Document B5rJXQvjpQgQeB9b0k6RRy2EL

/)R m - 0 1 % 3 Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Experiment No.: Conducted At: Dosing Period: Study Director: 0681TR0362 Safety Evaluation Laboratory Riker Laboratories Inc. St. Paul, Minnesota September 28, 1981 through October 8, 1981 E. G. Gortner E. G. Gortner Date Senior Research Technologist Animal Teratology Reproduction 7-i*-sa E.. GG.. Lamprecht, DVM, PhD Date Research Veterinary Pathologist M. T. Case, DVM, PhD Date Manager, Pathology-Toxicology Safety Evaluation Laboratory ' U A t t i M M. M. C. McCormick, MS Toxicologist Toxicology Services 7-Z0-9* Date 004281 Table of Contents Z*31 Table of C o n t e n t s ........................................... 1 Summary ................................................... 3 Introduction ............................................ 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 Grose Fetal Lens Findings For Individual Lena and Fetus Experimental Units in Two Strains of Rats and Two Dose Levels of T - 2 9 9 9 C o C ...................... 14 3* Ratios and Percent of Freehand Section Gross Lens Findings Confirmed Microscopically for Individual Lens and Fetus Experimental Units in Two Strains of Rats and Two Dose Levels of T-2999CoC . . . . 15 4. Ratios and Percent AgreeaMint of Gross Individual Lens Findings With Microscopic Lens Findings in Two Strains of Rets and Two Dose Levels of T - 2 9 9 9 C o C ....................................... 16 5. Number and Percent of Fetal Lenses With Microscopic Findings Listed by Lens Region in Two Strains of Rats. Two Levels of T-2999CoC and Two Tissue Processing Procedures With an Index of Lens Sectioning Proficiency................ .. . . 17 004282 Table of Contents (Concluded) Appendices Page I. Procotol for Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats . . . . . . . 18 Amendment for Special Lens Oral Teratology Stud? of T-2999CoC in Two Strains of Rats . . . . 20 Protocol Deviations ........................ . . 2 1 II. List of Principal Participating Personnel . . . . 22 111* Statement of Quality A s s u r a n c e .............. 23 IV. Individual Body Heights (g) With Mean Body Weights and Standard Deviations for Pregnant A n i m a l s .......................................24 V. Gross and Microscopic Lens Observations of Individual Petuses Sectioned Across the Lenses. . 28 VI. Microscopic Lens Observations of Individual Fetuses Not Sectioned Across the Lenses . . . . . 36 VIZ. T-2999CoC Composition, Purity and Stability . . . 44 004283 SUMMARY In a previous rat teratology study of a fluorochemical alcohol, FM-3U22, gestation day 20 gross lens findings on uncoded freehand sectioned fetuses vere interpreted as compound and dose related abnormalities. The objective of this current study was to compare the histological appearance of fetal rat lenses that had been freehand sectioned to the histological appearance of lenses which had not been freehand sectioned. Groups of thirty time-mated Crl:CD(SD)BR and CDF*(F-3**U)/CrlBR rats were orally dosed at 0 and 25 mg/kg/day with T-2999CoC, a comparable alcohol, on days 6 through 15 of -gestation. Four fetuses from each of 20 litters per group were selected. The identities of the fetuses were concealed from the investigators by coding. Gross lens observations were made on freehand sections of two fetuses per litter. Microscopic lens observations were made on all four coded fetuses following microtome sectioning and tissue staining. T-2999CoC caused maternal toxicity (low body weight gain) in the pregnant dams of both rat strains. The compound did not affect either the gross or microscopic lens appearance of fetuses from treated dams. The compoundrelated occurrence of lens findings reported in the previous rat teratology study could not be repeated when the fetuses were 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 between 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. 004284 Introduction A Toxic Substances Control Act (TSCA) Section 8(e) notice was filed by the 3M Company based on rat teratology study results on FM-3^22Si. fhrtly 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 was reported as a teratogenic change because it was found only in compound treated* dose groups and haul a dose-related incidence of occurance. T-2999CoC (FM-3921*) was selected for use in the current study because it is more representative of present production than is FM-3^22. Further, T-2999C0C potentially presents greater risk than FM-3^22 because of the somewhat higher content of more soluble lower homologues. The protocol for the current study was originally designed with two objectives. The first objective was 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 in utero to dosing and animal handling procedures used in teratology studies with the incidence of lens changes in the CD 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 was amended to test this hypothesis. The objective of this study was changed to compare 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 which have not been freehand sectioned before being processed for microscopy. The study was sponsored by 3M Commercial Chemical Division, St. Raul, Minnesota and was conducted by the Safety Evaluation Laboratory, Riker Laboratories Inc., St. Raul, Minnesota. The two compound administration groups were dosed between September 28, 1981 and October 8, 1961. 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) regulations^, and Safety Evaluation Laboratory Standard Operating Procedures. 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 Laboratory's record archives. 2FM-3^22 is at least 96% CsFifSOgHtCpHOCHgOH (Riker Experiment No. 068OTR0010) and T-2999CoC (FM-392M) is about 88% C8Fi7S02N(C2H5)CH20H ; in the remaining 11%, the perfluoroalkyl group contains three to seven carbon atoms. ^Federal Register, Vol. **3, No. 237, December 22, 1978 pp 60013-60025. 004285 5 Materials and Methods Ninety Crl:CD(SD)BR rats (CD) and sixty CDPeCF-BUlO/CrlBR rats (CDF) were assigned cages according to a computer-generated random numbers table. They were sexually mature time-mated females weighing between l63-229g and 127-l63g respectively (Appendix IV). 'Hie rats were housed individually in suspended, 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 CD non-treated group was identified only by a number on the outside of the cage. FeedS. and water were -available ad libitum. The lights were on a 12 hour light/dark cycle. The rat was chosen because previous teratology studies on FM-3^22 were 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 composition, purity and stability were determined by the 3M Commercial Chemical group (Appendix VII). The test article was suspended in corn oilX daily. The test article solutions and the corn oil control article were administered by oral intubation to rats on days 6 through 15 of gestation& according to the following experimental design for dosing: Rat Strain and Dose Group T-2999CoC Dose Level CD corn oil control article 0 mg/kg/day CDF corn oil control article 0 mg/kg/day CD T-2999CoC in corn oil 25 mg/kg/day Group Size 30 30 30 CDF T-2999Co C in corn oil CD Non-treated group 25 mg/kg/day rats not dosed 30 30 Supplied by Charles River Breeding Laboratories, Inc., North Wilmington, MA. Supplied by Charles River Breeding Laboratories, Inc., Stoneridge, NY. Purina Laboratory Chow, Als t o n Purina Company , 3t. Louis, MO. Supplied by Welch, Holmes and Clark, Harrison, NJ. - NF Quality. Day sperm detected is day 0 of gestation. 004286 The T-2999CoC treated and control group animals were observed daily from day 3 through day 20 of gestation for abnormal clinical signs. The dams 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 CD 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 -CD and CDF T-2999CoC 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-L0X dissecting microscope, were recorded. The head sections bisecting the eye bulges were processed for histological observations after paraffin embedding the cut eye bulge surface down (Tables 2, 3, 1* and Tissue Processing Procedure #2 of Table 5/ Heads of the remaining 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 evaluation of the same surface seen under the dissecting microscope. The specimens sliced behind the eye bulges were microtome sectioned to provide a histological evaluation of the center of the lens (Tissue Processing Procedure #1 of Table 5)* The embedded specimens were sectioned at 5-6 microns and stained with hematoxylin and eosin. Histology observations were recorded. Fetuses from the CD non-treated group were not evaluated. The following is an experimental design outline for tissue processing and reporting fetal lens observations: Rat Strain 2 Fetuses of 2 Fetuses of T-2999CoC 20 Litters 20 Litters Dose Level Freehand Microtome Ho. Lenses mg/kg/day______ Sectioned______ Sectioned________Evaluated CD 0 X ------------- 80 0 X ------------X ------------- 80 25 X ------------- 80 25 X ----------- X - - - - - - - 80 CDF 0 0 X ------------- 68il X ------------X -------------- 66hi 25 X ------------- 80 25 X ----------- X ------------- 80 bjData from fetuses of dams K1R7521, 7523 and 752U were not evaluated (Appendix I). iData from one fetus of dam N1R7533 was lost in transcription. 004287 7 i i ! The individual lens, rather than the fetus, vas the experimental unit. The results of the fetus as a possible experimental unit were also reported and compared with the results of the individual lens experimental unit. The chi-square test vas used to statistically compare the incidence data generated by gross and microscope observations and to evaluate the affect of T-2999CoC on fetal lenses. Tie Dunnett's T test vas used to evaluate body veight data. Statistical comparisons vere made vithin each respective rat strain. The level of statistical significance vas p < 0.05. Results The administration of 25 mg/kg/day of T-2999CoC to pregnant dams vas maternally toxic in both strains of rats. Tieir mean body veights vere significantly lover than that of controls on gestation days nine until termination for the CD Btrain and on gestation days 9 12 and 15 in the CDP strain (Table l). T-2999CoC administration to pregnant dams did not affect the gross appearance of the pre-term fetal lens. Gross findings of a lens cleft at the anterocentral region and a dark streak in the center of the lens vere reported in this study (Table 2). The percent of lenses vith clefts or dark streak findings vere not significantly different betveen the control and treated groups for the CD strain. In the case of the CDP 25 mg/kg/day group, the dark streak vas visualized significantly more often than in the CDP control group. The individual lens vas a more representative experimental unit than the individual fetus for reporting gross lens observations. Greater than 71* percent of the time, the lens cleft and streak findings vere reported unilaterally in individual fetuses vithin each study group (Table 2). The range of agreement betveen the gross lens findings and the microscopic findings vas highly variable. In general, there vas a vithin strain trend for a high proportion of microscopic confirmations of a dark streak and for a lev proportion of confirmations of the lens cleft (Table 3). In utero exposure to T-2999CoC did not affect the ratios of confirmation of gross vith microscopic findings in either strain of rat. When the number of gross lens findings confirmed microscopically vere compared vith the total number of microscopic observations, lov ratios of agreement resulted (Table U). Tvo factors vere largely responsible for the lov ratios of agreement for the embryonal nucleus finding; the large numbers of microscopic observations of that structure (Table U) and the small numbers of gross observations of the dark streak (Table 3). The same tvo factors vere responsible for the lov ratios of agreement for the lens vesicle space finding vith one exception. Ten gross lens clefts and eight microscopic lens vesicle spaces vere observed in the CDP 25 mg/kg/day dose group. Hovever, only tvo of the gross observations vere confirmed microscopically. The gross evaluation of freehand sectioned fetal lenses has limitations as a technique for assessing lens morphology. In the CD fetuses, over half of the lenses vere sectioned across the embryonal nucleus (Table 5) end vere therefore at risk of having the gross observation of a dark streak. Only a small portion of the lenses at risk had a dark streak on gross examination vhile a majority of the lenses vere reported as having 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). 004288 Lenses noted on the gross examination to have a dark streak were not different In appearance microscopically from the lenses sectioned through the embryonal nucleus. The dark 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 transecting the fetal lenses. Between 85 and 100 percent of the lenses were transected in this study (Table 5). The remainder were lost in tissue processing, shattered beyond interpretation or had foreign material debris rendering them not readable. There was a trend for the combined freehand and microtome sectioned lenses to be lost, shattered or contain foreign material more often than lenses only microtome sectioned. To a limited degree the method of tissue processing did influence the Incidence of microscopic lens observations. Identifying the region of sectioning within the lens is possible based microscopically on morphological differences between the lens regions. One distinguishing characteristic of 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-teim lenses of their fetuses (Table 5)* Discussion The embryonal origin of the lens is undifferentiated ectoderm. The tip of the optic vesicle, presumably the 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 formation would be aborted by accidents during the early phases of induction. While the nature of the inductive influence remains unknown, there are indications that substances may be transferred from the presumptive neural retina to the overlying ectoderm during induction. A prolonged period of inductive interaction not only increases the probability that lens induction will occur successfully in the face of interference, but provides a mechanism for continuously adjusting the size, shape, position and orientation of the lens to that of the retina. 1 During the early stages of the Inductive process, the ectodermal cell* immediately overlying the tip of the optic vesicle elongate perpendicularly to the body surface to form a 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. Ttaward the end of lens placode formation, acidophilic fibrils appear in the apices of the lens placode cells. At about this time, the placode imraginates to form the lens cup. Ibis invagination is independent of the concomitant invagination of the underlying optic vesicle, and is probably due to forces operating within the lens ectoderm. As the lens cup deepens, its lens pore opening becomes progressively constricted until its lips meet and fuse, cutting off the lens vesicle internally and re-establishing continuity in the overlying ectoderm. 004289 Closure of the lens pore is attended by, and possibly accomplished by, a local and temporary restricted 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 ectoderm, which do not elongate further, form the lens epithelium^. 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 growing 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 newer 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 become pyknotic and finally disappear. Hie 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 cells1 . The microscopic appearance of the gestation day 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 anterior lens epithelium and the posterior lens capsule. Hie lens bow is a prominent line of viable cell nuclei originating at the lens equator and coursing' through the acentric region toward the anteriocentral portion of the lens. Hie acentric region lies between the equatorial region and the region containing the embryonal nucleus. When sectioned through the acentric region, the lens is characterized by a regularity of curved fibers also extending 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. Hie lens bow of the acentric region iB 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 embryonal nucleus. Hie lens bow in the eabryonal nucleus progressively disappears as the nuclei lose viability. Hie lens cells are in various stages of degeneration within the embryonal nucleus. A vestige of the lens vesicle covers a majority of the anterior embryonal nucleus surface. Hie lens vesicle vestige serves as the line of apposition between the embryonal nucleus and the lens epithelial cells. The embryonal nucleus of gestation day 20 fetal lenses is an area of normal lens cell degeneration. 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, granular-appearing cytoplasm and nuclear pyknoais. 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. Hie embryonal nucleus was occasionally visualized and reported on gross observation to be a dark streak in the lens. 0429o 10 Spurious microscopic observations of fetal lens findings, which are not hereditary or are not caused by in utero compound exposure, have been reported in rats. Varying stages of lens cell degeneration have been detected in the tern Sprague-Dawley rat lens.^ 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 aid experimental influences. The incidence reported in gestation day 19 and 20 fetuses was less than U percent. None of the findings were considered deleterious to the developing rat fetus or lens. A 3.6 percent incidence of fetuses with congenital degenerative lens findings was reported in gestation day 18 'Chbb:TH0M (SPF) rat fetuses.1* These findings included individual lens cells 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 making observations 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.^ 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 contained the 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 the lens vesicle vestige to separation once transected. Low ratios of 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. Gross observations 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 teratology study of the FM-3^22 compound: l) all fetuses were coded before freehand sectioning for gross and microscopic observation, 2) all fetuses provided for in the protocol were evaluated microscopically, and 3) the lens, rather than the fetus, was t h e experimental unit.J. Compound related gross lens findings were reported in the 25 mg/kg/day dose group in the previous teratology study when the fetuses were read uncoded. These compound-related occurrences were not repeated when the fetuses were coded before freehand sectioning end gross evaluation. JjRiker Experiment Number 0680TR0010. 004291 11 T-2999CoC administration to pregnant dams 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 day 21 pups similarly exposed to T-2999CoC at the same dose level.*. jtRiker Experiment Humber 068OTR0020. 04292 References 1. Coulambre AJ: Tlie e, in DeHaan RL, Ursprung H (eds): Organogenesis. New York, Holt Rinehart and Winston, 1965, pp 227-232. 2. Hartman HA: Naturally occurring cataracts in the term fetal rat. JAVMA 153(7); 632-81*0, 1968. 3. Mann I: Development Abnormalities of the Eye, 2nd ed. Philadelphia, JP Lippincott Co., 1957. U. Weisse I, lfiggeschulze A, Stotzer H: Spontaneous congenital cataracts in rats, mice and rabbits. Archiv Puer Toxikologie 32: 199-207, 197U. WP:rm/26n 004293 TABLE 1 Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Bats. Mean Maternal Body Weights(g) With Standard Deviations Listed By Gestation Day in Two Strains of Rats and Two Dose Levels of T-2999CoC. Bat Strain CD Dose (fflg/hg/day) 0 Number Of Dams 2k 25 28 Gestation Day 3 9 12 15 20 202 226 2U7 277 303 378 +l6 16 +18 19 +21. +30 197 221 237- 2585: 28o5. 351- +11 +15 +16 +19 21 +25 CDF 0 25 26 1U2 155 161 171 177 209 +6 *8 7 +9 16 +20 27 1U0 15* 156 156^ 163^ 201 +7 +7 7 +9 +12 +17 -- The treatment group weights were significantly lower than the control group weights within each strain of rat (Dunnett's T Test p < 0.05). WP:ra/26*5 004294 TABLE 2 Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Hats 14 Number and Percent^ of Freehand Section Gross Fetal Lens Findings For Individual Lens^ and FetuaS. Experimental Units In Two Strains of Rats and Two Dose Levels of T-2999CoC. Hat Strain CD* Dose (ag/kg/day) 0 mg Lens Cleft^ 15 (19) FREEHAND SECTION GROSS FINDINGS^ (Lens Experimental Ifoit) No Visible Lens Dark Streak^ Change Not Sectioned ITT5) 60TT5) CD* CDF* 25 o5 20 (25) 6 (9) 9 (11) 5 (8) U2 (53)^ 1*0 (61) 15 (19)^ 15 (23) CDF* 25 10 (13) IT ( 2 1 U3 (51*) lU (18) (Fetus Experimental Unit) Fetuses With Dark Streak or Lens Cleft Findings CD* 0 11 (28) 3 (8) 11 (28) CD* 25 IT C**3) 8 (20) 19 (1*8) CDF* oft 5 (15) 5 (15) 8 (21*) CDF* 25 8 (20) 1U (35) 18 (1*5) -- Percent in parentheses ( ). -- n * 80 lenses. -- n * 1*0 fetuses. -- It is possible to have one or both gross findings (lens cleft or dark streak) in one or both lenses of an individual fetus. -- n 66 lenses and n * 33 fetuses. -- A lens cleft is aspace atthe anterocentral region of the fetallens. - A dark streak isa dark colored area in thecentral region of the lens. -- The treatment group was significantly different from the control group within the experimental unit and within the strain of rat (chi-square with Yates correction p < 0.05). WP:rm/26m2 004295 TABLE 3 Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats 15 Ratios^a- and Percentb^ of Freehand Section Gross Lens Findings Confirmed Microscopically For Individual LensS- and Fetus^ Experimental Units in Two Strains of Rats and Two Dose Levels of T-2999CoC. Rat Dose Strain (s/hg/day) CD 0 25 Freehand Section Gross Findings^ Lens CleftH t)ark Strcakt Individual Individual Lens Fetus Individual Individual Lens Fetus 8/15 (53) 9/20 (U5) 6/11 (55) 9/17 (53) H A (100) 6/9 (67) 3/3 (100) 6/8 (75) CDF as. 25 2/6 (33) 2/10 (20) 2/5 (HO) 2/8 (25) 2/5 (HO) 11/17 (65) 2/5 (HO) 11/lH (79) -- Ratios are the number of gross observation findings recorded following freehand sectioning and confirmed microscopically, divided by the number of gross observation findings. -- Percent in parentheses ( ). c -- n * 80 lenses. -- n * Ho fetuses. ~ n * 66 lenses and n * 33 fetuses. t -- It is possible to have one or both 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 rat (chi-square with Yates correction p < 0.05). -- A lens cleft is an observed space in the anterocentral region of the fetal lens. It is confirmed microscopically by the presence of a space between the subcapsular epithelium and the lens cells of the embryonal nucleus. Microscopically the space is a separation at the lens vesicle vestige. -- A dark streak is an observed dark colored area in the central region of the lens. It is confinaed microscopically by the presence of the embryonal nucleus. WP:nn/26ml 004296 TABLE U Special Lens Oral Teratology 9tudy of T-2999CoC in Two Strains of Ikts 16 Ratios^ of and Percent^- Agreement of Gross Individual Lens Findings^- With Microscopic Lens Findings in Two Strains of Rats and Two Dose Levels of T-2999CoC. Rat Strain CD CDP Dose 0 25 ______ Individual Lens Microscopic Findings^- Lens Vesicle Spaced Embryonal nucleus** 8/27 (30) J*/1*0 (10) 9/27 (30) 6 / H (il*) 2/10 (20) 2/8 (25) 2/20 ( 10) 11/28 (39) -- Ratios are the number of agreements between gross findings recorded following freehand sectioning and microscopic findings 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 findings vere not significantly different from the control group ratios within each strain of rat (chi-square with Yates correction p < 0.05). -- A lens vesicle space is an open space created by a separation of the embryonal nucleus lens cells from the subcapsular epithelium at the lens vesicle vestige. The ratio is the number of agreements between the gross finding of a lens cleft and the microscopic finding of a lens vesicle space divided by the number of lens vesicle spaces observed. & An embiyonal nucleus is an area of nonaal degenerative change located in the center of the lens. The ratio is the number of agreements between the gross finding of a dark streak and the microscopic finding of an embryonal nucleus divided by the number of embryonal nuclei observed. WP:rm/26m3 004297 TABLE 5 Special Lens Oral Teratology Studyof T-2999CoC in Two Strains of Rats a Number and Percent-- of Fetal Lenses-- With Microscopic Findings Listed by Lens Region in Two Strains of Rats, Two Levels of T-2999CoC and TVo Tissue Processing Procedures With an Index of Lens Sectioning Proficiency. Microscopic Findings And Lens Region Sectioned Central Region Tissual Lens Lens Shattered^ Index of Lens- fet Dose Processing Vesicle^ Qnbryonal-- Acentric Equatorial Lens or Foreign Sectioning Strain (ng/kg/day) Procedure Space Nucleus Region Region Missing Material Proficiency CD* 0 1 18 (23) 33 (fcl) **5 (56) 2 (3) 0 (0) 2 27 ( 3 M UO (50) 32 (HO) U (5) U (5) 2 (3) 5 (6) (100) (95) CD* 25 1 23 (29) 39 (**9) 33 (kl). k (5) 2 (3) 2 27 ( 3 M hk (55) 26 ( 3 3 P 6 (8) 2 (3) 6 (8) . 12 (15)1 (95) (95) CDF* 0 I* 9 (13) 20 (29) kk (65) 2 (3) & 10 (15) 20 (29) 37 (51*) 6 (9) 0 (0) 2 (3) 10 (15) 15 (22) (100) (93) CDF* 25 1 k (5) 27 (3*) U5 (56). 5 (6) 2 (3) 2 8 (10) 28 (35) 31 ( 3 9 P 9 (11) 6 (8) 11 (1U) 18 (23) (96) (85) ^ Percent in parentheses ( ) -- n * 80 lenses. -r n * 68 lenses. -- Tissue Processiijg Procedure #1 tissues were processed for microscopy. The lenses were not freehand sectioned. Tissue Processing Procedure #2 lenses were freehand sectioned before being processed for microscopy. i w-rfo.-.. ,,n n-nino fr~ r . v , i r q noTmrqt.tnn of* tVip pmhrvonfll nuolpiis le n s c e l l s from th e 004298 BEST copy AVAILABLE 18 Appendix I TITLE: Protocol for Special Lens Oral Teratology Study of T-2999CoC- in Two Strains of Rats (Riker Experiment Number 0681TR0362). OBJECTIVE: Changes were detected in day 20 fetal lenses of Sprague-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, Riker Laboratories, Inc., St. Paul, Minnesota. STUDY DIRECTOR: E. G. Gortner START OF DOSING: Fourth quarter, 1981 TEST SYSTEM: Ninety sexually mature, tin sated 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 libitum. 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 computer generated random numbers table. CONTROL ARTICLE: Corn oil TEST ARTICLE: T-2999COC - FM-3924 004299 19 Appendix I (Continued) 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 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 Fisher Sprague-Dawley Fisher Sprague-Dawley 25 mg/kg/day 25 mg/kg/day 0 mg/kg/day 0 mg/kg/day No treatment 30 30 30 30 30 The oral route of administration will be used because it is route of exposure in previous studies in which lens changes observed. No dietary contaminants are known to interfere with the test article. The T-2999CoC 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 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 days 3 through 20 of gestation. The females will be killed on day 20 of gestation. All of their pups will be fixed in Booin'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 PINAL 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, 1981). 004300 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 procedures 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 consistent with studying the lens change as an artifact. OBJECTIVEi (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 conparison 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 AMD 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 lens observations will be recorded. The head sections bisecting the eyes will then be processed for histological observation after embedding with the brain side down. The remaining tiro 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 fay paraffin embedding with the brain side down. The embedded specimens will be sectioned at 5-6 microns and stained with hematoxylin and eosin. Histology observations will be recorded. DATA ANALYSIS AND FINAL REPORT: (Amendment replaces this section of the protocol.) The experimental unit is the individual lens. The statistical test for incidence data will be the Chi-square test. The proposed date for the final report is the first quarter, 1982. 004301 Appendix X (Concluded) Protocol Deviations CDF animals N1R7521, 7522, 7523 and 7524 were mistakenly dosed with T-2999CoC at 25 mg/kg/day on gestation day 13. Fetuses of animals NXR7521, 7523 and 7524 were selected for lens evaluation. Since they were control group fetuses and they had been exposed to T-2999CoC in utero, their data was excluded from analysis. The fetuses affected by this protocol deviation are listed by dam as follows: NXR7521 225 226 227 228 NIR7523 89 90 91 92 NXR7524 85 86 87 88 This deviation left 68 lenses subject to microscopic evaluation in the CDF 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 CDF 0 mg/kg/day group whose gross observation data were lost in transcription. Gross and microscopic lens observations rare not made on CC^ non-treated group fetuses. 004302 2 Appendix 1Z Special Lens Oral Teratology StuV of T-2999CoC in Two Strains of Rats List of Principal Participating Personnel Haas Edwin G. Gortner Elden G. Laaprecht Wm. c. McCoraick Gary C. Pecore Xnara Porietis Loren O. Wiseth Function Study Director Veterinary Pathologist Toxicology Services Representative Supervisor of Animal Care Histopathology Technologist Technician 004303 APPENDIX III STATEMENT OF QUALITY ASSURANCE STUDY NUMBER: 0681TR0362__________________ . TITLE: Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats 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 23 co Riker Laboratories, Inc. 004304 Appendix IV Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Individual Boty Weights (g) with Mean Body Weights and Standard Deviations for Pregnant Animals CD DRV 2 6 9 12 15 20 0 MG.'KG.'0RV NiF: NIF: N1R NIF: NiF: NiF: NiF' NiF: - NiF: NiF: NiF: NiF.: NiF: NiF: NiF: NiF: NiF: NiF: NiF: N1R NIF: NiR NIF: NiF: 7802 7802 7S0-1 7805 7816 7818 7819 7820 7821 7812 7824 7820 7846 7847 7848 7849 7850 7861 7862 7863 7864 7879 7880 194 181 199 196 175 201 201 178 229 206 215 176 198 204 189 218 211 220 212 219 209 182 202 225 218 202 221 222 280 229 219 198 258 234 241 207 222 216 4 r` 231 246 244 246 237 209 222 229 224 216 229 227 218 255 2S7 221 284 257 264 222 228 240 240 270 255 270 264 262 268 225 243 261 268 247 271 27 4 248 288 264 243 218 289 292 266 265 266 271 299 281 302 203 289 288 254 272 292 298 27i 302 200 272 227 292 255 345 304 221 290 300 290 302 316 299 321 323 217 220 279 299 217 379 327 380 369 249 422 361 326 451 385 296 363 283 235 372 247 377 395 414 400 415 363 271 391 HERN 202 226 247 277 382 278 STRN. DE V 15. 7 16. 3 18. 2 19. 4 20. 9 29. 6 NON PREGNRNT RN1MRLS NIR NIR NIR NIR NIR NIR 7801 7817 7865 7876 7877 7878 171 2 Id' 202 208 210 209 201 241 215 229 238 222 214 256 224 253 243 242 210 278 226 2 1"2 259 245 224 270 228 262 271 252 227 286 252 284 291 258 004305 Appendix IV (Continued) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Individual Body Weights (g) With Mean Body Weights and Standard Deviations for Pregnant Animals CD DRV 3 6 9 12 15 26 25 MG/KG/DAV 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 01R 7806 7807 f80 S 7809 7810 7822 7823 7824 7825 7836 7837 7838 7839 7840 7851 7852 7853 7854 7855 7866 7867 7868 7869 7870 7881 7882 7883 7885 210 188 184 193 211 210 180 163 185 187 215 186 187 192 205 191 216 193 198 211 201 198 227 213 203 198 187 191 232 213 did 216 239 230 209 181 268 208 243 210 208 207 234 212 234 213 223 244 227 227 252 239 226 219 212 213 254 226 224 227 264 251 219 197 232 232 261 224 222 216 250 236 259 230 241 237 234 240 268 252 243 241 226 234 280 252 248 249 283 277 240 217 261 243 294 251 249 233 262 252 281 252 257 283 258 264 288 272 260 260 245 231 309 279 266 258 307 308 263 237 280 254 326 275 269 255 279 274 306 275 270 312 270 271 305 290 281 277 268 263 390 344 33 c* 323 288 82 334 297 361 318 389 337 329 316 353 346 382 343 344 388 348 354 383 370 362 345 326 348 MERN 197 221 237 258 280 351 STAN. D E V 13. 7 15. 3 16. 2 18. 6 21. 0 25. 3 NON PREGNANT ANIMALS 01R 7821 197 220 237 236 252 259 01R 7884 207 232 248 244 245 253 004306 Appendix IV (Continued) Special Lens Oral Teratology Study of T-2999COC in Two Strains of Rats Individual Body Weights <g) With Mean Body Weights and Standard Deviations for Pregnant Animals CDf* DAY < <5 C| 12 15 20 0 MG/KG/DAY NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR NlR 7521 7522 7522 7524 7525 7521 7522 7522 7524 7525 7541 7542 7544 7545 7551 7552 7552 7554 7555 7561 7562 7564 7565 7571 7572 7572 7574 7575 121 126 152 144 126 14? 125 143 122 146 144 12? 145 144 128 142 140 147 144 135 140 152 146 120 148 14? 126 14? 148 133 170 15? 14? .*3 145 15? 148 162 157 154 161 15? 150 154 152 15? 157 142 152 166 158 142 161 160 152 15? 152 147 172 162 155 168 151 167 158 16? 165 160 166 161 15? 160 157 161 162 151 155 172 162 150 166 166 160 165 164 158 184 172 162 15? 177 170 181 17? 175 185 168 16? 160 172 171 171 15? 14? 179 172 158 174 180 172 177 181 15? i?6 182 146 185 171 1?1 182 192 164 186 196 178 182 161 184 167 179 154 128 192 178 156 185 18? 182 192 222 168 224 204 186 225 201 21? 214 180 214 21? 229 217 226 164 226 201 202 185 182 228 215 182 226 221 217 222 MEAN 142 155 161 171 177 20? S T A N . D E V 6. 2 8. 0 6. 7 ? 0 15. 6 20. 2 NON PREGNANT ANIMALS N1R 7542 127 124 142 144 142 147 NlR 7562 125 14? 152 152 150 14? 004307 Appendix IV (Concluded) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Individual Body Weights (g) With Mean Bo<Jy Weights and Standard Deviations for Pregnant Animals CDF* DRV - 6 9 lif! 15 20 25 MG/KG/DRY OIF: DIP. OIF: OIR 0F: 01R 01R 01R OIF: 01R OIF: OIR OIR OIR 01R 01R 01R 01R OIR 01R OIR OIR OIF: OIR OIR OIR OIR 7526 7527 7528 752? 7520 7526 7527 752? 7540 7547 7548 754? 7550 7558 755? 7560 7566 7567 7568 756? 7570 7576 7577 7578 757? 7580 147 127 142 135 128 141 140 128 14? 12? 12? 127 124 121 142 162 142 127 146 146 127 142 151 127 128 124 142 161 142 15? 151 151 155 152 151 162 155 144 14? 148 146 156 177 154 151 158 15? 150 152 162 147 152 147 152 160 141 166 150 152 157 157 157 162 162 148 152 14? 151 158 178 152 156 160 164 152 14? 166 152 152 152 157 15? 120 171 156 155 166 156 15? 162 166 151 155 150 148 156 170 155 155 159 142 152 157 164 162 152 155 140 156 120 177 160 165 170 168 168 170 180 161 161 162 160 161 182 166 156 161 155 174 165 172 174 151 165 127 200 142 210 202 200 212 208 211 210 212 1?2 196 197 202 195 222 214 201 206 185 201 20? 212 215 186 205 174 HERN 140 154 156 156 162 201 STflN. D E V 7. 2 7. 1 7. 4 8. 9 11. 5 16. 5 27 NON P R E GNRNT RNIMflLS OIR OIR OIR 7546 7556 7557 141 12? 148 152 148 159 150 146 152 141 146 150 128 148 152 151 158 158 c 04308 004309 a* 0 SKjAg/day Fetus Nuafeer 3 4 29 30 1 44 57 H 151 152 154 155 1*1 1*3 1*9 171 1*1 1*4 It* 1M Dark Streak Appendix V Special Lena Oral Teratology Study of T-2999COC In Two Strain of Rata Gross and Microscopic Lens Observations^ of Individual Fetuses Freehand Sectioned Across the Lenseab Gross Findings bens Cleft t> L L NVC R XL XL L XL XL X RL X X m, XL XL XL RL RL XL RL XL RL Lens Not Sectioned L R Central Region Lena Vesicle Space Esferyonal Nucleus RR L RL R RL L RL LL LL L RL LL RR L RL XL RL RL LL Microscopic Findings Acentric Equatorial Region Region RL RL L L Lens Hlesing R R R R RL RL R L R Rh Lens Shattered or Foreign Material L L 1 - R right lens l left lens Tissue processing Procedure #2 of Table S to 00 004310 a* 0 ng/kg/day Fetus Nuafcer Dark Streak 21 219 235 236 2S9 R 260 262 264 R 277 27 291 263 2 2M 295 296 RL 297 300 319 320 4 - right lens L - left lens Appendix V (Continued) Special Lens Oral Teratology Study of T-2999CoC in Two Strain of Rata Gross and Microscopic Lens Observations-- of Individual Fetuses Freehand Sectioned Across the Lenseab Gross Findings Lens Cleft RVC Lens Not Sectioned Central Region Lena Vesicle Space Rsbryonal Nucleus Microscopic Findings Acentric Equatorial Lens Region Region Missing Lens Shattered or Foreign Material tR RL RL RL RL RL RL RL RL RL RL RL RL RL RL RL RL M. RL RL RL RL LL LL L R RL RL LL R RL RL LL RR RL RL RL RL R R R R L R L RL RL RL R RL RL RL L -- Tissue processing Procedure 92 of Table 5 ' R L RL t VO *s4 I 004311 CO* 25 eg/kg/day Fetus ifusfcer 5 7 9 11 2* 29 3* 40 77 7* 7 M iu 112 114 11 122 124 141 14) Dark Streak L 11 KL .>|xciai Lens Oral 'i'eratoluqy Study ot T-2999COC in Two Strains of Rats Cross and Microscopic Lens Observations- of Individual Fetuses Freehand Sectioned Across the Lenses- Cross Findings Lens Cleft MVC KL KL KL KL KL k RL kL LR KL RL UL RL L L KL L R XL Lsns Not Sectioned RL R R R L Central Lens Vesicle Space RL RL RL RL L R R RL RL L Eaferyonal Nucleus RL RL L RL R ML RL R RL R L L RL RL L R R Microscopic Findings Acentric Region L Equatorial Region Lens Hissing R * R L L L R R RL R L L L R Lens Shattered or Foreign Material RL RL L R -- R * riiit lsns I, left lsns -- Tissue processing Procedure 2 of Table 5 i Ul o COM 25 soAu/day Fetus ifueber 173 17 177 17 1M 19 7 20R 21S 21 221 223 2*9 291 302 303 306 30S 309 310 Harte Streak R L R R R f L l U i t- *.i. H ' M l l 1 U - * / ' l T-2999CoC in Two Strains of Rats Gross and Microscopic Lens Observations-- of Individual Fstussa Freehand Sectioned Across the Lenses-- Gross Findings Lens Cleft NVC R RL R R RL L RL K. R m. RL * RL L L RL RL lR R Lens Not Sectioned L L L R L L RL L Central Region Lens Vesicle Space Eafcryonal Nucleus RR R RR R RL LL L RL RR L RL R RL L ML RL RL RR Microscopic Findings Acentric Equatorial Lens Region Region Missing RL RL L L L RL RL R L RL LR L L Lens Shattered or Foreign Material R RL RL R 004312 - I right lens L left lens - Tissue processing Procedure 12 of Table 5 CDF* o g / kg/day Fetus tyuatoer 18 19 33 35 49 S3 85-- n ns 91S94 98 102 103 >118 119 130 132 133 135 Dark i Streak L R R Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Cross and Microscopic Lens Observations* of Individual Fetuses Freehand Sectioned Across the Lenses-- Gross Findings Lens Cleft MVC RL R RL R RL R R L L Lens Not Sectioned L L RL L R L RL RL RL RL RL RL RL RL Central Reqion Lena Vesicle Space Esbryonal Nucleus LL LL LL RR LL L LL R L R LL RR L L Microscopic Findings Acentric Region RL Equatorial Region R Lens Missing RL L RL R LR R L R RL L R L R R R R Lens Shattered or Foreign Material RL RL RL L R L RL R L ' - M right lens L left lens Jr Tissue processing Procedure 12 of Table 5 -- Data excluded fro* analysis in Tables 2,3,4 8 5 according to a Protocol Deviation (Appendix I) -- Groes findings data , lost in transcription 004313 K> COF* 0 mq/kq/ay Fetus ifuafeer 137 139 157 1*0 1*5 1*7 1*9 192 193 195 209 210 22* -- 227 -- 231 232 2*t 2M 275 27% Dark Streak R R RL R Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rata Groat and Microscopic Lens Observations- of Individual Fetuses Freehand Sectioned Across the Lenses^ Gross Findings Lens Cleft MVC Lens Not Sectioned Central Raqion Lena Vesicle Space Esfcryonal MAcleua Microscopic Findings Acentric Equatorial Lena Raqion Raqion Nissinq Lens Shattered or Foreiqn Material RL LR L RL L LR LR RL RL RL LR RL RL RL RL RL L LR LR LL LL RL L LL R RR RL LL L RL R R RL RL R RL R L RL L R LR RL RL RL RL RL M m H 004314 -- R riqht lens L - left I m a -- Tisana proceasinq Procedure 92 of Table 5 -- Data excluded fron analysis in Tables 2,3,4 to a Protocol Deviation 25 egAg/day Fetus Ijisber 14 IS 23 24 44 44 S3 SS Cl C2 cs M 70 71 73 7S 3 M 107 100 Dark Streak R L R L R RL L RL Appendix V (Continued) Special Lena Oral Teratology Study of T-2999COC in T M Strain* of Rat* Gross and Microscopic Lana Observations- of Individual refuses Freehand Sectioned Across the Lenses^ gross Findings Lana Cleft NVC Lana Not Sectioned Central Ragion Lens Vesicle Space tofcryonal Nucleus Microscopic Findings Acentric Equatorial Lens Region Region Missing Lens Shattered or Foreign Material L RL RL I> R RL RL R RL RL L LR LR LR LR RL L R tR RL RL R L L L RL L R L LL L LL L RL R RL LL L RL R R R RL R R R L LR RL R R R RL RL RL R R RL 1 004315 - I right lens l left lens -- Tissue processing Procedure 93 of Table S STCfroo CDF* 25 eg/kg/day Fetus tyuaber Dark Streak 127 12S L 143 147 1 301 R 203 237 L 340 243 244 243 24 RL 249 232 233 234 29 372 L 313 314 R i . right lens L . left lens Appendix V (Concluded) Special Lens Ural Teratology Study of T-2999CoC in Two Strains of Rats Gross and Microscopic Lena Observations-- of Individual Fetuses Freehand Sectioned Across the Lenses- Gross Findings Lana Cleft NVC Lens Not Sectioned Central Region Lens Vesicle Space tafcryonal Nucleus Microscopic Findings Acentric equatorial Lens Region Region Missing Lens Shattered or Foreign Material RL R RL RL L RL R RL LR RL LR L LR RL RL LR LR R RL L L RL RR R L L LL RL RL R L L RL R RL LR RL L L LR R L RL , RL RL RL RL R R RL R b -- Tissu procsssing Procedure #2 of Table 5 u> tn Appendix VI Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Microscopic Lens Observations-- of Individual Fetuses Not Freehand Sectioned Across the Lensesk 36 2 mg/kg/day ?tus jnber 1 2 31 32 42 43 59 60 49 50 53 56 62 64 70 72 32 33 35 37 Central Region Lens Vesicle Space Embryonal Nucleus Acentric Equatorial Region Region Lens Missing Lens Shattered or Foreign Material RL RL RR R R RL RL RL RL RR RR LL LL RL RL RR L L RL RL L RL L RL L RL R RL R RL L - left lens * right lens issue Processing Procedure #1 of Table 5 004317 Appendix VI (Continued) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Microscopic Lens Observations-- of Individual Fetuses Not Freehand Sectioned Across the Lenses*! 37 mg/kg/day tus jnber :17 220 133 .34 257 258 261 263 279 280 282 284 286 287 293 294 298 299 217 318 Central Region Lens Vesicle Space Embryonal Nucleus Acentric Equatorial Region Region Lens Missing Lens Shattered or Foreign Material LL RL RL R R L RL L L LL LL L L RR R L L R R RL R R R R RL LR R RL RL RL L RL L - left lens R - right lens Tissue Processing Procedure # 1 of Table 5 00431S Aopendix VI (Continued) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Microscopic Lens Observations-- of Individual Fetuses Not Freehand Sectioned Across the Lenses*! J 5 mg/kg/day -tus ;mber 6 8 10 12 25 27 37 39 78 80 99 100 109 110 113 115 121 123 142 144 Central Reqion Lens Vesicle Space Embryonal Nucleus LL RL RL L LL LL R RL R RL RL RL RL R RL RL RL Acentric Equatorial Region Region R R RL R RL R L RL RL RL RL RL RL Lens Missing Lens Shattered or Foreign Material R RL L * left lens R right lens Tissue Processing Procedure 1 of Table 5 004319 i Appendix VI (Continued) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Microscopic Lens Observations-- of Individual Fetuses Not Freehand Sectioned Across the Lenses*! 39 CD 25 mgAg/day Fetus Number Central Region Lens Vesicle Space Embryonal Nucleus Acentric Equatorial Region Region Lens Missing Lens Shattered or Foreign Material 174 RL 175 LL R 179 RR L 180 LL R 197 LL R 200 205 RL 206 213 LL R 214 RL RL 222 L L 224 RL 290 LL R 292 RL 301 RL RL 304 L R 305 L R 307 L R 311 RL 312 L RL L - left lens R " right lens Tissue Processing Procedure #1 of Table 5 004320 Appendix VI (Continued) Special Lens Oral Teratology Study of T-2999C oC in Two Strains of Rats & Microscopic Lens Observations-- of Individual Fetuses Not Freehand Sectioned Across the Lenses^. 40 00 IO CDF* 0 mg/kg/day Fetus Number 17 20 34 36 50 51 88^ 90s92-- 93 95 101 104 117 120 129 131 134 136 Central Region Lens Vesicle Space Embryonal Nucleus Acentric Equatorial Region Region Lens Missing Lens Shattered or Foreign Material L L R L RL RL LL R RL R RL RL RL R L RL R RL LR RL RL RL RL R RL RL L RL - L left lens R - right lens - Tissue Processing Procedure 9 ^of Table 5 Data excluded from analysis in Table 5 according' to a Protocol Deviation (Appendix I) 004321 t Appendix VI (Continued) Special Lens Oral Teratology Study of T-2999CoC in Two Strains of Rats Microscopic Lens Observations-- of Individual Fetuses Not Freehand Sectioned Across the Lenses** 41 CDF* 0 mg/kg/day Fetus Number 138 140 158 159 166 168 190 191 194 196 211 212 225228229 230 265 267 273 274 Central Region______ Lens Vesicle Space Embryonal Nucleus Acentric Region Equatorial Region Lens Missing Lens Shattered or Foreign Material LL L LL RL L L RL L RL L RL R R LL L R RL R R RL L R RL L L RL RL RL RL R RL RL R L RL - L - left lens R * right lens - Tissue Processing Procedure #1 of Table 5 Data excluded from analysis in Table 5 according*to a Protocol Deviation (Appendix I) 004322 ADpendix VI (Continued) Special Lens Oral Teratology Study of T-2999Co C in Two Strains of Rats Microscopic Lens Observations-- of Individual Fetuses Not Freehand Sectioned Across the Lenses*?. 42 OF* 5 mg/kg/day etus amber Central Region_____ Lens Vesicle Space Embryonal Nucleus Acentric Equatorial Region Region Lens Missing Lens Shattered or Foreign Material 13 L R 16 LL R 21 RL 22 LL R 45 RL 47 RL 54 L R 56 RL 63 L R 64 L R 66 RL 67 L R 69 RL 72 L R 74 L R 76 RL 81 RL 82 R L .05 RL .06 RL RL RL L * left lens R right lens Tissue Processing Procedure #1 of Table 5 004323 ADpendix VI (Concluded) Special Lens Oral Teratology Study of T-2999COC in Two Strains of Rats & Microscopic Lens Observations-- of Individual Fetuses Not Freehand Sectioned Across the Lenses^. 43 CDF* 25 mg/Jtg/day Fetus Number Central Region Lens Vesicle Space Embryonal Nucleus Acentric Equatorial Region Region Lens Missing Lens Shattered or Foreign Material 125 126 146 148 202 204 238 239 241 242 247 248 250 251 255 256 270 271 315 316 RL L RL L RL L R R RR RL RR L R RL RL R R RL R L L RL L RL RL R L RL RL L R RL K -- L left lens R right lens -- Tissue Processing Procedure #1 of Table 5 004324 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 #16474, 18319, Analytical Report #318 A light, straw-colored wa*y solid labeled FM-3924, lot 547 was received for analysis for R1ker teratology study #0681TR0362. The material has 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 0.4 0.7 0.4 0.7 Amide 0.5 0.6 0.6 0.6 0.2 c 3 1.2 1.3 1.2 1.3 C4 10.6 for 1.8 1.7 1.8 2.2 C5 c6 cH7ighc8Boilers a ll low boilers 88.9 - 1.5 4.8 88.9 0.6 1.6 4.3 88.9 0.8 1.5 4.8 88.9 0.6 1.9 4.9 88.9 0.2 All values are within experimental and integration parameters. Values meet QC specifications. C H H S F 0 by difference Elemental Analysis % Theor. 1 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 9.0 ' X Found 5/27/82 25.0 1.8 2.7 6.1 55.3 9.1 004325 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: Initial 38-48*0 Recheck 38-48*0 5/28/82 38-48*0 Spectroscopic: Infrared spectra 16474-2,3,4 and 18318-1 are Identical to 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 H838S, H8387, F8390, F8391 were obtained on 12/1/80 and 12/2/80. These were to confirm structure and not repeated. Structure was confirmed. The Isomer distribution was found to be 69.61 straight chain, 10.91 (CF3 )2CF branched, and 19.51 backbone branching. Conclusions: FM-3924, Lot 547 has an analysis within specifications, Is stable and is 004326 eu : M. T. Cuuc! --- .. E. G. Gortner (original -t 1). . .. -&-- Gjtiffith/W. C. McCormick F. Keller E. G. Lamprecht ... ,,_ W. 11. Pearlaun R. A. Prokop V. Pothapragada/L. L. 0. Wiseth Winter TITLE: Protocol for Special Lens Oral"Tera'tology Study of T-2999CoC-- in Two Strains of Rats (Riker Experiment Number 0681TR0362). OBJECTIVE: Changes were detected in day 20 fetal lenses of Sprague-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, Riker Laboratories, Inc., St. Paul, Minnesota. 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 libitum. 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 computer generated random numbers table. CONTROL ARTICLE: Corn oil TEST ARTICLE: T-2999COC 004327 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 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 Fisher Sprague-Dawley Fisher Sprague-Dawley 25 mg/kg/day 25 mg/kg/day 0 mg/kg/day 0 mg/kg/day No treatment 30 30 30 30 30 The oral route of administration will be used because it is route of exposure in previous studies in which lens changes observed. No dietary contaminants are known to interfere with the test 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 exams have been completed (approximately fourth quarter, 1981). E. G. Gortner Date Senior Research Technologist Animal Teratology-Reproduction Study Director 1- 3- 8) E. G. Lamprecht, DVM, PhD Date Research Veterinary Pathologist _____ 7/$/$/ M. T. Case, DVM, PhD V Date Manager, Pathology-Toxicology Safety Evaluation Laboratory xJ lQPjm *. 'tlu*. W. C. McCormick, MS Toxicologist Sponsor Representative 004328 q/rtf a Dane