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    BIS (2-ETHYLHEXYL) PHTHALATE

    Explanation

         Bis(2-ethylhexyl)phthalate (DEHP) has not previously been
    evaluated for an ADI for man by the Joint FAO/WHO Expert Committee on
    Food Additives.

         DEHP is used as a plasticizer in plastic film used in food
    packaging. Dietary exposure to DEHP occurs through migration of the
    plasticizer from packaging to food, and from the environment, where it
    exists as a contaminant in drinking water and fish.

         It has the following chemical formula:

    CHEMICAL STRUCTURE 1

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Metabolism

         DEHP was hydrolyzed to the mono-ester by preparations derived
    from the intestine and liver from several animal species (rat, baboon,
    ferret) and from human intestine (Lake et al., 1977; Rowland et al.,
    1977; Kaneshima et al., 1978). Homogenates of rat kidney, lung and
    liver, as well as liver microsomes and mitochrondria, hydrolyse DEHP
    to MEHP and 2-ethylhexanol. Hydrolysis was more rapid with liver
    preparations than lung or kidney preparations. Liver microsomal
    preparations were more active than mitochondrial preparations, whereas
    the cytosol fraction was inactive (Carter et al., 1974). DEHP was
    rapidly hydrolysed by pancreatic lipase, and at a slower rate by rat
    liver homogenate, plasma, and preparations from kidney and lung. No
    o-phthalic acid could be detected in the reaction products (Daniel &
    Bratt, 1974; Albro & Thomas, 1973).

         The in vitro absorption of mono- and DEHP was studied using
    isolated perfused rat intestins. All DEHP was converted to mono
    (ethylhexyl) phthalate (MEHP) before it reached the serosal perfusing
    solution.  No metabolic transformation of MEHP was reported (White et
    al., 1980).

         Adult male CD rats were given two oral doses of 14C-DEHP at
    24 hour intervals and 24 hour urine samples collected. Five major
    metabolites were present after the initial dose, the initial metabolic
    product, mono (2-ethylhexyl)phthalate being metabolized to 5-keto-2-
    (ethylhexyl)phthalate and 2-carboxyl-2-(ethylpentyl)phthalate,
    5-hydroxy-2-(ethylhexyl)-phthalate and 2-carboxymethyl-butylphthalate.
    Less than 5% of the administered compound was hydrolyzed completely to
    o-phthalic acid. Various tests for conjugation were negative (Albro
    et al., 1973).

         Male, 4-month old, CD rats, CD-1 mice, Syrian golden hamsters and
    Hartley albino guinea pigs were administered by gavage 2 doses of DEHP
    at 24-hour intervals, at levels ranging from 20-360 mg/kg b.w. Urine
    was collected for 48 hours after the initial dose. No metabolites were
    detected in urine from hamsters or guinea pigs that were not also
    present in rat or mouse urine. In the mouse, guinea pig and hamster, a
    significant proportion of DEHP metabolites were present as glucuronide
    conjugates. Conjugates of taurine and sulfate were not detected.
    Metabolites of DEHP in rats were not conjugated. Omega and omega-1
    oxidation of the ethylhexyl moiety was less efficient in guinea pig
    than in the other species, but guinea pigs appeared to produce
    significantly more MEHP (Albro et al., 1982a,b). The enzymatic
    processes normally associated with omega, omega-l, alpha- and
    beta-oxidation of fatty acids could account for the major urinary
    metabolites of DEHP in the rat. However, the structures of recently
    identified minor metabolites of DEHP suggest that oxidation of
    the aliphatic side chain can occur at positions more distant than
    omega-1 from the methyl terminus. This type of oxidation at multiple
    sites in an aliphatic side chain occurs commonly when alphatic
    hydrocarbons, rather than fatty acids, are metabolized by microsomal
    oxidases (Albro et al., 1983).

         In separate experiments, single gavage doses of 14C-DEHP
    were given to Fisher 344 rats, CD mice, Syrian golden hamsters and
    Hartley albino guinea pigs and the urine analyzed for 11 different
    metabolites. Species differences were noted in the distribution of
    urinary metabolites.  In the rat, 3 metabolites, not including MEHP
    (trace), comprised 81% of all urinary metabolites of DEHP, while in
    mice, 75% of all urinary metabolites were accounted for by 5
    compounds, including MEHP (18.6%). In the guinea pig, MEHP accounted
    for 71.2% of all urinary metabolites, while in the hamster, 92.4%
    of administered radioactivity was distributed among 5 urinary
    metabolites, not including MEHP (4.5%). Unreacted DEHP occurred at
    levels of 0.5% or less in all species. 100% of urinary metabolites

    were not-conjugated in rats, while 85% were non-conjugated in
    hamsters. Mice and guinea pigs excreted 35-36% of urinary metabolites
    in the non-conjugated form (Albro et al., 1982b). In other studies,
    dogs excreted 3 major urinary metabolites, and pigs excreted 5 (Ikeda
    et al., 1980).

         In a study with African Green monkeys infused with palsma
    containing about 5 mg of 14C-DEHP, more than 50% of the 14C was
    excreted in the urine by 4 hr, and more than 70% by 24 hr. The major
    metabolites were the 5-ethyl, isohexanol monoester of phthalic acid
    and MEHP. More than 80% of the urinary metabolites were conjugated to
    gluconide (Albro et al., 1981).

         Two cancer patients received, by platelet concentration infusion,
    94.7 mg DEPH in 4 hr and 174.3 mg DEHP in 1.5 hr. More than 50% of the
    infused dose appeared as DEHP metabolites in the urine within 6 hr.
    The predominant metabolite was the 5-ethyl, isohexanol monoester of
    phthalic acid. Approximately 80% of urinary metabolites were
    glucuronide conjugates. Eight other metabolites, including MEHP, were
    identified. The half-life of DEHP in plasma was 30  12 min.,
    apparent distribution volume was 2819  83 ml/m2 and clearance was 78
     20 ml/min-m2 (Peck et al., 1978; Albro et al., 1981).

    Absorption, distribution and excretion

         Rats were given by gavage single doses of 14C-DEHP
    ranging from 2.6 to 1900 mg/kg b.w. 35-55% of the labeled dose was
    excreted in the feces and 42-57% in the urine. At low dose levels
    (2.6-2.9 mg/kg b.w.), 9-14% of the dose appeared in the bile. No
    significant amounts of 14C-C02 could be found in the expired air. At
    high dose levels (1000 mg/kg2 b.w.) during an 8 day period, fecal
    excretion accounted for 6.5-10.5% of the administered dose of 14C as
    metabolites and 24.0-38.8% as unchanged DEHP. Most fecal metabolites
    had been excreted by 48 hours after dosing. 53.5-70.2% of the dose was
    excreted as MEHP or metabolites in the urine. Some 14C remained in
    fatty tissue 8 days after dosing, but none was found in 10 other
    tissues. In liver, kidney and skeletal muscle, radioactivity has
    essentially disappeared between 48 and 96 hours after dosing (Daniel &
    Bratt, 1974; Schulz & Rubin, 1973; Williams & Blanchfield, 1974a).

         Beagle dogs, miniature Hormel swine and male Sprague-Dawley rats
    were fed DEHP (650, 750 and 750 ppm, respectively) for 21-28 days and
    then given an oral dose of 14C-DEHP. In rats, 85% of the administered
    radioactivity was excreted in the urine and feces 24 hours after
    dosing excretion of metabolites predominated in dogs and urinary
    excretion predominated in pigs, while in rats, metabolites were
    distributed equally between urine and feces. Elimination of
    administered 14C-DEHP was 84% complete in rats by 24 hours after
    dosing; elimination of metabolites in dogs and pigs was slower
    (50% after 24 hr), but was complete in all species by 4 days after

    dosing.  Serum half-life for 14C-DEHP and metabolites was 1.2 hr in
    dogs and 5.4 hr in pigs. Analysis of tissue and excreta 4 days after
    dosing with 14C-DEHP indicates that DEHP was metabolized 96% in rats,
    63% in dogs and 71% in pigs (Ikeda et al., 1980).

         Male mice were given single oral doses of Di-(2-ethylhexyl)
    (14C)phthalate (6.72 mg/animal) and examined by whole-body
    autoradiography at intervals from 1 hr to 7 days after treatment. Most
    of the radioactivity was excreted in the urine and feces in the first
    24 hr. Excretion was complete within 3-5 days. Following absorption,
    14C was widely distributed in the carcass, but none was found in the
    central nervous system, skeleton, or thymus, and only rarely in the
    testes. There was no evidence of tissue storage (Gaunt & Butterworth,
    1982).

         Groups of 3 male Fischer 344 rats were given daily gavage doses
    of 1.8, 18 or 180 mg/kg b.w. 14C-DEHP, and sacrificed 1, 3, 10 and
    12 days after initiation of treatment. Residual 14C in testes and
    liver, as percent of administered dose, decreased with duration of
    treatment and magnitude of dose, except for testes at day 12 (all dose
    levels) in which 14C levels were higher than at day 10. In
    experiments with a single dose (1.8-1000 mg/kg b.w.) of DEHP, no
    accumulation occurred in the liver at doses below 400g/kg b.w. (Albro
    et al., 1982b).

         Wistar rats, 200 g, were administered a single dose of DEHP or
    MEHP (10,000 mg/kg b.w.) by gastric intubation and samples of blood
    and tissue collected 1, 3, 6, 24, 48 and 96 hr post-intubation. Levels
    of DEHP in liver declined with a half life of 1 day, while levels in
    epididymal fat declined more slowly (t1/2 = 6.5 days), t1/2 values for
    DEHP in other tissues were less than that observed in liver. MEHP
    levels declined much more slowly (t1/2 = 50 hr) than DEHP (8.3 hr) in
    testes, but more rapidly (t1/2 = 67.6 hr for MEHP vs. 156 hr for DEHP)
    in epididymal fat. In other tissues, levels of the two phthalates
    showed similar rates of decline. In heart and lungs, the highest
    levels of DEHP and MEHP occurred within 1 hr of dosing, while, in
    other tissues, the highest levels occurred 6-24 hours after dosing
    (Oishi & Hiraga, 1982).

         Groups of 24 female rats were fed diets containing either 0.1% or
    0.5% ppm 14C-DEHP for 35 and 49 days, respectively. In rats fed 0.1%,
    DEHP 14C residues reached steady state levels of 40-50 ppm in liver
    in 3 days, while steady state levels in fat (7-9 ppm), were attained
    by 2 weeks. 14C levels in brain never exceeded 1 ppm, but in the
    heart, levels ranged from 1 to 6 ppm. At the high dose level,
    equilibrium levels of 14C were attained in the rats after 9-14 days
    of feeding. Steady state levels of 14C in the rat tissues were 120
    ppm in liver, 80 ppm in fat, 2-3 ppm in brain and 15-20 ppm in heart.
    After removal of rats from the test diet, 14C declined with a half
    life of 1-2 days in liver, and 3-5 days in fat (Daniel & Bratt, 1974).

         4 male C57BL mice, 20 g, were given oral doses of 14C-DEHP
    (2-ethylhexyl -1-14C, 9.6 mg/kg b.w.; and carbonyl-14C, 3.6 mg/kg
    b.w.) and sacrificed 5, 14 or 30 days later. Whole body
    autoradiography showed that in the males, either labeled form of DEHP
    was retained at low levels in bone. The (carbonyl -14C) DEHP but not
    the other labeled form, displayed marked retention in the skin,
    cartilage and tendons. In a study with pregnant mice, administration
    of the 14C labeled compounds at day 8 or 16 of gestation resulted in
    14C-DEHP being concentrated mainly in the yolk sac. DEHP (carbonyl-
    14C was present in the neuroepithelium of the embryo. Dosing at day
    18 of gestation resulted in a more general distribution of the 14C
    labeled compounds in the fetuses, 4 hr post-dosing, but 24 hr post-
    dosing the 14C was primarily located in the renal pelvis, urinary
    bladder, intestines and skeleton of the fetus (Lindgren et al., 1982).

         Two adult men ingested single doses of 10 g and 5 g, respectively
    of DEHP.  Phthalic acid equivalent to approximately 4.5% of the
    administered dose was recovered from the urine in the 24 hr period
    after dosing, the major portion being excreted 5 to 7 hours after
    dosing (Schaffer et al., 1945).

    Effects on enzymes and other biochemical parameters

         When rats were fed DEHP (0, 1, 2 or 4%) for 4 weeks, liver
    glycogen decreased markedly, while hepatic fatty acid synthesis
    increased 2-fold. Treatment-related alterations in levels of
    intermediates in carbohydrate metabolism indicate that gluconeogenesis
    was inhibited at a point between 3-phosphoglycerate and fructose
    1,6-diphosphate (Sakurai et al., 1978).

         When male Wistar rats (80-95 gm) were fed 0.5% DEHP for 10 days,
    livers of treated animals were enlarged, and showed reduced levels of
    glucogen and triglycerides and increased levels of phospholipids.
    These treatment-related changes were suggestive of metabolic
    transformation from glycolysis to lipolysis as an energy source
    (Yanagita et al., 1978).

         Male Fisher-344 rats were fed diets containing either 100 ppm or
    1.0% DEHP for 11 days. On days 1, 6 or 10, rats were given a tracer
    dose of 14C DEHP and sacrificed 10 or 1 day later, respectively.
    Purified protein, RNA and DNA fractions were obtained from the liver
    and assayed for bound radioactivity.  Binding was detectable only when
    the ethylhexyl moiety of DEHP was labeled (Albro et al., 1982b).

         Male F-344 rats, 150-180 g, fed 2% 2-ethylhexanol for 3 weeks
    showed increased liver size, increased activity of hepatic peroxisome-
    associated enzymes (catalase and carnitine acetyl transferase) and
    hypolipidemia. These DEHP-induced effects are similar to those
    produced by hypolipidemic drugs such as clofibrate (Moody & Reddy,
    1978).

    Special studies on peroxisomal proliferation

         Adult male, Sprague-Dawley rats were fed for 2 weeks with diets
    containing 2% DEHP or DEHP metabolites (MEHP, phthalic acid,
    2-ethylhexanol and 2-ethylhexyl benzoate).  At sacrifice, livers were
    removed and samples taken for electron microscopy and subcellular
    fractionation (mitochondria and microsomes). DEHP and MEHP stimulated
    peroxisome enzymes (palmitoyl CoA oxidation, 350-600%), mitochondria
    (protein, 238-272%; carnitine-acetyl transferase, 1000-2900%) and
    microsomes (cytochrome P-450, 63-68%; NADHP-cytochrome C reductase,
    59-73%). Cytochrome C oxidase was stimulated by MEHP and EH (75-91%)
    but not by DEHP. 2-elkyl hexanol stimulated mitochondrial protein
    synthesis by 75%. Phthalic acid and ethylhexyl benzoate had little or
    no effect on marker enzymes. Ultrastructural examination revealed
    induction of mitochondrial and peroxisomal proliferation by DEHP and
    MEHP, without induction of the endoplasmic reticulum. Other
    metabolites had no ultrastructurally visible effect (Ganning et al.,
    1982, 1983; Ganning & Dallner, 1981).

         Rats (120 g)  fed diets containing 2% (w/w) di-(ethylhexyl)-
    phthalate (DEHP) for up to 4 weeks, showed a marked increase in
    activities of enzymes of peroxisomal beta-oxidation and of catalase
    (liver). The time required to reach 50% maximal induction for enzymes
    of peroxisomal beta-oxidation was 5-7 days, whereas that for
    catalase was 3 days. After withdrawal of DEHP, activities of enzymes
    of the beta-oxidation system and of catalase decrease to the control
    level with a half-life of 2-3 days. Mitochondrial marker enzymes
    (3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase
    (non-specific) and acetoacetyl-CoA specific thiolase) also were
    markedly increased in treated animals. These activities decreased
    after withdrawal of treatment (Miyazawa et al., 1980; Ohno et al.,
    1981).

    Special studies on mutagenicity

         In a number of laboratories, DEHP did not include reverse
    mutations of the base pair substitution type in Salmonella
    typhimurium with or without metabolic activation (Kirby et al.,
    1983; Kozumbo et al., 1982; Ruddick et al., 1981; Simmon et al., 1977;
    Warren et al., 1982; Yoshikawa et al., 1983; Zeiger et al., 1982).
    Neither was 8-azaguanine resistance in S. typhimurium affected by
    DEHP (Seed, 1982). Tomita et al., (1982) reported a weak positive
    reaction for DEHP in the S. typhimurium system.

         No mutations (tryptophan reversion) occurred in Eschericia coli
    exposed to DEHP in vitro (Tomita et al., 1982; Yoshikawa et al.,
    1983) or in DNA repair capability of Bacillus subtilis (Tomita et
    al., 1982) exposed to DEHP.

         DEHP did not show mutagenic effects in a number of mammalian cell
    systems: L5178Y mouse lymphoma (forward mutation), Kirby et al.
    (1983); rat hepatocyte (unscheduled DNA synthesis), Hodgson, (1982);
    human lymphocyte (chromosomal aberrations), Turner et al. (1974);
    human fetal lung cells (aneuploidy), Stenchever et al. (1976); Chinese
    hamster fibroblast (chromosomal aberrations), Abe & Sazaki (1977),
    Ishidate & Odashima (1977); Chinese hamster ovary (sister chromatid
    exchange, sister chromatid exchange HGPRT reversion), Phillips et al.
    (1982).

         DEHP, MEHP and 2-ethylhexanol were tested for mutagenicity in the
    following systems: Ames Salmonella/microsome plate test, mouse
    lymphoms forward mutation assay, in vitro transformation of Balb/3T3
    cells (with and without activation by primary rat hepatocytes), mouse
    micronubleus test, and unscheduled DNA synthesis in rat hepatocytes.
    No mutagenic activity was observed (Anonymous, 1982a-m).

         MEHP was shown to be mutagenic in S. thyphimurium (histidine
    reversion), in E. coli (tryptophan reversion) and in B. subtilus
    (DNA repair) (Tomita et al., 1982). The same workers also found MEHP
    to be mutagenic in Chinese hamster embryo cells (chromosome
    aberration, 8-azaguanine resistance, 6-thioguanine resistance, sister
    chromatid exchange).  Phillips et al. (1982) found MEHP to be
    mutagenic to Chinese hamster ovary cells (chromosomal damage, sister
    chromatid exchange), but non-mutagenic toward HGPRT reversion. Kirby
    et al. (1983) found MEHP to be non-mutagenic in the L5178Y mouse
    lymphoma system (forward mutation).

         2-ethylhexanol was shown to be weakly mutagenic in
    S. typhimurium (8-azaguanine resistance, Seed, 1982), but was
    non-mutagenic in L5178Y mouse lymphoma (Kirby et al., 1983), rat
    hepatocyte (Hodgson et al., 1982) and Chinese hamster ovary (Phillips
    et al., 1982).

         Pregnant Syrian golden hamsters were given DEHP orally at 3.75,
    7.5 or 15 g/kg b.w. or MEHP at 0.375, 0.750 or 1.5 g/kg b.w. on day 11
    of gestation. In embryonic cells removed on day 12 and examined after
    15-20 days of culture in vitro, there were significant increases in
    aberrant metaphase cells from all treatment groups except the one that
    received the lowest dose of DEHP. Aberrations included single
    chromatid gaps, isochromatid gaps, single chromatid or isochromatid
    breaks and chromatid exchanges. There were also significant increases
    in the rate of morphological transformation of the cells at the two
    highest doses of DEHP and the two lowest doses of MEHP, and a marginal
    increase in 8-azaguanine and 6-thioguanine resistance (Tomita et al.,
    1982).

         No significant increases in clastogenic changes were observed in
    the bone marrow cells of male Fischer 344 rats that had received
    0.5-5.0 g DEHP/kg b.w./day, 0.01-0.14 g MEHP/kg b.w./day or

    0.02-2.21 g 2-ethylhexanol/kg b.w./day by gavage for 5 days (Putman
    et al., 1983). Dominant lethal assays using male mice given DEHP
    (2.5-9.9 g/kg), MEHP (50-200 mg/kg) or 2-ethylhexanol (250-1000 mg/kg)
    by gavage in corn oil for 5 days produced negative results: fertility
    indices and the average numbers of dead and total implants per
    pregnancy were within normal ranges in all cases (Rushbrook, 1982).

    Special studies on carcinogenicity

         Groups of 50 male and female B6C3F1 mice were fed diets
    containing 0, 0.3 or 0.6% Di(2-ethylhexyl)phthalate (DEHP) for 103 wk.
    Treatment with DEHP did not affect survival or food consumption. Mean
    body weight gains of treated female mice (0.3 and 0.6%) were less than
    those of the corresponding controls. No other clinical signs of
    toxicity were reported. At termination of the studies, chronic
    inflammation of the kidney and seminiferous tubular degeneration were
    observed in male mice (0.6% diet). No other nonneoplastic lesions were
    detected in the treated groups at incidences greater than in the
    corresponding controls.

         Treatment with DEHP caused liver tumors in both sexes of mice. 
    Incidence data are as follows: for males, hepatocellular carcinoma
    9/50, 14/48 and 19/50, hepatocellular adenomas 6/50, 11/48 and 10/50,
    and for females hepatocellular carcinoma 0/50, 7/50 and 17/50 and
    hepatocellular adenoma 1/50, 12/50 and 18/50, for the 0, 0.3% and 0.6%
    groups, respectively.  20 of the 57 hepatocellular carcinomas
    diagnosed in treated mice (sexes and doses combined) had metastisized
    to the lung. No pulmonary tumors were observed in any control rats.
    The incidences of liver tumors in control mice was comparable to that
    in recent historical controls. The incidences of both male and female
    mice with hepatocellular carcinomas were significantly greater
    (p<0.05) at the 0.6% dose than in controls, and in female mice at the
    lower dose (p<0.05) by pairwise comparison, and trend tests detected
    significant (p<0.05) dose related effects in both sexes. The
    incidence of hepatocellular adenomas in both male and female did not
    differ from that of controls. However, the incidences of combined
    adenomas and carcinomas was significantly increased for both sexes at
    both doses (p<0.05), by pairwise comparisons and showed significant
    dose related trends (Kluwe et al., 1982).

         Groups of 50 male and female Fischer 344 rats were fed diets
    containing 0, 0.6 or 1.2% di(2-ethylhexyl)phthalate (DEHP) for 103
    consecutive weeks. Treatment with DEHP did not affect survival rates
    or food consumption. Mean body weight gains of treated male rats
    (0.6 and 1.2% diet) and female rats (0.2%), were less than those of
    the corresponding controls. No clinical signs of toxicity were
    reported. At autopsy, seminiferous tubular degeneration and
    hypertrophy of cells in the anterior pituitary were observed in
    high-dose male rats. Other nonneoplastic lesions were detected in the
    treated groups at incidences similar to the corresponding controls.

         Incidence data are as follows: for males, hepatocellular
    carcinoma 1/50, 1/49 and 5/49, neoplastic nodules 2/50, 5/49 and 7/49,
    and for females, hepatocellular carcinoma 0/50, 2/49 and 8/50 and
    neoplastic nodules 0/50, 4/49 and 5/50 for the 0, 0.6 and 1.26 groups,
    respectively. The incidence of female rats with hepatocellular
    carcinomas the 1.2% group was greater than that in control by pairwise
    comparison (p<0.01) and there was a significant (p<0.05) dose
    related trend effect. The incidence of female rats with neoplastic
    nodules was greater in the 1.2% than in controls (p<0.05) and showed
    a significant (p<0.05) dose related trend effect. For male rats the
    incidence of hepatocellular carcinomas showed a dose related trend
    effect, but was not significantly increased by pairwise comparison.
    The incidence of neoplastic nodules in the male rats did not show a
    significant increase using either pairwise comparison or trend tests
    (Kluwe et al., 1982).

    Special studies on promotion of carcinogenicity

         Weanling male B6C3F1 mice received a single i.p. injection
    (80 mg/kg) of diethylnitrosamine (DEN) at 4 weeks of age, followed by
    oral administration of phenobarbital (PB) or di(2-ethylhexyl)phthalate
    (DEHP) 2 weeks after DEN injection and continued for up to 6 months.
    PB was administered in drinking water at 500 ppm and DEHP in the feed
    at 0.3%, 0.6% or 1.2%. Groups of mice were sacrificed at 2, 4 and 6
    months after DEN exposure and the livers were examined. Few
    preneoplastic foci were seen at 2, 4 or 6 months, in mice exposed to
    DEN, PB or DEHP alone, while numerous foci and neoplasms were seen in
    mice given DEHP or PB after DEN. In DEHP-exposed mice, the number of
    foci did not increase between 4 and 6 months, but the foci increased
    in mean diameter and volume throughout the experiment. Foci and tumors
    appeared earlier in mice given higher dietary levels of DEHP than in
    those given lower doses. By the end of the study the number of foci
    per unit volume of liver was similar in mice given any dose of DEHP,
    but their volume was dose-related; basophilic foci and neoplasms
    predominated. The latter were more malignant in appearance than
    neoplasms in PB-exposed mice. At 6 months, the neoplasms in high dose
    DEHP-exposed mice were significantly larger than those in PB-exposed
    mice. Histochemistry, however, revealed similarities between lesions
    in mice exposed to PB or DEHP. PB given continuously for 6 months
    revealed no initiating activity of DEHP given once by gavage and
    followed by PB in drinking water (Ward et al., 1983).

    Special studies on the metabolites

         Male Sprague-Dawley rats, 250-350 gm, were given 14C-mono
    (ethylhexyl) phthalate (MEHP), 69 mg/kg, by gavage. MEHP is a primary
    metabolite of DEHP. The resulting urinary metabolites of MEHP were
    similar to those of DEHP, and included an alcohol, a ketone, and an
    acid from the side chain oxidation of MEHP, as well as a small amount
    of o-phthalic acid (Chu et al., 1978).

         Phthalic acid, a compound from which DEHP is formed by
    esterification, was fed by intubation to male Wistar rats at dose
    levels of 3.3 or 40 mg/kg b.w. 0.15% (as 14C02) of the administered
    dose was detectable in expired air. Only unchanged phthalic acid could
    be found in urine, feces or tissue. Elimination was complete by 24
    hours after dosing (Williams & Blanchfield, 1974b).

         After oral administration to rats, 2-ethylhexanol was rapidly
    absorbed and excreted. Radioactivity from (1-14C)-2-ethylhexanol was
    detected in the urine (80-82%), feces (8-9%) and expired C02 (6-7%).
    The route of metabolism of 2-ethylhexanol appears to involve oxidation
    of 2-ethylhexanoic acid, followed by w- and (w-1)-oxidation.
    Urinary metabolites included 2-heptanone, 4-heptanone, 2-ethyl-5-
    hydroxyhexanoic acid, 2-ethyl-1,6- hexanedioic acid and 2-ethyl-5-
    hexanone. The rate of elimination of 2-ethylhexanol was the same for 
    a 9 ug as for an 83 mg dose, with essentially all of the 14C being
    recovered within 28 hours after administration (Albro, 1975).

         Groups each of ten Weanling Sprague-Dawley rats were fed diets
    containing 0, 25, 100, 400, 1,600 or 6,400 ppm of mono-2-ethylhexyl
    phthalate for 28 days. Decreased growth rate was observed in the
    highest dose group. Increased heart and liver weights were observed in
    animals of the 1,600 and 6,400 ppm groups. Minor alterations in serum
    biochemical values included decreased SDH and calcium levels, and
    elevated alkaline phosphatase activity in some treated groups (Chu et
    al., 1981).

         In 3-month and 6-month feeding studies, groups of ten male and
    female Weanling Sprague-Dawley rats were fed diets containing 1, 5,
    25, 125 or 625 ppm of mono-2-ethylhexyl phthalate. Growth rate and
    food consumption were not affected at any dose level or time interval.
    Relative organ weights of rats of both sexes were not altered in the
    3-month period, but the liver weights of female rats were increased in
    the 6-month study. Changes in clinical chemistry and hematological
    values were minimal. These included lower LDH, SGOT, hemoglobin, and
    hematocrit values in male rats at the 3-month period and reduced
    potassium content at the 6-month period. Histological changes occurred
    in treated male and female rats at both time intervals in the liver,
    heart, and adrenals. Alteration in the liver consisted of midzonal and
    perioportal eosinophilic cytoplasmic inclusions and vacuolations with
    isolated binucleated and necrotoc hepatocytes. There was a mild
    enlargement of myocardial nuclei and segmental deregistration of
    myocardial striations in test animals. The adrenal glands exhibited
    vacuolation of the zone fasciculata which was less severe in the
    6-month study than the 3-month counterpart (Chu et al., 1981).

    Special studies on reproduction and teratology

    Mouse

         Groups of 10-22 pregnant mice (ddY-Slc (SPF) strain, 8-9 weeks
    old) were administered single oral doses (0, 50, 100, 500 mg/kg b.w.)
    of DEHP on day 7 of gestation, and sacrificed on day 18. Dose-related
    decreases were noted in fetal body weights, number of live fetuses and
    implantations per dam. Gross and skeletal anomalies exhibited
    dose-related increases. The only treatment effect noted in fetuses
    from dams fed 50 mg/kg DEHP was decreased body weight (Nakamura
    et al., 1979).

         Groups of 3-8 pregnant mice (ddY-Slc (SPF) strain, 8-9 weeks
    old) were administered single oral doses of DEHP ranging from 100 to
    30,000 mg/kg b.w. on days 6, 7, 8, 9 or 10 of gestation. MEHP was
    administered to a separate group of pregnant mice at levels from
    100-1000 mg/kg b.w. on days 7, 8 or 9 of gestation. Dams were
    sacrificed on day 18 of gestation.  With DEHP-treated dams, 
    treatment-related decreases were noted in fetal body weight
    (days 6-10) and number of live fetuses (days 7-9). Because of the
    variance in dose levels, no clear-cut treatment effect was evident on
    implantations per dam. Treatment-related increases were seen in gross
    anomalies (days 7-10) and skeletal anomalies (days 6-8 and day 9, high
    dose only).

         With MEHP-treated dams, treatment-related decreases were noted in
    the number of live fetuses (days 7-8) and increases in gross anomalies
    (days 8-9) and skeletal anomalies (day 8). Decreases in fetal body
    weight were only marginally related to treatment except for the high
    dose dams on day 8 and day 9. No clear-cut treatment effect could be
    seen on the number of implantations per dam (Yagi et al., 1980).

         Teratogenic effects were observed in CBA mice following a single
    oral administration of DEHP in doses representing 1/3-1/12 of the
    acute LD50 dose (26.9 g/kg b.w.) on days 6 to 10 of gestation. Excess
    fetal deaths were observed when higher doses were given on day 7 but
    not when given on day 9 or 10. A significant number of external and
    skeletal malformations were found in the group given 7.5 g/kg b.w. on
    day 8 (Yagi et al., 1976).

         Pregnant female mice (ICR-JCL strain, 8-16 weeks of age) were
    distributed in groups varying from 8 to 15 and fed diets containing
    0, 0.05, 0.1, 0.2, 0.4 and 1.0% DEHP throughout gestation. Dams were
    sacrificed on day 18 of gestation. At dose levels above 0.1%, maternal
    weight gain was significantly depressed, although mean food intake was
    unaffected. Fetal mortality was also significantly increased in this
    dose range. At dose levels above 0.05%, the percentage of resorptions
    and dead fetuses exhibited a dose-related increase, and was 100% at
    dietary DEHP levels of 0.4% and 1%. Several cases of neural tube

    defects (spine bifida and exencephaly) occurred in fetuses from dams
    fed 0.2% DEHP. Deficient lumbar rib and ossification of sternabrae was
    noted in fetuses from dams fed 0.1% and 0.2% DEHP. Fetal survival was
    too low at higher doses to permit meaningful data on incidence of
    anomalies. No other skeletal anomalies or visceral anomalies could be
    related to treament (Shiota et al., 1980).

    Rat

         Pregnant Wistar rats, 175-200 g, were randomly distributed into
    groups of 15 and dosed by gavage with 0, 225, 450, or 900 mg MEHP/kg
    b.w. on days 6-15 of gestation. Another similar experiment was
    performed at lower doses (0, 50, 100 and 200 mg/kg b.w.) of MEHP.
    Dose-related maternal mortality occurred at the higher doses (225,
    450, 900 mg/kg b.w.). In addition, the number of litters per treatment
    group decreased significantly at higher doses. Litter size and pup
    weight were not significantly affected by treatment. Disturbances in
    the placement of the sternebrae plates were noted in fetuses from dams
    treated with 450 mg/kg b.w. MEHP. No other skeletal or visceral
    anomalies could be related to treatment. In the low-dose experiment,
    the only treatment-related effect was a reduction in maternal weight
    gain at MEHP doses of 100 and 200 mg/kg b.w. The no-effect level for
    MEHP in this study was 50 mg/kg b.w. (Ruddick et al., 1981).

         Groups of 20 female Wistar rats (80-120 g) were given daily doses
    of 0, 0.34 and 1.70 g/kg DEHP by gavage. After approximately 3 months
    of treatment, 10 rats (150-180 g) per dose level were mated. During
    gestation, test compound was discontinued. In a separate experiment,
    groups of 10 female rats (150-180 g) received comparable doses of
    DEHP, but only during gestation. All fetuses from treated dams were
    live; no skeletal abnormalities were found. No examination of fetuses
    for soft tissue abnormalities was carried out. In dams treated during
    gestation, litter size was unaffected but resorptions increased
    significantly and fetal weights decreased significantly at both
    treatment levels.  No significant effects on resorptions, litter size
    or fetal weights occurred in dams treated prior to gestation
    (Nikoronow et al., 1973).

    Rabbit

         Virgin female New Zealand, white rabbits (3.9 + 0.5 kg) were
    artificially inseminated and injected with sodium monoethylhexyl
    phthalate (MEHP, 1.14, 4.69 or 11.38 mg/kg) on days 6-18 of gestation.
    When fetuses were removed on day 30, no treatment effect was evident
    on sex ratio, fetal weight or litter size. The number of corpora lutea
    or resorptions were not affected by treatment, nor were weights of
    maternal adrenals, liver, kidneys, heart or lung. No skeletal or
    visceral abnormalities occurred that could be ascribed to treatment.
    However, the 2 high dose groups of dams experienced increased
    mortality (22% and 33%) relative to controls (Thomas et al., 1979).

         When pregnant rabbits were injected with MEHP (11.4 mg/kg) on
    days 6-18 of gestation, phthalate levels were less than 1 ug/ml in
    fetuses and in homogenates of placenta and uteri. Maternal serum
    levels failed to reveal any bioaccumulation, and there was no
    significant evidence of histological changes in fetal organs,
    including liver, lungs and kidneys (Thomas et al., 1980).

    Special studies on testicular pathology and effect on zinc
    and testosterone levels

         Groups of 10 male JCL:ICR mice and rats were fed a diet
    containing 2% DEHP or 2% MEHP for 1 week. Both test compounds
    depressed growth, testicular weight, and liver weight. Levels of
    testosterone and zinc were depressed in the testes, and kidney zinc
    levels were elevated in both treatment groups. Liver zinc content was
    depressed in the DEHP treatment group, but unchanged in the MEHP group
    (Oishi & Hiraga, 1980a,b). Similar effects were observed with
    Sprague-Dawley rats given oral doses of DEHP or MEHP (Gray et al.,
    1982; Oishi & Hiraga, 1980c,d).

         Male Fischer 344 rats and B63CF mice were distributed into groups
    of 50 and fed diets containing 0, 0.6% or 1.2% DEHP (rat); or 0, 0.3
    or 0.6% (mice) for 103 weeks. High dose rats exhibited a 90% incidence
    of severe seminiferous tubular degeneration and testicular atrophy in
    comparison to an incidence of 5% or less in low dose rats or controls.
    The tubules in the affected animals were devoid of spermatocytes and
    germinal epithelium. Only Sertoli cells were observed on the basement
    membrane. High dose mice experienced a 14% incidence of seminiferous
    tubular degeneration, compared to an incidence of 4% or less in low
    dose mice or controls (Kluwe et al., 1982). In another study,
    0.2% DEHP fed to male CDE rats for 17 weeks, caused marked
    histological changes in the testes (Gray et al., 1977).

         Groups of 10 male Wistar rats of varying age (4, 10 or 15 weeks)
    were given 2.8 g/kg b.w. DEHP by gavage for 10 consecutive days.
    Treated 4 week-old rats showed uniform seminiferous tubular atrophy
    comprising a loss of spermatids and spermatocytes. 10 week old rats
    experienced a 5-10% incidence of seminiferous tubular atrophy, while
    in 15-week old rats, no adverse effects of this type were reported. If
    treatment was stopped prior to puberty, normal testicular weight and
    histology were regained within 12 weeks. Recovery was slower and less
    complete when treatment was discontinued after puberty (Gray and
    Butterworth, 1980).

         Groups of 7-8 Syrian hamsters (DSN strain) were given oral doses
    of 4200 mg/kg b.w./day DEHP or 1000 mg/kg b.w./day MEHP for 9 days.
    MEHP reduced mean testicular weight to 73% of control values and
    induced atrophy in less than 50% of the seminiferous tubules. DEHP had
    no significant effects on these parameters or on urinary zinc
    excretion (Gray et al., 1982).

    Acute toxicity

                                                              

    Animal         Route      LD50      References
                          (mg/kg b.w.)
                                                              

    mouse          p.o.      33,500     Krauskopf, 1973
    rat            p.o.      26,000     Patty, 1967
    rabbit         p.o.      34,000     ibid
                             33,900     Shaffer et al., 1945
    Guinea pig     p.o.      26,300     Krauskopf, 1973
                                                              

    Short-term studies

         A study was conducted on the effect of oral administration of
    di-(2-ethylhexyl)phthalate (DEHP) at a dose level of 25,000 ppm for
    7 and 21 days in young male and female Wistar albino rats. DEHP
    increased liver size in both sexes and reduced the relative weight of
    testes in male rats. Liver enlargement was accompanied by increases in
    several marker enzyme activities (7-ethoxy coumarin-0-deethylase,
    cytochrome P-450, alcohol dehydrogenase, aniline-4-hydroxylase, and 
    succinate dehydrogenase). However, glucose-6-phosphatase activity was
    decreased in all treatment groups. DEHP produced no hepatic
    histological changes in either sex but ultrastructural studies
    indicated proliferation of the smooth endoplasmic reticulum, an
    increase in the number of microbodies (peroxisomes), and mitochondrial
    changes (Mangham et al., 1981).

         When female rats were fed 5000 ppm 14C-DEHP for 49 days,
    relative liver weights increased progressively during the first week
    of treatment to a value approximately 50% above normal. Only a slight
    increase in smooth endoplasmic reticulum was visible by electron
    microscopy. When treatment was discontinued, liver weights returned to
    normal within one week. Rats fed for 35 days on a 1000 ppm diet did
    not experience alterations in liver weight relative to controls
    (Daniel & Bratt, 1974).

         Groups of 10 male and 10 female Wistar rats, 90-120 g, were
    administered daily gavage doses of DEHP at levels of 0, 3400 ppm and
    34,000 ppm for 90 days. At autopsy, gross pathological observations
    were made: liver, kidney and spleen were weighed and examined
    microscopically. High-dose rats displayed uncertainty of movements,
    drowsiness, diarrhea and rapid weight loss, with a 75% mortality.
    Gross autopsy revealed congestion of the small intestine and loss of
    mucosa in the stomach and of some parts of the intestine. A
    treatment-related increase in mean liver weight of low-dose rats was
    observed, but no gross or microscopic alterations were seen in the
    liver, kidneys or spleen (Nikonorow et al., 1973).

         Groups of 15 male and 15 female rats were given diets containing
    0 (control), 0.2, 1.0 or 2.0% di(2-ethylhexyl)-phthalate (DEHP) for 17
    weeks. At the two higher treatment levels there was a reduced rate of
    body-weight gain and food intake. A paired-feeding study showed that
    the reduced food intake did not account fully for the reduced growth
    rate. Hematologic studies showed decreased hematocrits in both sexes
    in the 1 and 2% DEHP group, as well as lower hemoglobin levels in
    males at these dose levels. Renal concentrating and diluting ability
    was reduced in the females receiving 2% DEHP. The relative testes
    weight of rats on the (2%) diet was markedly decreased and
    histopathological examination revealed severe seminiferous tubular
    atrophy and cessation of spermatogenesis. Although testes weight of
    the rats fed 0.2% DEHP was not reduced, histological studies showed
    evidence of decreased spermatogenesis. There were no other
    histopathological changes attributable to DEHP treatment (Gray et al.,
    1977).

         Groups of 22-24 male and female "hybrid" guinea pigs, 49 days
    old, were fed diets containing 0, 0.04 or 0.13% DEHP for 1 year.
    Histopathological examination was performed on kidney, liver, lung,
    spleen and testes; only liver and kidney were weighed.  No treatment
    effects were reported on mortality, growth, food consumption,
    microscopic pathology or incidence of neoplasms. Treated females had
    significantly increased relative liver weights (Carpenter et al.,
    1953).

         Groups of 6-7 male albino ferrets, 18 months old, were fed diets
    containing 0 or 1% DEHP for 14 months. Treated animals experienced
    significant growth depression and liver enlargement, with altered
    liver morphology. Microscopic examination of sections of brain, heart,
    adrenals, thyroid, trachea, esophagus, lung, kidney, and bladder did
    not reveal any treatment effect. Mean testicular weights of treated
    animals were reduced and 3/7 test animals exhibited a nearly complete
    absence of germinal epithelium in testes. Significant treatment-
    related effects were seen on activities of marked enzymes for
    microsomes, and in drug metabolizing enzymes. Significant changes were
    seen in mitochondrial marker enzymes of treated animals (Lake et al.,
    1976).

         8 dogs (4 Cocker Spaniels, 4 Wire-haired Terriers), 14-17 months
    old, were randomly distributed by sex and breed into 2 groups (one
    control, one administered DEHP by capsule). The treatment group
    received 30 mg DEHP/kg b.w./day, 5 days a week, for 19 doses; then
    60 mg/kg b.w./day for 240 additional doses. There was no effect on
    growth. At autopsy liver and kidney weight were within normal range. 
    Gross and histopathological examination of lung, heart, liver,
    stomach, small intestine, colon, cecum, spleen, adrenal, gonad,
    bladder and thyroid gland of treated animals showed no treatment
    related effects (Carpenter et al., 1953).

    Long-term studies

         Groups of 32 and female Sherman rats, 60 days old, were fed diets
    containing 0, 0.04, 0.13 and 0.4% DEHP for up to 2 years (P1). At week
    12, animals from the control and high dose levels were bred and their
    offspring used to establish an F1 generation composed of 32 males and
    32 females. After one year of treatment, groups of 9 to 17 rats from
    each sode level (P1 generation) were sacrificed for "tissue and organ
    weight" data. Remaining rats were culled to 8 males and 8 females per
    group and survivors sacrificed after 2 years of treatment. The F1
    generation was placed on test at 15 days of age and sacrificed after
    1 year. Histopathological examination was performed on adrenal, heart,
    kidney, intestine, liver, lung, spleen, testes, ovary and gross
    lesions; only kidney and liver were weighed. At the 0.04% level of
    DEHP, both P1 and F1 males exhibited growth depression and an
    increased mean relative weight of liver and kidney. F1 females also
    showed increased liver and kidney weights at this dose level. No
    treatment related effects were reported on survival, food consumption,
    microscopic pathology, incidence of neoplasms, hematology or fertility
    (Carpenter et al., 1953).

    OBSERVATIONS IN MAN

         No information available.

    Comments

         Orally ingested DEHP is probably absorbed as MEHP, since DEHP is
    rapidly hydrolyzed to MEHP and 2-ethylhexanol by extracts from the
    intestine from several animal species and man. In addition, studies
    with isolated perfused rat intestine showed that all DEHP was
    converted to MEHP before it reached the serosal perfusing solution.
    DEHP metabolites are rapidly distributed throughout the body and reach
    a steady state level in the tissues after a period of exposure of up
    to 2 weeks, with some accumulation in liver and fat. Tissue residues
    are rapidly depleted when DEHP is removed from the diet. There is no
    definitive information on the chemical nature of the tissue residues,
    since the available distribution studies reflect only a distribution
    of 14C, derived from 14C labeled DEHP. In experimental animals the
    absorbed material is excreted almost equally in the urine and feces,
    with very little being expired as CO2. Biliary excretion ranges from
    9-11%. The urine contains up to 5 major metabolites, MEHP, and
    4 oxidation products (beta-oxidation) of the MEHP. Only small amounts
    appear as o-phthalic acid. In species other than rat (including monkey
    and man), the metabolites are excreted mainly as glucuronides. Species
    differences were also noted in the distribution of other urinary
    metabolites. MEHP appeared in only trace amounts in rat urine, as
    did unchanged DEHP in the urine of all species. In monkey and man,
    MEHP is one of the major urinary metabolites. In rats, 6.5-10.5% of

    administered DEHP appeared as fecal metabolites; most had been
    excreted by 48 hr after dosing. Rats fed low amounts (10 ppm) of DEHP
    had no detectable residues in feces. The secondary metabolites in
    feces have not been identified.

         DEHP and MEHP cause an increased liver weight with marked
    proliferation of mitochondria and peroxisomes in the liver. The
    peroxisomal enzymes that are increased the most are those associated
    with beta-oxidation systems. There is less induction of microsomal
    enzymes. These effects are reversible when DEHP is removed from the
    diet.

         DEHP was teratogenic in mice, but not in rats and rabbits.  DEHP
    at high dietary levels caused severe seminiferous tubular degeneration
    testicular atrophy and cessation of spermatogenesis in rats and mice.
    Similar effects were observed with MEHP. Reduced zinc levels in serum,
    liver and testes were also reported. Other short-term effects of
    dietary DEHP include liver enlargement and effects on the heart and
    adrenals. DEHP and 2-ethylhexanol were not mutagenic in the Ames test
    and a number of mammalian cell systems. In contrast, MEHP was
    mutagenic in a number of systems. In lifetime feeding studies in rats
    and mice, DEHP caused a significant increase in liver tumors in both
    species.

    EVALUATION

         DEHP is a hepatocarcinogen in both rats and mice.

    Provisional acceptance

         The level of DEHP in the food contact material and the extent of
    its migration into food should be kept at the lowest levels which are
    technologically possible.

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    OISHI, S. & HIRAGA, K. (1980b) Effects of phthalate acid monoesters on
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    OISHI, S. & HIRAGA, K. (1980c) Testicular atrophy induced by phthalic
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    OISHI, S. & HIRAGA, K. (1980d) Testicular atrophy induced by phthalic
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    PHILLIPS, B,J., JAMES, T.E., & GANGOLLI, S.D. (1982) Genotoxicity
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    PUTMAN, D.L., MOORE, W.A., SCHECHTMAN, L.M., & HODGSON, J.R. (1983)
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    RUDDICK, J.A., VILLENEUVE, D.C., CHU, I., NESTMANN, E., & MILLES, D.
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    SEED, J.L. (1982) Mutagenic activity of phthalate esters in bacterial
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    THOMAS, J.A., MARTIS, L., GIOVANETTO, S., McCAFFERTY, R.E., & DONOVAN,
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    WILLIAMS, D.T. & BLANCHFIELD, B.J. (1974a) Retention, excretion and
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    WILLIAMS, D.T. & BLANCHFIELD, B.J. (1974b) Retention, excretion and
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    12: 109-112.

    YAGI, Y., NAKAMURA, Y., TOMITA, I., TSUCHIKAWA, K., & SHIMOI, N. 
    (1980)  Teratogenic  potential of di- and mono-(2-ethylhexyl)
    phthalate in mice. J. Environ. Pathol. Toxicol., 4: 533-544.

    YANAGITA, T., KOBAYASHI, K., & ENOMOTO, N.  (1978) Accumulation of
    hepatic phospholipids in rats fed di-(2-ethylhexyl) phthalate.
    Biochem. Pharmacol., 27: 2283-2288.

    YOSHIKAWA, K., TANAKA, A., YAMAHA, T., & KURATA, H. (1983)
    Mutagenicity study of nine monoalkyl phthalates and a dialkyl
    phthalate using Salmonella typhimurium and Escherichia coli. Food
    Chem. Toxicol., Apr; 21(2): 221-3.

    ZEIGER, E., HAWORTH, S., SPECK, W., & MORTALMANS, K. (1982) Phthalate
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    Mutagenesis Test Development Program. Environ. Health Perspect.
    


    See Also:
       Toxicological Abbreviations