Document KJXaJD3br4r5Lqg4vkzpn1z1o

Special Lens oral Teratology Study of T-2999CoC in Two Strains of Rats Experiment No.: Conducted At: Dosing Period: Study Directort 0681TR0362 Safety Evaluation Laboratory Riker Laboratories, Inc. St. Paul, Kinnesota September 28, 1981 through October 8, 1981 S. G. Gortner Gortner Date Senior Research Technologist Animal Teratology Reproduction 7- 1(-A%,2 X. G. LwWr*cht, DVM, PhD Date Research Veterinary Pathologist 7 acit_ M. T. Case, VM, PhD g,*V-t4 Date Manager, Pathology-Toxicology Safety Evaluation Laboratory 6 --- - We C. McCormick, Us Toxicologist toxicology Services Date --- -------- Table of Contents Table of Contents I Suvimry o . o o a 3 :Cntroduction o a 4 Materials and Methods 5 Results 7 Discussion 8 References 12 Tables 1. Mean Maternal Body Weights (g) With Standard Deviations Listed by Gestation Day in Two Strains of Rats and Two Dose Levels of T-2999CoC . . . . 13 2. Number and Percent of Freehand Section Gross fttal Loans Findings For Individual Lane and IP*tus Experimental Units in Two Strains of Rats and Two Dose Levels of T-2999CoC 14 3. Ratios and Percent of Freehand Section Gross IAns Findings Confirmed Microscopically for Individual Lens and Fetus Experimental Units in Two Strains of Rate and Two Dome Levels of T-2999CoC * , o . 15 4. Ratios and Percent Agreement of Gross Individual lane Findings With Microscopic Lens Findings in Two Strains of PAto and Two Do** Levels of T-2999CoC 16 !5. Number and Percent of Petal Lenses With kicroscojpic Findings Listed by Lens Region in Two Straine of Uts, Two Levels of T-2999CoC and Two TiBsu* Processing Procediwes With an Index of Lens Sectioning Proficiency 17 2 Table of contents (Concluded) Appendices Procotol for special Lens Oral Toratology Study I* of T-2999COC in Two Strains of Rats . . . . . . o Amendment for Special Lens oral Toratology & 20 StudW of T-2999CoC in Two Strains of Rats . . . protocol Deviations * o o . o 0 * 0 a a 0 0 o 21 List of Principal Participating Personnel o 9 o 9 22 ii. 111. Statement of Quality Assurance o o o o * o * a * 23 IV* individual Body Weights (9) With Mean Body Weights and Standard Deviations for Pregnant Animals o a o o o e . a o o o o o 9 o 9 0 0 a * . 24 Gross and Microscopic lans Observations of ve individual Fetuses Sectioned Across the Lenses. . 28 Microscopic lans observations of Individual VI. Fetuses Not Sectioned Across the Lenses a 36 T-2999CoC Composition, Purity and Stability . . . 44 vii. 3 SUMMARY In a previous rat teratology study'of a.fluorochemical alcohol, FM-3422, ,s on uncoded freehand sectioned fetuses gestation day 20 gross lens finding related abnormalities. The objective ol' vere interpreted as compound and dose this current study vas to compare the histological appearance offetal mt lenses that had been freehand sectioned to the histological appearance of lenses vhich had not been freehand sectioned* Groups of thirty time-mated Crl:CDO(SD)BR and CDFO(F-344)/CrlBR rats were orally dosed at 0 and 25 mg/kg/day with T-29ggCoC, a comparable alcohol, on days 6 through 15 of -gestation. Four fetuses from each of 20 litters per group vere selected. The identities of the fetuses vere concealed from the investigators by coding. Gross lens observations vere made on freehand sections of tvo fetuses per litter. Microscopic lens observations vere made on all four coded fetuses folloving microtome sectioning and tissue staining. T-2999CoC caused mternal toxicity (lov body veight gain) in the pregnant dams of both rat strains. The compound did not affect either the gross or microscopic lens appearance or fetuses from treated dam. The compoundrelated occurrence of lens findings reported in the previous rat teratology study could not be repeated when the fetuses vere coded before freehand sectioning and gross evaluation. The gross finding of a lens cleft and the microscopic finding of a space at the lens vesicle vestige are artifacts created independently by freehand and microtome sectioning. They represent a separation betveen the embryonal nucleus lens cells and the lens epithelium. The gross finding of a lens dark streak is a normal observation of the embryonal nucleus. The embryonal nucleus is an area of normal lens cell degeneration located in the central region of the lens. Lens cell degeneration was present microscopically in every lens sectioned across the central region of the lens. 4 Introduction the 3M t (TSCA) Section 8(e) notice was filed by A Tbxic Substances Control Ac Company based on rat tergLtologY study results on FM-342M. Partly responsible for filing the 8(e) notice was the observation of a fetal lens finding which was found only in the treatment groups. The observation vas reported as a teratogenic change because it vas foundonly in compound treate(f dose groups and had a dose-related incidence of occurance. T-2999CoC (FM-3924) vas selected for use in the current study because it is -more representative of present production than i FM-3422. rurther, T-2999CoC potentially presents greater risk than FM-3422 b:cause of the somewhat higher content of more soluble lower homologues. The protocol for the current study, was originally designed with two objectives. 7ne first objective vas to report the effect of T-2999CoC treatment and the strain of rat on the incidence of the lens change. The second objective was to compare the incidence of lens changes of fetuses not exposed.Ln utero to dosing and animl handling procedures used in teratology studies with the incidence of lens changes in the CDs control group. The animals were dosed and the dams were terminated. During this time, the laboratory, gained insight into normal and abnormal lens development. As the result of discussions with scientists from industry, government and academia, the lens change originally reported in previous studies was thought to be an artifact of freehand sectioning, and the protocol vas amended to test this hypothesis. The objective of this study was changed to empare the histological appearance of gestation day 20 fetal rat lenses that have been freehand sectioned and then processed for microscopy to the microscopic appearance of gestation day 20 fetal rat lenses vbich have not been freehand sectioned before being processed for microscopy. The study was sponsored by 3M Commercial Chemical Division, St. Paul, t4innesota and was conducted by the Safety Evaluation laboratory, Riker Laboratories Inc., St. Paul, Mirmesota. The two compound administration groups were dosed between September 28, 1981and October 8, 1981. The protocol and list of principal participants can be found in Appendices I and II respectively. All portions of the study were conducted according to the Good Laboratory Practice (GLP) regulationsk and Safety Evaluation laboratory Standard OperatinPgrocedures. Appendix III contains the Quality Assurance Unit statement. The storage location for specimens, raw data and a copy of the final report is maintained in the Safety Evaluation IALboratoryls record archives. ,LLYM-34i2s2at least 96%C8F,Tso2N(c2HZ)CH20H (Riker Experiment No. o68OTROO10) and T-2999COC (FM-392) is about88% CBFlTS02N(C2H5)CH2oHi ih the remaining 11%, the perfluoroalkyl group contains three to seven caruoti atoms. 3@Federal Register, Vol. 43, No. 237, December 22, 1978 pp 60013-60025. 5 Materials ar@d Methods Ninety Crl:CDO(SD)BR rats.E (CDO) and sixty CDFO(F-344)/CrlBR ratsd- (CDFW) were assigned cages according to a computer-generated random numbers table. Iliey were sexually mature time-mated females weighing between 163-229g and 127-163g respectively (Appendix IV). The rats were housed individually i-nsuspended, wire mesh, stainless steel cages in a temperature and humidity controlled room. They were identified by ear tags with that identification number indicated on the outside of the cage* The CDO non-treated group was identified only by a number on the outside of the cage. Feede-and water were -available ad libitum. The lights were on a 12 hour light/dark cycle. The mt was chosen because previous teratology studies on FM-3422were conducted in this species* The 25 mg/kg/day dose level was selected because it gave a positive response to the lens finding in the previous teratology study. The T-2999CoC emposition, purity and stability were determined by the 3M Commercial Chemical group (Appendix VII). The test article was suspended in corn oilf daily. The test article solutions and the corn oil control article were administeredby oral intubationto rats on days 6 through 15 of gestationELaccording to the folloving experimentaldesign for dosing: Rat Strain and Dose Gr -@-2-999CoCDose Levveell CDO corn oil controlarticle 0 tg/kg/day CDP* corn oil controlarticle C)mg/kg/day CDO T-29ggCoC in corn oil 25 mg/kg/day CDFO T-29ggCoC in corn oil 25 uLg/kg/daY CDO Non-treated group rats not dosed uGroup Size 30 30 30 30 30 ESupplied by Charles River Breeding Labomtories, Inc. . North Wilmir)gton.t4A. AWpplied by Charles River Breeding Iabomtories. Inc., Stoneridge,NY. iftr ina IALboratoryChov, F&Iston Pdrina CompLrW.St. LOUA.SL No. LWpplied by Welch,,Holmes and Clark,,Harrison.NJ. - NF Q=lity. AD&y sperm detected is dav 0 of gestation. 6 The T-2999CoC treated and control group animals were observed day 3 daily from through day 20 of gestation for abnormal clinical signs. The dam were weighed on days 3, 6, 9, 12, 15 and 20 of pregnancy. They were dosed according to the most recent body weight using a constant dose volume of 5 ml/kg of body weight. The CDV non-treated group was observed only for a live/dead check from days 3 through 20 of gestation. All dams were terminated by cervical dislocation on day 20 of gestation. All of their fetuses were fixed in Bouin's solution. At least 23 dams in each group had four or more fetuses. Four fetuses from each of 20 litters from the -CDO and CDF40 T-29ggCoC and corn oil control article groups were selected. The identities of the fetuses were concealed from the investigators by coding. Heads of two of the coded fetuses from each of the 20 litters were sliced in frontal-section through the center of the eye bulges and through the widest portion of the head. Freehand section gross lens observations, made with the aid of a 10-40X dissecting microscope, were recorded. The head sections bisecting the eye bulges were processed for histological observations after paraffin embedding the cut eye bul surface down (Tables 2, 3, 4 and Tissue Processing Procedure #2 of Table 5r. Heads of the remining two coded fetuses from each of the 20 litters were sliced behind the eye bulges. The eye bulge-containing specimens were processed for histological observation by paraffin embedding the brain surface down. The specimens bisecting the eye bulge by freehand slicing were microtome sectioned to provide a histological The evaluation of the same surface seen under the dissecting microscope specimens sliced behind the eye bulges were microtome sectioned to @rovide a (Tissue Processing Procedure histolozica.1 evaluation of the center of the lens #1 of T@ble 5). The embedded specimens were sectioned at 5-6 microns and stained with hematoxylin and eosin. Histology observations were recorded. Fetuses from the CDO non-treated group were not evaluated. The following is an experimental design outline for tissue processing and reporting fetal lens observations: Rat Stmin CDO CDFO T-2999CoC Dose Level 0 0 25 25 0 0 25 25 2 Fetuses of 20 Litters Freehand Sectioned 2 Fetuses of 20 Litters Microtome Sectioned No. lenses Evaluated x - - - - - - - 80 x - - --- - - x - - - - - - - 80 x - - - - - - - 80 x - - - - - - x - - - - - - - so x - - - - - - - 6ah- x - - - - - - x - - - - - - - 6ai x - - - - - - - 80 x - - - - - - x - - - - - - - 80 hdata from fetuses of dams NM521, 7523 and T524 were not evaluated (Appendix I). -izata from one fetus of dam NIR7533 was lost in transcription. 7 he experimental unit. The The individaal lens. rather than the fetust was t so reported and us as a possible experimental unit were al results of tt,tfet the individual lens experimental unit* The compared with the results Ofstatistically compare the incidence data generated chi-square test was used to rvations and to evaluate the affect of T-2999CoC by gross and microscope obse s T test was used to evaluate body weight data. on fetal lenses. The Dunnettl stmin. The Statistical comparisons were made within each respective rat level of statistical significance was p < 0.05. Results The administration of 25 mg/kg/day of T-2999CoC to pregnant dsms was maternally toxic in both strains of rats. Their mean body weights were significantly lower than that of controls on gestation &Lys nine until termination for the CDQPstrain and on gestation days 9, 12, and 15 in the CDFO strain (Table 1). T-2999CoC administration to pregnant dams did not affect the gross appearance of the pre-tem fetal lense Gross findings of a lens cleft at the anterocentral region and a dark streak in the center of the lens were reported in this studv (Table 2). 'Thepercent of lenses with clefts or dark streak findings were not significantly different between the control and treated groups for the CEO strain. In the case of the CDP% 25 mg/kg/day group, the dark streak was visualized significantly more often than in the CDFID control group* The individual lens was a more representative experimental unit than the individual fetus for reporting gross lens observations. Greater than 74 percent of the time, the lens cleft and streak findings were reported unilaterally in individual fetuses within each study group (Table 2). The range of agreement between the gross lens findings and the microscopic findings was highly variable. In general, there was a within strain trend for a high proportion of microscopic confirmations of a dark streak and for a low proportion of confirmations of the lens cleft (TeLble3). In utero exposure to T-2999CoC did not affect the ratios of confirmation of gross @rt@h microscopic findings in either strain of mt. When the number of gross lens findings confirmed microscopically were Compared with the total number of microscopic observations, low ratios of agreement resulted (Table 4). Two factors vere largely responsible for the low ratios of agreement for the embryonal nucleus finding; the large numbers of inicroseopic observations of that structure (Table 4) and thetwosmaflalctonrusmbewresre of gross observations of the dark streak (Table 3). The aft responsible for the lov ratios of agreement for the lens vesicle space finding with one exception. Ten grime lens CleftIsLnedightaicroscopic lens vesicle spaces vere observed in the CDP* 25 mg/kg/day dose group* However, only two of the gross observations were confirmed microscopically. The gross evaluation of freehand sectioned fetal lenses has limitationsas a technique for assessing lens morphology. In the CDC fetuses. over half of the lenses were sectioned across the embryonal nucleus (Table 5) and were therefore at risk of having the gross observation of a d&rk streek. Only a small portion of the lenses at risk had a dark streak on gross examination while a majority of the lenses were reported as baying no visible change (Table 2). A majority of lenses reported to be not sectioned based on gross observations had microscopic findings of sectioned lenses (Appendix V)o 8 rent Lenses noted on the gross examination to have a dark streak were not diffe oseopically from the lenses sectioned through the embryonal innucalpepuesa.raTnchee mdiaerrk streak was not an abnormality. Rather, its presence was indicative of the embryonal nucleus being occasionally visualized when the lens was freehand sectioned through the central region. The gross lens cleft and microscopic lens vesicle!space are artifacts generated by either freehand or microtome sectioning. The sectioning artifacts appear to be generated independently by both methods of sectioning. Both freehand sectioning and microtome sectioning were very effective in -transectingthe fetal lensese Between 85 and 100 percent of the lenses were tmnsected in this study (Table 5). The remainderwere lost in tissue processing, shattered beyond interpretation or had foreign material debris rendering them not readablee There was a trend for the combined freehand and microtome sectioned lenses to be lost, shattere.d or contain foreign material To a limited degree the more often than lenses only microtome sectioned method of tissue processing did influence the incidence of microscopic lens observations. Identifyingthe region of sectioningwithin the lens is possible based microscopically on morphological differences between the lens regions. One distinguishingcharacteristicof every gestation day 20 fetal lens sectioned through the central region was the presence of the embryonal nucleus; an area of lens cell degenerative change. The lens vesicle is an open space created by a separation of the embryonal nucleus lens cells from the subcapsular epithelium at the lens vesicle vestige. T-2999CoC administration to pregnant dams did not affect the microscopic appearance of,pre-ter-mlenses of their fetuses'(Table 5). Discussion The embryonal origin of the lens is undifferentiatedectoderm. The tip of the optic vesicle, presumablythe neural retina, plays the final role in inducing lens from overlying ectoderm and in aligning the lens precisely with the rest of the eye. Alternative or sequential action of tissues derived from endoderm (foregut)and mesoderm (heart) on the same target tissue decreases the probability that lens for=mtionwould be aborted by accidents during the early phases of induction. While the nature of the inductive influence remains unknown, there are indicationsthat substances my be transferred from the presumptive neural retina to the overlying ectoderm during induction. A prolonged period of inductive interactionnot only increases the probability that lens inductionwill occur successfully in the face of interference,but provides a mechanism for continuousv adjusting the size, shape, position and orientationof the lens to that Of the retinael During the early stages of the inductive process$ the ectodermal cell-s--immediatelyover3.,yintghe tip of the optic vesicle elongate perpendicularly to the body surface to form & thickened disc called the lens placode. The change .in cell shape is accomplished without change in cell volume. The number of cells, however, continues to increase during this period. Toward the end of lens pleldodeformation,acidophilic fibrils appear in the apices of the lens placode cells. At about this time, the placode invaginatesto form the lens cup. This invaginationis independent of the concoolitantinvagination of the underlying optic vesicles and is probably due to forces operating within the lens ectoderm. As the lens cup deepens, its lens pore opening becomes progressivelyconstricteduntil its lips meet and fuse, cutting off the lens vesicle internallyand re-establishingcontinuity in the overlying ectodem. 9 Closure of the lens pore is attended by. and possibly accomplished bYs a- 10cttl and temporary i-estricted wave of cell death. Following closure of the lens pore, the cells at the back of the lens vesicle continue to elongate, under the influence of the neural retina, to form the lens fibers. As the fibers grow the cavity of the lens vesicle is obliterated. The lens cells toward the ectodem, which do not elongate further, form the lens epitheliuml. The cuboidal lens epithelial cells which face the cornea continue to grow after the lens vesicle forms. As the cells rotate through the equator region, they take their places on the surface of the grovring fiber mass. These cells differentiate into secondary lens fibers at the equator and elongate rapidly toward the poles of the lens where they meet with other fibers in planes of junction called sutures. As secondary fibers grow, their nuclei become positioned at about the center of the fibers and form a convex lens bow outward. Since the never fibers are always deposited superficially, the oldest fibers in the lens come to lie centrally and are referred to collectively as the embryonal nucleus. With time the lens cell nuclei in this region beeme pyknotic and finally disappear. The cell fibers, however, are not broken down and removed but remain in place. Thus the size and shape of the lens are controlled by factors which control the number, size and shape of lens cellsl. The microscopic appearance of the gestation deq 20 fetal rat lens is dependent on the region sectioned. When sectioned at the equator region, the lens is characterized by, a regularity of curved fibers extending between the cuboidal &nterior lens epithelium and the posterior lens capsule. The lens bow is a prominant line of viable cell nuclei originating at the lens equator and courainiethrough the acentric region toward the anteriocentral portion of the lens. The acentric region 1,.esbetween the equatorial region and the region containing the enbryonal nucleus. When sectioned through the acentric region, the lens is characterized by a regularity of curved fibers also extendinv between the cuboidal anterior lens epithelium and the posterior lens capsule. Although there is posterior pole suture development at this point in development, it is not conspicuous. Suture development is not present at the anterior pole because of the anterior placement of the embryonal nucleus and because secondary lens cells of the fetal nucleus have not converged. The lens bow of the acentric region is located more anteriorly and appears less densely populated than at the equator because of lens cell elongation. Sections through the central region contain the more mature cells of the enbryonal nucleus. The lens bow in the embryonal nucleus progressively disappears as the nuclei lose viability. The lens cells are in various stages of degeneration within the embryonal nucleus. A vestige of the lens vesicle covers a mjority of the anterior embryonal nucleus surface. The lens vesicle vestige serves as the line of apposition between the embryonal nucleus and the lens epitheli&l cells. The embryonal nucleus of gestation day 20 fetal lenses is an area of normal ens cell degenerationo Lens cell degeneration was present in every lens sectioned across the central region. Some morphological components of the ' normal degenerative changes are as follows: loss of order of formerly linearly arranged lens fibers, smaller lens fibers, gmnular-appearing cytoplasm and nuclear py-knosis. Minor variations in the progression of lens cell degeneration are possibly due to differences in fetal age and the proximity of the section to the center or to the periphery of the embryonal nucleus. The embryonal nucleus was occasionally' visualized and reported on gross observation to be a dark streak in the lens. 10 Spurious microscopic observations of fetal lens findings* which are not hereditary or are not caused by in utero compound exposureg have been reported in rats. Var7ing stages of lens cell degeneration have been detected in the term SpraVe-Dawley rat lens.2 These findings were interpreted to be compatible with stages of normal lens degeneration previously reported as being congenital or were typical responses to various natural add experimental influences, The incidence reported in gestation day 19 and 20 fetuses was less than 4 percent. None of the findings were considered deleterious to the developing rat fetus or lens. A 3.6 percent incidence of fetuses with findings was reported in gestation day 18 congenital degenerative lens 4 These findings included individual lens cells -Chbb:THOM (SPF) rat fetuses. or small groups of lens cells with swelling, disintegration and formation of vacuoles in their cytoplasm. Since lens cell degeneration is a normal development change which initially occurs in the embryonal nucleus, the lens findings of these two reports could have been normal observations made on lenses sectioned through the embryonal nucleus. An increase in the incidence could result from mELkingobservations on older gestation age fetuses and with an increase in frequency of sectioning through the embryonal nucleus. Both reports labeled their findings as cataracts without seeing a lens opacity in pups following parturition. Technically the term cataract is applied to an obvious opacity or an opacity that can be seen by focal illumination with a slit lamp.3 The gross finding of a lens cleft and the microscopic finding of a space at the lens vesicle vestige are artifacts created by freehand and microtome sectioning. The spaces were observed microscopically only in sections which also cont@kinedthe embryonal nucleus. The absence of attachment of the embryonal nucleus cells to the lens epithelium plus the difference in consistency between the embryonal nucleus and secondary lens cells predispose Wv ratios of the lens vesicle vestige to separation once transected. confirmation and agreement of gross with microscopic occurrance of the separations suggest that the artifact is highly subject to tissue processing as well as being independently created by freehand and microtome sectioning. It is possible to freehand and microtome section through fetal lenses with an equally high level of proficiency. Gross observations of freehand sectioned fetuses however are unsatisfactory for evaluating;the lens when compared with microscopic observations. G@roseobservations are limited in not being able to define both the region in which lenses are sectioned and the morphological characteristics responsible for the lens findings reported. The experimental design for evaluation of fetal lenses in this study differed in the following three ways from the previous temtology study of the FM-3422 compound: 1) all fetuseswere coded before freehand sectioning for gross and microscopic obsez-vation,2) all fetuses provided for in the protocol vere 2.eally, ana3) thelens,ratherthanthe fea,.,*U,ass,the evaluated microsco experimental unit.1' Compound related gross lens findings were reported in the 25 mg/kg/day dose group in the previous tamtology study when the fetuses were read uncoded. These compound-related occurrences were not repeated when the fetuses were coded before freehand sectioning and gross evaluation. ,lriker Experiment Number o6Bc)TRoolo. T-29ggCoC admipistration to pregnant dam during the period of organogenesis did not affect either the gross or microscopic lens appearance of their pre-term fetuses. This conclusion is consistant with the absence of lens abnormalities observed in lactation dav 21 pups similarly exposed to T-29ggCoC at the same dose level.k @Xker Experiment Ymber o68oTRmo. 12 References Coulombre AJ: The Eye, in DeH&an RL, Ursprung H (eds): Organogenesir.. 1. New York, Holt Rinehart and Winston, 1965, pp 22T-232. HaLrtmanRA: HaturalV occurring cataracts in the tem fetal rat. JAVMA 2. L5-3(7). 832-840, 1968. Mann I: Develo-DmentAbnormalities of the Eye, 2nd ed. Philadelphia, JP 3. Lippincott Co.# 195T. Weisse 1, Niggeschulze A, Stotzer H: Spontaneous congenital cataracts in 4. mts, mice and rabbits. Archiv ruer Tbxikologie 32: 199-20T, 1974. WP: m/26n 13 TABLE 1 Special lans Oral TeratologY Study of T-2999COC in Two Strains of Pats. Rat Strain CDO CDFO Mean Maternal Body Weights(g) With Standard Deviations Listed By Gestation Day in Two Strains of Pets and Two Dose Levels of T-M9CoC* Dose Number Of (mg/kg/day) Dams 3 Gestatio 6 9 12 15 L,U 24 202 226 247 2T7 303 378 0 +16 +16 +18 +19 21 +30 28 197 221 2371 25a 280& 351t 25 +14 +15 +16 +19 21 +25 26 142 155 161 lTl 177 209 0 +6 +8 +7 +9 +16 +20 140 154 156S 15a 16A 201 25 27 T @9 12 +17 +7 +7 STne treatment group weights were significantly lower than the control group weights within each strain of rat (Dannettle T Test p < 0.05). WP:m/26m5 14 TABLE 2 Special ILens Oral Temtology Study of T-2999COC in Two Strains of Pats Fat Strain CDO CDO CDFS CDFIO CDO CDO CDFO CDP* Number and Pereent@@ of Freehand Section Gross Fetal Lens Findings For Individual Lenab and Fetuso- Experimental Units In Two ratmins of Rats and Two Dose Dose /_kg/day) 0 mg Levels of T-29ggCoC. d FREEHAND SECTION GROSS FINDINLGF- (Lens Experimental Unit) No Visible f Lens Cleftz- Dark StreakEL ChgLnge Not 15 (19) 4 (5) 60 (75) Lens Sectioned 5 (6) 25 20 (25) 9 (11) 42 (53)!! 15 (19)@L 02 6 (9) 5 (8) 40 (61) 15 (23) 25 10 (13) 17 (21)h- 43 (54) 14 (18) (Fetus Experimental Unit) Fetuses With Dark Streak or Lens Cleft Findings 0 11 (28) 3 (8) 25 17 (43) 8 (20) os 5 (15) 5 (15) 25 8 (20) 14 (35) 11 (28) @9 (48) 8 (24) 18 (45) E Percent in parentheses n - 80 lenses. n - 40 fetuses. It is possible to have one or both gross findings (lens cleft or dark streak) in one or botb lenses of an individual fetus. n - 66 lenses and n - 33 fetuses. A lens cleft is a space at the anterocentral region of the fetal lens. A dark streak is a dark colored area in the central region of the lens. The treatment group was significantly different from the control group within the experimental unit and within the stmin of mt (chi-square with Yates correction p < 0.05). WP: m/26m2 15 TABLE 3 special.Lens oral Teratology Study of T-2999COC in Two Strains of Rats Rat strain CDO CDFO fttios!@@and Pereentb- of Freehand Section Gross Lens Findings Mic roseOp jeelilyFor Individual Len&c-and Fetued- Experimental and Two Dose Levels of T-2999COC- Two strains of ]Rats Confirined Units in Dose (MR(-UL4m)- 0 25 FriterLlnan Section Lens Cie Individua1 Lens Individual Fetus 8/15 (53) 6/11 (55) 9/20 (45) 9/17 (53) Gross Findin SL& rk tre Individual Lens Individual Fetus 4/4 (ioo) 6/9 (67) 3/3 (100) 6/8 (75) 2/5 (40) 215 (40) a 2/6 (33) 2/5 (40) 2/8 (25) 11/17 (65) 11/14 (79) 25 2/10 (20) gross observation findings recorded following freehand Ratios are the number of microscopically. divided by the number of gross sectioning and confirmed observation findings. Percent in parentheses n - 80 lenses. n - 40 fetuses. e n a 66 lenses and n n 33 fetuses. It is possible to have one or bath gross findings (lens cleft or dark streak) in one or both lenses of an individual fetus. A fetus with one or two similar findings is counted only once. &-The treatment group ratios of gross findings confirmed microscopically were not significantly different from the control group ratios within each strain of Mt (chi-square with Yates correction p < 0.05). A lens cleft is an observed apsee in the anterocentral region of the fetal lens, It is confimed microseopicaiv by the presence of a space between the subcapsular epithelium and the lens cells of the embryonal nucleus. Hicroseopically the space is a separation at the lens vesicle vestige. ion of the A dark streak is an observed dark colored area in the central reg lens. It is confirmed microscopically by the presence of the embrvonal nucleus. ------- WP:rm/26ml 16 TABLE 4 Special Lens Oral TeratologY Study of T-2999COC in Two Strains of Pats R&tiosa-of and Pereent-bAgreement of Gross Individual Lens Findings-CWith Microscopic Lens Findings in Two Strains of FgLtsand Two Dose Levels of T-2999CoC. Rat Strain CDO Dose (mg/kit/day) 0 25 e Individual -ns Microscopic FindinAre@-- -];inVsesiclE_pacOl- Embryonal ucleus 8/2T (30) 4/40 (10) 9/2T (30) 6/44 (14) od CDP* 25 2/10 (20) 2/8 (25) 2/20 (10) 11/28 (39) Patios are the number of agreementsbetween gross findings recorded following freehandsectioningand microscopicfindings divided by the number of microscopic findings. Percent in parentheses n - 80 lenses. n - 66 lenses. The treatment group ratios of agreement between gross and microscopic lens findingswere not significantlydifferentfromthe controlgroup ratioswithin each strain of rat (chi-squarewith Yates correctionp < 0.05). A lens vesicle space is an open space created by a separationof the embryonal nucleus lens cells from the subcapsularepitheliumat the lens vesicle vestige. The ratio is the number of agreementsbetvaen the gross finding of a lens cleft and the microscopicfindingof a lens vesicle space divided by the number of lens vesicle spaces observed. An embryonal nucleus is an area of normal degenerativechange located in the center of the lens. The ratio in the number of agreements between the gross findingof a darkstreakand the microscopicfindingof an embryonal nucleus divided by the number of enbryonal nuclei observed. WP:rm/26m3 TABLE 5 Special Lens Oral TeratologY Studyof T-2999CoC in Two Strains of Rats Number and Percent.@@of Fetal b Lenses With Microscopic Findings Listed by Lens Region i Two Levels of T-29ggCoC and Two Tissue Processing Procedures With an Index of lans See RILT Dose Strain (mg/M/day) Microscopic Findings And Lens Region Sectioned Central Region Tissue-d Lens f Processing Vesicle- Enbryonal-LA.centric Procedure Space Nucleus Region Equatorial Region Lens Missing Lens Shattere or Foreign Material CDO 0 1 18 (23) 33 (41) 45 (56) 2 (3) 0 (0) 2 (3) 2 2T (34) 40 (50) 32 (40) 4 (5) 4 (5) 5 (6) CDO 25 1 23 (29) 39 (49) 33 (41). 4 (5) 2 (3) 6 (8) 1 2 27 (34) 44 (55) 26 (33)-:@-6 (8) 2 (3) 12 (15).!- CDFS 0 CDFO 25 1S 9 (13) 20 (29) 44 (65) 2 (3) 0 (0) 10 (15) 24 10 (15) 20 (29) 37 (54) 6 (9) 2 (3) 15(22) 1 4 (5) 27 (34) 45 (56) 5 (6) 1 2 (3) 6 (8) 11 (14) 18 (23) 2 8 (10) 28 (35) 31 (39):t9- (11) a Percent in parentheses n - 80 lenses. n - 68 lenses. Tissue Processing Procedure#1 tissues vere processed for microscopv. The lenses were n Tissue Processing Procedure #2 lenses were freehand sectioned before being processed for IB TITLE: Appendix I a in Two Protocol for special Lens Oral Teratology Study of T-2999Coc Strains of Rats (Riker Experiment Number 0681TRO362). OBJECTIVE: Changes were det cted in day 20 fetal lenses of Sprague-Dawley rats in teratolo; studies run on T-2999CoC. The Sprague-Dawley strain of rat may be predisposed to develop the lens change. in addition, the threshold for development of the lens change may be lowered by handling procedures used in conducting teratology studies. One objective of this study is to compare the incidence of the lens change in fetuses of both control and T-2999CoC treated Sprague-Dawley and Fisher strain dams; The second objective is to compare the incidence of lens changes of fetuses not exposed in utero to dosing and handling procedures used in teratology studies (Sprague-Dawley non-treated group) with the incidence of lens changes in the Sprague-Dawley control group. The study will be conducted according to the 1978 Good Laboratory Practice Regulations and Safety Evaluation Laboratory's Standard operating Procedures. SPONSOR: TESTING 3M Commercial Chemical Division, St. Paul, Minnesota. FACILITY: Safety Evaluation Laboratory, Riker Laboratories, St. Paul, Minnesota. Inc., STUDY DIRECTOR: S. G. Gortner START OF DOSING: Fourth quarter, 1981 TEST SYSTEM: Ninety sexually mature, time mated Sprague-Dawley derived female rats and sixty Fisher rats from Charles River Breeding Laboratory will be housed in hanging stainless steel cages with wire mesh floors and fronts in a temperature nd humidity controlled room. These strains of rats will be used ::cause time mated females are readily available and they will provide a comparison between two strains. Parina Laboratory Chow and water will be available ad libitum. The lights will be on a 12 hour ligbt/dark cycle. TEST SYSTEM IDENTIFICATION: Each animal will be ear tagged and that number will be Indicated on the outside of the cage. The Sprague-Dawley non-treated group will be identlfiod only by a number on the outsideof thecage. RANDOMIZATION: The animals will be assigned cages according to a computergenerated random numbers table. CONTROL ARTICLE: Corn oil TEST ARTICLE: T-29MoC FM-3924 19 Appendix I (Continued) ANALYTICAL SPECIFICATIONS: The test article composition and purity will be determined by the Sponsor (3M Commercial Chemical group). Ttie Sponsor is responsible for retaining a reference sample of the test and control articles, as required, for GLP compliance. DOSAGE LEVELS AND EXPERIMENT DESIGN: The test article will be suspended in corn oil daily. The test article and control article will be administered by oral intubation to the rats on days 6 through 15 of gestation according to the following: Strain Dose Level Group Size Sprague-Dawley 25 mg/kg/day 30 Fisher 25 mg/kg/day 30 Spracjue-Dawley 0 mg/kg/day 30 Fisher 0 mg/kg/day 30 Sprague-Dawley No treatment 30 The oral route of administration will be used because it is the route of exposure in previous studies in which lens changes were observed. No dietary contaminants are known to interfere with the test article. The T-2999Cc>C treated and control group animals will be observed daily from day 3 through day 20 of gestation for abnormal clinical signs. Body weights will be recorded on days 3, 6, 9, 12, 15 and 20 of pregnancy and the rats dosed accordingly using a constant done volume of 5 ml/kg of body weight. The Sprague-Dawley non-treated group will only be observed for a live/dead check from days 3 through 20 of gestation. The females will be killed on day 20 of gestation. All of their pups will be fixed in Bouin's solution and the heads will be free-hand sectioned by the Wilson technique. The presence of eye abnormalities will be determined using the dissecting microscope. Select eye sections could be sent to histopath for microscopic examination. DATA ANALYSIS AND FINAL REPORT: The proposed statistical method to be used for analysis of the data is the Fisher exact probability and Chi-square for percent of abnormalities. The proposed date for the final report is 2-3 months after the pup eye exams have been completed (approximately fourth quarter, 1961). 20 Appendix I (Continued) Amendment for Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats The subject of this study, a fetal lens change, was thought to be a strain-related phenomenon which was possibly precipitated by stress of handling piocedures used in teratology studies. Since the original protocol issued, the laboratory has gained insight into the development of the lens change. The change is now thought to be an artifact of freehand sectioning by the Wilson Technique. This amends the protocol consistant with studying the lens change as an artifact. OBJECTIVET (Amendment replaces the Objective section of the procotol.) Changes were detected in gestational day 20 fetal lenses of Sprague-Dawley rats in teratology studies run on T-2999CoC. The objective of this study is to report the comparison of the histological appearance of gestational day 20 fetal rat lenses that have been freehand sectioned by the Wilson technique and then processed for histology with the histological appearance of gestational day 20 fetal rat lenses which have not been freehand sectioned before being processed for histology. The comparison will be done on Sprague-Dawley and-Fisher rat strains. DOSAGE LEVELS AND EXPERIMENTAL DESIGN: (Amendment replaces the last three sentences.) All of the fetuses will be fixed in Bouin's solution. Four fetuses from each of 20 litters from the Sprague-Dawley and Fisher T-2999CoC and control vehicle treated groups will be coded before being processed for dissecting microscopic and histological evaluations. Two of the coded fetuses from each of the litters under consideration will have their heads freehand sectioned by the Wilson Technique. The freehand section len observations will be recorded. The head sections bisecting the e;es will then be processed for histological observation after embedding with the brain side down. The remaining two coded fetuses from each of the litters under consideration will have their heads freehand sectioned behind the eyes. The sections will be processed for histological observations by paraffin embedding with the brain side down. The em dded specimens will be sectioned at 5-6 microns and stained with hematoxylin and oosin. Histology observations will be recorded. DATA AN"YSIS ---- -- -----AND FINAL REPORT:- (Amendment replaces this sect:iorr-ot-th-o protocol.) The experimental unit in the individual lens* The statistical test for incidence data will be the Chi-square test. The proposed date for the final r" rt is the first quarter, 19OZ. 21 Appendix I (Concluded) Protocol Deviations CDFO animals NIR7521, 7522, 7523 and 7524 were mistakenly dosed with T-2999CoC at 25 mg/kg/day on gestation day 13. Fetuses of animals NIR7521, 7523 and 7524 were selected for lens evaluation. Since they were control group fetuses and they had been expos d to T-2999CoC in utero, their data was excluded from analysis. The fetu:es affected by Thi.p--arotocol deviation are listed by dam as follows: NIR7521 225 226 227 228 NIR7523 89 90 91 92 NIR7524 85 86 87 88 This deviation left 68 lenses subject to slicroscopid evaluation in the CDFO 0 mg/kg/day group. Table 5 reflects the absence of these twelve fetuses. Tables 2, 3 and 4 also reflect their absence plus the absence of fetus 94 from the CDE4100 mg/kg/day group whose gross observation data were lost in transcription. Gross and microscopic lens observations were not made on CDO non-treated group fetuses. 42 Appendix 11 Special Lens Oral Toratology Study of T-2999CoC in Two Strains of Rats List of Principal Participating Personnel Name Edwin G. Gortner Elden G. Lamprecht Wm. C. McCormick Gary C. Pecore Inara Porietis Loren 0. Wi*4th Plunction Study Director Veterinary Pathologist Tbxicology Services Representative Sapervisor of Animal Care Histopathology Technologist Technician 23 APPENDIX III STATEMENT oF QUALITY ASSURANCE STUDY NU@IBER: 0681TR0362 TITLE: loqy Special Lens oral Terato Two Strains of Rats Studv of T-2999CoC in Audits and/or inspections were performed by the Riker Compliance Audit unit for the above titled study, and reported to the study director and to management as follows: Date Performed Date Reported October 7, 1982 October 14, 1981 October 12, 1981 October 21, 1981 March 22, 1982 March 29, 1982 June 4,7,8, 1982 June 17, 1982 June 28-29, 1982 July 15, 1982 July 15, 1982 July 16,,1982 ---------------- .ILz@ raomMP4I:&LaC"@"Aud'Lt Ri or Laboratories, Inc. 44 24 Appendix IV Special Lens oral TeratologY Study of T-2999COC in Two Strains of Rats Individual Body Weights (9) with Mean Body Weights and Standard L)eviationsfor Pregnant Animals CV0 _-I :L.Ll :15 2 ci ----------------------------------------------- 14IR e-802 1-14F-.'7 e.Cl:@ 8 '--14, NIP' 7E.*05 NiF*. 78iE. t4lF, 7 8 1 IZ-@ t4iP 7819 NIP 7820 t4lF,. 78 <:I NiR 7832 NIR 781-..-;@ NIP 7834 NIP 78'-75 NIF'. 7846 t4lR 7847 t4lP 7848 tllr 7849 NIP 7850 141P 7861 NIR 7862 NIP 78L-N3 NiP 7864 NIF' 7879 N:LR 7880 194 218 181 2 r-I 199 221 19"-' 223 175 2CIO 201 229 201 219 178 198 22? 25e. 206 214 215 241 176 207 198 Z22 204 C-@1'2 18'-; Z16 21S 247 211 221 A-@'20 246 212 244 219 246 209 237 ie2 209 202 222 225 229 224 268 2 1 I=-.2,t7 2a9 271 2 --7k 274 2:18 2 4 1--' 255 288 2a7 2L;4 221 243 284 1 S" a-L57 289 264 293 232 266 2:.,@.8 265 240 2 t:, 24C-i 270 299 255 281 270 --<02 264 303 262 289 260 288 225 254 243 272 26.1 ;@92 298 2 301. C-10 272 327 292 255 345 304 321 290 300 29E, 3EI2 316 299 321 3a3 i17 320 279 299 317 379 380 IE 349 422 ---@I':1---4,.26 451 385 a96 aS--@ ::<35 ---;.7Z@. Z47 377 395 414 400 415 363 371 391 MEFft-4 202 226 247 277 303 a78 E.T'FiN.E)EV 15. 7 16.3 18.2 :L9.4 2C'.9 29.6 ----------- T-JON PPEGt4Fit4T 814IMFiLS NIP 7801 l(@i 201 214 210 224 21-:7 NIP 7elf" C'@'12241 256 278 270 286 NIP 78L;5 7-,. 215 224 238 253 NiP 7876 C-@'00 229 253 272 262 284 N:LP 7E:77 'C-IL*C-1 230 2 4 259 27:1 291 t4lP 781-8 Z09 211-: 242 245 252 258 25 Appendix IV (Continued) SP*cial Iens Oral TeratologY Study of T-2999COC in Two Strains of Rats individual Body Weights (g) With Mean Body Weights and Standard Deviations for Precjnant AniM&ls CDO 9 12 15 20 ---- ---------------------------------------- 25 MGe*KG,q)AY Ct:LF.OiF' CI:LR O:LR CI:IF.' CI:LP OiR OIR OIR OIR CI:LP OiR OiF' O:LF.'. OIF.O:LR C)I.R OiF! OJLR OiF'. OIR OIR OIR OIP OIR OIR OIR OIR 7 o C,E,. 7$C'(7C-IC,@-2, 78CI9 781C, 7c,'.22 7823 7824 782!, 7836 7E.--,.7 78----:8 7839 i@840 7851 7852 7A-'51-. 7854 78!f-5 7866 7867 7868 7869 7870 7881 7882 7883 7885 21C' 23;Ll .1-54 i C-: 2i.3 226 :1,---t4 2:1 224 19:@ -.r-;1.tD, 227 2i:l 2-@9 264 2IC1 22C, 251 :LSO 209 IE-1 181 2:L-q 197 io--5 2C48 232 187 20e- 232 215 243 261 1 e-,: 210 224 l@-*7 208 222 192 2CI7 216 205 234 250 191 2J.2 236 2iC- 234 259 :t*-;3 21-@ 230 19e. 223 241 211 244 237 201 227 234 198 227 24C4 227 252 268' 213 239 252 203 226 24--4: 198 219 241 _i87 .212-.-226 191 213 234 28C4 252 248 249 28:e 277 240 217 261 243 294 251 249 233 262 252 281 252 257 263 250 264 288 272 260 260 .245 231 309 279 266 258 3C'7 308 26@< 237 280 254 326 275 269 255 279 274 306 275 270 312 270 271 305 290 281 277 2618 263 390 344 36 --2< :'-88 382 334 297 361 318 389 3---,,.7 329 Z"16 1-:53 34 :1-82 343. 344 388 a48 354 370 362 345 326 348 MEAN 197 221 237 258 280 351 STFit4. DEV 13.7 15.3 16.2 18. 6 21. 0 25.3 NOt4 PREGt4Ht4T At4lMFILS O:IP, 7821 :197 c-;.,220::,7 2,36 252 OIF' 788,4 207 2--<2 248 244 245 259 25--,: 26 p,ppendixIV (Continued) special Lens oral Teratology Study of T-2999COC in Two Strains of Rats Individual Body Weights (9) with Mean Body Weights and Standard Deviations for Pregnant Animals CDFO 12 1 2L-1 DAY ------- -------------------------------------- o mrilKc,..,EIAY t4lp Mi.P. .4i.P NIP NIP N:LFN:LP t4:LP t4:LP t4:LP NIP Nip lqj.P NiP. Nip N:LP NJP. Ni-P N:LP Nip t4iP N:LP NJP t4lP NIP Nip Nip Nip 752:L 7522 752; 7524 7525 753J. 7532 7533 71!.24 7'f-3'!, 7c-;4:L 7543 7544 74545 7'f,51 7552 755a 7!f,54 7555 7561 756.-75C,4 7565 7571 7572 7572 7574 71575 :1:,!. :1 152 :t44 :L26149 :L2!143 i33 144 1,19 145 144 13e !4c14C, 147 144 135 14A152 146 120 14e 149 136 149 :L4E' I,--- :L7C4 159 14? :LE--@ 145 :L5S' :L4e 157 :L54 i'E-:L :159 :L5L' 154 :15:@' 159 :157 :L4:Y if-3 i6C, i5e 142 161 160 153 1,59 :L72 iiE.:? :L55 :L6e :L5:1 :LC-7 e. iC-o 1-,E.4 :L58 :LE44 17:1 :LC,2 :L7e ilf-9 177 7,D ;Lei :L79 175 :Le-5 :Lci i5g !CID 157 :1#--L.: :L6A. ic-9 :L6A 172 :L71 I-C-2 171 :L--Ii :L-"9 J.'-5'; 149 173 179 16.2 172 150 1'-KS 166 174 166 Igo 160 172 -:LiC.5 1-77 IF-ti 223 :159 :LEE' iqC, 2 '.4-' :LP-.2 146. 4 e-@ 204 !eE- 171 iQi 183 lg--,- iC,4 4-C46 201 219 2J-4 4--Po@-;14 21Q :196 17e 4 L42 JC-i 232:L-f, 2 4r-A :LL=14 226 :LC,7 20L I :L79 203 I --4* ie.5 1-?8 le3 :Lc-k2 22e. 17e 215 I -% 1IB2 226 199 231 le3 217 193 222 MEFTN 142 155 161 17i 177 209 STAt4. DEV 6.2 E-.0 6.7 S. e 15.6 2 C-4. Nn-N PPEGNRt4T PNIMRLS NIP NIP 754*-*- 127 756-1 1?5 i--,A :L4-Cl:-L44 :L42 :147 149 i5l- 152 i5C 149 27 Appendix rV (Concluded) special Lens Oral Teratology Study of T-2999Cc>C in Two Strains of Rats Individual Body weights (g) With Mean Body Weights and Standard Deviation& for Pregnant Animals CDEO E,,PY 6 9 :12 J-5 2 C-1 ---------------------------------------------- 25 MG/KC-./DFIY OiR* OIP n-IF: OIP O:LF: QIP OiP OiP OIP OiP OiP OIP OIF: CRIP OiP OIP O:Lp OIP OIR OiR OiP OiP OiF' OIP OIP QIP p,IiP 7'!*,CE-.@',147 16:1 iEO 159 1'-6R 2C-10 752f- :L'.:-;7142 :14:1 i2C4 -1-30 142 7!-2 e. 142 159 166 171 177 210 7!-r-29 5 i5i 150 156 i6O 20@-I 753C' 1 151 15a 155 165 200 7'!3,,E. i4i 15 g-%':L!!-7i6C- 170 213 7'T-37 140 152 157 15o-:t:E-e20E.' 7,@1. -'ei2e :Lfi I "-7; 159 i6e. 2ii 752f4 149 16-1* i6l-, 163 170 210 754L-1 129 155 16.2' !C-6 :!P0- 21!- 7547 129 :L44 14 ---* if-:L :L61 ISLI 754E-' :L*!-7@ 149 !T-2 155 i6:L :L9,E. -f'549 17.4 148 149 150 162 197 755C-1 1-;..l 146 151 !4e :L60 203 75f-q- 142 io-;6 i5-P- 156 161 195 755? 1 C.--<' 177 :L7e 170 182 2---:.--- 7560 142 :L54 1'f-2 :L55 166 214 7566 137 i5i 156 155 156 201 7567 14,5 i5P- :L6C 159 161 206 756e 146 :159 164 142 155 te5 7569 137 150 152 152 174 201 7570 i43 1'53 149. 157 165 209 7576 151 162 :L66 164 :L72 213 7577 137 147 152 162 174 215 757e i3e i5a i53 t5l. 151 le6 7579 134 i47 152 155 165 205 7-r%PG 14'-?- 15:? --157 14@ 7 i74 MEAN 140 154 156 i-K6 163 201 STAN. DEV 7.3 7.1 7.4 8.9 li. 5 16. 5 NON PPEGNAt4T FINIMALS OIR 7546 14i 152 150 OIR 7556 139 14e 146 OIP 75517 4-de 159 !52 141 i2e 151 146 14e 158 150 152 158 CDO 0 mg/kg/day Fetus Number 3 4 29 30 41 44 57 Dark Streak 151 2S2 154 iss 161 163 169 171 101 184 196 Is$ Appendix V Special L4mm Oral TeratoloqY StudY Of T-2999COC in Two Strains of Rate Gross and plicroscopic Lens ObservationES of Individual Fetuses Freehand sectioned Across the Loneent Gross rirdinp_ Lane cleft NVC lans Not sectioned Central Region Lens vesicle Space Embryonal Nucleus a L ItL FAL L AL ML L R im L R L a im IRL ML RL OL IRL ML Y4L iRL R L ML VIL L RL L L L T& L ItL L L R R L ML IRL RL n L L microscojpic Findings Acentric ftgion R IRL tquatorial P*gion L L*ns Missing L R ML L L R right I@ns 16 left long Tissue processing Procedure #2 of Table 5 CDO 0 mg/kq/day Iretus N-' r 219 219 23S 236 259 260 262 264 277 270 261 203 Dark Streak R R 2W 295 2% "7 300 319 320 A L right lane left Ions Appendix V (Continued) Special Lens Oral Terstology Study of T-2999CoC In Two Strains of Rats Gross and microscopic L*ns observationsa of individual Fetuses Freehand Sectioned Across the Lenstsb Gross rindin loom C]Left IM 14no not Sectioned Centr&I-Regicn lane vesicle Space Embryonal itucleus Hicroscoj?icFindings Acentric Region squatorial Region R IL RL R L RL L L ML ML VIL PL RL ML OL L L L L L R lm IRL L L R IRL RL L L R ML IRL RL R R R R L R L ML RL ML R RL RL IRL L Tinew proc sming P. dure #2 of Table 5 tans missing L ctp 2S mg/kg/d*Y F*tus ifumber Dark streak 26 29 39 40 77 97 ML 112 114 116 122 IL24 141 IL43 ,Iwcial Lens orai 'I'Pratoiu,;'y,LULIY01 T-2999COC in Two Strains of PAts Gross and Microscopic toonsC)bservationg@of Indivbidual Fetus*s rreehand Sectioned Across the lanses GrossFindings_ lans Cleft wic lang "Ot sectioned Central Region Lens vesicle Space Zubryonal mucl*us Hicrognote Find- LA s Lane Acentric Equatorial 0 Region Region missing L R RL PL PL RL UL IRL L RL RL m FAL R L ML RL RL lm R L R EL y4L R ML ItL IL L R L R EL RL ML RL L R L RL R L It - right lons a left Ion$ Tissue processing Procedure 12 of Table 5 cup 2S wq/kq/d&Y Fetus ilusbor 173 176 177 179 199 207 209 Dark St reak R L R 216 221 223 2" 291 302 R 303 "6 309 309 310 T-2999COC in Two Strains Of R&ts Gross and Microscopic Long Observations@-of Individual Fetuses Freehand sectioned Across the Ltn!iiesb Gross rindin C*ntral Region microscopic Findings L40M Cleft NYC R L R R L L R L ML R ML R L ML Lens not sectioned L lans vesicle space Embryon ^I )Rucleuo L R L R R R R ML L L L L Acentric Region ML RL zquatorial lang Region missing L ML RL RL L L ML UL L R R L L L AL RL R R - rILght Ions L - left less Tiesue processing Proc*ftro 12 of Table 5 CDVO o mg/kq/d&y Fetus Dark streak is 19 33 35 49 S2 L ass. 86C egs R gis 946 96 102 103 119 IL30 132 133 135 it right lOnB L left@ Ions spo-cial I.-nsoral Teratt)loqy %tUdY Of T-2999CoC in Two Strains of P&ts Gross and Microscopic Lens observations@ of individual Fetuses rreeh&M sectioned Across the Lensesb Gross Findings lans Cleft w4c RL R IRL R OL tans Hot sectioned L L Central Region Lens vesicle Space Embryonal 14*cleus L L L L Microscopic Findings Acentric Region squatorial Region Le Sh L*ns or missing me IRL R L R L L L OL ML R L RL AL PtL L L L L RL R L L R L L p R R R b Tissue processing Procedure 02 of Table S S Date excluded from analysis in Tables 2,3.4 5 according to a Protocol Deviation lappendix 1) Gross firm lost in CDVO 0 mg/kg/day retus ifonber 137 139 157 160 IGS 167 169 192 193 195 209 210 22gs 227 231 232 26+ 2" 27b 27% Dark Streak OL R Si@pcial tens Oral Teratoloqy Study of T-2999CoC In Two Strains of Rats Gross and microscopic lens Observationas of Individual Fetuses lpreehand Sectioned Across the Lensesb Gross irindings_ tcain@sft L WiC L L ML L L L Yah Lens Not Sectioned R R R L L R INL ML ML L L L L it L p Central Region L*ns vesicle Space owbryonal N6cleus L L L L OL L L L R R RL L L L Microscopic rindings Acentric Region RL R a RL rapotortal Region s L*ns 0 Missing RL R W. R L RL L L RL IL ML AL R - right Ions L - left Ions Tioouo p.oc asing Procaftre 12 of Table S c Data excluftd from analysis in Tables 2.3,4 to a Protocol Deviation (Appendis I). 5 according Appendi3t V (continued) Special Lens Oral TetatOloqy StudY Of T-299900C in Two Strains of PAts Gross and Microscopic LenB Observationst of IndiVidU&l TOtUaeg lpreehand sectioned Across the Lenses!! ODr* 25 mg/kg/d&Y Fetus qumber 14 is 23 24 46 49 53 Dark Str*ak R L 61 R 62 GS 70 L 71 73 79 R 63 RL 94 L 107 109 IRL r*rose rindinqo Loons cleft IM L ML PL L R RL ML IR a R L L L L R L ML L Lane "ot Sectioned L R R R R L R NL Central PAgion TA"W vesicle space Ubryonal lkwleus ML R L L L RL L it L L L L L L L F& R ML L L microscopic Findin" Acentric neqion ZqmtOri&l Mqion s Lane 0 14i@slnq L ML R R ItL R R RL R T14ht 14mg L left Ions Tissue proc*osinq Proc*ftre 92 of Table S cwo 2S mg/kq/day Fetus ?I'm' r 127 128 14S 147 201 203 237 240 243 244 24S 246 249 252 253 254 269 272 313 314 Durk streak L a L ML L R Appendix V (Concluded) Si,ecial Lens oral Teratoloqy Study Of T-2999CoC in Two Strains of Rats Gross and Microscopic L*no Observations@ of Individual retus*s Freehand Sectioned Across th* Lenseeb Gross Findings KA"W cl*ft mm RL im L ML ML L Lens not soctiomd R ML A R Central Region L*ns Vesicle Space tabryonal Nucleus L IRL L microscopic Findings Acentric Region IRL Z"torial Region R L L ItL AL L R Lang Missing L R LL R L a L R L R Im L R L n L n L L R a W. L RL L RL R - right tens L - left lens Tissue processing Procedure 12 of Table 5 36 Appendix VI Special Lens Oral Teratology StudY Of T-2999CoC in Tvo Strains of Rats Microscopic Lens Observationss of Individual Fetuses Not Freehand Sectioned Across the Lensest mg/kg/day -tus itber I 2 31 32 42 43 59 60 49 50 53 56 62 64 70 72 32 33 35 37 Central Region Lens Vesicle Space Evibryonal Nucleus Acentric Region Equatorial Region Lens missing RL RL R R L R L RL RL R L RL RL RL RL RL R R L RL R R L RL L L R RL L L R RL RL RL R R L Lens Shattered or Foreign material R left 1*ns right lens 'issue Processing Procedure #1 of Table 5 37 Aopendix VI (Continued) Special Lens oral Teratology StudY of T-2999Cc>C in Two Strains of Ratg Microscopic Lens Observations!t of Individual Fetuses Not Freehand Sectioned Across the Lenses!! mg/kg/day -.tus jnber '1-7 '@20 '33 '34 258 261 ',63 '@79 I:eo 82 284 286 -@'87 293 "@94 298 299 318 Central Region Lens vesicle Space Embryonal Nucleus L L RL RL R R L RL L L L L L L L L R R Acentric Equatorial Region Region Lens Missing R Lens Shattered or Foreign Material L L R R R RL R R R R RL L R R RL RL RL L RL L - left lens R - right lens Tissue Processing Procedure 0 1 of Table 5 38 Avoendix VI (Continued) Special Lens Oral Teratology Study Of T-2999CoC in Two Strains of Pats Microscopic Lens Observations!t of Individual Fetuses Not Freehand Sectioned Across the Lensesb 5 mgAg/day -tus -,nber 6 8 10 12 25 27 37 39 78 80 99 100 1.09 iio 113 115 121 123 142 144 Central Region Lens vesicle Space Embryonal Nucleus L L RL RL L L L L L P. RL R RL RL RL RL R RL RL RL Acentric Region Equatorial Region R Lens Missing Long Shattered or Foreign Material R RL R RL R L RL RL RL RL R RL RL RL -- - - - - - - - - L - left lens R - right le,ns Tissue processing procedure #I of Table 5 39 Aooendix VI (Continued) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Microscopic Lens Observations@i of Individual Fetuses Not Freehand Sectioned Across the Lensest C:D 25 mg/kg/day Fetus ';,amber Central Region Lens Vesicle Space Embryonal Nucleus 174 175 L L 179 R R 180 L L 197 L L 200 205 RL 206 213 L L 214 RL RL 222 L L 224 290 L L 292 301 RL RL 304 L 305 L 307 L 311 RL 312 L RL Acentric Equatorial Region Region Lens missing Lens Shattered or Foreign Material RL L R L R R RL RL R R RL R RL R R R L - left lens R - right lens Tissue Processing Procedure # 1 Of Table 5 40 Appendix VI (Continued) Special Lens Oral Teratology Study of T-2999CoC in 7wo Strains of Rats Microscopic Lens Observationss of Individual Fetuses Not Freehand Sectioned Across the Lenses!i CDEO 0 mgAg/day Fetus Number 17 20 34 36 50 51 87' go@E 92-S 93 95 101 104 117 120 129 131 134 136 Central Region Lens Vesicle Space Embryonal Nucleus L L R L RL RL L L R Acentric Region Equatorial Region Loens missing RL R RL RL RL R L R L R RL L R RL RL RL RL Lens Shattered or Foreign Material RL R RL RL L L a left lens R - right lens Tissue Processing Procedure Data excluded from analysis #plof Table 5 in Table 5 accord ini to a Protocol DOVi&Lion (&pl,endix 1) Appendix VI (Continued) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Microscopic Lens observationss of Individual Fetuses Not Freehand sectioned Across the Lenses!! CDE'O 0 mg/kg/day Fetus Number 138 140 158 159 166 168 190 191 194 196 211 212 225C 22,c 229 230 265 267 273 274 Central Region Lens vesicle Space Embryonal Nucleus L L L L L RL L L RL L RL L RL R R L L L Acentric Region Equatorial Region Lens missing Lens Shattered or Foreign Material R RL R R RL RL RL L R R L RL L L RL RL RL RL RL R - L - left lens R - right lens - Tissue Processing Procedure Data excluded from analysis #1 of Table 5 in Table 5 according'to a Protocol Devi&tior, (Appendix I) 42 ADDendix VI (Continued) Special Lens Oral Teratology Study of T-2999COC in Two Strains of Rats Microscopic Lens observationss of Individual Fetuses Not Freehand Sectioned Across the Lensesb :)FO 5 mg/kg/day etus amber 13 16 21 22 45 47 54 56 63 64 66 67 69 72 74 76 81 82 @05 @-06 Central Reli2n Loens vesicle Space Embryonal Nucleus L L L L L L L Acentric Equatorial Region Region Lens Missing L R R RL R RL RL R RL L R R RL L R RL R L R RL Lens Shattered or Foreign Material R L RI. RL RL RL ---- - - - - - L - left lens R - right lens TisSue Processing Procedure of Table 5 43 Aooendix VI (Concluded) special Lens Oral Teratology StUdY Of T-2999COC in Two Strains of RAts microscopic Lens Observationss of Individual Fetuses Not Freehand Sectioned Across the Lenses!! CDEO 25 mg/kq/day Fetus Number 125 126 146 148 202 204 238 239 241 242 247 248 250 251 255 256 270 271 315 316 Central Lens vesicle Space Region Embryonal Nucleus RL L RL L RL L R R R R RL R R L R Acentric Equatorial Region Region Lens missing Lens Shattered or Foreign material RL R R RL RL RL R L L R RL L RL L RL RL R L L - left lens R a right lens Tissue Processing Procedure 01 of Table 5 44 Appendix VII Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats T-2999CoC Composition, Purity and Stability - References: Commercial Chemicals Analytical Requests 116474, 18319, Analytical Report #318 A light, straw-colored waxy solid labeled FM-3924, Lot 547 was received for analysis for Riker teratology study #0681TRO362. The materialhas been analyzed with the following results: Gas Chromatography of Sample As Received All in Area % QC(14310 12/3/80 3/31/81 3/5/82 5/27/82 Inerts Amide C3 C4 CS C6 CI,C8BO, N gh lers 0.5 10.6 for all low boilers 88.9 - 0.4 0.6 1.2 1.8 1.5 4.8 88.9 0.6 0.7 0.6 13 1:7 1.6 4.3 $8.9 O.s 0.4 0.6 1.2 1.8 1.5 4.8 88.9 0.6 0.7 0.2 1.3 2.2 1.9 4.9 88.9 0.2 All values are within expe.riment.aland integration parameters. meet QC specifications. Values c H N s F 0 by difference Elemental Analysis % Theor. % Found 3/5/82 25.2 1.75 2.45 5.6 56.66 8.4 24.6 1.5 2.6 5.3 57.0 9.6 % Found 5/27/82 25.0 1.8 2.7 6.1 55.3 9.1 45 Appendix VII (Concluded) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats T-2999CoC Composition, Purity and Stability MW 571 Melting Point: Initial38-48*C Recheck 38-48*C 5/28/82 38-480C Spectroscopic: Infrared spectra 16474-2,3,4and 18318-1 are identicalto each other and match reference spectra. 16474-2 was obtained 16474-3 was obtained 16474-4 was obtained 18318-1 was obtained 11/14/80 3/31/81 3/5/82 5/28/82. NMR spectra H8385, H8387, F8390, F8391 were obtained on 12/l/80 and 12/2/80. These were to confirm structure and not repeated. Structure was confi'rmed. The isomer distributionwas found to be 69.6% straight chain, 10.9% (CF3)2CF branched, and Ig.S% backbone branching. Conclusions: FM-3924, Lot 547 has an analysiswithin specifications,is stable and is representative c rcialmaterial. RMP/hc cc: M. T. Cauu E. G. Gortner (original + 11 C. McCormick F. Keller E. G. Lamprecht W. it. ijt--ariuuti R. A. Prokop V. Pothapragada/L. L. 0. Wiseth Winter TITLE: Protocol for Special Lens Oral-Tera:tology Strains of Rats (Riker Experiment Number 0681TR0362). Study of T-2999CoC-E in Two OBJECTIVE: Changes were detected in day 20 fetal lenses of Spracjue-Dawley rats in teratology studies run on T-2999CoC. The Sprague-Dawley strain of rat may be predisposed to develop the lens change. In addition, the threshold for development of the lens change may be lowered by handling procedures used in conducting teratology studies. One objective of this study is to compare the incidence of the lens change in fetuses of both control and T-2999CoC treated Sprague-Dawley and Fisher strain dams. The second objective is to compare the incidence of lens changes of fetuses not exposed in utero to dosing and handling-procedures used in teratology studies (Sprague-Dawley non-treated group) with the incidence of lens changes in the Sprague-Dawley control group. The study will be conducted according to the 1978 Good Laboratory Practice Regulations and Safety Evaluation Laboratory's Standard Operating Procedures. SPONSOR: 3m Commercial Chemical Division, St. Paul,, Minnesota. TESTING FACILITY: Safety Evaluation Laboratory, St. Paul, Minnesota. Riker Laboratories, Inc., STUDY DIRECTOR: E. G. Gortner START OF DOSING: Fourth quarter, 1981 TEST SYSTEM: Ninety sexually mature, time mated Sprague-Dawley derived female rats and sixty Fisher rats from Charles River Breeding Laboratory will be housed in hanging stainless steel cages with wire mesh floors and fronts in a temperature and humidity controlled room. These strains of rats will be used because time mated females are readily available and they will provide a comparison between two strains. Purina Laboratory Chow and water will be available ad libit=. The lights will be on a 12 hour light/dark cycle. TEST SYSTEM IDENTIFICATION: Each animal will be ear tagged and that number will be indicated on the outside of the cage. The Sprague-Dawley non-treated group will be identified only by a number on the outside of the cage. RANDOMIZATION: The animals will be assigned cages according to a computergenerated random numbers table. CONTROL ARTICLE: Corn oil TEST ARTICLE: T-2999CoC FM-3924 ANALYTICAL SPECIFICATIONS: The test article composition and purity will be determined by the Sponsor (3m Commercial Chemical group). The Sponsor is responsible for retaining a reference sample of the test and control articles, as required, for GLP compliance. DOSAGE LEVELS A14D EXPERIVIENT DESIGN: The test article will be suspended in corn oil daily. The test article and control article will be administered by oral intubation to the rats on days 6 through 15 of gestation according to the following: Strain Dose Level Group Size Sprague-Dawley 25 mg/kg/day 30 Fisher 25 mg/kg/day 30 Spracjue-Dawley 0 mg/kg/day 30 Fisher 0 mg/kg/day 30 Sprague-Dawley No treatment 30 The oral route of observed. the test route of administration will be used because it is the exposure in previous studies in which lens changes were No dietary contaminants are known to interfere with article. The T-2999CoC treated and control groups animals will be observed daily from day 3 through day 20 of gestation for abnormal clinical signs. Body weights will be recorded on days 3, 6, 9, 12, 15 and 20 of pregnancy and the rats dosed accordingly using a constant dose volume of 5 ml/kg of body weight. The Sprague-Dawley non-treated group will only be observed for a live/dead check from day 3 through 20 of gestation. The females will be killed on day 20 of gestation. All of their pups will be fixed in Bouin's solution and the heads will be free-hand sectioned by the Wilson technique. The presence of eye abnormalities will be determined using the dissecting microscope. Select eye sections could be sent to histopath for microscopic examination. DATA ANALYSIS AND FINAL REPORT: The proposed statistical method to be used for analysis of the data is the Fisher exact probability and Chi-square for percent of abnormalities. The proposed date for the final report is 2-3 months after the pup eye exa-A have been completed (approximately fourth quarterf 1981). E. G. Gortner Senior Research Technologist Animal Teratology-Reproduction Study Director Date q@&@ -4 9- 3-W) E. G. Lamprecht, DVM, PhD Date Research Veterinary Pathologist M. T. Case, DVM, PhD Manager, Pathology-Toxicology Safety Llvaluation Laboratory Date W. C. McCormick, MS Toxicologist Sponsor Representative D Ve