Dimethoxyethyl phthalate: embryopathy, teratogenicity, fetal metabolism and the role of zinc in the rat.

A single intraperitoneal injection (0.6 ml/kg) of dimethoxyethyl phthalate (DMEP) was given to groups of Wistar strain rats on day 10, 11, 12, 13 or 14 of gestation. Control rats received 0.6 ml/kg of physiological saline intraperitoneally. In phthalate-treated rats, embryopathy was manifested by a high incidence (12-79%) of fetal deaths and fetal resorptions. Fetotoxic effects were expressed by a significant reduction in fetal weights. Hydrocephalus interna, a congenital malformation of the brain, was caused by DMEP. Congenital skeletal deformities (66-96%), with multiple skeletal (14-64%) and appendicular malformations (25-57%), were also induced by DMEP. Control rats exhibited no congenital malformations of the brain and no appendicular or multiple skeletal deformities. DMEP caused a significant decrease in the zinc content of the fetus. Fetoplacental metabolism 1 and 4 hr after intravenous administration of 14C-DMEP suggested rapid transfer of the parent compound to the fetus across the placenta and that DMEP is a teratogenic moiety. The possible role of zinc in phthalate-induced teratogenesis in rats is also discussed.


Introduction
Plastics have become an integral part of our everyday lives. Phthalate esters are plasticizers widely used in the manufacture of plastics (1). In the United States, an estimated one billion pounds of phthalate esters are produced annually (2,3). There are approximately 20 different phthalate compounds sold as vinyl plasticizers, and one of the most toxic of these is dimethoxyethyl phthalate (DMEP) (1)(2)(3).
Phthalate esters are found in the composition of floor tiles, various types of home furnishings, waterproof clothing, industrial tubing, food wrap-*This paper was not presented at the Symposium on Phthalate Esters. tToxicology  ping materials (4,5), and a variety of medical and paramedical devices, including heart valves, vascular grafting material, intrauterine devices, catheters, dialyzing units, blood sets and disposable syringes. Residues of phthalates have been found in milk (6), human tissues and blood plasma (7), bovine tissues and the hearts of dogs, rabbits and rats (8). Phthalates, in general, are colorless, high-boiling liquids, soluble in organic solvents but immiscible in water, and they are degraded very slowly in the ambient environment. Diethylhexyl phthalate (DEHP), for example, closely resembles organochlorine pesticides (DDT, PCB) in rate of uptake, storage, and biomagnification in a variety of aquatic organisms (9).
The U.S. Department of Health and Human Services, particularly the Food and Drug Administration (4,10) and the National Institute of Environmental Health Sciences (11)(12)(13) has, therefore, a continuing interest and concern regarding the safety of phthalates. This concern is reflected in the sponsoring of conferences on phthalate esters in 1972 (3,4,9,13) and in 1981. Several reviews (14)(15)(16)(17)(18)(19)(20)(21)(22)(23) presented the toxicity of phthalate esters. Although DMEP has been shown to be one of the most toxic phthalates, little is known regarding the teratogenicity caused by a single maternal injection of DMEP during the organogenetic period in the rat.
Dietary supplementation of zinc has prevented teratogenic effects in rats induced by the maternal administration of a chelating agent, ethylenediaminetetraacetic acid (24). Dietary deprivation of zinc, on the other hand, could induce embryopathic and teratogenic effects in the rat, which was otherwise resistant to the teratogenicity of EM-12, a stable analog ofthalidomide (25). Although phthalate esters bear similarities to thalidomide in inducing congenital malformations in the chick embryo (26), the role of zinc, if any, in phthalate-induced teratogenicity has not been reported.
Metabolism studies on phthalates using radiotracer techniques have been conducted in adult rats (27)(28)(29)(30)(31). There is only one report concerning maternalfetal transfer of radiolabeled phthalates. It deals with 14C-DEHP and diethyl phthalate (32). Fetal metabolism of 14C-DMEP, however, is unknown. The present studies, therefore, were designed to determine whether (a) maternal administration in rats of a single dose of DMEP during the organogenetic period induces embryopathic, fetotoxic or teratogenic effects; (b) multiple congenital skeletal malformations significantly higher than that of the control could be induced by DMEP; (c) uptake of 65Zn by and the zinc content in the fetus are altered by administration of DMEP; and (d) DMEP reaches the fetus as a metabolite or as the unaltered parent compound.

Materials and Methods
Teratological Studies PARKHIE, WEBB AND NORCROSS with each male in a separate cage. The dropping of the mating plug from the female on the morning following mating was counted as day zero of the pregnancy, at which time the females were allocated to control and treatment groups. All animals had free access to a high protein pelleted rat diet (supplied by BP Nutrition, Stepfield, Witham, Essex, England) and water and were housed in a temperature-controlled (22 + 2°C) animal facility.
DMEP of analytical grade was administered intraperitoneally (IP) at a dose of 0.6 ml/kg body weight on day 10, 11, 12, 13 or 14 of gestation. The control animals received the same volume (0.6 ml/kg) of physiological saline intraperitoneally. The number of animals in each treatment group is presented in Table 1.
Pregnant rats were anesthetized by chloroform on day 20, and the fetuses were removed by cesarian operation. More than half of the fetuses were assigned for evaluation of skeletal abnormalities (33,34); the rest were assigned for detection of organ malformations. The fetuses were prepared for evaluation of soft tissue abnormalities after fixation in Bouins fluid for one week (35) and then in 70% ethanol for 5 days. Wilson's technique was used for sectioning the fetuses and to evaluate gross organ pathology (36). The fetuses were prepared for evaluation of the skeletal abnormalities by the modified rapid clearing technique using the KOH-Alizarin Red S method of Staples and Schnell (37). Modifications included using 70% industrial methylated spirit (ethanol 94.5%; methanol 4.0%, water 1.5% vlv) in place of ethanol for fixation, leaving fetuses for 3-4 days in 1% KOH containing 0.01% Alizarin Red S stain for maceration and staining and keeping fetuses in clearing fluid for 2 to 3 days before transferring to an ethanol:glycerol (1:1, vlv) mixture for evaluation. The skeletous were evaluated and were classified as normal or retarded (38,39).
Outbred female rats of the Wistar strain weighing 240-280 g were used. One female was placed Uptake of 65Zn by and Zn Content in the Placenta and Fetus DMEP or physiological saline was injected IP on day 13 of gestation at 0.6 ml/kg 4 hr prior to the administration of carrier-free 65ZnCl2 in the coccygeal vein at a dose of 15 ,Ci/ml per kilogram of body weight. The animals were sacrificed by decapitation at 15 min, 1 hr and 4 hr after administration of the radiotracer zinc. The uptake of 65Zn was measured with a Packard autogamma counter. The concentrations of zinc in the placental and fetal tissues were measured by atomic absorption spectrometry with a method (40) which was reliable and repeatable to 0.2 ppm of zinc. This experiment included 24 pregnant female rats, with four rats at each time period within a treatment group.
Fetoplacental Metabolism of '4C-DMEP 14C-DMEP, specific activityO.912mCi/mmole (2.025 x 107 DPM/mmole), was prepared from [carbonyl '4C] phthalic acid (supplied by Radiochemical Centre, Amersham, Bucks, England). Radiochemical purity of the labeled 14C-DMEP was : 99% as confirmed by gas-liquid and thin-layer chromatography. Chemical identity was confirmed by NMR and GC/MS. Rats were injected intravenously on day 13 of gestation with 1C-DMEP or physiological saline at 0.6 ml/kg body weight, and the animals were sacrificed by decapitation at 1 and 4 hr after treatment. Each treatment group had three animals. Placentae and fetuses were dissected out. For measurement of total phthalate concentration, pooled portions of placental (0.33 ± 0.10 g) and fetal tissues (0.50 0.12 g) were dissolved in 1 ml Soluene and 10 ml of Instagel (both obtained from Packard Instrument Co.). For acid and neutral extractions of DMEP, portions of the placentae (0.40 ± 0.08 g) and the fetuses (0.42 ± 0.18 g) were homogenized with 1 ml of 0.9% NaCl in a ground glass homogenizer. The homogenates were extracted three times with ethyl acetate (2.5 ml). The extractions were separated into organic and aqueous phases by centrifugation (4000 g, 5 min). The residual aqueous phase was acidified to pH 1.0 with 2N H2SO4 and then reextracted with ethyl acetate. The neutral and acid extracts were made to 10 ml with ethyl acetate. A portion (5 ml) of each extract was transferred to a scintillation vial and was evaporated under a stream of N2 at 37°C to a volume of -1 ml before adding 10 ml of Instagel for counting. The chemiluminescence of the samples was minimized by storing the samples in a dark, cold storage room for 4-7 days. 91 Counts, counting efficiency, and the original weight of the sample were used to calculate the radioactivity of each specimen in terms of disintegrations per minute per gram of tissue. These values were used in conjunction with specific activity of injected '4C-DMEP to estimate molar concentrations of the teratogen in the fetus and the placenta.

Statistical Procedures
The reduction in mean fetal weight following administration of DMEP was compared with the normal saline controls by using Dunnett's procedure (41). Chi-square with Yates's continuity correction (42) was used to test the hypothesis that the percentages of dead and/or resorbed fetuses are equal in treatment and control groups and to compare the percentages of skeletally malformed fetuses with the control. Since the expected frequencies of the congenital deformities of the brain were small, Fisher's exact test was used to test these data. Student's t-test was used to compare the mean uptake of 65Zn by and the Zn content of the placentae and the fetuses.

Results
Embryopathy, Fetotoxicity and Teratogenicity of DMEP Maternal administration of DMEP in rats caused a significant (p < 0.01) reduction in the mean weight of living fetuses as compared to the corresponding value for the physiological saline-treated controls ( Table 1). A single injection of DMEP induced a pronounced (p < 0.01) fetotoxicity during the organogenetic period regardless of the day of injection. The fetotoxic (Table 1) and the embryopathic ( Table  2) effects were greater during the early stages (days 10 or 11 of gestation) of organogenesis than during the later stages (days 12, 13 or 14 of gestation).
Embryopathic effects were evaluated by percentage of dead and resorbed fetuses ( Table 2). Reproductive toxicity of DMEP, in this regard, was four to seven times higher upon injection on day 10 of gestation as compared to days 11-14; 79% of the fetuses were dead and resorbed following maternal administration of DMEP on day 10. Intraperitoneal administration of normal saline to pregnant rats resulted in fetal death and resorptions in 7.6% of fetuses.
In comparison to controls, DMEP induced higher rates of congenital deformities of the brain. None of the fetuses from the control group exhibited hydro- cephalus interna, a malformation of the CNS, as opposed to a 13-26% rate of malformation in DMEPtreated animals ( Table 3). The incidence of congenital skeletal malformations caused by DMEP is presented in Table 4. Control rats exhibited an 18% incidence of skeletal malformations whereas DMEP-induced skeletal deformities were found in 66-96% of the fetuses. This rate of skeletal dysmorphogenesis was 3.5to 5-fold higher than that in the controls. Fetuses in the control group showed no multiple malformations or appendicular dysmorphogenesis, while maternal administration of DMEP caused congenital multiple skeletal malformations in 14-64% of the fetuses and appendicular deformities in 25-57% of the fetuses. Malformations in the appendicular fibula were represented by its retardation, or by its absence. DMEP caused significantly higher (p < 0.005) rates of congenital skeletal deformities and multiple malformations in fetuses at all stages of gestation.
Malformations in the fibula were observed upon administration of DMEP on day 12 or 13. The skeletal deformities induced by DMEP included complete loss of thoracic ribs, their lack of articulation from the spinal column and forked ribs with bending, cracking and cessation of the vertebral column at the lumbar or at the lower sacral region and the appendicular deformities as represented by the absence or marked shortening of the fibula.

Adverse Effect of DMEP on Maternal-Fetal Zinc Metabolism
The uptake of 65Zn by placentae and fetuses and the zinc content in these tissues 1 hr post administration of 65Zn are presented in Figures 1 and 2. There was no significant difference (p < 0.05) in 65Zn uptake of the placentae and fetuses and the placental zinc content between DMEP and physiological saline treated animals. Fetal zinc content of rats treated with DMEP was, however, significantly lower (p < 0.01) than of those treated with physiological saline. The uptake and content of zinc in placentae and fetuses in other treatment groups (15 min and 4 hr) were not significantly different from the control.
Fetoplacental Metabolism of 14C-DMEP 14C-DMEP activity associated with the placenta and the fetus differed with respect to extraction ( Table 5). Placental activity was associated mostly with the acidic metabolic products, since less than 10% (mean 6.4 + 0.8) of the total activity was extractable from the neutral homogenates with ethyl acetate; while the remainder was recovered from the tissue residue after acidification. In contrast, 31-44.0% (mean 37.3 + 4.5) of the fetal activity    Mean uptake of 'Zn by and the mean zinc content in 1 g of placenta 1 hr following administration of 'Zn. Rats were pretreated with CMEP or physiological saline (PS) 4 hr prior to 6Zn administration. Zinc content is in ,ug/g wet tissue. SEM is the standard error of the mean value based on four rats per treatment group. NS stands for not significantly (p >0.05) different from PS group.
could be extracted, presum-^ly as the unchanged diester, from the neutral homogenates with ethyl acetate 4 hr following administration of DMEP. These preliminary results suggest, therefore, that the unmetabolized DMEP is transferred rapidly across the placenta to the fetus and the DMEP may be the teratogenic moiety.

Teratogenicity of DMEP
The reported fetal deaths and resorptions caused by a single maternal administration of DMEP on day 10 of gestation in rats ( Table 2) are in general agreement with the embryotoxicity resulting from multiple administrations of DMEP (22). A significant (p < 0.01) reduction in fetal weight (Table 1) has also been observed with other phthalate esters such as DEHP and dibutyl phthalate in rats (43) and in mice (44).
DMEP has been shown to be the most embryo-and fetotoxic member of the phthalate ester family (3,   16,20). In part, this may be due to its high aqueous solubility compared to other phthalate esters, although the aqueous solubility of all phthalate esters is extremely low. DMEP, for example, is about 143 times (0.73 ml/100 ml H20) more soluble in water than DEHP (0.0051 ml/100 ml H20) and is the most soluble phthalate ester (3,21). Fetal deaths and resorptions caused by DMEP were as high as 79% upon a single injection on day 10 of gestation (Table  2).
Congenital malformations of the brain involving hydrocephalus interna similar to those caused by DMEP (Table 3) have also been induced by dietary deficiency of zinc in rats (45)(46)(47)(48)(49)(50)(51). This is the first report to document hydrocephaly caused by the maternal administration of DMEP in rats.
Phthalate esters have been studied for their congenital skeletal dysmorphogenetic effects in rodents, but in this regard, these studies are the first to report skeletal malformations following a single injection of DMEP. Phthalate esters are considered compounds related to thalidomide (26). Congenital multiple skeletal malformations and appendicular dysmorphogenesis, which are considered "typical" of the thalidomide syndrome (39), have been difficult to produce in rats. These studies are the first, however, to report congenital multiple skeletal malformations and appendicular dysmorphogenesis following maternal administration of DMEP. Congenital multiple skeletal malformations upon a single intravenous maternal administration of thalidomide were also reported in our laboratory for the first time (52).
Intraperitoneal administration of sterile normal saline to pregnant animals resulted in some dead and resorbed fetuses and also caused skeletal malformations in control animals. Such effects have been reported earlier (53). The reproductive toxic-ity of DMEP, however, was several times higher than the normal saline control. The teratogenic effects of DMEP reported here have greater significance than the results where teratogenicity of phthalates is compared to untreated controls (22).
The malformations reported here are several hundredfold higher than the sporadic malformations found in rats, and are, therefore, due to the teratogenicity of DMEP. Major skeletal malformations in rats occur at a frequency of 0.41% (54), while those caused by DMEP were 161-234 times higher than this background level (Table 4). Craniofacial organ deformities such as hydrocephaly occur sporadically in rats at a frequency of 0.0506% (54), while DMEP induced this CNS malformation (Table 3) in 13-26% of the fetuses (257-514 times higher than the sporadic occurrence).

Role of Zinc in DMEP-Induced Teratogenesis
The conclusion that DMEP alters zinc metabolism in rats is based upon significantly (p -0.01) decreased zinc content in the fetuses of DMEPtreated rats as compared to that in the controls (Fig. 2).
Pregnant rats cannot mobilize zinc sufficiently from their body stores including that from the liver and bone (50,51), to supply the needs of their fetuses. As a result of this insufficient zinc supply, a transient deficiency of zinc may have been produced following DMEP administration which, in turn, is reflected by the decreased zinc content in the fetal tissue. The teratogenic effects reported here are in agreement with those caused by the decreased zinc content in fetuses when rats were placed on zincdeficient diets during gestation (51).
Permeability of the placenta to zinc increases after day 18 of gestation (55). However, this has not negated the validity of the results reported here because fetal zinc concentrations relate to maternal administration of DMEP or of physiological saline on day 13 of gestation. The fetal zinc concentration (micrograms per gram of wet tissue) reported here is far lower than that reported by Hurley and Swenerton (51) in 19-and 21-day-old fetuses. This may be due to the increased permeability of the placenta to zinc after day 18 of gestation and due to maternal supplementation of dietary zinc at 100 ppm, as well as other factors (54).
The results of the fetoplacental metabolic studies of zinc (Figs. 1 and 2) are also in agreement with the pharmacokinetic aspects of placental transfer of drugs using a single compartment model for dam and fetus, and in agreement with the first-order kinetics described by Levy and Hayton (56).
The adverse effects of DMEP on zinc metabolism and associated fetal anomalies reported here (Tables 1-4, Figs. 1, 2) permit one to draw an analogy between the phthalate-induced testicular damage in rats and the associated adverse effects on zinc metabolism caused by phthalates (57,58). The authors postulate that the protection provided by zinc in phthalate-induced testicular damage (57, 58) may be analogous to the protection offered by zinc against certain teratogens in rats (24). Singh et al. (32) reported that first order kinetics describe the elimination of 14C-labeled phthalates from maternal blood, amniotic fluid and fetal tissue with time. Also, zinc (59) and phthalates (32) follow, in general, a similar time course and concentration kinetics in maternal blood plasma and fetal tissue. Therefore, the authors propose a role of zinc in DMEP-induced teratogenesis in rats.
The reduced content of zinc in the target organ, the fetus (Fig. 2), following administration of DMEP, may be due to the rapid turnover rate of zinc following administration of phthalates (57,58). Coadministration of zinc was found to offer a substantial measure of protection against testicular atrophy and loss of organ weight caused by dibutyl phthalate (57).
The protective effect, if any, of coadministration of zinc and the underlying mechanism of zinc against the fetotoxic and teratogenic effects of DMEP is unknown, as is the protective mechanism of zinc therapy in phthalate-induced testicular atrophy. There are many common parallels, however, between congenital abnormalities caused by zinc deficiency in rats (45, 47-50, 60, 61) and those produced by DMEP (Tables 1-4). These include the manifestation of fetotoxicity by a marked reduction in fetal weight (Table 1); embryopathy by fetal resorption (  (Table 3); and skeletal malformations ( Table 4). The incidence of hydrocephalus interna produced by DMEP (Table 3) was similar to that produced by maternal zinc deficiency (51,60,61), although, in general, its severity was much less.
Fetoplacental Metabolism of 14C-DMEP Although no specific study has been reported which identifies the nature of DMEP or its metabolites in the rat fetus or placenta, the results reported here are in general agreement (28)(29)(30)(31)(32)62) with the metabolism of phthalates in nonpregnant rats as obtained by using radiotracer techniques.
There is a relationship between teratogenicity and carcinogenicity (63). Such a relationship for DMEP, is not unequivocally established. Although further studies are required to confirm whether or not DMEP is a carcinogen, there are examples of a carcinogen having teratogenic potential and a teratogen having carcinogenic potential (63). This is so because congenital malformations occur through genetic defects, or through embryotoxic effects on prenatal development, or both. For example, methotrexate has carcinogenic properties and possesses teratogenic potential, while 6-mercaptopurine, cyclophosphamide, and nitrosamides are teratogenic and have carcinogenic potential (63). Several researchers (3,10,12,16,26,64,65) have therefore cautioned about the toxicity and possible health threats of phthalates.

Conclusions
The studies described here establish that the maternal administration of dimethoxyethyl phthalate (DMEP) in a single dose during the organogenetic period (days [10][11][12][13][14] in the rat causes: (a) embryopathic, fetotoxic and teratogenic effects several times higher than reported by the widely studied plasticizer, diethylhexyl phthalate (DEHP); (b) congenital deformities of the brain, such as hydrocephalus interna; (c) congenital skeletal malformations (66-96%), including multiple skeletal (14-64%) and appendicular deformities (25-57%); (d) adverse effects on maternal-fetal metabolism of zinc; and (e) teratogenicity, as mentioned above, due to DMEP in the fetus. James Colaianne and Dr. Robert J. Condon, BVM, for their advice on statistical procedures; Dr. Maurice Zeeman for constructive suggestions, to Miss Kathy Briscoe for secretarial help; and to Mr. Malcolm King of the MRC Toxicology Unit for his excellent help and cooperation in the breeding, management, and handling of the laboratory animals used in this study.