Document 50bxw9OpOpEmR0R4Oz157og64

PPG Industries, Inc. One PPG Place Pittsburgh, Pennsylvania 15272 (412) 434-2801 James A. Barter, Ph.D. Manager, Industrial Health, Hygiene and Toxicology Environmental Affairs Chemicals Group February 23, 1990 Janine Mifsud Information Scientist Syracuse Research Corp. Merrill Lane Syracuse, NY 13210 Dear Ms. Mifsud, You recently requested a copy of the report on the Inhalation Developmental Toxicity Study of 1,1,l-Trichloroethane which is available from the Washington, DC office of the Halogenated Sol vents Industry Alliance. During this conversation, we also dis cussed other developmental toxicity studies that have been con ducted on this material in the last few years, and you requested the key references in this area. Several past studies had shown no significant developmental toxici ty effects due to this chemical. In 1984, Dapson et al. (Teratol ogy 29 25A, 1984) reported cardiac malformations in 21-day old rats born to dams exposed through the drinking water. Subsequently, the National Toxicology Program and the National Institute of Environ mental Health Sciences contracted to conduct a study to determine the repeatability of the effects reported in the Dapson study. This study has been completed and the results of this work showed no evidence for reproductive toxicity or teratogenic effects produced by 1,1,1-trichloroethane. The results from the NTP/NIEHS study were first reported in 1987 at the 26th Annual Meeting of the Society of Toxicology (Toxicologist 7 (1), 175, 1987). A copy of the abstract of the presentation is enclosed. This work has been published recently by j. d. George et al. (Fundamental and Applied Toxicology 13 641-651, 1989). A copy of the paper is enclosed. The complete work is available as two reports: George, J.D., Price, C.J., Marr, M.C., Morrissey, R.E., and Schwetz, B.A. Developmental Toxicity Evaluation of 1,1,1Trichloroethane (CAS No. 71-55-6) Administered to CD Rats. SL 037261 Janine Mifsud Syracuse Research Corp. February 23, 1990 Page 2 Part I. Postnatal Evaluation, Final Study Report. NTP/NIEHS Contract N01-ES-55080, Aug. 28, 1987. NTIS Accession No. PB88131321/AS. George, J.D., Price, C.J., Marr, M.C., Morrissey, R.E., and Schwetz, B.A. Developmental Toxicity Evaluation of 1,1,1Trichloroethane (CAS No. 71-55-6) Administered to CD Rats. Part II. Teratological Evaluation, Final Study Report. NTP/ NIEHS Contract N01-ES-55080, Sept. 23, 1987. NTIS Accession No. PB88134101. As you can see, the results of the NTP/NIEHS study confirm the conclusions of the earlier studies, and the findings of the Dapson study, which are inconsistent with the earlier studies, were not verified by the NTP/NIEHS study. Please feel free to contact me if you want to discuss this issue further. Yours truly. ames A. Barter, Ph.D D.A.B.T. jabmarl7 19-20 SL 037262 (o^sCol crfCCTS OF AFLATOXINS B1 AND Gl ON P0ST1MPLAN^ TAT I ON RAT EMBRYOS. E.A. Maull, B.A. Clement, K.D. Hcidelbaugb and T.D. Phillips. Veterinary ublic Health, Texas A&M University, College tation, TX. In vitro studies were performed to determine the potential for interactions between Aflatoxin B1 (AFB1) and Aflatoxin Gl (AFG1) in postimplanta tion embryos. Embryos were collected on day 10.5 of gestation, dissected from uterus and decidua and rolled continuously for 45 hours in gassed rat serum. Viability was assessed by yolk sac diameter, crown-rump length and somite number. Optimal concentrations of AFB1 (5 pM) and AFG1 (10 pM) were tested with and without an S9 hepatic metabolizing system. The combined effects of AFB1 and AFG1 resulted in an increase in yolk sac diameter when compared to the two toxins alone; while, the crown-rump length and somite number remained unchanged. Surprizingly, the addition of an S9 fraction to the treatment groups provided some protection from the toxic effects. This finding suggests that prior meta bolism may result in nonspecific binding and/or inactivation under the experimental conditions employed. Thus, the parent compounds may or may not require activation in the yolk sac or embyro for toxicity. Further investigations (employing monoclonal antibodies) are ongoing to quantita tively assess yolk sac and embryonal dosimetry. (Supported by USOA Project 84CRSR-2-Z434, TAES H6215 and AH 6830.) Mi Mb..' 700 DEVELOPMENTAL TOXICITY OF 1,1,1-TRICHLOROETHANE '1TCEN) IN CD RATS. R.D4 George, C.J. Price, M.C. Marr, B.A. Schweiz and R.E. Morrissey, Research Triangle Institue and 'NTP/NIEHS. Research Triangle Park, NC 1,1,1-Trichloroethane (TCEN) is an industrial solvent that has been found in contaminated water supplies. Cardiac malformations have been re ported in 21-day old rats born to dams exposed prenatally and throughout lactation to 10 ppm TCEN in the drinking water (Dapson et al.. Teratology 29, 25A, 1984). In the present study, male and female CD rats (>20/group) were given deionized/filtered water, 0.05S Tween 80 with 0,9 ppm 1,4-dioxane (vehicle control), 3, 10, or 30 ppm TCEN for 2 weeks prior to mating and during mating. Sperm-positive females were treated at the same dose levels throughout gestation and lactation. TCEN blood levels were determined in the F0 males and females after pre mating and mating, and in the F0 females after lactation. Pups were evaluated for visceral malformations and for TCEN blood levels on post natal day 21. There was no evidence of an in crease in cardiac or other visceral malforma tions in the pups at any dose level of TCEN. These results are in contrast to the published preliminary findings noted above. Supported by NTP/NIEHS Contract No. N01-ES-55080. 9 TERATOGENIC EFFECTS OF ORGANOPHOSPHORUS INSECTI CIDES IN Xgnopus laevls. J.E, Snawder and J.E- Chambers. Dept, of Biological Sciences, Mississippi State University, Mississippi State, MS Since organophosphorous insecticides have been shown to be teratogenic in developing chicken embryos, they have been studied for their teratogenic potential in Xenopus laevis embryos. Eggs were exposed to monocrotophos, dicrotophos, malathion, maLaoxon, parathion or paraoxon for 96 h. Concentrations of compounds ranged from 0.1 mg/1 to 25 mg/1 in water. At the end of 96 h the embryos were collected, measured, examined for morphological defects, and assayed for their concentrations of NAD. All the insecticides examined caused morphological defects and lowered NAD levels in a dose-dependent manner. The IC50 values in mg/1 for each compound were: dicrotophos, 2.5; monocrotophos 10.0; malathion, 0.25; malaoxon, 0.25; parathion, 1.0; and paraoxon, 0.1. The LC50 values in mg/1 were: malathion, 12.5; malaoxon, 1.0; parathion, 15.0; and paraoxon, 1.0. LC50 values for dicrotophos and monocrotophos are in excess of 10.0 mg/1. These results suggest that the organophosphorous insecticides do have teratogenic potential at concentrations that may be found In the environ ment, especially in temporary pools where many aphlblan species reproduce. 701 THE EFFECT OF PHENYLETHYL ALCOHOL. APPLIED DERMALLY TO PREGNANT RATS. R.A. Ford, A.M. Api, Research Institute for Fragrance Materials Inc., Englewood Cliffs, NJ and T.M. Palmer, Huntingdon Research Centre Ltd., Cambridgeshire, England. Sponsor: O.E. Easterday. Phenylethyl alcohol (PEA) is an important and widely distributed fragrance ingredient due to having the characteristic odor of roses. It Is also a natural constituent of roses. A study of the potential effects of dermally applied PEA on pregnant rats was conducted by applying undiluted PEA under 24 hr./day occlusion in doses of 0.14, 0.43 or 1.4 ml/kg daily during days 6 to 15 of pregnancy. The high dose resulted in maternal toxicity accompanied by a broad spectrum of fetal effects including morphological abnormalities in virtually all fetuses. The mid-dose did not produce maternal toxicity or fetal effects other than very small cervical rib buds in 30/129 fetuses. The low dose did not produce any definitive evidence of adverse effect. The 0*14 ml/kg dose is about 250 times higher than the calculated 90Zlle exposure dose from the use of PEA in fragranced products. Comparative rat/huoan blood level concentrations Increase this safety factor to approximately 10,000. SL 037263 175 IlINDAMI ntal and applied toxicology 13, 641-651 (1W| Developmental Toxicity of 1,1,1 -Trichloroethane in CD Rats1 Julia D. George,*-2 Catherine J. Price,* Melissa C. Marr,* Brian M. Sadler,* Bernard A. ScHwETZ,t Linda S. Birnbaum,! and Richard E. MoRRissEYf *Chemistry and Life Sciences. Centerfor Life Sciences and Toxicology. Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709-2194, and iNational Toxicology Program. National Institute ofEnvironmental Health Sciences, Research Triangle Park. North Carolina 27709 ReceivedSeptember 16,1988; accepted May 9, 1989 Developmental Toxicity of 1,1,1-Trichloroethane in CD Rats. George, J. D., Price, C. J., Marr, M. C., Sadler, B. M., Schwetz, B. a., Birnbaum, L. S., and Morrissey, R. E. (1989). Fundam. Appl. Toxicol. 13,641-651. 1,1,1 -Trichloroethane (TCEN), a major industrial and household solvent, was evaluated for pre- and postnatal developmental effects in SpragueDawley rats. This study was designed to assess the repeatability of a report (S. C. Dapson, D. E. Hutcheon, and D. Lehr, Teratology 29,25A, 1984) that indicated that 10 ppm TCEN in drink ing water caused cardiac malformations in developing rats. In the present study, TCEN (97% pure) was administered in the drinking water at target concentrations of 3, 10, and 30 ppm, using 0.05% Tween 80 as an emulsifying agent. Two control groups, one receiving deionized/ filtered water and the other receiving a vehicle control solution containing 0.05% Tween 80 and 0. 9 ppm 1,4-dioxane, a stabilizing agent found in the bulk chemical, were also included. Male and female breeders (more than 30 per group) were exposed to the control solutions or test compound for 14 consecutive days prior to cohabitation and for up to 13 days during the cohabi tation phase. Sperm-positive females (24-29 per group) continued to be exposed to these formu lations during pregnancy and lactation to Postnatal Day (PND) 21. Parental animals exhibited a slight aversion to the 30-ppm drinking water during the premating exposure. No significant effect on reproductive competence ofthe parental animals or postnatal growth and development ofthe offspring to PND 21 was noted. A slight increase in mortality from implantation to PND 1, possibly due to high mortality in one litter, was observed in the 30-ppm dose group. There was no indication of an increase in the incidence of cardiac or other malformations in PND 21 pups. In summary, TCEN administered at 3, 10, and 30 ppm in the drinking water had no significant effect on the morphological development ofCD rats. isot Society ofToxicoiogy. 1,1,1 -Trichloroethane (TCEN) is a saturated lipophilic, trichlorinated hydrocarbon used primarily as an industrial solvent for cleaning metals (Stewart, 1968). It is also used in dry cleaning, in vapor degreasing, as an aerosol propellant, as a developing solvent for printed circuit boards, and as a solvent in household products (IARC, 1979). In 1985, 1 Presented at the 26th Annual Meeting of the Society ofToxicoiogy, Washington, DC, February 24-27, 1987, Toxicologist 7(1), 175,1987. 2 To whom all correspondence should be addressed- the United States produced 869 million pounds of TCEN (C&E News, 1987). The current threshold limit value recommended for TCEN is 350 ppm (1900 mg/m3), deter mined as a time-weighted average for an 8-hr exposure period (ACGIH, 1987). Several laboratory investigations of TCEN have indicated no significant adverse effect on reproductive capacity. Pregnant SpragueDawley rats and Swiss Webster mice exposed by inhalation to 875 ppm commercial-grade TCEN (Chlorothene VG, 94.5% TCEN, 5.5% inhibitors and impurities) for 7 hr daily on I 0272-0590/89 $3.00 Copyright 1989 by the Society ofToxicoiogy. All rights of reproduction m any form reserved. SL 037264 642 GEORGE F.T AL, Gestation Days (GD) 6-15 (mean exposure 2404 mg/kg/day for rats or 3500 mg/kg/day for mice) exhibited no significant changes in maternal body weight, fetal viability, or mor phological development at term, when com pared with concurrent controls (Schwetz et al., 1975). In a multigeneration reproduction study conducted with ICR Swiss mice, TCEN (97% pure, stabilized with 3% 1,4-dioxane) was administered in the drinking water (con taining 1% Emulphor EL-620 emulsifier) at concentrations selected to provide nominal doses ofO, 583, 1749, and 5833 ppm [mean exposure 87-870 mg/kg/day (Lane el al., 1982)]. A deionized/filtered water control group was also included. Exposure was con tinuous throughout the experiment. Expo sure to TCEN produced no significant evi dence of reproductive or developmental tox icity in the parental generation (F0) or any offspring (i.e., the Fu, Flb, and F,c litters, or the Fja and F2b litters produced from the Fib litter). In 1981, in Santa Clara County, California, a well was removed from service 3 weeks after contamination with TCEN and other sol vents. At that time, the TCEN level in the wa ter had reached 1700 ppb. This was followed by anecdotal reporting of increased inci dences of spontaneous abortion and congeni tal malformations including cardiac defects. As a result, the California Department of Health Services (CDHS) conducted two epi demiological studies: (1) an examination of pregnancy outcome of residents in the con taminated area compared with residents in a demographically similar area having an un contaminated water supply; and (2) an exam ination of the incidence ofcongenital cardiac defects in the contaminated area compared with incidence rates for the entirety of Santa Clara County (CDHS, 1982). Both studies fo cused on women who conceived during the period January 1, 1980, to December 31, 1981. No direct correlation could be drawn between exposure to the TCEN-contaminated water and the increased incidence of spontaneous abortion or congenital anoma lies. Shortly thereafter. Dapson et al (1984) reported an increase in cardiac anomalies in TCEN-exposed developing rats. In that study, male and female Sprague-Dawley rats received either drinking water containing 10 ppm TCEN (97% pure, stabilized with 3% 1,4-dioxane) dispersed with 0.05% Tween 80 or vehicle control water containing 0.05% Tween 80 for 7 days prior to cohabitation and up to 21 days after cohabitation and prior to conception (mean total exposure to TCEN for males and females prior to conception was 20.4 days). Sperm-positive females con tinued to receive treatment throughout the gestational and lactational periods. Offspring from five treated litters and six control litters were evaluated for growth and physical devel opment on Postnatal Days (PND) 21-23. No significant differences were observed in mean body weights or in the mean wet heart weights of the two groups; however, cardiac abnormalities consisting of persistent ductus arteriosus and right and/or left atrial hypo plasia or displacement were observed in ap proximately 30% (15/52) of the TCEN-ex posed offspring. This incidence was signifi cantly higher than observed in the control offspring. The mean TCEN level in the serum from eleven 78-day-old pups from one TCEN-treated litter continuously exposed to 10 ppm TCEN was 0.03 Mg/ml (30 ng/ml) (Dapson, personal communication). Because of the importance of TCEN as an industrial and household chemical and the preliminary results of Dapson el al. (1984), which were in conflict with other laboratory studies (see above), additional studies were conducted in our laboratory using a study de sign similar to but more extensive than that of Dapson et al. and under experimental con ditions more controlled than those used by Dapson et al. (1984), Offspring were evalu ated at term (GD 20) or on PND 21 (George et al., 1987a,b). In the teratology study, TCEN (99% pure) was administered in the drinking water at a target concentration of 3, 10, or 30 ppm, using 0.05% Tween 80 as an emulsifying agent. Two control groups, one DEVELOPMENTAL TOXICITY Ol- l.l.l-TRICHLOROETHANE 643 receiving deionized/filtered water and the other a vehicle control solution containing 0.05% Tween 80, were also included. Male and female breeders (more than 30 per group) were exposed to the control solutions or test compound for 14 consecutive days prior to cohabitation and for up to 6 days during co habitation. Sperm-positive females (26-30 per group) continued to be exposed to these formulations through GD 20. TCEN had no significant effect on parental body weight, food consumption, or water consumption, compared with the vehicle control group. In addition, TCEN had no effect on prenatal vi ability or growth, and did not alter morpho logical development of fetuses observed on GD 20. Further details of this study can be found in George el al. (1987b). The postnatal evaluation of TCEN is described in the pres ent article. MATERIALS AND METHODS3 Animals and animal husbandry. Sprague-Dawley rats (Crl:CD BR VAF/Plus outbred albino rats)4 were 8-12 weeks ofage at the time ofarrival, and were quarantined I week prior to study initiation. Rats were housed on AbSorb-Dri cage litter5 in solid-bottom polycarbonate cages (19 X 10) X 8 in.) with stainless-steel wire lids6 and molded filter tops.7 Males were housed singly except dur ing cohabitation; females were group housed (maximum of three per cage) during quarantine and singly housed after cohabitation. Food8 *a9nd10d*ri*n1k3ing water (control, vehicle control, or TCEN-containing) were available ad libitum throughout the study. Animal rooms were equipped with automatic light cycles (lights on from 7. 00 am to 7:00 I'M). Relative humidity and temperature were maintained at 60 6% and 71 PC, respectively.'' Estimated exchange rate for air in each animal room was 12 to 14 times per hour. Male and female rats were randomly distributed into three equal cohorts (A, B, or C), with exposure to the compound beginning by cohort on 3 successive days. An imals were assigned to dose groups in cohorts so that body weight on the first day of the premating exposure was not significantly different across dose groups within individual cohorts, or for the entire study. Test chemical and ireatmem. 1,1,1-Trichloroethane (CAS No. 71 -55-6) of 97% purity stabilized with 3% 1,4dioxane10-11 was dissolved in deionized/filtered drinking water containing 0.05% Tween 80 as an emulsifying agent. Verification by gas chromatography with flame ionization detection of the concentration ofTCEN in pi lot batches of drinking water formulations at room tem perature, in drinking water bottles on empty rat cages, indicated that despite the presence of 1,4-dioxane as a stabilizing agent, the TCEN mixtures were still relatively unstable. TCEN concentration decreased to 82-90% of the nominal concentration after 72 hr (George et al., 1987a). Therefore, each batch ofdrinking water formula tion was administered to the animals for 2-3 days, the shortest possible period that could be scheduled within the study design. Formulations were mixed at 4, 15, and 40 ppm TCEN to allow the concentration of TCEN in the formulations to approach the target concentrations of 3, 10, or 30 ppm during the period of administration. A deionized/filtered water control and a vehicle control containing 0.05% Tween 80 and 0.9 ppm 1,4-dioxane,13 the stabilizing agent found in the bulk supply of TCEN, were also used. Nevertheless, due to the volatility of the compound, prodosing concentrations of TCEN ranged from 85 to 137% ofthe nominal concentration, although 81 % ofthe assayed formulations were within 90-110% of 3 This study was conducted in accordance with the Food and Drug Administration's Good Laboratory Prac tice Regulations for Nonclinical Laboratory Studies (FDA, 1978). Copies of the final study report (in two parts: George et al., 1987a,b) are available from the Na tional Technical Information Service, U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22151 [NTIS Accession Nos. PB88131321/AS (Part I) and PB88134I01 (Part II)]. 4 Charles River Laboratories, Inc., Raleigh, NC. 5 Laboratory Products, Garfield, NJ. 6 Laboratory Products, Rochelle Park, NJ. 7 Ancare Corporation, Manhasset, NJ. 8 Purina Certified Rodent Chow (5002), Ralston Pu rina Company, St. Louis, MO. 9 Datapod Model DP220 Electronic Hygrothermograph, Omnidata International, Inc., Logan, UT. 10 Obtained by Research Triangle Institute from the manufacturer, Aldrich Chemical Company, Milwaukee, WI (Lot No. 8725 LL). " Purity determinations were conducted at Research Triangle Institute (RTI), Research Triangle Park, NC (NTP/NIEHS Contract N01-ES-45061) and involved mass, infrared, and nuclear magenetic resonance spec trometry, Karl Fischer titration, and gas chromatogra phy. Identity and purity were confirmed at RTI (NTP/ NIEHS Contract N0I-ES-55080) using infrared spec trometry and gas chromatography, respectively, follow ing completion ofthe study. 13 Obtained by Research Triangle Institute from the manufacturer, Aldrich Chemical Company, Milwaukee, WI (Lot No. C 2514 KM). SL 037266 644 GEORGE ET AL. the nominal concentration. Postdosing concentrations blood. Each pup was necropsicd. with special attention of TCEN ranged from 30 to 108% of the predosing con to the heart and surrounding vasculature. The uterus of centration. To estimate average TCEN consumption, each dam was stained to reveal the number of implanta time-weighted averages of the TCEN concentration in tion sites (Salcwski, 1964). the dosing solutions were calculated based on the predos Blood analysis. Samples from adult animals were col ing concentrations and the decay curves for the 15- and lected directly from the vena cava into vacuum tubes and 40-ppm formulations (George el al.. 1987a). immediately placed on ice. Pup samples were collected Male and female rats (more than 30 per sex per group) by cardiac puncture into a syringe, and then transferred were exposed to TCEN for 14 days prior to cohabitation to a vacuum tube and put on ice. The iced samples were and throughout cohabitation (up to 13 days). This expo transported to the analytical laboratory where they were sure scheme was adopted to allow mating of the required extracted with isooctane. All blood samples were ex number of females (total of 117 sperm-positive females) tracted on the day they were collected. In addition, to within a reasonable amount oftime, while still providing account for possible loss of TCEN from the stored ex approximately the same mean exposure period used by tracts and variation in instrument response, on each day Dapson el al. (1984). Sperm-positive females (24-30 per samples were received, spiked control blood samples group) continued to be exposed through PND 21 as in were extracted and the extracts stored as a set with the the Dapson et al. study (1984). study sample extracts. The control and sample extracts Observations. Animals were observed daily for clinical were stored under refrigeration and analyzed in sets for signs oftoxicity. Food and water consumption and body TCEN at a later time, using gas chromatography with weight were determined on selected days during the pre- electron capture detection. mating, gestational, and postnatal periods, but not dur Statistical analyses. Analysis of selected data was car ing cohabitation. Blood samples were taken from the ried out using the general linear model (GLM) procedure vena cava from sentinel male and female rats (two/sex/ in the SAS software library (SAS Institute, Inc., 1982a,b). dose group/cohort) prior to cohabitation, to determine Prior to analysis, an arcsine-square root transformation the premating levels ofTCEN in whole blood. Cohabita was performed on all litter-derived percentage data tion continued until 24 to 29 sperm-positive females were identified in each dose group. Male breeder rats and sperm-negative female rats were killed after the cohabita tion period was completed, and blood samples were (Snedecorand Cochran, 1967), and Bartlett's test for ho mogeneity of variance was performed on all data to be analyzed by ANOVA (Winer, 1962). Dose-response re lationships for selected measures were evaluated using a taken from the vena cava of one or two animals from test for linear trend. Analysis of variance (ANOVA) was each treatment group in each cohort for the determina used to determine whether significant dose effects had oc tion ofpostcohabitation level ofTCEN in whole blood. curred. When ANOVA revealed significant differences Dams were maintained with their own litters through among groups, then Dunnett's test (Dunnett, 1955, out lactation to PND 21. During the postnatal period, 1964) and Williams' test (Williams, 1971, 1972) were the dams were weighed and food and water consumption used to compare each TCEN-treated group with the vehi was determined on PND 0, 7, 14, and 21. Individual lit cle control group for that measure (a - 0.05). Nominal ters were evaluated for the length of the gestational pe scale measures were analyzed by a test for linear trend riod and litter size. Pups were weighed on PND 1 and 4. on proportions, and a x2 test for independence among On PND 4, litter size was noted, and perinatal mortality treatment groups (Siegel, 1956). When x2 revealed sig was determined (Oser and Oser, 1956). Litters contain nificant (p < 0.05) differences among groups, then a one- ing more than 10 pups were culled to a litter size of 10, tailed Fisher exact probability test (a - 0.05) was used giving attention to equal sex distribution, ifpossible. Lit for pairwise companions between each TCEN-treated ters with less than 10 pups remained in the study undis group and the vehicle control. In addition, for all data turbed. Culled pups were sacrificed by ip injection of T- analyzed by parametric statistics, a comparison between 61 Euthanasia Solution13 and examined for visceral mal the deionized/filtered water control and the vehicle con formations (Staples, 1974; Stuckhardt and Poppe, 1984). trol group was conducted using Student's t test (SAS In Growth and survival of the remaining pups were evalu stitute Inc., 1982b). Nonparametric statistics were used ated to PND 21, when the pups were killed by ip injection to analyze selected data that violated the assumptions of with T-61 Euthanasia Solution. Dams were anesthetized the parametric tests. The Kruskal-Wallis (Siegel, 1956), with C02 and then killed either by exsanguination or cer Mann-Whitney U (Siegel, 1956), and Jonckheere vical dislocation. Blood samples were taken from the (Jonckheere, 1954) tests were used to examine the experivena cava of selected dams and up to two male and two mentwise effect of dose, the pairwise effect of dose, and female pups in at least four litters per dose group per co the dose-response trend, respectively, when data were hort for the determination of TCEN levels in whole13 continuous or approximately continuous but not nor mally distributed. In addition, for all such data, a com parison between the deionized/filtered water control and 13 American Hoechst, Somerville, NJ. the vehicle control group was conducted using the Mann-Whitney U test. DEVELOPMENTAL TOXICITY OF l.l.l-TRICHLOROETHANE 645 TABLE 1 Postnatal Evaluation: Water Consumption ry Mall anii Female CD Rats Exposed to 1,1,1-Trichloroethanf Water consumption (g/kg/day) Controls Deionized/ filtered water 0.05% Tween 80 + 0.9 ppm 1,4-dioxane 1 , 1,1-Trichloroethane" 3 ppm 10 ppm 30 ppm Premating Day 1 to mating Day 1 (14 days)* Males* Females' GD0 to parturition4 PND 1 to 21' 96.7 3.1 118.1 3.6 134.5 4.7 237.3 + 5.9 96.0 3.0 119.3 3.0 134.4 4.1 222.0 7.6 98.5 3.6 115.7 3.2 128.1 3.9 222.9 8.1 92.8 2.8 116.5 3.1 135.4 4.4 232.1 4.3 88.1 2.1 109.1 2.5 129.0 4.3 218.5 6.9 " Target concentration. * Includes all animals assigned to study; means SEM. * Test for linear trend involving the Tween 80 control group and the TCEN-treated groups; p < 0.05. d Includes all confirmed-pregnant dams delivering live litters; means SEM. ' Includes all dams with litters surviving to PND 21; means SEM. RESULTS No deaths, morbidity, or distinctive clini cal signs were exhibited by either the male or the female breeder rats during the premating or cohabitational periods. For males, there was no significant effect of treatment on body weight, body weight gain, or food consump tion during the premating period. Premating water consumption (g/kg/day) for male breeders exhibited a decreasing trend, involv ing primarily the 30-ppm TCEN group, sug gesting a slight aversion to the TCEN-treated water at the 30-ppm dose level (Table 1). Av erage calculated TCEN consumption for males during the premating period, based on time-weighted averages of TCEN concentra tion, was 0.3, 0.9, and 2.6 mg/kg/day for 3-, 10-, and 30-ppm groups, respectively. For females during the premating period, there was no effect of treatment on body weight, body weight gain, or food consump tion. Water consumption exhibited a de creasing trend suggesting that the females also disliked the TCEN-treated water (Table 1). Mean TCEN consumption during the pre mating period for breeder females, based on time-weighted averages of the concentration of TCEN in the 3-, 10-, and 30-ppm TCEN groups was calculated to be 0.3, 1.3, and 3.3 mg/kg/day, respectively. During the gestational period, no maternal deaths, morbidity, or distinctive clinical signs were observed. There was no effect of treat ment on body weight, body weight gain, food consumption, or water consumption (Table 1). Mean maternal TCEN intake from GD 0 to parturition based on time-weighted aver ages ofthe TCEN concentration in the dosing formulations indicated that females in the 3-, 10-, and 30-ppm TCEN dose groups received an estimated 0.3, 1.2, and 3.5 mg/kg/day, re spectively. For dams delivering live litters, there was no effect of treatment on maternal body weight, maternal body weight gain, or food or water consumption during the lactational period (PND 1-21). Neither was there any SL 037268 646 GEORGE ET Al.. TABLE 2 Postnatal Evaluation: Reproductive Outcome in CD Rats Exposed to LLI-Trichloroethanefrom Prematinu to Postnatal. Day 21 Controls Deionized/ filtered water 0.05% Tween 80 + 0.9 ppm 1,4-dioxane L, 1,1 -Trichloroethane" 3 ppm 10 ppm 30 ppm Percentage confirmedpregnant dams with live litters Number ofimplantation sites per dam**17 Length ofgestation (days)4' Number oflive pups per litter on PND I4c PND lbc PND44f Percentage mortality4 ' Implantation through PND \* Implantation through PND 4* Average pup body weight (g)4-f PND 1 PND 21 96.0 (24/25) 15.5 0.3 21.5 0,1 14.4 0.3 14.1 0.3 7.7 1.9 9.7 2.2 6.0 0.1 40.3 0.8 100.0 (22/22) 14.3 1.2 21.3 0.1 13.9 0.9 13.4 0.9 4.9 1.5 14.5 5.5 6.0 0.1 41.8 1.3 100.0 (24/24) 16.6 0.8 21.5 0.1 100.0 (25/25) 15.5 0.7 21,4 0.1 100.0 (22/22) 15.6 + 0.7 21.5 0.1 15.0 0.7 14.5 0.7 9.3 2.2 12.4 2.4 14.2 0.5 13.8 0.5 7.2 1.8 9.7 2.1 13.4 0.7 12.9 0.9 15.9 3.2* 19.6 4.7 6.0 0.1 40.2 0.8 6.1 0.1 40.8 0.7 6-0 0.1 40.4 1.1 ' Target concentration. 4 n - 18-25 for each group, since some data were inadvertently not collected or not available. ' Presented as means SEM. d Jonckheere's test, involving the Tween 80 control group and the TCEN-treated groups; p 0.05. ' Jonckheere's test, involving the Tween 80 control group and the TCEN-treated groups; p < 0,01. * Mann-Whitney U test, comparing the TCEN-treated group with the Tween 80 control group; p as 0.05. effect on the number of implantation sites tween the deionized/filtered water group and per dam or the length of the gestational pe the vehicle control group for any measure of riod (Table 2). When the vehicle control pup toxicity (Table 2). There was no effect of group was compared with the TCEN-treated treatment on maternal water consumption groups, the only significant effect noted was (Table 1). Mean maternal TCEN consump an increase in the percentage mortality from tion from PND 1 to 21, based on the timeimplantation to PND 1 due solely to a sig weighted average of the concentration of nificant increase above the vehicle control at TCEN in the formulations, was estimated to 30 ppm TCEN, This increase appeared to be be 0.6,2,0, and 5.9 mg/kg/day for the 3-, 10-, due primarily to 61% mortality in one 30- and 30-ppm TCEN dose groups, respectively. S* ppm TCEN-treated litter on PND 1. There Visceral examination of pups culled on was no effect of treatment on the number of PND 4 indicated that out of a total of 482 live pups per litter on PND 1 or 4, or average pups examined, only one (3-ppm dose group) pup body weight on PND 1 or 21. In addi had patent ductus arteriosus. Of those pups tion, there was no significant difference be found dead on PND 1, a total of 10 pups in SL 037269 DEVELOPMENTAL TOXICITY OF 1,1,1-TRICHLOROETHANE 647 TABLE 3 Summary OF Malformations Observed ON Postnatal Day 21 in CD Rat Pure Exposed to I,I,I-Trichloroethane from Conception to Postnatal Day 21" Controls Deionized/ filtered water 0.05% Tween 80 + 0.9 ppm 1,4-dioxane 1.1, l-Trichloroethane'' 3 ppm 10 ppm 30 ppm Number of pups examined Number oflitiers examined Number of pups with malformations Number of litters with one or more malformed pups Number of pups with external malformations (no tail) Number of pups with visceral malformations Hydronephrosis Small kidney 238 24 1 1 0 1 0 203 2L 2 2 1 0 I 229 246 24 25 21 21 204 21 ` 0 0 100 1 10 00 0 " No significant treatment-related effects were noted. h Target concentration. r All pups in one litter were missing and presumed dead on pnd 4. the TCEN-treated groups (6 from four litters at 3 ppm; 1 at 10 ppm; 3 from two litters at 30 ppm) were observed upon visual examina tion to have patent ductus arteriosus, with no Ioccurrence of patent ductus arteriosus in ei ther the deionized/filtered water group or the vehicle control group. Statistical analysis of the incidence by litter of patent ductus arte riosus indicated no significant effect of treat ment on this parameter. Patent ductus arte riosus was not observed in any pup that died between PND 2 and 21. When pups were evaluated for external and visceral malformations on PND 21, there was no evidence of cardiac malformation in any dose group (Table 3). An external malfor mation (no tail) was observed in one pup in each ofthe vehicle control (1/203) and 3-ppm (1/229) TCEN groups, hydronephrosis was observed in one pup in the deionized/filtered water group (1/238) and the 3-ppm (1/229) and 10-ppm (1/246) TCEN groups, and small kidney was observed in one pup (1/ 203) in the vehicle control group (Table 3). Blood samples from a total of 60 animals (6 males and 6 females per dose group) after the premating exposure, 45 animals (5 or 6 males and 2-6 females per dose group) after the postcohabitational exposure, and 61 dams (12-14 per dose group) and 127 pups (up to 2 male and 2 female pups from each of their litters) on PND 21 were analyzed. More than 90% ofthe samples analyzed for each pe riod contained TCEN at or slightly below the limit of detection (5.0 ng/ml). Validation of the analytical method indicated that had the compound been consistently present in the calibrated range (5-50 ng/ml) it would have been detected. DISCUSSION In the present study, TCEN administered in the drinking water at target concentrations of 0, 3, 10, or 30 ppm to CD rats caused no significant toxicity in male and female breed ers after a 14-day premating exposure, and had no effect on measures of maternal toxic ity during the gestational period. Measures of food and water consumption throughout the study indicated that the TCEN-dosed drink i-jf . *, 1 -*> , SL 037270  648 GEORGE ET AL. ing water was well tolerated by both male and female animals, although water consumption was slightly decreased at 30 ppm, suggesting decreased palatability at the high dose. In ad dition, measurements for the vehicle control and deionized/filtered water groups indicated that the two control groups were comparable, with only very minor differences for maternal body weight or water consumption. TCEN had no effect on fertility, length ofgestational period, live litter size, pup body weight, or pup survival from PND 1 to 21. A slight but significant increase in the mortality from im plantation to PND 1 was observed in the 30ppm TCEN dose group. The occurrence of this effect may have been influenced by a high mortality in one treated litter, however, since PND 1 litter size was not significantly affected, and no indication of compromised prenatal survival had been observed in the teratology study, the significance ofthis result is unclear. There was clearly no indication of an increase in the incidence of cardiac mal formations or visceral malformations of any other organ system in pups on either PND 4 or 21, even at concentrations as high as 30 ppm TCEN, This is in contrast with the re sults of Dapson et at. (1984), who indicated a significant increase in persistent ductus arte riosus and other cardiac malformations in surviving PND 21 offspring exposed to 10 ppm TCEN in the drinking water. In the pres ent study, patent ductus arteriosus was ob served in 10 TCEN-treated pups (3, 10, or 30 ppm) found dead on PND 1 and in 1 pup (3 ppm) culled on PND 4. Statistical analysis of these data did not confirm a treatment effect. In addition, studies ofthe time course ofpost natal closure of the ductus arteriosus in rats indicate that a functional closure of the vessel (i.e., constriction of the smooth muscle wall) occurs within the first 1-3 hr of birth (Homblad, 1969). This constriction may be main tained by prostaglandins or by the increased oxygen tension in the blood, and has been shown to be reversible at this stage (Homblad, 1969). The second stage of closure of the ductus arteriosus involves permanent sealing of the lumen of the vessel, and may take up to 5 days in the rat (Heymann and Rudolph, 1975). For those pups found dead on PND 1, observation of a patent ductus ar teriosus may have been the result of the ab sence, after death, ofchemical (prostaglandin or oxygen) control of construction of the ves sel. Patent ductus arteriosus was observed only in one living pup on PND 4, i.e., during the postnatal period when constriction of the ductus is still reversible (Hornblad, 1969), Observation of a patent ductus arteriosus in this animal may have been the result ofa low ered oxygen concentration in the vessel, due to cyanosis caused by the method of euthana sia. Therefore, the presence of patent ductus arteriosus in PND 1 or 4 animals as observed in this study was not attributed to TCEN ex posure; however, further experimentation may be warranted to fully elucidate the origin of this observation. The lack ofdetection ofTCEN in the blood ofanimals in the postnatal study was surpris ing in light of the results reported by Dapson et al. (1984 and personal communication); however, subsequent kinetic modeling of TCEN after ingestion in drinking water indi cated that detection of TCEN in blood using the present study design was unlikely, as ex plained below. TCEN is rapidly absorbed from both the lungs and the gastrointestinal system (Holmberg et al., 1977; Stewart, 1968). The com pound is then rapidly redistributed from the blood into the tissues. The majority of the systemically absorbed TCEN is eliminated via the lungs (Hake et al., 1960). Kinetic modeling of the disposition of TCEN in the postnatal study female breeders at the end of the premating period, based on timeweighted levels of TCEN in the drinking wa ter (see Appendix), suggested that based on measured water consumption, the highest blood levels that could be expected in these animals at the time ofsacrifice would be 0.51, 1.57, and 4.73 ng/ml blood. In actuality, with staggered times of sacrifice, the blood TCEN levels would likely be less. Based upon the SL 037271 * DEVELOPMENTAL TOXICITY OF LU-TRICHLOROETHANE 649 vailable pharmacokinetic information on fCEN, it seems unlikely that the TCEN con centration in the blood of the animal would exceed the 5 ng/g limit of detection, using the exposure regimen and sacrifice schedule in the present study. Since relative water consumption (i.e., based on body weight) for male breeders at the end of the premating period was actually less than for female breeders, this pattern would be expected to hold true for the male rats as well, in addi tion to being indicative of blood TCEN lev els at the end of the cohabitation period. A kinetic model for TCEN disposition during lactation was not developed in the present study; however, it might be assumed that al though relative water consumption for lactating animals is greater than for their nonlactating counterparts, the lactating females would exhibit steady-state levels of TCEN that were similar to those of nonlactating fe males, and that the remainder of the TCEN consumed would be evenly distributed through the milk to the dam's litter. Alterna tively, TCEN may be preferentially distrib uted through the milk due to high lipid solu bility of the test compound. Further experi'ments may be warranted to define the disposition of orally administered TCEN in the pregnant rodent. The results of the present study and the companion teratology study (George et al., 1987a,b) support the conclusions of Lane et al. (1982) and Schwetz et al. (1975), who showed that TCEN was not a reproductive toxicant or teratogen even at doses 300- to 1300-fold higher, however, our results are in contrast with the preliminary results of Dapson el al. (1984) both with regard to the in duction of malformations in exposed off spring and with respect to the measured blood levels of TCEN. Data from the present study support the conclusion that, under the conditions of the study, exposure to TCEN does not present a selective risk to the devel oping organism, and does not induce cardiac or other malformations. APPENDIX Pharmacokinetic Modeling of Blood Levels of TCEN To clarify the reason for the low TCEN val ues obtained in the blood analysis, a pharma cokinetic model of TCEN in the blood after administration in the drinking water was gen erated using data from the premating period of the postnatal evaluation. Standard formulas for a two-compartment open model with oral absorption were used (Wagner, 1975). Pharmacokinetic parameter estimates for the distribution and elimination of TCEN in rats after exposure by inhalation were obtained from Schumann et al. (1982). Values for the bioavailability (/) and the first-order absorption rate constant (kj for the oral route of exposure were estimated to be 0.50 and 1.0 min-1, respectively (Dallas et al., 1987). To simulate the intermittent na ture of the drinking behavior of rats (Arm strong, 1980), 28% of the daily dose was di vided into 24 equal parts to represent poten tial consumption outside feeding periods. The remaining 72% was divided evenly be tween two nocturnal feeding periods evenly spaced during the dark cycle. Since the dark cycle in the present study was from 7 pm to 7 am these feeding periods were estimated to be at 11 pm and 3 am. Simulations were per formed using SAS software running on an IBM 3081 mainframe computer at Triangle Universities Computation Center (TUCC), Research Triangle Park, North Carolina. Based on estimates of TCEN consump tion, the drinking habits of rats, and other data describing the kinetic behavior of TCEN, maximum blood levels of TCEN were predicted for females after the premat ing exposure. At 9:00 am, the earliest time of sacrifice, animals receiving the 3-, 10-, and 30-ppm formulations would have blood TCEN levels of approximately 0.51, 1.57, and 4.73 ng/g, respectively. Subsequently, the blood levels would fluctuate around the steady-state levels for each treatment group, s*v ' ' ' 037272 SL 650 GEORGE ET AL. i.e., 0.33, 1.04, and 3.13 ng/g blood for the 3-, 10-, and 30-ppm groups, respectively. When a "worst case" was simulated for the 30-ppm group, i,e,, the entire daily dose of TCEN consumed at 7:00 am, and the animals were all killed at 9:00 am, the TCEN blood level was predicted to be 23.64 ng/g blood at 9:00 am, decreasing to 7.06 ng/g blood by 1:00 pm, the last possible time of sacrifice. ACKNOWLEDGMENTS The present study was conducted at Research Triangle Institute (RTI), Research Triangle Park, North Carolina, under contract to the National Toxicology Program and the National Institute ofEnvironmental Health Sciences (NTP/NIEHS Contract N0I-ES-55080). The authors ex press their appreciation to the following RTI personnel who contributed to the completion of this investigation: Ms. Doris J. Smith, Ms. Gwendolyn McNeill, Ms. Dorene L. Bigelow, Mr. Philip V. Piscrchia, Ms. Polly E. Sanderson, Ms. Mertie V. Snead, Ms. Margaret R. Shil ling, Mr. Steven C. Morgan, Ms. Vickie I. Wilson, Mr. Fred D. Cole, Ms. Billie M. Sumrelt, Ms. Melody P. Gower. We thank Ms. Susan H. Harris and Ms. Nathelle J. Gross for their secretarial assistance. REFERENCES ACGIH(1987). Threshold Limit Values and Biological Exposure Indices for 1987-1988. American Confer ence of Governmental Industrial Hygienists, Cincin nati, OH. Armstrong, S. (1980). 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