Document omo3BEj9gv136MaXpw2pzee5R

AR226-2773 ^ ^-^ Bio/dynamicsInc. Division of Biology and Safety Evaluation PROJECT NO. 81-2571 EMBRYOFETAL TOXICITY AND TERATOGENICITY STIinY IBYINHALATION IN THE RAT Final Report Submitted to: E. I. DuPont neNemours Wilmlngton, ne1aware 19898 Attention: Date; Dr. H.M. Solomon December 31 1981 tSkanpanSyanitized. Does net contain TSCA CB) Bio/aynamics Inc. PROJECT NO. 81-2571 EMBRYOFETAL TOXICITY AND TERATOGENICITY STUDY OFIPERFLUOROBIJTYLETHEME (PFBE)JINBHAYLATION IN THE RAT ABSTRACT o'^hBBBBlHHL to^^jat This pilot study was conducted for E.I. DuPont nejtemours and Company foevaTuate the" eniBryatoxic and/or terartogeni c potentt<)1 I'BHft1'1 the pregnant rat. Mated cng female rats (seven females/group) were exposed by chanber Inhalation dose levels of 1,000 and 70,000 ppm. Animals were exposed for six hours/day during the Day 6-15 gestation interval. Included In the study was a sham-air, chamber-exposed control group (seven mated females). All females were sacrificed on Day 21 of gestation and fetuses recovered at this time were weighed, sexed and evaluated for external, soft tissue and/or skeletal malformations. Fetuses processed for skeletal evaluation were also examined for the presence of ossification variations. No maternal mortality was encounte ed during the study and all females, control and treated groups were pregnant. No treatment-related effect was evident in body weight or food con sumption data for the low-dose group. At the high-dose level, mean body weight gain for the Day 6-16 gestation interval (exposure period) was significantly lower than control. Mean food consumption data for the high-dose group was lower than control for the exposure (Day 6-16) and post-exposure (Day 16-Z1) Intervals; however, only during the exposure period was the difference from control data statistically significant. No adverse maternal effects were seen In physical in-life examination data, liver weight data or gross postmortem examination data. Likewise, corpora lutea and uterine Implantation data, fetal weight and sexing data and ossifica tion variation data did not appear to be adversely affected by treatment. No increase In malformation rate was seen in the treated groups during the fetal external or skeletal evaluations. During the soft tissue evaluations, a slight Increase In the incidence of fetuses with malformations was seen only In the low-dose group. The most co,.nnon observation noted during this evaluation Involved distentlon and/or tortuous condition of the ureter. Similar type observations were seen In the concurrent control group with a lower Incidence, and are noted frequently in our historical data for this strain of rat. In that a similar response was not noted at the higher exposure level, no treatment-related effect was Indicsi d. Company Sanitized. Does not contain TSCA C8J P.O. Box 43 East Millstone. New Jersey 08873 (201)873.2550 Telex: 844597 Cable: B10/DYN d) ntiinli'B Jn< 81-2571 TABLE OP CONTENTS I. INTRODUCTION..................................................... 1 II. MATERIALS........................................................ 1 A. Test Substance............................................... 1 1 B. Test Animals................................................. III. METHODS A. Husbandry.................................................... 2 3 B. C. MAssaigtnimnegnt..to..................................................... -3 D. Identification GofroAunpism*a..l.s......................................................................... 3 E. F. TEexspterSimubesn^tainl ceOutline......................................... Administration................................ 3 3 IV. EVALUATIONS A. In-Life...................................................... 4 4 B. 1P. osStamcriofircteeMmet.h.o.d...a.n.d..M..a.t.e.r.n.a.l...P.o.s.t.m..o.r.te.m...P..r.o..c.e.d..u..r.e............. 4 2. 3. EExxaammiinnaattiioonn poff the Reproductive System................... 5 5 C. Statistical Fetuses................................ Evaluations...................................... 6 V. RESULTS AND DISCUSSION A. 1M.ateMrnoarl taDlitayta................................................ 7 7 2. Pregnancy Rate........................................... Rates.......................................... 7 3. Body Food CWoenigsuhmt pntioanta......................................... 7 8 4. 5. Physical In-Life DObasetrav.a.tion.Da.t.a............................................ 9 6. Reproduction Data --Corpora Lutea and Uterine 7. Implantation Liver Weight Data....................................... 9 11 8. Gross PostmortDematOab.s..e.r..v.a..t.io..n..s..................................................... 11 B. 1F.etalFeDtala tBaod.y. .W. .e.ig. .h.t . .a.n.d. .S.e.x. .D. .is.t.r.i.b.u.t.i.o.n. . .D. .a. .t.a............ ............... 11 11 2. 3. Fetal Fetal ESxotfet rnTaislsuEexaEnx'-aimatiinoantioDn ata.......................... 13 13 4. Ossification Variation Data....................... Data*...*........................ 15 5. Fetal Skeletal Examination Data.......................... 16 CompanySanitized. Does not contain TSCA CM Rto -m-25y/idynamics Jnc TABLE OF CONTENTS (CONT.) TABLES 1. Mean Maternal Body Weight Data - fiestation................... 7 2. 3. MCoearnpoFraoodLuCteoansaunmdptUiotneriDneatImap.l.a.n..ta.t.i.o.n.......................... 8 10 4. Liver Weight Data.................. Data............................................ 11 5. Mean Fetal Body Weights and Fetal Sex Distribution Data...... 12 6. Fetal Soft Tissue Evaluation - Summary of 7. M Fe ent tal o r-mat Skel ion eta l DEaxatmai.n.a.t.i o..n. . .- . .S.u.m. moa.r.y . .o.f. .M..a. l f o. r.n. a. .ti.o.n. . D..a. t.a........ 14 16 APPENDICES BA.. ISntdaitviisdtuicaal l FAemnalae lByosdyisW.e.i.g.h.t..a.n.d...B.o.d.y..W..e.i.g.h.ot .C.h.a.n.g.e...D.a.t.a..-... -A-1 C. InGdievisdtuaaltiFoemna.l.e..R.e..p.r.o.d.u.c.t.i.o.n................................... B-1 C-l D. Individual Female Liver Weight Data.......................... Data.......................... 0-1 E. F. Individual Female Food Adult Gross Postmortem Consumptli-.i Data...................... E-1 F-1 Observations.......................... G. H. Summary of Fetal Soft Fetal External Examination Tissue Examination Data................... G-1 H-1 I. Fetal Skeletal Examination Data........................... nata.............................. 1-1 J. Fetal Ossification Variation Data Summarized................. )-1 K. Inhalation Report............................................ K-1 L. M. QPuearlistyoAnsnsueraln.c.e.................................................. L-1 M-1 Statement.................................. Company Sanitized. Does not contain TSCA CBI I. INTRODUCTION: ^^^^c--- -1- 81-25^10 ydytiaintcb Inc Revised: ^ ^. - -. Date: //j/^ This pilot study, conducted for E.I. DuPont DeNemours and Company, was designed to determine the fceratogenic potential and/or degree of enibryotoxi- city ^nUHI^BlH^flfl'n the pregnant rat. Test material was admini stered by chamber inhalation to mated rats (seven females/group) at dose levels of 1,000 end 70,000 ppm. Included in the study was a chambered, sham-air control group. Species and strain of test animal, dose levels and route of test substance administration were established by the sponsor. Testing was conducted at Bio/dynamics, Inc., Mettle rs Road, East MillFt.one, Mew Jersey 08B73. -Raw data to include fetal specimens were sent to sponsor for storage. II. MATERIALS: A. Test Substance: Supplier: Lot No.: Description: Date Received: Amount Rece"' ?d; E. I. OuPont DeNemours Uilmington, Delaware 19898 CB----Q Hiasskkeell *14JOO Colorless liquid 6 July 1981 Container l?1: 24.900 kg Container j?2: 26.475 kg Container #3: 26.650 kg Container #4: 26.500 kg Sto-age: Room Temperature B. Test Animals: Species: Strain: Supplier: Rat (albino) Cr1:Cn (SO) BR Charles River Breeding Laboratories Inc. Wilmington, Massachusetts Conwwy^S'W^-"? H^nq > i*n>t?n "re/n ifin -2- 81-i?57T"y^,,^,...,,,.. B. Test Animals (cont.): Justification for Animal Selection: Number of Animals Purchased: Placed on Test: Date Received: Age at Initiation of Mat i ng: III. METHODS: A. Husbandry; Housing: Food: Water: Environmental Conditions: Generally recognized laboratory animal, This strain of albino rat was selected because this rat was used In recent acute and subacute testing. 5? females 21 females 30 July 1980 (56 days of .ge) Females - 69 days of age Males from an in-house mating colony of the same strain of rat were used .'or breeding purposes only. Females were housed individually (except during overnight mating inter vals) in stainless steel, suspended cages with wire mesh floors. Certified Purina Rodent Chow <?5002 - available ad libitum during the non-exposureTniervaYs. Tap water (Ellzabethtown Water Company) available via automatic watering system ad libitum during the non-exposure Tntervals. During the exposure Intervals, animals did not have access to either food or water. Temperature: 70 - 78F Humidity: 42 - 70% Photo period: 12 hours light/dark cycle. Gbmp^ny S&httt^. Dses tte^ontataTSCAcai (Ijo/ -M-2571 /clynaioiRte Jic . B. Mating: Females selected for mating were placed with male rats nightly In a 1:1 ratio. Vaginal smears were taken early in the morning and females were considered mated if sperm were observed microscopically in the vaginal rinse. The day evidence of mating was observed was designated Day 1 of gestation. Dates of Mating: 11 August to 15 August 1981. C. Assignment to Groups: Females which mated were assigned to groups daily in such a way as to most nearly equalize the Day 1 body weights. D. IdentKicationof.Anjjnals: Each. mated fern? Ie was identified with a metal eartag bearing it's animal number. The individual animal number plus the Rio/dynamics project number comprised a unique identification number for each female. E. Experinental Outline: Group No. Test Substance3 Dose Level TOT" ITNo. of Females ,TCf ^crificed Day 21 of Gestation Treatment Schedule6 ^Gestation Days) I Sham-air 0 7 7 (Chambar- 1g^ " in l Control) 1.00D 7 7 70.00D 7 7 ""CffaBi i.er F. Inhalation ^- 6 ^ours/day. Test Substance Administration: Route: Chamber Inhalation 6-15 6-15 6-15 Treatment Schedule: Mated females were exposed daily, 6 hours/day from Day 6 to Day 15 of gestation, a 10-day treatment period. Chamber Operation and Atmospheric Monitoring: See Appendix K. CompanySanHizeA Doesnotwiflgin-RCACfll IV. EVALUATIONS: A. In-life: Gross Physical Observations; Detailed Physical Examinations: Body eights; -^ HI"/t/l> UmillC't Jnr. Bl-^b/1 Twice dally (morning; afternoon) females were evaluated for obvious signs of phannacologic or toxicologic effects and mortality. Each female was given a detailed physi cal examination on Days 1, 5, 9 12, 15, 18 and 21 of gestation. During the treatment period, these examinations were performed In the afternoon following exposure. Females were weighed on Days 1, 6, 9. 13, 16 and 21 of gestation. Food Consumption: B. Postmortem; Food consumption was recorded during the exposure (Days 6-9. 9-13 and 13-16) and post-exposure (Days 16-21) periods.' Food spillage was weighed and recorded during the same Intervals that feeder jars were weighed. 1. Sacrifice Method and Maternal Postmortem Procedure: All females were sacrificed with an overdose of carbon dioxide on Day 21 of gestation. The abdominal and thoracic cavities were opened and each female was given a gross postmortem examination. The uterus was then removed Intact (to Include the ovaries) from the abdominal cavity and weighed. Likewise, the liver was dissected free and weighed for each female. Maternal Identifications were coded at time of sacrifice. Maternal sacrifices and subsequent fetal evaluations were performed blind in respect to dose group. ^t was intended that food consumption be recorded during the pre-exposure Interval (Days 1-3, 3-6 of gestation); however, due to technical error, these data w not recorded for either the control or treated groups. ^^wSS.^^^^^C8ft -5- 81-2571'"% ~~ /dynamic.. Inr '_____________________ B. Postmortem (cont.): 2. Examination of the Reproductive System; Ovaries - each ovary was dissected free of the uterus, cleaned of connective tissues and evaluated for the presence and number of corpora lutea. Uterus - the number and relative location of the following were recorded for each uterine horn; Live fetuses Dead fetuses (dead fetus with no visible degeneration) Late resorptions (recognizable dead fetus undergoing degeneration regardless of size) Early resorptions (evidence of Implantation but no recognizable fetus) Implantation sites (represents the sum total of fetuses [live and dead] and resorption sites [early and late]). Once fetuses have been removed from uterus and a11 implantation sites Identified, the placental/extra-embryonic tissues were removed and the uterus re-weighed (empty weight). Dates of Maternal Day 21 Sacrifices: 1 September to 4 September 1981 3. Examination of Fetuses: Upon removal from the uterus, each fetus was given a gross exter nal examination for malformations, weighed, sexed and tagged for future Identification. Fetal Soft Tissue Examination: Approximately one-half of the fetuses In each litter were evalua ted for soft tissue malformations by the Staples microdlssectlon technique.2 Staples, R.E. (1974). Detection of Visceral Alteration In Mammalian Fetuses. Teratology. 9:A37 (abstract). ^mpanysanftfeedn^ ^"^^nrscAca, -6-_____________________81-257f"yd.vna.n)CH In.- B. Postmortem (cont.): 3. Examination of Fetuses (cont.): Fetuses so processed were decapitated and heads fixed In Bouins solution to be processed by a razor blade slicing technique (Wi1son3). At completion of the soft tissue evaluation, the decapitated, eviscerated fetal specimens were processed for staining of the skeletal structures using an Alizarian Red S staining procedure.4 Stained fetal specimens were subsequently evaluated under a dissecting microscope for malformations and ossification variations. Fetal Skeletal Examinations: The remaining fetuses in each Utter were eviscerated (external sexing data confirmed by internal inspection of the gonads) and processed for staining of the skeletal structures using an Alizarian Red S staining procedure.4 Stained fetal specimens were evaluated under a dissecting microscope for skeletal malformations and ossification variations. C. Statistical Evaluations: Data were compared between the control and each treated group. Statistical evaluations used for this study are discussed in Appendix A. teratology, J.G. wilson and J. Warkany, eds. (The University of Chicago Press, 1965) pp. 267-277. ^.U. Crary, Stain Technology, 37 (1962) pp. 124-125. Company Sanitized. Does not contain TSCA CBI m"l/id.vDainlCh )n- V. RESULTS AND DISCUSSION; A. Maternal Data: 1. Mortality Rate: No mortality occurred In the control or treated groups. 2. Pregnancy Rates: Pregnancy rates were 100( In eacf 'f the control and treated groups. All females (control and treated groups) were pregnant. 3. Body Weight Data: Mean maternal body weights and weight gain data for the control and treated groups are presented In Table 1. These data are summarized from,data for Individual females presented In Appendix B. Table 1 - Mean Maternal Body Weight Data - Gestation J l Group ____Mean Body Weight (grams) Gestat<on bays TppmT __ .1. _L ^ ^ ---- 1-fi Actual Corr.8 I 201 224 235 256 275 337 274 23 (0) II 204 230 239 257 275 338 (1,000) 277 26 III 206 234* 237 253 270 338 (70.000) 277 29 Mean Weight Gain (grams) Gestation Days 6-16 16-21 6-21 Actual Corr.8 Corr.* 51 62 -1 50 45 63 3 47 36** 68 6 42 Difference from the control group statistically significant; *p<0.05; **p<0.01. ^orr. " Corrected nay 21 body weight * final Day 21 body weight""- -- gravid uterine weight + empty uterine weight. Mean body weight data were generally comparable between the control and each treated group throughout gestation; however, on Day 6 of gestation, mean body weight for the high-dose group was significantly higher than control. Company Sanitized. Does not contain TSCA CBI -8- 81-2MA/ ________________________________ /dynamics Inc. 3. Body Weight Data (cont.); Mean body weight gain during the several gestation Intervals were considered comparable between the control and low-dose group. In the high-dose group, mean weight gains were comparable to control during the pre-exposure (Days 1-6) and post-exposure Intervals (Days 16-21 using actual and corrected Day 21 weight data). During the exposure interval (Days 6-16), mean weight gain for the high-dose group was significantly lower than control, ^<ean weight gain for the high-dose group during the Day 6-21 interv-1 (using corrected Day 21 weight data) was also lower than control; however, this difference from control was not statistically significant. 4. Food Consumption Data: Mean food consumption data for the exposure and post-exposure gesta tion intervals are presented In Table 2. These data are summarized from indi vidual female data in Appendix E. Table 2 - Mean Food Consumption Data T W Group I (0) II (1,000) III (70.000) Mean Food Consumption (gram/ammal/day) Gestation Days 6-16------------------------16 - 21 21.6 25.6 20.4 24.8 18.7** 23.0 Difference from the cont. al group statistically significant: **p<0.01. /^- Company Sanitized. Does not contain TSCAC52 tllK/ -=5=------------------------------------------------------g^257/<l.M>a.,,l<i^m 4. Food Consumption Data (cont.): Mean food consumption data during the exposure period (Days 6-16) and post-exposure period (Rays 16-21) were considered comparable between the control and low-dose group. In the high-dose group, mean food consumption during the exposure interval (Day 6-16) was significantly loh^r than control values. During the Day 16-21 interval, mean food consumption for the high-dose group was lower than control; however, this difference was not statistically significant. 5. Physical In-life Observation Data: A similarity was seen between the control and treated groups in the types and incidences of observations noted during the detailed physical in-life evaluations. No treatment-related effect was evident. 6. Reproduction Data - Corpora Lutea and Uterine Implantation Data: Corpora lutea and uterine implantation data for the control and treated groups are presented in Table 3. These data are summarized from indi vidual female data In Appendix C. The mean numbers of corpora lutea. Implantations and fetuses (live and dead) were comparable between the control and each treated group. Likewise, the mean percentage of resorptions to Implants and the mean percentage of resorptions plus dead fetuses to Implants were comparable between these same groups. The one dead fetus seen during the study was recovered from the titter of low-dose female No. 2579. No treatment-related effect was evident in corpora lutea or uterine implantation data. Company Sanitized. Does not contain TSCA CS8 Hlo/ -=W-----------------------------------81^2571 /tjyiiaii)if;i iiit- Table 3 - Corpora Lutea and Uterine Implantation Data Parameter; No. Pregnant Females: Mean No./Pregnant Female - Corpora lutea: Implantations: Resorptlons Total No.: Mean/pregnant female: Mean % of Implants: No. of dams with 100% resorptlons: Live Fetuses Total No.; Mean/pregnant female: Dead Fetuses Total No.: Mean/pregnant female: Mean X fetal deaths per Implant: Mean % resorption and fetal deatns/Implant: I W 7 14.9 13.3 2 0.3 2.0 0 91 13.0 0 0.0 0.0 2.0 Group (ppm) II 11,000) 7 13.0 12.7 5 0.7 5.7 0 83 . 11.9 1 0.1 1.0 6.7 III (70,000) 7 15.0 12.6 3 0.4 3.2 0 85 12.1 0 0.0 0.0 3.2 No statistically significant differences from the control group. .Company Sanitized. Does not contain TSCA cm .U------------------------------------&1-257H1 it/.l/u.yvn"u"m""l<ctr Jnc 7. Liver Weight Data: Mean Hver weight data (absolute and relative to Day 21 corrected body weights) are summarized In Table 4. Liver weight data for individual females are presented in Appendix D. Table 4 - Liver Weight Data Group Tppm) Mean Liver Weight (grams)____ Mean LIv.-r/Body Weight Ratio3 _______(x 100)______ I 14.6 (0) 5.34 II 14.0 5.06 (1,000) HI (7U.OOU) 14.9 5.38 No statistically significant differences from the control group, ^sing the corrected Day 21 body weights. Mean liver weights, both absolute and relative to Day 21 corrected body weights, were comparable between the control and treated groups. 8. Gross Postmortem Observations: Observations noted during the gross postmortem evaluations of females sacrificed on Day 21 of gestation, are presented In Appendix F. r . No treatment-related effect was evident from these evaluations. B. Fetal Data: 1. Fetal Body Weight and Sex Distribution Data: Mean fetal body weight and fetal sex distribution data are presented In Table 5. These data are presented for Individual litters in Appendix C. ICompanySanitized. Does not contain TSCA CSS. Hinf/t,iynnwtfh Inr Table 5 - Mean Fetal Body Weights and Fetal Sex Distribution Data Group Tpprnf I (0) 11 (1,000) III (70,000) Mean Fetal Weight (grams) ------^---------- 3.49 3.65 3.57 Sex Distribution^6 ""Ho:------RaTT---- 40/51 0.8 46/37 1.2 48/37 1.3 Percent Male Fetuses 44.0 55.4 56.5 No statistically significant d1fferinces from the control group. 'Total Number of Male Fetuses/Total Number of Female Fetuses. "Data not analyzed statistically. excludes data for the one dead fetus recovered In this group. Mean fetal weight data were considered comparable between the control and treated groups. No stunted fetuses'* were recovered In either the control or treated groups. Likewise, no fetuses were calculated as being stunted6 In respects to Uttennates 1n these same groups. Fetal jex distribution data revealed a preponderance of males In each of the treated groups with an increase In number of females 1n the control group. The differences In sex distribution are attributed to normal biological variability noted with this parameter and the relatively small group sizes Involved. No treatment-related effect was evident In fetal body weight or fetal sexing data. JStuntedfetuses fetuses that weigh 1 gram or less. "Calculated stunted fetus (see Appendix A: page A-5). Company Sanitized. Does not contain TSCA CB& -13-_____________________________gl-^r/'.V""'"!'-'" !"< 2. Fetal External Examination Data: Observations noted during the fetal external examinations are summar ized In Appendix G. No external malformations were seen In the 84 low-dose fetuses (seven litters), or 85 high-dose fetuses (seven litters). In the control group, one fetus had a darkened, raised area on Its back (thoracic region). No other external malformations were seen in the 90 remaining fetuses from this group (seven litters). 3. Fetal Soft Tissue Examination Data: Observations noted during the fetal soft tissue evaluations are summarized in Appendix H. These malformation data are mmanzed in Table 6. Sanitized. Does not contain TSCACBl Company -I4-_______________________"l-^/dyn.,...^ inc. Table 6 - Fetal Soft Tissue Evaluation - Summary of Malformation Data Group T^W I (0) II (1,000) III (70,000) Observation Ureters - tortuous: unilateral: bilateral: Total affected: Ureters - tortuous: unilateral: bilateral: Ureters - tortuous and distended: unilateral: Liver - Sinai1! dark discolored area on ----m--edFiaontaIloabttee;c_te_d_: Ureters - tortuous and distended: unilateral: Ureters - tortuous: bilateral; ""Total affected: Fetuses Affected/ Total Examined -TC:------------T 1/48 Z.I 1/48 2.1 '2748' T7 2/43 4.7 2/43 4.7 2/43 1/43 uVS 4.7 2.3 75T7 1/44 .2.3 1/44 2.3 ZjVi T^ Litter Affected/ Total Examined "Ro:------------T ^1/7 1 / ' 14.3 1/7 14.3 'ZB3 2/7 28.6 1/7 14.3 2/7 28.6 1/7 "S77 14.3 7T77 1/7 14.3 1/7 14.3 T77 TO No statistically significant differences from the control group. The most common observations noted during the soft tissue evaluations Involved di'stentlon and/or the tortuous condition of the ureter(s). These types of observations were seen In two of 48 control fetuses (4.2(), six of 43 lowdose fetuses (14.P?) and two of 44 high-dose fetuses (4.5$). The significance of these types of ureter observations Is unclear. In these same fetuses with ureter observations, the bladders were noted as filled (Indicating a patency between ureter and bladder) and no kidney malformations (e.g., severely dis- eo^sa".^.^"0'"""'"TM*"' ^5.--_------------------------^-_^^/dynmnlfB liif tended renal pelvis, absence of renal papillae) were seen. These types of ureter observations (I.e., distended, tortuous condition) are seen frequently in the term rat fetuses; historically, the incidence of such ureter observations is 8.7( (representing 1067 fetuses from 100 litters). In that no dose relationship was apparent in the incidence of these ureter observations, no treatment-related effect Vias indicated. With the exception of ureter observations, the only other observation noted during the soft tissue evaluations was a small, dark, discolored area on the medium lobe of the liver. This observation was seen in one low-dose fetus. No treatment-related effect on the fetus was evident from data collected during the soft tissue evaluations. 4. Ossification Variation Data; The types of ossification variations seen during the skeletal examination and the Incidence of fetuses (litters) affected are presented in Appendix J. Ossification variations were seen in 87/91 control fetuses, 75/84 low-dose fetuses and 77/85 high-dose fetuses, an incidence of 95.6%, 89.3X and 90.62, respectively. These incidences were comparable between the control and treated groups. The mean percentage of fetuses per litter with ossification variations for the control, low- and high-dose group was 95.3%, 89.It and 91.0%, respectively. These data were comparable between the control and each treated group. The types and incidence, of ossification variations seen were similar between the control and treated groups. No treatment-related effect was evident in these data. Company Sanitized. Does not contain TSCACB? Ulii/ ^______________^,257r^""'""'''' J>" 5. Fetal Skeletal Examination Data: Observations noted during the fetal skeletal evaluations are summarized In Appendix I. Malformation data are summarized In Table 7. Table 7 - Fetal Skeletal Examination - Summary of Malformation Data ^ ^ . Group I (0) II (1,000) III (70,000) Malformation ---------------------- AnguTated Ribs and Shortened filbs: --------Total: Fused Sternebrae --------------Total: Fused Sternebrae and midllne cleft through the sternum: ------a------Total; Fetuses Affected/ Total Examined -US-'----------T" 1/91 1.1 "TM 17T 1/84 1.2 TTST 177 1/85 1.2 TTB? 177 Litter Affected/ Total Litters Evaluated 'V------~T 1/7 14.3 177 777? 10 1/7 T4.3 177 1/7 14.3 177 TO No statistically significant differences from the control group. ^Ho, of litters containing a malformed fetus divided by the number of litters evaluated. The Incidence of skeletal malformations, both on a per fetus and per litter basis, was comparable between the control and treated groups. In the control group, one fetus had an angulated/shortened rib defect. No othe' skeletal malformations were seen In this group. Fused Sternebrae were seen In two treated group fetuses. In the low-dose group, one fetus from the Utter of, female No. 2580 had a fusion between the first and second sterr-brae. The fused Sternebrae seen In one high. dose fetus, rom the litter of female No. 3578, Involved the second through the fifth stemebra. A mid-line cleft was also evident through this fused segment of the sternum. ^ Company Sanded. Does tin! con^n^ [-2Srj/dyt,anU' Jnr. The sternebra! fusion defects seen in a treated groups were dissimilar in single fetus from each of the respects to the sternebrae involved and were not considered to represent a treatment-related effect. ThuSiIjUUHlHUfadministered During the external, soft tissue and skeletal tions were seen in four of 91 control evaluations, malforma fetuses (4.4%), eight of 84 low-dose fetuses {9.5%) and three of 85 high-dose fetuses (3.5%). The of malformed fet mean percentages es per litter for the control, low- and high-dose groups were 4.3<, 9.82 and 3.3< respectively. low-dose group, diffen ;es froni Though these values were increased in ^e Likewise, control were not statistically significant. no dose-relationship was evident in these values. levels of 1,000 and by chamber inhalation at dose 70,000 ppm during the Day 6-15 gestation appear to be embryotoxic or teratogenic in the interval, did not per dose level. seven pregnant rats evaluated ha-^LSX _ft_aryUmuotrti . - - . . ~> ywy-i-^'---- Study DirEe. ctSocrnroeder, ^.S. ^/3dh Company Sanitized. Does not contain TSCA CC Company Sanitized. Does not contas; tSCACBI A-l Appendix A -- -- o y Embryofetal Toxicity and JTeratogeni city Study inhalation in the Rat Statistical Analysis 81-2571 Statistical Analysis of Data: Results of analysis by the indicated statistical procedure were reported for the following; Parameter Method 1. Body Weight - Days L 6. 9, 13; 16 and 21 of gestation factual and corrected by subtracting gravid uterus): A 2. Body weight change - Days 1-6. 6-16, 16-21 and 6-21 of gestation (actual and corrected by subtracting gravid uterus): A 3. Reproduction Data a Mean number of corpora lutea A b Mean number of implantation sites: A c Mean number of resorptions: A d Mean number male find female fetuses: A e Mean number of 'ive fetuses: A ' f Mean fetal might: A 4. Mean Percentage of Resorptions to Implants A 5. Mean Liver weights (absolute and relative to Day 30 corrected body weights); A 6. Food Consumption Data - Days 3-6, 6-16 and 16-21; A 7. Number and percentage of fetuses with external, soft tissue-or skeletal malformations: B 8. Number and percentage of litters containing at least one fetus with a malformation: B 9. Incidence of fetuses with ossification variations: B 10. Mean percentage of malformed fetuses per litter. A 11. Mean percentage of fetuses with ossification variations per litter. A C.^nySanWzed.D^no.'o""'"130*0111 A-2 ly Appendix A (cont.) Embryofctal Toxicity and.Teratogenicfty Study Inhalation in the Rat 81-2571 Statistical Analysts (cont.) Statistical Analysis of Data (cont,) Method A3: Statistical evalration of equality of means was made by th; appropriate one-way analysts of variance technique, followed by a multiple comparison pro cedure ft needed. First, Bartlett's test was performed to detenwne-+f groups If had equal variances. the variances. Mere equal,- parametric procedures were I f used; not, ^onoacaroetric procedures^were used. The parametric procedures were-tne standard one-way ANOVA using the F-distribution to assess significance, If significant differences among the means were indicated, "unnett's test was used to determine which means were significantTy different from the control. If a nofiparametric procedure for testing equality of means was needed, the Kruskal- if Wallis test was used, and differences were indicated, a summed rank test (Dunn) was used to determine which treatments differed from control. When statistically significant differences were identified by the Dunnett's test, a standard two-way analysis of variance was used to detect interaction between breeding Tots and test groups. A statistical test for trend in the dose level was also performed. In the parametric case (i.e. equal variance), standard regression techniques with a test for trend and lack of fit were used. In the nonparametric case, Jonckheere's test for monotonic trend was used. The test for equal variance (Bartlett's) was conducted at the IX, two-sided risk level. All other statistical tests were conducted at the 5% and IX twosfrfpd risk levels. 'Snedecor, G.w., and Cochran, w.S., Statistical Methods, 6th ed.,Iowa State univ. Press (1967), Hollander and Wolfe, NonparametrTE' Statistical Methods. John l/iley and Sons, New York (1973), Ounnett, C.w.,J. API. Sta, Assn.. vol. 50 (1955) and Biometrics. Vol. 20 fl964). -------------------- Bartletts's Test ANOVA . Dunnett's Test Kruskal-Wallis Summed Rank Test (Dunn) Regression Analysis Trend Lack of Fit Jonckheere's Statistic Arc Sine pp. 296-298 pp. 277-279 pp. 1096-1121, 482 pp. 7'4-116 pp. T3? pp. 149-152 pp. 160-164 pp. 120-123 pp. 327-329 S&C S&C 0 HSU WW S&C SfcC Hw S&C C^any Sanded. Does no, contain TSCA CB, A-3 Appendix A (cont.) Embryofetal Toxiclty and Teratogenlcity Study Inhalation in the Rat 81-2571 Statistical Analysis (cont.) Statistical Ana Tysis of Data (cont.) Method 6^- Statistical analysts of incidence data was performed using contingency tables. First, a standard cht-square araiysi's was performed to determine If the proportion of Incidences differed between the group-; tested. In keeping wHh standard statistical practice. If any, one cell had an (expected value of less than 5^, thIs-stepws-Trot reported.. (text, each treatment group was compared to the control group usfng a 2x2 Fisher Exact test and the significance level was corrected via the Bonferroni inequality to assure an overall test of the stated significance level. Thirdly, Amn'tage's test for linear trend in the dosage groups was performed. U., ^hi-square: Snedecor, 6.W., and Cochran, fcf.G.. Statistical Methods, 6th ed.. Iowa State Univ. Prfe s. Ames. Iowa, 1971. pp. Z50-Z53. Fisher Exa^ Test: Sradley. J.V.. Distribution Free Statistical Tests. entice Hall Englewood CHffs. l^ft. pp. ^5-2fl3. Bonferroni Inequality: Miller, R.S., Jr,, Simultaneous Statistical Inference McGraw-HilI Book Co., N.^. l^o. p. 15.-------------- Armltage's Test: Armitage, P., "Tests for Linear Trends in Proportions and Frequencies", Biometrics. Sept. 1955. pp. 375-386. Company Sanitized. Does not contain TSCACBI A-4 Appendix A fcont.) Empryofetal Toxicit^afltLTeratogenicity Study by Inhalation in the Rat 8I-?571 Statistical Analysts fcont.) Statistical Analysis of Data (cont.) Statistical notations RoTsTg- Symbol p<ft.05 p^O.01 A+ A++ ** K- K+ K++ t tt L L+ F F+ J J+ )? - H Statistical Statement Absolute Pata No statistical differences among the means (paranetrfc ANOVA). The means differ significantly (parametric AWV/\). Significantly different from control (Dunnett's) No statfsffcaT differences among the means (KruskaT-WalHs. nonpa rametnc) The means differ significantly (K-ruskal-Ua Ill's, non-parametric). Significantly different from control (Dunn's Rantc Sun). The response is linearly related to the dose levels. Lack of fit. Jonckheere's test. Incidence data indicates diffe rence from control by the Fishers Exact test. Company Sanitized. Does not contain TSCA CBf A-5 Appendix A (cont.) ^eratogenicity Study >y Inhalation in the Rat 81-2571 Statistical Analysts (cont.) Procedures for calculating stunted fetuses, mean Titter weight and naximum stunted weight calculations.""" Stunted fetuses (S) - Fetuses that weigh 1 gram or less are considered stunted. Calculatedstunted fetuses (Sc) - Mean Titter weight and maximum stunted weight must be calculated to Identify jifhich fetusas ^PC stunted by calculation (based upon cfflipArlsoftt^ the weTgfft" of Its litter mates only). Mean litter weight WLW) is determined by adding the weights of all live fetuses excluding ''stunteds" and "calculated stunteds" and dividing this sum by the number of live fetuses except "stunteds" and "calculated stunteds," Maximum stunted weight (MSW) i'5 determined by calculating the mean weight of a7^ live fetuses excluding Erjntods" and the smallest of the remaining fetuses, and multiplying this sum by 0.666o This product Is then compared to the welgnt of the fetus that was exclud'-'l. If the fetus weighs less than or equal to the above figure It is labeled as "calculated stunted" (Sc). If the fetus weighs more than the figure, then this value Is the maximum stunted weight (MSW). If the lightest fetus was a $c then the next lightest fetus must also be analyzed. In this step the Sc and the next lightest fetus are excluded from the calculations. Again, the mean value is multiplied by 0.666 and this product is compared to the weight of the fetus under examination. This procedure is repeated until a MSW larger than the next fetus to be excised is found. Lastly, the MLW must be recalculated after excluding all ?' '-'fuses in the litter. Staples, R. E. (January 1978). Tgratology Laboratory Technioues, collated from information and training prov1"'"' d to Research Yriangle Institute by R. E. Staples. Company Sanitized. Does not contain TSCA CBI ?s' --1C -n 3-- 01 CT.,? M i n n a tt a n M& ^ rr f p o 1 O ^ ^2?s2-- B-22 0 N* <??? 55S .^s a^Mrp * ?<rS ?--- 5-.- oS.." <;?,. M-<B3 + ?-------- s^ ipr 'A --- --0- i1"? .0,^ 2P,- < 0 c^T y ^ 1 -- o g -g 5 19 X"^ a -!- c r -* a 5 i i jt o= ^"" Ig-S 1 g a rnft 3 u> e m CB m CB u> w ui <u 5> S w id r5--SCO^O 01 w> m < m 5! n rr. 3 Mcc --GC0aiO'O<B'x'--0i->i a w 3 0'X j ~j-j a> ai o n m u> m c= a Pa -- <a 0 <C ~j S> <l o jw (M u -jfMa o^Ro>'o^s<>--me *SaiS-^*--^ -- -- ijrj IM PJ rM IM >M ni u m fu u w 01 u * m u (d "G i . -^ -^ '.^ --<ro j IM l 10 m 10 B -- SG SG a-- --"" So o-- IA M n> <\> IM ru n> m Uwb->^n*j<ki<e>(Nwiu s> o -w A> -- ro x '4W& wofr r> o otooM ' -- o M o wu > ^ c*oj fi r nImMaIMiM-M^imv^<rfuourour--Nj r*- ftj W - M u ->j.ku (UMAf.wteiM-ki&MUMfnM* wo* IM f) .M M o'v Mw ro Mt^ wm ix---"-< n* U ' -^ *uru'^ pa aH>ra*nl,n-jun>wn* drvi--^scimnoutrmwufulumt ~aaaaa'n*o ro LJ ru u> -JIMO< nj (u n* m fu <u n> *C>>b*UU J> IM 9 IoMkrvu crujiKu>inaiirouin.Ai ^ ru OIOIA o o ^ i . m ^4 X t> 0 aivi'^MjOr>oOnBOpatOIMicrno IM 'C --' 0110 fa) 1M r\> -^ h ^ Ul K^J4m^^luimo^nj*o(k^I^M U: U U1 * fM -- Jk ru j JNm foru ru ru (M ru IM ^^'w-^Ni^crosrot & nifW-MIV^'XM; U ...&J UwcUL(JVU^iU**UurU*i W J Ui<*<-W^^(jJ0Lk^JfU\fW(v U cj Uu&UdWAf\UiUfiuUUna^ 3 ^ucj S3?SS2SS? OCDU>M>>M 2 '^.-i .'SSSSS'Sg (uooB~jaw- 'S ~ju SS3SSS3 A-daauo>--i A ^0^ cy> ffi f^ 03 ^ ^ ^ -no^mo^o 0 <A ^*rsaff ^ A 0 ^J i9l CJt 3* rs -- (/T . ^ Ot <n ~- ^po^. ^40iaA^>J^U19C --^*oootrfryv0i 'JOU* ^> w m o> a vi ui ->i o en <ji o> m unji en <i -^ o .n ui&aio>o>o<uo' -^x.'iSe "* !O2B r>* -- " o "o fwa " <r pl* *o "* "a * "* u "* aa " u 'u*'Ma "* -x u u c-- j-rr><M^-'ur>ivi--M or3a a u rfjkoi IM m * <j o auu -- c? Ji o> <o '*<yft r^ojpkc-- rm01 wui u^iu=1i wi -xra-- ^3-^5mooiSwo>BjkT*? j ^ | -- > a <a f ;a. < fa ! ? 5? 3 I? - -- 01 " (1 < K g~ 3 n (< ^ ? 9 ft <^2 a " 3 i fs"" ? kI'o r< ,. i < ' '' 3 '-unaa ^<0 0&or/ui A 01 3B o* ^j w i-< 01 m 0 -- -^ -< t-i 3 0 01 iwu 4WU 01 a j>moni^jjCo>Boi>Muo> U 'J w w 0> rM 3 e> ~iruru ^ .-^ j-ni o.aou<imafu^<ujtieunt< a>U)nii3a>^'.M* .? i n e | i? fs i g2S 1 ] n ? n 2r 51 = a i s K j- 7 ; C 0 3 j^f i 8" Vs ? i ^^0 ^^ -- rr 1 j^ CO ^^^1 0 ^^^ 1 5- ' ? 1^53- ^-5'c t -- 3 ? ^1 f is& 3 < ^ 3 = ^^ rr c s y-s- y ff f s ? a [ 01 1 r\ w -t.fcra U U 01 Jk LJ ft* ui ua -- u aa o> --i Jk >UoA<U^IU-oI--*--x>3 U -<rMo PJJ>Jk'U01AUa><\u) <0u<i 0 l <0 9 5 S 3 ^rf r a Company Sanitized. Does not contain TSCA CBf C-1 B--------PHMHHBpJ' Appendix C Enipryofetal Toxicity and J:erato9eni city Study Inhalation 1n the Rat Bl-2571 Individual Female Reproduction Data Female N0. of Corpora Resorptions *to. lutea No. of Implants I of Fetuses HNaB, .a """" Implants ' N0. live Ho, Hale ' Female Total "ead I A- 1576 15 A- A-. A- A- A- II (0) 1577 21 (Chanber 15 0 0.0 4 1E 7 - 11 0 1578 15 Control) 1579 13 15 0 1 (3 0 6.7 5 9 14 0 0.0 9 6 15 0 0.3 9 4 13 1580 14 1581 13 1134 01E 7.1 2 11 13 00 158Z 13 1 12 0 0.0 5 A 13 0 0.0 6 6 12 0 Total : 104 93 2 *tean : S.E.M.: 14.a 1.1 13.3 0.3 13.8 2.0 40 51 5.7 91 0 N 7 0.6 0.,2 7 7 1.3 7 1.0 7.3 13.0 0.9 0.5 0.0 0.0 II 2576 12 12 1E 7 7 7 7 (1,000) 2577 12 11 1E 8.3 9 2 11 0 2578 13 13 1E 9.1 4 A 10 n 2579 14 14 D 7.7 7 5 12 0 2380 15 2501 K 13 1E 0.0 7 7.7 7 R 13 5 1(7.1)0 2582 '1 1124 01E 7.1 3 10 1132 00 0.0 9 3 12 n Total ; 91 89 5 Hean : S.E.M.: 13.0 o.s 12.7 0.7 359..79 466.S 357.3 1K13 .9 1 N : 7 0.4 0.2 7 7 1.5 7 0.9 1.0 0.4 0.1 0.1 7 7 7 7 ni 3576 14 i 70,000) 3577 16 13 1E 7.7 7 5 12 n .15 1E 6.7 11 3 14 3578 14 3579 17 13 0 16 0 0.0 5 0 13 0 3580 15 35S1 16 6 0 0.0 7 9 16 0 13 1E 0.0 1 5 S 0 35S? 13 12 0 7.7 8 4 12 n 0.0 9 3 12 Total ; 105 3 Mean ; S.E.H.; 15.0 0.5 12.6 0.4 232..12 468.9 357.3 85 0 N ; 7 1.2 0.2 7 7 1.5 7 1.2 12.1 0.9 1.2 0.0 0.0 7 7 7 7 Mean Fetal Weight foraBd A- 3.59 3.32 3.11 3.31 3.61 3.51 3.98 24.43 3.49 0.11 7 . 4.01 3.53 3.71 3.44 3.44 3.46 3.98 25.57 3.65 0.10 7 3.87 3.3S 3.13 3.42 3.25 3.77 3.40 24.99 3.57 0.09 7 For explanation of ?1 statistically statistical symbol (A-) see Appendix 3{ ' Early significant 'esorption. differences fron the A. control group. "Hunker in (nrpnthesis represents the perceiitage of dead fetuses to implants. .Company Sanitized. Does not contain TSCA CBI D-l Appendix D KI-idtiyjFRTAL TuIIulTy A,\'P TF.HATjGE;--li;:ry ^TUPy -.-'2 i ^rQ------plyNH".LATl^ IrJ THE H; ^^P^VIDUAL FEilALK Ll</F;h r/E;Gnl IV.; M TRrif^NAL AN BOpy Ml.9 NO tG.^ L.VKA xl. IGH/ GH3 b uh3/Ti3h (^ m ) GAJJP i - ^0 PPh> 157c ^.i- ^/J 1:;,'^ ^oO ^o1 15^2 A- 2/1 .0 2o6,.0 2'^66,,..00 27?..U 26;. y 26^. 0 A- ^ ^ 1 u,.2 , 5.. ':, ^.^ 't^. o 13. 1 ^. 5 A- 5..;1 5.6& 5. 41 5.2;. 5.3^ 'i.91 5.Dj MEAN ;>r i>E ij Mx 11 niX 27.. 7 6. 9 -^ ^ : 25^.0 266.0 '^.6 ^O 0.^ 7 1;,.1 ^.2 5.S'* 0.24 0.09 7 H.9i 5.65 For explanation-of statistical sjmbolf A-} see Appendix A. ^Corrected Day 27 body weights (see Appendix B). "Liver weight divided by corrected Day 21 body weight X 100. Company Sanitized. Does not contain TSCA CBI D-2 Appendix D (contj 2';: Rf-iBfiyOFET uF 1^ ;i(jiy /iNp TEfiriruGr.'.viciry ^rupy LIV IKHALA'.uGi^ IN l.JE ti/.T ^PIVjrUrtL FEMALE LIVF.h riRJGHT PAr/, ]Ert.'in'<AL Ajrf oGpy r. a Nu iG;-i/ L^VE/t rt3ril MI. tGn; C.hu/rBnb f x 100 ) uhj'uP ^005 PPM/ <3 i 0 2^// Z-73 Z5: ^ ;^jJ 25o1 25->2 2u^.O 2 7 3. ^ 2 7 y . C/ C; ^ I . U ^'..0 coO.O 2,"^.C 1'^,.6 ^. i 'S,.^ ^j,,c' ^j.,3 ^.2 ^.77 &.33 ^.;-. 5.22 3.^^.62 ^. o5 riE A;JP SR U &/,' .n ^ .2 ^ .6 1 ^.0 i i ^ 0.4 5.35 0.2o O.11 1-iiii 26^.0 riAX 2^7.0 ^^ ^.S *<.77 5.^7 "Corrected Day 21 body weights (^ee Appendix 3.). "Uver weight divided b^ corrected Day 21 body weight X 10D. Company Sanitized. Does not contain TSCA CB9 D-3 Appendix D ^cont.) OF|yjby RMBRy&FETAL iox:c:ry Arip TEHATjGr'..ii:c;Ty ^rury -LUHALAT:^ :.< IriK tim ;i^PiVlPUAL FKf-i.-.Lfi LJVRh nr^Gili PrtI'-. TEAii.NnL AN 6i;py rtl.3 NO IUPJJ LIVF.ri MGriI w-I. GhS/Iiiw0 (6M) (X 100 ) GHC-UP 11 j. - 170000 PPf-il -576 j5i'i :5~la jS7^ .5->0 ^j1 ;jiJ2 262.0 2?;.0 272.0 2y-;.0 2^i-..J 2u^.O 2'fj.Ci ^ ^ * ( . ^ 15.0 ''5.3 "'o.c ^.^ ''J.2 ^.'^-j 5.27 5.51 5..;^ 5.o-< 5.3" 3.6; Dhan 276.0 "'^.^ s.-.s oP '^.J 1._ 0.27 ^ 5.1 0.5 0.''0 N 7 filN 262. u MAX 2^.0 7 7 '2.7 0.6 ^.o5 5.6^ aCo^rected Day 21 body weights (see Appends; B). "Liver weight divided by corrected Day 21 body weight X 100. Company Sanitized. Does not contain TSCA CBI E-1 ^Bnbr.yofeta_r^xAp1pce<ndti^x AEMLTeratogen1c1ty Study Individual Female Food Consumptic Day: 3-6 I (0) (Chamber Control) 1576 1577 1578 1579 1580 1581 1582 II (1,000) Total : Mean : S.E.M.: N : 2576 2577 2578 2579 2580 2581 2582 III (70,000) Total : Mean : S.E.M. : N 3576 3577 3578 3579 3580 3581 3582 Total : Mean ; S.E.M.: N 17.0 18.0 35.0 17.5 0.5 2 18.7 18.7 18.7 0.0 1 18.3 18.3 18.3 0.0 1 A++L+ 20.7 21.1 21.4 21.1 21.2 20.7 24.7 150.9 21.6 H.5 7 20.7 18.3 19.6 22.2 23.9 17.5 20.7 142." 20.4 0.8 7 18.2 - 18.1 19.4 20.0 18.3 19.2 17.6 130.8 18.7** 0.3 7 A- 26.4 2?. 4 26.6 22.4 23.0 25.6 29.8 179.2 25.6 0.9 7 24.8 22.8 24.6 27.4 20.8 25.6 27.6 173.6 24.8 0.9 7 26.2 20.0 19.2 26.6 27.6 17.4 24.0 161.0 23.0 1.5 7 For explanation of statistical symbols (A-, A<"<-, L+) see Appendix A. Company Sanitized, Does not contain TSCA CBS F-1 Appendix F EmbryofetalToxi city and Teratogenicity ofM----py Inhalation in th^ Rat ('athology Report Study Summary of Adult Gross Postmortem Observations 81-2571 All of the animals on test survived to the termination of the study when they were killed for postmortem examination. Morphologic abnormalities observed grossly r 'd sporadically in the treated and control animals; they did not appear to elated to the administration of the test article. ^ W 'C^.? Sanitized. Does "ot contain TSCA CB! F-2 AHppppeenndoiixx Fr (itcoont.) ,^Eiiibrvofetay[flxllt^nd^JreratogenlcUy Study by OBIB^^^^^^^^^^^^^^^^^^^^^^^^^^^B Inhalation In the Rat 81-2571 Adult Gross Postmortem Observations Group TPp^T Fema Ie No. Type of Heath" I 1576 T (0) 1577 T 1578 T 1579 T 1550 T 1581 T 1582 T II 2576 T (1,000) 2577 T 2578 T 2579 T 2580 T 2581 2582 T T , III 3576 T (70,000) 3577 T 3578 T 3579 T 3580 T 3581 T 3582 T Pregnancy Status0 Obscrvatlonfs)0 + N.O.A. + N.O.A. + Kidney CR): moderately hollow. + ^.O.fl. + N.O.A. + N.O.A. + N.O.A. + N.O.A. + N.O.A. + N.O.A. + N.O.A. + Lungs; small and dark red in TOX color; diaphragmatic hernia. + Kidney (R): moderately hollow. + "-- + N.O.A. + N.O.A. + N.O.A. + N.O.A. + N.O.A. + N.O.A. + N.O.A. ^T = "+ c CR s Temilnal (Day 21 Pregnant. Right. of Gestation). Doeslto^eonW| ^TSCe G-l Appendix G .Embryofetal Toxicitv and JTeratnoen^city Study |by Inhalation in the Rat 81-2571 Summary of Fetal External Examination nata Group (PPm) I13 (0) II (1,000) III f70,000) Female No. Fetus No. Malformationa 1578 R Upper mid-dorsal - dark, blue-black, raised areas. A total of 84 fetuses were evaluated representing 7 litters. A total of 85 fetuses were evaluated representing 7 litters. ^Represents only fetuses wit<- malformations. "A total of 91 fetuses were evaluated representing 7 litters. Company Sanitized. Does not contain TSCA CBi H-1 Appendix H Embryofetaj__[axlcllY_aiid-Jeratogeni' ci ty Study """"^^""""^^^"^^^^^Ihy Inhalation in the Rat fll-257) Petal Soft Tissue Examination Oata Group TPW I0 lit1 (1,000) Female Fetus Fetus No. No. Sex ________Malformationfs)3^________ 1579 7 1580 13 2576 3 7 11 2577 2578 11 M Ureter (I) - (S) tortuous. F Ureters (8) - (S) distended and tortuous. M Ureter fR) - (S) tortuous; Ureter (L) - {H) tortuous. ---------- M Liver - small, dark discolored area on median Tobe near cleft (approx. 0.3 cm in dian.). Ureters (B) (S) tortuous. Ureter (L) (S) tortuous. Ureter (R) (S) tortuous. 2579 2581 Ureter (L) ureter (R) (S) di-- ended and tortuous. (S) distended and tortuous. Ill6 3578 (70,000) ureters (B) (S) tortuous. Ureter (S) distended and (H) tortuous. represents faR Right; total of total of A total of only fetuses with observable malformations. Left; Both; Slightly; Moderately. 48 fetuses were evaluated representing litters. 43 fetuses'were evaluated representing litters, 44 fetuses were evaluated representing litters. Sanitized. Does not contain TSCACBJ Company T W Group ^ (0) II0 (1,000) 11 It* (70,000) Embryofetal 1-1 Appendix I Toxicitv ancLJeratooemci'tv |by Inhalation Study In the 81-2571 Rat Fetal Skeletal Examination Data Female No. -------- 1579 2580 3578 Fetus No. ------ 1 10 4 _--_--_--_--__--_--_--H--a--lf--or--m--a--t--io--n--fs--)--8_--_--_--_--_ Ribs (Right side) - 6th-8th, 11th and 12th a.;gulated; 9th and 10th .ribs shortened. 1st and 2nd sternebrae fused. 2nd-5th sternebrae fused; cleft evident down ventral md-line of fused sterne brae. ^Representsonly fetuses with observable mat format ions. "A total ^A total of 91 of 84 fetuses fetuses were were evaluated evaluated representing representing 7 7 li,- litters. "A total of 85 fetuaes were evaluated representing 7 litters. c^^-^^^^ J.I Appendix J ._fa0ryofetal Toxicltyand Jeratogenlclty Study 81-2571 fetal Ossification Variation Diti Suimarlied tirouo: (P("Il) No. of Fetuses Examined Intact: without heads: Total: No. of Litters Evaluated: No. of Fetuses Witn it Least One Ossification Variation: Ho. of Litter: Containing at Least One Fetus with an Ossification Variation: Mean X Fetuses with Variation/Litter; TOT 43 8 91 7 87 7 95.3 (,11.0100.) 41 43 84 7 75 7 89.1 III (70.000) 41 44 85 7 77 7 91.0 "3: wr No. of Fetuses fLItters) 1th Ossification Variationsa".b^.'c -Ro- 115- r^e of Ossification Variations: Craniil Ossifications'*; Parietal Incompletely Ossified: Malar Incompletely Ossified Unilateral: Bilateral: (1) 2.3 0.0 0.0 I P/ 2.3 0 0.0 0 0.0 2 (2) 4.7 0 0.0 0 0.0 Hyoid - Incompletely Ossified: Unosslfied: 0 0.0 9 (3) 20.9 I (1 ) 2.4 5 (2 i 12.2 1 1 2.4 I I ) 2.4 Sternebrae Asymmetric (small) Sternebrae No. I 2 3 4 5 6 Unosslfied Stcrnebrae No. 1 2 3 4 5 6 0 IS (5) 7 (5) 27 (0 0 57 (7) 0.0 16.5 7.7 29.7 0.0 62.6 0 0.0 0 0.0 0 0.0 0 0.0 26 6) 28.6 ? 1) 2.2 - 3 F2 15 17 8 15 26 (7 0 56 (7) 3.6 17.9 9.5 31.0 0.0 66.7 0 1 (1) 0 0 9 (3) Z( 1) 0.0 1.2 0.0 0.0 10.7 2-4 2 (2) 19 6 8 6 19 7, 0 55 (7) 2.4 22.4 9.4 22.4 0.0 64.7 0 0 0 0 19 3) 6 2) 0.0 0.0 0.0 0.0 22.4 7.1 Ribs: Hudimentary Structures6 Unilateral: til lateral: 12 Kib pairs: 14 Rie pairi. 3 2) 3.3 2 1) 2.2 2 1) 2.2 0 0.0 1 () 1.2 0 0.0 0 0.0 0 0.0 5 (3) 5.9 2 (1) 2.4 0 0.0 ' 0 1.2 uata not analyzed statistically. ''This Is (unmary of representative ossification variations as noted during the skeletal evaluations. It is not the Intent to present every type of osslficttlw variation observed during the study. c* No. of ftftuses with the variation/Total No. of fetuses evaluated. "I No. of fetuses witn tne variation/Total No. of intact fetuses evaluated. Small discrete ossification(c) adjacent to the last thoracic or first lumbar vertebral transverse process(es). ' ^Pany SanJfeed. poes notcnn^ ^ ^ ^(iflBlF^ J-2 f Einbryofetal Appendix J (cont.) Toxici ty jind.Jeratogenic 1ty Study Inhala. Ion In the Rat 81-2571 Fetal OtsKtcitloo Variation Data Sunnarlred >'""""/ Verteora) Column;^ Wo. ef FeTttuos:e--s --flitters)' with Ossification Variations*'^ __ ^--------2--------B?----------s- Cervical V. Trans. Proc. Incompletely Ossified - Unilateral: Bilateral: 11 (1) 1.1 0 0.0 0 Cervical Ossifications (1) 1.1 0 0.0 1 (1) 1.2 Unilateral; - 1 () 0.0 Tnoracle V. Centra 1.1 0 0.0 0 0.0 - Incompletely Ossified: Unossified: (.unibar V. Trans. Proc. Incompletely Ossified: Sacral V. Trans. Proc. Fused to Centra - 43 (7) 0 47.3 0.0 I (1) 1.1 41 (7J1 48.8 2(2) 2.4 I 0 1,? 33 (;1) 0 38.8 0.0 0 0.0 Unilateral: Bilateral: Incompletely Ossified - Unilateral: Bllate-al; unosslfied 4 (Z) 4.4 2 W 2.2 7 (41 7.7 6 (3) 6.6 Unilateral: Bilateral: 3 (3) 3.3 1 (1) 'I Pelvic Ossifications Puois Incompletely Ossified - 3(3) 5 (2) 1 0 3 (1) 1 (1) 4 (2)- 3.6 6.0 1.2 3.6 1.2 4.8 0 0 > ('} 0 0 0 0.0 0.0 1.2 0.0 0.0 0.0 Unilateral: 0 } Bilateral: Puofs Unossified 11 (1) 10..0' 01 (1) 05..02 00 00..00 (bilateral): 0 1 0.0 i m 1.2 0 Distal Phalanges Unossiffed 0.0 - Fore limbs: Hindi imbs: Retacarpals Unossified: 4 (3 ) 40 ((66,) (1) 4.4 44.0 1.1 12 (1) 30 (5) 1 (I) 14.3 35.7 1.2 5 13) 23(6) 0 5.9 27.1 0.0 "Uata "This not analyzed is a statistically. the summary of representative skeletal evaluations. It is ossification variations as noted during ossification variation observed not the intent during the to present every type of study. '-I, No. of fetuses with One or more vertebrai the variation/Total No. of fetuses 'lements evaluated. Transverse Process). affected (Key: V.-Vertenral; Trans. Proc.* CompanySanitized, Does not contain TSCA OBI Appendix K PROJECT NO. 81-257] EMBRYOFETATLOXICIT/ AND TERATOGENICITr OFfarilBy STUOr INHALATION IN THE RAT Company Sanitized. Does not contain TSCA CS Appendix K (cont.) -1- 81-2571 J. INTRODUCTION Tne purpose of this study was to determine the teratogenic potential and degrpe of embryot 0x1 city ofJUHBBHUU^ln the pregnant rat. Two groups of female rats were exposed to atmospheres ^ontafmng j ffor- 6 hr/day from Day 6 through Day 15 of gestation. A ; oocui-rent control group was exposed only to houseline air. Target concentrations selected for testing were 7000, and 70,000 parts per .in'lion (ppm). 11. METHODS A' Test Material Delivery The test material was placed in a 50 emitter (ml) glass syrir'je mounted on a Ssge syringe pump (Model #362) for the low exposure (Group II). For tne high exposure (Group III;, approximately 1600 mis ofJUNwasp^ced fn a 2000 ml flask connecteo 10 an FMI Lab Pump (Model 6-20). From tnese reservoirs, the test material was metered to a 4-neck round-bottom flask that was maintained at a temperature of 45C using a water bath. Houseline dry air, at a flow rate of 7.0 liters per minute (1pm), was directed into the flask. The resultant vapor-iaden airstream was delivered into the exposure chamber. B. Chamber Operation Tne animals were exposed in a glass chamber with a volume of !".7 liters. The average airflow rate through the char.ber was 7.0 Ipm. !his flow is calculated to provide one complete air change every 2.8 minutes and a 99% equilibrium time of 12.9 minutes. C. Atmospheric Sampling Chamber concent itions were determined in chamber including the cof'.rol every half hour using a COW-MAC Model f750 gas chromatogrjph. A six foot 1/8" Chromosorb 102 column, at a temperature of ISO^, was employed for the chroniatographic separation and determination. Chamber concentrations were Sanitized. Does not contain TSCA CB1 Company Appendix K (cont.) -2- 81-2571 II. METHODS (Coot.) C. Atmospheric Sampling (cont.) III, II, determined by comparing the integrated G.C. area to responses obm'ed primary gas standards. The G.C. sampling sequence was Groups I, offfffin etc. However, because of the carry-over the sampling lines high dose to control, this sequence was reversed on Day 2 and on Day 6 sampling tun,- wn'> increased from 8 to 9 minutes. with I, from the Chamber oxygen was monitored daily (4 times per day for the first two days and twice daily thereafter) using an MSQ Portable Oxygen Indicator (Type E). Chamber air temperatures were recorded hourly during the exposures. i ?an5t5ze&,Does not -'-"rilcun JSQA C^i | Company Sanitized. Does not ?-? Append!A K (cont.) -3- fil-2571 JIJ. RESULTS AHD DISCUSSION The mean nominal concentrations were 1050 and 69,600 pomflfffffor Groups II and III, respectively. The cumulative analytical concentrations of ^IBr"^ 9S14 and 70,700 pfri (Table 1). The dlfferef..es between nominal and analytic"' are within the limits of experimental error. For the first two days of the exposure, concentrations of approximately 25 "(" were observed for samples taken from the control chamber. This was attributed to the carry-over In the sampling lines from high dose to control. This problem was eliminated by reversing the sampling sequence and Increasing the sampling time. During the remainder of the control exposures, peak areas observed were either "very small" or not present at all. The oxygen concentrate n In the exposure chambers ranged from 19.0 to 20.8%. The mean chamber temperatures and ranges are presented below: Group Mean Range I (Control) II (900 ppr) III ( 70,000 pom) 24.6 25.0 24*6 20.0-28.0 21.5-28.0 22.0-26.5 In some cases, higher thermometer readings were noted but these were due to Intimate contact between the rats and the thermometer. These findings were shown to be much higher (1-8C} than the atmospheric temperature of the exposure chamber. When this occurred, a second temperature reading was taken after the thermometer was free of physical contact. High chamber temperatures, not associated with-Improper placement of the thermometer, were lowered by placing Ice-bags on the chamber. ,fcw^/?,,^4^^ J,unes B. Tern ll, Ph.tT. Scudy Director, Inhalation ^A/^ /)a^e Company SanHizeA Does not contain TSCA CB( Appendix K (cont.) -4- Table 1 EmbryofetalTpxicity and Teratogem'city Study oij--gby Inhalation in the Rat Group I (Control) B1-2571 Date 8/17/81 8/18/81 8/19/81 8/20/81 8/21/81 8/22/81 8/23/81 8/24/61 8/25/81 8/26/81 8/27/81 ri/28/81 8/29/81 Test JISZ- 1 2 3 4 5 6 7 8 9 10 11 12 13 Exposure Number 1 2 3 4 5 fi 7 8 9 10 11 12 13 ""BaTTr Analytical1____ Concentra-ti.o.n Cumulative Mean TppS} Concent ration TpW 25.2 26.1 7.60 3.65 6.10 2.<0 1.00 0.00 6.12 3.35 0.00 0.00 0.81 25.2 25.7 19.6 15.6 13.7 11.9 10.4 9.06 8.73 8.19 7.45 6.83 S.36 *mThuelsteipocortncseanmtrpalitniognssyswteerme . attributed to carry-over of test material in the Company Sanitized. Does not contain TSCACBI Appendix K (cont.) -5- Emt)^yo^f(etfllTtoxJi^aacDbftieyle 1I (cont.)/ o>f f]liqbyrlrit" and 'Teratogenfclty Inhalation "^^roup II (1000 n the pprn) Rat Study B1-257I 8/17/yi 8/la/m 8/19/81 8/20/yi 8/21/B1 8/22/8} 8/23/yi 8/24/B1 8/25/81 8/26/Hi /?7/tf] tf/28/81 8/29/81 1 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13 922 942 966 993 rose 1010 1070 1000 1020 1020 995 1000 993 922 932 943 556 975 981 985 987 990 993 993 994 994 995 1080 1050 TOTO 1040 996 1040 1040 1100 1020 1070 1060 lior Mean: 1050 CompanySanitized. Does not ^ainrsCACBI Appendix K (cont.j -5- EmbryofetaljoTxaibclietyI (cont.) ofQpfby and Teratogenicity Study Group InJJhJala(7t0io,0n00Inppthffel) Rat 8/17/81 8/18/81 8/19/81 8/20/81 8/21/81 8/22/81 8/23/81 8/24/81 8/25/81 8/26/81 8/27/81 8/P8/81 8/29/81 2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 8 9 10 11 12 13 74000 75300 69200 72100 69500 71200 69300 69500 69100 70400 69900 70200 69600 7400C 74700 72800 72700 72000 71900 71500 71300 71000 71000 70900 70800 70700 72000 72200 69300 70700 72200 65500 68200 69100 70100 69100 69100 68700 68100 Mean: 69600 ^'''""^""-.D,^, ""iltiTscACBj L-l Appendix L 81-2571 ^ f ^ l l f l l f ^ f ^ , ^^intu-aafetaJ^Qjdl^^Afl(L-T(eratoyen^city by Inhalation Study in the Rat PERSONNEL DEPARTMENT/TITLE NAME DEGREE STUDY DIRECTOR STUDY MONITOR Raymond . Schroeder Joann Breyan M.S. SMALL ANIMAL: REPRODUCTION & TEKATOLOGYSECTION MANAGER SUPERVISOR Raymond E. Schroeder Maureen Kenny M.S. B.A. TECHNICIAN-IN-CHARGE VETERINARIAN PATHOLOGY PATHOL )GIST QUALITY ASSURANCE MAMAfiEK COMPUTER OPERATIONS MANAGER INHALATION MANAGER SUPERVISOR TECHNICIAN-IN-CHARGE Kathryn Sloan Henry F. Bolte B.S. D.V.M., Ph.D. Henry F. Bolte D.V.M., Ph.D. Craig M. Lamb B.A. Crai'g M. Lamb B.A. iJames Terrill Ph.D. Mary R. Melcon B.A. Joan M. Claytor - Company Sanitized. Does not contain TSCA CBj M-l Appendix M QUALITY ASSURANCE STATEMENT innnniinnnnnmnnnnnnnnnnnnnnnnnniinniinnnnnm STUDY WO. ^.^L'^Zl-.--TEST STUDY SPONSOR....i^.0!!^06S'?U!B'S!1T!A'N!.C..E^/A..R..T. ICLE. Lifd df by h btflow r avlity that thl tud^ df wre rvporfd toAthuvrSnctudyUnit nd fh w Pifctor nd incpcfd finding* Hngnnt *>ATE(S) OP INSPECTION REPORTED TO STUDY DIRECTOR REPORTED TO MANAGEMENT 9/1/81 9/4/81 11/23-11/25/81 12/9/81 & 12/10/81 9/2/81 9/4/81 12/3/81 12/17/81 9/21/81 6 10/9/81 9/8/81 & 9/22/81 12/30/81 1/5/82 12/18/81 & 12/23/81 company Sanitized. Does not contain TSCA CBI ^BuS^^ i^'^ DATE""*-