Hamster, and Mouse When Given by

Transcription

1 En vironinentl Helth Perspectives Vol. 30, pp , 1979 Dipterex Tertogenicity in the Rt, Hmster, nd Mouse When Given by Gvge by R. E. Stples*t nd E. H. Goulding* Dipterex ws tertogenic fter dministrtion by gvge (t.i.d.) t dose level of 480 mg/kg-dy to the CD rt on dys 6 through 15 of gesttion, but not when dministered only on dys 8 or 10 of gesttion. A positive tertogenic response lso occurred in the hmster fter dministrtion on dys 7 through 11 of gesttion t 400 mg/kg-dy; the pprent no-effect level for the criteri studied ws 200 mg/kg-dy. Embryotoxicity, but not tertogenicity, occurred fter dministrtion t 400 mg/kg-dy on dy 8 of gesttion. In both species, the tertogenicity seen ws not merely due to reduced mternl food consumption during the period of exposure. The mouse ws less susceptible to Dipterex thn were the rt nd hmster, but significnt increse in the incidence of cleft pltes resulted from exposure on dys 10 through 14, or on dys 12 through 14 of gesttion. Dipterex (0, 0-dimethyl-l-hydroxy-2,2,2-trichloroethyl phosphonte) ws found to be tertogenic when consumed by rts on dys 6 through 15 of gesttion but not fter dministrtion by gvge, once dily (i.d.), for the sme period of gesttion (1). Since drugs, pesticides, nd other environmentl gents often re tested for tertogenic potentil only by gvge, it ws considered importnt to demonstrte whether the tertogenic potentil of Dipterex could be detected in the rt by gvge through multiple dily dministrtion s mens of ttining higher dily dose levels of Dipterex more comprble to tht chieved through incorportion in the diet. In the rt the tertogenicity of Dipterex ws demonstrted only t dose levels tht decresed mternl food consumption nd tht were fr in excess of expected levels of routine humn exposure. To determine whether the tertogenicity of Dipterex is unique to the rt, its tertogenic potentil lso ws investigted in the hmster nd the mouse. * Environmentl Toxicology Brnch, Ntionl Institute of Environmentl Helth Sciences, P. 0. Box 12233, Reserch Tringle Prk, North Crolin t Present ddress: Hskell Lbortory for Toxicologicl nd Industril Medicine, E. I. dupont denemours & Compny, Inc., Elkton Rd., Newrk, Delwre Mteril nd Methods Rts Nonprous, femle rts (CD strin) were housed overnight with mture mles of the sme strin in qurters lighted for 12 hr of ech dy nd mintined t 20 C (50% RH). Mting ws confirmed by detection of spermtozo in the vginl lvge the following morning (dy 1 of gesttion). Between dys 1 nd 6 of gesttion, ll rts were cged individully nd hd free ccess to wter nd Wyne Lb Blox. On dy 6 of gesttion, the rts were ssigned to the experimentl group or to the pir-fed control group (CPF). Dipterex ws given by gvge on dys 6 through 15 of gesttion t dily dose on pr with those tht were tertogenic in the first study (1). Preliminry study reveled tht if the rts were gvged three times dily (t.i.d.) dily dose of 480 mg/kg ws tolerted quite well. The rts of the experimentl group were gvged (t.i.d.) with 160 mg/kg body weight in 10 ml/kg of 0.5% methylcellulose in distilled wter; the CPF group ws gvged similrly with only the vehicle. To determine whether the tertogenicity of Dipterex could be reveled by gvge (t.i.d.) on single dy during orgnogenesis, dditionl rts were given Dipterex only on dy 8 or 10 of gesttion. In this prt of the study, the rts in the respective con- June

2 trol groups (Cv) were gvged with vehicle (t.i.d.), but they were not pir-fed to the experimentl groups. Hmsters Nonprous golden hmster femles rrived from Lkeview Frms, New Jersey on dy 3 of gesttion. The morning fter mting ws considered to be dy 1 of gesttion. They were housed individully in plstic cges with corn cob bedding nd hd free ccess to Wyne Lb Blox nd wter. Room temperture ws mintined t 20 C (50%o RH) nd light ws provided for 12 hr of ech 24 hr. Dipterex ws dministered by gvge (t.i.d.) in 10 ml/kg body weight of distilled wter from dy 7 to dy 11. It ws estblished by preliminry study tht the hmster would tolerte 400 mg/kg-dy for this period of gesttion. The Dipterex solution ws freshly prepred ech dy nd the dily dose ws dministered in thirds t 9:00, 12:30, nd 16:00 hr of ech exposure dy. Concurrent control groups included untreted controls (C) nd nother group tht ws given only distilled wter by gvge (t.i.d.) t 10 ml/kg-dy on dys 7 through 11 of gesttion (Cv). Lgging one dy behind ws third control group (CPF) tht ws pir-fed to the group given Dipterex t 400 mg/kg-dy. The CPF group lso ws gvged (t.i.d.) with distilled wter on dys 7 through 11 of gesttion. Food consumption ws mesured dily, nd mternl body weights were recorded on dys 3, 7, 9, 11, 13, nd 15 of gesttion. Upon completion of the experiment described bove, lower dose levels were dded (100, 200, 300 mg/kg-dy) to determine the pprent no-effect dose level with respect to the criteri under study, nd the 400 mg/kg-dy dose level ws dministered by gvge (t.i.d.) to dditionl hmsters only on dy 8 to determine fetl sensitivity to exposure on single dy of gesttion. Mice Nonprous, femle mice (CD-1 strin) were cohbited overnight with mture mles of the sme strin (2W/cj) in qurters lighted for 12 hr of ech dy nd mintined t 20 C (50%o RH). Mted femles detected by the presence of copultory plugs in their vgins (considered to be dy 1 of gesttion), were housed in lrge plstic cges nd hd free ccess to Wyne Lb Blox nd wter. All femles were housed individully beginning with the first scheduled dministrtion of Dipterex nd therefter to permit mesurement of food consumption until 106 two dys fter the lst dy of exposure to Dipterex. It ws estblished by preliminry study tht pregnnt mice could tolerte Dipterex in doses up to 600 mg/kg-dy for four consecutive dys if given by gvge (t.i.d.). On this bsis groups of pregnnt mice were given Dipterex t dose level of 600 mg/kg-dy on dys 6 through 10 nd on dys 10 through 14 of gesttion. Agin the pprent noeffect dose level ws determined by study of dditionl dose levels (300, 400, nd 500 mg/kg-dy) dministered for the sme periods, nd fetl sensitivity to shorter exposure periods ws explored by dministrtion of Dipterex t the dose level of 600 mg/kg-dy on dy 8, dys 8 through 10, dys 10 through 12, nd dys 12 through 14 of gesttion. The Dipterex solutions were prepred ech dy in distilled wter such tht the desired dosge could be dministered in 10 mi/kg body weight t 9:00, 12:30, nd 16:00 hr dily. Groups of control femles (Cv) were given the sme volume of distilled wter by gvge (t.i.d.) for the sme periods of gesttion s for the groups given Dipterex; other groups of control femles (C) were not gvged. Mternl body weight ws mesured on dy 1 of gesttion, on the first dy of Dipterex dministrtion, every other dy during dosing, the second dy fter cesstion of Dipterex dministrtion, nd on the dy of scrifice. On dy 21 of gesttion for the rts, on dy 15 for the hmsters, on dy 18 for the mice, ll femles were coded nd scrificed (by CO2 inhltion for the hmsters, nd by CO2 inhltion followed by cervicl disloction for the rts nd mice), nd their reproductive sttus ws determined. Implnttion sites in ech uterine horn were counted nd ech conceptus ws recorded s being live or ded nd s to generl condition. Live fetuses were weighed individully, sexed externlly nd internlly, nd exmined for externl mlformtions. Those weighing 2.0 g or less for rts, 1.0 g or less for hmsters, nd 0.5 g or less for mice, nd those weighing less thn two-thirds the men of their lrger litter mtes were termed "stunted." At lest one-third of the fetuses of ech litter, ll "stunted" fetuses, nd those hving externl mlformtions were dissected nd exmined for viscerl ltertions (2); their heds were fixed in Bouin's solution nd sliced to revel developmentl ltertions. All fetuses live t scrifice were clered for visuliztion of skeletl ltertions (3). Sttistics Nonprmetric test procedures were used in the dt nlysis. The litter ws considered to be the experimentl unit nd incidence dt were expressed s men percent ffected fetuses per litter. Experimentl groups were compred to controls (Cv nd CPF) by Mnn-Whitney U tests (4). One- Environmentl Helth Perspectives

3 sided tests were used, nd differences significnt t the 0.05 level were noted. Results Rts Signs of cholinesterse inhibition were evident fter ech dministrtion of Dipterex by gvge s ws reported previously (1). The effects did not ccumulte from dy to dy nd s result only three of the 34 femles given Dipterex (t.i.d.) for 10 dys died between dys 13 nd 15 of gesttion. None of those given Dipterex only for one dy died. Dipterex dministrtion to rts by gvge (t.i.d.) on dys 6 through 15 of gesttion ws followed by substntil, lthough not sttisticlly significnt, decrese in the mintennce of pregnncy, n increse in the fetl mortlity rte (p = 0.06) mong rts in which pregnncy ws mintined, nd significnt decrese in the body weight of the surviving fetuses versus vlues for the sme criteri mong pir-fed control rts (Tble 1). Furthermore, n verge of 76.4% of the fetuses in ech litter of the experimentl group ws mlformed. The mlformtions occurred in ll of the 19 litters exposed to Dipterex nd consisted mostly of generlized edem, vrious types of hernition of the brin nd cerebrospinl fluid through the skull, internl hydrocephly, micrognthi, cleft plte, severely shortened rdius nd uln, nd hypophlngism nd syndctyly. Other ltertions noted were hemtoms, extensive doubling of the thorcic centr, fusions of the sternebre nd of the ribs, umbilicl hernis, smll kidneys nd common truncus. The mlformtions seen in the CPF group consisted of two fetuses with wvy ribs nd nother with doubled thorcic centr; these fetuses were from three litters. On the other hnd, dministrtion by gvge (t.i.d.) of the sme dily dose of Dipterex on dy 8 or 10 of gesttion significntly decresed mternl food consumption, on temporry bsis (one dy period), but did not hve significnt dverse effect on the remining criteri studied (Tble 1). Hmsters Ten minutes fter dministrtion of Dipterex t 400 mg/kg-dy by gvge (t.i.d.) the hmsters exhibited signs of cholinesterse inhibition, e.g., ptylism, exophthlmos, loss of equilibrium, nd musculr tremors. In generl, they recovered by the time the next dose ws to be dministered, but three of the 30 femles given Dipterex (dys 7 through 11) t this level died; one fter the first nd two fter the third dy of dosing. None of the control nimls died before scheduled scrifice. The mintennce of pregnncy ws not dversely ffected by Dipterex dministrtion on dys 7 through 11 of gesttion, but food consumption nd mternl weight gin were significntly reduced below the vlues for both the Cv nd CPF groups nd the incidence of fetl deth, stunted, nd ml- Tble 1. Effect of Dipterex dministrtion by gvge (t.i.d.) to C-D rts for vrious periods of gesttion. CPP Dys mg/kg-dyb Dy 8 or 10 Cvc Dy 8, 480 mg/kg" Dy 10, 480 mg/kg" Totl number Mortlity (%) 1(9) 3(9) 1(10) 0 0 Number pregnnt (%) 8(80) 20(65)Y 9(100) 13(93) 12(86) Food intke, ge 17.5 t t t 0.8f 23 ± 0.5f Weight gin, ge I ± ± ± ± 1.82 Totl number Fetl deth, % ± SE 14(21 t 8.6) 71( )9 1( ) 5(2.6 ± 1.62) 4( ) Number stunted Weight, g f ± Number mlformed (%) 5(6 ± 4.1) 86(76.4 ± 8.4)' 6(6 ± 4.7) 2(1 ± 0.8) 2(2 ± 1.7) CPF: group given only the vehicle by gvge on dys 6 through 15 of gesttion nd pir-fed to the corresponding group given Dipterex. b Dily dosge of Dipterex given in mg/kg-dy. c Cv: combintion of two groups of rts given only the vehicle by gvge on dy 8 or 10 of gesttion. d One femle hd 13 resorptions but no fetuses. e Included the period gvged plus two more dys (X + SE). 'p < 0.05 compred to controls. op = JunIe

4 Tble 2. Effect of Dipterex dministrtion by gvge (t.i.d.) to hmsters on dys 7 through 11 of gesttion C Cvb mg/kg-dy mg/kg-dy mg/kg-dy mg/kg-dy CPF' Totl number (2) 10 30(3) 16 (deths) Number pregnnt Food intke, gd ± ± ± ± 0.50e 7.0 ± 0.38e 7.2 ± 0.47e Weight gin, gd ± ± ± ± 3.14e 5.7 ± 2.1Oef 12.5 ± 1.5le Totl number Number per femle 10.9 ± ± ± ± ± ± 0.55ef 11.3 ± 0.45 Fetl deths 17/12 10/8 5/2 13/8 16/5 77/19 11/6 (no. ded/no. litters) Fetl deth, % 17( ) 10(4.6 ± 1.46) 5(9.1 ± 4.98) 13(5.0 ± 1.48) 16(12.6 ± 5.08) 77(24.5 ± 4.50)ef 11(5.5 ± 2.28) Number stunted Weight, g 1.90 ± ± ± ± ± 0.059e 1.57 ± 0.047ef 1.86 ± Number mlformed Percent mlformed 0.39 ± ± ± ± ± ± 3.947e 0.44 ± C: group not gvged. b Cv: group gvged (t.i.d.) with distilled wter for the dys indicted c CPF: group gvged nd pir-fed to the Dipterex group given 41 d Included period gvged plus two more dys (X ± SE). " p < 0.05 compred to Cv. f p< 0.05 compred to CPF. formed fetuses ws significntly incresed mong the litters of the Dipterex group (Tble 2). Most of the mlformtions consisted of edem, cleft plte, ptgium, nd fused ribs (Tble 3). The hemimelic fetus hd no rdius or uln in the left front limb, nd the tibi in the left hind limb ws only one-hlf the length of tht in the right hind limb; the distl portion ws missing. In ddition, this stunted fetus hd cleft plte, bilterl cleft lip, micrognthi, nd both fused nd doubled thorcic nd lumbr centr. The pulmonry trunk of nother fetus led from the right ventricle under the ortic rch nd inserted into the right subclvin rtery insted of into the descending ort vi the ductus rteriosus. The ectrodctyly seen in both the Cv nd Dipterex groups consisted of missing thumbs. Administrtion of Dipterex on dys 7 through 11 of gesttion t dose level of 300 mg/kg-dy lso resulted in significntly lower food consumption, mternl weight gin, nd fetl weight upon comprison to the vlues for the Cv group (Tble 2). Fetl lethlity ws incresed, but the difference from tht of the control group ws not sttisticlly significnt. Only one mlformed fetus ws seen mong the fetuses of the group given Dipterex t 300 mg/kg-dy, but it hd severl severe mlformtions (Tble 3). In the group given Dipterex t 200 mg/kg-dy, two femles died between the end of the dosing period nd scheduled scrifice. During the dosing period signs of cholinesterse inhibition, while still present, were not s severe s those seen fter dministrtion t 400 mg/kg-dy. Additionl sttisticlly significnt differences were not seen between the 200 mg/kg group nd the Cv group (Tbles 2 nd 3). After Dipterex dministrtion only on dy 8 of gesttion t 400 mg/kg-dy, mternl food consumption ws significntly decresed nd fetl lethlity ws significntly incresed upon comprison to the corresponding Cv group (Tble 4). None of the remining criteri studied differed significntly between the two groups. Mice Dipterex ws dministered to mice t 600 mg/kgdy, but even t this dose level overt signs of cholinesterse inhibition were not noted. Four of the femles given Dipterex t this level for some period of gesttion died before scheduled scrifice but no more thn one occurred in ech group. Higher dose levels were not studied becuse fter dministrtion t 600 mg/kg-dy for five dys both mternl food intke nd rte of gin in body weight were significntly reduced (Tble 5). Although mternl food consumption nd weight gin were significntly reduced fter dministrtion of the highest levels of Dipterex on dys 6 through 10 of gesttion, dverse effects were not detected on mintennce of pregnncy, fetl survivl, or on incidence of stunting (Tble 5). Fetl weight ws significntly reduced t dose levels of 400 mg/kgdy or more, but the totl incidence of developmentl mlformtions ws not incresed bove control vlues (Tbles 5 nd 6). Administrtion of 108 Environmentl Helth Perspectives

5 Tble 3. Incidence of ltertions mong fetuses of hmsters given Dipterex by gvge (t.i.d.) on dys 7 through 11 or on dy 8 of gesttion. Dys 7 through 11 Dy C' CvO mg/kg-dy mg/kg-dy mg/kg-dy mg/kg-dy CPP Cvb mg/kg Number of fetuses/number of litters exmined Externl ltertions 356/31 218/20 54/5 251/22 121/10 302/27 192/16 161/14 225/22 Viscerl ltertions 144/31 83/20 21/5 95/22 43/10 119/27 73/16 63/14 96/22 Skeletl ltertions 356/31 218/20 54/5 251/22 121/10 302/27 192/16 161/14 225/22 Number of fetuses ffected/number of litters ffected Externl exmintion Edem 3/2 Cleft lip 1/1 Cleft plte 1/1 3/3 Ectrocrdi 1/1 Micrognthi 1/1 Microphthlmi 1/1 Hemimely 1/1 Ectrodctyly 1/1 1/1 2/1 Ptgium 4/4 Tiled kinked 1/1 Hemtom 1/1 Viscerl exmintion Pulmonry trunk displcement 1/1 Innominte, bsent 1/1 1/1 Pncres genesis 1/1 Spleen genesis 1/1 Kidney, smll 1/1 1/1 1/1 1/1 Lungs, smll 1/1 Skeletl exmintion Vertebre centr fused 1/1 1/1 Vertebre centr doubled 1/1 Vertebre centr misligned 1/1 Ribs fused 2/2 2/2 1/1 3/3 Ribs brnched 1/1 Sternebre fused 1/1 1/1 1/1 Totl 1/1 3/2 1/1 4/4 1/ 1 13/10" 1/1 1/1 4/4 " C: group not gvged. b CV: group gvged (t.i.d.) with distilled wter for the dys indicted. ' CPF: group gvged nd pir-fed to the Dipterex group given 400 mg/kg. d p < 0.05 compred to Cv. e p < 0.05 compred to CPF. Dipterex on dys 10 through 14 ws ccompnied by similr mternl nd fetl effects, however, the incidence of stunting ws significntly incresed t the 600 mg/kg-dy dose level nd t both the 500 nd 600 mg/kg-dy levels the incidence of development ltertions ws significntly incresed (Tble 7). The increse in developmentl ltertions consisted lrgely of cleft pltes (Tble 6); in the 500 mg/kg dose group only one of the cleft pltes occurred in fetuses weighing less thn 0.75 g, but in the 600 mg/kg dose group three of the five fetuses with cleft pltes were stunted nd only one weighed more thn 0.75 g. In the 300 mg/kg dose group ll ltertions except the doubled centr occurred in only one fetus. After dministrtion of Dipterex to mice t 600 mg/kg for three or less dys of gesttion sttisticlly significnt increse in cleft plte occurred mong nonstunted fetuses in the group dosed on Tble 4. Effect of Dipterex dministrtion by gvge (t.i.d.) to hmsters on dy 8 of gesttion. CvI"' 400 mg/kg Totl number (deths) Number pregnnt Food intke, g" 10.2 t t 0.36 Weight gin, g' 6.0 t t 0.82 Totl number Number per femle 11.1 ± t 0.54 Fetl deth (no. ded/ 6/4 18/13 no. litters) Fetl deth, % 6(3.3 t 1.35) 18(7.1 t 1.64)' Number stunted 1 2 Weight, g 1.91 ± ± Number mlformed 1 4 Percent mlformed 0.64 t t "Cv-group gvged (t.i.d.) with distilled wter on dy 8. Included dy 8 plus two more dys (X ± SE). 'p < 0.05 compred to Cv. June

6 Tble 5. Effect of Dipterex dministrtion by gvge (t.i.d.) to CD-1 mice on dys 6 through 10 of gesttion C" Cv" mg/kg-dy mg/kg-dy mg/kg-dy mg/kg-dy Number pregnnt Food intke, g' 6.3 ± ± d 6.1 ± ± 0.25" 5.3 ± 0.11" Weight gin, gc 4.6 ± ± ± ± " 1.6 ± 0.35 Totl number(%) i44(1.1) 187(11.7) 53(10.6) 39(13.0) 46(11.5) 270(10.8) Fetl deth, % 12.7 ± ± ± ± 2.00 Number stunted (%) 0 1(0.53) 1(1.89) 0 1(2.17) 1(0.37) Weight, g 0.89 ± ± " 0.84 t 0.027" 0.82 ± 0.015" Number mlformed (%) 1(1.0 ± 0.96) 1(0.7 ± 0.69) 2(3.1 ± 3.08) 0 1( ) 3( ) " C; group not gvged. b CV: group gvged (t.i.d.) with distilled wter for dys indicted. 'Included the period gvged plus two more dys (X + SE). p < Tble 6. Incidence of ltertions mong the fetuses of CD-1 mice given Dipterex by gvge (t.i.d.) on dys 6-10 of gesttion. Dys 6-10 Dys mg/kg- mg/kg- mg/kg- mg/kg- mg/kg- mg/kg- mg/kg- mg/kg- C Cv dy dy dy dy C Cv dy dy dy dy Number of fetuses/number of litters exmined Externl ltertions 144/13 187/16 53/5 39/3 46/4 270/25 135/12 267/23 91/7 44/4 67/6 205/20 Viscerl ltertions 57/13 67/16 20/5 14/3 16/4 100/25 52/12 101/23 33/7 16/4 27/6 88/20 Skeletl ltertions 144/13 187/16 53/5 39/3 46/4 270/25 135/12 267/23 91/7 44/4 67/6 205/20 Externl exmintion Cleft plte 1/1 1/1 1/1 1/1 1/1 3/2* 5/5* Exencephly 1/1 Ablephri 1/1 Umbilicl herni 1/1 Kinked til 1/1 1/1 Clubbed limbs 1/1 1/1 Hemtom 2/1 1/1 Viscerl exmintion Hydrocephly 1/1 1/1 Testes smll 1/1 Lungs smll Skeletl exmintion Vertebre fused rch 1/1 Vertebre doubled 1/1 centr Ribs missing 1/1 Ribs wvy 2/2 Sternebre fused Totl ffected 1/1 1/1 2/1 0 1/1 3/3 1/1 2/1 2/2 1/1 413* 7/7* 110 dys of gesttion. Three fetuses in the group given Dipterex only on dy 8 of gesttion hd severl structurl ltertions, but their incidence did not differ significntly from the Cv group in which three nonstunted litter mtes hd cleft pltes. Comprison of the femles given 10 mi/kg of wter t.i.d. (Cv) for vrious periods of gesttion to those not gvged or hndled severl times dy during pregnncy (C) reveled no significnt chnge in food consumption, mternl weight gin, mintennce of pregnncy, fetl survivl, or fetl development to dy 18 of gesttion (Tbles 5 to 10). Discussion In previous study (3), Dipterex ws found to be tertogenic in the rt if consumed t dose levels of 432 or 519 mg/kg body weight/dy on dys 6 through 15 of gesttion, but not if given by gvge once dily for the sme period even t doses tht were lethl to Environmentl Helth Perspectives

7 Tble 7. Effect of Dipterex dministrtion by gvge (t.i.d.) to CD-1 mice on dys 10 through 14 of gesttion C Cvb mg/kg-dy mg/kg-dy mg/kg-dy mg/kg-dy Mortlity 1 1 Number pregnnt Food intke, gc 6.8 ± ± ± ± ± 0.22d 5.5 ± 0.1 de Weight gin, ge ± ± ± I.Old 6.0 ± 0.95d 5.3 ± 0.48d' Totl number 135(11.2) 267(11.6) 91(13.0) 44(11.0) 67(11.2) 205(10.2) Fetl deth, % 12.9 ± ± ± ± ± Number stunted (%) (1.95) Weight, g 0.95 ± ± ± 0.019d 0.84 ± 0.041d 0.80 ± 0.026d 0.80 ± 0.017de Number mlformed (%) 1(0.7 ± 0.69) 2(0.8 ± 0.79) 2(2.0 ± 1.33) 1(2.5 ± 2.50) 4(6.5 ± 3.56)d 7(4.0 ± 1.28)' C: group not gvged. b Cv: group gvged (t.i.d.) with distilled wter for the dys indicted. c Included the period gvged plus two more dys (X ± SE). d p <0.05. Significnt dose response. Tble 8. Effect of Dipterex dministrtion by gvge (t.i.d.) to CD-1 mice for dy 8 nd dys 8-10 of gesttion. Dy 8 Dys 8-10 C Cv' 600 mg/kg-dy C Cv' 600 mg/kg-dy Mortlity 1 Number pregnnt Food intke, gr 6.3 ± ± ± ± ± ±.18d Weight gin, gr 0.54 ± ± ± ± ± ±.38 Totl number 122(11.1) 149(11.4) 293(10.5) 121(11.0) 156(11.1) 200(11.1) Fetl deth, % 13.6 ± ± ± ± ± 1.82 Stunted, (%) 0 0 2(0.68) 0 0 1(0.50) Weight, g 0.96 ± ± ± ± ± ± Mlformed, (%) 0 5(3.5 ± 2.42) 4(2.1 ± 1.26) 1(0.8 ± 0.75) 1(0.9 ± 0.89) 0 C: group not gvged. b Cv: group gvged (t.i.d.) with distilled wter for the dys indicted. c Included the period gvged plus two more dys (X ± SE). d p < Tble 9. Effect of Dipterex dministrtion by gvge (t.i.d.) to CD-1 mice on dys nd dys of gesttion. Dys Dys Cv 600 mg/kg-dy Cb Cv 600 mg/kg-dy Mortlity Number pregnnt Food intke, gr 6.3 ± ± ± ± ± 0.18d Weight gin, g' 5.3 ± ± 0.72d 6.9 ± ± ±.47 Totl number 23(11.5) 49(12.2) 156(11.1) 185(11.6) 306(11.8) Fetl deth, % 17.8 ± ± ± ± ± 2.0 Number stunted (%) 0 0 1(0.64) 0 1(0.33) Weight, g 0.92 ± ± 0.026d 0.96 ± ± d Number mlformed (%) (0.4 ± 0.44) 4(1.2 ± 0.58) CV: group gvged (t.i.d.) with distilled wter for the dys indicted. b C: group not gvged. c Included the period gvged plus two more dys (X ± SE). dp < June 1979 illl

8 Tble 10. Incidence of ltertions mong the fetuses of CD-I mice given Dipterex by gvge (t.i.d.) on dy 8, dys 8-10, dys 10-12, nd dys Dy 8 Dys 8-10 Dys Dys C" Cvb mg/kg C" Cvb mg/kg-dy Cv' mg/kg-dy C'" Cv" mg/kg-dy Number of fetuses/number of litters Externl ltertions 122/11 149/13 293/28 121/11 156/14 200/18 23/2 49/4 156/14 185/16 306/26 Viscerl ltertions 50/11 55/13 117/28 47/11 61/14 80/18 8/2 18/4 61/14 72/16 114/26 Skeletl ltertions 122/11 149/13 293/28 121/11 156/14 200/18 23/2 49/4 156/14 185/16 306/26 Externl exmintion Cleft plte 3/1 41' Exencephly 1/1 Ablephri 1/1 Umbilicl herni Kinked til Clubbed limbs Hemtom Viscerl exmintion Hydrocephly 1/1 Testes smll Lungs smll 1/1 Skeletl exmintion Vertebre fused rch 1/1 Vertebre doubled centr Ribs missing Ribs wvy 1/1 Sternebre fused 1/1 1/1 Totl ffected 0 3/1 3/3 1/1 1/ /1 4/4 "1 C: group not gvged. b CV: group gvged (t.i.d.) with distilled wter for the dys indicted. ' p < some of the dms (150 to 250 mg/kg-dy). In the current study positive tertogenic response roughly equivlent to tht obtined in the former study ws chieved by dministrtion of Dipterex by gvge (t.i.d.). By dividing the dily dose into three equl portions, given roughly 3 hr prt, Dipterex could be dministered t 480 mg/kg-dy on dys 6 through 15 of gesttion without cusing significnt increse in mternl lethlity. Use of pir-fed control group demonstrted tht the tertogenicity seen ws not due to decresed food intke during the dosing period or to the stress of extended t.i.d. dministrtion. The positive tertogenic response ws not obtined if Dipterex ws given (t.i.d.) for only one dy of gesttion (on dy 8 or 10). Hence, the positive tertogenicity seen fter dministrtion of this chemicl with trnsient phrmcologic ction lso could be demonstrted fter dministrtion by gvge, provided the dily dose ws given in frctions. This is n importnt considertion for those responsible for determining the tertogenic potentil of environmentl chemicls. For the hmster, the mximum dose of Dipterex tolerted by gvge (t.i.d.) on dys 7 through 11 of gesttion ws 400 mg/kg-dy. A positive tertogenic response ws obtined t this dose level which included edem, cleft pltes, digitl mlformtions, fused ribs, nd even one cse of the rrely seen bsence or shortening of the long bones of the extremities. The incidence of mlformed fetuses per litter ws not s high s seen in the rt but the spectrum of mlformtions obtined ws very similr in both species. Agin comprison to pir-fed control group reveled tht the mlformtions were not merely due to reduced food intke. Sttisticlly, the pprent "no-effect" dose ws 200 mg/kg-dy, in tht significnt differences from the control group were not obtined in the dms or their offspring for the criteri under study. Embryotoxicity, but not tertogenicity, ws evident fter dministrtion of Dipterex t 400 mg/kg-dy by gvge (t.i.d.) on dy 8 of gesttion. Fused ribs were seen, but they occurred in three littermtes mong the 22 litters exposed s well s in one fetus from the control group. Hence, Dipterex hd to be given by gvge for more thn one dy to demonstrte its tertogenicity in the hmster. The mouse ws less susceptible to Dipterex thn the rt or hmster upon considertion of the dosge dministered without disply of cholinesterse inhibition. The specific reson for this species differ- 112 Environmentl Helth Perspectives

9 ence ws not scertined in the present study, but the trend lso ws evident in the offspring. A positive tertogenic response ws obtined t the highest doses tested but only when the Dipterex ws given on dys 10 through 14 or 12 through 14 of gesttion. Dipterex relted mlformtions consisted mostly of cleft pltes present in one-qurter to one-sixth of the litters exposed, respectively. Therefore, the tertogenicity of Dipterex ws not unique to the rt but ws present lso in the hmster nd to lesser extent in the mouse. In ech species dverse effects on the conceptus occurred only t doses tht decresed mternl food consumption, however, t lest in the rt nd hmster, the tertogenicity ws not merely due to the decresed level of food consumed. The uthors thnk R. G. Kelim for providing excellent technicl ssistnce for the portion of this study deling with rts, nd J. K. Hsemn for providing sttisticl ssistnce. The fetuses ws exmined for defects by Reserch Tringle Institute under Contrct #NIH-74-C-390. The Dipterex used ws kindly donted by Chemgro, Inc., Knss City, Missouri. REFERENCES 1. Stples, R. E., Kelim, R. G., nd Hsemn, J. K. Developmentl toxicity in the rt fter ingestion or gvge of orgnophosphte pesticides (dipterex, imidn) during pregnncy. Environ. Helth Perspect. 13: 133 (1976). 2. Stples, R. E. Detection of viscerl ltertions in mmmlin fetuses. Tertology 9: No. 3, A37 (1974). 3. Stples, R. E., nd Schnell, V. L. Refinements in rpid clering technic in the KOH-Alizrin Red S method for fetl bone. Stin Technol. 39: 62 (1964). 4. Seigel, S. Nonprmetric Sttistics for the Behviorl Sciences. McGrw-Hill, New York, 1956, pp , June