Document jyOM9Mep1RLmGdK3Rq5p3GqEy

AR226-2776 (SS INHALATION: EMBRYO-FETAL TOXICITY AND TERATOGENICITY STUDY IN THE RAT HASKELL LABORATORY REPORT NO. 881-81 Copies to: Company Sanitized. Does not contain TSCA CBI FOR DU PONT USE ONLY HASKELL I. E. DU LABORATORY PONT DE NEMOURS & CO., INC. FOR TOXICOLOGY AND INDUSTRIAL MEDICINE CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT ELKTON ROAD, P. 0. BOX 50 NEWARK, DELAWARE 19711 JjB INHALATION: EMBRYO-FETAL TOXIC ITYAND TERATOGENICITY STUDY IN THE RAT HASKELL LABORATORY REPORT NUMBER 881-81 Dates: Initiation (breeding date) - April 27, 1981 Completion (sacrifice date)- July 9, 1981 Date Written: Date Issued: December 30, 1981 January 14, 1982 - 1 - ggmpan^.SanjfoeDdoe.s not contain TSCA CB8 _INHALATION: EMBRYO-FETAL TOXICITY ID TERATDGENIC-ITY STUDY IN THE RAT HLR 881-81 REPORT BY: W - ^ R. E. S^Tples, Ph.D. Stuay Director Staff Teratologist Teratology Section Toxicology & Pathology APPROVED BY: i^s^ucQ J. G.^/Aftosmis, D.V.M. Associate Director Toxicology & Pathology REVIEWED BY: CAftM^uQ^^'^aAj!^ C. M. Barba Auditor Quality Assurance Committee RES/ms/mie - 2 - ^inpanx^iHzed. Does not contain TSCA CBI HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT ELKTON ROAD, P. 0. BOX 50 NEWARK, DELAWARE 19711 QUALITY ASSURANCE DOCUMENTATION STUDY: }jflifiWQ HLR 881-8^ dpiplnhalation: Embryo-Fetal Toxicity and Teratogenicity Study in the Rat QUALITY ASSURANCE AUDITS Audited: July/August 1981 Date Findings Reported to Management and Study Director: August 14, 1981 Reported by; CMB /mie 1/8/82 (y}A^usun^fwJwJ C. M. Barba Quality Assurance- Auditor - 3 - .Company Sanitized. Does no! contain TSCA CB1 EMBRYO-FETAL UU INHALATION: TOX[ICCiITTyT^^ADNrB1T]TERATOGENICITY STUDY IN THE RAT HASKELL LABORATORY REPORT NUMBER 881-81 TABLE OF CONTENTS TSiigtlneatuPraegePa.g.e......................................... 1 2 TQaubalelityof ACssounrtaennct.se. .D' .o.c.u.m.e. n. .ta.t.i.o.n. ........................................... .. 3 4 .................................... I. Summary ........................................ 7 II. Introduction .................................. 7 ^ v A. B. Background Protocol ................................ 7 8 C. Purpose ...........................-.-..-.,.................................. 8 III. Materials and Methods ......................... 8 A. B. Test Material Animals ............................. 8 9 C. D. SExtapetirsimtiec.na.tla. .l E.D.v.ea.slu.ig.an.ti.oa.nn.d. .P. .ro.c.e.d. .u.re.s. . .............. .. 10 15 .................... IV. Results ....................................... 15 A. B. Eye Examination of Prospective Parents Maternal Exposure to C-8 .... 15 16 C. Clinical Maternal FSeigends COonbssuemrvpetdion. ........................ .. .. .. .. .. .. 16 17 D. E. Maternal Body Weight Gain .. ............................... 17 18 F. G. MGraotsesrnEaxl aDmeiantahtsion..o.f..M..a.t.e.r.n.a.l..O.r.g.a.n.s..a.t.... H. R S ep acrifice roductiv e .E..ff.e.c.t.s. . a.n.d. . B. .o.d.y . .W.e.i.g.h.t . .o.f . . . . 18 I. the Fetal AOlftfesrpartiniogns....'................................................ 19 20 J. Pup Alterations ........................... 20 V. Discussion .................................... 21 VI. Conclusion .................................... 23 - 4 - Company .Sanitized. Does not contain TSCA CBI _[NHALATION: EMBRYO-FETAL TOXICITY 3D TERATOGENICITY STUDY IN THE RAT HLR 881-81 TABLE OF CONTENTS (CONT) VII. VIII. Acknowledgements References ...... Page 23 24 TABLES: I. Experimental Design: Exposure Levels and Distri bution of Mated Females Among Test Groups ... 26 .......... N II. FeDedaysCo6n-s1u5mpotfionGeisntaRtiaotns Exposed to from 27 III. Reproduction and^Fetal Development in Rats Exposed to ^H[from Days 6-15 of Gestation . . 28 IV. Fetal Malformations in Rats Exposed to jjfma7j from Days 6-15 of Gestation ................. 31 V. Fetal Variations in Rats Exposed tpj^JKfrom Days 6-15 of Gestation ....................... 33 VI. Reproduction and ipment of Offspring of Rats Exposed to [from Days 6-15 of Gestation 39 APPENDIX A. Protocol 42 Amendment to the Protocol for Inhal Teratogenicity Study in Rats with 52 Amendment to the Protocol for Inhala-tj, Teratogenicity Study in Rats with 57 1 individual animal data available upon request - 5 - Company Sanitized. Does not contain TSCA CBI INHALATION: EMBRYO-FETAL TOXICITY D TERATQGENICITY STUDY IN THE RAT HLR 881-81 TABLE OF CONTENTS (CONT) ATTACHMENTS 1. In-House Rgport: Inhalation Exposure ^Results Teratology Study - Part I and Part II ......... 60 2. Letter, from J. M. Clinton to R. E. Staples, 4/24/81 ....................................... 73 3. Letter, from J. M. Clinton to R. E. Staples, 5/5/81 ........................................ 74 4. Memorandum, from W. D. Kerns to R. E. Staoles, 12/18/81 ...................................... 75 5. Memorandum, from W. D. Kerns to R. E. Staples, 12/21/81"...................................... 77 6- Letter, from.J. M. Clinton to R. E. Staples, 6/5/81 ........................................ 82 - 6 ppjmpan^Sanitized. Does not contain TSCA CBI (Jj INHALATION: EMBRYO-FETAL TOXICITY^fiND TERATOGENICITY STUDY IN THE RAT HASKELL LABORATORY REPORT NUMBER 881-81 I. SUMMARY ^l^----------f--|was administered to rats by inhalation as a dust (whole body exposure) from Days 6 through 15 of gestation at nominal concentrations of 0, 0.1, 1.0, 10.0, and 25.0 mg/m3. The actual mean concentrations achieved were Oa'nd'about 0.14 , 1.2, 9.9, and 21.0 mg/m3, respectively. Maternal deaths occurred at the 25.0 mg/m3 concentration only, but overt toxicity was evident among the surviving dams and among those of the 10.0 mg/m3 group. A teratogenic response was not demonstrated upon sacrificeof the dams on of^d Day 21 of gestation at any concentration tested. Embryo-fetal toxicity was noted only at the^S^O mg/m3 concentration which was overtly toxic to the dams. Other than for a temporary reduction in the body weight of the pups raised by additional dams in the 25.0 mg/m3 group, no adverse effect that was concentration-related was noted among the till dams or their offspring weaning. At the 10.0 mg/m3 lesvcheel,dyuilVedffwsaascrsiftiicUe after toxic but not lethal to the dams; however, no-^dversefSS^ related effect was noted among their offspring. Hence, in this study, gptjwas not demonstrated to represent a unique hazard to the conceptus. II. INTRODUCTION A. Background ^------^gy^^^g^^^^^^Q 3M Company for use ^n the manufacture of a variety oif fluoropolymer dispersions, including some of Du Font's Teflon products. A study of^the embryo-fetal toxicity and teratogenic potential ofUjpfwas requested by E. D. Champney, Polymer Produces Department, at a meeting held at Haskell Laboratory on April 9, 1981. This request was initiated in response to TSCA, Section 8(e)'s filed bv^SM between the last part of 1980 and March 20, 1981 onfUBlTfand on several related chemicals.1 The possTb% teratogenic activity reported to us by 3M included lens changes in the eyes of the - 7 - .CompanySanitized. Does not contain TSCA CBI ") l<----y[NHALATION: EMBRYO-FETAL TOXICITY .--AND TERATOOGGEE] NICITY STUDY IN THE RAT HLR 881-81 A. Background (cont) near-term offspring of rats exposed to the test chemi cals by gavage from Days 6 through 15 of gestation. It was not determined whether the lens changes persisted after birth of the rat offspring. B. Protocol (Appendix A) it A draft protocol was distributed on April 24, 1981, and an MR request was signed on May 6, 1981; the protocol was issued on June 11, 1981, and on December 3 and on .December 30 , 1981. was amended C. Purpose This study was designed^ to^determine whether the reported teratogenicity of[^BBJin the rat would be expressed after exposure by the inhalation route and, if so, to establish an apparent "no-effect" concen tration for the conceptus and to determine whether - cghaainnegdeswpasertsoisati,daftienr ebstiartbhli.shmTehnet information of workplace standards for women of childbearing potential. III. MATERIALS AND METHODS The study consisted of two experiments (Tabi^ I 1. Experiment I was a teratogenicity study in whichiffcwas given by inhalation, and the dams were sacrificed oir~the day before expected delivery (Day 21 G). The dams for Experi II ment were exposed as in the first experiment, but were till allowed to give birth and the offspring were maintained 35 days postpartum (Day 35 PP). A. Test Material 1. Physical charact ^ which sublimes at1 is nd its struct The""pur'lty of the sample contaminantspresent wer ^were presen TCS , formula ed was' a white powcular weight nd and or additfves - 8 - pompanySanitized. Does nol contain TSCA CR NHALATION: EMBRYO-FETAL TOXICITY AND TERATQGENICITY STUDY IN THE RAT ;R 4129-OOlf HLR 881-81 Source^- The\----(sample --------Q^as supplied by _ tE^Polymer Products Department. It was assigned Haskell Number 14,045. 3. Test concentrations - The Approximate Lethal Concentration of ^Bl^irT rats was 0.8 mg/L (800 mg/m3) after a single 4-hr exposure period by inhalation (head only) as a dust. Liver enlargement and corneal opacity resulted (1). ^--fwas administered to 7-8 week-old male Crl:(-D rats via inhalation as a dust at nominal concentrations of 0, 1, 8, and 80 mg/m3. Exposure (head only) was for 6 hr/day, 5 days/ week, for two weeks (2) . At the highest exposure level, some rats died and among the survivors, body weight was decreased, and liver weight was increased; these effects were not noted at the 8 mg/m3 concentration. In addition, the serum alkaline phosphatase level in the 8 and 80 mg/m3 groups was elevated significantly above the control value (3). Based upon this information and that gained from rats, a thberijeCflpperxeptoessut rein hon-piregnant female. levels selected for testing inthe current study were 25.0, 1.0, 0.1, and 0 mg/m3. After the first of two "Runs" was conducted, the 25.0 mg/m3 exposure concen tration was replaced by one at 10.0 mg/m3 in response to severe toxicity expressed at the initial concentration. B. Animals The rat was chosen 'J^or this test because previous toxicity testing oneg^Qwas conducted in this species. The Crl:CD (SD)BR sl?iain was selected because the pre liminary teratogenicity test on^l^B^conducted for 3M used the Sprague-Dawley (SD) derr^eST rat obtained from the same supplier, and because extensive background information from previous teratogenicity testing at Haskell Laboratory exists on this rat strain. 9 - g ^ Sanitized. Does not contain TSCA CB1 IINHALATION: EMBRYO-FETAL TOXICITY -AND TERATQfiENICITY STUDY IN THE RAT HLR 881-81 B. Animals (cont) II, Female rats about 55 days of age (nulliparous) were received from Charles River Breeding Laboratories, Inc., North Wilmington, Massachusetts. For Run I, they arrived on April 16, 1981, and weighed between 151.3 and 190.1 g. For Run they arrived on May 1, 1981, and weighed between 152.7 and 188.0 g. Male rats of the same strain and from the same supplier were used for-cohabitation with the ...females. .They ranged in.:age from, that .of the females to about one. month older. . . Upon arrival at Haskell Laboratory, each female rat was identified by a combination of toe clips and ear punches and by a cage card bearing its assigned number. The male rats were identified by ear slash and cage card. The rats were housed two per cage in suspended, wire-mesh, stainless steel cages. Purina Certified Rodent Chow 5002, Checkers and water from the Wilmington Suburban Water Corporation (WSWC) were supplied ad libitum. A lighting cycle of 12 hr light: 12 hr dark (dark period was from 6:00 P. M. to 6:00 A. M.) was maintained throughout the study. Animal room temperature was maintained between 72 and 77F, and relative humidity was maintained between 36 and 70%. Since the historical incidence of cataracts or opacities in adult CD rats is about 3% (personal communication with James Clinton, V.M..D? consultant ophthalmologist), all prospective parental rats were examined for these alterations before breeding. The eyes of each rat were dilated with 1% atropine ophthalmic solution and examined in-semidarkness by the_, consultant ophthalmologist using focal illumination, indirect ophthalmoscopy, and, when indicated,' slit- lamp microscopy. Affected rats were eliminated from the colony before the breeding began. C. Experimental Design and Procedures (Table I) The female rats were quarantined for 11 days (Run I) or 12 days (Run II) after arrival at Haskell Laboratory and then they were mated to .-he;._roal.RS identified under III.B. on an as-needed basis. Mating was verified by - 10 - Companysanitized. Does not contain TSCA CBI ^MINHALATION: EMBRYO-FETAL TOXICITY <TD TERATflfiENICITY STUDY IN THE RAT HLR 881-81 C. Experimental Design, and Procedures (cont) it detection of spermatozoa in the vaginal lavage each morning following overnight cohabitation. The day on which spermatozoa were detected was designated as Day 1 April of 28 gestation (Day 1 G). For Run I, Day 1 G and 29, 1981, for Breeding Lots A and B, was respectively; for Run II, was May 13, 14, and 15, 1981, for Breeding Lots A, B, and C, respectively. After the necessary number of females were bred for each "Run" and before exposure to tf^ began, the mated females were ranked by body weight and assigned to groups by rotation in order of rank. The exposure group that the first animal was assigned to was selected randomly.. II, For Experiment I (females sacrificed before parturition), a total of 24 mated, females were to have been assigned to each group (12 females/group/Run). However, due to the degree of maternal toxicity in evidence in the 25.0 mg/m3 group in Run I, tration was reduced to 10.0 mg/m3 for Run this concen and 15 mated females were assigned to this new group. Further more, two more control groups (six mated females/group) were added to Run II; one was pair-fed to the 25.0 mg/m3 group, and the other was pair-fed to the 10.0 mg/m3 group. For Experiment II (females allowed to give birth) in Run I, 12 mated female rats were distributed to each group. It was not intended that more dams be included in this experiment, but with the addition of the 10.0 added to mg/m3 both I group in Run the control and I , six mated females were the 10.0 mg/m3 groups. These were all the mated females available unless the exposure period was extended for at least six more days. The exposure of animals toMHyy inhalation was conducted by the Acute InvestigawLons Section, Haskell Laboratory. The inhalation route was selected because it to is be the route by which Du Pont employees exposed toQfQ The exposure period are was likely 6 hr/day from Days 6-15 G." Neither feed nor water was available during the exposure period. The procedures used for generation of^fyxposure levels and measurement of the concentrations and particle size attained are attached (Attachment 1). . 11 .Company Sanitized. Does not contain TSCA CB1 [INHALATION: EMBRYO-FETAL TOXICITY 'AND TERATQGENICITY STUDY IN THE RAT HLR 881-81 C. Experimental Design and Procedures (cont) The test groups were exposed (whole body) to the^----(\ in 150-liter-glass and stainless steel Rochester-type chambers within which the rats were housed individually in wiremesh modules. Breeding lots within exposure levels were rotated w^tlun the chambers daily. Chamber concentra tions ofI^Bflwere determined by gravimetric analysis either eacn" one-half hour (intermediate and high levels) or each hour (low level) , and by a spectrophotometric technique (on each low level sample, and on 5-6 samples per exposure day for the other levels tested). A cascade impactor was used to determine the particle size attained only for the high concentration group on the first and tenth exposure days for Run I, and on the seventh and tenth exposure days for Run II. 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 for each day during the exposure period. After each exposure session, the rats were housed in suspended, wire-mesh cages (two females/cage), and the racks holding these cages were placed in a walk-in hood. Since air-flow was vertical, the rats for each group were caged vertically such that the control group was on the end with the low level next to it. Baffles were present between cages, but, to further minimize the likelihood of airborne cross-contamination, a single baffle was placed between the rack containing the groups exposed to the two highest levels and the rack containing the low level group and the controls. For Experiment I, the dams were weighed on the day of arrival, before breeding, and on Days 1, 6, 9, 13, 16,. and 21 G. They were observed for clinical signs and changes in demeanor upon arrival at Haskell Laboratory, at breeding, and daily from Days 6-21 G. Feed consumption was measured during gestation (two females/cage due to till till till space restriction) . The dams were coded, from before sacrifice by cervical dislocation on Day 21 G, all maternal and fetal data were collected and all structural alterations noted among the fetuses were classified, so that personnel involved did not know the exposure group to which any dam or fetus belonged. The identity of each fetus was retained at least the report was written. After sacrifice, the dams were - 12 - I^nRanx|aniti2e(DLoes not contain TSCA CBI 04 INHALATION: EMBRYO-FETAL TOXICITY --AND ENICITY STUDY IN THE RAT HLR 881-81 C. Experimental Design and Procedures (cont) examined for gross pathologic changes, liver weight was recorded, and reproductive status 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 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 lutea. About one-half of the fetuses of each litter that 'were alive when removed from the dam were examined for visceral alterations (4) ; in addition, all stunted or malformed fetuses also were examined similarly. The heads of all fetuses examined for visceral alterations were fixed in Bouin's solution to permit examination as described by Barrow and Taylor (5), but only those from the 25.0 mg/m3 and control groups (Run I) were sectioned free-hand and examined under a stereoscope. This included examination of a vertical cross-section through the center of the eyes as well as of one in front of the eyes and another through the widest portion of the head. Sections containing the eyes of three fetuses from each litter of the 25.0 mg/m3 group and of two fetuses from each litter of the control group were processed one fetal histologically head from each for examination. In of four litters from Run the II, 10.0 mg/m3 group and the control group were examined under a stereoscope after free-hand sectioning in front of and behind the eyes, and through the widest portion of the head. The eyes were left intact to minimize processing artifacts. The slices containing the intact eyes were processed histologically and examined. In addition, the heads from all fetuses in the group pair- - 13 - Sfimpanx Sanitized. Does not contain TSCA CB1 CNHALATION: EMBRYO-FETAL TOXICITY ^AND TERATQGENICITY STUDY IN THE RAT HLR 881-81 C. Experimental Design and Procedures (cont) fed to the 25.0 mg/m3 group and the heads from two fetuses with dark eyes detected during external examina tion . (one from the 0.1 mg/m3 group and one from the 10.0 mg/m3 group) were processed by the method described for Run I. Histologic specimens were examined by light microscopy. All fetuses, except for the heads of those that were fixed in Bouin's solution, 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. On an as-indicated basis at sacrifice, some tissues were fixed in Bouin's solution for storage or for histologic evaluation. till For Experiment II, the procedures used Day 21 G were the same as for Experiment I, except that the dams were weighed less frequently during gestation (only on Days 1, 6, and 21), feed consumption was not measured, and the identity of each offspring within litters was not retained. Before expected parturition, each dam was housed in a 13" x 15" polycarbonate cage (with a wire- mesh lid)that contained Bed-0-Cobs (1/4" size). The bedding was changed weekly following the seventh day postpartum (Day 7 PP). The date of parturition was noted it and was termed Day 1 PP. Each parturition day was considered to begin at 9:00 A. M. The dams were weighed and examined for clinical signs on Days 1, 7, 14, and 22 PP. For each test group, a Fertility Index (% matings resulting in pregnancy) and a Gestation Index (% pregnant resulting in live births) were calculated, and for each litter, a Viability Index (% animals born that survived to Day 4 PP) and a Lactation Index (% animals alive at four days that survived to Day 22 PP) were calculated. All dams were sacrificed on Day 22 PP. The pups from each dam were counted, sexed, weighed, and examined for external alterations toward the end of Day 1 PP. Thereafter, each pup was weighed and inspected for adverse clinical signs on Days 4, 1, 14, and 22 PP; pups with adverse signs were marked for subsequent identi fication. Neither standardization of litters nor crossfostering was practiced. The eyes of the pups in all groups of Run I (Experiment II) were examined by a consul tant ophthalmologist on Days 15, 16, or 17 PP shortly - 14 - Does not contain TSCA CBI INHALATION: EMBRYO-FETAL TOXICITY TERATDGENICITY STUDY IN THE RAT HLR 881-81 C. Experimental Design and Procedures (cont) after the eyes opened. This examination was conducted with the exposure levels coded. The eyes were first dilated with atropine and then examined in a semidark room by focal illumination, indirect ophthalmoscopy, and, when indicated, by slit- lamp microscopy. At sacrifice on Day 35 PP. each pup was exsanguinated with a guillotine, and its eyes were removed and fixed in Bouin's solution. D. Statistical Evaluation The litter was used as the experimental unit for the purpose of statistical evaluation (6). The significance of differences in the incidence of pregnancy, clinical signs, and maternal death was determined by use of Fisher's exact probability test (7). A two-way analysis of variance was used to detect differences in feed con sumption among, breeding lots and among test groups. The significance of differences in feed consumption among groups was determined by a one-way analysis of variance. Dunnetf's test (8) was used to test the statistical signif icance of differences between the control and experimental 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 a concentration-related response in the incidence of structural alterations and other parameters was determined by Jonckheere's test (9). The significance of differences in incidence between the control group and individual experimental groups was determined by application of the Mann-Whitney U test (10). When more than 75% ties occurred in the data, the Fisher's exact probability test was applied (11). The level of significance selected was p<0.05. In addition, several reproductive indices were calcu lated for some results from Experiment II. IV. RESULTS A. Eye Examination of Prospective Parents The eyes of 291 male and 357 female rats were examined for Run I on April 24, 1981; 11 males and seven females - 15 Pfimpany .Sanitized. Does not contain TSCA CBf M NHALATION: EMBRYO-FETAL TOXICITY ANI7 'TERATOCENICITY STUDY IN THE RAT HLR 881-81 A. Eye Examination of Prospective Parents (cont) II, were removed from the colony because their eyes were not normal ophthalmoscopically (Attachment 2). For Run 210 females were similarly examined on May 5, 1981, and 13 were discarded (Attachment 3). The same males were used for both.runs. B. Maternal Exposure tofMBH The animal exposure levels achieved were reported in detail by the Acute Investigations Section (Attach ment I). In centrations brief, for for 6 hr/day Run I, from the Days nominal exposure con 6-15 G were 0, 0.1, 1.0, and 25.0 mg/m3; the average daily concentrations achieved (x + S.D.) by gravimetric measurement were 0, 0.14^ 0.06, 1.2 j^ 0.5, and 21.0 j^ 9.7 mg/m3, respectively. The average mean daily temperature in the chambers was about 76F and ranged between 72.0 and 78.5F. II, For Run the nominal exposure levels were 0, 0.1, 1.0, and 10.0 mg/m3; the values similarly achieved were 0, 0.12 +_ 0.04, 1.1 ^ 0.6, and 9.9 ^ 5.4 mg/m3, respectively. The average mean daily temperatures in the chambers were between 76.4 and 77.3F, and indi vidual temperatures recorded ranged between 72.2 and 78.6F. For the two runs, between 73 and 88% of the fUS particles in the chambers were respirable (Attacnmeht 1) C. Clinical Signs Observed In Experiment I, clinical signs noted during the period of exposure that were concentration-related appeared only in the dams of the 10.0 and 25.0 mg/m3 groups. In both groups most of the dams developed wet abdomens, which began in the perineal area, had chromodacryorrhea and chromorhinorrhea, and were unkempt. In addition, three of the 12 dams in the 25.0 mg/m3 group died, and four of the remainder became very lethargic toward the end of the exposure period. Focal alopecia was somewhat more prevalent among the dams of - 16 [5an/feed. Does not contain TSCA CBI NHALATION: EMBRYO-FETAL TOXICITY GENICITY STUDY IN THE RAT HLR 881-81 C. Clinical Signs Observed (cont) the 10.0 and 25.0 mg/m3 groups (5/15 and 4/12) than among those of the control group (3/24), but the differences were not statistically significant. In Experiment II, concentration-related clinical signs appeared only in the same two groups, and they were similar in type and incidence. Again, in the 25.0 mg/m3 group, maternal deaths occurred (2/12 females). No adverse clinical signs were noted among the dams of the pair-fed control groups. D. Maternal Feed Consumption II). Feed consumption During the period of was measured, onLv in exposure to[(B^,j the Experiment feed con I. sumption of the dams in the 10.0--rncf""25.0 mg/m3 groups was significantly less than that for the control group (Table Feed consumption returned to the control value in the post-exposure period. No significant differences were noted in this regard between the control group and J-heoroups exposed to the two lowest concentrations ofj----J The average amount of feed consumed by the pairfed control groups was not different statistically from that of the groups to which each was matched (Table II). E. Maternal Body Weight Gain 1. Experiment I III). The body weight gain of the dams exposed tci^^ly at the 25.0 mg/m3 concentration was significantly less during the exposure period (Days 6-15 G) than for the control group (Table Although the 10.0 mg/m3 group also gained less weight during the exposure period than the control group, the differ ence was not statistically significant. Body weight gain from D.ays^J.6-21 G was similar for the groups exposed tow--I/and fb'r the control group. The body 17 i^OTpMy.SanrtizedD.oes nof contain TSCA <"'?( <teBINHALATION: EMBRYO-FETAL TOXICITY ^-AND ENICITY STUDY IN THE RAT HLR 881-81 E. Maternal Body Weight Gain (cont) 1. Experiment I (cont) . weight gain achieved by both the 10.0 and 25.0 mg/m3 groups after Day 6 G was at least as much as that gained by their respective pair-fed control groups. 2. Experiment II From Days 6-21 G, the^naternal body weight gain of the groups exposed to--^gfchat were not sacrificed at term was not significantly different from the control value (Table VI). F. Maternal Deaths In this^study, five dams died; all were in groups exposed to ^^fet the 25.0 mg/m3 concentration (3/12 in Experiment I, and 2/12 in Experiment II). =o{--^ In Experiment I, the dams were found dead on Days 12, 13, and 17 G (Table IV). The first was not necropsied; both of the others had grossly observable liver changes, and their uteri contained only resorbed fetuses. The probable cause of death of the dam found dead on Day 17 G was circulatory collapse. All three dams died after showing considerable body weight loss, an unkempt appear ance, a wet abdominal surface, and chromodacryorrhea and cAhc^o-ommoorijl^iiinorrhea beginning on to^----rjbr shortly thereafter. the first day of exposure and In the Experim second ent wa s I I, au t one ops i dam was ed on D a found y 11 G dead when on s h D e ay b e 9 ca G m e moribund (Table VI). The uteri of both contained implantations. Their clinical signs-preceding death were similar to those described above. G. Gross Examination of Maternal Organs at Sacrifice In Experiment I, dark noted for two of the dams one dam in the 10.0 mg/m3 rectum and pelvic muscles 1.0 mg/m3 group. red mottling of the lungs was in the 0.1 mg/m3 group and in group. A hematoma between the .was noted in one dam in the - 18 SompanySanitized. Does not contain TSCA CBI INHALATION: EMBRYO-FETAL TOXICITY ,AND TERATOGENICITY STUDY IN THE RAT HLR 881-81 G.;. Gross Examination of Maternal Organs at Sacrifice (cont) III). In addition, in the 25.0 mg/m3 group, actual liver weight was significantly increased (Table The liver weights of the control groups that were pair-fed to the 10.0 and 25,^.mg/m3 groups were significantly less than those for the^tltflgroups to which they were paired, and than that for tne "control group (Table III) . On a relative weight basis (using corrected Day 21 G maternal body weights) , groups exposed ttohJeBliSvle&rt weights (x +_ S.E.M.) 10.0 and 25.0 mg/m3 for the (5.42 +_ 0.125, and 6.46 + 0.222 ,f^spect2.vely) were still significantly more (MWU - two-tailed) than that for their respective pair-fed control groups (4.58 +_ 0.164, and 4.62 j^ 0.103). On the same basis, all groups differed significantly from the control group. II, In Experiment the dams were examined for clinical signs and weighed on Day 22 PP (Table VI); they were then sacrificed and discarded. H. Reproductive Effects and Body Weight of the Offspring In Experiment I, the maintenance of pregnancy and the incidence of resorptions among th^ surviving dams were not adversely affected by exposure toH^gat concentrations up to and including 25.0 mg/m3 (Table'llI) . In Experiment II, nsotraatdevdersthe aet ffweac^t^--o--rL--,refcpornocdeuncttriavteiopne-rrefolartmeadnc(eTawbales VdeIm) o. n Parameters examiTTed Acluded Fertility Index, Gestation Index, Viability Index, and Lactation Index. II, III). The mean body weight of fetuses in the 25.0 mg/m3 group was significantly decreased (p = 0.002) below the control value (Table III); but, this also was the case (p = 0.001) for the control group pair-fed to the 25.0 mg/m3 group (Table The mean weight of the fetuses in the 10.0 mg/m3 group, and in its pair-fed control group, were not significantly different from the control group (p>0.23). In Experiment the neonates in the 25.0 mg/m'7 group also were significantly smaller than those in the control group (p = 0.002) but by Day 4 PP, the difference was no longer statistically significant (Table VI). t eWa^San/feed. Does not contain TSCA CBI - 19 - J------JINHALATIOEMNB:RYO-FETAL TOXIC ITY .AND T.ERATQ.GENICITY STUDY IN THE RAT HLR 881-81 I. Fetal Alterations 1. Fetal Malformations jconcentration-related increase in the incidence of external, visceral, or skeletal malformations was not detected (Table IV). No malformations-were seen among the fetuses from dams exposed toQIRat 25.0 mg/m3. Similarly, coded stereoscopic andTiistomorpho logic examination of fetal eyes from heads that were fixed in Bouin's solution did not reveal concentration-related structural alterations (Attachments 4 and 5). 2. Fetal Variations The overall "percent fetuses with variations" per litter did not increase significantly with increased concentration offB9 (Table V) . This was the case also for the two specific components of the total incidence v.i.z. "developmental variations," and " "variations :-due to retarded development. " Among the "developmental variations" a positive concentrationresponse.;also was not obtained for the incidence of subcutaneous hemafcomas; however, in the 25.0 mg/m3 group, their incidence was significantly increased (p = 0.05), but only upon application of the onetailed Mann-Whitney U test. Among the "variations due to retarded development," only the incidence of partially ossified sternebrae in the 25.0 mg/m3 group was similarly increased (p = 0.04). Again, the incidence of partially ossified sternebrae was not concentration-related (Jonckheere's test). In the control group that was pair-fed to the 25.0 mg/m3 group (Table V), the incidence of partially ossified sternebrae was significantly increased (p = 0.04 by the two-tailed MWU test) as was the incidence of variations regarded as being due to retarded development (p = 0.02). J. Pup Alterations Only one pup was observed to be malformed externally. It occurred in the 0.1 mg/m3 group (Table VI). On - 20 - Sornp^ Sanitized. Does not contain TSCA CBI (jpBtflNHALATION: EMBRYO-FETAL TOXICITY ^.AND TERAIiQCENICITY STUDY IN THE RAT X----H--------U HLR 881-81 ^ -J J. Pup Alterations (cont) it it Day 19 PP, was noted to have severe hydrocephaly and an abnormal gait. Since it was moribund, was sacri ficed the next day. II On Days 15, 16, or 17 PP, coded examination of the pups' eyes in vivo from Run I did not reveal concen tration-related malformations. Eye changes were detected in six pups (Table Viand Attachment 6).; -three of. these occurred--in' the" control- .gro-up;. and the remaining three occurred in the 0.1 mg/m group. In'view of these nega tive" result's, similar in vivo examination of the eyes of the pups from Run w-a-s not conducted. II On Day 35 PP, all of the pups were sacrificed, and their eyes were fixed in Bouin's solution. Since concen tration-related eye changes were not detected t:o this stage, in either the fetuses or the pups, pathologic examination of the eyes of the pups of Runs I and was not conducted. V. DISCUSSION In this study, exposure t(^----(at a concentration of 25.0 mg/m3 for 6 hr/day from Days (?-15 G -was overtly toxic to rats in that 5/24 did not survive to term, and most of the survivors had'-:wet abdomens, reddish-brown discolor ation around the eyes, nose, and mouth, lethargy, decreased feed consumption and body weight gain during the exposure period, andan.unkempt appearance. None of the 21 dams exposed toj----Tttt 10.0 nig/in3 died, but they showed similar clftrtAA signs to a lesser degree than that seen at the 25.0 mg/n^J.evftl. Despite this degree of maternal toxicity, no evidence ofiff^relafced teratogenicity was detected. The fetuses from the 25.0 mg/m3 group were significantly smaller than those from the control group. This probably was due to the decrease in j^ateaaial feed consumption rather than to a direct response tcyBJPjsince the fetuses of the control group pair-fed to the 2-?.0*fflg/m3 group also were significantly smaller than the control fetuses. Similarly, decreased maternal feed consumption also was probably responsible for the significant decrease in weight of the neonates in Experi ment II. /^. - 21 - iCompanfSanitized. Does not contain TSCA CBI 1INHALATION: EMBRYO-FETAL TOXICITY 'AND TERATQCENICITY STUDY IN THE RAT HLR 881-81 V. DISCUSSION (cont) relatiMvaetewrneaigl hltivbearsisweiingdhitcactheadngeths aatmeoxnpgogsuroruepstc^onHlaS 10.0 mg/m3 or more resulted in significantly larger livers than in the control group. This effect also was noted in previous toxicity tests with||f|(1, 2, 12, 13, 14) and probably represented fatty degeneration accompanied by enzyme induction. This increase in liver weight occurred despite the significant decrease in body weight gain in these groups which significantly reduced the relative liver weights of the pair-fed control groups. ofjfjor The incidence of resorptions was not ycraased above the control value at any test concentration among the pair-fed control groups. The statist! callysignificant decrease obtained in the 1.0 mg/m3 group was not concen- Jbra,i on-related and hence was not'demonstrated to be due to --4 The incidence of partially ossified sternebrae was significantly increased among the fetuses of the 25.0 mg/m3 group by the Mann-Whitney U test (one-tailed). This varia tion is indicative of developmental delay. Since its inci dence to be was not concentration-related directly due to ^^ILlexposure. it was not demonstrated It also was probably related to the decreased feed consumption of the dams, since its incidence was even more frequent among the control dams pair-fed to the 25.0 mg/m3 group. Two fetuses were observed to have reddish eyes at sacri fice. Histomorphologic examination revealed hyphemia or the presence of blood in the anterior chamber of the eye (Attach ment 7). Their incidence was not concentration-related since one occurred in the 0.1 mg/m3 group and the other in the 10.0 mg/m3 group. . This alteration occurred in seven of- 6706 fetuses (0.10) from six of 681 litters (1.0%) among the control litters of past studies at Haskell Laboratory since March, 1976. Subcutaneous hematomas were detected in all groups including the control groups . The difference in incidence between the 25.0 mg/m3 group (7.6%) and the control group (4.0%) - 22 CompanySam-feed. Does nof contain TSCA CW NHALATION: EMBRYO-FETAL TOXICITY TERATOGENICITY STUDY IN THE RAT HLR 881-81 V. DISCUSSION (cont) was statistically significant but only upon application of a Mann-Whitney U one-tailed test. In addition, the incidence of fetuses with hematomas in the 25.0 mg/m3 group did not exceed the range of incidence contained among the control groups of past studies (4.6-9.0%) at Haskell Laboratory. Therefore, it is not likely that the magnitude of this difference would be repeatable. Moreover, even if the increase was repeatable, its occurrence would likely depend more upon the presence of t,fae~ covert toxicity in this group than upon a direct effect of^B^^er se. VI. CONCLUSION Cyfffwas not demonstrated to be teratogenic even when administered to rats at overtly toxic concentrations from Days 6 through 15 of gestation. Concentration-related embryo-fetal toxicity, expressed as decreased fetal weight, occurred only at the highest concentration tested (25.0 mg/m3) which was overtly toxic to the dams. This decreased body weight persisted to Day 1 postpartum but not to Day 4 postpartum. After exposure toQiqat the 10.0 mg/m3 concen tration, which, was still toxic buT'not lethal to the dams, no adversefli^yrelated effect was noted among their offspring. Among the fetuses of the 25.0 mg/m3 group, a marginally significant increase in the incidence of hematomas also jc<u,rred; the increase was not demonstrated to be due to 5er se. Hence, in this study,fWwas not demonstrated to represent a unique hazard to T'ne'conceptus. VII. ACKNOWLEDGEMENTS The concentrations offftf^for inhalation were generated, monitored, and analyzed by Vfe^cvite Investigations Section, Haskell Laboratory. Histologic specimens were prepared by the Pathology Section, Haskell Laboratory. Histomorphologic examination of the eyes and other tissues was conducted by William D. Kerns, D.V.M. , M.Sc. The in vivo eye examinations were conducted by James M. Clinton, V.M.D., Consultant in Comparative Ophthalmology. The remainder of the study was conducted by the Teratology Section, Haskell Laboratory. - 23 - S^QWiy. Sanitized. Does not contain TSCA CBI [NHALATION: EMBRYO-FETAL TOXICITY FAND TERATQGENICITY STUDY IN THE RAT HLR 881-81 VIII. REFERENCES 1. Unpublished Du Font Data, Haskell Laboratory: 2 .^Jnpublishf^d Dn Pont Data, Haskell Laboratory: 3 .U1--np--ub--li--sh--eCd--linD]uicaPol nPt aDthaotalo, gyHaRsekpeollrtL, a8b/o1r3a/t8o0ry. : 4. Staples, R. E., "Detection of visceral altera tions in mammalian fetuses." Teratology, 9:A3 7 (1974). 5. Barrow, M. V., and W. J. Taylor, "A rapid method for detecting malformations in rat fetuses." J. Morph., _12_7(3);291-306 (1969). 6. Haseman, J. K., and M. D. Hogan, "Selection of the experimental unit in teratology studies." Teratology, 3_2_:165-172 (1975). 7. Siegel, S., Nonparametrie Statistics for the Behavioral Sciences, McGraw-Hill, New York, pp. 96-104 (1956). 8. Steel, R. G. D., and H. H. Torrie, Principles and Procedures of Statistics, McGraw-Hill, New York, pp. 99-128 (1960). 9. Jonckheere, A. R., "A distribution-free K-sample test against ordered alternatives." Biometrika, 4JL:133-145 (1954) . 10. Mann, H. G., and D/ R. Whitney, "On a test of whether one or two random variables is sto chastically larger than the other." Ann. Math. Stat., 1^:50-60 (1947). 11. Haseman, J. K., and D. G. Hoel, ""Tables of Gehan's generalized Wilcoxon test with fixed point cen soring." J. Statist. Comput. Simul., 3:117-135 (1974) . - 24 - ^"Pany sanitized. Does notcontatn TSCA CB INHALATION: EMBRYO-FETAL TOXICITY TERATQSENICITY STUDY IN THE RAT HLR 881-81 VIII. REFERENCES (cont) 12. JJnpublished Du Font Data, Haskell Laboratory: 13 .Unpublished Du Font Data, Haskell Laboratory: 14 .Jnpublished Du Pont Data, Haskell Laboratory: Company Sanitized. Does not contain TSCA CW 25 - TAE?LE I EXPERIMENO."'AL DESIGN; EXPOSURE LEVELS AND DISTRIBUTION OF MATED FEMALES AMONG TEST GR RnrU-rKNT I-r- Exposure Levels (ing/m3) 0 -- --- ^Q 25.0 Number Mated Females/Group Experiment I1 Experiment II2 12 12 12 12 12 12 12 12 Exppsure Perio Days; Days Days; Days; 6-15 6-15 6-15 6-15 G3 / G /-- G 1I G (-- 0 12 I ^ I 0.1 12 RUN 12 II 15 (0 I PEXO;..4 6 I PF25.05 6 \ sf f 6 Days; 6-15 G ( Days, 6-15 G Days:6-15 G 6 Days:6-15 G Days 6-15 G l Days 6-15 G ( II 0 1 all Experiment I females sacrificed on Day 21 G (Runs I and II 2 all Experiment females allowed to give birth and to raise t o offspring to at least Day 21 postpartum (Runs I and II) B> G - day of gestation 3" 4 control group pair-fed to group givenM|1jat 10.0 mg,/m3 W 0 5 control group pair-fed to group given----W|aatt 25.0 mg;/m3 0 01 TABLE II FEED CONSUMPTION3 IN RATS EXPOSED TdflBgFRODMAYS 6-15 OF GESTAT Experiment I _________Exposure Concentrations (mg/m3)__________ 0.0 0.1 1.0 10.0 25.0 Pre-exposure period (Days 1-5 G) Exposure period'1 (Days 6-15 G) Post-exposure period'1 (Days 16-20 G) . 20.80.38 23.40.38 27.90.78 21.00.33 24.00.52 30.01.24 21.10.35 23.00.45 28.3t0.64 20.80.37 21.80.37(() 27.1t0.74 19.00.52 18.40.46(f' 27.4+0.52 ___ 20.3 20. 26. a - gtS.E.M.; values for non-pregnant rats were excluded b - nominal concentrations oEufHf c - on each day of gestation, UuTi'exposed dams were given the amount of feed consumed gestation day by selected rats in the corresponding exposure group; if the expo housed two per cage, then their average consumption for each day was the amount the pair-fed rat d - during the exposure and post-exposure periods, the rats were caged in pairs; the unit used was: total grams feed consumed per cage per day number of rats per cage (() - significantly different from control value by Dunnett's test (p^O.05) TABLE III REPRODUCTION ^\ND F ETAL DEVELOF MENT IN RATE EXPOSED T0< ^ FROM DAY S 6-15 OF Exposure (Soncentratioris8 (mg/m3) Experiment I 0.0 0.1 1.0 10.0 25.0 P 10.0 Females No. pregnant / no. mated 23/2 4 24/24 23/24 15/15 8/12 6/6 No. deaths 0 0 0 0 3 0 No. litters 23 24 23 15 7 6 Mean no. corpora lutea 14.50 ^ 14.30.51 15.60.58 15.20.65 15.11.42 13.80.48 1 Mean no. implants 14.00 .51 13.40.41 13.80.28 14.2i0.22 14.00.62 14.00.63 i\j 00 1 Mean liver , weight(g)8'" 15.20 .30 15.00.30 15.40.35 16.10.50 18.00.78++ 12.80.50 ^ Mean maternal 's weight gain (g) "0 Days 1-5 29.11 .99 29.1tl.01 32.511.00 29.01.39 27.21.97 26.21.11 f5 (/> Days 6-15 57.61 .69 57.62.06 56.71.95 50.92.21 36.4t5.330 34.93.21( I i' Days 16-21 71.42 .19 68.91.92 72.81.97 72.9t2.77 68.63.41 71.34.72 i <D Q. Days 6-21 129.03.39 126.53.28 129.53.12 123.8t2.86 105.017.26" 106.214.7 0 0 Days 6-210^ 56.71 .90 56.92,02 56.4t2.11 ^ 49.12.52 37.45.39 34.32.98 2 (0 3 0 5" 8 ffl 3 CO 0 > 0 5 TABLE III (CONT) REPRODUCTION AND FETAL DEVELOPMENT IN RATS EXPOSED TO tf------ROMDAYS 6-15 OF t ts.) \s l W |a3 (/ . (a 5 P- 0 0 (0 (A 3 0 " |S" g- -^ 0 > 0 S Experiment I Fetal Death Mean no. resorptions Mean % No. litters with total resorption Fetuses No. live Mean no. live No. stunted Mean weight (gf 0.0 1.00.15 6.61.02 0 299 . 13.00.47 1 4.00.04 . Exposure Concentrations 0.1 1.0 (mg/m3) 10.0 25.0 P 10.0 0.80.20 5.31.34 ++ 0.50.12 + 3.80.87 0.70.21 5.21.47 0.80.55 5.93.50 0.70.49 4.12.94 0 0 305 12.70.39 0 3.90.05 305 13.30.28 0 3.90.04 0 0 0 202 13.510.34 92 13.10.70 80 13.30.21 0 1 0 3.90.07 3.60.12 3.90.12 TABLE III (CONT) REPRODUCTION AND FETAL DEVELOPMENT IN RATS EXPOSED TO^BfcFROM DAYS 6-15 O nominal concentrations of flBB on each day of gestation, non-exposed dams were given the amount of feed consum gestation day by selected rats in the corresponding exposure group; if the ex were housed two per cage, then their average consumption for each day was the to the pair-fed rat all females had visible sign of pregnancy evident at autopsy except for one in group in which resorptlons were detected only by ammonium sulfide staining; d this female were excluded from all other calculations ^S.E.M. non-pregnant animals were excluded Day 21C body weight denotes the body weight of females excluding the products o (i.e.. Day 21 corrected body weight) ^ o Se stunted fe'tuses were excluded ' significant dose-related response detected by Jonckheere's test (p^O.05) <(> significantly different from control value by Dunnett's test (p^O.05) + significantly different from control value (one-tailed Mann-Whitney U test, p<0 ++ significantly different from control value (two-tailed Mann-Whitney U test, p<0 (A 0 > 0 9 5 I . w fi) L/-1 15. ^ ' a, 0 0 (D W 3 1 0 S" 5 -( w .^ a- i TABLE IV FETAL Mi^LFORMATK3NS IN RATS1 EXPOSED 1'cffK1^0^. DAYS 6-1;) OF GESTAT L.'-'J Experiment I Exposure Coiicentratiol-is (mg/m3) 0.0 0.1 1.0 10.0 External Malformations No. examined Microphthalmia 299/23 d 305/24 305/23 l/l6 202/15 Visceral Malformations No. examined 159/23 Great vessel malformation Innominate artery-none Renal papilla - none Kidney - very small ^ Head Malformations No. examined I/I3 90/17 161/24 2^ l/l1 161/23 110/15 I/I8 1/1 19/6 Skeletal Malformations No. examined Rib - fused - missing 299/23 l/l3 l/l1 305/24 305/23 202/15 25.0 92/7 51/7 51/7 92/7 Pair- 10.0 80/6 42/6 80/6 LJ N) t'ff 1 O f< w 1R' (D a. 0 0 (D Uf | " M". 3 -vt> 0 > 0 g TABLE IV (CONT) FETAL MALFORMATIONS IN RATS EXPOSED TO ------FROM DAYS 6-15 OF GESTATI Experiment I Total with Malformations Avg. 7, Malformed Fetuses per Litter (S.E.M.) Exposure Concentrations"1 (mg/m3) 0.0 0.1 1.0 10.0 25.0 2/2 3/3 1/1 1/1 Pair-Fe 10.0 0.60.44 1.00.53 0.40.40 0.40.41 nominal concentrations o||^fR if on each day of gestation, non-exposed dams were given the amount of feed consumed same gestation day by selected rats in the corresponding exposure group; the rats were housed two per cage, then their average consumption for each day was amount offered to the pair-fed rat c fetuses/litters d blanks represent zero incidence e fetus weighed 3.38 g; no variations detected f one fetus (3.44 g) also had a misaligned sternebra; the other fetus weighed 4.51 g fetus weighed 3.91 g and also had petechiae h fetus weighed 3.25 g; no variations detected i fetus weighed 3.78 g and also had slight hydroureter , J stunted fetus weighed 2.41 g and also had calloused, thickened, and partially os ribs, petechiae, partially ossified sternebra, misaligned sternebra, pulmonary arteries connected to main pulmonary artery by a common trunk k no head malformations were noted 1 fetus weighed 3.82 g; no variations detected TABLE 1V FIiTAL VARIATIONS! IN RATS EXPOSED TO | ^ ROM DAYS bC-1j.-I); ur ->T-1 u^Ti-.t^STATION Experiment I External Variations No. examined Hematoma Petechia Visceral Variations No. examined Renal papilla. -reduced Renal pelvis -large Pulmonary arteries -common trunk Head Variations No. examined Eye - blood in anterior chamber Exposure Concentrati ons a (mg/m3) 0.0 0.1 1.0 10.0 299/23 12/10 31/13 305/24 16/12 38/17 305/23 11/7 30/16 202/15 5/4 33/14 159/23 I/I13 1/1 5/2 161/24 e 1/1 161/23 4/4 110/15 3/3 25.0 92/7 7^ 9/5 51/7 1/1 90/17 1/1 1/1 19/6 1 /1 51/7 10. 80/ 4/3 16/6 42/6 2/2 fI UB W S. 1 P- 0 LJ^ ? 1 3 g y 0 2 FETAL VARIATION;3 IN RATS TABLE V (CC)NT) ]3XPOSED TOfl----kF'RDM IS.Ui'1 nAVQ JJJ1.I.Q 6u --J1- 5^ \Oj sF. C\-rFjj;STATION Experiment I Skeletal Variations No. examined Sternebra -misaligned(.1)^ -misaligned(2+)f Centrum -bipartite -dumbbelled Rib -extra ossification center -rudimentary -extra -beaded -calloused -wavy Exposure a Concentrations (mg/m3) 0.0 0.1 1.0 10.0 299/23 16/12 6/5 305/24 24/14 12/9 305/23 10/9 13/11 202/15 13/8 6/5 4/4 4/4 6/4 1/1 9/6 3/3 7/6 4/3 25.0 92/7 5/3 3/3 1/1 1/1 50/12 7/6 1/1 41/1511 3/3 1/1 3/2 1/1 39/151 3/3 26/6 3/3 1/1 1/1 22/7 2/2 2/1 10 80 3/ 1/ 2/ 11/4 TABLE V (CONT) FETAL VARIATIONS IN RATS EXPOST-n TO fl|| TTW DAYS 6-15 OF GESTATIO Experiment I Exposure Concentrations (mg/m3) 0.0 0.1 1.0 10.0 25.0 1 Skeletal Variations (cont) No. examined with heads 140/23 144/24 144/23 92/15 41/7 3 Skull bones partially ossifiedd -parietal 3/3 7/4 2/1 1/1 i -interparietal 2/2 12/4- 1/1 4/2 -supraoccipital 3/3 9/5 2/2 1/1 -squamosal 2/2 2/1 2/1 Zygoma -partially ossifieedd I/I 2/2 1/1 Maxilla -partially ossifieedd I/I 1/1 Hyoid .-partially ossifie.edd 11/6.- 9/4 7/5 7/4 -unossified 4/2 15/6"1 13/9 2/2 TABLE V (CONT) FETAL VARIATIONS IN RATS EXPOSED. TO ------ FROM DAYS 6-15 OF GESTATIO Experiment I Exposure Concentrations (mg/m8) 0.0 0.1 1.0 10.0 25.0 1 Skeletal Variations (cont) Subtotal - Developmental Variations No. affected 118/22 123/24 100/23 76/15 44/6 3 Avg. % Affected Fetuses per Litter (S.E.M.) 40.65.16 41.64.06 31.63.84 37.65.08 47.66.62 38. Sternebra -partially ossified 27/9 10/6 13/8 12/6 17/5-'1+ -unossified 3/2 3/2 4/2 Centrum -partially ossified 2/2 Rib -partially ossified 2/1 Ischium -partially ossified 1/1 2/2 Pubis -partially ossified -unossified 4/3 1/1 TABLE V (CONT) FETAL VARIATIONS IN RATS EXPOSED TO I----^ROM DAYS 6-15 OF GESTATIO .EgperimentI ^xg^sur^_Con^ent^at^m_s^(mg/m3) 0.0 0.1 1.0 10.0 25.0 1 Skeletal Variations (cent) Subtotal - Variations Hue to Retarded Development No. affected 45/14 45/11 34/14 17/7 24/6 Avg. 7, Affected Fetuses per Litter (S.E.M.) 14.34.01 14.24.35 10.92.84 8.43.03 25.38.50 8.5 TOTAL No. Fetuses with Variations Avg. % Fetuses with Variations per Litter (S.E.M.) 1^/22 140/24 123/23 87/15 55/6 3 48.75.07 49.44.42 40.13.43 42.85.14 58.45.98 42. TABLE V (CONT) . FETAL VARIATIONS IN RATS EXPOSED TOfflB^FROM DAYS 6-15 OF GESTATIO a - nominal concentrations of ----^ b - on each day of gestation, non-exposed dams were given the amount of feed consumed same gestation day by selected rats in the corresponding exposure group; if the exposed rats were housed two per cage, then their average consumption for each was the amount offered to the pair-fed rat c - fetuses/litters ' d - slight hydroureter also was detected 1-5 e - blank cells indicate zero incidence f - (1) and (2+) denote that 1, or 2 or more sternebrae g - one was located in the cervical region, all others h - 14 occurred in two litters i - 16 occurred in two litters j - 7 occurred in one litter k - 18 occurred in two litters occurred in one litter m - 11 occurred in two litters n - 4 occurred in one litter + - statistically significant difference detected only (p^O.05) were misaligned, were located in if Mann-Whitney respectively the lumbar re U test is one ++- statistically significant difference detected by two-tailed Mann-Whitney U test REPRODUCTION AND DEVELOPMENT OF OFFSPRING OF RATS EXPOSED TO tfkFP;OM DAYS 6-15 Experiment II Females a Exposure Concentrations (mg/m3) 0.0 0.1 1.0 10.0 No. pregnant/no, mated No. deaths No. litters Fertility Index W'1 Gestation Index (%)8 Mean maternal weight gain (g) Days 1-5 G Days 6-21 G Days 1 PP-22 PP 18/18 0 18 100 100 10/12 0 10 83.3 100 11/12 0 11 91.7 100 6/6 0 6 100 100 31.111.^ 112.5t4.33 -3.410.58 31.8t2.08 115.88.07 -2.80.75 31.72.16 116.27.77 -2.0t0.81 28.22.44 118.93.28 -3.10.78 Offspring At delivery (Day 1 PP) No. litters No. live pups/litter No. live pups (%) No. dead pups (%) 18 C 12.30.60, 222(99.6) 1(0.4) 10 12.10.80 121(99.2) 1(0.8) 11 11.21.27 123(98.4) 2(1.6) 6 / 13.30.67 80(100) 0(0) 1'ABLE VI (CONT) REPRODUCTION AND DEVELOPMENT 1OF OFFSPRING CF RATS EXPOS ED TO^^ FRCIM DAYS 6-1- Experiment II a Exposure Concentrations (mg/m3) 0.0 0.1 1.0 10.0 Offspring (cont) After delivery No. dead 1 I11 0 0 No. cannibalized 0 2 0 0 0 No. live (Day 22 PP) 221i 118 123 80 u No. males/females 115/106 62/56 70/53 44/36 ^W Viability Index W3 100 99.2 100 100 1 Lactation Index (7,)^ 99.5 98.3 100 100 R" (D Mean weight (g) 0. 4 0 Day 1 PP' Day 4 PP 3 6.80.11 10.310.25 7.00.18 10.90.32 6.70.18 10.90.39 6.60.23 9.90.43 & Day 7 PP 0 15.00.44 15.610.46 15.9t0.63 14.8t0.69 0 5" Day 14 PP 29.21.06 30.1t0.89 30.6tl.29 28.0tl.23 Day 22 PP -< W 50.11.82 51.4tl.73 52.0t2.46 48.4t2.15 0 > Alterations 0 5 No. examined externally 222/181 121/10 123/11 80/6 No. malformed l/l"1 No. eyes (pairs) examined in vivo 141/12 118/10 123/11 -- No. with alterations 3/3 3/2P 0 -- INHALATION: EMBRYO-FETAL TOXICITY AND TERATOGENICITY STUDY IN THE RAT ------^^ HLR 881-81 a 0 u ri E-l uSo^'a 'a M WS-T33 ^ '--^ 0 g -^J 0 U43T3 ^rt1^'"-1-1'^ ^ ( 0-^^^ OD. 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(c3ori-dl&coiijcQdJt3u0-^i3-i<a4ia-1i G K S T3-ri(U(U nl^ct) (Ucfl(U4J ^;J-J3!-lJ3^^4Jc! 0 D. 0 ri e n 0) h a J-i C 4-1 M a)ai,c;j^ a -(orij34J4J 4-14-lo 4Jn)'ril ('i0j ^3 Ci tf '0 0 3 tO'ClCLCOCOCO (UOOCd-ri-ri 4J.'^>i-ii-Oi(IC0i(UU^(IuHa-1) 'T3>C4J-ri 4J 4-i ix(-;i .a-ri^i an)(U(U(ua) c[j3tti4Jo)a1-4r-1i o3m3p<0&(U.hJ42JMOT3 u o^ CiM S ,c c ^: (3 Uo3>4-J,aMicO!(M0 (M r~)jS4J CL|(U(I)T-)>M}-IOhci,04Jc4icJ; J;?aj " p>(ac3 rC-Il)E(UeOi^)>U-l-rii-)rCrBi-4rJiC4-Ul g 4J[BP.M-r4iJ.roi<C^Mi'i-Jri&.r4j .jWCL(-UriJ4J3-4riJ-rUi-uri CH-)l|-| ri >-| 3 cO-riS (fl 4-1 0) C04-IM 4-1 ri . Q>C,t-rfi^4n)uJ3a.rir-1r4iCJ fl>,r'-^1O3m.Q,&3-4rJi-&riCUd4^-rIi4--ri-4 P-0 CIJ04J -ri-ri COCO Ifl ^3 0 T3 11 1 11 11 11 01 4-) OOJ^ ri T-)^i r-)e C!00. 1H- .CompanySanitized. Does not contain TSCA CBI 41 - CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ELK TON ROAD NEWARK, DELAWARE APPENDIX A PROTOCO. RATS WITH FOR INHALATION TERATOGENICITY STUDY IN & I I. BACKGROUND . . . - This study was requested by fJBMBBMW1^' Polymer Products Department tf--------i at a meeting held at Haskell Laboratory on April 9, 1981. The need for such a study * emanated from TSCA Section 8(e)'s filed by 3M between the last part of 1980 and March 20, 1981. The adverse effects seen in studies conducted for 3M consisted of lens changes in the eyes of term offspring from rats exposed to the test chemicals by gavage from Days 6 through 15 of gestation. For additional infonna- March 30, 1981. draft of HFaosrketlhle LdaebvoerlaotpomryenRt eopfortthftiasl--,p--ro--to--c--o--l, --t--he^! on the known toxicity ofj||----------BB||Bj|^^---------- ----Hg was reviewed, and inrorroation gleaned from 3M also was considered. II. MATERIALS AND METHODS The study consists of two experiments. Experiment I will be a full teratogenicity study by^inhalation with the dams sacrificed the day before expected delivery. The dams for Experiment II will be exposed as in the first experiment, but will be allowed to give birth and to raise their offspring to at least 22 days post partum. * Du Pont classified information Company Sanitized. Does not contain TSCA CBt - 42 - PROTOCOL -INHALATION TERATOGENICITY PAGE 2 II. MATERIALS AND METHODS (CONT) A. Test Chemical received from the Polymer Products Department, was assigned HasJcell Number 14,045. B. Animals Crl:CD(SD)BR female rats 48-54 days of age (nulliparous), weighing about 170 g will be ordered from Charles River Breeding Laboratories, Inc., North Wilmington, Massachusetts. The rats will be quarantined for at least 1 week after arrival by air-conditioned truck. Upon arrival each will be identified by a combination of ear punches and toe clips, and by a unique identification number on a cage card. They will be mated to mature males of the same strain. Mating will be verified by detection of spermatozoa in the vaginal lavage each morning (Day 1 of gestation) following overnight cohabitation. The rat was selected for this study because 3M reported a positive teratogenic response in this species, and we wish to duplicate this finding at Haskell Laboratory. In addition, the degree of toxicity of this chemical was determined in this species at Haskell Laboratory. The Cri:CD(SD)BR strain was chosen because extensive background teratogenicity data exists at Haskell Laboratory only on this rat strain. Since the historical incidence of cataracts or opacities in CD rats is 3%, according to consulting opthal- mologist ^----MI------HW all animals will be screened for these alterations before breeding. The screening procedure will include examination of both eyes and elimination of affected rats. C. Exposure by Inhalation The inhalation route was selected since it is the route by which Du Font employees are likely to be exposed Company Sanitized. Does not contain TSCA CBI 43 PROTOCOL -INHALATION TERATOGENICITY PAGE 3 C. Exposure by Inhalation (cont) will Days __ The test groups will be exposed to the --------------Bin Rochester Chambers; free moving Ta^:s be given whole body exposure for 6 hours/day from 6 through 15 of gestation. Chamber concentrations every hour by gravimetric analysis and at least twice daily by a spectrophotometric technique. Control rats will be exposed to in-house air in the same type of chamber for the same duration of gestation. After each exposure session, the rats will be housed in suspended wire-mesh cages (two females per cage), and the racks holding these cages will be placed in a walk-in hood. The rats for each group will be caged vertically starting with the highest exposure level and progressing to the lowest exposure level and then the control group. To minimize the likelihood of airborne cross-contamination, a baffle will be placed between the rack containing the groups exposed to the two highest levels and the rack containing the low level group and the controls. In addition, in the latter rack, baffles will be inserted, between each cage containing control rats and those containing rats exposed to the lowest concentration of the test chemical. D. Exposure Levels Concentrations recommended for testing are 25.0, 1.0, 0.1, and 0 mg/m3. Based on the limited toxicity data available at Haskell Laboratory and the results reported by 3M, these concentrations are expected to yield an "effect" level and an apparent "no-effect" level of exposure. E. Animal Distribution Before exposure, mated females will be ranked by body weight and assigned to groups by rotation in order of rank. The dose group the first animal is assigned to will be selected randomly. If the distribution process results in statistically significant differences in body weight among groups before exposure, then minimal switching within breeding dates will be used to alleviate the statistically significant differences. A total of 24 mated females will be assigned to each will be assigned to group for Experiment I. About each group for Experiment II. 12 females Mated rats ill that are or that do not gain weight properly"will be discarded before distribution - 44 - CompanySanitized. Does not contain TSCA CBt PROTOCOL -INHALATION TERATOGENICITY PAGE 4 F. Husbandry Upon arrival at Haskell Laboratory, the female rats will be housed two per cage in suspended wire-mesh steel cages. Purina Certified Rodent Chow, 5002, Checkers and water from Wilmtngton Suburban Water Corporation (WSWC) will be provided ad libitum. The potential effects of dietary contaminants were considered and, on the basis of the manufacturer's data, the contaminant levels are believed to be within acceptable ranges. No other contaminants reasonably anticipated to be present in the feed are expected to interfere with the results of this study. The potential effects of water contaminants reported by WSWC were considered and appear to be within acceptable ranges. To supplement the WSWC data, Haskell Laboratory initiated an analytical program that monitors these and other contaminants reasonably anti cipated to be present in its water supply. G. Records Maintenance Records will be maintained for each animal except that feed consumed daily will be noted as a total for the animals caged together. The final report and raw data will be forwarded to the Information Section of Haskell Laboratory for archiving. H. Safety Precautions and Disposal of Waste Material Access to the inhalation laboratory will be limited during the periods that rats are being placed into or removed from the chambers. Chamber exhausts will be filtered through cotton filters and MSA charcoal filters before renting the air to the hood. Waste material containing kB^B|||lBWtf----B1i^^------|will be packaged in polyethylene'^nedFiberpaks or plastic-lined waste bags for incineration at Stine Laboratory. All persons handling the test material or exposed to the study animals will wear the protective equipment and follow the safety procedures presented to the Process Hazards Review Committee. CompanySanded. Does notcontatnTSCACBt - 45 - PROTOCOL -INHALATION TERATOGENICITY PAGE 5 I. Parameters to be Studied Experiment I - Full Teratogenicity Study 1. Dams a. Body weight - weighed on the day of arrival, before breeding, and on the morning of Days 1, 6, 9, 13, 16, and 21 of gestation b. Feed consumption - the average amount of feed consumed daily per rat for each group will be determined for the pre-exposure, exposure, and postexposure time periods. The measurements will be taken at the same time each morning c. Clinical signs - observed upon arrival, at breeding,, and daily at least from Days 6 through 15 of gestation d. Liver weight - absolute weights will be taken and, if indicated, liver weight will be presented relative to the corrected maternal body weight at the time of sacrifice e. Uterine weight - the intact and empty uterus of each dam having one or more fetuses will be weighed to permit calculation of actual maternal body weight gain during gestation f. Corpora lutea - counted and recorded for each ovary g. Implantation sites - counted and recorded for each pregnant rat; the uterus of each apparently "non-pregnant" rat will be stained with ammonium sulfide to detect very early resorptions h. Resorptions - counted and recorded for each rat (not those detected by stain only) Company Sanitized. Does not contain TSCA CBI - 46 - PROTOCOL -INHALATION TERATOGENICITY PAGE 6 I. Parameters to be Studied (cont) 2. Fetuses a. Number, location, and condition recorded for each fetus b. Fetal weight - recorded for all live fetuses and those classified as "Dead" fetuses c. External alterations - detected and recorded for all live fetuses d. Soft tissue alterations - detected and recorded for the first live fetus and thereafter for every other live fetus of each litter; all stunted fetuses and all live fetuses with external malforma tions also will be examined for soft tissue alterations The heads of all fetuses examined for soft tissue alterations will be fixed in Bouin's solution. Each will be sliced in vertical cross-section in front of the eyes, through the center of the eyes, and through the widest portion of the head as described by Barrow and Taylor ('69).1 The sections containing the eyes of at least the control and high exposure levels will be processed by the Histology group for examination by a pathologist. Particular emphasis will be placed on lens structure during examination. e. Skeletal alterations - detected and recorded for all fetuses (including "Dead" fetuses); heads of fetuses examined for soft tissue alterations will be excluded. All groups will be coded from just before sacrifice until all raw data are collected. 1 Barrow, M. V., and W. J. Taylor. J. Morph., 12^:291-306 (1969) - 47 - CompanySanitized. Does not contain TSCA CBt PROTOCOL -INHALATION TERATOGENICITY STUDY WITH I. Parameters to be Studied (cont) Experiment II - Extended Teratogenicity Study 1. Dams a. Body weight - weighed on the day of arrival, before breeding, Days 1, 6, and 21 of gestation and 1, 1, 14, and 22 days post partum b. Clinical signs - observed upon arrival, breeding daily, at least on Days 6, through 15 of gestation, and on 1, 7, 14, and 22 days post partum. Adverse effects observed at any other time will be noted. c. Date of delivery - noted for each dam d. Reproductive indices For each test group: Fertility index (% matings resulting in pregnancy) Gestation index (% matings resulting in live births) For each litter: Viability index (.% animals born that survived 4 days :or more) Lactation index (% animals alive at 4 days that survived to 22 days post partum) Company Sanitized. Does not contain TSCA CBI 48 PROTOCOL -INHALATION TERATOGENICITY PAGE 8 I. Parameters to be Studied (cont) Experiment II - Extended Teratogenicity Study 2. Offspring a. The number of live pups per litter and the number of dead or cannibalized pups per litter will be recorded on 1, 4, 1, 14, and 22 days post partum. b. Sex ratio - recorded for each litter on the date of delivery and for each dead pup. The sex ratio of pups alive 22 days post partum also will be presented. c. Body weights - weighed 1, 4, 7, 14, and 22 days post partum d. Clinical signs - observed 1, 4, 7, 14, and 22 days post partum. Pups with adverse signs noted will be marked for subsequent identification within the litter. e. External alterations - detected and recorded for all live pups f. Soft tissue and skeletal alterations - pups will not be examined for these types of alterations unless otherwise indicated g. Eye examinations - the eyes of pups in all groups will be examined by an ophthalmologist shortly after the eyes open. If eye alterations are detected that appear to be compound-related, a second examination may be conducted. The groups will be coded for all examina tions. Pups with eye alterations will be marked for identification at sacrifice. At sacrifice, each pup will be exsanguinated and its eyes will be removed. All eyes will be fixed and processed by the Histology group for examination by a pathologist. The identity of eyes from pups previously marked will be retained. Otherwise, all eyes from pups will be identifiable only by dam number. 49 Company.Sanitized. Does not contain TSCA CBt PROTOCOL -INHALATION TERATOGENICITY STUDY WITH PAGE 9 III. STATISTICS Experiments I and II The litter will be used as the experimental unit. The Fisher's Exact test will be used to determine the significant differences in the incidence of pregnancy, and maternal pup mortality. Jonckheere's test will be used to determine the presence of a dose response. Dunnett's test will be used for testing the significance of differences in maternal body weight and body weight gain. A two-way analy sis of variance will be used to detect interaction between breeding lots and test groups. For all other parameters, the Mann-Whitney U test will be applied to detect significant differences between the control group and individual experimental groups. The level of significance will be p^O.05. In addi tion, the reproductive indices given earlier will be calculated. IV. CRITICAL DATES Starting Date (breeding): April 27, 1981 Completion: November 15, 1981 PpmpanySanKizetf. Does not contain TSCA CBI - 50 - PROTOCOL -INHALATION TERATOGENICITY STUDY WITH REPORT BY: RTE^Stap 16 s Study Director Staff Teratologist Teratology Section Toxicology and Pathology B. ^A. Burgess Research Toxicologist Acute Investigations Toxicology and Pathology ;^>^J^.-'G,A. - ^ APPROVED BY: / /r (---' ./^ J. G. Aftosilis Associate Director Toxicology and Pathology RES/BAB/mIe 6/9/81 jaZLjLAJ0 Y^^J^ G. L. Kennedy Chief .' Acute Investigations Toxicology and Pathology Company Sanitized. Does not contain TSCA CBl - 51 - CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE APPENDIX A (CONT) cc: December 3, 1981 MEMORANDUM TO; QUALITY ASSURANCE COMMITTEE - C. M. BARBA FROM: R. E. STAPLES SUBJECT: .PROTOCOL AMENDMENT FOR TERATOGENICITY STUDY study ofj_IA^nIHamBen^dHme^n^t ^toHtHh< note thal^TeviatTon #3 S^the study no longer pertains; all examined for alterations. mils atocol for the teratogenicity attached. Please audit report |HHB| for this skeletal specimens were RES/mIe Attachment Company Sanitized. Does not contain TSCA CBI - 52 - I. E. DU FONT DE NEMOURS & CO., INC. CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ELKTON ROAD NEWARK, DEALWARE AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY STUDY IN ^ ____,. ^^. \---- --/ The following changes were made after initiation of the study: Page 6 II. MATERIALS AND METHODS I. Parameters to be Studied Experiment I - Full Teratogenicity Study 2. Fetuses d. Soft tissue alterations As stated in the protocol, the heads of all fetuses examined for soft tissue alterations were fixed in Bouin's solution and retained for subsequent examination. For 0.0 I Run mg/m3 , al and l head specimens in 25.0 mg7m3 groups the were examined grossly after being sliced in vertical cross-section in front of the eyes, through the center of the eyes, and through the widest portion of the head. These specimens were embedded, and two specimens from each litter in the 0.0 mg/m3 group and three from each litter in the 25.0 mg/m3 group were examined by a pathologist. Since no compound-related effects were detected grossly or microscopically, the remaining specimens were not examined. f o u r For lit Run ters I Iin, one the specimen from 0.0 mg/m3 and each 10.0 of mg/m3 groups was examined grossly. They were sliced in vertical cross-section in front - 53 - Company Sanitized. Does not contain TSCA CBl AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY STUDY PAGE 2 d. Soft tissue alterations (cont) of and behind the eyes and through the widest portion of the head. The eyes were left intact to minimize processing artifacts. These specimens were pro cessed and examined microscopically by a pathologist. Because no compoundrelated effects were detected grossly or microscopically, the remaining specimens were not examined. Page 8 II. MATERIALS AND METHODS I. Parameters to be Studied Experiment II - Extended Teratogenicity 2. Offspring Study g. Eye examinations No compound-related effects were detected in vivo by the ophthalmologist among the pups from Run I, and the II highest exposure level in Run (10.0 mg/m3) was lower than that for Run I (25.0 mg/m3). Therefore, the eyes of pups from Run were not examined in II vivo by an ophthalmologist. As stated in the protocol, each pup was exsanguinated at sacrifice and its eyes were removed, fixed, and identi fied. However, since no compound--related effects were detected in the previous test specimens during clinical, gross, or microscopic examination, the eye specimens were not processed or examined but were retained for storage. - 54 - Company Sanded. Does not contain TSCA CBI AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY STUDY Page 9 II. MATERIALS AND METHODS J. Retention of Specimens All skeletal, head, and selected visceral specimens, as well as histologic preparations, will be retained till issuance of the final report. Thereafter, they will be retained for as long as the quality of the material affords proper evaluation. CompanySanitized. Does not contain TSCA C81 - 55 - AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY STUDY IN RATS WITH REPORT BY: / L-^l^-^ R. ^. Staples Study Director Staff Teratologist Teratology Section Toxicology and Pathology Research Tojxicologist Acute Investigations Toxicology and Pathology APPROVED BY: // J. G. Aftosmis ^--- Associate Director Toxicology and Pathology RES/mIe 12/3/81 ^ ^fc^^^y G. L. Kennedy^ Chief Acute Investigations Toxicology and Pathology Company Sanitized. Does not contain TSCA CBt - 56 - CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE APPENDIX A (CONT) B...:E:;Y .1 S E D December 30, 1981 MEMORANDUM TO: QUALITY ASSURANCE COMMITTEE - C. M. BARBA FROM: R. E. STAPLES /C<f\J FOR THE INHALATION TERATOGENICITY STUDY An amendment to the protocol for the teratogenicity RES/mIe Attachment(l) .noeTffotco"1*1'"'^^1" 57 - REVISED I. E. DU PONT DE NEMOURS & CO., INC. CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ELKTON ROAD NEWARK, DELAWARE AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY STUDY IN RATS The following changes were made after initiation of the study: II. MATERIALS AND METHODS D. Exposure Levels II), II. e After severe clinical signs and mortality occurred in females in the 25.0 mg/m3 exposure group (Run I), the exposure level was reduced to 10.0 mg/m3 for Run The 10.0 mg/m3 exposure group now consists of 12 of the females originally scheduled for the 25.0 mg/m3 group (Run and an additional nine females. Fifteen of these females will be used for Experiment I, :six, for Experiment II. Females in the 25.0 mg/m3 exposure group consumed significantly less feed on the exposure days than did the control group. To help determine whether the reduced intake of feed would be responsible for any adverse effects that might be noted among the fetuses, control groups pair-fed to the 25.0 and the 10.0 mg/m3 groups were added to Run II. Company Sanitized. Does not contain TSCA CBI 58 - AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY STUDY IN RATS WITH REPORT BY: R. E. staples Study Director Staff Teratologist Teratology Section Toxicology and Pathology W / "B. A/ Burgess -Research Toxieologist Acute Investigations Toxicology and Pathology APPROVED BY: ^ J.S. Aftosmis Associate Director Toxicology and Pathology RES/mIe 12/30/81 Section Supervisor Acute Investigations Toxicology and Pathology Company SanItFzed. Does not conWn TSfta cm - 59 FOR DU PONT USE ONLY ATTACHMENT 1 Haskell E. I. du Font de Nemours and Co., Inc. Laboratory for Toxicology and Industrial Elkton Road, P. 0. Box 50, Newark, Delaware 19711 Medicine HASKELL LABORATORY IN HOUSE REPORT Material Tested Haskell No. 14.045 Other Codes None Study Initiated/Completed 5/2/81-5/13/81 - Part I 5/18/81-5/29/81 - Part II Material Submitted by Washington Works Polymer Products Department IN-HOUSE REPORT: INHALATION EXPOSURE RESULTS FOR THE ^TERATOLOGY STUDY - PART I AND PART II I. Introduction This report summarizes the methodologies for generation and analysis of exposure atmospheres for the inhalation teratology study of4 "~~ ------------^------Hft This test was conducted in two parts. II. Procedures A. Protocol Pregnant Crl:CD rats for this study were supplied and cared for by II, Haskell's teratology group. Dams were mated in lots, two for Part I and three for Part by caging them overnight with males. Mating was verified by detection of spermatozoa in a vaginal lavage each morning following overnight cohabitation. Mated rats were assigned to the following test groups: Part I Group Group Group Group I - Air Control ,, IIIII (low) - Design Level of 0.1 mg/m (intermediate) - Design Level of 1.0 IV (high) - Design Level of 25.0 mg/m mg/ m Company Sanitized. Does not contain TSCA CBI - 60 - Part II Group V - Air Control ,, Group VI (low) - Design Level of 0.1 mg/m - Group VII (intermediate) - Design Level of l.Q mg/m Group VIII (high) - Design Level of 10.0 mg/m These groups were exposed to the indicated design concentrations for 6 hours a day on days 6 through 15 of gestation. Because of different breeding II, dates a total of 11 exposures were necessary for Part I, and 12 exposures for Part to expose all animals on these gestation days. Dams were exposed in 150 L glass and stainless steel chambers. During 'exposure, dams were housed in compartmentalized, stainless steel, wire mesh modules. These were stacked one over the other. X-Cold Bricks, placed on the outside surface of the chambers, were used to control chamber temperature. '. B. Atmospheric Generation airtight, two stage glass apparatus composed of a round bottom reservoir and a cyclone shaped elutriator. A generation air stream introduced 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. Attached to the lower reservoir was a surface contact vibrator, which vibrated the entire apparatus minimizing buildup on apparatus walls. The dust atmosphere was vented from the bottom of the chamber, passed through a hand-made cotton fiber filter, and an MSA cartridge filter. Air, thus filtered, was then exhausted into the hood. C. Atmosphereic Analysis Two methods were employed to determine chamber concentrations of Gravimetric samples were taken from all chambers at regular intervals (low @ 1 hr; intermediate and high @ 1/2 hour). A known Jff----Mjff^ volume of chamber air was drawn though preweighed Gelman glass fiber filters (Type AE, 25mm). Filters were reweighed andtf------BB concentration calculated from filter weight gain." Filters were weighed on a Cahn Model 26 Automatic Electrobalance. Chamber atmospheres were also analyzed by a spectrophotometric desorbed with an acidified aqueous methylene blue solution. The these conditions was extracted into chloroform and^UI^B concentrations determined spectrophoto- - 61 - Company Sanitized. Does not contain TSCA CBI metrically by comparison with standards prepared in the same manner. Samples were analyzed using a Bausch and Lomb Model 710 Spectrophotometer at a wavelength of 627.6 um. All samples taken from the low level were analyzed by this procedure. Due to the excessive labor involved however, only 5-6 samples per exposure were analyzed from the intermediate and high levels. Filter samples taken from the control chamber were also periodically analyzed. A mean concentration (+ standard deviation) was calculated for each exposure. Chamber temperature was monitored each hour with a thermometer. Oxygen depletion is not a problem with "all-air" systems, so oxygen was not monitored during these exposures. Due to the low design concentrations, particle size could be obtained from the high levels only. An eight stage Sierra Cascade Snpactor Model //218K was used to determine particle size. III. Results Test concentrations were not visible to the naked eye but particulate could be seen by turning off room lights and passing a flashlight beam through the darkened chamber. Control samples were indistinguishable from blanks. II. Particle size samples were taken from the first and tenth exposures during Part I. Mass median diameters for these samples were 1.4 and 2.8 u, with 90 and 887, total respirable content. Particle size samples were taken from the seventh and tenth exposures during Part Mass median diameter for these samples were 9.4 and 8.6 u, with 73 and 94% total respirable content. The data would indicate that these atmospheres were of highly respirable character. Analytical results are summarized in Tables I through IV (Part I) and V throught VIII (Part II): Company Sanitized. Does not contain TSCA CBI 62 TABLE I Low Level Analytical Data (Part I) Exposure // 1 2 3 4 5 6 7 8 9 10 11 Mean^ Chamber Concentration (mg/m + S.D.) Gravimetric Spectrophotometric 0.12 + 0.06 0.08 + 0.05 0.14 j^ 0.07 0.18 ^ 0.08 0.13 ^ 0.05 0.08 + 0.03 0.12 + 0.04 0.12 + 0.06 0.12 + 0.08 0.18 j^ 0.10 0.09 + 0.04 0.13 + 0.09 0.13 ^ 0.06 0.09 + 0.04 0.12 j^ 0.02 0.10 ^ 0.02 0.13 + 0.03 0.11 j 0.05 0.12 + 0.04 0.10 + 0.04 0.12 + 0.03 0.14 ^ 0.02 Overall Meant + S.D. 0.14 + 0.06 0.11 + 0.05 f Mean and S.D. of all samples throughout exposure period. Company Sanitized. Does not contain TSCA CBI - 63 - TABLE II Intermediate Level Analytical Data (Part I) Exposure // 1 2 3 4 5 6 7 8 9 10 11 Mean Chamber Concentration (mg/m"' + S.D.) Gravimetric Spectrophotometric 1.3. j^ 0.4 1.3 + 0.5 1.3 ^ 0.5 1.1 j^ 0.5 0.9 + 0.2 1.0 j^ 0.2 1.1 + 0.3 1.0 ^ 0.2 1.1 j^ 0.3 1.3 ^ 0.3 1.3 ^ 0.7 1.5 j^ 1.0 1.1 ^ 0.8 0.8 j^ 0.4 1.1 ^ 0.2 1.1 ^ 0.2 1.2 j^ 0.5 1.3 ^ 0.7 1.4 ^ 0.5 1.5 + 0.5 1.2 ^ 0.8 1.1 j: 0.2 Overall Meant + S.D. 1.2 + 0.5 1.2 + 0.5 t Mean and S.D. of all samples throughout the exposure period. .Company Sanitized. Does not contain TSCA CBI - 64. - TABLE III High Level Analytical Data (Part I) Exposure // 1 2 3 4 5 6 7 8 9 10 11 Mean Chamber Concentration (mg/m + S.D.) Gravimistr:Lc Spectrophiotc>metric 18.0 + 15,.7 24.6 j^ 2^(.2 15.9 ^ 5.,9 12.0 ^ 31.0 23.8 j^ 19.,4 18.2 ^ 5. 1 22.0 j^ 6. 5 23.0 ^ 4. 7 26.7 + 16i.8 18.8 j^ 1 .9 22.7 ^ 6 .6 19.9 j^ 5 .7 21.9 + 6. 4 21.0 ^ 8. 8 21.9 j^ 6 .6 18.5 j; 6 .4 21.7 ^ 7. 7 23.3 j^ 7 .4 21.7 + 10.0 14.8 j^ 5 .2 23.3 ^ 6. 2 24.1 j^ 7 .7 Overall Meant + S.D. 21.0 + 9. 7 20.5 + 10.0 T Mean and S.D. of all samples throughout the exposure period. Company Sanitfzed. Does not contain TSCA CBI - 65 - TABLE IV Mean Chambe r Teaipe;ratiire Data (F'+S .D.) ( Part I) Exposure # 1 2 3 4 5 6 7 8 9 10 11 Control 72.0 + 0.0 78.0 j^ 1.4 77.3 ^ 0.8 78.0 jb 1.1 77.3 j^ 1.5 76.6 j^ 0.9 77.4 j^ 0.5 78.5 j^ 0.8 76.4 + 1.1 77.3 j: 2.2 72.3 j^ 1.9 L iOW 77'.5 ^ C).6 77 .0 + 1 .7 75 .0 + 1 .3 76 .3 i.8 76 .2 j^ 1 .3 76 .6 + 0'.5 75.8 + 1 .6 77 .7 + 1 .5 76 .6 + 1 .8 75.8 + 1 .5 73.7 + 1 .5 Inte rmiediate 77.1 + 0.6 77.2 + 2.2 77.0 + 1.1 76.6 + 0.7 76.0 + 1.2 76.7 + 0.7 75.8 + 1.6 77.7 + 1.2 75.8 + 1.6 76.8 + 1.6 74.5 + 0.8 Hig:h 751.8 + 1.3 75 .8 j^ 2.9 75.5 + 1.0 76 .8 + 1.2 78 .2 + 1.2 76 .2 + 1.1 75 .8 + 2.5 77 .0 + 0.9 77 .6 + 1.1 77 .1 + 1.3 75 .0 + 1.1 Overall Meant 76.5 j^ 2.4 + S.D. 76.2 + 1.7 76.5 + 1.5 76.4 + 1.7 t Mean and S.D. of all values throughout exposure period. companysanw^- Does not contain TSCA CBI - 66 - TABLE V Low Level Analytical Data (Part II) Exposure // 1 2 3 4 5 6 7 8 9 10 11 12 3 Mean Chamber Concentration (mg/m + S.D.) Gravimetric Spectrophotometric 0.11 ^ 0.05 0.13 -\ 0.05 0.11 ^ 0.03 0.11 + 0.04 0.14^0.03 0.12 + 0.02 0.11 + 0.03 0.10 + 0.03 0.10^0.05 0.13 + 0.03 0.07 + 0.02 0.11 + 0.02 0.15 + 0.01 0.14 + 0.01 0.13 + 0.04 0.11 -\ 0.02 0.11 ^ 0.04 0.07 ^ 0.02 0.15^0.04 0.12 + 0.05 0.13 ^ 0.02 0.09 j^ 0.05 0.12 + 0.04 0.12 ^ 0.04 Overall Meant 0.12 + 0.04 0.11 + 0.04 t Mean and S.D. of all samples throughout exposure period. Company Sanftfzed. does nofcorrtam TSCA CBl - 67 - TABLE VI Intermediate Level Analytical Data (Part II) Exposure ft 1 2 3 4 5 6 7 8 9 10 11 12 Mean Chamber Concentration (mg/m + S.D.) Gravimetric Spectrophotometric 1.1 ^ 1.20 0.67 ^ 0.32 1.1 j^O.56 1.2 j^ 0.47 0.96^0.47 1.1 ^ 0.29 1.3 + 0.27 1.1 + 0.37 1.2 + 0.66 1.2 + 0.21 0.80 + 0.37 1.0 + 0.33 1.2 j^ 0.24 1.2 j^ 0.17 1.1 + 0.61 1.1 ^ 0.45 0.78 ^ 0.25 0.82 + 0.08 0.75 ^ 0.39 0.80 ^ 0.17 0.97 ^ 0.33 1.3 j^O.59 0.85 j 0.16 1.3 ^ 0.71 Overall Meant 1.1 + 0.56 0.98 + 0.37 t Mean and S.D. of all samples throughout exposure period. Company SanWzecT. Does not contain TSCA CBI - 68 - TABLE VII High Level Analytical Data (Part II) Exposure // 1 2 3 4 5 6 7 ,. 8 9 10 11 12 Mean Chamber Concentration (mg/m + S.D.) Gravimetric Spectrophotometric 8.4 + 3.74 10.3 ^ 2.45 8.8 ^ 3.16 8.1 + 2.58 8.3 + 3.80 10.0 + 4.99 10.8 + 6.56 11.1 ^ 7.69 9.5 ^ 5.07 9.9 + 6.60 12.5^6.58 13.6 + 6.27 10.3 j^ 5.30 11.8 + 6.09 10.9 j^ 8.66 11.1 j 4.97 9.9 ^ 6.41 14.6 + 7.35 9.8^5.19 8.7 j 3.84 12.5 + 6.21 7.9 ^ 3.86 10.7 ^ 1.35 12.2 ^ 1.65 Overall Meant 9.9 ^ 5.41 11.1 + 5.3 t Mean and S.D. of all samples throughout exposure period. Company Sanitized. Does not contain TSCA CBI - 69 - TABLE VIII Exposure // 1 2 3 4 5 6 7 8 9 10 11 12 Mean Chamber Temperature Data (F +S.D.) (Part II) Control Low Intermediate High 78.4+0.5 77.2+1.0 74.7+1.4 76.7^1.2 77.5+1.5 72.2+3.4 78.0^1.1 74.3+1.0 78.3^1.3 77.3+1.0 77.0 +_ 1.5 75.9+^2.6 75.6+1.4 76.8 +^ 1.7 77.1+^1.5 77.0+2.0 77.7 + 1.0 77.0+^2.3 78.4+0.8 78.1 +^ 1.3 76.8+^2.3 77.1+1.3 77.6+1.0 77.7+2.3 76.3+1.0 77.5+0.8 78.0+1.2 77.5+0.5 77.2+0.8 77.6+1.5 77.5+1.2 77.3+1.7 78.6+0.5 77.2+1.3 77.0+^1.7 77.9+^1.1 76.8+1.3 76.0+0.6 .75.9+^1.9 77.2 _+0.8 76.1+^0.7 77.6+^1.9 78.0+1.8 76.2+^1.8 75.3 + 0.8 74.8 + 0.8 76.0 + 0.9 75.3 + 1.0 Overall Meant 77.3 + 1.4 +S.D. 76.4+1.7 76.6+2.1 77.1+1.8 t Mean and S.D.of all values throughout the exposure period. .CompanySan.'fized. Does not contain TSCA CB( 70 - III, During exposure no clinical observations were noted in Groups I, II, V, VI, or VII. A dry red discoloration around the mouth was observed the morning following the first exposure in Group IV and VIII rats, which persisted for the remainder of the exposure period. No other overt clinical During Part I, four Group IV rats died during the exposure phase of the test. One rat was found dead each day, on the mornings following the 3rd, 5th, 7th, and 8th exposures. No deaths occurred during Part II. Summary The purpose of this study was to determine the teratogenic effects of in two parts, in which dams were exposed 6 hours/day on days 6 through day 15 of gestation. Atmospheric concentrations were determined gravimetrically and spectrophotometrically. Mean gravimetric concentrations were 0, 0.14, 1.2 and 21.0 mg/m in Part I and 0, 0.12, 1.1, and 9.9 mg/m in Part II. These results were confirmed by spectrophotometric analysis. In Part I, no clinical observations were noted in rats exposed-to 0, 0.14, or 1.2 mg/m . Four rats died following exposure to 21.0 mg/m of dry red discoloration around the mouth following each exposure. During Part II, a dry red discoloration was observed around the mouths of rats exposed to 9.9 mg/m following each exposure. No other clinical signs were noted in any other test group. '"P'n/S.n,,,,..,. o... ,,,,, ,,,,,,,,, ^ ^ - 71 - **Reference: L. F. Percival. "Determination of C- and Cg Dispersing Agents, Methylene Blue Method". E. I. du Font de Nemours & Company. Plastics Polymer Products Department Code No. W 240.240, issued 10/9/69. Washington Works Technical Library, 1st Circulation 5-20-68. ^ Work and Report by: sS^ Clarence Hutt Technician ^ Stephen D. Nastr Technician Supervised by: I^^M \n<^./Jk (- 'Joseph C. Hamill Technologist Reviewed by: Approved by: CH:SDN:tac:WP:12.2 Date Issued: December 18, 1981 ^Si^JL^.^------4 ^ Gerald L. Kennedy, 0r.| Section Supervisor Acute Investigations 72 S,n,,,z.a.D..sno,con,a,nTSC.CB, comply DIPLOMATE; AMERICAN COLLEGE OF VETERINARY OPHTHALMOLOGISTS ATTACHMENT 2 JAMES M. CLINTON. V. M. D. ANIMAL EYE CLINIC AT SOUTH JERSEY ANIMAL HOSPITAL ROUTE 541 ABOVE CHURCH ROAD -- P. 0. BOX US MEDFORD. NEW JERSEY 08055 TELEPHONE (609) 654-0304 Haskell Laboratories Robert E. Staples, Ph.D. Exam Date; 24 April 1981 Ophthalrnoscopic Summary Both eyes of all of the male and female rats in the colony were examined by focal illumination and indirect ophthalmoscopy. Mydriasis was achieved with 1% Atropine (1% Afcropisol,. Cooper, B3035, exp. 7/82), and darkness, maintained until the following morning. Both eyes of all rats were ophthalmoscopically normal except as follows: Rat Number 301112 301116 301335 301194 301305 301231 301251 FEMALE RATS Observations Left Eye; Right Eye Right Eye Right Eye Right Eye Right Eye Right Eye Posterior synechiae. Focal retinal degeneration. Focal retinal degeneration. Focal retinal degeneration. Anterior synechiae from 2 to 4 o'clock. Comeal opacities and subjacent anterior synechiae from 11 to 1 o'clock. Marked ventral enfcropion, blepharospasm. MALE PATS Rat Number 298494 298514 298429 298305 298299 298128 298083 298148 300058; 300130 300177 Observations Left Eye: Superficial corneal vascularization. Bilateral superficial comeal vascularizafcion. Right Eye: Anterior synechiae from 1 to 3 o'clock, Left Eye: Anterior synechiaee Left Eye: Anterior synechiae. Right Eye: Superficial cornea! vascularizafcion. Right Eye: Anterior synechiae from 9-11 o'clock. Right Eye: Anterior synechiae at 9 o'clock. Bilateral anterior synechiae, nasal quadrant. Left Eye: Anterior synechiae. Left Eye; Anterior synechiae. COMMENTS The 18 rats described above were removed from the colony and euthanatized by Supervisor Alice Erwin. All of those rats remaining are ophfchalmoscopically normal and suitable for use in the forthcoming study. ^^7^--^ w<' CfS- James M. Clinton, V.M.D. 73 Company SanRfeed. Does not contain TSCA C8I DIFLOMATE; AMERICAN COLLEGE OF VETERINARY OPHTHALMOLOGISTS ATTACHMENT 3 JAMES M. CLINTON. V. M. D. ANIMAL EYE CLINIC AT SOUTH JERSEY ANIMAL HOSPITAL ROUTE 541 ABOVE CHURCH ROAD -. P. 0. BOX 115 MEDFORD. NEW JERSEY 08055 TELEPHONE (609) 654-0304 Haskell Laboratories Robert E. Staples, Ph.D. Exam Date; 5 May 1981 Ophthalmoscopic Examination Both eyes of 210 female rats vere examined by focal illumination, indirect ophthalmoscopy and, when indicated, slit-lamp microscopy. Mydriasis was produced with 1% atropine solution and the eyes examined in subdued light. The subdued light was maintained until the following morning. With just 13 exceptions, both eyes of all of the rats in the colony were ophthalmoscopically normal. The exceptions are listed below: Rat Number Observations 302217 Left Eye; Very pale ocular fundus. 302240 Bilateral pale ocular fundi. 302194 302201 Right Eye; Right Eye: Focal retinal degeneration. Anterior synechiae 5-6 o'clock. 302248 Right Eye: Anterior synechiae, nasal quadrant., ".zith comeal''opacity. 302319 Right Eye:' Anterior synechiae from 11-1 o'clock. 302321 Left Eye: Very pale ocular fundus. 302339 Left Eye: Very pale ocular fundus. 302291 Right Eye; Anterior synechiae from 11-1 o'clock. 302356 Left Eye: Pale ocular fundus. 302357 Pale ocular fundi. 302351 Pale ocular fundi. 303360 Pale ocular fundi. Comments A-s soon as they were identified,'the 13 rats described above were removed from the colony. .""---^ M- &^-4-i^_ t--^ James M. Clinton, V.M.D. 74 Company Sanitized. Does not contain TSCA CB; tti.UIMfkT-0 HASKELL LABORATORY ATTACHMENT 4 December 18, 1981 MEMORANDUM TO: FROM: SUBJECT: R. E. Staples W. D. Kerns All animals listed in the attached table were evaluated microscopically for the presence of histomorphologic lesions in the fetal lens. There were no microscopic lesions detected in any of the sections that were evaluated. Frequently, lenses contained arti facts that were attributed to trimming and processing. WDK.-wfd Company Sanfffzed. Does notconfa?" "''^Cft CR 75 - SPECIMENS TO BE PREPARED FOR EVALUATION BY W. D. KERNS The following fetuses had no eye alterations detected during external examination: Dam Code 68 54 58 58 Fetus Number 1 7 9 11 Observations during Head Examination Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration CLL/as 9/30/81 jCompanySanitized. Does not contain TSCA CBl 75 - HASKELL LABORATORY ATTACHMENT 5 REVISED December 21, 1981 MEMORANDUM TO: FROM: SUBJECT: R. E. Staples / ^f^ ]}bh- W. D. Kerns ^ I' H-14045 - FETAL OPHTHALMIC PATHOLOGY There were no lesions present in either run that were considered to be related to the administration of the test compound. The fetal heads from run two produced much better histological preparations than those from run one. WDK:wfd Company Sanitized. Does not contain TSCA CB - 77 - H-14045 TABLE 1 INDIVIDUAL ANIMAL HISTOPATHOLOGY DATA Tissue*and Observation** Fetal Eyes Hyphema, focal Run: Exposure Level (ms;/m3) : Dam lumber; Fetus Number; I 0.00 40 35 35 38 12 12 15 15 11 11 03 03 13 13 03 11 11 05 03 10 0 NNNNNNNOON'O * Tissue Accounting Code; N = Both eyes (including lens) were within normal limits N'= Only one lens was present for histomorphologic evalu within normal limits. 0 = Lens was not present in the sections submitted for e L = Lesion observed ** Degree of Change (severity): 1 = Minimal 2 = Mild 3 = Moderate 4 = Marked 5 = Severe s i H^4Q^^ TABLE 1 (Continued) INDIVIDUAL ANIMAL, HISTOPATHOLOGY DATA Tissue*and Observation** Fetal Eyes Hyphema, focal 1 ~~j U3 1 Bun; I Exposure Level (mg/m3) : 0.00 Dam Number: 23 25 25 30 30 40 43 43 ; 38 45 9 Fetus Number: 09 05 11 03 09 05 01 09 05 09 01 N' N N' N' N N N N N N N - 1 Code: See Table 1. <rt 1 I 0 0 % S. 1 3 -A W 0 ? S H-14045 ^^^^ TABLE: 1 (Continued) INDIVIDUAL ANIMAL HISTOPATHOLOGY DATA Tissue* and Observation** Run: I Exposure Level (mg/rn3); 25.0 Dam Number: 3 3 6 6 6 19 19 19 221 22 22 2 Fetus Number: 07 13 01 05 11 01 11 13 03 07 09 0 Fetal Eyes Hyphema, focal 00 0 N' N' 0 fS' N1 0. N'. H N N N' N 'rt Code: "0 5 w 1. g 0. 0 0 S 3 1 S. 3 -1 W 0 0 S See Table 1. H-14045 TABLE 2 "INDIVIDUAL ANIMAL HISTOPATHOLOGY DATA Tissue*and Observation** Fetal Eyes Hyphema, focal II Run; Exposure Level (mg/m3) : 0.00 0.10 10 Dam Number: 78 75 101 55 99 105 74 Fetus Number: 04 06 13 07 09 02 05 N N N N L N N 2 Code: See Table 1. t^r-rummc,; ^Ivif-KtL^AM LULL.EGE OF V6TERINARY OPHTHALMOLOGISTS ATTACHMENT 6 JAMES M. CLINTON. V. M. D. ANIMAL EYE CLINIC AT SOUTH JERSEY ANIMAL HOSPITAL ROUTE 541 ABOVE CHURCH ROAD -- P. 0. BOX 115 MEDFORD, NEW JERSEY 080S5 TELEPHONE (609) 654-0304 Haskell Laboratories R. E. Staples, Ph.D. Exam Date: 5 June 1981 Ophthalmoscopic Examination Fl Generation of Ophthalmoscopically Nonnal Parents First Run Both eyes of all of the male and female rat pups obtained from. the parents already determined to be Ophthalmoscopically normal were examined by focal illumination, indirect ophthalmoscopy and, when indicated, slit- lamp microscopy. Mydriasis was produced with 1% afcropine ophthalmic so lution, and the eyes examined in semi-darkness. Semi-darkness was main tained until the following morning. The dose levels and group identities ^ere not disclosed to me. Both eyes of all of the male and female rat pups were Ophthalmoscopic ally normal except as follows; Litter No. Offspring No. Observations 301260 1 301154 301179 301117 301117 301072 Prominent filamentous opacities of eachposterior lens pole. Prominent filamentous opacities of each posterior lens pole. Prominent filamentous opacities of each posterior lens pole. Left Eye: Focus of pre-refcinal hemorrhage of 2 disc diameters. Right Eye: Incomplete mydriasis and red oval opacity on central endofchelium. Right Eye: Pre-refcinal hemorrhage at peripheral nasal quadrant. Comments In my opinion, the lesions noted might typically occur in any well managed colony of rats. Although some 'of these young rats' eyes v/ere too small to be completely visualized by the means described, I believe that the anterior and posterior segments of most of the eyes were readily visualized. At this juncture, I do not believe that the test material, as eval uated in this stud;'-, has produced ocular changes in the Fl generation. - 82 - -^ ^-\ ^-j-^wj^i cf!^^>^6'<<^%?A-<- - r James M. Clinton. V.M.D. Company Sanitized. Does not contain TSCA CB1