Developmental Toxicity Evaluation of Ethylene Glycol by Gavage in New Zealand White Rabbits

Artificially inseminated New Zealand white (NZW) rabbits wereadministered ethylene glycol (EG) by gavage on Gestational Days(GD) 6 through 19 at doses of 0, 100, 500, 1000, or 2000 mg/kg/day,with 23–24 inseminated animals per group. Clinical signswere recorded and water consumption was measured daily; doeswere weighed on GD 0, 6–19, 25, and 30. At necropsy (GD30), maternal liver, kidney, and gravid uterine weights wererecorded. Histopathologic examination was performed on kidneysfrom 10 does/dose and for all unscheduled deaths. Ovarian corporalutea were counted and uterine implantation sites (total sites,resorptions, dead and live fetuses) were recorded. All livefetuses were weighed, sexed, and examined for external, visceral,and skeletal malformations and variations. EG resulted in profoundmaternal toxicity at 2000 mg/kg/day (42% mortality; three earlydeliveries and one spontaneous abortion) associated with renalpathology and unaccompanied by any other indicators of maternaltoxicity. Renal lesions at 2000 mg/kg/day involved the corticalrenal tubules and included intraluminal oxalate crystals, epithelialnecrosis, and tubular dilatation and degeneration. No dose-relatedmaternal toxicity occurred at 100–1000 mg/kg/day. Therewas no indication of developmental toxicity at any dose tested,including no effects on pre- or postimplantation loss, numberof fetuses, fetal body weight, or sex ratio (% male fetuses)per litter, and no evidence of teratogenicity. The "no observableadverse effect level" (NOAEL) for maternal toxicity was therefore1000 mg/kg/day and the NOAEL for developmental toxicity wasat least 2000 mg/kg/day in this study. The sensitivity of NZWrabbits relative to that of Sprague—Dawley rats and Swissmice for maternal and developmental toxicity from gavage administrationof EG during organogenesis can be determined for maternal toxicity:rabbits>mice>rats, and for developmental toxicity, mice>>rats >> rabbits.     

Reproductive Toxicity of Sulfamethazine in Swiss CD-1 Mice during Continuous Breeding

Sulfamethazine (SMZ) was evaluated for reproductive toxicityin Swiss CD-1 mice using a continuous breeding protocol. SMZwas administered in the diet at 0, 0.25, 0.5, or 1% (w/w), whichrepresented an average daily intake of 0, 313, 625, or 1250mg SMZ/kg/day, respectively. Exposure of F0 male and femalemice to 1% SMZ for 126 days resulted in a significant decreasein the mean number of live pups per litter and the number oflitters produced (task 2); the percentage pups born alive to1% SMZ females showed a nonsignificant decrease versus controlfemales. The effects on fertility were rapid to onset (1 to4 weeks) and cumulative in nature. F0 male and female body weightswere slightly depressed from 3 weeks to the end of the study.The crossover mating trial (task 3) revealed that the adverseeffect on ferility involved both treated partners in that littersize decreased when either 1% SMZ males were bred to controlfemales or 1% SMZ females were mated with control males. Afterapproximately 155 days of exposure of F0 mice to 1% SMZ, theterminal body weight of 1% SMZ females was significantly decreasedand that of 1% SMZ males showed a nonsignificant decrease. Inaddition, the liver weight to body weight ratio of the maleswas increased. Further, the prostate and seminal vesicle weightto body weight ratios were decreased in 1% SMZ males relativeto control males. No treatment-related gross or histopathologicallesions were noted for the pituitary or reproductive organsof either sex. Sperm assessment indicated no significant differencein the epididymal sperm concentration or percentage motile orabnormal sperm. In conclusion, SMZ was found to be a reproductivetoxicant in the male and female Swiss CD-1 mouse, albeit atrelatively high dietary intake (1250 mg/kg/day), and in thepresence of mild systemic toxicity.     

The Developmental Toxicity of Orally Administered Oxytetracycline in Rats and Mice

The Developmental Toxicity of Orally Administered Oxytetracyclinein Rats and Mice. MORRISSEY, R.E., TYL, R.W., PRICE, C.J., LEDOUX,T.A., REEL, J.R., PASCHKE, L.L., MARR, M.C, AND KJMMEL, C.A.(1986). Fundam. Appl. Toxicol. 7, 434-443. Timed-pregnant CDrats and CD-1 mice were dosed by gavage with oxytetracyclinehydrochloride (OXT) in corn oil on gestational days (gd) 6-15(0, 1200, 1350, or 1500 mg/kg/day for rats; 0, 1325, 1670, or2100 mg/kg/day for mice). Deaths among treated females occurredin a dose-related manner in all OXT dose groups (2-7%, mice;5-24%, rats), but no maternal deaths occurred in the vehiclecontrol groups. Significant dose-related decreases in maternalweight gain during treatment, as well as for corrected gestationalweight gain (i.e., maternal gestational weight gain minus graviduterine weight), were observed at all doses in rats but notin mice. Gravid uterine weight was reduced in a dose-relatedmanner only in mice, with the high-dose group significantlyreduced compared to the control group. At termination (gd 20,rats; gd 17, mice), the status of uterine implantation siteswas recorded and live fetuses were weighed. Fetuses were examinedfor external, visceral, and skeletal abnormalities. There wereno significant effects of OXT in either species on the incidenceof postimplantation loss (resorptions plus dead fetuses) ormalformations. In both species, there was a significant trendtoward reduced fetal body weight, and each group of rats receivingOXT was significantly reduced compared to the control group.Administration of OXT during organogenesis at doses exceedingthe therapeutic range for humans produced maternal and fetaltoxicity, but did not produce any treatment-related increasein malformations.     

Assessment of the Developmental Toxicity of Ethylene Glycol Applied Cutaneously to CD-1 Mice

Ethylene glycol (EG; CAS No. 107-21-1) is teratogenic to miceby whole-body exposure to an aerosol at high concentrations,but results were confounded by possible exposure from ingestionafter grooming and/or from percutaneous absorption. Therefore,CD-1 mice were exposed to EG on Gestational Days (GD) 6 through15, 6 hr/day by occluded cutaneous application at 0, 12.5, 50,or 100% (undiluted) EG (0.1 ml/animal, equivalent to {smalltilde}0, 404,1677, or 3549 mg/kg/day, respectively) or by gavageon GD 6 through 15 at 3000 mg/kg/day [10 ml/kg, positive controlgavage group (PCGG)], 30 females/group. Dams were weighed andevaluated daily (including application site) for clinical signsand water consumption throughout gestation. On GD 18, maternaluterus, liver, and paired kidneys were weighed; kidneys of 0and 100% and the PCGG were examined microscopically. Corporalutea and implantation sites were recorded. Live fetuses wereweighed, sexed, and examined for structural alterations. Forcutaneously exposed dams, there was no treatment-related maternaltoxicity, no differences in pre- or postimplantation loss orin fetal body weights/ litter, and no increased incidences ofany fetal malformations. Two skeletal variations, increasedat 100%, may represent effects of restraint stress and/or findingsdue to chance. In the PCGG, 8 females (26.7%) died, water consumptionwas increased, fetal body weights/litter were reduced, and fetalmalformations and variations were increased. PCGG kidneys exhibitedtubular nephrosis and tubular cell degeneration, with no oxalatecrystals, documenting renal toxicity at this oral dose in mice.Minimal-grade renal tubular lesions observed in 3 mice (of 30)at 100% EG may represent treatment-related or incidental findings.Therefore, exposure of pregnant CD-1 mice to 0, 12.5, 50, or100% EG during organo-genesis by occluded cutaneous applicationresulted in minimal or no observable maternal or developmentaltoxicity at 100% ({small tilde}3549 mg/kg/day), the NOEL.     

Reproductive Toxicity Evaluation of Methylethyl Ketoxime by Gavage in CD Rats

Methylethyl ketoxime (CAS No. 96-29-7; MEKO; 2-butanone oxime),an antioxidant agent used in paints, resins, and adhesives,was tested for reproductive toxicity in a two-generation studywith CD (Sprague-Dawley) rats. Thirty-eight-week-old rats/sex/group(F0) were administered MEKO in water, by gavage, at 0, 10, 100,or 200 mg/kg/day (at a dosing volume of 2 ml/kg), 5 days/weekfor 10 weeks with vaginal cytology evaluation (VCE) of F0 femalesduring the last 3 weeks of the prebreed period. Animals weremated within groups for 3 weeks with dosing during mating, gestation,and lactation for 7 days/week. F0 parents and F1 weanlings,10/sex/dose, were necropsied (after a 2-week postwean VCE inF0 females) with hematologic evaluation (including methemoglobin)and histology of adult livers, spleens, and reproductive organs.F1 weanlings, 30/sex/dose, were dosed for 11 weeks and matedas described above. Because of poor reproductive performance,not treatment related, F1 animals with no F2a litters were rebredto produce F2b litters. F1 parents and F2a weanlings, 10/sex/dose,were necropsied and evaluated as described above. Inguinal mammaryglands were examined histologically from all nonselected F1and F2 (a and b) female weanlings. Adult toxicity was observedin both generations and both sexes at all doses. Treatment-relatedparental deaths occurred at 200 mg/kg/day. At 100 and 200 mg/kg/day,parents exhibited dose-related reduced body weights and weightgains, reduced feed consumption, clinical signs of toxicity,and anemia with concomitant extramedul-lary hematopoiesis andhemosiderosis in livers and spleens (and increased spleen weights).At 10 mg/kg/day, only adult liver and spleen histologic effectswere present. There was no evidence of reproductive organ ormammary gland pathology or of reproductive or postnatal toxicityat any dose tested. There was no adult "no observable adverseeffect level" (NOAEL) established; the NOAEL for reproductiveand postnatal toxicity was at least 200 mg/kg/day for rats inthis study.     

Evaluation of the Developmental Toxicity of Ethylene Glycol Aerosol in the CD Rat and CD-1 Mouse by Whole-Body Exposure

Ethylene glycol (EG) is a major industrial chemical, shown tobe teratogenic at high doses by gavage in rodents. Since oneroute of industrial exposure is to the aerosol at high concentrations,timed-pregnant CD rats and CD-1 mice were exposed, whole-body,to a respirable aerosol of EG (mass median aerodynamic diameter,2.3 µm) on Gestational Days (GD) 6 through 15 for 6 hrper day at target exposure concentrations of 0, 150, 1000, or2500 mg/m³ (analytical concentrations of 0, 119 ± 13,888 ± 149, and 2090 ± 244 mg/m³, respectively),with 25 plug-positive animals per species per group. Clinicalobservations and maternal body weights were documented throughoutgestation for both species. Maternal food and water consumptionwas measured in rats only throughout gestation. At schedulednecropsy (GD 21 for rats, GD 18 for mice), maternal animalswere evaluated for body weight, liver weight, kidney weight,gravid uterine weight, number of ovarian corpora lutea, andstatus of implantation sites, i.e., resorptions, dead fetuses,live fetuses. Fetuses were dissected from the uterus, counted,weighed, sexed, and examined for external, visceral, and skeletalmalformations and variations. All rat dams survived to scheduledtermination. Minimal maternal toxicity was indicated by a significantincrease in absolute and relative liver weight at 2500 mg/m³.Food and water consumption, maternal body weights and weightgain, and maternal organ weights (other than liver) were unaffectedby exposure. Gestational parameters were unaffected by exposure,including pre- and post-implantation loss, live fetuses/litter,sex ratio, and fetal body weight/litter. There was no treatment-relatedincrease in the incidence of any individual malformation, inthe incidence of pooled external, visceral, or skeletal malformations,or in the incidence of total malformations by fetus or by litter.There were no increases in the incidence of external or visceralvariations. Evidence of fetotoxicity, expressed as reduced ossificationin the humerus, the zygomatic arch, and the metatarsals andproximal phalanges of the hind-limb, was observed at 1000 and2500 mg/m³. All mouse dams survived to scheduled termination.One dam at 2500 mg/m³ was carrying a totally resorbed litterat termination. Maternal toxicity was observed at 1000 and 2500mg/m³, expressed as reduced body weight and weight gain duringand after the exposure period, and reduced gravid uterine weight.(Maternal effects may have been due, in part or in whole, toeffects on the conceptuses; see below.) Embryo/fetal toxicitywas also observed at 1000 and 2500 mg/m³, expressed as an increasein nonviable implantations/litter, a reduction in viable implantations/litter,and reduced fetal body weights (male, female, and total)/litter.The incidences of individual and pooled external, visceral,and skeletal malformations were increased at 1000 and 2500 mg/m³,as was the incidence of total malformations. Malformations werefound in the head (exencephaly), face (cleft palate, foreshortenedand abnormal face, and abnormal facial bones), and skeleton(vertebral fusions, and fused, forked, and missing ribs). Theincidences of many fetal variations were also increased at 1000and 2500 mg/m³ (and only a few at 150 mg/m³). The no observableadverse effect level (NOAEL) for maternal toxicity in rats was1000 mg/m³ (analytical concentration 888 mg/m³) and in micewas 150 mg/m³ (analytical concentration 119 mg/m³). The NOAELfor development toxicity in rats was 150 mg/m³ and in mice wasat or below 150 mg/m³, under the conditions of this study. Analysisof EG on the fur of rats and mice during and after the exposureperiod at 2500 mg/m³ indicated that much of the EG "dose" (65–95%)was potentially derived from ingestion after grooming and/orpercutaneous absorption. This contribution of the ingested and/orabsorbed chemical could have been sufficient, per se, to producethe teratogenic effects observed in mice. The definitive evaluationof the possible role of inhaled EG aerosol alone in teratogenesisrequires an exposure regimen which limits or precludes exposureby any other route.     

Evaluation of Exposure to Water Aerosol or Air by Nose-Only or Whole-Body Inhalation Procedures for CD-1 Mice in Developmental Toxicity Studies

This study was performed to evaluate the effects of nose-onlyrestraint versus whole-body exposure procedures in the absenceof test chemical, and to determine the appropriate control environment(water aerosol or air) for subsequent developmental toxicitystudies of test materials administered as aerosols. Timed-pregnantCD-1 mice, 30/group, were exposed to high concentrations ofwater aerosol or to air by whole-body or nose-only inhalationprocedures on Gestational Days (GD) 6 through 15 for 6 hr perday. The group exposed to air by whole-body procedures was designatedas the control group. Clinical observations and maternal bodyweights were recorded throughout gestation. At scheduled necropsyon GD 18, maternal animals were evaluated for body weight, graviduterine weight, liver weight, number of ovarian corpora lutea,and status of uterine implantation sites. Fetuses were counted,weighed, and sexed and were examined for external, visceral(including craniofacial), and skeletal alterations. Indicesof maternal toxicity were affected in both nose-only groups.Maternal body weights were reduced during and after the exposureperiod; maternal weight gain was reduced during the exposureperiod. Clinical signs observed, from animals struggling duringrestraint, were resolved by GD 18. At sacrifice on GD 18, maternalbody weights and maternal gestational weight gains (both correctedfor gravid uterine weights) and absolute liver weights werereduced in both nose-only groups. Four females died (13.3%,all pregnant) in the air nose-only group, and maternal liverweight (relative to body weight) was reduced in the aerosolnose-only group. Gestational parameters were unaffected by anyof the treatments. There were no statistically significant differencesin the incidences of any individual malformations or malformationsby category (external, visceral, or skeletal) or of total malformations.However, exencephaly, low set ears, cleft palate, and ventricularseptal defect were observed only in both aerosol-exposed groups(whole-body and nose-only exposed). The incidences of individualexternal or visceral variations or of variations by categoryor of total variations were unaffected. The incidence of oneskeletal variation, poorly ossified supraoccipital skull bone,was significantly increased in the aerosol nose-only group relativeto the air whole-body controls. There were also increased incidences(not statistically significant) of extra (14th) ribs in bothaerosol groups. Therefore, maternal restraint (in both nose-onlygroups) during organogenesis produced indications of maternaltoxicity, but restraint did not appear to affect normal embryo/fetalmorphologic development. Maternal exposure to water aerosol(by whole-body or nose-only procedures) resulted in small, biologicallysignificant (but not statistically significant) increases inthe incidences of fetal malformations and variations. In conclusion,for mice at least, nose-only exposures employing the presentprocedures can be used effectively, and water aerosol is anappropriate control environment for developmental toxicity studiesof test materials administered as aerosols at high concentrations.     

Developmental Toxicity Evaluation of Inhaled Methyl Isobutyl Ketone in Fischer 344 Rats and CD-1 Mice

Developmental Toxicity Evaluation of Inhaled Methyl IsobutylKetone in Fischer 344 Rats and CD-1 Mice. Tyl, R. W., FRANCE,K. A., FISHER, L. C., PRITTS, I. M., TYLER, T. R., PHILLIPS,R. D., and MORAN, E. J. (1987). Fundam. Appl. Toxicol. 8, 310–327.Pregnant Fischer 344 rats and CD-1 mice were exposed to methylisobutyl ketone vapor (CAS No. 108-10-1) by inhalation on GestationalDays 6 through 15 at concentrations of 0, 300, 1000, or 3000ppm (mean analytical values of 0, 305, 1012, and 2997 ppm, respectively).The animals were sacrificed on Gestational Day 21 (rats) or18 (mice), and live fetuses were examined for external, visceral,and skeletal alterations. In rats, exposure to 3000 ppm resultedin maternal toxicity expressed as clinical signs, decreasedbody weight and body weight gain, increased relative kidneyweight, and decreased food consumption, and in fetotoxicityexpressed as reduced fetal body weight per litter and reductionsin skeletal ossification. In mice, exposure to 3000 ppm resultedin maternal toxicity expressed as exposure-related increasesin deaths (12.0%, 3/25 dams), clinical signs, and increasedabsolute and relative liver weight, and in fetotoxicity expressedas increased incidence of dead fetuses, reduced fetal body weightper litter, and reductions in skeletal ossification. No treatment-relatedincreases in embryotoxicity or fetal malformations were seenin either species at any exposure concentration tested. Therewas no evidence of treatment-related maternal, embryo, or fetaltoxicity (including malformations) at 1000 or 300 ppm in eitherSpecies.     

The Developmental Toxicity of 2-Ethyihexanol Applied Dermally to Pregnant Fischer 344 Rats

Undiluted 2-ethylhexanol (2-EH) was administered by occludeddermal application for 6 hr per day on Gestation Days 6 through15 to pregnant Fischer 344 rats, in range-finding (R) and main(M) studies. The dermal route is considered to be the most relevantfor human exposure. Treatment levels were (R) 0.0, 0.5, 1.0,2.0, and 3.0 ml/kg/day (equivalent to 0, 420, 840, 1680, and2520 mg/kg/day) and (M) 0.0, 0.3, 1.0, and 3.0 ml/kg/day (equivalentto 0, 252, 840, and 2520 mg/kg/day). Controls (0.0 ml/kg/day,sham controls) received deionized water at 3.0 ml/kg/day. Dermal-positivecontrol groups received undiluted 2-methoxyethanol (2-ME) at(R) 0.5 and 1.5 ml/kg/day and (M) 1.0 ml/kg/day as a referencecompound in a similar regimen. An oral reference compound, valproicacid, was administered by gavage in the range-finding studyon Gestation Days 6 through 15 at 400 mg/kg/day. The range-findingstudy employed an untreated (naive) control group. Numbers ofplug-positive females per group were (R) 8 and (M) 25. Maternalweight gain was reduced for 2-EH at 1680 (R) and 2520 (R andM studies) mg/kg/day. Exfoliation and encrustation were seenat the application site in both studies at 840, 1680, and 2520mg/kg. Maternal liver, kidney, thymus, spleen, adrenal, anduterine weights, and gestational and fetal parameters were unaffectedby treatment with 2-EH. There were no treatment-related increasesin the incidence of individual or pooled external, visceral,and skeletal malformations or variations following the applicationof 2-EH. The NOAELs for the maternal toxicity of 2-EH were 252mg/kg/day based on skin irritation and 840 mg/kg/day based onsystemic toxicity. The developmental toxicity NOAEL was at least2520 mg/kg/day, with no teratogenicity. Administration of 2-MEat 840 mg/kg/day resulted in reduced maternal weight gain andfood consumption, increased postimplantation loss, reduced numbersof live fetuses per litter, and reduced fetal body weights perlitter. The incidence of fetal malformations and variationswas increased. Oral administration of VPA produced maternaltoxicity, developmental toxicity, and teratogenicity. The Fischer344 rat is thus susceptible to known rodent teratogens by boththe dermal and oral routes. It is concluded that 2-EH is notdevelopmentally toxic by the dermal route in the Fischer 344rat at and below treatment levels which produce maternal toxicity.     

Determination of a No-Observed-Effect Level for Developmental Toxicity of Ethylene Glycol Administered by Gavage to CD Rats and CD-1 Mice

Previous studies have indicated that ethylene glycol (EG) isa developmental toxicant in rats and mice primarily when ingested.This study was designed to establish no-observed-effect levels(NOELs) for developmental toxicity of EG administered by gavagein both rodent species. Dams were administered EG on GestationDays 6–15; rats were given 0, 150, 500, 1000, or 2500mg EG/kg/ day; mice were dosed with 0, 50, 150, 500, or 1500mg EG/kg/day. In rat dams given 2500 mg EG/kg/day, water consumptionwas increased during treatment and body weights were reducedthroughout gestation; liver and kidney weights were increasedat euthanization (Gestation Day 21). Relative liver weightswere also increased at 1000 mg/kg/day. Effects observed in ratfetuses at 2500 mg/kg/day included the following hydrocephaly;gastroschisis; umbilical hernia; fused, duplicated, or missingarches, centra, and ribs; poor ossification in thoracic andlumbar regions; and reduced body weights. Reduced body weights,duplicated or missing ribs, centra, and arches, and poor ossificationwere also ob served in rat fetuses at 1000 mg/kg/day. In mice,there was no apparent treatment-related maternal toxicity. Inmouse fetuses (Gestation Day 18), effects were observed at 1500mg/kg/day and included reduced body weights, fused ribs andarches, poor ossification in thoracic and lumbar centra, andincreased occurrence of an extra 14th rib. At 500 mg/kg/day,slight reductions in fetal body weight and increased incidencesof extra ribs were observed. Under conditions of these studies,NOELs for developmental toxicity were 500 mg/kg/day for ratsand 150 mg/kg/day for mice, indicating that mice were more susceptiblethan rats to the teratogenic effects of EG.