Document 3evgvRpEQ4Z232nQkGE29Qvb3

AR226-2776 fINHALATION : EMBRRYO-FET-]AL TOXICITY AND TERATOGENICITY STUDY IN THE RAT HASKELL LABORATORY REPORT NO. 881-81 Company Sanitized. Does not contain TSC A CB i FOR DU PONT USE ONLY E. I. DU PONT DE NEMOURS & CO., INC. HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT ELKTON ROAD, P. 0. BOX 50 NEWARK, DELAWARE 19711 . rHS>INHALATION : EMBRYO-FETAL TOXICITY AND 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: December 30, 1981 Date Issued: January 14, 1982 1- Company Sanitized. Does not contain TSCA CBS INHALATION: EMBRYO-FETAL TOXICITY it) TERATOGENICITY STUDY IN THE RAT Toxicology & Pathology APPROVED BY: Uw lQ J. GyAftosmis, D.V.M. Associate Director Toxicology & Pathology REVIEWED BY: O /A M n /n J '' O A baJ C. M. Barba Auditor Quality Assurance Committee R ES /m s/m ie 2- - Company Sanitized. Does noi contain TSCA CB I 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: HLR 881-8 fijpiJlnhalation: 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: _______________________________ C. M. Barba Quality Assurance Auditor CMB/mle 1/8/82 - 3Company Sanitized. Does not contain TSCA CBI tNHALATION: EMBRYO-FETAL TOXICITY ANBr TERATOGENICITY STUDY IN THE RAT HASKELL LABORATORY REPORT NUMBER 881-81 TABLE OF CONTENTS Page Title Page ............................................ Signature Page ......................................... Quality Assurance Documntation ...................... Table of Contents .,................................... ~ 3 4 I . Summary ............................... II. Introduction ......................... ,, A. Background ....................... i B. Protocol ............. . C. Purpose .......................... III. Materials and Methods ............... . A. Test Material .................... B. Animals .......................... C. Experimental Design and Procedures D. Statistical Evaluation .......... IV. Results ........................................ A. Eye Examination of Prospective Parents .... B. Maternal Exposure to C-8 .................. C. Clinical Signs Observed ................... D. Maternal Feed Consumption ................. E. Maternal Body Weight Gain ................. F. Maternal Deaths ............................ G. Gross Examination of Maternal Organs at Sacrifice ................................ H. Reproductive Effects and Body Weight of the Offspring ............................ I. Fetal Alterations .......................... J. Pup Alterations ........................... V. Discussion VI. Conclusion 7 7 7 8 8 8 8 9 10 15 15 15 16 16 17 17 18 18 19 20 20 21 23 -4- Company Sanitized. Does not contain TSC A CBI N.HALATION : EMBRYO-FETAL TOXICITY *-mpD TERATOGENICITY STUDY IN THE RAT TABLE OF CONTENTS (CONT) VII. Acknowledgements VIII. References .... Page 23 24 TABLES1 I. Experimental Design: Exposure Levels and D i s t n bution of Mated Females Among Test Groups ... 2b II. Feed Consumption in Rats Exposed to froin Days 6--15 of Gestation ...................... III. Reproduction aji Fetal Development in Rats Exposed to ^ p j f r o m Days 6-15 of Gestation .. ~7 28 IV Fetal Malformations in Rats Exposed to from Days 6-15 of Gestation ................. V. Fetal Variations in Rats Exposed to from Days 6-15 of Gestation .............. ........ VI. Reproduction and Dev^opment of Offspring of Rats Exposed to ^ P ^ f r o m Days 6-15 of G e s t a t i o n ..... .. ............................ 3-L APPENDIX A. Protocol fo Rats with <^njn 1 tv Study in Amendment to the Protocol for Inhale Lon Teratogenicity Study in [12/3/81 Amendment to the Protocol for Inhalation Teratogenicity Study i n R a t ^ i t h /30/81 42 52 57 1 individual animal data available upon request - 5- Company Sanitized. Does not contain TSC A CB1 [JINHALATION : EMBRYO-FETAL TOXICITY D TERATOGENICITY STUDY IN THE RAT MM!! HLR 881-81 TABLE OF CONTENTS (CONT) ATTACHMENTS 1. In-House Rgport: Inhalation Exposure bUX L. for Teratology Study - Part I and Part II 2. Letter, from J. M. Clinton to R. E. Staples, 4/24/81 ................................. 3. Letter, from J. M. Clinton to R. E. Staples, 5/5/81 .......................................... 4. Memorandum, from W. D. Kerns to R. E. Staples, 12/18/81 .............................. ` ....... 5. Memorandum, from W. D. Kerns to R . E. Staples, 12/21/81 ....................................... 6* Letter, from J. M. Clinton to R. E. Staples, 6/5/81 .............. Page 60 73 74 75 77 82 6- - figmpany Sanitized, Does not contain TSC A CBf ( 4 * 3 INHALATION: EMBRYO-FETAL TOXICITY^ND TERATOGENICITY STUDY IN THE RAT HASKELL LABORATORY REPORT NUMBER 881-81 SUMMARY ,, __Jwas 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 m g / m 3. The actual mean concentrations achieved were 0 and about 0.14 , 1.2, 9.9, and 21.0 m g / m 3, respectively. Maternal deaths occurred at the 25.0 m g / m 3 concentration only, but overt toxicity was evident among the surviving dams and among those of the 10.0 m g / m 3 group. A teratogenic response was not demonstrated upon sacrifice of the dams on Day 21 of gestation at any concentration o f f M R tested. Embryo-fetal toxicity was noted only at the^STl) m g / m 3 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 dams or their offspring till scheduled sacrifice after weaning. At the 10.0 mg/m3 level, w W ^ as st-ilj- toxic but not lethal to the dams; however, no^SverseflflB related effect was noted among their offspring. Hence, in this study, ^ p ^ w a s not demonstrated to represent a unique hazard to the conceptus. II. INTRODUCTION A. Background Du Pont o b t a i n s . ' f r o m 3M Company for use m t h ^ n a n u f a c t u r ^ o f a v a r ^ ^ ^ r f fluoropolymer dispersions, including some of Du Pont's Teflon products. A study of^tl^e embryo-fetal toxicity and teratogenic potential o f b p | w a s requested by * E. D. Champney, Polymer Products 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 b ^ 3 M between the last part of 1980 and March 20, 1981 o n n m j a n d on several related chemicals.1 The possible teratogenic activity reported to us by 3M included lens changes in the eyes of the Company Sanitized. Does not contain TSC A CBf INHALATION: EMBRYO-FETAL TOXICITY TERATOGENICITY 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) 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 it was amended on December 3 and on Qecembei: 30 , 1981. C . Purpose This study was designed* to^determine whether the reported teratogenicity o f M S R J i n 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 - changes persist,after birth. The information gained was to aid in establishment of workplace standards for women of childbearing potential. III. MATERIALS AND METHODS The study consisted of two experiments (Tahlp i L . Experiment I was a teratogenicity study in whichWflfcTiias given by inhalation, and the dams were sacrificed" oiT"the day before expected delivery (Day 21 G ) . The dams for Experi ment II were exposed as in the first experiment, but were allowed to give birth and the offspring were maintained till 35 days postpartum (Day 35 PP). . A. Test Material 1 Physical characte d^i^which sublimes at i s \ B r t n d its struct T h ^ "buxty of the sam ^contaminants present inhib W e r e presenil*. 8 j^mp^ny Sanitized. Does nol contain TSCA CR (j(^INHALATION: EMBRYO-FETAL TOXICITY V'AND TERATOGENICITY STUDY IN THE RAT Y^R 4129-00]/ HLR 881-81 2. Source - The M B sample as ssuuppifiied by the^Polymer Products Department. It was assigned Haskell Number 14,045. 3. Test concentrations - The Approximate Lethal Concentration `o F 'W M & I n rats was 0.8 mg/L (800 m g / m 3) after a single 4-hr exposure period by inhalation (head only) as a dust. Liver enlargement and corneal opacity resulted (1). SpBPfwas administered to 7-8 week-old male CrlrCDrats 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 a brief p retest in non-pregnant female, rats, the UjHgexposure levels selected for testing irrhe 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 chosgn 'for this test because previous toxicity testing o n s ^ w a s conducted in this species. The Crl:C D (SD)BR s Plain was selected because the pre liminary teratogenicity test on\|^yconducted for 3M used the Sprague-Dawley (SD) d e r i d i ? rat obtained from the same supplier, and because extensive background information from previous teratogenicity testing at Haskell Laboratory exists on this rat strain. -9- Cm^ 5 5 5 * Does not contain TSCA UNHALATION: EMBRYO-FETAL TOXICITY "AND TERATOGENICITY STUDY IN THE RAT HLR 881-81 B. Animals (cont) 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 II, 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 o n e . 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 --7 . u wic^Kcib 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,' slitlamp 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 theiroales identified under III.B. on an as-needed basis. Mating was verified by 10. - - ^ m p a n ^ f e e d . Does not contain TSCA CBI INHALATION: EMBRYO-FETAL TOXICITY tfD TERATOGENICITY STUDY IN THE RAT HLR 881-81 C. Experimental Design, and Procedures (cont) detection of spermatozoa in the vaginal lavage each morning following overnight cohabitation. The day on which spermatozoa were detected was designated as . Day 1 of gestation (Day 1 G ) . For Run I, Day 1 G was April 28 and 29, 1981, for Breeding Lots A and B, respectively; for Run II, it 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 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.. 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 in the 25.0 mg/m3 group in Run I, this concen tration was reduced to 10.0 mg/ m 3 for Run II, 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 m g / m 3 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 mg/m3 group in Run II, six mated females were added to both the control and the 10.0 m g / m 3 groups. These were all the mated females available unless the exposure period was extended for at least six more days. The exposure of animals t o M B ^ b y inhalation was conducted by the Acute Investigations Section, Haskell Laboratory. The inhalation route was selected because it is the route by which Du Pont employees are likely to be exposed to[ | B j The exposure period was 6 hr/day from Days 6-15 G. Neither feed nor water was available during the exposure period. The procedures used for generation of^JJ^xp o s u r e levels and measurement of the concentrations and particle size attained are attached (Attachment 1). . - 11 - .Company Sanitized. Does not contain TSCA CBi  INHALATION : EMBRYO-FETAL TOXICITY AND TERATOGENICITY STUDY IN THE RAT HLR 881-81 Experimental Design and Procedures (cont) The test groups were exposed (whole body) to the,, in 150-liter .glass and stainless steel Roches ter-type chaiu within which the rats were housed individually in wire- mesh modules. Breeding lots within exposure levels were rotated the chambers daily. Chamber concentra tions of Wp^Jtfere determined by gravimetric analysis either eHT 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 dtermine 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 m - h o u s e 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. 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 space restriction) . The dams were coded, from before sacrifice by cervical dislocation on Day 21 G, till all maternal and fetal data were collected and till 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 till'the report was written. After sacrifice, the dams were - 12 - (IPfimpany Sanitized. Does not contain TSC A CBI INHALATION: EMBRYO-FETAL TOXICITY TERATQfENICITY STUDY IN THE RAT HLR 881-81 C. Experimental Design and Procedures (cont) examined for gross pathplogic 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 histologically for examination. In Run II, one fetal head from each of four litters from the 10.0 mg/m 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 - Company Sanitized. Does not contain TSCA CB1  Q l N H A L A T I O N : EMBRYO-FETAL TOXICITY ^ A N D 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/ m 3 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. For Experiment II, the procedures used till 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 wiremesh lid)that contained Bed-O-Cobs (1/4" size) . The bedding was changed weekly following the seventh day postpartum (Day 7 PP). The date of parturition was noted and it 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 P P . 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, 7, 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 - fforopany Sanitized. Does not contain TSCA CBI INHALATION: EMBRYO-FETAL TOXICITY TERATOGENICITY 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 slitlamp 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. lhe significance of differences in feed consumption among groups was determined by a one-way analysis of variance. Dunnett'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 m 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 - Company Sanitized. Does no! contain TSC A CB I ^ENHALATION: EMBRYO-FETAL TOXICITY [a n d t e r a t o g e n i c i t y s t u d y i n t h e r a t nfl HLR 881-81 A. Eye Examination of Prospective Parents (cont) were removed from the colony because their eyes were not normal ophthalmoscopically (Attachment 2). For Run II, 210 females were similarly examined on May 5, 1981, and 13 were discarded (Attachment 3). The same males were used for both.runs. The animal exposure levels achieved were reported in detail by the Acute Investigations Section (Attachmerit I). In brief, for Run I, the nominal exposure con centrations for 6 hr/day from Days 6-15 G were 0, 0.1, 1.0, and 25.0 mg/m 3; the average daily concentrations achieved (x + S.D.) by gravimetric measurement were 0, 0.14 + 0.06, 1.2 + 0.5, and 21.0 + 9.7 mg/ m 3, respectively. The average mean daily temperature in the chambers was about 76F and ranged between 72.0 and 78.5F. For Run II, 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 t h e ^ M # particles in the chambers were respirable (Attachment 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/ m 3 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 / m 3 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 Sanitized. Does not contain t s c a c b i JNHALATION: EMBRYO-FETAL TOXICITY ^AND TERAIQGENICITY 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/m 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 _^"^sd consumption was measure^ on^y in Experiment I . During the period of exposure t q J H ^ T the feed con- " sumption of the dams in the 10. O ^ n c f ^ S .0 mg/m3 groups was significantly less than that for the control group (Table_II) Feed consumption returned to the control value in the post-exposure period. No significant differences were noted in this regard between the control group a n d h e groups exposed to the two lowest concentrations of 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 The body weight gain of the dams exposed t q i ^ P i at the 25.0 mg/m3 concentration was significantly less during the exposure period (Days 6-15 G) than fnrAt h e /C?ntro1 9rup (Table III). Although the 10.0 mg/m group also gained less weight during the exposure period than the control group, the differ ence was not statistically significant. Body weight g a m from I^y^J.6-21 G was similar for the groups exposed toH^Bjand for the control group. The body - 17 - Sanitized. Does not contain TSCA obij INHALATION : EMBRYO-FETAL TOXICITY TERATOGENICITY 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 m g / m 3 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, th<=mate.rnal body weight gain of the groups exposed to ^ B t h a t were not sacrificed at term was not s i g n i f i c ^ l y different from the control value (Table V I ) . F . Maternal Deaths In t h i s s tudy, five dams died; all were in groups exposed to W rjfct t_h_e _2_5_._0 .m.g./.m3 concentr.ation (3/12 : Experiment I, and 2/12 in Experiment II). In E*Perin3ent the dams were found dead on Days 12, G Iuable IV) ' The first was not necropsied; f . he<- herS had grossly observable liver changes, andu^ lr 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 s h o w m g considerable body weight loss, an unkempt appear ance, a wet abdominal surface, and chromodacryorrhea and ch^omoshinorrhea beginning on the first day of exposure t o W f c r shortly thereafter. P n Experiment II, one dam was found dead on Day 9 G was autopsied on Day 11 G when she became moribund (Table V I ) . The uteri of both contained ' Slsmilar at^o *those Td5eSsicrriCbieid^ iaGbaolves.ign s-preceding death were G ` Gross Examination of Maternal Organs at Sacrifice I ' dark red mottling of the lungs was noted for two of the dams in the 0.1 mg / m 3 group and in one dam in the 10.0 mg/m3 group. A hematoma between the ^ecturn and pelvic muscles was noted in one dam in the i .0 mg/m group. - 18 - Sanitized. Does not contain TSCA CBI f~~ - I n HAIATION: EMBRYO-FETAL TOXICITY fcAND_ TERATOGENICITY STUDY IN THE RAT HLR 881-81 g^oss Examination of Maternal Organs at Sacrifice (cont) Ji? addition, in the 25.0 mg/m3 group, actual liver weight was significantly increased (Table III). The liver weights of the control groups that were pair-fed t h ^ ^ h TM 0^311^ ^ 5^ ^ 71113 groups were significantly less " 5 4. ? fufr t h e l8BO groups to which they were paired, anf jj^an that for t^e Control group (Table III) . on a relative weight basis (using corrected Day 21 G maternal body weights), the,liver weights (x + S.E.M.) for the gr2U?SJ XPfed 10 * and 2570 mg/ m 3 (5.42 + 0 125 and 6.46 + 0.222 ,Respectively) were still significantly ' more (MWU - two-tailed) than that for their respective7 pair-fed control groups (4.58 +0.1 6 4 , and 4.62 + 0.103). n the same basis, all groups differed significantly from the control group. * J--LU1U . In ExPeriment II, 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 . . ExPeriment I , the maintenance of pregnancy and the incidence of resorptions among the. surviving dams were not adversely affected by exposure t c h M a t concentrations up to and including 25.0 mg/ m 3 (Tabl~III). In Experiment II no a v e r s e effec^ o i ^ r eproductive performance was demon- ' strated that wad||Boncentration-related (Table VI) . ^ t e r s examilT&d A c l u d e d Fertility index, Gestation Index, Viability Index, and Lactation Index. The mean body weight of fetuses in the 25.0 mg/m3 group was significantly decreased (p = 0.002) below the " " I ? 1' <Tjble " D ; but, this also was the case (p *00}) for the control group pair-fed to the 25 0 mg/m group (Table III). The mean weight of the fetuses m the 10.0 mg/m3 group, and in its oair-fed control group, were not significantly different from the control frnP (/ > `23)- In Experiment II, the neonates in the 5.0 mg/m group also were significantly smaller than those in the control group (p = 0.002) but by Day 4 PP ' iTablefV i r nCS ^ n l0nger statistically significant' QwPany Sanitize*Does nof contain TSCA CBt 19 M INHALATION: EMBRYO-FETAL TOXICITY AND TERATOGENICITY STUDY IN THE RAT HLR 881-81 I. Fetal Alterations 1. Fetal Malformations A pHconcentration-related increase in the incidence of external, visceral, or skeletal malformations was not detected (Table IV). No malformations^wgjre seen among the fetuses from dams exposed t o M a t 25.0 mg / m 3. Similarly, coded stereoscopic andliistomorphologic 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 of [ (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 hematomas; however, in the 25.0 m g / m 3 group, their incidence was significantly increased (p = 0.05), but only upon application of the one tailed Mann-Whitney U test. Among the "variations due to retarded development," only the incidence of partially ossified sternebrae in the 25.0 m g / m 3 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 . 0 4 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 / m 3 group (Table VI). On - 20 ^22JP^X5a^itized. Does not contain TSCA CB I ON : EMBRYO-FETAL TOXICITY 1ENICITY STUDY IN THE RAT HLR 881-81 J. Pup Alterations (cont) Day 19 PP, it was noted to have severe hydrocephaly and an abnormal gait. Since it was moribund, it was sacri ficed the next day. 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 VI and Attachment 6); -three of these occurred-in'th'control-group, and the remaining three Occurred in the 0.1 mg/m3 group. In' view of these nega tive'results, similar in vivo examination of the eyes of the pups from Run II w-a-s not conducted. 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 to this stage, in either the fetuses or the pups, pathologic examination of the eyes of the pups of Runs I and II was not conducted. V. DISCUSSION In this study, exposure t<?C?at a concentration of 25.0 mg/m3 for 6 hr/day from Day 6^15 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 discolore, ation around the eyes, nose, and mouth, lethargy, decreased feed consumption and body weight gain during the exposure period, anj-,an^nkempt appearance. None of the 21 dams exposed t o j B B P e t 10.0 mg/m3 died, but they showed . similar clini Afa signs to a lesser degree than that seen at the 25.0 m g / m 3 leveA. Despite this degree of maternal toxicity, no evidence of elated teratogenicity was detected. The fetuses from the 25.0 m g / m 3 group were significantly smaller than those from the control group. This probably was due to the decrease in j^atesaial feed consumption rather than to a direct response t^SSjpJsince the fetuses of the control group pair-fed to the Z S . O H ^ / m 3 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 C w P m ^ it iz e d . Does not contain tscacbi L INHALATION: EMBRYO-FETAL TOXICITY AND TERATOGENICITY STUDY IN THE RAT ~ HLR 881-81 DISCUSSION (cont) =.^ aternal l.iver weight changes among groups on a i ^ n ^ ?eight basis indicated that e x p o s u r e ^ f l f M f at t9h?a;n "m"9/^the cromntrroel rgersouulpted Tihnissignificantly l a r9& S ^lviveerrss previous toxicity telts i t h W u 2 a" e TM e 1L d S ? Sentel fatty | f i e ^ ^ i f i ^ t decrease accompanied by l - i - liver c o n t r o w i i n i ^ ? 06 f resorPtions w as not ipcreased above the controi vaiue at any test concentration o f / M f o r amona the fir~fed cntfo1 groups. The statisticall^finificant tration-relateded 1,0 mg/m3 9rouP was not concen- related and hence was n o t :demonstrated to be due to The incidence of partially ossified sternebrae was signincantly increased among the fetuses of the nm /3 ? S n PiSy -t5e " -Whitney 0 ? e s t ( o S e - S ^ d , W s ion is indicative of developmental delay Since its inei- oenbCe "?S nb c?ncentatijyi-related it was not demonstrated related to toe f tof l p i x p o s u r e . It also was probably its incidence decreased feed consumption of the dams, since am ong th e c o n tro 1 fice Tw.fjtusesuwfr e .^ s e r v e d to have reddish eyes at sacri- mpernetsin71e of "?tod0in9theen'"natOnnrer aled 1?Jthe anterior chamber of hyPhemia r the the eye (Attach- ?Se0 S / red n 1976 SUTgS -Sr" |nSea1 ?heleLatthdeSnCe PaSt StUdiES at HaSke11 O r a t o r y since Subcutaneous hematomas were detected in all crrouDs inoi nd ir,rr toe C25nt0r/9r=UpS ' The dlffe " in incidence b ^ t " " 9 g/m group (7.6s) and the control group (4.0%) - 22 - CompanySanitized Does not contain TSCA CBf 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!tSe incidence of fetuses with hematomas in the 25.0 exceed the range of incidence contained among the control of Pfst studies (4.6-9.0%) at Haskell Laboratory, herefore, it is not likely that the magnitude of this7 difference would be repeatable. Moreover, even if the increase was repeatable, its occurrence would likely depend more upon the presence of the evert toxicitv i n f h i c group than upon a direct effect o t W ^ e r se VI. CONCLUSION , not demonstrated to be teratogenic even when rats at overtly toxic concentrations from Days 6 through 15 of gestation. Concentration-related embryo fetal toxicity, expressed as decreased fetal weight nlY thS highest concentration tested ^ . ( ^ m g / m 3) wiiSh4-rfSS Vef"iY toxlc to the dams. This decreased body5 eight persisted to Day 1 postpartum but not to Day 4 postpartum. After exposure t o M M j a t the 10.0 m g / m 3 concen- ioaid?2rseSfcik^af !t^ 11^Joxicl^ n ot lethal to the dams, seB ^ r e l a t e d effect was noted among their offspring. significanf ' SS f ^ 5* mg/m3 ?roup, a marginally significant increase m the incidence of hematomas also J ^ jiperdsethe increase was not demonstrated to be due to ,, Hence, in this study, fl^Qwas not demonstrated to represent a unique hazard to'tReJ conceptus. VII* ACKNOWLEDGEMENTS _ Thf concentrations o f / i i f o r inhalation were generated anal^ zed hY te^lcute Investigations Section ' i-hikp 1ihL?brat0ry* Histologic specimens were prepared by ' examination^fS^h^"Ln ' Lab-- tory. His?omo?phologic W lilllTita^m Di. KVerfns,heD.eVy.eMs. a,nMd.Sct.herThteisisnuevsivwoaseyceonedxuacmtiendatbiyons were conducted by James M. Clinton, V.M.D., Consultant in c o n d ^ ^ S Pjthalmolgy- The remainder of the stuSy as conducted by the Teratology Section, Haskell Laboratory. - 23 S S M J S " " * Does not contain TSCA CBl ( ^ / I NNHHAALLAATTIIOONN:: EMBRYO-FETAL T O X I C I T Y ^ ^ T E R A T O G E N I C I T Y STUDY IN THE RAT HLR 881-81 VIII. REFERENCES 1 . Du Pont Data, Haskell Laboratory: 2 - -Du Pont Data, Haskell Laboratory: 3. I/npub^yshed Du Pont Data, Haskell Laboratory: Clinical Pathology Report, 8/13/80. 4. Staples, R. E., "Detection of visceral altera tions in mammalian fetuses." Teratology, 9:A37 5. Barrow, M. V . , and W. J. Taylor, "A rapid method for detecting malformations in rat fetuses " J . Morph., 127 (3):291-306 (1969). * 6. Haseman, J. K . , and M. D. Hogan, "Selection of the experimental unit in teratology studies " Teratology, 12:165-172 (1975). ' 7 Siegel, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill. N^w Vn-rV 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, 41:133-145 (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., 18:50-60 (1947). --------- ` 11. Haseman, J. K., and D. G. Hoel, "Tables of Gehan'1 generalized Wilcoxon test with fixed point cen soring." J. Statist. Comput. Simul., 3 : 1 1 7 - 1 3 5 (1974) . - 24 Gptopany Sanitized. Does not contain TSC A CBf fgfl INHALATION: EMBRYO-FETAL T O X I C I T Y -"ASlb TERATOGENICITY STUDY IN THE RAT J HLR 881-81 VIII. REFERENCES (cont) 12. Unpublished Du Pont Data, Haskell Laboratory: 1 3 Du Pont Data, Haskell Laboratory: 14.Unpublished Du Pont Data, Haskell Laboratory: Company Sanitized. Does not contain TSCA CBI 25 Company Sanitized. Does not contain TSCA CBI TABLE I ,,,,,, EXPERIMENTAL DESIGN: exposure levels and distributioh of mated females among test r.n,.pa Exposure Levels (mg/in 3) Number Mated Females/Group Experiment I1 Experiment II2 Exposure Period RUN I 0 0.1 1.0 25.0 12 12 12 12 12 Days; 6-15 G 12 Days 6-15 G 12 Days: 6-15 G 12 Days; 6-15 G RUN II 0 0.1 1.0 10.0 p e x o ..o;'1 PF 2 5 .0 5 12 12 12 15 6 6 Days; 6-15 G Daysi 6-15 G Days; 6-15 G Days;6-15 G Days 6-15 G Days 6-15 G j 311 ExPeriment I females sacrificed on Day 21 G (Runs I and III ^ o f f i p S I T t o ai femaien al lwed to give birth and to raise their 3 t t s p r m g to at least Day 21 postpartum (Runs I and II) G - day of gestation s contro1 group pair-fed to group given/wfclat 10.0 m g / m 3 control group pair-fed to group giver/w^^lat 25.0 mg;/m3 n ik-9!gtd t-3 *5 G D 01 K H td i-3 3 tGu f-3 W 33 CO 00 M td w y 3 X H o o1o h9 H H k; TABLE II FEED CONSUMPTION3 IN RATS EXPOSED Trif M J r DnM D AY,, , ------------ -- l ^ l JKUH UAY S 6-15 OF RESTATt h m Experiment T Pre- exposure period (Dayys 1-5 G) Exposure periodd (Days 6-15 G) 20.8+0 88 71 n+n n ' ' '33 ,, 20.80.37 25.0 19.010.52 Pair-Fed( 10.0 25.0 20.310. 64 21.911.10 23.4i0.38 24.0i0.52 23.0i0.45 21.8iO.37* 18.4*0.46* 20.6i0.57* 1 7 .2i0.77* tv) Post-exposure periodd 27 9+0 -J (Days 16-20 G) . 78 30.0il.24 28.3i0.64 27.110.74 27.4i0.52 26.610.54 26.411.: Ift) Company Sanitized. Does no! contain TSCA CB1 b - nominal'c o n c e n t r a t i o n s o f r" S aItcluded 0 f s t J Z d. d * -- tha * * d consumed on the same ^- unit used was: '>St ex!>osure Periods, the rats were caged in pairs; the experimental _otal_grams feed consumed per cape per Hoy $ - significantly different from ?atS PSr Cage ntrol value by Dunnett's test (p0.05) Cpmpany Sanitized. Does no! contain TSCA CBI Experiment I Females 0.0 Exposure Concentrationsa (mg/m3) 0.1 1.0 10.0 25.0 10 0 Fed1 25.0 No. pregnant0/ no. mated No. deaths No. litters Mean no. corpora 23/24 0 23 24/24 0 24 23/24 0 23 15/15 0 15 8/12 3 7 6/6 0 6 5/6 0 5 NJ 00 lutea 14.50.47d Mean no. implants 14.010.51 Mean liver 14.310.51 13.410.41 15.610.58 13.810.28 15.210.65 15.lll.42 14.210.22 14.010.62 13.810.48 14.010.63 14.411.29 14.011.10 weight (g)e,5i Mean maternal weight gain (g)e 15.20.30 15.010.30 15.410.35 16.110.50 18.010.78+t 12.810.50++ 12.710.48 tt Days 1-5 Days 6-15 Days 16-21 Days 6-21 Days 6-21Cf 29.11.99 29.111.01 32.5il.00 57.611.69 57.6i2.06 56.7il.95 71.4+2.19 68.911.92 72.811.97 129.03.39 126.5i3.28 129.513.12 56.71.90 56.912.02 56.412.11 29.Oil.39 27.2il.97 26.2il.ll 50.9i2.21 36.415.33^ 34.913.21^ 72.9i2.77 68.613.41 71.314.72 123.812.86 105.017.26^ 106.214.70^ 49.112.52 37.415.39 ^ 34.312.98 ^ 31.613.33 22.716.93^ 68.912.73 91.616.66^ 27.016.07^ i H ISS O l--l t21d 2o1 H n H K+3j gtd CO w +3 K GO DI K td H i-3 2! > t+ +3 tO W +3 td o 00 X 00 w H H>o 1 +3 H 00 3 H Kj ^INHALATION: EMBRYO-FETAL TOXICITY ^ ^ E R A T O G E N I C I T Y STUDY IN THE RAT HLR 881-81 o a oH CmM 3 x> 0) H CO w O 11 3 H tfl o FM o roH VO CO 1 <! Q s I aH o H o o CO H W M MXOPh W a CO E- aH swaH hwoJ w o eS 1(33 ao o vS o i-t cd 03 oe H JCJO JJ c 0) o o 1--1 a x o w 80 97 iono m o co +i +i 00 co om o 99 H-- ON o o +1 CM o d +i m CO lH CO On ON o CM +1 +1 i--i d o tmo too d oo o+oi +i on o in r--1 CM O rH +1 +1 CM d in o 4- +-r^~ 00 O i+n1 o +1 00 CO o CM coa o rH +1 +1 oo co om o o f--1 CM O +( o co co CM r--( o +1 ON co ro^. CM O +1 on ro McJo* CM O o m+1 CM co o vOH co Oh o o +1 On co oo CM in O Cd+O1 o CO co o o+1 ON CO ON co d m o f+^1. CO in o Od+Ni in CM rH O o Mao O rH H +1 O VO O /V--JV ON +| ON O o+1 o UE-f rH VO CM CO a rH Q O (X3 w "Hh r ik uu u o uo oocO cCO5 aaj cO0(3 aa> . Vi QJ a, x w co QJ Q e o H JJ Oa S '? COrH QJ cd JJ 4J o a rH JJ U *H JJ JCjX cad 003 QJ 0) iad <u rH r [ o o U ri w QJ S gs 0) >H QJ p> CO H 0Q3J 3 rH a JJ QJ o" FH o C o s JJ H 0) & (1) S " 2 9 - Company Sanitized. Does not contain TSC A CB I jlOM PAYS 6-15 OF GESTATION a - nominal concentrations of j w s t s ~ \ l l tatog the " " " f * - -- ^ " h. same as ^ rr^ h^ i" ;jL8^:rr2 7 iL 7 7 iat -- -- fo r ln the 25. 0 me/,, , d - x a i l l ! 8" 1' " ere excluded fTM a n other c a l c u l l t i ^ g ' 1'" SUlfide stalninS: data from f I "n_^ eenant animals were excluded OLO Ci.e Day fcorctefL d ^ w e i g h t r 1^ 11 ^ f6maleS eXCludin^ the Products'of conception g ^ stunted fetuses were excluded 0 1o 5 CB2)O. N s. aaO(1D0 3 Sa . 5 CA O > Oen t 1 ^ S " L 7 t l y 7 ; f : " :dfr7 Pc0 r o 7 l 7 g " kg ? - ' s test (p<0.05) tt - significantly different from c o n t r g S g;> I lis sO H* la h wc s2 ah H 3 tt KS en tu *a-3 o 0K 1 K *1 W H i-3 t->Hl # tu f-3 00 w Xo 00 tu H h-1> O o1o 1-3 Hi-3 M K! Company Sanitized. Does not contain f s c a rs f 1 TABLE IV FETAL MALFORMATIONS IN RATS EXPOSED Tc/ S fROM DAYS 6-1 5 OF GESTATTON Experiment I Exposure Concentrations3 (mg/m3^ 0.0 0.1 1.0 10.0 25.0 External Malformations No. examined Microphthalmia 299/23c d 305/24 305/23 1/1 202/15 92/7 Visceral Malformations No. examined Great vessel malformation 159/23 Innominate artery-none Renal papilla - none Kidney - very small k Head Malformations l/lj No. examined 90/17 161/24 2/2f l/l1 161/23 110/15 l/lg 1/1 19/6 51/7 51/7 Skeletal Malformations No. examined Rib - fused missing 299/23 1/1^ l/l1 305/24 305/23 202/15 92/7 Pair--Fedb 10.0 25.0 80/6 66/5 42/6 34/5 l/lh 80/6 34/5 66/5 (Tj td 2 o H 3 k; CO t-3 G O k; m w2 G 1-3 G 00 W 00 to I 1-3 Company Sanitized. Does not contain TSCA CBe TABLE IV (CONT) FETAL MALFORMATIONS IN RATS EXPOSED TO FROM DAYS 6-15 OF GESTATION Experiment I Total with Malformations Avg. % Malformed Fetuses per Litter (iS.E.M.) Exposure Concentrations3 (mg/m3)______ 0.0 0.1 ___ 10.0 25.0 2/2 3/3 1/1 1/1 0.60.44 1.010.53 0.40.40 0.40.41 Pair-Fed^ 10.0 25.0 1/1 I U) hJ a nominal concentrations o f H i o - on each day of ---- --------- I same gestation day by selected^ats i^the6^ ^ amount of feed consumed on the rats were housed twoper cagt thin 6XPSUre the exposed amount offered to the pair-fed rat rage consumption for each day was the c - fetuses/litters d ~ blanks represent zero incidence f _ !etU! Wei^ ed 3-38 85 no variations detected g - fetus weighed 3.91 g^nd^lso h i r J e K c h i a e " TM " " ' ^ ^ th" fetus "el8hed 4 -51 S ribs, petechiaef"partially " "staid fetus weighed 3.82 g; no variations detected 'i hickf ed " " partia11^ " l e d IO 1-3 3 W ''> h.3 >It1 O H3 OH WO 33 H n H t-3 M K g CO fO t-3 K 30 O1 C 3 M H i-"3 3 3 3 h3 3 3 H3 oo W oo O X H n oIo ^3 H t-3 k Company Sanitized. Does not contain TSCA CL TABLE V FETAL VARIATIONS IN RATS EXPOSED TO m ^ F R O M HAYS . OF GESTATION ______Experiment T External Variations No. examined Hematoma Petechia Visceral Variations I No. examined <jO U) Renal papilla, I -reduced Renal pelvis -large Pulmonary arteries -common trunk Head Variations No. examined Eye - blood in anterior chamber Exposure Concentrations 3 (mg/m3-) 0.0 01 1 .0 1 0 . 0 25.0 299/23 12/10 31/13 305/24 16/12 38/17 305/23 11/7 30/16 202/15 5/4 tt 33/14 92/7 7/5+ 9/5 159/23 l/ld 1/1 5/2 161/24 e 1/1 161/23 4/4 110/15 3/3 51/7 1/1 90/17 1/1 1/1 19/6 1/1 51/7 Pair--Fedb 10.0 25.0 80/6 4/3 16/6++ 66/5 3/3 11/3 42/6 34/5 2/2 34/5 S Company Sanitized. Does notcontain TSCA CBI TABLE V (CONT) f e t a l v a r i a t i o n s i n r a t s e x p o s i ii i .......... 6-15 OF GESTATTOM Experiment I Skeletal Variations N o . examined Sternebra -misaligned(l)f -misaligned(2+ ) f i Centrum CjO fc. -bipartite I -dumbbelled Rib Exposure Concentrations5 (mg/m3) 0 .1 1.0 10.0 299/23 16/12 6/5 4/4 9/6 305/24 24/14 12/9 4/4 3/3 305/23 10/9 13/11 6/4 7/6 202/15 13/8 6/5 1/1 4/3 92/7 5/3 3/3 1/1 1/1 -extra ossification . center -rudimentary -extra -beaded -calloused -wavy 5 0 /12s 7/6 1/1 41/15h 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 Pair-Fedb iO-O 25.0 80/6 3/1 66/5 6/3 4/3 1/1 1/1 2/2 1 / 1 11/4 3/28 1/1 1/ 1. If'I Wt-3 2x g> OH MO 3 2! H O H K-ja gw CO to 8 ?>-3 K >0 to H t-3 2> C -a to to t-3 oo 00 H to to > O X H O 010 t-3 H Kt-!3 Company Sanitized. Does not contain TSCA CBI TABLE V (CONT) FETAL VARIATIONS in RATS EXPOSED t o f r o m DAYS 6-15 OF GESTATTON Experiment I Skeletal Variations <Vnr,M 00 No. examined with heads Skull bones partially ossified -parietal -interparietal -supraoccipital -squamosal Zygoma -partially ossified 140/23 3/3 2/2 3/3 2/2 Maxilla -partially ossified Hyoid -partially ossified -unossified 1 1 /6, 4/2 Exposure Concentrations3, (mg/m3) U 1 1 . 0 1 0 . 0 144/24 144/23 7/4 12/41 9/5 1/1 1/1 9/4 15/6m 2/1 1/1 2/2 2/1 2/2 1/1 7/5 13/9 92/15 4/2 7/4 25.0 41/7 1/1 1/1 2/1 1/1 2/2 Pair-Fed^ 10.0 25.0 38/6 32/5 1/1 3/2 1/1 4/2 2/2 3/2 10/3 2/1 2/2 2/1 3/3 1/1 5/ 2n IsJ 88 ^ wO Ho 23 M n H >K-j3 gM CO to -3 k ; S? KJ ^ w M 1-3 3> 3f It*>-3 i W i-3 00 W OX COM H i-1 n o1o -3 H (-3 m k; TABLE V (CONT) -- TAL VARIATIONS IN RATS E X P O g m xo FROM DAYS 6-15 OF GESTATTON .Company Sanitized. Does not contain TSCA CBI Experiment I Skeletal Variations (cont) Pair-Fed^ 10.0 25.0 Subtotal - Developmental Variations No. affected Avg. % Affected 118/22 123/24 100/23 76/15 44/6 31/6 26/5 Fetuses per Litter I (S.E.M.) UO O '! Sternebra 40.6+5.16 41.64.06 31.63.84 37.65.08 47.66.62 38.75.14 38.85.98 I -partially ossified -unossified 27/9 10/6 13/8 12/6 17/5j '+ 1/1 25/4k,++ 3/2 3/2 4/2 Centrum -partially ossified Rib -partially ossified Ischium -partially ossified Pubis -partially ossified -unossified 1/1 2/1 2/2 4/3 1/1 1/1 272 1/1 TABLE V (CONT) IgTAL_VAKIATIONS IN RAT S EXPOSED TO 6-15 OF GESTATION i u> -j i O) 5' co o J> o 2 Experiment I Skeletal Variations (cont) Pair-Fed 10 .0 25.0 Subtotal - Variations Hue to Retarded Development No. affected Avg. % Affected Fetuses per Litter (S.E.M.) *5/14 u.34.01 45/11 14.214.35 34/14 10.912.84 TOTAL 17/7 24/6 8.413.03 25.318.50 7/3 31/5 8.513. 93 49.8+16.07*^ No. Fetuses with Variations Avg. % Fetuses with Variations per Litter (S.E.M.) 144/22 140/24 123/23 87/15 55/6 34/6 42/5 48.715.07 49.414.42 40.113.43 42.815.14 58.415.98 42.215. 91 64.510.42 I O N3g2 'IOGS It^---3i MO 25 S H O H K+a gw co to +3 K GD Ol K^ W H 1-3 2 ! >< G >-3 ro m H3 wO 00 00 to X H Mo 1 +3 H 00 i-3 H-1 K jPgmpany Sanitized, Does rwfconfa/n TSCA CBI FETAL VARIATIONS IN RATS EXPOSED TO I a ^ nominal concentrations of ^FROM DAYS 6-15 OF GESTATION c- same gestation d a ^ r s e - L e ^ " * ^ exposed rats were housed two per cage fered t0 the ^ 0 ^ ^ d i * am U n t f feed consumed n the then t h S ? ' d l n 8 exPosure group; if the rat >Vera8e cnsl""P" '> each day e " m 18,^ h^ roureter also was detected f _ r n nk *ndcate 2ero incidence r (1 ) and (2+) denote that 1 r.r- 9 ,, gg - one was located in the cervical r e g i ^ a l l ^ S ^ Were misallgn e d > respectively I hih - 14 16 occurred occurred in in two two litters litters j 7 occurred in one litter 8 ' 1 1 others were located in the lumbar region OJ 00 I k 18 occurred in two litters 1 5 occurred in one litter m 1 1 occurred in two litters n 4 occurred in one litter t 5t( p l o ^ f ly -" * " < < 1 7 ^ Mann-Whitney ,, test ls _ talled - statistically significant difference detected hy two-tailed Mann-Whltney 0 test (p<0.05, a iCpnjpany Sanitized. Does notconiato TSCA CBI m I CO KO I TABLE VI REPRODUCTION AMD DEVELOPMENT OF OFFSPRING OF RATS EXPOSED TO 10M DAYS 6-15 OF GESTATION _______Experiment II Females Exposure Concentrations 0.0 0.1 1.0 10.0 25.0 No. pregnant/no. mated No. deaths No. litters Fertility Index (%)d Gestation Index (%)e Mean maternal weight gain (g) 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 llb /12 2C 9 91.7 100 Days 1-5 G Days 6-21 G Days 1 PP-22 PP Offspring 31.11.19 f 31.82.08 112.54.33 115.88.07 -3.4i0.58 -2.80.75 31.72.16 116.27.77 -2.00.81 28.22.44 118.93.28 -3.0.78 34.82.38 97.08.79 -2.20.52 At delivery (Day 1 PP) No. litters No. live pups/litter No. live pups (%) No. dead pups (%) 18 f 1 2 . 3 0 . 60. 222(99.6) 1(0.4) 10 12.li0.80 121(99.2) 1 (0 .8 ) 11 ll.2il.27 123(98.4) 2 (1 .6 ) 6 13.3i0.67 80(100) 0 (0 ) 9 1 1 . O i l . 26 99(99.0) 1 (1 .0 ) H t-3 K CO H3 3 3 tr* -3 3 3 h= 00 W 00 3 C X H M> o 010 -9 H 1-3 I--1 k ! i LjnD A ,lU " r j l A l j XJ TERATOGENICITY STUDY IN THE Company Sanitized. Does not contain TSCA CBI TABLE VI (CONT) REPRODUCTION AND DEVELOPMENT OF OFFSPRING OF RATS EXPOSED TO FROM DAYS 6-15 OF GESTATION ______ Experiment II______ 0.0 Offspring (cont) After delivery No. dead No. cannibalized No. live (Day 22 P P ) No. males/females Viability Index (%)j Lactation Index (%)k Mean weight (g) ^ q 221i 115/106 100 Exposure Concentrations3 M(imigg//mm3)) 0.1 1.0 10.0 1,h 2 118 62/56 99.2 98.3 0 0 123 70/53 100 100 0 0 80 44/36 100 100 25.0 0 2 97 45/52 99.2 99.1 Day 1 Day 4 PP DaY 7 PP Day 14 PP Day 22 PP Alterations No. examined externally No. malformed No. eyes (pairs) examined in vivo11 No. with alterations 6.80.11 10.3+0.25 15.00.44 29.21.06 50.11.82 7.010.18 10.910.32 15.610.46 30.110.89 51.411.73 6.710.18 10.910.39 15.910.63 30.611.29 52.012.46 6.610.23 9.910.43 14.810.69 28.011.23 48.412.15 6.10.15++ 9. 70.33 1 4 . 3 0 . 38 29.00.55 49.00. 88 222/181 141/12 3/3 121/10 l/lm 118/10 3/2p 123/11 123/11 0 80/6 __ __ 99/9 97/9 0 It1 00 00 > I t-3h OO fHK Company Sanitized. Does not contain TSCA CBI a nominal concentrations of b ~ two females with i m p l a n t s ^ ? a*a s S S i r% d - 9 G (She had " implants)1" a"tS); * ^ " e included ^ e - Gestation Sdex"" the Day" " " ? ) 5 resultlne 1" Pregnancy (excludes - * 1 ^ 1 !" " g ~ to Day 22 pp pup with domed hparl T7 n o 't^ m v e t H i m ? 8 ^ " * M rth of ^ x -- one^urias neCVPSled n 20 ? it was not expected to survive the one pup It is wrnaismtn*eac^ropsi1 ed on Day y 19 as cit J - V i a b i l i t y i n d e x i s S ^ c e n L ^ f ^ ^ ^ a U o ^ ^ n d e x 15 ^ ^ ' l m ^ 3 STMup; ; s s r lstha~ =5 - - s~ expected toiurvive t lac hea,d ,and abnormal gait on Day 19 Si d - sc a& E i S " " V - significantly diffetent ftom wlth iMomplete mydrlasls and red vai ( ^ d ^ i t ^ ^ t L t , p<,,.0 5) LI o H W wn oh M3 s n H HW H< g CO <P0 h3 K< 01 K h) W H t-3 ** t"<i-3 fo w ^ 00 00 pa o X H|>n 010 >-3 i-H3 I--1 K) CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ELK TON ROAD NEWARK, DELAWARE APPENDIX A PROTOCOI^ F O R INHALATION TERATOGENICITY STUDY IN RATS WITH ' ----- N I . BACKGROUND hSirii This study was requested by Department W M at a meeting' foi such a Ss ^ a y ^ 0rat0ry n APril 9 ' 1981' The need ^ emanated from TSCA Section 8(e) 's filed bv M between the last part of 1980 and March 20, 1981. The adverse effects seen in studies conducted for 3M f lenf chan?es in the eyes of term offspring rom rats exposed to the test chemicals by gavage from Days 6 through 15_c_f gestation. For addition a l ^ TM - tion, see m e m o s : .. , " 1981, and March 26, March 30, 1981. draf<. of HFi ^ eTdvelPment of this protocol, the draft of Haskell Laboratqj-- " --- o n t h e known toxicity of ^ 5 .n lid a ? id eWSd' " d " formation II. MATERIALS AND METHODS t w i n k Th? study consists of two experiments. Experiment the^dL^ l teratogenicity study, by inhalation Sith The d TM * fJCri flC thS day before expected delivery. The dams for Experiment II will be exposed as in the first experiment, but will be allowed to give birth nd to raise their offspring to at least 22 days post pfrtu^? Du Pont classified information Company Sanitized. Does not contain TSCA CB I 42 PROTOCOL -INHALATION TERATOGENICITY II MATERIALS AND METHODS (CONT) A. Test Chemical . received from the Polymer Products Department, was assigned Haskell Number 14,045. ^ B Animals I CD (S D )BR female rats 48--54 days of age parous), 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 Mating will be verified by detection of spermatozoa in the vaginal lavage each morning (Day 1 of gestation) following overnight cohabitation. (nulli-- . 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 m this species at Haskell Laboratory. The Crl:CD(SD)BR ^ rafn Yas 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 all animals will be screened for these aiterations before breeding. The screening procedure 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 Pont employees are likely to be exposed Company Sanitized. Does not contain TSC A CBI 43 PROTOCOL -INHALATION TERATOGENICITY STUDY WITH PAGE 3 C. Exposure by Inhalation (cont) The test groups will be exposed to the in Rochester Chambers; free moving T a t s will be given whole body exposure for 6 hours/day from Days 6 through 15 of gestation. Chamber concentrations of will be determined about 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 m g / m 3. Based on the limited toxicity data available at Haskell Laboratory and the results reported by 3M, these concentrations are expected to yield an r'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 group for Experiment I. About 12 females will be assigned to each group for Experiment II. Mated rats that are ill or that do not gain weight properly will be discarded before distribution - 44 - jCompany Sanitized. Does not contain TSCA CBI PROTOCOL -INHALATION TERATOGENICITY STUDY WITH' 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 Wilmington 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 "Twill be packaged in polyethylene!uned Fiberpaks 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. Company Sanitized. Does not contain TSCA CBt 45 PROTOCOL -INHALATION TERATOGENICITY STUDY WITH 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 post exposure 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 a n d , 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 TSC A CBI 46 PROTOCOL -INHALATION TERATOGENICITY 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 7 :291-306 (1969). - 47 - Company Sanitized. Does not contain TSCA CBI PROTOCOL -INHALATION TERATOGENICITY 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, 7, 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 m o r e ) e Lactation index (% animals alive at 4 days that survived to 22 days - post partum) Company Sanitized. Does not contain T S C A CBI 48 PROTOCOL -INHALATION TERATOGENICITY 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, 7, 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 CB1 PROTOCOL -INHALATION TERATOGENICITY 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<0.05. In addi tion, the reproductive indices given earlier will be calculated. IV. CRITICAL DATES Starting Date (breeding): April 27, 1981 Completion: November 15, 1981 Company Sanitized. Does not contain TSCA CBI 50 PROTOCOL -INHALATION TERATOGENICITY Teratology Section Toxicology and Pathology B .(a . Burgess Research Toxicologist Acute Investigations Toxicology and Pathology APPROVED BY: J .'G . Aftosmis Associate Director Toxicology and Pathology RES/BAB/mle 6/9/81 G. L . Keniiec Chief . Acute Investigations Toxicology and Pathology Company Sanitized. Does not contain TSC A CBl 51 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE APPENDIX A (CONT) ----------- December 3, 1981 MEMORANDUM TO: QUALITY ASSURANCE COMMITTEE - C. M. BARBA FROM: R. E. STAPLES / v T / o J SUBJECT: ^PROTOCOL AMENDMENT FOR TERATOGENICITY STUDY : ^VVr^amendmer^^t^th^Drotocol for the teratogenicity study o f a t t a c h e c L Please note tha-t^aeviation#^^n^theaudi^report for this study no longer pertains; all skeletal specimens were examined for alterations. RES/mle Attachment Company Sanitized. Does not contain TSCA CBi 52 E. I. DU PONT DE NEMOURS & CO. , INC. CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ELKTON ROAD NEWARK, DEALWARE AMENDMENT TO THE 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 Run I , all head specimens in the 0.0 mg / m 3 and 25.0 mg7m3 groups 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 m g / m 3 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. For Run II, one specimen from each of four litters in the 0.0 m g / m 3 and 10.0 mg / m 3 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 Study 2. Offspring g. Eye examinations No compound-related effects were detected in vivo by the ophthalmologist among the pups from Run I, and the highest exposure level in Run II (10.0 mg / m 3) was lower than that for Run I (25.0 mg/m3). Therefore, the eyes of pups from Run II were not examined in 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 Sanitized. Does no con.ai,, TSCA CBI AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY STUDY IN RATS WITH PAGE 3 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. Company Sanitized. Does not contain TSCA CBI 55 AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY STUDY Staff Teratologist Teratology Section Toxicology and Pathology Toxicology and Pathology APPROVED BY: J. G. Aftosmis Associate Director Toxicology and Pathology R E S /mie 12/3/81 G. i. G. L.. Kenr dy. Chief' Acute Investigations Toxicology and Pathology Company Sanitized. Does not contain TSCA CM 56 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE APPENDIX A (CONT) R :E:.V ,1 S E D December 30, 1981 MEMORANDUM TO: QUALITY ASSURANCE COMMITTEE - C. M. BARBA FROM: R. E. STAPLES SUBJECT: PROTOCOL AMENDMENT FOR THE INHALATION TERATOGENICITY STUDY An amendment to the protocol for the teratogenicity study of is attached. RES/mle A t t a c h m e n t (1) f. Does not contain TSCA CBt REVISED E. I. 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 e After severe clinical signs and mortality occurred in females in the 25.0 m g / m 3 exposure group (Run I ) , the exposure level was reduced to 10.0 mg/m3 for Run II. The 10.0 mg/m3 exposure group now consists of 12 of the females originally scheduled for the 25.0 mg/m 3 group (Run II), 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 / m 3 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 m g / m 3 groups were added to Run II. . Company Sanitized. Does not contain T S C A CBI 58 AMENDMENT TO THE PROTOCOL FOR INHALATION TERATOGENICITY Staff Teratologist Teratology Section Toxicology and Pathology Toxicology and Pathology APPROVED BY: T 7 J. G. Aftosmis Associate Director Toxicology and Pathology R E S /mie 12/30/81 G . L . Kennedy Section Supervisor .Acute Investigations Toxicology and Pathology Company Sanitized. Does not contain r u n a c r i 59 FOR DU PONT USE ONLY ATTACHMENT 1 E. I. du Pont de Nemours and Co., Ine. Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P. 0. Box 50, Newark, Delaware 19711 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 fTERATOLOGY 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 off This test was conducted in two parts. II. Procedures A. Protocol Pregnant Crl:CD rats for this study were supplied and cared for by ' Haskell's teratology group. Dams were mated in lots, two for Part I and three for Part II, 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 I - Air Control , Group II (low) - Design Level of 0.1 mg/ni Group III (intermediate) - Design Level of ^.0 mg/in Group IV (high) - Design Level of 25.0 mg/in Company Sanitized. Does not contain TSC A 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.(j 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 dates a total of 11 exposures were necessary for Part I, and 12 exposures for Part II, 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 Dust atmospheres of were generated with an 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 volume of chamber air was drawn though preweighed Gelman glass fiber filters (Type AE, 25mm). Filters were reweighed a n d f l B B I B B f t concentration calculated from filter weight gain.Filters were weighed on a Cahn Model 26 Automatic Electrobalance. Chamber atmospheres were also analyzed by a spectrophotometric technique**. absorbed to filters was desorbed with an acidified aqueous methylene blue solution. The ^ B B B B t t B B V B B 0 0 methylene blue complex formed under these conditions was extracted into chloroform a n d ^ ^ B ^ H ^ 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 urn. 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 Impactor 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. 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 88% total respirable content. Particle size samples were taken from the seventh and tenth exposures during Part II. 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 3 Mean Chamber Concentration (mg/m + S.D.) Gravimetric Spectrophotometric 0.12 + 0.06 0.08 + 0.05 0.14 + 0.07 0.08 + 0.03 0.18 + 0.08 0.12 + 0.04 0.13 + 0.05 0.12 + 0.08 0.12 + 0.06 0.09 + 0.04 0.18 + 0.10 0.13 + 0.09 0.13 + 0.06 0.09 + 0.04 0.12 + 0.02 0.10 + 0.02 0.13 + 0.03 0.10 + 0.04 0.11 + 0.05 0.12 + 0.03 0.12 + 0.04 0.14 + 0.02 Overall Meant + S.D. 0.14 + 0.06 0.11 + 0.05 t Mean and S.D. of all samples throughout exposure period. Company Sanitized. Does not contain TSC A CBI 63 TABLE II Intermediate Level Analytical Data (Part I) Exposure // 1 2 3 4 5 6 7 8 9 10 11 3 Mean Chamber Concentration (mg/m + S.D.) Gravimetric Spectrophotometric 1.3- + 0.4 1.3 + 0.5 1.3 + 0.5 0.9 + 0.2 1 .1 + 0.3 1.1 + 0.5 1.0 + 0.2 1.0 + 0.2 1 . 1 + 0.3 1.3 + 0.3 1.3 + 0.7 1.5 + 1.0 1.1 + 0.8 1 .1 + 0.2 1.2 + 0.5 0.8 0.4 1.1 + 0.2 1.3 0.7 1.4 + 0.5 1.5 + 0.5 1.2 + 0.8 1.1 ^ 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 TSC A CBI 6* TABLE III High Level Analytical Data (Part I) Exposure # 1 2 3 4 5 6 7 8 9 10 11 3 Mean Chamber Concentration (mg/m + S.D.) Gravimetric Spectrophotometric 18.0 + 15.7 24.6 + 23.2 1 5 . 9 + 5.9 12.0 + 3.0 23.8 + 19.4 26.7 + 16.8 18.2 + 5.1 18.8 + 1 . 9 22.0 + 6.5 2 3 . 0 + 4.7 22.7 + 6. 6 19.9 + 5.7 21.9 + 6.4 2 1 .0 + 8. 8 21.9 + 6 . 6 18.5 + 6.4 21.7 + 7.7 23.3 + 7.4 21.7 + 10.0 14.8 + 5.2 23.3 + 6.2 24.1 + 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 Sanitized. Does not contain TSCA CBI 65 TABLE IV Mean Chamber Temperature Data (F+S.D.) (Part I) Exposure # Control 1 72.0 + 0.0 2 78.0 + 1.4 3 77.3 + 0.8 4 78.0 + 1.1 5 77.3 + 1.5 6 76.6 + 0.9 7 77.4 + 0.5 8 78.5 + 0.8 9 76.4 + 1.1 1 0 77.3 + 2.2 1 1 72.3 + 1.9 Low 77.5 + 0.6 77.0 + 1.7 75.0 + 1.3 76.3 + 0.8 76.2 + 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 Intermediate High 77.1 + 0.6 75.8 + 1.3 77.2 + 2.2 75.8 + 2.9 77.0 + 1.1 76.6 + 0.7 75.5 + 1.0 76.8 + 1 . 2 76.0 + 1.2 78.2 + 1.2 76.7 + 0.7 76.2 + 1.1 75.8 + 1.6 75.8 + 2.5 77.7 + 1.2 77.0 + 0.9 75.8 + 1.6 77.6 + 1 . 1 76.8 + 1 . 6 77.1 + 1.3 74.5 + 0.8 75.0 + 1.1 Overall Meant 76.5 + 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. Company Sanilizad. 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 0.07 + 0.02 0.13 + 0.03 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 0.13 + 0.02 0.12 + 0.05 0.09 + 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 Sanitized. Does notcontain TSCA CBt 67 TABLE VI Intermediate Level Analytical Data (Part II) Exposure # 1 2 3 4 5 6 7 8 9 10 11 12 Mean Chamber Concentration (mg/in + S.D.) Gravimetric Spectrophotomet 1.1 + 1.20 0.67 + 0.32 1 . 1 + 0.56 1 . 2 + 0.47 1.3 + 0.27 1 . 1 + 0.37 1.2 + 0.66 1 . 2 + 0.24 0.96 + 0.47 1.1 + 0.29 1.2 + 0.21 0.80 + 0.37 1.0 + 0.33 1.2 + 0.17 1 . 1 + 0.61 1.1 + 0.45 0.78 + 0.25 0.82 + 0.08 0.75 + 0.3? 0.80 + 0.17 0.97 + 0.33 1.3 + 0.59 0.85 + 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. Qompany Santfizeef. Does not contain TSC A CBI 68 TABLE VII High Level Analytical Data (Part II) Exposure it 1 2 3 4 5 6 7 8 9 10 11 12 3 Mean Chamber Concentration (mg/m + S.D.) Gravimetric Spectrophotometric 8.4 + 3.74 8 . 8 + 3.16 10.3 + 2.45 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 + 5.30 10.9 + 8. 66 11.8 + 6.09 11.1 + 4.97 9.9 + 6.41 14.6 + 7.35 9.8+5.19 12.5 + 6.21 8.7 + 3.84 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 Mean Chamber Temperature Data (F +SD.) (Part II) Exposure // 1 2 Control 78.4 + 0.5 77.2 + 1.0 Low 74.7 + 1.4 76.7 + 1.2 Intermediate High 77.5 + 1.5 78.0 + 1.1 72.2 + 3.4 74.3 + 1.0 3 78.3 + 1.3 75.9 + 2.6 77.1 + 1.5 77.0 + 2.3 4 77.3 t 1.0 75.6 1.4 77.0 + 2.0 78.4 + 0.8 5 77.0 + 1.5 76.8 + 1.7 77.7 + 1.0 78.1 + 1.3 6 76.8 + 2.3 77.7 + 2.3 78.0 + 1.2 77.6 + 1.5 7 77.1 + 1.3 76.3 + 1.0 77.5 + 0.5 77.5 + 1.2 8 77.6 + 1.0 77.5 + 0.8 77.2 + 0.8 77.3 + 1.7 9 78.6 + 0.5 77.9 + 1.1 75.9 + 1.9 77.6 + 1.9 10 77.2 + 1.3 76.8 + 1.3 77.2 + 0.8 78.0 + 1.8 11 77.0 + 1.7 76.0 + 0.6 76.1 + 0.7 76.2 + 1.8 12 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. Company Sanitized. Does not contain TSCA CBI 70 During exposure no clinical observations were noted in Groups I, II, III, 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 signs were noted in rats exposed 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 8 th exposures. No deaths occurred during Part II. Summary The purpose of this study was to determine the teratogenic effects of on pregnant Crl:CD rats. The test was conducted 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 spectrophotom^trically. 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/in in Part II. These results were confirmed by spectrophotometric analysis. In Part I, n^ 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/^ of Rats surviving exposure to 21.0 mg/m exhibited dry red discoloration around the mouth following each exposure. During Part II, a dry ^ed discoloration was observed around the mouths of rats exposed to 9.9 mg/in following each exposure. No other clinical signs were noted in any other test group. Company Sanitized. Does not contain TSCA CBI 71 **Reference: L. F. Percival. "Determination of Cg and Cg Dispersing Agents, Methylene Blue Method". E. I. du Pont 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: Clarence Hutt Technician // Stephen D. Nash Technician Supervised by: l~ei e.U tili ( j Joseph C. Ramili Technologist Approved by: CH:SDN:tac:WP: 12.2 Date Issued: December 18, 1981 Gerald L. Kennedy7 . j Section Supervisor 's Acute Investigations '' - 72 Sanitized. Does not contain TSC A C8 Company DIPLOM ATE; AM ERICAN C O LLEG E O F VETERINA RY O PHTHALM O LO GISTS ATTACHMENT 2 JAMES M. CLINTON. V. M. D. --------- AN IM AL E Y E C L IN IC AT SO U TH J E R S E Y AN IM AL H O SPITA L R O U T E 5 4 1 A B O V E C H U R C H R O A D - P. O . BO X 115 MEDFORD. NEW JE R S E Y 0 8055 Telephone (6 0 9 ) 654-0304 Haskell Laboratories Robert E. Staples, Ph.D. Study: Exam Date; 24 April 1981 Ophthalmoscopic Summary ^Both eyes of all of the male and female rats in the colony were examined by focal illumination, and indirect ophthalmoscopy. Mvdriasis was achieved with 1% Atropine (1% Atropisol. Cooper, B3036, 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 E5^e: Right Eye : Right Eye: Posterior synechiae. Focal retinaldegeneration. ' Focal retinaldegeneration. Focal retinaldegeneration. Anterior synechiae from 2 to 4 o'clock. Corneal opacities and subjacent anterior synechiae from 11 to 1 o'clock. Marked ventral entropion, blepharospasm. MALE RATS Rat Number 298494 298514 298429 298305 298299 298128 298083 298148 300068; 300130 300177 Observations Left E y e : Superficial corneal vascularization, Bilateral superficial corneal vascularization. Right Eye : Anterior synechiae from 1 to 3 o'clock. Left Eye: Anterior S37nechiae. Left E y e : Anterior synechiae. Right Eye : Superficial corneal vascularization. Right Eye;: Anterior synechiae from 9 - 1 1 o'clock. Right Eye: Anterior synechiae at 9 o 'clock, Bilateral anterior synechiae, nasal quadrant. Left Eye: Anterior synechiae. Left E y e : Anterior synechiae. . ' COMMENTS The 18 raus aescri bed above were removed from the colour and euthanatized by Supervisor Alice Erv in. All of those rats remaining are ophthalmoscopically no rma1 and uicable for use in the forthcoming study. - 73 - X dames M. Clint KJLi V.M.D. Company Sanftizecf. Does not contain TSCA CBI DIPLO M ATE: AM ERICAN C O L LE G E O F VETERIN A RY O PHTHALM O LO GISTS ATTACHMENT JAMES M. CLINTON. V. M. D. AN IM AL E Y E C LIN IC AT SOUTH J E R S E Y AN IM AL H O SPITA L R O U T E 54 1 A B O V E C H U R C H R O A D - P. O . B O X 115 MEDFORD. NEW JE R S E Y 0 8 0 5 5 TELEPHONE ( 6 0 9 ) 6 5 4 - 0 3 0 4 3 Haskell Laboratories Study : 9K^ Robert E. Staples, Ph.D. Exam Date; 5 May 1981 Ophthalmoscopic Examination Both eyes of 210 female rats v.?ere 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 E3'-e: Very pale ocular fundus. 302240 Bilateral pale ocular fundi. 302194 Right Eye: Focal retinaldegeneration. 302201 Right Eye: Anteriorsynechiae 5-6 o'clock. 302248 Right Eve: /ulterior synechiae, nasal quadrant, with corneal opacity. ' 302319 Right Eve:' 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 As soon as they were identified,'the 13 rats described above were removed from the colony. --- ,Zicr^_ James M. Clinton, V.M.D. - 74 - Company Sanitized. Does not contain TSCA CBi > HASKELL LABORATORY ATTACHMENT 4 December 18, 1981 MEMORANDUM TO: R. E. Staples FROM: W. D. Kerns SUBJECT: _ 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 Sanitized. Does not confa? t qc;a c r 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 C L L /as 9/30/81 .Company Sanitized. Does not contain T S C A CBI 76 (IPP) HASKELL LABORATORY MEMORANDUM ATTACHMENT 5 REVISED December 21, 1981 SUBJECT: 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. WDKrwfd Company Sanitized. Does not contain TS C A CBl 77 TABLE 1 INDIVIDUAL ANIMAL HISTOPATHOLOGY DATA Company SanHted-00 I 00 1 CP 3 a oo a a3 'A O > r> * Tissue Accounting Code: N N Both eyes (including lens) were within normal limits for a developing fetus, Only one lens was present for histomorphologic evaluation and it was within normal limits. 0 Lens was not present in the sections submitted for evaluation. L -- Lesion observed ** Degree of Change (severity): 1 = Minimal 2 = Mild 3 = Moderate 4 = Marked 5 = Severe iradwoCk H Tissue*and Observation** Fetal Eyes Hyphema, focal I "4 I Code : See Table 1. TABLE 1 (Continued^ INDIVIDUAL ANIMAL. HISTOPATHOLOGY DATA Part 2 TABLE 1 (Continued) INDIVIDUAL ANIMAL HISTOPATHOLOGY DATA Part 3 Tissue* and Observtion** Run: Exposure Level (mg/m3); Fetal Eyes Hyphema, focal i 00 0 1 Code: See Table 1. N' N' It' N' 25.0 N' N N N N' N N N N N ftiedutCL H - 14045 TABLE 2 INDIVIDUAL ANIMAL HISTOPATHOLOGY DATA Tissue*and Observation** Run ; Exposure Level (mg/m3) Dam Number; Fetus Number: 0.00 0.10 . 10.0 78 04 75 06 101 55 13 07 99 09 105 74 __ 64_ 93 02 05 07 07 64 14 Fetal Eyes Hyphema, focal i 00 H 1 NNNNLNNNN L 22 Code: See Table 1. Company Sanitized. '---t.m m c ..- n n c m u n L U L L E G E O F VETERINARY OPHTH ALM OLOGISTS JAMES M. CLINTON. V. M. D. AN IM AL E Y E C LIN IC AT SOUTH J E R S E Y A N IM A L H O SPITA L R O U T E 54 1 A B O V E C H U R C H R O A D - P. O . B O X 115 MEDFORD. NEW JE R S E Y 0 8 0 5 5 Telephone (6 0 9 ) 6 5 4 -0 3 0 4 Haskell Laboratories R. E. Staples? Ph.D. Exam Bate: 5 June 1981 . Ophthalmoscopic Examination rl Generation of Ophthalmoscopically Normal Parents First Run TL^ * J-liunanation, indirect ophthalmoscopy and, wb-n Indi'catPd" tai'ned unt-1'1 t-i-a ~* 5 ^ * 3 3 ^>-em_-aar?kn^ess.l x bemi-aarkness was main- a i clileS to m0rnlnSV d Se l e W l s and roup identiiLs ally nornalLixcept^s foil!*?*1* ^ remale rat pups were ophthalmoscopic- Litter N o . 301260 301154 301179 301117 301117 301072 Offspring No. T-L 1 1 1u. 2 .1 Observations Prominent filamentous opacities of ^arh posterior lens pole. '' Prominent filamentous opacities of ea^h posterior lens pole. ~ Prominent filamentous opacities of each posterior lens pole. ' Left Eye: Focus of pre-retinal hemorrhage Ol 2 disc diameters. '6 Right Ej^e: Incomplete mydriasis and rod oval opacity on central endothelium. " Right Eye: Pre-retinal hemorrhage at peripheral nasal quadrant. Comments In my opinion , :he lesions noted might typically occur in any well managed colony of rats. small ho be r-omnlp-oiV #Although some of these young rats' eyes were f-oo vtihseualnitz'eedr.Lr S ^ o'l t L S r ^siegnmeen^tss L0 1? m6os"t! "or! the eyes werIe brreiaedviely thi? u a t e d L / S L ^ S y ^ h a J p L d u c e i o c S l r ^ th ?*8t material> eval nauges in the El generation. - 82 - James M. Clinton, V.M.D. Company Sanitized. Does not contain TSCA CBI