Document 4KybMRD8p8BM6LEaoemGjq3Q

AR226-2818 The Embryo-Fetal Toxicity and Teratogenic Potential of Ammonium Perfluorooctanoate (APFO) in the Rat (Work Done for Hire) Robert E. Staples, Bruce A. Burgess, and William D. Kerns Haskell Laboratory for Toxicology and Industrial Medicine E. r. du Font de Nemours and Company, Inc. Elkton Road, P. 0. Box 50 Newark, Delaware 19711 Teratogenic Potential of APFO in the Rat Index Terms Chemical name: Octanoic acid, pentadecafluoro-, ammonium salt Generic name; ammonium perfluorooctanoate Manufacturer's name: Other: rat; teratogenicity; embryo-fetal toxicity; postpartum effects; eye; inhalation; gavage - 2 - Company Sanitized. Does not contafn TSCA CEt ABSTRACT The Embryo-Fetal Toxicity and Teratogenic Potential of 9 Ammonium Perfluorooctanoate (APFO) in the Rat. Robert E. Staples, Bruce A. Burgess, and William D. Kerns (1983) Fundam. Appl. Toxicol. APFO II^HB^IH wa s administered to Sprague Dawley rats from Days 6 through 15 of gestation by inhalation as a dust (whole body exposure) for 6 hr/day at 0, 0..1, 1, 10, and 25 mg/m , or by gavage at 100 ing/kg body weight/day in corn oil. Maternal deaths occurred in the groups given the highest level of APFO by each route and overt fcoxicity was evident among the surviving dams of these groups and among those of the 10 mg/m group. The fetuses were examined for external, visceral, and skeletal alterations and for APFO-related macroscopic and microscopic alterations of the eyes. In the postpartum period, pups from additional control and experimental dams were examined externally and ophthalmoscopically, and the usual fertility and viability indices were calcula-fced. A teratogenic response was not demonstrated. Toxic effects on the conceptus were noted only in the groups given the highest level of APFO by each route. Hence, APFO was not demonstrated to represent a unique hazard to the conceptus of the rat. ' 3 " Doesnotconta.nTSCACB ^ n a n v S a n r t - t ^ d, - D o - INTRODUCTION 2 Ammonium perfluorooctanoate (APFO) is a representative of an important class of compounds, the perfluorocarboxylic acids and their salts. Their teratogenic properties have not been studied extensively. Its toxicity in several animal species was reported by Griffith and Long (1980). The oral LD- in the rat was 540 mg/kg with the liver being the most sensitive target. In the rhesus monkey the gastrointestinal tract and the reticuloendothelial system were the sites of toxic effects after dietary exposure. By inhalation, the approximate lethal dose (ALD) in the male rat after a 4 hr, head only, exposure I I was 80 mg/m (unpublished Du Pont data). bu Pont purchases APFO for use in the manufacture of a variety of fluoropolymer resins, dispersions and elastomers. Between the last part of 1980 and March 1981, the manufacturer reported to the U.S. Environmental Protection Agency (EPA) under Section 8(e) of the Toxic Substances Control Act (TSCA) that APFO and several related chemicals had demonstrated teratogenic activity in rats. The reported teratogenic activity consisted of lens changes in the eyes of near-term offspring of rats exposed to the test chemicals by gavage from Days 6 through 15 of gestation.. The changes included macroscopically evident discoloration of the fetal nucleus of the lens, apparent abnormal arrangement of lens cells to a - a. Company Sanitized. Does not contain TSCA CB? varying degree, and clefts in the anterior portion of the lens. Significant dose-responses were reported with incidences of up to 100%, with 0% incidence in the control groups. It was not determined whether the lens changes persisted in fetuses born and raised to weaning age. As a precautionary measure, female employees in both companies were removed from exposure to APFO and additional teratogenicity testing was initiated in animals. At Haskell Laboratory (Du Pont) the inhalation route was tested to determine whether the reported teratogenicity of APFO in the rat would be expressed after exposure by this route and, if so, to establish an apparent "no-effect" concentration for protection of the conceptus. The information gained was to aid in establishment of workplace standards for women of childbearing potential. Rats also were given APFO by gavage to confirm or refute the preliminary findings reported to the EPA, and if confirmed, to determine whether the changes observed persist after birth. MATERIALS AND METHODS Test material The APFO sample |^HBB^^^^HH|^ wa s obtained from Du Font's supply previously purchased from the manufacturer. Its . . - ,, - ,, t^0111r""' c^ed.Do^^^3'"^^08' >----- purity wasg^------the contaminants present were or additives were present. Degradation of APFO is insignificant unless temperature exceeds 250C (unpublished Du Pont data). Generation and sampling of concentrations of APFO Dust atmospheres of APFO were generated with an airtight, two-stage glass apparatus composed of a round bottom reservoir and a cyclone-shaped elutriator. A generation air stream introducfced at the bottom of the reservoir carried dust particles upward to the elutriator. Dilution/carrier air entered tangentially into the top of the elutriator and swept airborne dust particles into the exposure chamber. To determine chamber concentrations of APFO' gravimetric samples were taken- from all chambers at regular intervals (low at 1 hr; intermediate and high at 0.5 hr). A known volume of chamber air was. drawn through preweighed Gelman glass fiber filters (Type AE, 25 nun).- The filters were reweighed and APFO concentration was calculated from the gain in filter weight. As a back-up system the APFO collected on the filters was extracted and analyzed spectrophotometrically (Percivaly 1968). All samples taken from the low concentration were analyzed by this procedure, as were 5 or 6 samples per exposure period from the intermediate and high concentrations. Filter samples taken from the control chamber also were analyzed periodically. 6 - Co^ny Sanitized. Does not contain TSCA CBI / Chamber temperatures were monitored each hour. Particle size at the high concentration was determined during Trials I and' II through use of an 8-stage Sierra Cascade Impactor Model #218K. Administration of APFO by gavage In the preliminary study conducted by the manufacturer the APFO was suspended in corn oil and given to pregnant rats by gavage. Hence, for the current study stripped corn oil was used. During the dosing period (Days 6-15 of gestation), suspensions were prepared daily in corn oil such that 100 mg APFO/kg body weight was contained in 5 mL of suspension/kg body weight. The body weight most recently recorded was used to calculate the dose to be given to each dam. A sample of the suspension remaining after completion of each day's dosing was stored at about 4C to be available for analysis of concentration and of uniformity of mixture. The control group in each experiment received 5 mL stripped corn oil/kg body weight for the same period of gestation. Corn oil suspensions of APFO were analyzed for fluorine content by decomposition in an oxyhydrogen flame using the standard Wickbold apparatus (Bock, 1979). The fluoride ion content in the condensed vapors was measured by thorium nitrate 'CompanySanitised. Does not contain TSCA CBl titration (Williams, 1979). A standard sample of m-carboxybenzotrifluoride was burned and titrated with each batch to verify accuracy of the analytical method for fluoride, Animals At arrival, nulliparous, female rats' (Sprague-Dawley derived Crl:CD(SD)BR strain) weighed between 151 and 198 g and were about 55 days of age.. Male rats of the same strain from the same source ranged from the same age as the females to 1 month older. The rats were conditioned to the laboratory environment for a minimum of 10 days. A standard laboratory Q diet and water from the Wilmington Suburban Water Corporation were supplied ac[ libitum. The animal rooms were lighted from 6:00 a.m. to 6:00 p.m. daily, and were maintained between 22-25C, and 36-70% relative humidity (The 95% confidence interval was 50.3-52.0% in the a.m. and 48.8-50.4% in the p.m ). Since cataracts or opacities occur among adult CD rats, all prospective parental rats were examined for these alterations before breeding. The eyes of each rat were dilated Q with 1% atropine ophthalmic solution and examined in semidarkness by a consultant ophthalmologist using focal illumination, indirect ophthalmoscopy, and, when indicated, slitlarop microscopy. Rats with eye lesions were eliminated from the colony before the breeding began. 8 - Company Sanitized. Does not contain TSCA CB1 Experimental design and procedures For the inhalation route (Table 1) the concentrations of APFO selected for study were 0, 0.1, 1, and 25 ing/m . The selection was based upon available toxicity data and upon information gained from a pilot study with non-pregnant rats. The design included 2 trials with 12 mated female rats per group per trial. It was anticipated that for Experiment I(Teratology) the data from both trials might be combined. However, for Trial II the 25 mg/m exposure concentration was reduced to 10 mg/m in response to severe toxicity seen at 25 mg/m . Also, 2 groups (6 dams/group) pair-fed to the 10 and 25 mg/m groups were added to Experiment I(Teratology), and 2 groups (6 dams/group) were added to Experiment II(Dams allowed to litter). The test groups were exposed (whole body) to APFO in 150 L glass and stainless steel chambers within which the rats were housed individually in wire-mesh modules. The location of breeding lots in each chamber was rotated daily. Control rats were exposed to in-house air in the same type of chamber for the same duration of gestation. The temperatures of each chamber were recorded hourly each day during the exposure period. After each exposure, the rats were housed in suspended, wire-mesh cages (2 females/cage), and the racks holding these cages were placed in a walk-in hood. 9 - Company Sanitized. Doss not contain TSCA CBI For the gavage portion of this study (Table 1), the 100 nig/kg/day dosage level was judged to be the maximum that the dams could tolerate based upon a preliminary study with pregnant rats. For both routes of administration the females were mated on an as-needed basis. The day on which spermatozoa were detected in the vaginal lavage, following overnight cohabi-tation, was designated as Day 1 of gestation (Day 1G). After the necessary number of females were bred, they were ranked within breeding days by body weight and assigned to groups by rotation in order of rank. For Experiment I(Teratology), the dams were weighed on the day of arrival, before breeding, and on Days 1, 6, 9, 13, 16, and 21G. They were observed for abnormal clinical signs and changes in demeanor upon arrival at Haskell Laboratory, at breeding, and daily from Days 6-21G. Feed consumption was measured during gestation, but for the inhalation route the dams were housed 2 per cage due to space restriction. To limit possible bias in the examination of maternal and fetal specimens, the dams were coded (group designation unknown to examiner) from just before sacrifice until all maternal and fetal data were collected and until all structural alterations noted among the fetuses were classified. After sacrifice of the dams by cervical dislocation on Day 21G, abnormalities were identified macroscopically, liver Compan-y cS-amnitit-,i7z6e--1i. D^"oie"s> not contain TSCA CBS weights were recorded, and the reproductive status of each animal was determined. The number of corpora lutea and implantation sites were counted, and the number and position of all live, dead, and resorbed fetuses were recorded. The uterus of each apparently "non-pregnant" rat was stained with ammonium sulfide (Salewski, 1964) to detect very early resorptions; data collected were used only to determine the incidence of pregnancy. The weight of the intact and empty uterus for each dam was recorded to allow calculation of actual maternal gain in body weight. All live and dead fetuses were weighed and sexed externally and internally, and the live fetuses were examined at a magnification of 2.5X (Ednalite) for external alterations. The Ednalite also was used to count the corpora A lutea. About one-half of the fetuses of each litter that were alive when removed from the dam were examined for visceral alterations (Staples, 1974); in addition, all stunted or malformed fetuses were examined similarly. For the inhalation route, the heads of all fetuses examined for visceral alterations were fixed in Bouin's fluid to permit examination as described by Barrow and Taylor (1969), but only those from the 25 mg/m3 and control groups (Trial I) were sectioned free-hand and examined under a stereoscope. This included examination of a vertical cross-section through the center of the eyes. Sections containing the eyes of 3 fetuses from each litter of the 25 mg/m group and of 2 fetuses - 11 - Company S- am.t,'.z_e-d,.. nD^.-r--'ro<?- c^ovntain TSCACBS from each litter of the control group were processed for examination by light microscopy. In Trial II, 1 fetal head from each of 4 litters from the 10 mg/m group and the control group were examined under a stereoscope. The eyes were left intact to minimize processing artifacts. The slices containing the intact eyes were processed and examined by light microscopy. In addition, the heads from all fetuses in the group pair-fed to the 25 mg/m group were processed by the method described for Trial I. For the gavage route, the heads of all fetuses examined for visceral alterations and sufficient of the remainder to total two-thirds of each litter were fixed in Bouin's fluid. Examination of 2 of the fixed fetal heads of each litter included slicing through the center of each eye in vertical cross-section^ The heads of 3 additional fetuses from each litter were not cut through the eyes before being processed to permit examination by light microscopy. All histologic specimens were coded for examination, and particular emphasis was placed upon the structural integrity of the lens. All fetuses, except for the heads of those that were fixed in Bouin's fluid, were fixed in 70% ethanol, eviscerated (if not done previously), macerated in 1% aqueous KOH solution, and stained with alizarin red S to permit examination for skeletal alterations. For Experiment II(Dams allowed to litter), the procedures used until Day 21G were the same as for Experiment ' " co.np.n,^"-"0*""-78""' I(Teratology), except that the dams were weighed on Days 1, 6, an 21G (and twice between Days 9 and 16G, for the gavage portion), feed consumption was not measured, and the identity of each offspring within litters was not retained. At least 2 days before expected parturition, each dam was housed in a 33 x 38 cm polycarbonate cage outfitted with a water bottle, and a wire-mesh lid. The bedding (Bed-0-Cobs; 1/4" size) was changed weekly. The date of parturition was noted, and it was termed Day 1 PP. The dams were weighed and examined for clinical signs on Days 1, 7, 14, and 22PP. sacrificed on Day 23PP. All dams were The pups from each dam were counted, weighed, and examined for external alterations toward the end of Day 1PP. Pups with external alterations were marked for subsequent identification. Thereafter, each pup was weighed and inspected for adverse clinical signs on Days 4, 1, 14, and 22PP. Neither standardization of litters nor cross-fostering was practiced. The eyes of the pups from Experiment II were examined by an ophthalmologist between Days 15 and 17PP (inhalation-Trial 1) or between Days 27 and 31PP (gavage). All pups were sacrificed on Day 35PP. The litter was used as the experimental unit for the purpose of statistical evaluation (Staples and Haseman, 1974; Haseman and Hogan, 1975). The significance of differences in the incidence of pregnancy, clinical signs, and maternal death - 13 - Company San-^zed,,. Dr--^5i-.ct contain TSCACBl was determined by use of Fisher's exact probability test (Siegel, 1956). A two-way analysis of variance was used to detect differences in feed consumption among breeding lots and between groups. Dunnett's test (Steel and Torrie, 1960) was used to test the statistical significance of differences between the control and APFO groups in maternal body weight, in body weight gain, and in feed consumption when the one-way analysis of variance was significant. The presence of concentration-related responses for the inhalation portion was determined by Jonckheere's test (Jonckheere, 1954). The significance of differences in incidence of structural alterations between the control group and the APFO group was determined by application of the Mann-Whitney U test (Mann and Whitney, 1947). When more than 75% ties occurred in the data, the Fisher's exact probability test was applied (Haseman and Hoel, 1974) The level of significance selected was p<0.05. Variability about means was expressed as Standard Error of the Mean (S.E.M.) unless stated otherwise. In addition, several reproductive indices were calculated for some results from Experiment II(Dams allowed to litter). 'CompaSnaynitized. Does not contain TSCA CB1 RESULTS A. inhalation Route The exposure levels of APFO achieved (+S.D.), as measured gravimetrically, for nominal exposure concentrations of 0, 0.1, 1, 10, and 25 ing/in3 were 0, 0.13+0.020, 1.1+0.16, 10+5.4 and 21+9.7 mg/m , respectively. Spectrophotometric data were virtually identical. For both trials (Table 2) between 77 and 90% of the atmospheric particulate was <10 m; mass median diameters of aerodynamic particles ranged from 1.4 to 3.4 m. The average mean.daily temperatures in the chambers were between 24.5 and 25C, and individual temperatures recorded ranged between 22.5 and 26C. 1. Experiment I(Teratology) Clinical signs that were concentration-related appeared only in the dams of the 10 and 25 mg/m groups. Most of the dams developed wet abdomens, which began in the perineal area, had chromodacryorrhea and chromorhinorrhea, and were unkempt. In addition, 4 of the dams in the 25 mg/m group that survived to Day 21G became very lethargic toward the end of the exposure period. No adverse clinical signs were noted among the dams of the pair-fed control groups. - 15 - Company Sanitized. Does not contain TSCA CBi Feed consumption of the dams in the 10 and 25 ing/in groups from Days 6-15G was significantly less than that for the control group (21.8^0.46 g vs. 23.4^0.38 g, respectively); no significant differences existed between the consumption of the pair-fed groups and the APFO exposed groups to which they were matched. Similarly, the body weight gain of the dams in the 25 mg/m group from Days 6-15G was significantly less than that for the control group, but the difference for the 10 mg/m was not statistically significant (Table 3). On Day 21G, actual liver weight of the dams in the 25 ing/in group was significantly increased above the control value (Table 3). The liver weights of the control groups that were pair-fed to the 10 and 25 mg/m groups were significantly less than those for the APFO groups to which they were paired, and than that for the control group (Table 3). On a relative weight basis (using corrected Day 21G maternal body weights), the liver weights (x ^ S.E.M.) for the groups exposed to APFO at 10 and 25 mg/m (5.42 + 0.125, and 6.46 + 0.222, respectively) were still significantly larger (MWU - two-tailed) than that for their respective pair-fed control groups (4.58 + 0.164, and 4.62 ^ 0.103). The maintenance of pregnancy and the incidence of resorptions among the surviving dams were not adversely - 16 Company Sanitized. Does not contain TSCA CBl affected by exposure to APFO at concentrations up to and including 25 mg/m (Table 3). The mean fetal body weight in the 25 mg/m group was significantly (p = 0.002) decreased (Table 3); but, this also was the case (p = 0.001) for the control group pair-fed to the 25 mg/m group. The mean weight of the fetuses in the 10 mg/m group, and in its pair-fed control group, were not significantly different from the control group (p ^0.23). Neither coded stereoscopic and light microscopic examination of fetal eyes from heads that were fixed in Bouin's fluid, nor detailed examination of the remainder of the fetuses, revealed a concentration-related increase in the incidence of fetuses with malformations or variations (Table 4). In the control group that was pair-fed to the 25 mg/m group, the incidence of fetuses with partially ossified sternebrae was significantly increased (p = 0.04 by the two-tailed MWU test), as was the incidence of those with variations regarded as being due to retarded development (p = 0.02) vs. the control value. In the 25 mg/m group the incidence of fetuses with partially ossified sternebrae also was increased, but the difference from the control value was statistically significant only if the one-tailed MWU test was employed. - 17 - Company Sanitized. Does not contain TSCA CB( 2. Experiment 11(Dams Allowed to Litter) During the prenatal period, clinical signs that were concentration-related appeared only in the 10 and 25 mg/m groups; they were similar in type and incidence to those seen in Experiment I. In the prenatal period maternal body weight gain of the 25 mg/m group was less than that for the control group (Table 5), but the difference was not statistically significant (p>0.05). No adver'se concentration-related effect on reproductive performance was demonstrated among the does that survived to term, but the weight of the neonates from the 25 mg/m group was significantly less (p = 0.02) than the control value (Table 5). By Day 4PP, the difference -was no longer statistically significant. Coded external examination of all of the postpartum pups in Experiment II-Trials I and II, and ophthalmoscopic examination of the eyes of those in Teratology IT-Trial I did not reveal concentration-related alterations. No further eye examinations were conducted in view of these negative results and those for "Experiment I-Teratology," which included light microscopic examination of fetal eyes in the group exposed to APFO at 25 mg/m . ~ 18 ~ eSoanitized, Duooie-s not contain TSCACBi Company B. Gavage Route Five of the 14 suspensions of APFO in corn oil prepared during the study were analyzed. Calculations based upon fluoride ion measurement indicated that the APFO content of individual suspensions ranged from 2.04 to 3.14%; an APFO content of 2.13% was expected. 1. Experiment I(Teratology) TtTree of tlie 25 dams given APFO died as opposed to 0 of the 25 dams given only corn oil (Table 3). Those that died had the clinical signs described earlier under "Inhalation." During the dosing period the APFO group consumed significantly less feed than the control group (17.2 _^ 0.37 vs. 21.9 _H).48 g, respectively), and gained about one-third less body weight (p<0.05). Those that survived to Day 21G remained pregnant, the incidence of resorptions was not adversely affected, and mean .fetal weight was not significantly different between the 2 groups (Table 3). No malformations were detected among the fetuses of the dams given APFO by gavage and the overall incidence of variations was not significantly different from the control value (Table 4)- Neither malformations nor variations were revealed by stereoscopic examination of the bisected eyes from 2 fetal heads per litter (Bouin's - 19 - TSCACB H n^ oes not contain Sanded. Company fixed) from each group, or by light microscopic examination of the eyes of an additional 3 fetuses per litter per group. Microscopically, lesions of the fetal lens were not observed. 2. Experiment II(Dams Allowed to Litter) Again, 3 of the dams in the APFO group died, but the reproductive criteria studied were not adversely affected among the dams that survived to term (Table 5). Neither external examination of the neonates nor in vivo examination of their eyes between Days 27 and 31PP demonstrated adverse effects related to APFO administration. DISCUSSION APFO-related teratogenicity was not demonstrated in this study after administration of the fluoropolymer to rats, by gavage or by inhalation, throughout the period of major .organogenesis even at exposure levels that included those lethal to some of the dams. Embryo or fetal toxicity, expressed as decreased fetal weight, was demonstrated, but only after inhalation of APFO at 25 mg/m which was the highest exposure level tested. This probably was due to decreased maternal feed consumption rather than to a. direct response to - 20 - Company Sanltizsrf. Does -" ""^ wa ^ APFO, since the fetuses of the control group pair-fed to the 25 mg/m group also were significantly smaller than the control fetuses. Results from additional APFO-exposed dams revealed that the significant difference was temporary, since it did not persist to Day 4PP. The types of lens changes previously reported to the EPA by the manufacturer of APFO were detected in several fetuses. However, they were determined not to be related to APFO administration because they occurred at similar incidences among all groups including the control group. Lens clefts were determined to be postmortem artifacts that were caused by cutting through the center of the eyes of Bouin's fixed coronal sections. When the fetal eyes were bisected, the fetal nucleus of the lens (which is normally shifted anteriorally at this stage of gestation) was torn from its loose attachment to the anterior lens capsule and a void (cleft) was formed. The cleft was surrounded anteriorally by the lens capsule, laterally by lens' sutures, and posfceriorally by the anterior surface of the fetal nucleus. This artifact was essentially eliminated by processing Bouin's fixed fetal heads that were trimmed on either side of the orbit, instead of through the center of the eye. Examination of the eyes of offspring using focal illumination, indirect ophthalmoscopy, and, when indicated, slitlamp microscopy also did not detect APFO-related alterations. Company Sanitize Do^con^TSCACBS Therefore, the results obtained in this study did not confirm the teratogenicity of APFO in the rat as previously reported to the U.S. Environmental Protection Agency (EPA) under Section 8(e) of the Toxic Substances Control Act (TSCA) , On the basis of the results of this study and additional studies conducted by Du Font and the manufacturer of APFO, female employees were permitted to return to their original workplace on a voluntary basis. ACKNOWLEDGMENT The authors wish to acknowledge the following departments and personnel who participated in this study: the Analytical Chemistry Section (CR&D), James M. Clinton (V.M.D.), Joseph C. Hamill, Joan A. Wolfe, Alice E. Parks, Carol L. Lamontia, and Blaine C. McKusick (Ph.D.). - 22 - Company Sanitize. Does not contain TSCA CBl FOOTNOTES Present address: Smith, Kline and French Laboratories, 1500 Spring Garden Street, L60, P. 0. Box 7929, Philadelphia, PA (19101). Octanoic acid, pentadecafluoro-, ammonium salt; ammonium Section 8(e) #373 and #374 on November 19, 1980; Section 8(e) 1394 on March 20, 1981. 5 3M, 3M Center, St. Paul, Minnesota (55144). CAS Registry Number 8001-30-7; Item 13266, Lot No. Eastman Kodak Company, Rochester, New York. D4-45, 7 Charles River Breeding Laboratories, Inc., North Wilmington, Massachusetts (01887). " ^^y <^iz.d. Does not contain TSCA CBI p Purina Certified Rodent Chow 5002, Checkers, Ralston Purina Company, Checkerboard Square, St. Louis, MO (63188). Q Atropisol, NDC 0058-0705-15, Cooper Laboratories (P.R.), Inc., San German, P.R. 00753 U.S.A. 10 James M. Clinton, V.M.D., 300 Brookmead Drive, Cherry Hill, New Jersey (08034). - 24 - Company Sanitized. Does not contain TSCA CB( REFERENCES Barrow, M.V., Taylor, W.J. (1969). A rapid method for detecting malformations in rat fetuses. J. Morph., 127(3), 291-306. Bock, R. (1979). Decomposition Methods in Analytical Chemistry, International Textbook Co., Ltd., London, pp. 185-186. Griffith, F.D., Long, J.E. (1980). Animal toxicity studies with ammonium perfluorooctanoate. Am. Ind. Hyg. Association, 41(8), 576-583. Haseman, J.K., Hoel, D.G. (1974). Tables of Gehan's generalized Wilcoxon test with fixed point censoring. J_ Statist. Comput. Simul., 3, 117-135. Haseman, J.K., Hogan, M.D. (1975). Selection of the experimental unit in teratology studies. Teratology, 12, 165-172. Jonckheere, A.R. (1954). A distribution-free K-sample test against ordered alternatives. Biometrika, 41, 133-145. Mann, H.G. , Whitney, D.R. (1947). On a test of whether one or two random variables is stochastically larger than the other. Ann. Math. Stat., 18, 50-60. Percival, L.F.. (1968). Determination of Cn and Cg Dispersing Agents, Methylene Blue Method. Washington Works Technical Library, E. I. du Font de Nemours & Company. - 25 Company Sanitized. Doss not contain TSCA C3l Salewski, E. (1964). Farbemethode zum makroskopischen Nachweis von Implantztionsstellen am Uterus der Ratte. Archiv. Path. Exp. Pharmakol., 247, 367. Siegel, S. (1956). Nonparametrie Statistics for the Behavioral Sciences, McGraw-Hill, Mew York, pp. 96-104. Staples, R.E. (1974). Detection of visceral alterations in mammalian fetuses. Teratology, 9, A3 7. Staples, R.E., Haseman, J.K. (1974). Selection of appropriate experimental units in teratology..Teratology, 9, 259-260. Steel, R.G.D., Tbrrie, H.H. (1960). Principles and Procedures of Statistics, McGraw-Hill, New York, pp. 99-128. Williams, W.J. (1979). Handbook of Anion Determination, Butfcerworth & Co, Inc., Boston, pp. 349-350. - 26 - Company Sanitized. Doss r.oi contain T3CA CBS Route Inhalation0 TRIAL I Exposure bevels (o>e/m3) 0 0.1 1 25 TABLE 1 , Experimental Deaifin Number Mated Females/Group Experiment Ifferatolqgy) . Experiment II (Dams Allowed 12 12 12 12 12 12 12 12 Inhalation0 TRIAL II 0 12 6 0.1 12 1 12 10 15 6 PF10" 6 PI^6 6 Uavoge 0 25 12 100 nig/kg 25 12 All Experiment I females sacrificed on Day 21 of gestation ^11 Experiment II females were sacrificed on Day 23 postparcua} the offiipring were sacrificed on Day 35 postparci 6 hr/day from Days 6 through 15 of gestation Control group pair-fed to group exposed to^^^^Hat 10 ing/in E ^^^^---- 3 Control group pair-fed Co group exposed to^^^^Rat 25 nig/in Vehicle used waa 5 mL corn oil/kg body weight/day from Days 6 through IS of gestation TABLE 2 Inhalation Route; Particle Size Data for APFO Aerodynamic Dust In Exposure Chambers T1HAL ; TRIAL II Exposure 1 10 :.Has a Median Diameter (Um) 1.4 2.8 3.ill Geometric Standard Deviation '1.5 6.0 A.3 Z Resplrable Particles {<,l0 \u 90 88 77 eohlpany 8ar TABLE 3 Reproduction and Fecal Development in Rats Exposed Co APFO by Inhalation or by Gavage from Days 6-15 of G Experiment I (Teratology) 0 Females p No. pregnant: / no, mated No. ileatlis 23/24 0 No. liLtera 23 Mean no. corpora lutea Mean no. Implants Mean liver weight(g)"'" 14.5 t 0.^ 14.0 1 0.51 15.2 t 0.30 Mean maternal weight galn(g) naya 6-15 57.6 t 1.69 Days 16-21 71.4 t 2.19 Pays 6-21^ 56.7 t 1.90 Fetal Death Mean % resorptlona per Jam 6.6 1 1.02 Inh station* (mg/B }) Pair--Fed" 0.1 1 10 25 10 25 24/24 23/24 15715 8/12 6/6 5/6 0 0 0 3 0 0 24 23 15 7 6 5 14.3 t 0.51 13.4 0.41 15.0 1 0.30 .15.6 t 0.58 13.8 0.28 15.4 t 0.35 15.2 t 0.65 14.2 t 0.22 16.1 1 0.50 15.1 t 1.42 14.0 t 0.62 18.0 t 0,781 13.8 t 0.4B 14.0 t 0.63 12.8 t 0.501 Id.4 1.29 14.0 t 1.10 12.7 t 0.481 57.6 t 2.06 68.9 1 1.92 56.9 1 2.02 56.7 t 1.95 72.8 t 1.97 56.4 t 2.11 50.9 1 2.21 72.9 t 2.77 49.1 1 2.52 36.4 1 5.333 68.6 t 3.41 37.4 t 5.39'1 34.9 t 5.31-7 71.3 t 4.72 34.3 t 2.M'1 22.7 6.93'1 68.9 1 2.73 27.0 t 6.01'1 5.3 1.34 3.8 1 0,87'' 5.2 1.47 5.9 I 3.50 4.1 i 2.94 5.3 t 3.54 Company S TADLE 3 (Coat.) Reproduction and Fetal Development In Kata Exposed to APFO by Inhalation or by Gavaga from Days 6-15 of Inhalation* (ing/m3) Pair-Fed" (Teratology) 0 0.1 1 10 25 10 25 Fetuses No. live 299 305 305 202 92 80 66 Mean no. live 13.0 t O./i7 12.7 t 0,39 13.3 t 0.28 13,5 t 0.34 13.1 t 0.70 13.3 t 0.21 13.2 t 0.97 ^^-- 0 0 0 0 No. stunted Mean weighing)11'" I 4.0 t 0.04 3.9 t 0.05 3.9 * 0.04 3.9 t 0.07 1 3.6 t 0.123 0 3.9 t 0.12 3.5 t 0.09'1 Nominai concentratJonB ^UHi^U 11 if On each day of gestation, non-eitpuaed dams were given the amount of feed consumed on the Bnmo gestation day by selected rata exposucii group; the exposed rats were housed 2 per cage, then their average consumption for each day was the amount offere ' p i All females In numerator had visible sign of pregnancy evident at autuopay except for 1 in the 25 mBgg//nc)i group in wwhhich implant detected only by tunnionluiii sulflde staining; data from tills female were excluded from all other calculations "Died on Days 12, 13, or 17 of gestation; the first waa not necropaled. but the other two had reaorptions in utero '''One ili'eil on Day 11, and 2 more on Day 12 of gestation; not recorded whether pregnant ''X t S.E.M. 'Non-pregnant animals were excluded "Significant dose-related response detected by Joncklieere's teat (p<0.05) 1 Significantly different from control value (two-tailed Mann-Hhitney I) test, p<0.05) 'f Significantly different from control value by Dunnett's test (p<0,05) 1< Uay 21C body weight denotes the body weight of females excluding the products of conception (i.e.. Day 21 corrected body we L Significantly different from control valne (one-tailed Mann-Whitney U test, p^O.05) Mean fetal weight/litter; stunted fetuses were excluded icom^ TABLE 4 Fetal Alterations In Rats Exposed to APFO by Inhalation or by Gavage froi^ Days 6-15 of Gestation Experiment I (Teratology) * 0 Inh alatlon* (mg/Bl"') 0.1 1 10 25 Pair -Fed 10 25 No. Examined Ifet.uaes/} (tiers) External Visceral. lleail & Eyes Skeleton 299/23 159/23 90/17 299/23 305 / 24 161 IU 1/1 305/24 305/23 161/23 0 305/23 202/15 110/15 19/6 202/15 92/7 5:1/7 5.1/7 92/7 80/6 42/6 C 80/6 66/5 34/5 34/5 66/5 Total with ,, Variations Avg. X Fetuses with Variations/ Litter (t S.E.M.) 144/22 <l8.7 t 5.07 14.0/24 123/23 87/15 55/6 49.4 t 4.42 40.1 t 3.43 42.8 t 5.14 58.4 5.98 34/6 42.2 t 5.91 42/5 64.5 t 10.42 Total with Malformations 2/2 3/3 1/1 1/1 E G 1/1 Avg. 7. Malformed Fetuses/Litter (t S.E.M.) 0.6 t 0.44 1.0 t 0.53 0.4 t 0.40 0.4 0,41 1.4 1.42 ^^^ Nominal concentrations ofAHIBR) If "11On each day of geatatlon. non-expoaed dama Mere exposure group, the exposed rats were hoiiaed (to fetusea examined . given 2 per - the amount cage, then - of th f^ ee eir d . a co ver n a sum ge . ed co on nsu t m - he pti o sa n me fo r .gesta each . .tion day . .day by Has the s . . . . . elected rats amount offere .In d .t Does not Include variations present in malformed fetuses. If present !; No malformed fetuses detected Company Sanitiz TABLE 5 Re product ton and Di^yeicipmeht: of Offspl:tng of RaCB Exposed to APFO by Inhalation or by Gavage from Rays 6-15 o Experiment- II (Dams Allowed to Litter) Females No. pregnant/no, mated No. deaths No. llttera Mean maternal weight galn(g) Days 6-21^ Days 1 PP-22 PP0 Offspring At delivery (Day 1 PP) -no. live pups/litter -no. dead pups (X) After delivery (co Day 22 PP) -no. -no. -no. -no, dead cannibalized live males/females 0 18/10 0 18 112 t 4.3'' -3.4 t 0.58 12.3 1 0.60 1(0.4) 1 0 2211 115/106 Inhalation* (mg/ni ) 0.1 1 10 10/12 11/12 6/6 10 11 6 116 t 8.1 -2.8 0.75 116 t. 7.8 -2.0 A 0.81 119 3.3 -3.1 t 0.78 12.1 1 0.80 1(0.8) 1" 2 118 62/56 11.2 1: 1,27 2(1.6) i 0 0 123 70/53 1:1.3 t 0.67 0 0 80 44/36 25 9B/12 2 9 Gavage (m 0 12/12 12 97 1 8.8 -2.2 t 0.52 129 4.2 9.6 t 4.28 11.0 t 1.26 1(1.0) 12.8 t 0.60 02 97 45/52 01 152 76/76 1 eonipan^Sanit^e^. TABLB 5 (Cont.) Reproduction and Development; of Ofliapring of Rats Exposed to APFO by Inhalation or by Gavage from Days 6-15 o Experiment 11 (Dams Allowed to Litter) Viability Index W3 Lactation Index (X)" Mean Weight (g) Day 1PP1' Day 4PP Day 22PP 0 100 99.5 0.1 99.2 98.3 Inhalation* (ins/in3) 1 10 100 100 100 100 . , 25 99.2 99.1 Gavage (m 0 100 99.5 6.8 ^ 0.11 l0.3t0.25 50.1 t 1,82 7.0 t 0.18 10.9t0.32 51.4 t 1.73 6.7 * 0.18 10.9*0.39 52.0 t 2.46 <6.6 t 0.2? 9,9 t 0.43 48.4 * 2.15 6.1 t 0.15" 9.70.33 49.0 t 0.88 6.9 t 0.12 10.410.3 0 49.5 t 1.6 8 Alterations No. exnroined externally -no. malformed No. eyea (pairs) examined in vivo1' No. with alterationa ,, 222/18" 141/12 3/31' 121/1Q u 1/1 118/10 ^/2Q 123/11 123/11 80/6 99/9 97/9 152/12 152/12 I" ., .'-.'- ',/^'^ /: :-..' :;^i^i^:.;.^:;;- ).' . '.l,::;;;ii...;?^-:.:"^,'so?l"paiw;Sda> e TABLE 5 (Cont.) . . . . ,, - , *, Nominal concentrations ofplBDMUl .---- B"D.. oes not Include 2 females that-did not survive the exposure period, but they had Implants in utero at necropsy Pregnancy atacua not determined for the 3 females that did not survive to scheduled sacrifice Blanks Indicate zero Incidence G gestation ''Mean t S.E.M. ' f1 PP postpartum ' ' "Pup with abnormal gait and domed head sacrificed on Day 20 PP. 11; was hydrocephalic lpup sacrificed as control for pup from 0,1 nig/in group ''X of pups born that survived to Day 4PP or longer v'?. of pups alive on Day W that survived to Day 22PP ''Significant exposure-related response detected by Jonckheere's teat (p^O, 05) tor the Inhalation route "Significantly different from control value (two-tailed Hann-Htiltney U test, p^O.05) ^upa/littera "Examined on Days 15-17PP the eyes of all live pups from Experiment II, Trial I, and on Daya 27-31PP all from fem Opacities of posterior lens pole ^ with pre-retinal hemorrhage and 1 with incomplete roydrlasiB and red oval R! male pup had a band of focal retinal degeneration ventral to the disc in s! female pup had corneal edema with superficial vascularizatlon present in opacity on the central endothellum the right eye the temporal quadrant of the left ey Gocnpany ^ani