of normal saline. On day 21 of the
    experiment, each male was housed with one virgin female for seven
    days. Female mice were sacrificed on day 13 of gestation: total
    number of corpora lutea, implantations, early fetal deaths, and
    viable fetuses were determined. The number of preimplantation
    losses, an indirect indication of mutagenesis, was calculated from
    the difference between the number of corpora lutea and
    implantations in each animal. Increases in the incidences of
    preimplantation losses and early fetal deaths were observed in
    DEHP-treated groups. The authors concluded that calculated
    mutagenic indices for control and DEHP-treated mice suggest a
    dominant lethal mutation effect in treated mice.

         Agarwal et al. (1986) investigated the effect of DEHP on
    reproductive performance of male rats. Following dietary exposure
    to DEHP and return to a normal diet for five days, twenty-four male
    rats from each of five DEHP-dosage groups (0 to 20,000 mg/kg diet;
    ppm) were housed individually with two sexually mature virgin
    females (untreated) for five days. Mated females were housed
    separately and allowed to litter naturally. Sixteen of the twenty-
    four rats from each of the DEHP-dosage groups were maintained on a
    normal diet for an additional 65 days, then mated for the
    assessment of reproductive performance as described above. From
    these experiments, the effects of DEHP on the incidence of
    pregnancy, litter size, litter weight, and growth of pups up to 7
    days of age were determined. The incidence of pregnancy, mean
    litter weight on day 1, frequencies of stillbirths and neonatal
    deaths  and mean litter growth up to 7 days of age were unaffected
    by DEHP treatment; however, mean litter size was significantly
    reduced at 20,000 ppm DEHP. The authors suggested that these data
    indicate a lack of reproductive dysfunction in F344 male rats at
    DEHP doses below 20,000 ppm, a dose which also produced measurable
    testicular degeneration and effects on sperm morphology.

         Reproductive toxicity of DEHP in mice was assessed using the
    National Toxicology Program (NTP) Fertility Assessment by
    Continuous Breeding Protocol (Lamb et al., 1986; Melnick et
    al., 1987). Male and female CD-1 mice were fed diets containing 0,
    0.01, 0.1, or 3.0% DEHP during a 7-day pre-mating period and a
    subsequent 98-day cohabitation period. For the continuous breeding
    phase, males and females from the same dose group were randomly
    paired and cohabitated for 98 days, with one breeding pair per
    cage; the control group consisted of 40 pairs and each dose group
    consisted of 20 pairs. After cohabitation, the breeding pairs were
    separated and continued individually on treatment for 20 more days.
    On each day of delivery, offspring were counted and weighed by sex,
    then removed and killed. Continuous dietary exposure of CD-1 mice
    to DEHP during the experiment resulted in complete suppression of
    fertility in the 0.3% dose group and significant reduction of
    fertility in the 0.1% group compared to the control group. In
    addition, breeding pairs in the 0.1% treatment group had fewer male
    and female live pups per litter and a lower proportion of pups born
    alive per litter than did the breeding pairs in the control group.
    There was no effect of DEHP on fertility in the 0.01% treatment
    group.

         In a crossover mating trial in which high-dose (0.3%) males
    and females were randomly bred with control mice (animals were not
    exposed to DEHP during this trial), the proportion of detected
    matings did not differ among treatment groups; however, fertility
    was significantly reduced for the 0.3% DEHP-treated males paired
    with control males (0% fertility) versus the control  control
    mating group (90% fertility). In addition, the proportion of pups
    born alive was significantly lower in the 0.3% DEHP-treated males
    paired with control females compared to the control  control
    mating group. The authors concluded that, under the conditions of
    these studies, DEHP was a reproductive toxicant in both male and
    female CD-1 mice (Melnick et al., 1987).

         In 1987, Melnick et al. (1987) reported the results of
    studies by the National Toxicology Program (NTP); developmental
    toxicity of DEHP was evaluated in Fischer 344 rats and in CD-1 mice
    (Tyl et al., 1987). DEHP was administered to pregnant rats in the
    diet at concentrations of 0, 0.5, 1.0, 1.5, or 2.0% on gestational
    days 0 through 20. On gestational day 20, dams were killed and
    evaluated for ovarian corpora lutea count, gravid uterine weight,
    and status of uterine implantation sites. Live fetuses were
    evaluated for viability, body weight, sex, gross morphological
    abnormalities, and visceral or skeletal malformations. On
    gestational day 20, mean absolute maternal weight gain (body weight
    gain during gestation minus gravid uterine weight) was
    significantly lower in the 1.0, 1.5. and 2.0% close groups compared
    to the control group, and the mean gravid uterine weight was
    decreased in the 2% dose group. There were no significant
    differences in the number of implantation sites per litter in the
    DEHP-treated pregnant rats compared to controls; however, the
    percent resorptions per litter was significantly increased in the
    2.0% dose group.

    Treatment with DEHP did not affect the ratio of males to females,
    but did cause a dose-related decrease in mean fetal body weight per
    litter for both males and females. The number of fetuses malformed
    per litter and the percent malformed fetuses were not significantly
    different between DEHP-treated groups and the control groups. The
    authors conclude that, at dose levels that caused maternal and
    fetal toxicity (1.0 to 2.0%), DEHP did not produce increases in the
    incidence of malformed rat fetuses.

         In the NTP mouse developmental toxicity study, DEHP was
    administered in the feed at concentrations of 0, 0.025, 0.05, 0.10,
    or 0.15% on gestational days 0 through 17. Dose selection was based
    on results from a preliminary toxicity study in pregnant CD-1 mice
    in which 0.3% or higher DEHP in the diet caused maternal toxicity
    (decrease in maternal weight gain) and 100% resorptions. Mean
    absolute maternal weights were lower in the 0.10 and 0.15% dose
    groups than in the control group. There were no dose-related
    differences in the number of implantation sites per litter in the
    DEHP-treated pregnant mice compared to controls; however, the
    percent of resorptions per litter was significantly increased in
    the 0.10 and 0.15% dose groups, the mean fetal body weight was
    significantly reduced only in the 0.15% dose group compared to
    controls. The percentage of fetuses with malformations and the
    percentage of malformed fetuses per litter exhibited a significant
    positive trend with 0.05-0.15% doses of DEHP. Gross defects
    included eye and tail defects and exencephaly; visceral
    malformations were predominantly aortic and pulmonary arch defects;
    skeletal malformations included rib and thoracic central defects.
    The authors concluded that DEHP was teratogenic to CD-1 mice when
    administered in the feed at dose levels which produced maternal and
    other fetal toxicity (0.10 and 0.15%) and at a dose level (0.05%)
    which did not produce maternal or other fetal toxicity. At 0.025%
    DEHP in the feed, there was no significant maternal or fetal
    toxicity, including teratogenicity (Melnick et al., 1987).

         Shiota & Mima (1985) reported on the teratogenicity of DEHP
    and its principle metabolite, MEHP, in mice. Ten to 18-week old
    female ICR mice were placed overnight with a male of proven
    fertility. On days 7, 8, and 9 of pregnancy, DEHP (0, 250, 500,
    1000, 2000 mg/kg bw) or MEHP (0, 50, 100, or 200 mg/kg bw)
    dissolved in olive oil were administered by gavage or by
    intraperitoneal injection. On day 18 of pregnancy, female mice were
    killed and the number and position of implantations, resorptions,
    and dead fetuses were recorded. Live fetuses were weighed, sexed,
    and inspected for gross external abnormalities. Examination of live
    fetuses for skeletal and internal soft tissue abnormalities was
    omitted in this study because DEHP has been reported not to
    increase these kinds of abnormalities in ICR mice (Shiota &
    Nishimura, 1982). The authors reported that MEHP was more toxic to
    female mice than DEHP. Among females that maintained their
    pregnancy until term, there was no significant difference in the
    average number of implants between control groups and groups given

    DEHP or MEHP. The average weight of viable fetuses at term, however,
    showed a dose-related decrease in groups receiving DEHP by gavage 
    (the decrease was significant at 100 and 2000 mg/kg). Oral
    administration of DEHP increased the incidence of malformed viable
    fetuses in a dose-related manner; anterior neural tube defects were
    the malformations most commonly induced. The authors reported that
    intraperitoneal administration of high doses of DEHP was
    abortifacient, but that DEHP administered orally failed to exert
    any notable teratogenic effects at doses below the abortifacient
    doses. In addition, the authors reported that there was no
    indication that either oral or intraperitoneal administration of
    MEHP was teratogenic.

         Tomita et al. (1986) reported that MEHP exerted embryo/
    fetotoxic effects similar to those of DEHP at lower doses.  Oral
    administration of MEHP (1 ml/kg) to mice at 8 days of gestation
    resulted in less than 32% live fetuses, all of which were
    deformed. A single injection of MEHP (25 or 50 mg/kg), but not DEHP
    (500 mg/kg), into pregnant mice induced a significantly higher
    incidence of somatic mutations in the coat hair of the offspring.
    The authors suggested that this data indicates that MEHP could be
    responsible for the embryotoxic and fetotoxic effects observed with
    DEHP. In contrast to this theory, Ritter et al. (1987)
    hypothesize that DEHP exerts its teratogenic effects by  in vivo
    hydrolysis to 2-ethylhexanol, which in turn is metabolized to
    2-ethylhexanoic acid, the proximate teratogen. When these three
    agents were administered to Wistar rats on day 12 of gestation,
    teratogenic responses indicated that they act through a common
    mechanism and that, on an equimolar basis, DEHP was least potent,
    2-ethylhexanol was intermediate in potency, and 2-ethylhexanoic
    acid was the most potent teratogen.

    Special studies on peroxisome proliferation and hepatocarcinogenicity

    General hepatic effects of peroxisome proliferators

         Dietary administration of compounds known to cause peroxisome
    proliferation in the liver also cause liver hyperplasia and
    hypertrophy. Enlargement of the liver begins shortly after the
    peroxisome proliferator is first fed to the test animal. In
    rodents, there is a gradual increase in liver size for 2 to 3
    weeks; this final increased size is maintained for as long as the
    peroxisome proliferator is administered. The liver returns to
    normal weight within days after the peroxisome proliferator is
    discontinued. Radioactive labeling and mitotic indices suggest that
    the increase in liver weight is a genuine hyperplastic response
    associated with the synthesis of new DNA. Both DEHP and its
    principal hydrolysis product, MEHP, produce liver peroxisomal
    proliferation and hypertrophy (National Toxicology Program, 1983;
    Nair & Karup, 1986).

         Smith-Oliver & Butterworth (1987) have reported the
    correlation of the carcinogenic potential of DEHP with induced
    hyperplasia rather than with genotoxic activity in the liver of
    male mice. Genotoxicity was determined by the extent of DNA repair
    (unscheduled DNA synthesis) and cell replication was determined by
    the percentage of cells undergoing scheduled DNA synthesis.
    Unscheduled and scheduled DNA synthesis were determined by
    autoradiographic quantification of radiolabeled thymidine
    incorporation in primary hepatocyte cultures treated directly or
    isolated from adult (28-33 g) male B6C3F1 mice treated  in vivo.
    For the  in vivo studies, DEHP was administered in the diet at a
     concentration of 6000 ppm or given by gavage as a corn oil
    solution (500 mg/kg); for the  in vitro studies, cells were
    incubated for 18 hours (1.5 hours after attachment) with DEHP
    (0, 0.01, 0.1, or 1.0 然) or MEHP (0, 0.1, 0.2, or 0.5 M) in
    dimethyl sulfoxide. DNA repair was not detected in mouse
    hepatocytes treated  in vitro with DEHP or MEHP. DNA repair also
    was not induced in hepatocytes isolated from mice treated with DEHP
    for 12, 24, or 48 hours before sacrifice. However, a 15-fold
    increase in the percentage of cells undergoing scheduled DNA
    synthesis was observed in cells from male mice with 6000 ppm DEHP
    in the diet for 7, 14, and 28 days did not stimulate the induction
    of DNA repair in hepatocytes, however, the percentage of cells
    undergoing scheduled DNA synthesis increased to over 9% at day 7
    but returned to control values (0.3%) by day 14. The liver to body
    weight ratio at day 28 was greater for DEHP-fed mice (8.3%) than
    for control mice (4.8%).

         The responses related to hyperplasia observed in DEHP-treated
    B6C3F1 mice (Smith-Oliver & Butterworth, 1987) were greater than
    those previously reported for F344 rats tested under similar
    conditions (Butterworth et al., 1984). While a single dose of 500
    mg/kg DEHP caused 0.6% of the cells to undergo scheduled DNA
    synthesis in the rat, the same regimen caused 3.1% of the cells to
    undergo scheduled DNA synthesis in the mouse. Female rats fed 6000
    ppm DEHP in the diet for three weeks exhibited a liver to body
    weight ratio that was 138% of controls, while the liver to body
    weight ratio of male mice subjected to the same regimen was 173% of
    controls.

    Effects on liver peroxisomes, other subcellular organelles,
    proteins, and enzymes

         In addition to inducing liver hypertrophy and hyperplasia,
    DEHP and other peroxisome proliferators have been reported to
    affect liver peroxisomes, cytosol, mitochondria, microsomes, and
    endoplasmic reticulum. Peroxisome proliferation is induced by a
    wide variety of chemicals and conditions: hypolipidemic drugs,
    phthalate esters, high-fat diet, and vitamin B deficiency, potency
    of different proliferating agents varies over a 30-fold range.
    Electron microscopy studies revealed a dose-related increase in rat
    liver peroxisomes at DEHP dietary levels of 0.1% and above in males
    and 0.6% and above in females (Chemical Manufacturers Association,
    1987).

         Mann et al. (1985) identified the major short-term hepatic
    effects associated with administration of DEHP in the diet,
    including midzonal and periportal accumulation of small droplets of
    lipid, hepatomegaly accompanied by an initial burst of mitosis,
    proliferation of hepatic peroxisomes and of the smooth endoplasmic
    reticulum accompanied by induction of peroxisomal fatty acid
    oxidation, damage to the peroxisomal enzymes as evidenced by
    increased leakage of catalase to the cytosol, and centrolobular
    loss of glycogen and decreases in glucose-6-phosphatase and low-
    molecular weight reducing agents.

         Tomaszewski et al. (1987) have asserted that acyl CoA
    oxidase is a sensitive marker for early hepatic peroxisomal changes
    caused by treatment of F344 rats with DEHP. Groups of five male
    rats (250-300 g) were given 2.0 g/kg bw DEHP in corn oil daily for
    1, 2, 3, 4, 7, or 14 days. Control rats received corresponding
    amounts of corn oil (5 ml/kg). Acyl CoA oxidase activity was
    increased 2.5-fold after 1 day and 8-fold after 14 days, enoyl CoA
    hydratase activity increased 2-fold after 2 days and 6-fold after
    14 days, there were no significant increases in hydroxyacyl CoA
    dehydrogenase or catalase activities after 3 days of treatment with
    DEHP. In a second experiment, rats were dosed with DEHP (0.06, 0.2,
    0.6, 2.0, or 4.0 g/kg) dissolved in corn oil for 1, 3, or 7 days. At
    the lowest dose, there was no significant increase in the relative
    liver weight during the treatment period, at higher doses, increases
    in relative liver weight were dose-related and significant. The
    authors concluded that the apparent no-observable-effect level for
    liver weight changes in response to DEHP treatment was 0.06 g/kg/day.

         The effects of prolonged administration of DEHP on rat liver
    were reported by Ganning et al. (1985). Male rats (180 g) were
    fed DEHP in the diet (0, 0.02, 0.2, or 2.0%) for two years. Rats
    were killed at intervals throughout the experiment, and changes in
    KCN-insensitive palmitoyl-CoA dehydrogenase activity were monitored
    as an indicator of general changes in peroxisomal beta-oxidation of
    fatty acids. At the highest DEHP concentration (2.0% in the diet),
    enzyme activity increased approximately 20-fold during 20 weeks;
    slower but continuous elevations in activity were also obtained
    with 0.2 and 0.02% DEHP, although activities after two years had
    not reached the level achieved upon administration of 2.0% DEHP.
    Among mitochondrial enzymes, those participating in fatty acid
    transport (for example, carnitine-acetyl transferase) were markedly
    induced during treatment with DEHP; other mitochondrial enzymes (NADH-
    and NADPH-cytochrome c reductases, cytochrome b5, glucose-6-
    phosphatase, and ATPase) were influenced only to a limited extent
    or not at all. The cytochrome P-450 system was induced with the
    highest dose of DEHP (2.0%), particularly during the early phase of
    treatment. The authors emphasized that it is incorrect to speak of
    threshold values for phthalate esters since intake of low amounts
    for long periods of time can produce pronounced biological effects.

         Preliminary studies have suggested that peroxisome
    proliferation induced by MEHP, the principal metabolite of DEHP,
    may be due to an initial biochemical lesion of fatty acid
    metabolism resulting in increased intrahepatic lipid (Elcombe &
    Mitchell, 1986). Evidence for this hypothesis comes from the
    observation that increased omega- and beta-oxidation of fatty
    acids, acyl CoA hydrolases, CoA, and carnitine are among the most
    common observations following administration of peroxisome
    proliferators to rodents and that similar changes in rodent liver
    are affected by high-fat diets, where peroxisomal beta-oxidation
    may be increased 8-fold (Reddy & Lalwani, 1983).

         Watanabe et al. (1985) reported that DEHP induces marked
    changes in the profile of some hepatic proteins. Reddy et al.
    (1986a) reported that peroxisome proliferators ciprofibrate,
    clofibrate, and DEHP selectively increased the rate of
    transcription of the first two enzymes of the peroxisomal fatty
    acid beta-oxidation genes (fatty acyl-CoA oxidase and enoyl-CoA
    hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme) but
    not of the catalase gene. Reddy et al., concluded that the
    rapidity of the translational response (one hour after
    administration of the test compounds) suggests that these agents
    act directly on liver cells, and is reminiscent of receptor-
    mediated responses. In partial support of this hypothesis, Lalwani
    et al. (1983) have reported that the peroxisome proliferator
    nafenopin binds to a cytosolic receptor in liver cells.

         Current evidence indicates that maximal peroxisomal
    proliferation is a tissue-specific phenomenon, restricted largely
    to the hepatocyte but also reported to occur to a limited extend in
    kidney and intestine. Reddy & Rao (1987) argue that the tissue-
    specific nature of the biological response suggests that
    interaction of these structurally dissimilar xenobiotics with a
    receptor(s) might be the mechanism responsible for peroxisome
    proliferation and the selective increase in the rate of
    transcription of peroxisomal fatty acid beta-oxidation enzyme
    system genes without significantly affecting the transcriptional
    rate of peroxisomal marker enzyme catalase gene. They further
    conclude that hepatic carcinogenicity of peroxisome proliferators
    is not directly attributable to the chemical but to the adaptive
    responses of the host (Reddy & Rao, 1987).

         Nair & Kurup (1986) reported that dietary administration of
    DEHP (2.0% w/w) for 30 days caused proliferation of hepatic
    mitochondria in rats but not in Wistar mice. Inhibition of
    respiratory activity on administration of DEHP was observed in
    mitochondria isolated from the livers of rats, but not of mice.
    DEHP administration increased the activity of alpha-glycero
    phosphate dehydrogenase in hepatic mitochondria of the rat
    (6-fold) and of the mouse (20%). Although the activity of carnitine
    acetyltransferase was increased in liver mitochondria of both rats
    and mice, the increase was greater in the rat (54-fold) than in
    mice (36-fold). Nagi et al. (1986) reported that administration
    of DEHP in the diet (2% v/w) for 8 days to male Sprague-Dawley rats

    resulted in more than a three-fold increase in activity of acetyl
    CoA-dependent hepatic mitochondrial fatty acid elongation, and that
    this DEHP-sensitive activity was not due to peroxisomal contamination
    of the mitochondrial fraction.

         Ganning et al. (1985) reported that both decreased breakdown
    and increased incorporation of amino acid precursors into rat liver
    protein contribute to induction of mitochondria by DEHP
    administration. The authors reported that DEHP appears to decrease
    protein breakdown by interfering with membrane protein turnover,
    and concluded that extensive induction of hepatic peroxisomes and
    mitochondria during DEHP treatment is a complex process
    characterized by both increased synthesis and decreased breakdown
    of macromolecules.

         Gollamudi et al. (1985) suggested that DEHP administration
    may alter the composition of microsomal phospholipids in the liver
    of male Sprague-Dawley rats: DEHP inhibited  UDP-glucuronyl-
     transferase activity of rat liver  in vivo and  in
     vitro, but did not affect the activities of these cytosolic enzyme
    N-acetyltransferase or microsomal P-450  in vitro.

         Cook et al. (1986) demonstrated marked, yet differential,
    stimulation of short-chain trans-2-enoyl CoA hydratase and
    beta-ketoacyl CoA reductase activities in the liver peroxisomal,
    microsomal, and cytosolic fractions from Sprague-Dawley rats
    (200-250 g) treated with DEHP (2.0% v/w in the diet for 8 days).

         Lake et al. (1987) studied the effects of prolonged
    administration of clofibric acid and DEHP on lipid peroxidation in
    the rat. Male Sprague-Dawley rats were fed diets containing either
    0.05% clofibric acid (CA) or 2.0% DEHP for 2 years. Both compounds
    produced liver enlargement accompanied by the formation of liver
    nodules. Lipid peroxidation, as measured by whole homogenate
    conjugated dienes, was increased to 620 and 640% of control levels
    in livers from CA- and DEHP treated rats, respectively. The authors
    concluded that prolonged peroxisome proliferation can result in
    increased lipid peroxidation.

         Goel et al. (1986) reported increased lipid peroxidation,
    assessed by the measurement of conjugated dienes, in rats fed
    ciprofibrate and Wy-14,642 - potent peroxisomal proliferators - for
    6 months or longer. However, Prince et al. (1985) concluded that
    lipid accumulation in the livers of male Wistar albino rats treated
    with 25 mg/kg/day chlorpromazine did not induce peroxisome
    proliferation by substrate overload.

         Lake et al. (1987) observed that non-nodular regions of
    DEHP-treated rat livers contained large deposits of mature
    lipofucsin; Reddy & Lalwani have suggested that the formation of
    lipofuscin is the result of sustained oxidative stress to the
    hepatocytes (caused by increased generation of hydrogen peroxide),

    which is manifested in enhanced lipid peroxidation (Reddy & Lalwani,
    1983). In support of this hypothesis, peroxisome proliferators are
    known to increase hepatocyte hydrogen peroxide levels (Goel et al.,
    1986), peroxisomal fractions from treated animals exhibit an increased
    capacity to generate highly reactive hydroxyl radicals (Elliot et
    al., 1966), and antioxidants inhibit the carcinogenicity of the
    potent peroxisome proliferator ciprofibrate (Rao et al., 1984).

         Conway et al. (1987a, b) reported the results of experiments
    designed to quantitate increases in conjugated dienes and
    lipofuscin in the liver with long-term treatment of F344 rats with
    peroxisome proliferators. Conjugated dienes and lipofuscin were
    measured in livers from rats treated with DEHP and Wy-14643.
    Conjugated dienes were increased about 45% in livers from rats
    treated for 151 days with Wy-14,643; DEHP-treatment had no effect
    on conjugated dienes. Autofluorescent lipofuscin was quantitated by
    morphometry in sections of liver from control and treated rats.
    Statistically significant increases in lipofuscin were observed
    after 18 and 39 days of Wy-146432 and DEHP treatment, respectively.
    At all time points up to 151 days following initiation of
    treatment, the amount of lipofuscin in livers from Wy-14643-treated
    animals was 5- to 10-fold more than in livers from DEHP-treated
    animals. The authors suggested that these initial studies clearly
    show a close association between the carcinogenicity of these two
    peroxisome proliferating compounds and the accumulation of
    lipofuscin: Wy-14643 in the diet caused nearly a 100% incidence of
    hepatic cancer after 60 weeks (Rao et al., 1984), while DEHP in
    the diet only caused a 10% incidence of hepatic cancer after two
    years (National Toxicology Program, 1982).

    Comparative effects of phthalates and phthalate metabolites
    in various species

         Significant increases in the number of peroxisomes and in the
    activity of the hydrogen peroxide-generating peroxisomal fatty acid
    beta-oxidation system occur in liver parenchymal cells of mice,
    rats, and other species exposed to several structurally dissimilar
    hypolipidemic drugs and certain phthalate ester plasticizers,
    including DEHP (Reddy & Rao, 1987). Compared to other classes of
    peroxisome proliferators, phthalate ester plasticizers, such as
    DEHP, are relatively weak inducers of peroxisomal proliferation
    (Moody & Reddy, 1978; Reddy et al., 1986b).

         Mitchell et al., (1985a) reported the identification of
    proximate peroxisome proliferators derived from DEHP. The authors
    administered DEHP to rats and isolated the urinary metabolites;
    major metabolites were ones resulting from initial omega or omega-1
    oxidation of MEHP (metabolites were named according to the system
    of Albro et al., 1973). These metabolites together with MEHP and
    2-ethylhexanol, were added to primary rat hepatocyte cultures and
    their effects on peroxisomal enzyme activity were observed.

    Omega-oxidation products I and V and 2-ethylhexanol had little or
    no effect on CN-insensitive palmitoyl-CoA oxidation (a peroxisomal
    marker enzyme for beta-oxidation). Omega-1 oxidation products VI
    and IX, as well as MEHP, produced large (7- to 11-fold) inductions
    of peroxisomal enzyme activity. Similar results were observed for
    the induction of cytochrome P-450-mediated lauric acid hydrolase
    and increase in cellular coenzyme A content. The authors concluded
    that the metabolites producing positive results in the
    aforementioned assays represent proximate metabolites for DEHP-
    induced peroxisome proliferation in rodent liver. Mitchell et al.
    (1985b) also reported that oral administration of MEHP (150-250
    mg/kg) to guinea pigs did not induce proliferation of hepatic
    peroxisomes, and that addition of MEHP and other active metabolites
    to primary guinea pig hepatocyte cultures failed to induce
    peroxisomal beta-oxidation enzymes.

         Mann et al. (1985) compared the short term effects of DEHP
    to those of two straight-chain analogs in rats. It had previously
    been reported that peroxisome proliferation was not induced in rats
    administered free phthalic acid, nor in cultured hepatocytes
    treated with n-hexanol (Gray et al., 1982a); the authors were
    concerned with the question of whether straight-chain phthalates
    differ in their hepatic effects from those of the branched chain
    phthalates. Male Wistar albino rats (approximately 4 weeks old)
    were allocated to 12 groups: Three control groups with 6 animals
    each and 9 treatment groups with 4 animals each; three treatment
    groups were assigned to DEHP, di(n-octyl)phthalate, and di(n-
    hexyl)phthalate (administered in the diet at 20 g/kg; 20,000 ppm).
    Groups of animals were killed 3, 20, and 21 days after the
    beginning of treatment. The authors identified the major short-term
    effects of administration of DEHP in the diet, many of which were
    apparent 3 days after the initiation of treatment: (1) midzonal and
    periportal accumulation of small droplets of lipid, (2)
    hepatomegaly accompanied by an initial burst of mitosis, (3)
    proliferation of hepatic peroxisomes and of the smooth endoplasmic
    reticulum accompanied by induction of peroxisomal fatty acid
    oxidation, (4) damage to the peroxisomal membranes as evidenced by
    increased leakage of catalase to the cytosol, and (5) centrolobular
    loss of glycogen and decreases in glucose-6-phosphatase
    (endoplasmic reticulum-associated enzyme) and low-molecular weight
    reducing agents. In contrast, diets containing the two straight-
    chain phthalates induced (1) accumulation of large droplets of fat
    around central veins leading, by 10 days, to mild centrolobular
    necrosis, and (2) a very slight induction of catalase in the
    peroxisomal fraction and a small, and late-appearing, increase in
    liver weight; other changes induced by DEHP were not significantly
    affected by the two straight-chain phthalates.

         Benford et al. (1986) investigated differences in peroxisome
    proliferation induced by MEHP and three longer branched chain
    phthalates in hepatocytes isolated from adult male Wistar albino
    rats and marmoset monkeys. Twenty-four hours after seeding, 0-0.6


    然 MEHP, monoisonoyl phthalate, monoisodecyl phthalate, or di-isononyl
    phthalate were added to the hepatocytes cultures; cultures were
    analyzed for peroxisome proliferation, using increases in
    peroxisomal palmitoyl-CoA oxidation as a marker. MEHP, monoisononyl
    phthalate, and monoisodecyl phthalate produced marked dose-response
    increases in peroxisomal palmitoyl-CoA oxidation in rat hepatocytes,
    with MEHP producing the greatest response. In contrast, marmoset
    peroxisomes showed only minimal changes with poor dose-response for
    monoisononyl phthalate and monoisodecyl phthalate, and no change with
    MEHP.

         Lake et al. (1984) reported the results of comparative
    studies of the hepatic effects of di- and mono-n-octyl
    phthalates, DEHP, and the hypolipidemic drug clofibrate in male
    Sprague-Dawley rats. DEHP (1000 mg/kg/day), its straight-chain
    isomer di- n-octyl phthalate (DHOP; 1000 mg/kg/day), mono- n-
    octyl phthalate (MNOP; 715 mg/kg/day), the straight-chain isomer of
    MEHP, and clofibrate (500 mg/kg/day) were administered to 35 day
    old rats by gavage for 14 days; control animals received 5 ml/kg bw
    of the corn oil vehicle. All rats were starved overnight following
    the last dose, then killed on day 15 of the experiment. Oral
    administration of DNOP, MNOP, DEHP, and clofibrate to rats for 14
    days produced significant increases in relative liver weight.
    Marked peroxisome proliferation was observed in liver sections from
    rats treated with DEHP and clofibrate, but not from rats treated
    with DNOP and MNOP. DEHP and clofibrate, but not DNOP and MNOP,
    produced marked increases in the hepatic activities of cyanide-
    insensitive palmitoyl-CoA oxidation, a specific peroxisomal marker
    enzyme, and carnitine acetyltransferase, located in mitochondrial,
    peroxisomal, and microsomal fractions of liver cells. Only DEHP and
    clofibrate treatment increased total (mitochondrial and
    peroxisomal) and heat-labile (peroxisomal) enoyl-CoA hydratase
    activities. While none of the compounds affected microsomal protein
    content of the liver, both DEHP and clofibrate induced cytochrome
    P-450 content; these two compounds, but not DNOP and MNOP, also
    produced changes in the spectral properties of rat hepatic
    microsomal hemoproteins.

         Reddy et al. studied the induction of hepatic peroxisome
    proliferation in nonrodent species, including primates (Reddy
    et al., 1984). The hypolipidemic drug ciprofibrate, known to
    induce peroxisome proliferation in rodent liver, was used in these
    experiments. Male cats (500-700 g) were given ciprofibrate orally
    in gelatin capsules (10-200 mg/kg bw) daily for up to 4 weeks;
    pigeons were administered ciprofibrate by gavage (300 mg/kg bw) for
    3 weeks; chickens received ciprofibrate (25-150 mg/kg bw) orally in
    capsules for 4 weeks; adult male rhesus monkeys were allowed to eat
    graded doses of ciprofibrate mixed with fruit jelly and bread 
    (50-200 mg/kg bw) for 7 weeks, and male cynomolgus monkeys were given
    ciprofibrate in jelly and bread (400 mg/kg bw) for approximately 4
    weeks. Treatment induced peroxisome proliferation in the livers of
    all treated animals: cats at a dose greater than 40 mg/kg bw for 4


    weeks; chickens at a dose greater than 25 mg/kg bw for 4 weeks;
    pigeons at a dose of 300 mg/kg bw for 3 weeks; rhesus monkeys at
    a dose of 50-200 mg/kg bw for 7 weeks; and cynomolgus monkeys at a
    dose of 400 mg/kg bw for 4 weeks. In all five species studied, a
    marked but variable increase in the activities of peroxisomal
    catalase, carnitine acetyltransferase, heat-labile enoyl-CoA
    hydratase, and the fatty acid beta-oxidation system was observed.
    The authors concluded that peroxisome proliferation can be induced
    in the livers of several non-rodent species, including primates,
    and that it is a dose-dependent and not a species-specific phenomenon.

         Hepatocyte transplantation systems are being used to evaluate
    species differences in response to xenobiotic-induced peroxisome
    proliferation (Reddy et al., 1984; Rao et al., 1986).
    Enzymatically dissociated hepatocytes have been transplanted to the
    interscapular or inguinal fat pads or to the anterior chamber of
    the eye of a syngeneic host of an athymic nude mouse. The
    transplanted hepatocytes responded to the peroxisome proliferative
    effect of peroxisome proliferators administered in the diet of the
    host. The degree of proliferation in transplanted liver was
    comparable to the response observed in the parenchymal cells of the
    homotropic liver. Hepatocytes from feline and canine livers, when
    transplanted into athymic nude mice, appeared to respond to the
    peroxisome proliferators administered to the nude mouse host, but
    the magnitude of response was lower than that noted in the
    hepatocytes of rats and mice.

         In 1986, Elcombe & Mitchell reported that the exposure of
    cultured rat hepatocytes to MEHP (0 to 0.5 mM) for 72 hours
    resulted in marked induction of peroxisomal enzyme activity
    (cyanide-insensitive palmitoyl CoA oxidase, an enzyme of the
    peroxisomal beta-oxidation peroxisomes). Similar treatment of
    cultured guinea pig, marmoset, or human hepatocytes revealed little
    or no effect of MEHP. Identified proximate peroxisome proliferators
    derived from MEHP (omega-1 oxidation products VI and IX, according
    to the numbering system established by Albro et al. in 1973) were
    also without effect in cultured guinea pig, marmoset, or human
    hepatocytes.

    In 1983, Gariot et al. (1983) reported that fenofibrate, a
    triglyceride-lowering drug that also induces peroxisome
    proliferation in rodent liver, failed to induce a similar response
    in human liver. Ten patients (7 males, 3 females) with
    hyperlipoproteinemia received fenofibrate at a daily dose of 300 mg
    (6 subjects, 400 mg (2 subjects), and 600 mg (2 subjects) for from
    16 days to 7.45 months (mean duration of treatment was 9.01
    months). Thirteen patients in the control group (12 males, 1
    female) affected by hyperlipoproteinemia were treated by diet only.
    Liver biopsies were examined by light and electron microscopy; no
    morphological differences were noted between the fenofibrate-
    treated and control groups. The authors concluded that the
    difference between this result and those consistently obtained in
    rodents may be due to the relatively low doses given to human subjects

    or to species dependent differences in liver response to peroxisome
    proliferators. Similar examination of human liver biopsy material
    from patients receiving peroxisome proliferating drugs gemfibrizol
    and clofibrate also demonstrated marginal or no increases in
    peroxisome numbers or volume densities (Blumcke et al., 1983; De
    la Iglesia et al., 1982; Hanefeld et al., 1983).

         In contrast top these results, Gunning et al. (1984)
    reported slight peroxisome proliferation in human liver during
    renal dialysis. However, the study did not present quantitative
    data and patients were those with chronic renal dysfunction, which
    has an unknown effect on liver peroxisomes.

    Hepatic carcinogenesis: Initiation and promotion

         It has been shown that seven hypolipidemic peroxisome
    proliferators, including DEHP, induce hepatocellular carcinomas in
    both rats and mice (Reddy et al., 1980; Reddy & Lalwani, 1983;
    Rao et al., 1984). Liver rumours induced by peroxisome
    proliferators are usually multiple, varying in size from small foci
    of approximately 1 mm in diameter to large tumours of 20 to 40 mm
    in diameters, and metastases in lungs are generally encountered
    (Reddy & Rao, 1987). Rao et al. (1982) have shown that lesions
    produced by peroxisome proliferators do not express gamma-glutamyl
    transpeptidase activity; in addition, neoplastic liver lesions
    induced in rats by peroxisome proliferators do not express the
    placental form of gluthathione-s-transferase (Rao et al., 1986),
    which has been shown to be a universal positive marker expressed in
    liver lesions induced by genotoxic hepatocarcinogens (Sato et
    al., 1984; Tatematsu et al., 1985). Reddy & Rao (1987) concluded
    that expression of these two enzymes is not essential to the
    initiation or promotion of liver carcinogenesis.

         Reddy et al. (1986b) reviewed comparative morphometric and
    biochemical data from rats treated with varying doses of
    ciprofibrate, DEHP, and di(2-ethylhexyl)adipate (DEHA) and
    concluded that the data indicate that the hepatocarcinogenic
    potency of these agents is correlatable with their ability to
    induce peroxisome proliferation, peroxisomal beta-oxidation, and
    PPA-80, a peroxisome proliferation-associated 80,000 molecular
    weight polypeptide. They also proposed a receptor mechanism for
    induction of hepatic peroxisome proliferation by these and other
    compounds.

         Reddy et al. (1986) attempted to correlate the degree of
    peroxisome proliferation in rat liver with the eventual
    carcinogenic response. When rats were fed ciprofibrate (0.02%) or
    DEHP (1.0%) in the diet for 30 days, peroxisome proliferation
    induced by ciprofibrate was approximately double that induced by

    DEHP. After treatment for one year, ciprofibrate in the diet (0.02%)
    caused a 100% incidence of rats with hepatic carcinomas. The authors
    suggested that the degree of peroxisome proliferation may be
    predictive of the eventual carcinogenic response.

         Researchers at CIIT further tested this hypothesis by
    comparing the peroxisome proliferating effects and carcinogenicity
    of Wy-14634 and DEHP in rats (Conway et al., 1987a). Wy-14643 in
    the diet caused nearly a 100% incidence of male F344 rats with
    hepatic neoplasia after 60 weeks (Rao et al., 1984); DEHP in the
    diet only caused a 10% incidence of hepatic cancer after two years
    (National Toxicology Program, 1982). When male F344 rats were fed
    1.2% DEHP or 0.1% Wy-141643 in the diet for 1, 2, 4, 8, 39, 77, and
    151 days, a near doubling of absolute liver weight was observed
    within ten days. Similar increases in cell replication were
    observed during the first days of treatment as measured by
    autoradiographic quantification of nuclear labeling indices of
    hepatocytes. Surprisingly, the induction of peroxisomal beta-
    oxidation activity in liver homogenates by DEHP and Wy-14643 was
    nearly identical: Both compounds increased peroxisomal beta-
    oxidation activity by about 15-fold over controls after 18 days of
    treatment and this level of induction was sustained until the end
    of treatment (151 days). The authors concluded that, in direct
    conflict with the suggestion of Reddy et al. (1986b) that the
    degree of induction of peroxisome beta-oxidation enzymes can be
    used to predict the relative carcinogenic effects of various
    peroxisomal proliferators, effects of chronic DEHP and Wy-14643
    treatment on the peroxisomal beta-oxidation system were not
    predictive of the carcinogenicity of these two compounds.

         Kluwe et al. (1985) and Kluwe (1986) studied the
    relationship of the structure of phthalic acid esters and related
    compounds to their carcinogenic potential in rats and mice. Groups
    of 50 weanling F344 rats and B6C3F1 mice of each sex were exposed
    to phthalic anhydride (PAn) [rat (male and female): 0, 7500, and
    15,000 ppm; mouse (male): 0, 16,000, and 33,000 ppm; mouse
    (female): 0, 12,000, and 24,000 ppm], DEHP [rat (male and female):
    0, 6,000, and 12,000 ppm mouse; (male and female): 0, 3,000 and
    6,000 ppm], DEHA [rat (male and female): 0, 12,000, and 25,000 ppm;
    mouse (male and female): 0, 12,000, and 25,000 ppm], butyl benzyl
    phthalate (BBP) [rat (female): 0, 6,000, and 12,000 ppm); mouse
    (male and female): 0, 6,000, and 12,000 ppm], or 2-ethylhexyl
    sulfate (EHS) [rats (male): 0, 10,000, and 20,000 ppm; mouse
    (male): 0, 5,000, and 10,000 ppm; mouse (female): 0, 10,000, and
    20,000 ppm] in the diet or diallyl phthalate (DAP) [rat (male and
    female): 0, 50, and 100 mg/kg; mouse (male and female): 0, 150, and
    300 mg/kg] or tris (2-ethylhexyl) phosphate (TEHP) [rat (male): 0,
    2,000 and 4,000 mg/kg; rat (female): 0, 1,000 and 2,000 mg/kg;
    mouse (male and female); 0, 500, and 1,000 mg/kg] with corn oil by
    gavage (once per day, five days per week) for 104 weeks. Highest
    doses given were estimated to be the maximum tolerable doses from
    the results of 13-week studies. Except for DEHP (males: 10%

    at high dose compared to 2% in controls and low-dose animals,
    females: 16% at high dose compared to 4% in mid-dose and 0% in
    control animals), none of the chemicals increased the incidence of
    liver carcinomas in rats. However, the other three chemicals 
    with a 2-ethylhexyl moiety exhibited varying degrees of
    hepatocarcinogenic activity in mice: DEHA increased the occurrence
    of liver carcinoma in both male (24% for low and high doses
    compared to 14% for controls) and female mice (28% for low dose and
    24% for high dose compared to 2% for controls), although the
    response was greater in females. TEHP caused a small but
    significant increase in hepatocellular carcinomas in female mice
    (0, 8, and 14% in control, low-, and high-dose groups,
    respectively), while EHS equivocally increased hepatocellular
    carcinomas in female mice (0, 2, and 7% in control, low-, and
    high-dose groups, respectively). DEHP increased hepatocellular
    carcinomas in mid(24%) and high-dose male mice (36%) compared with
    male controls (18%), and in mid- (4%) and high-dose female mice
    (34%) compared with female controls (0%). The 2-ethylhexyl compound
    that evidenced the greatest hepatocarcinogenic response in mice
    (DEHP) was also hepatocarcinogenic in rats; similarly, those
    compounds with a relatively greater effect in female mice were also
    active in male mice. The authors concluded that, although all of
    the 2-ethylhexyl-containing compounds studied possessed some
    hepatocarcinogenic activity, the results of these studies did not
    reveal common neoplastic or non-neoplastic lesions suggestive of
    structural correlates of toxic activity. Instead, sex and species
    differences in 2-ethylhexyl-induced hepatocarcinogenesis in rodents
    are probably quantitative rather than qualitative.

         None of the carcinogenic peroxisome proliferators interact
    with or damage DNA, suggesting that formation of a peroxisome
    proliferator-DNA adduct is not an essential step in carcinogenesis
    by this class of hepatocarcinogens (Von Daniken et al., 1984;
    Goel et al., 1985; Gupta et al., 1985).

         Gupta et al. (1985) attempted to identify peroxisome
    proliferator-DNA adducts in rat liver cells under  in vivo and
     in vitro conditions. Clofibrate (250 mg/kg), ciprofibrate (50
    mg/kg), Wy-14643 (50 mg/kg), and DEHP (2000 mg/kg) were
    administered by gavage in 0.3 ml dimethyl sulfoxide to groups of
    three F344 male rats (150 g) at 0, 24, and 48 hours after the
    beginning of the experiment. Control rats received dimethyl
    sulfoxide alone. Rats were killed 50 hours after the beginning of
    the experiment. DNA isolated from the livers was analyzed for
    possible carcinogen-DNA adducts by the 32P-postlabeling technique
    which can detect one adduct in 100 billion nucleotides. Known DNA-
    binding agents AAF and AAp served as positive controls. No adducts
    were detected in the DNA isolated from livers of rats treated with
    any of the peroxisome proliferators. For  in vitro studies,
    hepatocytes from male F344 rats were isolated, suspended in
    chemically defined media containing 0.001 M clofibrate,
    ciprofibrate, Wy-14643, or DEHP, and incubated for 4 hours.

    Hepatocytes incubated in the presence of dimethyl sulfoxide served
    as negative controls; hepatocytes incubated in the presence of AAF
    (0.001 M) or N-OH-AAF (0.00001 M) served as positive controls. The
    fact that adducts were not found in the DNA of hepatocytes exposed
     in vitro to peroxisome proliferator-DNA adducts in hepatocytes by
    this sensitive technique supports the contention that adduct
    formation is not an essential step in carcinogenesis induced by
    peroxisome proliferators.

         Fahl et al. (1984) demonstrated DNA damage related to
    increased generation of hydrogen peroxide by hypolipidemic drug-
    induced liver peroxisomes. Peroxisomal-containing light
    mitochondrial fractions were prepared from pooled livers of control
    and hypolipidemic drug-treated rats (100 g male F344 rats were fed
    0.1% Wy-14643 in the diet for 4 weeks). Drug treatment of animals
    induced a large increase in the level of liver peroxisomal fatty
    acid oxidation and an associated increase in the generation of
    hydrogen peroxide. Hydrogen peroxide generation was the result of
    an increase in the specific activity of peroxisomal hydrogen
    peroxide-generating enzymes (6.3-fold) as well as an increase in
    the number of peroxisomes per unit of liver volume (4.8-fold by
    peroxisome recovery or 11-fold by monomorphic analysis of liver
    sections). As a result, the overall peroxisome production of
    hydrogen peroxide was increased 30- to 70-fold in the hypolipidemic
    drug-treated rat livers, depending on the method used to assess the
    numerical increase in peroxisome density. Peroxisomes isolated from
    drug-treated rat liver, but not from control rat liver, induced
    strand breaks and altered the electrophoretic mobility of
    supercoiled SV40 DNA. Pure hydrogen peroxide also was able to
    induce single strand nicks in a dose-dependent manner in SV40 DNA.
    In incubations containing peroxisomes from drug-treated rats, a time-
     and enzyme-dependent conversion to nicked SV40 DNA was observed
    that paralleled the level of hydrogen peroxide production in the
    peroxisome incubations. Catalase (400 units/ml) had only a marginal
    quenching effect on the peroxisome-initiated DNA damage. The
    authors suggested that these results are consistent with a
    mechanism of hepatocarcinogenesis in which hepatocellular genetic
    damage is introduced by the by-products of peroxisomal fatty acid
    beta-oxidation, an oxidative pathway that is dramatically increased
    in hypolipidemic drug-treated livers.

         Rao et al. (1984) tested the hypothesis that antioxidants
    could retard or inhibit neoplasia by scavenging active oxygen
    (superoxide radicals, hydrogen peroxide, hydroxyl radicals, and
    singlet oxygen). Groups of 25 male F344 rats were fed synthetic
    antioxidants 2(3)-tert-butyl-14-hydroxyanisole (0.5% w/w) or
    ethoxyquin (0.5% w/w) with or without the peroxisome proliferator
    ciprofibrate (10 mg/kg bw) for 60 weeks. Rats fed ciprofibrate in
    the diet or fed a diet with no added chemicals served as controls.
    Ethoxyquin markedly inhibited the hepatic tumorigenic effect of
    ciprofibrate, as evidenced by decreased incidence of rumours,


    decreased number of tumours per liver, and reduced tumour size.
    Administration of 2(3)-tert-butyl-14-hydroxyanisole also caused
    a significant decrease in the incidence and number of hepatocellular
    carcinomas larger than 5 mm. The authors postulated that the
    inhibitory effect of these synthetic antioxidants on ciprofibrate-
    induced hepatocarcinogenesis may be due to their hydrogen peroxide
    and free radical-scavenging properties, since these antioxidants do
    not prevent peroxisome proliferation and the induction of hydrogen
    peroxide-generating peroxisomal enzymes in livers of rats fed
    ciprofibrate.

         Conway et al. (1987b) used oxidized glutathione (GSSG)
    efflux into bile as an indicator of possible increases of hydrogen
    peroxide concentrations in the extraperoxisomal compartment of
    livers treated with nafenopin. Ackerboom et al. (1982) have
    demonstrated that glutathione peroxidases form GSSG from reduced
    glutathione during the detoxification of hydroperoxides and
    hydrogen peroxide. Because glutathione peroxidases are located
    outside the peroxisome in the cytoplasm and mitochondria, changes
    in the efflux of GSSG into the bile can be used as a qualitative
    measure of the diffusion of hydrogen peroxide out of peroxisomes.
    Conway et al. infused various fatty acids into perfused livers
    isolated from F344 male rats (150-300 g) treated with methyl-
    cellulose (control; 1.0%) or nafenopin (80 mg/kg/day by
    gavage for 7 days); GSSG efflux into the bile was measured. When
    oleate (the predominant fatty acid found in rat blood) was infused,
    a large efflux of GSSG into the bile of livers from nafenopin-
    treated rats, but not control rats, was observed. Fatty acids with
    varying substrate specificities for peroxisomal beta-oxidation were
    tested: octanoate, laurate, and oleate excellent substrates - all
    caused large increases in GSSG efflux from perfused livers of
    nafenopin-treated, but not vehicle-control, rats; butyrate,
    linoleate, and arachidonate with low activity for peroxisomal beta
    oxidation - had no effect on GSSG efflux in perfused livers. Conway
    et al (1987b) also demonstrated that bromooctanoate, an inhibitor
    of fatty acid beta-oxidation, completely inhibited fatty acid-
    induced increase in GSSG. The authors concluded that the data
    suggest that some hydrogen peroxide produced by fatty acyl coenzyme
    A oxidase during high rates of peroxisomal beta-oxidation in livers
    from nafenopin-treated rats escapes detoxification by catalase and
    diffuses into the cytoplasm to be metabolized by glutathione
    peroxidase. The authors further suggested that these results
    demonstrate a good correlation between substrate-specificity for
    peroxisomal beta-oxidation and fatty acid-induced increases in GSSG
    efflux.

         Goel et al. (1986) have suggested that hydrogen peroxide
    diffusing from the peroxisomes may be responsible for the
    lipofuscin and conjugated dienes observed in liver after chronic
     in vivo treatment with peroxisome proliferators.

         Tomaszewski et al. (1986) reported increased peroxisomal
    steady-state hydrogen peroxide levels in rats and mice treated with
    peroxisome proliferators. Groups of 5 male F344 rats (250-350 g)
    and female B6C3F1 mice (18-28 g) were dosed once per day for 14
    days with DEHP (2 g/kg bw), DEHA (2 g/kg bw), or nafenopin (0.25
    g/kg bw) dissolved in corn oil. Activities of enzymes responsible
    for the production [peroxisomal palmitoyl CoA oxidase (PCO)] and
    degradation [catalase (CAT) and glutathione peroxisome (GSHPx)] of
    hydrogen peroxide were assayed in liver homogenates prepared from
    treated and control animals. Activities of peroxisomal enzymes PCO
    and CAT were enhanced 5- to 25-fold and 1.5- to 3-fold respectively
    by treatment with peroxisome proliferators. The activity of
    cytoplasmic GSHPx was reduced 40-60% in liver homogenates prepared
    from treated animals compared to control animals. Treatment of rats
    with peroxisome proliferators caused increases in steady-state
    hydrogen peroxide in liver homogenates. The greatest increase
    (approximately 13-fold) was produced by nafenopin; DEHA caused only
    a 2-fold increase and DEHP produced a 7-fold increase relative to
    control. In mouse liver homogenates, DEHP caused the greatest
    increase (10-fold) in steady-state hydrogen peroxide relative to
    control; nafenopin produced a 5-fold increase and DEHA caused a 
    2-fold increase. The authors also reported decreases in
    concentrations of diene conjugates in the livers of treated
    animals: In rats, the decrease corresponded with the increase in
    steady-state hydrogen peroxide, but in mice this correspondence was
    not observed. The authors concluded that the results of these
    studies support the hypothesis that increased peroxisomal hydrogen
    peroxide is involved in the hepatocarcinogenesis of peroxisome
    proliferators.

         Ward et al. (1983, 1986) reported on a number of studies on
    the tumour initiating and promoting activities of DEHP  in vitro and
     in vivo. An initiation-promotion system for liver used male
    B6C3F1 mice: To test for promotion, mice were injected with
    diethylnitrosamine (DEN; 80 mg/kg bw) intraperitoneally once at 4
    weeks; two weeks after injection, mice were placed on diets
    containing 0, 3,000, 6,000, or 12,000 ppm DEHP or given water
    containing phenobarbital (PB) at 500 ppm. At 2, 4, 6, 8, 10, or 18
    months, groups of mice were killed; at selected times, hepatic DNA
    synthesis and mitotic indices of hepatocytes were measured. To test
    for initiating activity by DEHP, mice received one intragastric
    dose (25 or 50 g/kg) at 4 weeks of age followed by PB (500 ppm in
    drinking water) for 6 weeks; mice were killed at 6 and 18 months,
    the authors reported that there was no evidence of liver tumour
    initiation in mice by DEHP after 6 or 18 months of subsequent
    exposure to the liver tumour promoter PB. However, both PB and DEHP
    were effective tumour promoters. The focal hepatocellular
    proliferative lesions (FHPL) in DEN-initiated mice that received
    DEHP at 12,000 ppm were significantly larger in mean focus volume
    at 6 months than those of mice in other groups; by 18 months, 25%
    of the mice given 6000 ppm DEHP had hepatocellular carcinoma
    metastatic to the lung.

         In a separate experiment (Ward et al., 1984), DEHP was fed
    in the diet at 3000 ppm or PB was given in the water at 500 ppm for
    1, 7, 28, 84, or 168 days, beginning one week after DEN injection
    at 4 weeks of age. All mice were killed at 168 days. Additional
    groups of treated animals received DEHP or PB for 168 days and were
    killed 84 days later to observe possible regression of hepatic
    proliferative lesions. The authors concluded that DEHP was an
    effective liver tumour promoter after 28, 84, and 168 days while PB
    was effective only after 168 days of exposure. At 84 days after the
    most prolonged period of exposure (168 days), however, FHPL in mice
    given either PB or DEHP had not regressed but had increased in
    size. The authors also reported that lung tumours were induced by
    DEN in all groups of mice, but incidence of these tumours was not
    affected by subsequent administration of either PB or DEHP.

         Ward et al. (1986) also reported on the effectiveness of
    DEHP as a tumour promoter in rat liver. Groups of 10 female F344
    rats (5 weeks old) were injected intraperitoneally with N-
    nitrosodiethylamine (282 mg/kg). Two weeks later, rats were placed
    on diets containing 12,000 DEHP or on drinking water containing PB
    at 500 ppm. After 14 weeks of exposure to the promoter, rats were
    killed. By standard hematoxylin/eosin histology and histochemical
    staining for gamma-glutamyl transpeptidase, DEHP failed to increase
    the number or size of FHPL after 16 weeks, while PB was
    significantly effective at the same doses used in mice.

         Popp et al. (1984) also reported that DEHP lacked promoting
    activity in rat liver. When DEHP was used in the promotion phase of
    a rat (female CDF rats; 6-8 weeks old) hepatic initiation-promotion
    system, no promoting activity could be demonstrated after 3 or 6
    months of feeding DEHP at a dietary concentration of 1.2%. No liver
    neoplasms or nodules were identified; in addition, DEHP did not
    increase the number of foci or the mean volume of foci when foci
    were identified by six different histologic stains.

         Williams et al. (1987) reported that prolonged dietary
    administration of DEHP (12,000 ppm for 24 weeks) to male F344 rats
    (some of which had been initiated by N-2-fluorenylacetamide) did
    not demonstrate initiating activity, significant sequential
    syncarcinogenic activity, or a promoting effect on liver
    carcinogenesis under conditions in which numerous agents with such
    activities have been identified.

         Garvey et al. (1987) reported that no initiating activity
    was found when DEHP was administered in a single, oral dose (10
    g/kg) or after 12 weeks of feeding by a promotion regimen. Animals
    used were female F344 rats (150-180 g at the beginning of the
    experiment). No liver tumours were found and there was no increase
    in number of mean volume of loci when liver sections were examined
    using multiple histologic markers.

         DEHP was also tested for the ability to promote DMBA-induced
    tumours in mouse skin (Ward et al., 1986). CD-1 mice initiated by
    a single topical application of DMBA (50 痢) to the dorsal skin
    received DEHP (98.1 痢 in acetone, 0.2 ml total volume) or TPA (10
    痢 in 0.2 acetone) twice weekly for 40 weeks. To test for second-
    stage promoting activity, female SENCAR mice were given DMBA once
    (20 痢), and then TPA (2 痢 twice a week for two weeks), followed
    by DEHP (100 痢, twice weekly) or TPA, mezerein, or acetone weekly
    for up to 26 weeks (Diwan et al., 1985). To test for complete
    promoting activity by DEHP in SENCAR mice, DEHP was given twice
    weekly after a single dose of DMBA (20 g) (Diwan et al., 1985).
    The authors reported that DEHP did not promote the development of
    skin tumours after DMBA initiation in CD-1 mice, nor was it an
    initiator or complete skin carcinogen after 40 weeks. In female
    SENCAR mice, however, DEHP was a weak second-stage promotor (6.4
    papillomas per mouse compared to 0 for control mice initiated by
    DMBA) and a weaker complete promoter of skin carcinogenesis (0.9
    papillomas per mouse). Mezerein was a stronger second-stage
    promoter (23 papillomas per mouse) and TPA was a stronger complete
    promoter (26.4 papillomas per mouse).

         Ward et al. (1986) and Diwan et al. (1985) also reported
    on the results of  in vitro studies designed to test promoting
    activity of DEHP. Cultures of three epidermis-derived cell lines of
    the JB6 mouse (C141, C121, and R219) were suspended in medium
    containing DEHP (1.3-51.2  10-3 M in acetone), MEHP (1-5  10-3
    M in DMSO), or 2-ethylhexanol (EH; 4-7  10-4 M in DMSO), Suspensions
    were layered over agar; colonies in agar were counted 14 days later.
    These cell lines have previously been shown to be promoted to
    anchorage independence by tumour promoting phorbol esters and also by
    mezerein, nezoyl peroxide, and epidermal growth factor. DEHP showed
    activity for promotion of transformation in all three of these cell
    lines: C141 gave the most pronounced response to DEHP, with nearly
    32% of cells giving rise to colonies after treatment with 2.7  10-7
    M DEHP. MEHP was also shown to be an effective promoter; EH,
    however, failed to promote transformation in this system.

         Numoto et al. (1984) described the effects of clofibrate and
    nafenopin on the rat liver cell membrane enzymes gamma-glutamyl
    transpeptidase and alkaline phosphatase during the early stages of
    hepatocarcinogenesis. Male F344 rats (8 weeks of age) were fed
    diets containing 0.02% N-2-fluorenylacetamide (FAA) for 8 weeks to
    induce hepatocellular altered foci, and were than given no
    chemical, equimolar amounts (0.03 mmol/kg diet) of clofibrate or
    nafenopin, or 0.07% of the liver promoter phenobarbital (PB) in the
    diet for 24 weeks. PB had a marked enhancing effect on FAA-induced
    loci, while clofibrate produced only slight enhancement and
    nafenopin produced none. Nafenopin suppressed histochemical gamma-
    glutamyl transpeptidase activity in the abnormal hepatocytes of the
    foci as well as in periportal hepatocytes. In homogenates of lives
    from rats fed nafenopin and, to a lesser extent, clofibrate,


    activity of this enzyme was reduced, whereas PB enhanced its
    activity. The authors suggested that these results reveal
    significant cell membrane effects of nafenopin and clofibrate and
    suggest that their involvement in hepatocarcinogenesis is more
    complex than a promoting action.

         DeAngelo & Garret (1983) and DeAngelo et al. (1984) reported
    the inhibition of phenobarbital- and dietary choline deficiency
    promoted preneoplastic lesions on rat liver by DEHP. Gamma-glutamyl
    transpeptidase positive (GGT+) preneoplastic foci were initiated in
    the liver of Sprague-Dawley male rats given a single dose of
    diethylnitrosamine (DEN) following partial hepatectomy. Promotion
    of loci occurred by one of three methods: (1) a choline-deficient
    diet (CD), (2) a choline-supplemented diet containing 0.6%
    phenobarbital (CS+PB), and (3) a Cd diet containing 0.06%
    phenobarbital (CD+PB). In rats receiving DEN, each promoting
    regimen effectively increased the number of GGT+ loci above levels
    in control rats fed only the CS diet. Inclusion of 2.0% DEHP in
    each of the dietary promotion treatments, however, effectively
    inhibited the appearance of the loci. However, DeAngelo et al.
    later reported that DEHP was unable to inhibit the promoting effect
    of the CD diet at a concentration of 0.1% (DeAngelo et al.,
    1985b), and that di-n-octyl phthalate, a relatively ineffective
    peroxisome inducing straight chain isomer of DEHP, enhances the
    development of initiated GGT+ loci in rat liver (DeAngelo et al.,
    1986).

         DeAngelo et al. (1985a) postulated that DEHP may inhibit
    emergence of GGT+ foci in initiated rats by blocking the response
    of initiated cells to stimulation by epidermal growth factor: DEHP
    in the diet of rats (2.0%) blocked the ability of epidermal growth
    factor to enhance the phosphorylation of its receptor protein in
    isolated liver plasma membranes.

         Perera & Shinozuka (1984) and Perera et al. (1987)
    investigated the relationship between a CD diet and peroxisome
    proliferators on liver rumour promotion. A CD diet, Which is an
    efficient liver tumour promoter, induced peroxidative damage of
    liver cell membrane lipids. By modifying components of the CD diet,
    Perera et al. were able to demonstrate that the efficacy of the
    promotion is correlated with the extent of lipid peroxidation. Both
    an antioxidant and hypolipidemic peroxisome proliferators BR931 and
    DEHP suppressed CD diet-induced lipid peroxidation and promoting
    effects in rats initiated with diethylnitrosamine.

         However, Hagiwara et al. (1986) reported that butylated
    hydroxianisole (BHA), an antioxidant, had no effect on liver tumour
    promotion by DEHP. Four week-old male B6C3F1 mice were given a
    single intraperitoneal injection of N-nitrosaodiethylamine (DEN; 80
    mg/kg) followed one week later by oral exposure to DEHP (6000 ppm
    

    in the diet) and/or butylated hydroxianisole (7500 ppm in the diet) for
    29 weeks. DEHP and BHA (alone and together) increased the incidence of
    DEN-initiated focal hepatocellular proliferative lesions, including
    both microscopic hyperplastic foci and hepatocellular adenomas.

    COMMENTS AND EVALUATION

         DEHP-induced testicular atrophy in rats is an age-dependent
    response, younger rats being more susceptible. Although zinc-
    deficient rats showed enhanced susceptibility to the gonadotoxic
    effects of DEHP, testicular atrophy was not prevented by the 
    co-administration of zinc with DEHP. The co-administration of
    testosterone with DEHP to adult male rats appears to prevent
    testicular injury otherwise induced by DEHP. Mono-2-ethylhexyl
    phthalate (MEHP), a metabolite of DEHP, is likely to be the active
    metabolite of DEHP affecting rat testes both  in vivo and  in
     vitro. There is a partial reversibility of DEHP-induced testicular
    atrophy in rats following cessation of exposure to DEHP.

         The hepatocarcinogenicity in rats and mice attributed to DEHP
    and other phthalate esters is preceded by hepatocellular peroxisome
    proliferation. Suggestions as to the probable mechanism of
    peroxisome proliferation have been advanced but remain unproved.

         It was noted that the lowest DEHP level in food contact
    materials is a function of its effectiveness in plasticizing
    (softening) plastic materials. Technologically optimum DEHP levels
    range from 20 to 50% of the weight of the plastic material.
    Migration levels of DEHP into food are influenced by its level in
    the packaging material and factors such as the composition of food
    and time and temperature of processing and storage of packaged
    food. As a consequence of its extreme solubility in lipid
    materials, the use of DEHP-containing materials in some countries
    is limited to foods of high water content (non-fatty foods).

         After reviewing all the information available, the Committee
    reiterated its recommendation that human exposure to DEHP is food
    be reduced to the lowest level attainable. The Committee considered
    that this might be achieved by using alternative toxicologically
    acceptable plasticizers or alternatives to plastic materials
    containing DEHP.

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    See Also:
       Toxicological Abbreviations
       bis(2-ETHYLHEXYL)PHTHALATE (JECFA Evaluation)