Document 1QR3N8XyBMVROne2O0DE17qbX

Teratogenicity of Phthalate Esters in Rats ^ A. R. SINGH, W. H. LAWRENCE, and J. AUTIANiAi Don' Abstract In (he past, it was demonstrated that small quantities of phlbalalcs may Ik released from polyvinyl chloride items to physio logical solutions. To ascertain the effect these plasticizers might have upon the fetus, a teratogenic study was undertaken in rats using eight phthalate esters. Six of the phthalates, whose acute intraperitoncai 1.15.* values were-below It) nil,/kg., were administered to rats at doses representing one-tenth, one-fiTth. and one-third of the acute 1.15.* dose on the Sth. 10th. and 15th day or gestation; the two less toxic compounds were injected at levels .of 5 and It) ml./kg, Distilled water and normal saline were tested at 10 ml./kg., and cottonseed oil was tested at 5 and 10 ml./kg. Untreated animals were used for control values. The number of corpora lutea ranged from 52 to 65 per group of live rats, with no apparent distribution according to treatment. Embryo-fetal toxicity ranged from 0"' to a high of 98.2%. Fetal malformations ranged from 0 to 100?/. both for gross and skeletaI abnormalities, with skeletal defects generally being mol'd common'. Fetuses from all phthalate-trealed groups were smaller (/> < 0.01) than the untreated controls. Absence of tail, anophthalmia, and twisted hind legs were the most common gross abnormalities, while elongated and fused ribs and abnormal skull bones were the most common deformities found in stained skeletal specimens. Key phrases L) I'hthalates embryo-fetal toxicity, teratogenic effects, rats Teratogenicity phthalates. rats Plasticizers possible leaching of phthalates, teratogenic effects, rats Numerous devices and materials made from polyvinyl chloride are available for medical, dental, and para medical applications. To provide the desirable physical properties, particularly flexibility, these materials con tain significant quantities of plasticizer, up to 40% of the finished product. Presently, one commonly used group of plasticizers is the esters of phthalic acid, of which di-2cthylhexyl phthalate (often referred to simply as dioctyl phthalate or DOP1) is probably the most widely used. Ev idence (1,2) has been accumulating to indicate that small quantities of plasticizer may be leached from the mate rial by blood or various injectable solutions. However, toxicologic implications were discounted by many based upon animal feeding experiments and the.magnitude of difference between the quantities- so leached and the acme toxicity of these compounds. More recently, Jaeger and Itubin (3) reported finding di-2-cthylhexyl phthalate. in-the tissues of two patients who had received transfusions of blood stored in plastic bags. They found concentrations ranging from 2.5 to 27 mg- % (dry weight) of di-2-ethylhexyl phthalate in the spleen, liver, lung, and abdominal fat, with the spleen containing the least and abdominal fat the most. In vitro experiments, which they conducted using the perfused rat liver, indicated this organ would accumulate di-2-cthylhcxyl phthalate but would not hydrolyze it, while a related plasticizer, butyl glycolylbutyl phthalate, was hydrolyzed by this system. Haberman and his coworkers (4 -6) reported terato genic effects (crania bifida, anophthalmia, "cross-beak," exophthalmia, and malrotation of left leg) from some phthalate esters when the compounds were injected into the yolk sac or allantoic cavity or were applied to the chorioallantoic membrane of developing chick embryos. After hatching, the chicks were examined for gross congenital malformations, symptoms of toxicity, and neuromuscular abnormalities. Chicks were kept 3 weeks and observed for adverse effects on development, particularly neurological damage. In addition to their use in plastics, some of these esters are ingredients in insect rcpclhnt formulations.They generally have a low order of oral toxicity, although ingestion can cause GI disturbances, irritation of mucous membranes and eyes, and even CNS depres sion (7). Toxicity studies on phthalate esters were con ducted by a number of investigators (8 -15). Some parenteral toxicity data were presented, but the majority of the reports involved oral feeding experiments. The present study was undertaken to determine the teratogenic potential in mammals of some phthalate esters. MATERIALS AND METHODS Materials--Eight phthalate esters were included in this study: dimethyl phthalate2, dimethoxyethyl phthalate2, diethyl phthalate2, dibutyl phthalate2, diisobutyl phthalate2, butyl carbobutoxymethyl phthalate3, dioctyl phthalate3, and di-2-ethylhexyl phthalate3. Test animals were adult, virgin female rats of the SpragueDawley strain4, weighing between 200 and 250 g. Male rats of the same strain and age were utilized as the "stud pool." Procedure--Acute toxicity of the phthalates was determined by administering graded doses or these specific samples by intraperitoneal injection in rats and observing the animals 7 days for mor tality. I.Dm values of 95% confidence limits were calculated by Cornfield ami Mantel's modification (16) of Kurina's method or by the method of Weil (17). Female rats were selected for experimentation only idler olrservation of at least two complete 4- or 5-day extras cycles. Occur rence of cslrus was determined by daily vaginal smears. Vaginal smears were obtained by introducing 0.2 ml. of fresh, clean tap water into the vagina with a smooth, clean, sterile medicine dropper, withdrawing a part of the liquid, and transferring it to a clean microscope slide. The slide was examined microscopically while fresh, and the stages of the estrus cycle were recorded according to cell types found in the vaginal smear. . Five female rats were housed with one male in a large cage and kept in a room at 22-27. Laboratory chow (Purina) and fresh, clean tap water were provided ad libitum. The onset of gestation was established by the presence of sperm in the vaginal smear and was designated as Day 0. The following day was recorded as Day 1 of the gestation period. At this time, the female rats were moved to > Common use of the term DOP or dioctyl phthalate in referring to the compound with the branched chain octyl group, 2-ethylhexyl, often creates confusion concerning the exact chemical identity between . the straight-chain plasticizer (dioctyl phthalate) and its branched-chain isomer (di-2-cthylhcxyl phthalate). .. . ... .-- ._ 2 Eastman Chemical Products, Kingsport, Tcnn. 'Matheson, Coleman and Bell, Cincinnati, Ohio, 4 Sprague-Dawley Inc., Madison, Wis.- i ' .. . Vol. 61, No. I, January 1912 51 STLCOPCB4093628 Table 1 --Acute Toxicity in Rats DISCUSSION Compound LIXo, 95 % Confidence . ml./kg. Limits, ml./kg. Dimethyl phthalate Dimethoxyethyl phthalate Dielhvl phthalate Dibutyl phthalate Diisohulyl phlhalale Butyl carbobutoxymethyl phthalate Dioelyl phthalate Di-2-cih\ihexyl phthalate 3.3751 2.5338-4.4955 3.7355 3.0496-4.5744 5.0579 3.7973 6.7370 3,0496 2,0688-4.4957 3.7498 2.5000-5.6249 6.8892 5.1720 9.1766 >50 ml./kg. >50 ml./kg. individual capes where the)1 were kept undisturbed except for speci- lied injections. Each group was composed of live female rats. All treatments were ndministerd by intrnpcriloneal injections on the Mh. IO|h, titul 15th day of gestation. The sis more toxic phthalates were administered at three dosage levels: one-tenth, one-tilth, and one-third of the nciilc I D .o in eneli of the three in- Jwtions". The other two phtlmlates (dioelyl phlhalale and di-2- elhylhexyl phthalate) and cottonseed oil were administered at Iwo- dosage levels of 5 and 10 ml./kg., while normal saline and distilled water were used only at the lO-ml./kg. level. An untrenled control was also included. On the 20th day of gestation. I day prior to expected parturition, each rat was placed in an inhalation chamber and killed with an overdose of ether. The uterine horns and ovaries were surgically exposed to permit count ing and recording of the numbers of corpora lutea, resorption sites, and viable and dead fetuses. Fetuses were removed, blotted dry, and weighed to the nearest tenth of a milli gram'1. All fetuses, both viable and nonviable, were examined for gross malformations. A-randomly selected mtmiter of fetuses (30-50",j of the total), ex cluding whenever possible those showing gross evidence of mal formations. were prepared as transparent specimens to permit visualization of their skeletal system for evaluation of skeletal malformations. This was accomplished by the procedure of Staples nnd Schncll (18). Hrielly. this procedure involves fixing in 70% ethanol, dehydrating with acetone, clearing with 1% potassium hydroxide, nnd selective staining of bone with nii/nrin red S stain. Then the specimens ure rinsed in cold water, gently blotted, nnd immersed in a well-stirred mixture of 70% aqueous ethanol, glyc erol, nnd benzyl nlcohol. The cleared specimen may be preserved, if desired, in pure glycerol with the addition of a crystal of thymol to inhibit bacterial and mold growth. RESULTS The following parameters of adverse effects were investigated: (a) embryo-fetal toxicity, as evidenced by resorptions and stillbirths, (b) gross (external) malformations of fetuses, (c) skeletal malforma tions, and (d) fetal size. In all evaluations, both viable and nonviable fetuses were considered. The study encompassed 27 groups, each containing five female rats. The number of corpora lutea observed ranged from 52 to 65 per group, with a mean-of 58.15. Of the 1570 corpora lutea observed in this study, 1535 (97.8%) were accounted for as live fetuses, dead fetuses,-or resorption sites. The acute intrapcritoneal LD;o values and 95% confidence limits for the phthalate esters are presented in Table I, As shown, LDm values were obtained for six of the phthalates employed in this study, but the specific samples of dioctyl phthalate and di-2-ethylhcxyl phthalate used failed to produce 50% mortality at doses con siderably in excess of 50 ml./kg. The results or this investigation are presented in Tables II and III for the untreated controls and the groups treated with distilled water, normal saline, cottonseed oil, and the eight phthalate esters. Table II presents the data for the number of corpora lutea observed, resorption sites, dead fetuses, live fetuses, and fetal weights accord ing to treatment groups. Table III presents a comparison of the ** number and percent of resorptions, gross abnormalities, and skeletal abnormalities. Compound-dependent nnd dose-related cllecis upon embryonic and felnl growth and development were generally observed with these phthalates. The ejects produced by normal saline should not be too surprising, since Nishimura (19) indicated that abnormalities can be produced in animals by hypertonic saline (1.9 3.75 g./kg.) when given til a critical time of fetal development. Resorptions -There were no resorptions, dead fetuses, and gross or skeletal abnormalities in the untreated control, group. The intermediate doses of diethyl phthalate nnd dimethyl phthalate did not produce any resorption sites. The reason for this finding is not clear, since both higher nnd lower doses of both compounds pro duced some resorptions. The intermediate dose of dimethyl phlhal ale, however, did show one fetid death. All other substances tested showed some resorptions, with the highest incidences occurring with the two highest doses of dimclhoxyclhyl phthalate:-96.5 and 89,7%. respectively, The lowest incidences-or resorptions occurred in the groups treated with diethyl phlhnlalc (high dose), dihtityl plitlialnlc (middle dose), and dioelyl phlhalale (low dose). With each of these last three compounds, t(vo resorption sites were observed, represen ting 3.6, 3.6, and 3.8%, respectively. In this category, di-2-cthylhcxyl phlhalale showed greater activity than dioctyl phthalate; this trend, however, was reversed when one considers fetal abnormalities. Fetal Deaths--Fetal deaths were observed with less frequency and in smaller number than the other criteria considered. Although most of the treated groups revealed some resorption sites, dead fetuses were found in groups treated with only three of the com pounds (dimethyl, dimethoxyethyl, and diisobutyl phthalates); of these, only dimethoxyethyl phthalate showed a high level of fetal toxicity at all dose levels tested. A comparison of fetal deaths for these three compounds is presented in Table IV. As shown, the low dose of dimethoxyethyl phthalate produced the greatest number of fetal deaths: however, this finding reflects the more potent early toxicity of the higher doses of the compound as seen in the large numbers of resorptions produced. For instance;'the high dose pro duced only one fetal death, but only two of the 57 fertilized ova developed into fetuses (50% fetal death). The intermediate dose produced four fetal deaths (66.7%), but only six of the 58 fertilized ova developed into fetuses. Thus, the final column of Tabic IV presents percent of fetal deaths plus resorptions! This comparison exhibits a dose-response relationship for the high, medium, and low doses of dimethoxyethyl phthalate of 98.2, 96.6. and 56.9%. re spectively. Teratogenicity--Gross Abnormalities--At the time of sacrifice, all fetuses, both viable and nonviable, were removed and examined grossly for structural abnormalities. A significant number of mal formations were observed. They were not uniformly distributed throughout the treatment groups but tended to cluster around certain phthalates and to show a dose-related response pattern when considered as percentage of fetuses showing malformations. One malformed fetus was seen in each of the following groups: normal saline, cottonseed oil (10 ml./kg.), dimethoxyethyl phthalate (0.374 ml./kg.), and butyl carbobutoxymethyl phthalate (2.296 and l. 378 ml./kg.); this result represented from 1.8 to 2.4% of the fetuses from the various groups. Two malformations were noted for dimcthoxyelhyl phthalate (1.245 ml./kg.) and diisobutyl phthalate (0.750 ml./kg.). Due to the high incidence of resorptions for this dose of dimethoxyethyl phthalate, this result represented 100% of fetuses, whereas it represented only 3.9% for the diisobutyl phthalate group. The greatest number of gross malformations (15 fetuses or 27.3%) was seen with dioctyl phthalate (10 ml./kg.). A complete tabulation of the number of fetuses in which gross abnormalities were noted, as well as percentage of the total, is presented in Table m. v The most common gross abnormalities found in the phthalatetreated animals were absence of tail, anophthalmia, twisted hind legs', and hematomas (hemangiomas). Thus, there appears to be some similarity between the responses observed in rats in this study and the observations reported by Bower et al. (6) who used chicks to study the response to phthalates when injected into the incubating egg; they observed anophthalmia along with other abnormalities. * Sec Table I for LD> values. * Using a Mcttlcr H6T balance. . 'Twisted hind legs are considered an abnormality here since all fetuses were delivered surgically, thus avoiding the possible trauma of natural birth. 52 Journal ofPharmaceutical Sciences %. r - '-- V. . DSW 588293 STLCOPCB4093629 :onic with t not lilies /kg.) and The did not orohalsled vith >' m lie ink* Osc cn* *>l (Ul icy igh ad mof tal Jbr ;HV of ly Jtc .'a *c d V h .v . - i i i i Table II IVnitogenie I'llVcis of n Group of IMitlmlnle listers on Kill Fetus" Treatment Groups Volume Injected, nil./kg. Number or Corpora Lulcu Number ofResorptions'' Number of Dead Fetuses'1.' Number of Live Fetuses'' Average Weight of Fetus'1 Untreated controls Hist i lied water Normal saline Cottonseed oil Dimethyl phthalate Dimethoxyethyl phthalate Diethvl phthalate Dihutvl phthalate Diisobutyl phthalate Hutyl carhohuloxymethyl plithalate Dioctyl, phthalate Di-2-clhylhexyl phthalate None 10.00 10.00 lO.tX) 5.00 1.125 0.675 0.338 1.2-15 0.7-I7 0.37-1 1. 686 I.OI2 0.50(i 1 .017 0.610 0.305 1.250 0.750 0.37,5 2.29(> 1.378 0.689 10.00 5.00 10.00 5.00 . 60 59 62 59 54 55 55 65 58 58 59 57 59 65 64 56 56 64 55 52 57 56 55 60 53 56 61 0 4 ( 6.8%) 7(11.5%) 4 ( 6.8%) 3 ( 6.4%) 17(32.1%) 0 21(33.3%) 55 (96.5%) 52(89.7%) 16(27.6%) 2 ( 3.6%) 0 28 (44,4%) 23 (36.5%) 2 ( 3.6%) 4 ( 7,3%) 16(25.8%) 3(5.5%) 5 ( 9.6%) 13(24.1%)8(14.5%) 4 ( 7.8%) 5( 8.3%) 2 ( 3.8%) 15(26.8%) 5( 8.2%) 0 0 0 0 0 5( 9.4%) 1(19%) 0 1 ( 1.8%) 4 ( 6.9%) 17(29,3%) 0 0 0 0 0 0' 0 2 ( 3.6%) 0 0 0 0 0 0 0 0 59(100.0%) 55 ( 93.2%) 54( 88.5%) 55( 93.2%) 44 ( 93.6%) 31 ( 58.5%) 52( 98.1%) 42( 66.7%) K 18%) 2 ( 3.4%) 25 ( 43.1%) 54 ( 96.4%) 57(100.0%) 35 ( 55.6%) 40 ( 63.5%) 53 ( 96.4%) 51 ( 92.7%) 46 ( 74.2%) 50 ( 90.9%) 47( 90.4%) 41 ( 75.9%) 47 ( 85.5%) 47 ( 92.2%) 55 ( 91.7%) 51 ( 96.2%) 41 ( 73.2%) 56 ( 91.8%) 4.83 0.01 4.40 0.33 4.10 0.13* 4.45 0.17 '4.65 0.19 2.20 0.18* 2.60 0.01* 2.38 0.13* 1.75 0.001.88 0.38' 2.19:1:0.25' 2.85 1: 0.27' 2.85 O.I9' 2.63 0.24" 3.60 0. 10* 3.65 0.10' 3.70 0.09' -2.00 0.46' 3.50 0.21' 3.60 0. 13' 3.05 0 .30' 3.30 0.05' 3.95 0.09' 3.40 0.13' 4.00 0.18' 3.50 0.18' 3.55 0.17' " Five pregnant female rats were injected in each group on Days 5, 10, and 15 of pregnancy. 6 Numbers in parentheses indicate percent values based upon total number of implantations. ' Also see Tabic IV, where fetal deaths are expressed as a percentage of total fetuses. * Numbers represent the average values (grams) St'for each group. * Significantly dill'erent from untreated controls at 99% level (p < 0.01) by Student's t test (20). Skeletal Abnormalities--Generally, 30-50% of the fetuses were prepared for visualization of the skeletal structure. When possible, fetuses were selected that did not show gross abnormalities. How ever, in a few instances, in which the resorption rate was very high, all fetuses were used. Even with this typeof specimen selection, there was generally a higher incidence of skeletal abnormalities than gross abnormalities. Skeletal abnormalities were seen with -saline, cottonseed oil (10 ml./kg. but not with 5 ml./kg.), and all three dose levels of dimethyl, dimethoxyethyl, diethyl, dibulyl, diisobutyl, and butyl carbobutoxymelhyl phthalatcs. No skeletal abnormalities were observed with either dose level or dioctyl and di-2-cthylhexyl phthalatcs. Also, there were no abnormal bone structures in the untreated controls or llie distilled water-treated animals. The percentage of skeletal mal- Tablc 111--Gross and Skeletal Malformations Produced by Phthalate Esters Treatment Groups Volume Injected, ml./kg. Number of Resorptions0 Number of Gross ' Abnormalities6 Number of Skeletal Abnormalities' Untreated controls Distilled water Normal saline Cottonseed oil Dimethyl phthalate Dimethoxyethyl phthalate Diethyl phthalate Dibutyl phthalate Diisobutyl phthalate Butyl carbobutoxymethyl phthalate Dioctyl phthalate Di-2-ethylhexyl phthalate . None 10.00 10.00 10.00 5.00 1.125 0.675 , 0.338 1.245 0.747 0.374 1.686 1.012 0.506 1.017 0.610 0.305 1.250 0.750 0.375 2.296 1.378 0.689 10.00 5.00 10.00 5.00 0 4 ( 6.8%) 7(11.5%) 4 ( 6.8%) 3 ( 6.4%) 17(32.1%) 0. 21 (33.3%) 55 (96.5%) 52(89.7%) 16(27.6%) 2( 3.6%) 0 28 (44.4%) 23 (36.5%) , 2(3.6%) 4 ( 7.3%) 16(25.8%) 3 ( 5.5%) 5 ( 9.6%) ' 13(24.1%) 8(14.5%) 4 ( 7.8%) 5(8.3%) 2(3.8%) 15(26.8%) 5 ( 8.2%) 0 0 1( 1.9%) H 1.8%) 0 4 ( 11.1%) 4 ( 7.5%) 4 ( 9.5%) 2(100.0%) 5 ( 83.3%) 1( 2.4%) 0 0 0 0 0 0 0 2 ( 3.9%) 0 1( 2.4%) K 2.1%) 0 15 ( 27.3%) 8 ( 15.7%) 9(22.0%) 0 0 0 4 ( 14.3%) 3 ( 10.7%) 0 9 ( 75.0%) 6 ( 35.3%) 4 ( 25.0%) 2(100.0%) 4(100.0%) 13 ( 92.9%) 13 ( 81.3%) 8 ( 47.1%) 5 ( 26.3%) 8( 33.3%) 7 ( 24.1%) 6 ( 20.7%) 8 ( 33.3%) 5 ( 17.2%) 4 ( 14.8%) 5 ( 21.7%) 4 ( 16.0%) 4 ( 13.8%) 0 0 0 0 Numbers in parenthescs.represent percent resorption based on total number of implantations.6 Numbers in parentheses indicate percent gross abnormalities based on total number of fetuses. Numbers in parentheses represent percent skeletal abnormalities based on total number of stained fetuses. - Vd. 61, No. I, January 1972 53 DSW 588294 STLCOPCB4093630 Table IV Comparison of Total Deaths After visualizing the skeleton, the bones of the hind legs appeared to be very weak and delicate and were poorly formed. In the low dose Compound and Dose Level, ml./kg. Fetal Deaths + --Fetal Deaths0--. Resorp- Num- tions\ ber Percent % group, one fetus was without a tail and seven of 14 visualized fetuses did not have tail bones. Six of these 14 had elongated and fused ribs. Another compound of this series, dimethyl phthalate, produced marked teratogenic effects. In the group that received the high dose of this phthalate, three fetuses were observed that had neither tails nor Dimethyl phthalate: 1.125 0.675 0.338 Dimethoxyethyl phthalate: 1.245 0.747 0.374 5 13.9 41.5 1 1.9 1.9 00 33.3 1 50.0 98.2 4 66.7 96.6 17 40.5 56.9 eyes, and one fetus had a normal tail but was without eyes. Txamination or skeletal structure revealed four of 12 fetuses had elongated and fused ribs, two did not have tail bones, and three had abnormal and incomplete skull bones. In the middle dose group, four fetuses had abnormally small tails and six of the 17 stained fetuses had complete or incomplete elongated and fused ribs (four complete and two incomplete, i.e., only on one side). At the low dose, two fetuses were observed with short tails and two were without eyes. Of the 16 stained fetuses in this group, two had elongated and fused ribs on Diisobutyl phthalate: both sides, one had unilateral elongation of the ribs, and one was 1.2-50 0.750 0.375 00 25.8 2 3.8 9.1 00 9.6 without tail bones. . Similar gross and skeletal abnormalities were observed in groups treated with other phthalate esters. Diethyl and dibutyl phlhalates Percent fetal deaths = (dead fctuscs/total fetuses) X 100, 6 Fetal deaths -f resorptions = (dead fetuses + resorption sitcs/totnl fetuses + resorption sites) X 100. did not produce any gross malformations or dead fetuses; however, a number of skeletal abnormalities were noted. In the high dose group treated with diethyl phthalate, 12 of 16 stained fetuses showed complete or incomplete elongated and fused ribs nnd one fetus had curved nnd elongated upper and lower jaw bones. In the middle (lose formations tanged front 13.8",; for the low dose of butyl turbo* bntnxymoilnl plulmlale to 100",; for the two highest tloses of tlinu'ihoxsetlnl phthalme and d.'.d",; for the low dose of this coin group, eight of 17 fetuses revealed abnormal skeletal sliiielmes, especially elongated and fused libs and incomplete sVull bone for mation, while live of 19 Ictuses in the low dose group had elongated pound, I lie nest most active pliilmlnies in this regard were dimethyl and diethyl phthnlatox, IVtcenlngo of skeletal inull'onnutions protlueed by the three doses of dimethyl phlhalate were 75,0, 35..1, unci anil fused ribs. Dibutyl phlhululc-lrenled animals produced fetuses with elongation and fusion of the l ilvs with a fieiiucncy of eight of 24, seven of 29, and six of 29 in the high, medium, nnd low dose 25.0%, respectively; for diethyl phlhalate, they were 81.3, 47.1, and groups, respectively. 26.3%, respectively. There were no gross abnormalities or dead fetuses in the groups The skeletal abnormalities most commonly encountered were treated with diisobutyl phlhalate except for the middle dose, in elongated and fused ribs (bilateral and unilateral"), absence of tail which two dead fetuses were found; both were without eyes due to bones, abnormal or incomplete skull bones, and incomplete or incomplete formation of the head. After visualization and staining, f > missing leg bones. The missing leg bones were most often the ulna in eight of 24, five of 29, and four of 27 were seen to have partially the fore legs and the fibula in the hind legs and, less often, the radius elongated and fused ribs in the high, medium, and low dose groups, in the fore legs and the tibia in the hind legs. Observation of incom respectively. ! plete skull bones may merely represent n developmental defect in The high and medium dose groups of butyl carbobuloxymcthyl Which delayed ossification may be secondary to general retardation phlhalate each contained one very small, abnormal fetus, weighing Of growth and development of the fetus. . only I g., but there were no dead fetuses. No gross abnormalities i'l'hit Site fetuses from all groups of phthalaie-irealed rats were were seen in the low dose group. Alter staining, however, elongated ;i hnmller than the untreated controls. This dill'erenee was significant at the 99% level </> < 0.01). Although fetuses from ircnlmcnls other and fused ribs were seen in five of 23, four of 25, and four of 29 ' fetuses in the high, medium, and low dose groups, respectively. than plnhalates were slightly smaller than the controls, thcdilferencc As a result of their low acute toxicity, dioctyl and di-2-elhylhcxyl was not significant at (his level except for the saline-treated group. phlhalates were injected at two fixed dosage levels of 5 and 10 ml./kg. The control fetuses averaged 4.83 g. each, while those from the high each. Twenty-three grossly malformed fetuses were seen with dioctyl and middle doses of dinicthoxyetliyl plUluilutc-trcutcd rats had a phthalate, 15 of 55 at the high dose and eight of 51 at the low dose. mean fetal weight of 1.75 and 1.88 g., or 36.2 and 38.9% of the Twisted hind legs was the most commonly seen feature. No ab controls. Only a small number of fetuses were obtained from these normalities were noted in the animals treated with 5 ml./kg. of di-2- groups (two and six, respectively). The mean fetal weight was 2.00 g. ethylhexyl phthalate, but nine of the 41 fetuses in the I O-ml./kg. dose (41.4% of control) for those animals treated with the high dose of group exhibited hemangiomas of the legs and one had twisted hind diisobutyl phthalate; this finding, however, was based upon 46 .legs. Although resorptions were seen in all four groups treated with fetuses. Although fetuses from all phthalate-treated rats were sig these two phthalates, no dead fetuses were present. nificantly smaller than from the controls, there was a progressive increase in fetal size up to maximum mean of 4.00 g. (82.8% of controls) for the low dose of dioctyl phthalate. These data are SUMMARY AND CONCLUSION presented in Table II. . As indicated previously, all phthalate esters utilized in this study ex Teratology, Summary Comments--A significant number of mal ert a deleterious effect upon the developing embryo and/or fetus. At formed fetuses were'observed in most phthalate-treated animals. A ' one or more of the dose levels employed, each compound produced few were also found in the groups treated with normal saline and some or all of the following effects: resorptions, gross abnormalities, cottonseed oil (10 ml./kg.). However, none was found in the un skeletal malformations, fetal death, or decreased fetal size. Un treated, distilled water-treated, cottonseed oil-treated (5 ml./kg.), or treated control animals.were used to determine "normal" fetal size; di-2-ethylhexyl phthalate-treated (5 ml./kg.) groups. within this group, there were no resorptions, gross or skeletal ab Dimethoxyethyl phthalate was an interesting member of the normalities, or fetal deaths. . 1 r phthalate series because of the high level of activity exhibited by this compound on the developing embryo and fetus. Most of the ferti The regimen of phthalate administration did not interfere with fertility, as reflected by the ratio of corpora lutea to implantation lized ova were detected only as resorption sites in the two higher dose sites, but there were significant effects upon embryonic and/or fetal levels. One dead fetus and one live fetus were delivered in the high development. The observed incidence of adverse effects was gen dose group, and both of these had a short or compressed head and erally dose related and compound dependent, with the more water- neck. At the middle dose level, two fetuses had no tail or eyes, an soluble compounds tending to be most active. Of the phthalate esters other was missing a tail, and two dead fetuses had twisted hind legs. included, dioctyl and di-2-ethylhexyl were the least soluble and tended to exert the least deleterious effect on embryo-fetal develop Bilateral or complete fused ribs were noted spread throughout the rib cage, whereas unilateral or incompletely fused ribs were generally observed in the lower ribs. . ment. In view of the widespread use of di-2-ethylhexyl phthalate and, to a lesser extent, butyl carbobutoxymethyl phthalate (butyl glycolyl- 840 Journal ofPharmaceutical Sciences DSW 588295 A, - STLCOPCB4093631 xdto ' dose Muses I ribs. Juccd ose of s nor xain83 led rnml :t\iscs had c nnd loses he 16 on : was oup times ever, dose owed > had : d0SC lures, e forgated .(uses :ht of dose oops e. in it lo fling, daily ups, ethyl diing lilies ;ated f 29 . ' lexyl ./kg. ?clyl .ose. ab- di-2dose hind with ycxi. Al jeed tics, Untizc; ab- with lion fetal geniteriters and lop- 1, to JM- bulyl phthalatc) as plasticizers in medically used devices, the ques tion arises as to the potential danger to the human reproductive process from plasticizers leached from these devices by blood or parenteral solutions. While the doses of di-2-cthylhcxyl phthalate employed in this study were greatly in excess of the 5-7 mg. % which have been reported in whole blood stored in di-2-ethylhcxyl phthalatc-plasticizcd blood bags (3), the possible cumulative nature of the plasticizer in the body must also be considered. Jaeger and Rubin (3) found from 2.5 to 27 mg. % (dry weight) of this plasti cizer in tissues of patients who were known to have received blood transfusions: in addition, they found that while the perfused rat liver could hydrolyze the plasticizer butyl glycolylbutyl phthalate, it did not hydrolyze di-2-ethylhcxyl phthalatc but tended to sequester nnd accumulate it. ' further investigations arc needed to determine the fate of these small, but possibly repeated, quantities of plasticizers that may enter the patient ns a result of medical or surgical treatment and to deter mine wltnl, if any. effect on reproduction or other physiological processes piny result. It ICI<'lClt ICNC.'ICS (1) A. S. Trimble, H. S. Goldman, J. K. Yao, L. K. Kovats, and W. G. Iligelow, Surgery, 59, 857(1966). . (2) W. L. Guess, J. Jacob, nnd J. Autian, Drug Intel., I, 120 (1967). (3) R. J.-Jaeger and R. J. Rubin, Science, 170, 460(1970). . (4) W. L. Guess. S. Haberman, D. F. Rowan, R. K. Bower, and J. Autian, Atner. J. Hosp. Pharm., 24, 494(1967). (5) S. Haberman, W. L. Guess, D, F. Rowan, R. O. Bowman, and R. K. Bovver, SPE (Sac. Plant. Eng.) J., 24, 62(1968). (6) R. K. Bower. S. Haberman, and P. D. Minton, J. Pharmacol. Exp. Ther.. 171. 314(1970). (7) "The Merck Index," 8th cd;, Merck & Co., Inc., Rahway, N. J,, 1968. (8) H. C. Hodge, Proc. Sac. Exp. Biol. Med., 53, 20(1943). (9) C. B. Shaffer, C. P. Carpenter, and H. F. Smyth, Jr., J. hid. Hyg. Toxicol., 27, 130(1945). (10) J. H. Draizc, E. Alvcrcz, M. F. Whitcscll, G. Woodard, E. C. Hagan, and A. A. Nelson, J. Pharmacol. Exp. Ther., 93, 26 (1948). (11) C. P, Carpenter, C. S. Weil, and H. F. Smyth, Arch. hid. Hyg. Occup. Med., 8, 219(1953). (12) A. J. Lehman, Ass. Food Drug Offic., V. S. Quart. Bull., 19, 87(1955). (13) R. S. Harris, H. C. Hodge, F.. A. Muynnrd, nnd H. J. Ulnnchct, Arch. Ind. Heultli, 13, 259(1956). . (14) J. McLaughlin, Jr., J. P. Marline. M. J. Verrelt, M. K. Mutchlcr, and O. G. Fitzhugh, Toxicol. Appl. Pharmacol., 5, 760 (1963). . (15) D. Callcy, J. Autian, and W. L. Guess,V. Pharm. Sci., 55, 158(1966). (16) J. Cornfield and N. Mantel, Amer. Statist. Ass. J45, 181 (1950). (17) C. S. Weil. Biometrics, 8, 249(1952). (18) R. E. Staples and V. L. Schnctl, Slain Technol., 39, 61(1964). (19) II. N.ishimuru, "Chemistry unci Prevention of Congenital Abnormalities," Churlcs C Thomas, Springfield, 111., 1964. (20) H. Bancroft, "Introduction to Bioslatistics," Harper & Row, New York, N. Y., 1963, chap. 15. ACKNOWLEDGMENTS AND ADDRESSES Received March 29, 1971, from the Materials Science Toxicology Laboratories, College of Dentistry and College of Pharmacy, Uni versity of Tennessee Medical Units, Memphis, TN 38103 Accepted for publication August 27, 1971. Presented at The Society of Toxicology Meeting, Washington, D. C,, March 1971. Supported in part by Research Grant DE02944-02 from the National Institute of Dental Research, Bethesda, MD 20014 . A To whom inquiries should be directed. Synthesis and NMR Spectral Characteristics of Bisnorargemonine Isomers CHUNG-HSIUNG CHEN* and T. O, SOINEA Abstract Two structural isomers of bisnorargemonine, 3,9-dihydroxy-2,8-dimcthoxy-/V-mcthylpavinane (la) and 2,8-dihydroxy3,9-dimcthoxy-A^-mcthylpavinanc (16), were synthesized employing the Pictct-Gams modification of the Bischler-Napieralski cyciization to construct the required isoquinoline intermediates. These were qualcrnized to the methiodides, followed by partial reduction to the N-mcthyl-1,2-dihydroisoquinolines, which were then cyclized under acidic conditions to provide la and 16. Comparison of the NMR spectra of la and 16 with similar compounds revealed inter esting aspects of the spectral properties pertaining to this ring systern. Kcyphrascs Bisnorargemonine isomers--synthesis, NMR charac teristics 3,9-Dihydroxy-2,8-dimethoxy-Af-methylpavinane--synthesis, NMR characteristics 2,8-Dihydroxy-3,9-dimethoxy-Armclhylpavinanc--synthesis, NMR characteristics NMR spectros copy--characteristics of bisnorargemonine isomers The recent synthesis of ( )-bisnorargemonine (1, 2) confirmed Structure Ic assigned to it previously on the basis of its unique NMR spectrum (3). Two alternate structures, la and lb, could have been considered but were rejected due to a predictable nonconformity in their NMR spectra. However, it was of interest to syn thesize la and lb, not only to verify the predicted NMR patterns but also to seek a practical synthetic route to provide these phenolic bases for projected oxidative coupling studies. The incidental provision of spectral and other data could also be of value in the future in assigning structures in the event that either or both of these diphenols are found to be naturally occurring, a not unlikely possibility. > Pursuant to a suggestion by Stermitz1, it is believed appropriate to introduce a new type of nomenclature for this ring system since further extension of the "ar- gemonine" nomenclature seems inadvisable if not im possible. Accordingly, it is proposed that the term "pav- * Private communication from Dr. F. Stermitz, Colorado State Uni versity, Fort Collins, Colo., which forms the basis for the proposed nomenclature. Vol. 61, Ho. 1, January 1972 55 DSW 588296 STLCOPCB4093632