Document VJqLDbEm5veN4m7Z2D6xBMn5K

AR226-2788 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HASKELL LABORATORY REPORT NUMBER 441-84 Copies to: Company Sanitized. Does not contain TSCA CB1 FOR DU FONT USE ONLY I. E. DU FONT DE NEMOURS & CO., INC. HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT NEWARK, DELAWARE 19714 C-8 ALTERNATES': MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HASKELL LABORATORY REPORT NUMBER 441-84 Dates: Initiation (breeding date) - July 13, 1981 Completion (sacrifice date) - August 19, 1981 Notebook Numbers: Date Written; September 26, 1984 Date Issued: October 2, 1984 No. pages in this report: 72 SamUzcd.Soos^containTSCA Company - 1 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 Report by: Study Director Staff Teratologist Haskell Laboratory Approved ^~^ & y^' by ^~--^f^- - s^ J. G. Aftosgfis^ D.V.M. Associate Director Haskell Laboratory RES:jy:MR4130.2 ,.^ contain TSCACBl Company San^d. DCC.-, not C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HASKELL LABORATORY REPORT NUMBER 441- TABLE OF CONTENTS Title Page. 1 Signature Page 2 Table of Contents 3 ......................................................... I. SUMMARY 6 ................... II. INTRODUCTION. 6 A. Background 6 ................. B. Protocol. 7 .................................. III. MATERIALS AND METHODS. 7 A. Test Chemicals. 7 . . . . . . . . . . . . . . B. Animals and Husbandry 8 C. Dose Selection. 9 . . . . . . . . . . . . . . . D. Animal Breeding and Distribution. 10 . . . . . . . . . . . . . E. Test Chemical Administration 10 . . . . . . .................. F. Maternal and Fetal Examination and Sacrifice. 11 ............... G. Statistical Tests. 12 . IV. RESULTS 12 . . . . . . . . . . . . . . A. Eye Examination of Prospective Parents. 12 . . . . . . . . . . . . . . . . . . . B. Maternal Clinical Signs Observed. 12 . . . . . . . C. Maternal Feed Consumption 13 D. Maternal Body Weight Gain 14 . . . . . . . . . E. Gross Examination of Maternal Organs at Sacrifice 14 . . . . . . . . . . . . F. Reproductive Effects and Fetal Body Weight 14 ................ G. Fetal Alterations. 14 . . . . . . V. DISCUSSION 15- .............. VI. CONCLUSION 16 ................... VII. ACKNOWLEDGEMENTS 16 ...................'................................. VIII. REFERENCES 17 Company Sanitized. Does not contain TSCA CM 3 - 1 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINAR^STUDY. BY GAVAGE) HLR 441-84 TABLE OF CONTENTS (CONT.) TABLES Page I. Feed Consumption in Rats Given Test Chemicals by Gavage from Days 6-15 of Gestation 18 . . . . . . . . . . . . . II. Reproduction and Fetal Development in Rats Given Test Chemicals by Gavage from Days 6-15 of Gestation 19 . . . . . . . . III. Fetal Malformations in Rats Given Test Chemicals by Gavage from Days 6-15 of Gestation 21 IV. Fetal Variations in Rats Given Test Chemicals by Gavage from ............. Days 6-15 of Gestation 22 . . . . . . . . . . . . . APPENDIX A. Protocol for Maternal and Developmental Toxicity Study in Rats A m af end ter men t Adm to i t nis he tration Protoc b ol y Gavage ... w . ith . C- . 8 Alt .. ernates . *.... . . 28 39 ATTACHMENTS 1. *Letter, from J. R. Barnes to R. E. Staples, 3/20/81 42 . . . . . 2. Letter, from J. M. Clinton to R. E. Staples, 7/7/81 54 . . . . . 3. Memorandum, from W. D. Kerns Co R. E. Staples, 8/19/81 55 .. .. .. .. 4. 'Memorandum, from W. D. Kerns to R. E. Staples, 1/29/82 56 * Du Font classified information c^---------01^"^" 4 - C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT .(PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 TABLE OF CONTENTS (CONT.) ATTACHMENTS (CONT.) Page 5. Memorandum, from W. D. Kerns to R. E. Staples, 10/5/81 including Memorandum from C. L. Lamontia to N. L. Chromey, 10/1/81, and the code for rats assigned to this study. 57 . . . . . . . 6. Memorandum, from W. D. Kerns to R. E. Staples, 12/18/81. 67 . . . 7. Memorandum, from R. E. Staples to Teratology Section File, 10/30/81 71 8. Tabulation of Results of Dose Selection Based Upon Comparison of ................................. LD50 or ALD Values 72 Comply Sanded. Doss not contain TSCA CB1 - 5 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT [PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 I. SUMMARY f||^BH|^^^^^^^^^^^^^^^^^^^^^^Hwere administered to rats by gavage at 100, 500,250,or500 mg/kg"body weight/day, respectively, from Days 6 through 15 of gestation to determine the _degree ^f hazard to the developing conceptus The dosage of^BQ B--------^Kivenwas lethal to six of the seven rats tested, and^ffie rat ^na^survived did not yield live fetuses at term. Therefore, its potential hazard to the conceptus was not assessed. The remaining chemicals were tested at levels that were less toxic to the dams. None of the test chemicals were demonstrated to represent a unique hazard to the maintenance of pregnancy or to the developing conceptus. II. INTRODUCTION A. Background Du Font obtains ammonium perfluorooctanoate (C-8) from 3M Company Hji for use in the manufacture of a variety of fluoropolymer dispersions, including some of Du Font's Teflon products. A study of the ^_ _ embryotoxicity and teratogenic potential of C-8 was requested by ftJiUBI^H^U Polymer Products Department, and by^^BHHIHQ ^henuSaJ^'md Pigments Department, at a meeting ne^^a^Haskell Laboratory on June 11, 1981. This request was initiated in response to TSCA, Section 8(e)'s filed by 3M between the last part of 1980 and March 20, 1981 on this and several related chemicals. The possible teratogenic activity relayed to us by 3M included lens changes in the eyes of the near-term offspring of rats exposed to the test chemical by gavage from Days 6 through 15 of gestation. Inhalation of C-8 was not demonstrated to be teratogenic in the rat after exposure from Days 6 through 15 of gestation (1) even though the concentrations tested included those that were overtly toxic to the dam. No additional adverse effects were noted among similarly exposed dams or their offspring when maintained through weaning. ^^.Ooe3.c.con>..nTS=.CB. mpany C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT .(PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 A. Background (Cont.) In addition, a report (2) on the C-8 portion of the current study was issued on January 14, 1982. The current report presents preliminary data on the maternal and developmental toxicity of four similar chemicals that were collected concurrently with the C-8 data. The request for a proposal .developmental toxicity study o, J^^H^Hp was received fromjB__ TpnT^?March 23, 1981 (Attachment araUvre quested that the deve III. study for comparison to C-8. B. Protocol (Appendix A) An MR request was sent to PPD on July 17, on July 28, 1981. The protocol was issued on was amended on December 3, 1981. 1981; it July 23, was authorized 1981, and it MATERIALS AND METHODS A. Test Chemicals* 1. C-8 is a white powder which sublimes at 110C^ Its molecular weight is 431, and its structural formula is' The purity of the sample used wasi contaminants present were] isffmffjand Cts CAS Registry Number it was assigned Haskell Number 14,045. The sample was received from the Polymer Products Department. ^^iBUmBH^ 'dHf-s a white solid with greater than. 10^ solubility in water. Its structural formula ^ts molecular weight is 463, its CAS Registry Number is and the Haskell Number assigned is 14,075. The sample was received from| PP&R, Washington Works. * Du Pont classified information San^d.D^500'.conia^^ ;oinpa"y- C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 A. Test Chemicals (Cont.) 3. ^IB|(LS a white solid, with greater than 10% solubility in water. Its structural formula isF^" f r o m B " " Its molecular weight is 525 and the Haskell Number assigned is 14053. The sample was received PP&R, Washington Works, with a purity of] 2.0, pH 7-SLan formula is |is an off-white powder with a density of Llitv in water.' Its structural Iwith- a molecular weight of 550. Its assigned^Haskell Nu ber is 14,068 and the sample was received from^ Polymer Products, E353/318 Experimental Station. ___|is a beige powder with a density of about ', pH 4-5 in water, and 1-10^ solubility in water. Its , Its is molecu 14,008. l ar T h w e eight samp l is e 540andits wasJ^^^B^ ass Btf) i gne the d-_^H a s k e l l Number SSL 269/306. B. Animals and Husbandry The Crl:CD(SD)BR strain of rat was chosen for this test because previous toxicity testing on this class of chemicals was conducted in this species and strain, and because extensive background information from previous developmental toxicity tests at Haskell Laboratory exists on this rat strain. Female rats about 56 days of age (nilliparous) were received from Charles River Breeding Laboratories, Inc., North Wilmington, Massachusetts. They arrived on July 2, 1981, and weighed 170j0.7 g (S.E.M.). Individual weights ranged from 154 to 198 g. Male rats of the same strain and from the same supplier were used for cohabitation with the females. They ranged in age from one to two weeks older than the females and weighed 278_+_2.8 g (S.E.M.) with a range of 224 to 342 g. Compan, Sanitized. Does not contain TSCACBI C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 B. Animals and Husbandry (Cont.) Upon arrival at Haskell Laboratory, each female rat was identi fied by a combination of toe clips and ear slashes and by a cage card bearing its assigned number. They were quarantined for 11 dyas. The male rats were identified by ear slashes and cage cards. The rats were housed two per cage in suspended, wire-mesh, stainless steel cages. Purina Certified Rodent Chow 5002, Checkers and water from the Wilmington Suburban Water Corporation (WSWC) were supplied ad libitum. The water was provided by an automatic watering device. A lighting cycle of 12 hr light: 12 hr dark (dark period was from 6:00 P.M. to 6:00 A.M.) was maintained throughout the study. Before mating, the temperature of the animal rooms was maintained at 74^+_2F. After -mating, the animal room temperature was maintained between 75 and 78F, and relative humidity was maintained between 40 and 80%. Since the historical incidence of cataracts or opacities in adult CD rats is about 37a (personal communication with James M. Clinton, V.M.D.; consultant ophthalomologist), all prospective parental rats were examined for these alterations before breeding. The eyes of each rat were dilated with 1% atropine ophthalmic solution and examined in semidarkness by the consultant ophthalmologist using focal illumination, indirect ophthalmoscopy, and, when indicated, slit lamp microscopy. Affected rats were eliminated from the colony before the breeding began. C. Dose Selection In a pretest, two nonpregnant female rats were administered C-8 by gavage at 150 mg/kg/day which was the highest dose used in the preliminary study for 3M. The first rat, which weighed 278 g, showed severe clinical signs of toxicity by the fourth day and was found it dead on the morning of the fifth day by which time had lost about 40 g body weight. The second rat, which weighed 260 g, lost about 11 g by the third day. Two additional nonpregnant female rats were then given C-8 at 100 mg/kg/day, the second highest dose level used for the 3M study. After five days of dosing, adverse clinical signs were not noted in one rat that lost about 6 g and were minimal in the other which lost about 14 g. On this basis, the 100 mg/kg/day dosage level was judged to be the maximum that the dams could tolerate for the planned exposure period of ten days. ----,------oenolcnatoTSCACBl C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) 1\ HLR 441-84 C. Dose Selection (Cont.) Dose selection for the other chemicals was based upon LD50 and ALD data relative to that for C-&_The calculated dosages were 100, --two rats per group, the dose levels for the last two chemicals were reduced to 250 and 500 mg/kg/day, respectively. D. Animal Breeding and Distribution The female rats were mated on an as-needed basis. After the necessary number of females were bred, the mated females were ranked by body weight and assigned to groups by rotation, in order of rank, in proportion to the total number of rats to be allocated to each group. The dams were weighed on the day of arrival, before breeding, and on the morning of Days 1, 6, 9, 13, 16, and 21G. Day 1G was the day that proof of mating was detected. They were observed for clinical signs and changes in demeanor upon arrival at Haskell Laboratory, at breeding, and daily from Days 6-21G. After mating, the rats were housed individually in suspended, wire-mesh, stainless steel cages. Feed consumption was measured during gestation. E. Test Chemical Administration The test chemicals were given by gavage in corn oil. Stripped corn oil was purchased in 400-g cans from Eastman Kodak Company, Rochester, New York. Most of the tocopherols present in the refined corn oil had been removed by stripping off the most volatile fraction by molecular distillation. The amount of test chemical required was removed from a sealed plastic bag, which was contained in a Fiberpak carton. During the dosing period (Days 6-15G), suspensions were prepared daily such that the dose was delivere'd in 5 mL of suspension/kg body weight. The ' body weight most recently recorded was used to calculate the dose to be given to each dam. The dams were dosed between 1:30 and 3:30 P.M. daily. The control group received 5 mL stripped corn oil/kg body weight for the same period of gestation. To minimize oxidation during the dosing period, the corn oil from opened cans was stored at about 4C in red bottles with ground glass stoppers. This practice previously was shown to limit peroxide concentration to <25 ppm after storage for one year(3). Aflatoxin concentration contained in stripped corn oil received previously from the same source (Lot D 4-25) was determined to be <2 ppb(3). CAS Registry Number 8001-30-7; Item 13266, Lot No. D 4-45. _ ^ icontaiR"^^ C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT .(PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 F. Maternal and Fetal Examination at Sacrifice To prevent bias in the examination of maternal and fetal specimens, the dams were coded from just before sacrifice till all maternal and fetal data were collected, and till all structural alterations noted among the fetuses were classified. After sacrifice of the dams by cervical dislocation on Day 21G, gross pathologic changes were sought, liver weight was recorded, and reproductive status was determined. The number of corpora lutea and implantation sites were counted, and the number and position of all live, dead, and resorbed fetuses were recorded. The uterus of each apparently "nonpregnant" rat was stained with ammonium sulfide to detect very early resorptions; data collected were used only to determine the incidence of pregnancy. The weight of the intact and empty uterus for each dam was recorded to allow calculation of actual maternal gain in body weight. All live and dead fetuses were weighed and sexed externally and internally, and the live fetuses were examined at a magnification of 2.5X (Ednalite) for external alterations. The Ednalite also was used to count the corpora lutea. About one-half of the fetuses of each litter that were alive when removed from the dam were examined for visceral alterations(4); in addition, all stunted or malformed fetuses were examined similarly. The heads of all fetuses examined for visceral alterations and sufficient of the remainder to total two-thirds of each litter were fixed in Bouin's solution. Two of the fetal heads of each litter that were fixed in Bouin's solution were sliced in vertical cross-section in front of the eyes, through the center of the eyes, and through the widest portion of the head(5). The remainder that were fixed in Bouin's solution were sectioned immediately in front of and behind the eyes (rather than through the eyes) and through the widest portion of the head. Three of the fetal heads of each litter that were fixed in Bouin's solution, but not cut.through the eyes, were processed, and the eyes were examined histologically via light microscopy by a pathologist. The histologic specimens were coded for this examination, and particular emphasis was placed upon the structural integrity of the lens. ^^CAC1& . A DOS50- CO^33""" - 11 - C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 F. Maternal and Fetal Examination at Sacrifice (Cont.) All fetuses, except for the heads of those that were fixed in Bouin's solution, were fixed in 70% ethanol, eviscerated (if not done previously), macerated in 1% aqueous KOH solution, and stained with alizarin red S to permit examination for skeletal alterations. On an as-indicated basis at sacrifice, some tissues were fixed in Bouin's solution for storage or for histologic evaluation. The identity of each fetus was retained at least till the report was written. G. Statistical Evaluation The litter was used as the experimental unit for the purpose of statistical evaluation (6). The significance of differences in the incidence of pregnancy, clinical signs, and maternal death was determined by use of Fisher's exact probability test(7). A two-way analysis of variance was used to detect differences in feed consumption among breeding lots and between groups. Dunnett's test(8) was used to test the statistical significance of differences between the control and each experimental group in maternal body weight, in body weight gain, and in feed consumption when the one-way analysis of variance was significant. The significance of differences in incidence of structural alterations between the control group and each experimental group was determined by application of the Mann-Whitney U test(9). When more than 75% ties occurred in the data, the Fisher's exact probability test was applied(lO). The level of significance selected was p^O.05. IV. RESULTS A. Eye Examination of Prospective Parents The eyes of the male and female rats were examined on July 7, 1981; four males and four females were removed from the colony because ocular lesions were identified (Attachment 2). B. Maternal Clinical Signs Observed In the control group, the only clinical sign noted was focal alopecia which developed in one. dam. Comply SanttW."^"'""'"3""5"081 12 C-8 ALTERNATES: MATERNAL AND DL/SEJ VVEl-tLljLO/JPLMi'UEjIN.tXTnAjLL* TOXICITY IN THE RAT (PPRREELUIMMIDNAIARRYY;STUDY. BY GAVAGE) HLR 441-84 B. Maternal Clinical Signs Observed (Cont.) Six of the 37 dams given C-8 by gavage were either found dead (five) or had to be killed (one) in view of a moribund state before scheduled sacrifice, as opposed to none of the 37 dams given only corn oil. All but one of the dams that subsequently died (during the dosing period) had wet perineal areas and were lethargic. Two also had chromodacryorrhea and chromorhinorrhea. Among the remaining dams, four developed alopecia, one had lung noise, and another developed diarrhea. No deaths or clinical signs were^hserved in the (|BBgroup. Similarly, no deaths occurred in the^^^roup and only one female had clinical signs which consisted of red discharge from the left eye from Days 6-13G and yellow stain of the perineal area from Days 10-16G. Um^JB In the group overt toxicity was observed after the second day of dosing. Dosing was discontinued after Breeding Lot A received eight daily dosages, but even so five of the. seven females died between Days 11-14G, and one was sacrificed in extremis on Day 12G. The only female that survived to scheduled sacrifice had no visible sign of being pregnant but nidations were detected after staining of the uterus with ammonium sulfide. Hence, fetal data for the I^B^^------groupare not available for tabulation. ^^^^^^^^B^B^B^^B^ Q^UUP In the group five of^hedamshad clinical signs similar to those seen in the C-8 and flUUmgroups. Therefore, dosing was discontinued after breeding Lot A received eight daily dosages. One of the seven females in the group was found dead on Day 13G and two others were sacrificed in extremis before scheduled sacrifice (Days 12 and 14G). Three of the four that remained had live fetuses. Microscopic evaluation of the pulmonary parenchyma of the rats that did not survive to scheduled sacrifice did not reveal evidence of iatrogeni'c pulmonary injury, hence the deaths were not found to be related to improper gavaging (Attachment 3). C. Maternal Feed Consumption ' ^^^J3unng the dosing period, the groups given C-8, uHBI^m^^y^ ^^I^^^Bconsumed significantly less feed than the control group I/. "(Table Feed consumption was similar to the control value inthe post-exposure period. Insufficient data were available for thejBy^ ^tmninnraJSgi-onp to permit meaningful statistical comparison. C.W^S""11"". 1-c0c . ^ " ' " " " ^ 0 8 1 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 D. Maternal Body Weight Gain The mean gain in body weight from Days 1-5G was significantly less in the group designated to be given C-8 than in the control II). group (Table However, from Days 6-15G, the group receiving C-8 gained about one-third less (p^O.05) than the control group, and during the post-treatment period (Days 16-21G), the body weight gain of the C-8 group significantly exceeded (p^O.05) the control value. During the dosing period, the body weight gain of theldpgroup was not significantly different from that of the control group, but during the postdosing period, the^^^jgroup gained significantly more than did the control group. The ^|and ------------^groupsgained little during the dosing period, but, therear^^^^^B1lgroup gained significantly more body weight than the controlgroup. E. Gross Examination of Maternal Organs at Sacrifice At sacrifice, one of the dams given C-8 was observed to have several red areas on the visceral surface of the median lobe of the liver. The relative weight of the liver for the ^Bijgroup was significantly higher than that for the control group (Table II). F. Reproductive Effects and Fetal Body Weight The maintenance of pregnancy, the incidence of resorptions, and fetalbodyweight were not adversely affected by administration of G. Fetal Alterations No more than one fetus was malformed in each of the groups given III). test chemicals (Table In the control group, two were mal formed. None of the chemicals were demonstrated to increase the frequency of malformed fetuses. -However, the incidence of fetuses with variations, was signifi cantly ^^ - increased ^a above the control value in both th^^e^^^HBH^BHmBHH^H^H^^U^^ --^--------groups (Table IV). The increase for the ^jgroupwas statistically significant only if a one-tailed test was applied. In both groups the increase was not due to a statistically significant increase of any individual type of variation. 14 - Company Sanitized. Dees not contain TSCA CB1 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 G. Fetal Alterations (Cont.) I I I S t e r e o s c o p i c heads per litter malformations or alteration noted examination of the bisected eyes from two fetal (Bouin's fixed) from each group did not reveal variations (Tables and IV). The only eye among the fetuses was a focal area of redness beneath the cornea in a fetus from the C-8 group and in two fetuses of thelBjigroup that was detected during routine examination of the live fetuses for external alterations (Table IV), The redness was due to the presence of blood in the anterior chamber of each of the affected eyes (Attachment 4). In addition, histomorphologic examination of the eyes of three fetuses per litter per group did not reveal any pathologic lesions. A postmortem artifact was recognized in the central anucleate portion of the fetal lens which was equally distributed in incidence among the experimental and control groups (Attachments 5-7), V. DISCUSSION Dose selecton based upon comparative LD50's or ALD's was not reliable even among chemicals with some similarity in chemical activity (see Attachment 8). Due to the maternal toxicity observed in the teii------IK^jB--ijjBi6ji|^ groups extra unmated female rats from thesamesn^S^^^^^^^^^enlower dosages by gavage than were used in this study (4/group). Administration ofjH|HB^gat 50 mg/kg or |H|Him&at 150 mg/kg for a period oT ten days still resulted into^nucnbody weight loss to serve as the maximum tolerated doses. Therefore, it is recommended that dosage levels of 35 and 100 mg/kg, respectively, be the highest tested should additional Coxicity testing be undertaken. ^^^C-8 was previously evaluated in Haskell Laboratory Report No. l^m^nd will not be repeated here; the data on C-8 are presented in this report for comparative purposes only. ^Piqwas given at a dosage- that reduced feed consumption but not to a degree sufficient to significantly reduce body weight gain during the dosing period. However, the dosage administered was toxic as indicated by the significant rebound effect measured after completion of the dosing period. Neither the reproductive outcome of the dams nor the survival, or body weight, of their fetuses were demonstrated to be adversely affected by exposure to HA A slight, but statistically significant, increase in developmental variations was detectable only if a one-tailed rather than a two-tailed statistical test was applied. This effect was largely due to an increase in the incidence of extra ossification sites next to the vertebrae Chat was not significantly increased above the control value Xp>ff.05). In view of the concurrent Coxicity present among the dams, yEBj^as not demonstrated to represent a unique hazard to the conceptus Company ^Snanili-lrse-eda-Duc^o-s not contain TSCA Cff C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY (PRELIMINARY STUDY, BY " IN THE RAT GAVAGE) HLR 441-84 V. DISCUSSION (CONT.) ^mjalso was given at an effect level for the dam as evidenced by a significant decrease in feed consumption and in body weight gain during the dosing period, and by a significant increase in actual and relative liver weight. Neither the reproductive endpoints evaluated for the dams nor the developmental endpoints used for the fetuses were demonstrated to be adversely affected. The dose level at whichflmUJHwas given was too high to permit evaluation of toxicity to the dam or their concept!. Subsequent range-finding studies in non-pregnant rats indicated that if the dosage level should not exceed 35 mg/kg body weight, daily for 10 days, by gavage, in corn oil. given The (^^H^^^ljj also was given at a dose level that was more toxic than desired. Three of the seven mated females in the group did not survive but three of the remainder did yield live young at scheduled sacrifice. Despite significantly reduced feed consumption and body weight gain vs. that for the control group, no additional adverse effects were demonstrated among the dams and changes detected among their offspring were very minor. The average percent of fetuses with variations per litter was significantly increased for developmental variations (p<0.05; two-tailed test) and for total variations (p<0.05; one-tailed test). These differences were due to slight increases in the degree of sternal ossification and in the number of misaligned sterebrae; the differences from control incidence for these ind^idual variations were not statistically significant (p>0.05). CBHBHA^1^ not P08^ a ""^q116 hazard to the rat conceptus at the toxic level tested. 71. CONCLUSION unique hazard to the dunna^heperiod of '^Jl^mm^as given assess its potential conceptus after administration by gavage to rats major organogenesis at dosages toxic to the dam. at a dose level that was too toxic to the dam to hazard Co the conceptus. VII. ACKNOWLEDGEMENTS Histologic specimens were prepared by the Pathology Section. Histomorphologic examination of the eyes and other tissues was conducted by William D. Kerns, D.V.M., M.Sc. The in vitro eye examinations were conducted by James M. Clinton, V.M.D., Consultant in Comparative Ophthalmology. The remainder of the study was conducted by the Teratology Section, Haskell Laboratory. Company Sanitized. Doss not contain TSCA OW 16 C-8 ALTERNATES: MATERNAL AND 1s DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY. BY GAVAGE) HLR 441-84 VIII. REFERENCES 1. Unpublished Du Font Data, Haskell Laboratory: 2. Unpublished Du Font Data, Haskell Laboratory: 3. Unpublished Du Font Data, Haskell Laboratory: 4. Staples, R. E., "Detection of visceral alterations in mammalian fetuses." Teratology. 9^A37 (1974). 5. Barrow, M. V., and W. J. Taylor, "A rapid method for detecting malformations in rat fetuses." J. Morph., 127(3):291-306 (1969). 6. Haseman, J. K., and M. D. Hogan, "Selection of the experimental unit in teratology studies." Teratology, l2_: 165-172 (1975). 7. Siegel, S., Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York, pp. 96-104 (1956). 8. Steel, R. G. D., and H. H. Torrie, Principles and Procedures of Statistics. McGraw-Hill, New York, pp. 99-128 (1960). 9, Mann, H, G., and D. R. Whitney, "On a test of whether one or two random variables is stochastically larger than the other." Ann. Math. Stat., l8_: 50-60 (1947). 10. Haseman, J. K., and D. G. Hoel, "Tables of Gehan's generalized Wilcoxon test with fixed point censoring." J. Statist. Comput. Simul.. 3^:117-135 (1974). ^^con^nTSCACM -17-ConP^sa^ilizec, sD HLR 441-84 TABLE I C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOX1CITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FEED CONSUMPTION IN RATS GIVEN TEST CHEMICALS BY GAVAGE FRCM DAYS 6-15 OF GESTATION (G) Corn oil (5 mL/kg/day) C-8 (100 mg/kg/day) C i R (100 mg/kg/day) \ U f \ (500 mg/kg/day) 0 (250 __^ mg/kg/day) Days 1-5G Days 6-15G 21.0j_0.59 21.9_t_0.48 20.6_H).32 17.2j0.37' 21.8__0.79 18.5j0.25' 21.5j_0.52 15.7_K).40''' 19.5__0.0 11.7_K),0 Days 16-20G 28.1_H).58 29.0+0.52 30.5j_1.04 27.9_t_0.52 18.5_KI.O N Q , u mber of dams grams/rat/da significantl y y I ncluded _tS.E.M,; differen t v alues from 24 for cont r no ol n-pre valu g e n a b n y t 22 female Dunne+ s t' s were tes t e x ( 7cluded p<0.05) 6 1 HLR 441-84 TABLE 11 C-8 ALTERNATES: T<3XICITY IN THE RAT MATERNAL AND DEV'ELOPMENTAL (PRELIMINARY STUDIY, BY GAVAGE) REPRODUCTION AND PETAL DEVIELOPMENT IN RATS CilVEN TEST CHEMICAL.S BY GAVA6E FROM CIAYS 6-15 OF GESTA Corn oi1 (5 inL/kg/day) C-8 (100 mg/kg/day) Females a . No. pregnant /no. mated ^ No. deaths ' No. 1itiers 2 7, -o ' U fd \(fl ' 8 Mean no. corpora 1utea Mean Mean Mean no. implants a liver weight (g)- q weight galn(g) \'fS. ' R' Days 1-5 0. Days 6-15 ^ Days 16-21 (0 3 Days 6-2) iS- h o Days 6-21C s. 3 .^Fe+al Death yi ^Mean no.. resorptIons/dam .SNO. litters with total resorp+lon 25/25 0 248 le.np.ss^ 15.6+0.57 15.4+0.54 55.'2+1.12 56.7+2.54 72.6+1.51 '~~ 129.2j2.68 57.8+2.55 0.7+0. 16 22/25 5'' 22 16.7+0.87 15.8+0.64 16.2+0.44 <!> 50.0+J.09' 58.5+2.89' J> 84.5+5.00' '--~ 122.8+4.26 49. B+2.75 0.6+0.15 c ^ (100 mg/kg/day) 7/7 0 7 15.5+J.19 14.6+0.65 17.5+0.68 55.4j2.08 44.4+5,25 A 86.6+4.69' -- 151.0+7.78 56.4+5.69 1.0+0.58 UKO (500 mg/kg/day) c(250 -mg^/kg/day) 6/7 0/7 0 6 6 0 15.5+0.67 -- 15.2+0.54 -- <!> 22.9j1.20' -- 52. 1+2.75 .---- <t) -1.5+8.95' ------ <!> 92.6+5.99' <!> 91.0+4.29' - 21.8+2.721' . 0.8+0.17 -- T HLR 441-84 TABLE II (CONT.) C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) REPRODUCTION AND FETAL DEVELOPMENT IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF GE Corn oil C-8 . L^fel^ ( flB 3 (5 mL/kg/day) (100 nig/kg/day) (100 mg/kg/day) (500 nig/kg/day) (500 Fetuses No. I ive - 322 292 95 86 Mean no. Iive 13.4+0.32 13.3+0.67 13.6+0.95 14.3+0.61 1 0 0 0 No. stunted Mean weight (g) 3.8+0.08 4.0+0.05 3.9+0.14 g all females had visible sign of pregnancy evident at autopsy one was noted as being dead on Day 116, and two more on Day 12G c rats were found dead on Days 10, 11, 12, 13, and 14G and one was sacrificed 0 0 3.3+0.09 In extremis on Day 12 that survived had resorp+ions detected by stain only one rat was found dead on Day 13G; the others were sacrificed In extremis on Days 12 and 14G e one female had only two early resorptions In utero on Day 21G x+S.E.M. g -- females without a litter were excluded Day 21C body weight denotes the body weight of females excluding the products of conception (I.e. , corrected body weight) significantly dif'ferent from control value by Dunne+t's test (p<0.05) HLR 441-84 TABLE III C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETAL MALFORMATIONS IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF G Corn oil (5 mL/kg/day) External Malformations3 No. examined 322/241' i M i--> i I 0) ^ w s> N' W p- 0 0 n> <o g. 0 0 oT W 0 > 0 2 Visceral Malformations No. examined Great vessels Innominate - none Kidneys - none Kidneys - small Spleen - small Brain - ventricles enlarged Head Malformations No. examined Skeletal Malformations No. examined Sternebrae - fused Total with Malformations Avg. % Malformed Fetuses per Litter (j^S.E.M.) 171/24 c 1/1^ l/^ 1/1^ l/^ 221/24 322/24, l/^ 2/1 0.7^0.70 no malformations were noted f> fetuses/litters blanks represent zero incid ence occurred in a single f 'etus C-8 (100 mg/kg/day) 292/22 155/22 198/22 292/22 (100 mg/kg/day) 95/7 49/7 1/1 1/1 65/7 95/7 1/1 l.lj1.10 (500 mg/kg/da 86/6 45/6 60/6 86/6 TABLE- IV C-8 AL IERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETAL VARIATIONS a IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF GE Corn oil (5 mL/kg/day) Developmental Variations External No. examined Hematoma Petechia Eye - red '..) 0 Visceral 1 No. examined Renal papilla (/> -reduced B> Renal pelvis 5: N" -large (B Q. Pulmonary arteries to -common trunk 0 TO 01 Head1 3 2, No. examined rt > 3. (U 5' ^(ft 2 S , 322/241' 15/10 60/19 c 171/24 l/l6 l/^ 3/2 221/24 C-8 (100 mg/kg/day) 292/22 10/8 34/15 l/l3 155/22 2/lg 9/6 198/22 V (100 w \ ing/kg/day) 95/7 6/3 22/6 49/7 3/2 65/7 ^3 \ (500 mg/kg/da 86/6 6/3 1161/6-^, 45/6 3/2 60/6 HLR 441-84 TABLE IV (CONT.) C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETAL VARIATIONS'^ IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF GE Corn oil .(5 mL/kg/day) Deivelopmental Variations ( Cone.) j> 0 0 o u 3 *< W ID H' ra a a 0 0 (.1 ;- a. 0 0 :j s3' 5} Q v a 00 ..." ikeletal No Examined Sternebrae -misaligned(l)-1 . -misaligned(2+)-] -bipartite Centrum -bipartite -dumbbelled Rib -extra ossification center -rudimentary -extra -thickened -calloused -wavy 322/24 14/11 7/7 1/1 3/3 2/2 45/16 2/2 1/1 1/1 C-8 (100 mg/kg/day) 292/23 6/6 9/8 4/4 76/16* 11/5 1/1 1/1- l/l3 ^ (100 mg/kg7day) 95/7 2/2 2/2 1/1 2/2 30/16 1/1 (500 mg/kg//ddaa 86/6 5/4 3/3 14/5 1/1 2/1 4/1 2/1 ^) TABLE IV (CONT.) HLR 441-84 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETAL VARIATIONS3 IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM Corn oil (5 mL/kg/day) C-8 (100 mg/kg/day) ^ W (100 mg/kg/day) DAYS 6-15 OF GE (500 mg/kg/da Developmental Variations (Cont.) Subtotal - 3 Developmental Variations -No. Affected -Avg. % Affected Fetuses/Litter OjS.E.M.) 0 0 Variations Due to t'> Retarded Development 3. w Visceral |. ' Renal papilla -slightly reduced p. Skeletal 00 Sternebra 1ro 01 -partiall ossified 1 Rib -unossified 0 -partially ossified -- S. Ischium -partially ossified Centrum -partially ossified 3i -unossified 119/22 36.3^3.92 51/13 10/6 1/1 128/21 43.2^4.53 1/1 22/8 1/1 2/23 1/1^ 51/7 53.7^9.41* 40/6 46.7^6.87 2/2 6/4 2/1 2/1 1/1 1/1 HLR 441-84 TABLE IV (CONT.) C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETAL VARIATIONS'1 IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF GE Corn oil (5 mL/kg/day) Va riations Due to R.etarded Development (Cone.) Skeletal (Cont.) - Pubis -partially ossified 0 No. examined with heads '0 0) ;3 Skull bones i< w partially ossified -parietal & R' -interparietal 2 & -supraoccipltal -squamosal -frontal -zygoma P &0 Maxilla -partially ossified 5" , a Hyoid ^ -partially ossified -unossified 2/2 104/24 1/1 4/3 1/1 1/1 3/3 5/4 C-8 (100 mg/kg/day) 2/21194/22 2/2^ 5/5^ 5/5^ 2/2^ 1/1" ^K l/^ 2/2 4/3"- cfltfl (100 mg/kg/day) 30/7 f 3 (500 mg/kg/da 26/6 3/1 1/1 1/1 1/1 2/2 HLR 441-84 TABLE IV (CONT.) C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETAL VARIATIONS3 IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF GE Variations Due to Retarded Development i (Cont.) [0 a> Subtotal - Variations ' Due to Retarded Development 0 o ' 3 W 1. -No. Affected -Avg. % Affected Fetuses per Litter (+S.E.M.) N" Ci) TOTAL 13. 0 0 M No. Fetuses with til 3 Variations a 0 0 Avg. 7, Fetuses with 3, Variations per Litter S2. 5" (+S.E.M.) -W4 2 V 8 Corn oil (5 mL/kg/day) - (100 nig/kg/day) 70/14 ^ 20.6+4.61 35/12 10.9+3.08 154/23 46.5+5.18 145/21 48.5+4.35 --3 C (100 mg/kg/day) 4/7 '4.9+2.82 53/7 56.0+8.80 C ^ 3 (500 mg/kg/da 14/6 16.5+4.36 41/6 47.9+6.84 HLR 441-84 TABLE IV (CONT.) C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETAL VARIATIONS3 IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF GES 1-0 "' g 0 h i 'aT3 | P) J-, k (Is01 A rtA 1 R1 ra p. C3 0 ro ui 3 Q does not include those present in malformed fetuses as these fetuses were previously ta malformed fetuses/litters blanks represent zero incidence blood subsequently noted in the anterior chamber of the eye at histomorphologic examin this fetus weighed 2.89 g; hydroureter was not detected at visceral examination this fetus weighed 3.58 g; hydroureter was not detected at visceral examiantion one fetus (4.02-g) with left kidney affected had associated hydroureter; the other, a had both kidneys affected without associated hydroureter, but a slightly reduced papil the right side one fetus (3.47 g); both renal pelves slightly enlarged no fetuses with developmental variations of head were detected (1) and (2+) denote 1, or 2 or more sternebrae were misaligned, respectively one fetus (3.26 g) had each of these variations statistically significant difference by one-tailed Mann-Whitney U test (p^O.05) statistically significant difference detected by two-tailed Mann-Whitney U test (pj^O.0 0 5' -(rit 0 > 0 CO ^"^ r- c^^e^T^o^e^ m m > cc ES- 575 REV. 1-75 ______ EST&BUSHED 1802 E. I. DU FONT DE NEMOURS S COMPANY INCORPORATED WILMINGTON, DELAWARE 19898 CENTRAL RESEARCH & DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ArraiNuiA A July 23, 1981 MEMORANDUM. n------3 To: POLYME^TRODUCTS DEPARTMENT CUSTOMS HOUSE SQUARE 314 CHEMICALS AND PIGMENTS DEPARTMENT BRANDYWINE BUILDING 6210 ^ m FROM: R. E. STAPLES SUBJECT: C-8 AND PROSPECTIVE ALTERNATES: PROTOCOL FOR TERATOGENICITY TESTING C-8 and The protocol its alternates fisor atthtaechteedra^^p--g--en--ici--ty--t--es^ti^nI^gf of you have questions concerning this study^p^asej-et me know (366-5302). RES/mIe Attachment(1) Company,SaniUzod. Does not conlain TSCA CB1 - 28 - BETTER THINGS FOR BETTER LIVING . . . THROUGH CHEMISTRY I. E. DU FONT DE NEMOURS & CO. CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ELKTON ROAD NEWARK, DELAWARE PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTRR ADMTNTSTRATION BY GAVAGE WITH I. BACKGROUND study on (Haskell was made for I that ,A_request for a proposal to conduct a_ teratogenicity response to a TSCA 8(e) notice from the 3M Company Qn_imn--LLj--1^81, E. D. Champney orally requested ^BI|m^mmH^^^^^^^^o^e^esTed in ?o'^^secondTSCA8^^no^^eIrom the 3M Company for The oral LD50 and ALD estimates for the rat were by the Acute Section at Haskell Laboratory on June 12, ^_____The purpose of this study is to test the teratogenicity adm^ffl^^^^onby gavage to determ^e^mether we can repeat the preliminary findings reported by 3M, and to conduct a preliminary teratogenicity test on the other chemicals listed above for comparative purposes. II. MATERIALS AND METHODS This study consists of two experiments. Experiment I is a teratogenicity study by gavage with the dams sacrificed the day before expected delivery. The dams for Experiment II will be dosed as in the first experiment, but will be allowed to give birth and to raise their offspring to at least 22 days post partum. c.^san^.Do^o^"^"1 - 29 - PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTER ADMINISTRATION BY GAVAGE WITH' PAGE 2 A. Test Chemical The following chemicals were received from the Polymer Products Department and assigned Haskell Numbers: B. Animals Crl:CD(SD)BR female rats (nulliparous) 50-60 days old weighing between 170-180 g will be ordered from Charles River Breeding Laboratories, Inc., North Wilmington, Massachusetts. The rats will be quarantined for at least one week after arrival by air-conditioned truck. Upon arrival, each will be assigned a unique identification number that will be written on a cage card and by a combination of ear punches and toe clips. The females will be mated by overnight cohabition to mature males of the same strain and mating will be verified by detection of spermatozoa in the vaginal lavage each morning (Day 1 of gestation). The rat was selected for this study because 3M reported a positive teratogenic response to C-8 in this species and we intenj^o__confirm this findjjg. Also, the degree of toxicity of C-8, [BBHBHMHHBHHiB"83 determined in this species at - HaskeTl Laboratory. The'CrT:CD(SD)BR strain was chosen because extensive background teratogenicity data exists at Haskell Laboratory on this rat strain. Since the historical incidence of eye alterations including cataracts and lens opacities in CD rats is 3% (according to consulting opthalmologist James Clinton) all prospective parents for this study will be screened for these alterations before breeding. During the screening procedure, both eyes will be examined and all affected rats will be eliminated. - 30 - ^conta.nTSCAC^ Con^y Sanife^ ^si PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTER__ADMINISTRATION BY GAVAGE PAGE 3 C. Route of Administration Administration of the test chemicals will be by gavage since this was the route used in the 3M study. The test chemicals will be suspended in stripped corn oil1just before being administered each morning from Days 6 through 15 of gestation. The body weight most recently recorded will be used to calculate the dose to be given to each rat. Periodically, samples of each solution will be retained for possible analysis of concentration and uniformity of mixture. A vehicle control group in each experiment will receive 5 ml stripped corn oil/kg body weight for the same period of gestation. To minimize oxidation during the dosage period, the corn oil from opened cans will be stored at 4C in red bottles stoppered with ground glass tops. D. Dose Levels The recommended dosage levels are; Experiment I 5 ml/kg 100 mg/kg 100 mg/kg 500 mg/kg 250 mg/kg 500 mg/kg Stripped corn oil C-8 Experiment II 5 ml/kg Stripped corn oil 100 mg/kg C-8 The C-8 level is recommended because it is the maximum level that can be tolerated by the dams by gavage as determined by range-finding testing; the level selected for-each of the alternate chemicals was determined similarly. E. Animal Distribution Before exposure, mated females will be ranked by body weight and assigned to groups by rotation in order of rank. The dose group the first animal is assigned to will be selected randomly. If the distribution process results in statistically significant differences in body weight among groups before 1 Eastman Kodak, Rochester, NY, nolco-ai.iTSCA"3' Con-P^""^-^5 - 31 - PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTER ADMINISTRATION BY GAVAGE PAGE 4 exposure, then minimal switching within breeding dates will be used to alleviate the statistically significant differences. For Experiment I, about 25 mated females will be assigned to the vehicle control group and to the test group receiving C-8. Each of the remaining test groups in this experiment will be assigned about 7 mated females. About 12 mated females will be assigned to each test group for Experiment II. F. Husbandry Upon arrival at Haskell Laboratory, the female rats will be housed two/cage in suspended wire-mesh steel cages. Purina Certified Rodent Chow #5002, Checkers, and water from Wilmington Suburban Water Corporation (WSWC) will be provided ^d libitum. The potential effects of dietary contaminants were considered and, on the basis of the manufacturer's data contaminant levels are believed-to be within acceptable ranges. No other contaminants reasonably anticipated to be present in the feed are expected to interfere with the results of this study. The potential effects of water contaminants reported by WSWC were considered and appear to be within acceptable ranges. To supplement the WSWC data, Haskell Laboratory initiated an analytical program that monitors these and other contaminants reasonably anticipated to be present in its water supply. G. Records Maintenance Records for parameters in the protocol will be maintained for each animal. When the study is completed and the final report is issued, the raw data will be forwarded to the Information Section of Haskell Laboratory for archiving. H. Safety Precautions and Disposal of Waste Material All personnel will wear flock-lined latex gloves2 when handling test compounds or mixtures. Kevlar gloves will be worn when study animals are handled and latex examination gloves will be worn during autopsy. Contaminated waste material will be packaged in polyethylene-lined Fiberpaks for incineration at Stine Laboratory. 2 Golden Thumb Glove Company, Glove ^L-61,18 mils thick Company,Sanitized.Doss not contain TSCA CBI 32 PROTOCOL FOR TERATOGENICITY STUDY IN RATS Af.l-ER AnMTNTSTRATION BY GAVAGE ^ITti_l^^^^Bf----------------l |oRir! PAGE 5 I. Parameters to be Studied Experiment I - Teratogenicity Study 1. Dams a. Body weight - weighed on the day of arrival, before breeding, and on the morning of Days 1, 6, 9, 13, 16, and 21 of gestation b. Feed consumption - the average amount of feed consumed daily per rat for each group will be determined for the pre-exposure, exposure, and post-exposure time periods. The measurements will be taken at the same time each morning. c. Clinical signs - observed upon arrival, at breeding,and daily at least from Days 6 through 15 of gestation Viscera of dams will be examined immediately after sacrifice by cervical dislocation. Liver weight - absolute weights will be taken and,if indicated, liver weight will be presented relative to the corrected maternal body weight at the time of sacrifice f. Uterine weight - the intact and empty uterus of each dam having one or more fetuses will be weighed to permit calculation of actual maternal body weight gain during gestation g- Corpora lutea ovary counted and recorded for each h. Implantation sites - counted and recorded for each pregnant rat; the uterus of each apparently "non-pregnant" rat will be stained with ammonium sulfide to detect very early resorptions i. Resorptions - counted and recorded for each rat (not those detected by stain only) 2. Fetuses a-- Number, location, and condition recorded for each fetus in each litter Does ^ .1 contain TSCACB^ Compa"y Sanded. - 33 - PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTER ADMINISTRATION BY GAVAGE WITH 1l------------------BB--------------------f^ 101 PAGE 6 b. Fetal weight - recorded for all live fetuses and those classified as "Dead" fetuses c. External alterations detected and recorded for all live fetuses. d. Soft tissue alterations - detected "and recorded for the first live fetus and thereafter for every other live fetus of each litter; all stunted fetuses and all live fetuses with external malformations also will be examined for soft tissue alterations . The heads4of all fetuses examined for soft tissue alterations and sufficient of the remainder to total two-thirds of each litter will be fixed in Bouin's solution. Two of the Bouin's fixed heads of each litter will be sliced in vertical cross-section in front of the eyes, through the center of the eyes, and through the widest portion of the head; the remaining Bouins's fixed heads of each litter will be sectioned immediately in front of and behind the eyes, rather than through the eyes to permit processing for histologic evaluation if desired. It is expected that at least three of the Bouins's fixed heads with uncut eyes from each litter of the C-8 and Control groups will be processed and examined histologically by a pathologist. Particular emphasis will be placed on lens structure during examination. e. Skeletal alterations - detected and recorded for ail fetuses (including "Dead" fetuses); the fetal heads that were fixed in Bouin's solution will be excluded All groups will be coded from just before sacrifice until all-raw data are collected. 'Staples, R. E. , Teratology, 9:A37-A38 (1969). 4 Barrow, M. V., and W. J. Taylor, J. Morph., 127:291-306 (1969) . contain TSCA CBI wnsnv Sanl^zsd. Doas nsl 34 - PROTOCOL FOR TERATOGENICITY STUDY IN KATS .AFTER ADMINISTRATION BY GAVAGE WT~" ----------------------------- PAGE 7 Experiment II - 1. Dams Extended Teratogenicity Study a. Body Weight - weighed on the day of arrival, before breeding. Days 1, 6, and 21 of gestation and 1, 7, 14, and 22 days post partum b. Clinical signs - observed upon arrival, at breeding, daily from Days 6 through 15 of gestation, and on 1, 7, 14, and 22 days post partum. Adverse effects observed at any other time will be noted. c. Date of delivery - noted for each dam d. Reproductive indices For each test group: o Fertility index (% matings resulting in pregnancy) o Gestation index (% matings resulting in live births) For each litter: o Viability index (% animals born that survived 4 days or more) o Lactation index (% animals alive at 4 days that survived to 22 days post partum) 2. Offspring a. The number of live pups. per.litter and the number of dead or cannibalized pups per litter will be recorded on 1, 4, 7, 14, and 22 days post partum. b. Sex ratio - recorded for each litter on the date of delivery. The sex ratio of pups alive 22 days post partum also will be presented. The sex of each pup found dead will be recorded. c. Body weights - weighed 1, 4, 7, 14, and 22 days post partum -rcCACBl. - 35 - ;omp3,,^,. canine - PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTER ADMINISTRATION BY GAVAGE WITH {------^^fUH--------------^11 IORITSALTEI PAGE 8 d. Clinical signs - observed 1, 4, 1, 14 and 22 days post partum. Pups with adverse signs noted will be marked for subsequent identification within the litter. e. External alterations for all live pups detected and recorded f. Soft tissue and skeletal alterations - pups will not be examined for these types of alterations unless otherwise indicated g. Eye examinations - the eyes of pups in all groups will be examined by an ophthalmologist shortly after the eyes open. If eye alterations are detected that appear to be compound-related, a second examination may be conducted. The groups will be coded for all examinations. Pups with eye alterations will be marked for identification at sacrifice. At sacrifice, each pup will be exsanguinated and its eyes will be removed. All eyes will be fixed to permit processing by the Histology group for examination by a pathologist if indicated. The identity of eyes from pups previously marked will be retained. Otherwise, all eyes from pups will be identifiable only by dam number. At least the eyes of two pups from each litter will be processed and examined by a pathologist. III. STATISTICS Experiments and II The litter will be used as the experimental unit. The Fisher.'s exact test will be used to determine the significant differences in the incidence of pregnancy, and maternal pup mortality. Jonckheere's test will be used to determine the presence of a dpse response. Dunnett's test will be used for testing the significance of differences in maternal body weight and body weight gain. A two-way analysis of variance will be used to detect interaction between breeding lots and test groups. For all other parameters, the Mann-Whitney u test will be applied to detect significant differences between the control group and individual experimental groups. The level of significance will be p < 0.05. In addition, the reproductive indices given earlTer will be calculated. c^.----------------'"5" - 36 - " ' ' -- PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTRT? a nMTA7-rc'mi-> ?"->- '-'" ----- IV- CRITICAL DATES Starting Date (breeding): Completion: July 13, 1931 January 18, 1982 PAGE 9 37 - Compan-.yy Sanitized, Does nsl contain TSCA CB1 PROTOCOL FOR TERATOGENICITY STUDY IN RATS AF iR ADMINISTRATION BY GAVAGE JfITH PAGE 10 Prepared by: Teratology Section Toxicology and Pathology _R__. E__._S_t_a_p_ _le_ s__ _T_^^Sy t : af f . 'T_e_r_a_t_o_lo__g_is_t_ Study Director Teratology Section Toxicology and Pathology Approved by: Associate director Toxicology and pathology ^conialnTSCACBl ^P^^^-0085 38 - \ CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE M E M. 0 R A N D U M TO: QUALITY ASSURANCE COMMITTEE - C. M. BARBA FROM: R. E. STAPLES SUBJECT: PROTOCOL AMENDMENT FOR TERATOGENICITY STUDY: C-8 AND PROSPECTIVE ALTERNATES^ testing An amendment of C-8 and its to the protocol for the alternates is attached. teratogenicity RES/mIe Attachment(l) - 39 TSCACBI Company Sanded. Does ^contain I. E. DU FONT DE NEMOURS & CO., INC. CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ELKTON ROAD NEWARK, DELAWARE AMENDMENT TO THE PROTOCOL FOR TERATOGENICITY TESTING IN RATS AFTER ADMINISTRATION BY GAVAGE WITH^J )R ITS The following changes were made after initiation of the study: Page 8 II. MATERIALS AND METHODS I. Parameters to be Studied Experiment II - Extended Teratogenicity Study 2. Offspring g. Eye examinations II Because no compound-related effects were detected grossly or microscopically in the fetal eyes from Experiment I, the eyes from all offspring in Experiment were fixed, identified, and retained but none were processed or examined microscopi cally by a pathologist. J. Retention of Specimens till All skeletal, head, and selected visceral speci mens , as well as histologic preparations, will be retained issuance of the final report. There after, they will be retained for as long as the quality of the material affords proper evaluation. ^.^----'nTSCAC8> Compaq - 40 - AMENDMENT TO THE PROTOCOL FOR TERATO- WITHmUUBB^*\ GENICITY TESTING IN RATS AFTER ADMINIS TRATION BY GAVAGE PAGE 2 PREPARED BY: /7sZV^ / L_. r/. cTCcL^uUi)--^/^hh^^- C. L. Lamontia - Biologist Teratology Section Toxicology and Pathology /^^J^^_________ R. E. St'a^le'g'^Staff Terato legist Teratology Section Toxicology and Pathology APPROVED BY: J. aS^Aftosmis - Associate Director Toxicology and Pathology CLL/RES/mIe 12/3/81 not cor CpniP3^- San^-0052 41 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT cc: HASKE.LL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ATTACHMENT 1 itESYAPLES iAR 2 3 1381 March 20, 1981 MEMORANDUM TO : ^7 FROM: R. E. Staples J. R. Bames TERATOGENIC STUDY WITH Polymer Products Department would like Haskell Laboratory to pre pare a protocol and cost estimate for an oral teratology study with H^IH^H^B^HIHL The purpose of the study is to determine whether HHI^^BH^I^IP reduces effects similar to those reported by the 3M Company forH------RLn a TSCA 8(e) notice. Some details of the 3M Company protocol were obtained by PPD and are attached for your infor mation. The project .should be addressed to M|IHB|^^BP PPD, Customs House Square-314. JKB:mjh Attach. ,,------'" 42 - 2-5^ REV 12-79 ESTABLISHED 180Z . E. I. DU FONT DE NEMOURS 51 COMPANY INCORPORATED WiLMINGTON, DELAWARE 19898 . POLYMER PRODUCTS DEPARTMENT February 11, 1981 TO: FROM: E. D. CHAMPNEY D-11070 J. B. ARMITAGE CPIS-314. TSCA SECTION 8(e) REPORT FROM 3M Under the Freedom of Information Act, we obtained. [rom the EPA the attached TSCA 8^g) notice on .^^fijjjHj ^IIIIBIIIHB|JBIH|B|HBHlB|^submitted by 3M, which wa^ioted^u^t^December^^^Wsu letter to J. R. Barnes. Some details of the oral teratology protocol are given, which may already have been disclosed to McKusick and Kennedy by 3M .at their January 16, 1981 .meeting in Chicago, but of which I was unaware. JBA/skd Attachment DOS2 ^^^SCACa C. o.T^ftf^"S' a^.,-,."^.d - 43 There's a world of things we're doing something about .^N REC'-D OFFICE OF PESTICIDES ANO TOXIC SUBSTANCES Mr. J. B. Armitage E.I. duPont de Nemours & Company, Inc. Patents and Regulatory Affairs Division Wilmington, Delaware 19899 Re: Freedom of Information Request Act (A-101) RIN-247-81 Dear Mr. Annitage: In response to your letter dated January 23, 1981, enclosed is a nonconfidential copy of substantial risk notification OEHQ-1180- 0374S, submitted by 3M Company in compliance with Section 8(e) of the Toxic Substances Control Act. There is no charge for the enclosed material. In accordance with Agency regulation, we charge $.20 per page for duplication and $5.00 per hour for search time if a total charge of $10.00 or more is incurred. If you have any questions, please contact Ms. Janet L. T'7est of my staff at (202) 755-8050. Please use the reference number RIN-247-81 when inquiring about your request. Sincerely, - fM^J) 0- ^u IK. Edward J. Cult, Chief Information Control Branch Enclosure nolcon^nTSCACBl ^^^ ' 44 0 /^^ /v <. - ^'0 .-,"' J-' 5 ;C'-.d,^r.eMral <0-.M'flcl^3ast <3M Ce-'tified y.dil - P.et'^r" P.3';: =i-: i ? ::/. _.-; ^ ; 2M Canlef St. Paul. Minnesota 55101 612/73311^0 /- '- November 19, 1980 " ^ - ^ ^ ''--"-'<''! '^ru-B-.'in-Ji'M-j'-U' Document Control Officer Chemical Information Division Office of Toxic Substances (WH-557)- ' Envlroni-iental Protection Agency ^01 M Street, S. W. / Washington, D. C. 20^60 <yS^l//^-^J/^ ^ Gentlemen: r= 3 ^ ^ P -^ C; ^ = . . ^ ^ - ^ O / ^ Subject: Section 0(o) Toxic Subutances Control Acl^ 5 Potassium Salt of Perfluoroalk.yl Sulfonat'es ..'" \i .: 0 : 5 Please find attached the following information relating-: to the subject chemicals: (1) Protocols for Oral Teratology Study in Rats. (2) Preliminary report, 3M memo, dated November 12, 1980. (3) 3M Technical Report entitled "Analysis of Selected Decatur Employee Serum for Sulfonic and Carboxylic Fluorochcmicala". Preliminary information from the ongoing teratology study cited in (2) above indicates that the subject chemicals are teratogenic in rat^s. This information and che findings desc"ri5ed~"i'h~0')~above, indicating the presencfc' of fluorocneraicalsj.n the_blood of some of our_Dlanfc emnloye^es, leads us to submit this. information pursuant to'Section 8(e) of the Toxic Substances Control Act and EPA's statement of interpretation published in the FEDERAL REGISTER, March 16, 1978. As noted on page 584 of an article published in the October I960 American Industrial Hygiene Journal, entitled "Health Scatus of Plant Workers Exposed to Fluorochemicals-A Preliminary Report", 'this publication and the attached information reflect, in part, 3H's testing and monitoring program designed to "...evaluate the overall impact of exposure of fluorochemicals on the health of workers". Al^hcu^h unis preliminary information cited in (2) i.-r-Jica^es t'.-s.': the subject chemicals are m^st_p_robaol^__^:'j:_i^l ^eratcgens, our employee records and the epidemiolop:^'. dasa "described in the aforementioned publicASfiiSi"^^^^'5 zr.3.1 to date no human health prO(b-.,e^sa.Rr?e been observed nor disease "patterns dete'cean^^Kch are attributab^~0?--q .-s^rs s'^ r^ .'-'-.-. - 45 - ... "p-'fti-.^-'"' i"''"*'- Environmental Protection Agency Page Two November 19, 1980 related to fluorochemical exposure. This publication also described the industrial hygiene measures undertaken in further reducing employee exposure. "Potassium salts of pcrflu-roalkyi uulfonates" la a (sunur'lc chemlca'l~"n'ame"Tor"'a.mISTure~o'f-'ho^iologs which can be expressed by the general formula C Pp -SO^K. These hornolo^ were reported on the TSCA inventory This homologous mixture, or the corresponding ammonium salts, is current'ly'solcT as various products containing from 100_percent ^solids" to 0.58 percent of the mixture. These" produc'fcsr and"~"heTr~~ customer""uses' "are de's crib^ed as follows: PLUORAD'^ Brand Etching Bath Additive PC-93 (correponding ammonium salt of the subject chemical) Electronic Manufacturing PLUGRAD1^Brand Fluorochemical Surfactanfc, PC-95 Chrome PluLiri[; PLUORAD1^Brand Fluorochemical Surfactanfc, PC-99 (corresponding amine salt of subject chemical) LIGHT WATER1^Brand Aqueous Film Forming Foam PC-203 Chrome Platinp; Fi'i.''e Suppression LIGHT WATER11Brand Aqueous Film Forming Foam FC-203A Fire Suppression LIGHT WATER1^Brand Aqueous Film Forming Foam, FC-206A Fire Suppruuaiori LIGHT WATER1^Brand Aqueous Film Forming Foam Alcohol Type Concentrate, FC-600 Fire Suppr-ess-ion APPF 6% Concentrate, FC-780B Fire Suppression Approximately of perfluoroalkyi sulfonafces are pro duced per year domestically at our Decafcur production facillt; located at P.O. Box 2?o6, Decafcur, Alabama 3560?, with '18 employees potentially exposed on an intermittent basis. Chemical reaction occurs in a closed system. Approximately of the are processed at our Chemolifce production site located at Highway 6l & Washington County Rd. 19, St. Paul, Minnesota 55133, with 37 employees potenfcAg^W exposed on an intermittent basCiso.mP^53"1112. ^n.oss^00 -46- Environmental Protection Agency Page Three November 19, 1980 We plan so infor:";, by mid-December, all those customers and 3M employees who have a potential, through.certain uses and/or processing, of. significant exposure to the subject chemicals. At that time we will summarize these findings and outline our recommendations for handling and using thes products. We are by copy of this letter advising NIOSH of these new but preliminary terafcogenic findings. When significant new information becomes available to us, we p"-". to advise those cusLo;ners anc s;jiiploye-;;a accordingly. i In line with our ongoing testing and monitoring program in fluorochemlcals, this is to advise the Agency of the following work planned for initiation in the near future: (1) A second teratogenic study designed to further evaluate these findings in both rats and rabbits. (2) Developing industrial hygiene procedures designed to further reduce the exposure to plant employees. Since certain of the information provided herein is con sidered confidential business information, we are providing a sanitized version of this report for the public file. In addition, we have deleted.from the confidential submission inconsequencial information such as the names of 3?'i employees for the purpose of protecting their privacy. The teratology study referred to herein is based on feeding PC-95, the mixture of potassium salts of perfluoroalkyi sulfonates described as above. When the study is complete, a final written report will be provided to EPA. In the interim, should additional correspondence. be necessary on this matter, please contact: Larry Magi 11 Manager, Regulatory Affairs Department Commercial Chemicals Division 3M 3M Center, 223-63-0^ Saint Paul, MN 551^ Telephone: 612/733-7062 ^ Yours truly, /^ f)/'1^' -. ' _ L. D. DeSimone Group Vice President cea Attachments - 47 - c: Anthony. Ro'cbir.s, :.1 Director, NZGSH Par't Lawn Building 5600 Fishers Lane Rockville,MD 2085! Company Sanitised. Dos3 E'.OS contain 7SCACBI ->.?4 lH- |'(> ll ! . . . 11 i November 12, 1980 cc: TO; . . FKOM: . SUBJECT: Teratogenicity of Two Fluorochemical Compounds in Rats (Study numbers 680TR0008 and 680TR0010) I'roll mi nary results from rat teratology studies on PC-95 and indicate* a teratogenic effect on the embryonal eye. The developmental eye abnormality . was seen at all dose levels except the control groups. The range of morphologic .ipiK.'artU-tces observed under the dissecting microscope varied from a slight- dis coloration on the lens near the anterior margin to a dark colored oval area, often containing a cleft, extending from beneath the lens epithelium to halfway through the lens posteriorly. Histologically, the discolorations were primarily due to the presence of lens vesicle remnants or abnormal primary lens fibers in the embryonal nucleus of the lens. ,^3notconta,nTSCACm 48 - company san;&y: TiTLfc;; Protocol Number for Oral Teratology Study of PC-9S in stats 0680TR0008). (Zjtperitwnt OBJECTIVE: A teratology study will teratogenic effct of to U9d too ovaluaf fcha eabryotoxic and durina th priod of orally a<&Binia6rdfC-95 organogaeaiia. 'S> to preqnant rats the general r9oa----4tioiui <"GuidliiMr of && ?BA ozocochara ccapliea with iaMuod ia January, 1966 for ayycdttctlc S&sdlaa for Safaty Bvaluation of Drugs Gfooord HluaabaoaraUt--or"y). PrTahceticsetude-yagwuiialflcittsoaa conducted according to the 1978 Standard ^wraelag ?roc^ur. am) Safety evaluation 3-aboratory' s SPONSOR: 3M CcaaaTcrial Cheaicjii Divljioo, 86. Paul, TESTING FACILITY( Safoty Evaluatloo Laboratory St. Paul, Minnsota. Minoaaota. STUDY DJHSCTO . STAST OF DOSINGt Wd July, 1980. TL'ST SYSTl^tt Eighty-eight saxual.ly aatusra, feauilft rat9 frca Charlos tia fsatod Spraqua-Sawlay derived in hanging atainlaaa Mvar Ss-^wSing Laboratory atael cagea with wir cseah will be housed in a will teaperatura ba uaed and huaidity controllad roca. floors and f rones l^ia strain of rats females are bcaua of historical control readily available. Purina data and tiee giaced will be available ad llbitua. Laboratory Chew and water The lights will be on a 12 light/dark cycle. hour TEST SYSTEM IDZWI7ICATION? Each aniaial will be ear tagged and indicated on the outaide of the cage. RANDOMIZATION: ranT<h3ooannuiaMublesr wtaibllleb. e assigned cages according to that nufBber will b<* a compute r-qeneftitfd CONTROL AaTICLBt Corn Oil. Tfc:ST ARTICLE i PC-95. ANALYTICAL SPECIFICATIONS: detsrained by The teat article, composition and purity will h>- the atari of ththeesStupdoynsaonrd f3aNt Cthoesaeenrdcioafl dCohaeianigc.al group) prior ro DOSAGE LKVELS AND EXPERIMENT DESIGN: oil daily. The The teat article will be suspended in --orr> administered by test oral article suspension intubation to the and control article will be gestation according to che following: rats on days 6 throuqh 15 of ny -- Sanitised. Does not contain TSCA CB1 (Company Pose Group Dose Level High Mid LOW Control JO rogAg/day 5 ngAg/day I agAg/day 0 agAq/day 22 ? 22 ? 22 ? 22 ? Tshtuedoieraal shroowueted orafdaiodl&aabianliaadtra?tCii-i9a5 wwialal wbalul saedbaboertcwauds. e Noof daeietatabroylisra contaainant3 ara kncwi to intsrfar'a with (AA Seat article. The aniaala will i>a ci^aerved daily ^rca gestation for abnoraal c2iaicl ai^na. racordad on day 3, 5, 9, 15, 15 and 20 doed accordingly winy a ccnatwt dos weight. day 3 throigh day 20 i2ody waighta vill be of pregnancy and the voliaae of 5 sal/teg of of racs body DATA The its faale will b Slillad cai contants will b axaaind day to 20 and tha ovaries, utarus and nu-r.ber of fatusaa illi/9 dotarsiina: and dead), nuaber of nussber of corpora raaorpticn aitas, luCea, nuRber of inplantaticn aitca, pup weight Biately one-third of the pupa uill and grooa abnormalitiaa. b< fiaad in Approxi- subsequent free-hand sectioning by tha ftilacn Souin'3 solution for technique to determine any visceral abnoraalities uainq a diaaactinq aicroacope. The remaining approxiaataly two-thirds of the pupa alcohol for subsequent skeletal o^ajninafcion will be fixed in ethyl staining with alisarin red. aftar clearinq and ANALYSIS AND FINAL REPORTi The proposed statistical laathods to be use/i fo analysis of the data are; nuaiber of fetuses, nuober Dunnett'a t tast of resorption for daa and pup w*iqht3, sitas The and number of corpora lutea; Chi sites, naber of iiapl<int-atio square Cor percent abnormalities, p1u9p80ep)x.roapmoisneadtiodnastehafover btheeen fcinoamlpleretepdort(apispr2o-a3icmato?nltyhs foauftrethr qdueutaritleerd, ^/3^/^ Date I .^^/^ Drttr 0 ^Date y - 50 --^'^a.r?/^ Company Sanaaed. Does not contain TSCA CB1 3SYJ TliCIHilNiiCJ^L HISPdRT Report Number: Data; October 4, 1979 TITl-Si Analysis of Selected Decatur ESnployee Serun for Sulfohic and Carboxylic Fluorochenicalg AUTHORi ABSTRACT: The.precencs of sulfonic acids was detected in certain blood saiaples using botn electron capture and aicrowave plasiaa detector methods CBL ^,-.-"--"11"' ,, Roport24o. ----------_.------- Date- ---- - . -^"^I-ifc -1979-- - -- Subject: Characterization of Decatur RF Values requestor:--... _^--------- ?,squeat No. --..-..^....---- Report: ' . Dept. Name ..Co^^S^l.J^^^lsProj. No.,.. Dated --c2b^r,^^9^..... . The serua of 10 saiecssd 5H Dacatur eaployeea was submitted (A72754) la June, 197 for analysis of organic fluorida levels and the total organic fluoride values wer reported in Analytical Report 7194. Subsequent to that work, it becane of iatare co further characterise cha fluoride serua value and acceapt; co idencixy che pras of 3M fluorochenicala. Initial analysis (7/18/79) by the microwave plasma detector (MPD) established the presence of the expected perfluoro sulfonata and perfluorooctanoate anions in the serum extract of --25 and . -22. However, the observation of perfluoro- sulfonace anion in che above extracts was unexpected and it therafore bacara< necessary co spend additional analytical development cine ia order to measure Che; 3 fluorocheaicais in the saae serum extract saiapla. During this tine, the method was evaluated on the electron capture (SC) gas chrosatograph. Ths EC detector would be expected to have greater sensitivity than the MPD for these fluorochsmicc but with leas specificity since the MPD can be operated on the fluorine channel eliminating any confusion by other EC active compounds.. We selected the 5 highea.t fluorine containing Decacur samples and analyzed the serum extract for parfluoro sulfonace, perfluoro sulfonata, and perfiuor occanoace anion. We used both the MPD and EC detection systems and report chu following values: X FOUND^ Person -22 -23 -24 -25^ -26 RF (ppm) 10.1 5.7 9.4 11.8 4.1 Perfluorosulfonate 52 15X 5S 5Z . 10X Perfluoro- sulfonate 602 702 - 802 . 5^ 652 Perfluorooctanoate Tocal 30X 202; "152; 202 25Z 95:2 105^ "' "1QQ7. 302 100^ ^-The analysis waa conducted availability of sanpla and of she EC and ^0?D values. AR 6962, 7028. 7194 on a single determination (1 ml of serum) due tiae and represents our interpretation of the No internal standard was used; expect 10-20^ to liniiL, average accuracy '"'Previously identified as -21(2) due to mis-labeling by medical personnel. Con.panySani^Dce3"otconlainTSCAG - 52 - . .;o. 7230 October 4, 1979 P^sa 2 The above results ara necessarily soaewhac approxiaaca due co Cha lack of an iacer acandard and continual refiaeia&ncs in the derivacisacioa and GC of th& correspond! meshyl estar. Sufficianc saaple remains for .additicaal analysis. ?ur;her improve in she analysis are and will continue Co be aade ad tisa paraita. Wa thoughc It best Co reporc chasa valuaa now, especially aiaca wa idaocifiad thft pr&aeace of perfluoro sulfonata in husftan sarun extract acd no toxicology scudiaa to daca have been coaplQCed on Chia fluorocheical. Three yaara 030 (10-1S-76) wa reporsad thac Dscatur saploya . aerua eatracc concained parfluoro sulfonata (baaed on ?/^H5L) and a aiaor fluorina containin componeoc now idenciflad as perfluorooctanoacs. Therefore, che presence of per- riuoro sulfonats ia this iadividuai has baen known for scna cine. Now, vich iaproved analytical sethoda, we hava sraatar s^nsicivicy and can datamine che presence of perfluoro sulfonaCa in addition to parfluoro sulfonace anion nta.nTSCACBl Oc^0100" 33- co^^53,,^^ c^- 'e^ /}^%^<; AMERICAN course C-^ OF VETERINARY OPHTHALMOLOOISTS ATTACHMENT 2 JAMES M. CLINTON. V. M. D. ANIMAL EYE CLINIC AT SOUTH JERSEY ANIMAL HOSPITAL ROUTE 541 ABOVE CHURCH ROAD - P. 0. BOX 115 MEDFORD. NEW JERSEY 0805S TELEPHONE (609) 654.0304 R. . STAF-LbS ,IUL 2-1981 RECEIVES Haskell Laboratories Study C-8 and Alternates Exam Date: 7 July 1981 Robert Stapels, Ph.D. Initial Ophthalmoscopic Examination Both eyes of all of the male and female rats in the colony were ex amined by focal illumination, indirect ophthalmoscopy and, when indicated, slit-lamp microscopy. Mydriasis was achieved with 1% Atropine (1% Atropisol, Cooper Laboratories, lot B3039, 12/82), and the eyes examined in subdued light. Semi-darkness was maintained until the following morning. Ocular lesions v-ere identified in female rats 306514, 306518, 306440 snd 306448 and in male rats 305275, 305289, 305184 and 304846. They were removed from the colony and euthanatized by technologists S. Vivian and E. T o11enburg. ^ < ^ , ^ ^ _ The remaining male and female rats are ophfchalmoscopically normal and suitable for use in the forthcoming study. 7;,, James M. Clinton, V.M.D. , Does not contain TSCA CBi 54 Company San^eo - L..&K iKALi CJLSCARL<n AINU Ui vi^ijurru^u x i-'riin-Lvrujnj HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ATTACHMENT 3. p.. E. STAPLES A'uu 2 n981 August 19, 1981 TO : ^ ,FROM: R. E. Staples W. D. Kerns .{ .' H-14045 MEMORANDUM PULMONARY PATHOLOGY The pulmonary parenchyma was evaluated microscopically from several female rats (Table I) in order to determine if the death of the animals was technique related. There was no microscopic evidence of iatrogenic pulmonary injury in any of the rats that were examined. TABLE I Animal No. 306409 306498 306477 306469 306508 306529 306346 306330 306363 306401 306551 306519 306338 306354 306486 WDK:mjh .c^50^61 -GRO1" sari^-00' 55 - ^?a^-sa!l- H.UWT*t-f HASKELL LABORATORY MEMORANDUM ATTACHMENT 4 R. E. STAPLES JAN 29 "i982 SSCEiVSS^ DATE: January 29, 1982 TO: R. E. Staples V l | m n FROM: JJ^^Cerns SUBJECT: ^ ^ /,,4"l ^ Three fetuses were examined microscopically (12-12, 13-6, 30-6) for a histoaorphological correlate to a red area in either the right or left eye. All three fetuses had focal areas of hyphema. This lesion was consistantly located adjacent to the drainage angle in all fetuses. WDK:smk - 56 - Company Sanitized. Doss not contain TSCACBI ES-3606 <MMP HASKELL LABORATORY ATTACHMENT 5 cc; October 5, 1981 MEMORANDUM TO; FROM; R. E. Staples W. D. Kerns RE. : Fetal lenses from this gavage study (Table 1) were evaluated for the presence of microscopic lesions. The evaluations were completed without knowledge of group assignments or dose. There were no pathological lesions in any of these fetal eyes. A peculiar postmortem artifact was recognized and it was equally distributed among the high-dose and control groups. This alteration was present in the central anucleate portion of the fetal lens. In this area, the lens fibers were more eosinophilic and there was separation from the anteriorlens capsule in some cases. In others, the lens material in this area had been fractured by the microtome. Six additional fetuses |fl|||^H7ere evaluated for the presence of microscopic lesions. Five orthese^mimals had macroscopic observations of brown discoloration in the central lens (Table 2). Microscopically, (Table 3) alterations in these animals were similar in their morphological characteristics to the postmortem alterations that occurred in the gavage (C-8) study. The lens alterations in these animals additionally contained brownish-green birefringent particulates similar to acid hematin. This pigmentation may correlate with the macroscopic observation of brown discoloration. I suspect that this alteration is in some manner an effect of acidic Bouin's fixation and is similar to a brownish pigment (acid hematin) produced in tissue fixed in nonbuffered formalin. All microscopic lens alterations seen thus far .are interpreted as postmortem artifact and cannot be associated with test agent administration. WI>K:ljm - 57 - Company Sanitized. Does nol contain TSCACBl TABLE 1 Dam Code 2 4 5 6 7 8 9 10 14 15 ^y 18 . 19 .21 22 23 24 25 26 29 30 32 33 Ft3tU;3 . N1jmbesr 1, 5, 9 3, 5, 9 3, 4, 7 1, 8, 13 1,. 5, 11 1, 5, 11 3, 7, 13 4, 7, 12 5, 8, 11 3, 5, 15 5,. 9, 15 4, 9, 15 1, 8, 13. 3, 5, 7 1, 4, 11 1, 5, 9 4, 7, 11 1, 7, 11 5, 9/ 13 4, 9, 15 1, 11. 15 5, 7, 9 3, 8, 13 Dam Code 34 37 38 39 . 40 41 42 43 45 46 49 50 51 53 55 57 59 60 61 62 64 65 66 Fietus N'mnbesr 3, 7, 12 3, 1, 9 1, 8, 11 .1, 8, 11 5, 11, 15 3, 8, 17 1, 7, 12 5, 11, 15 4, 13, 15 3 1, 9, 12 4, 9, 11 1, 1, 13 5, 9, 11 1, 5, 12 1, 9, 13 3, 9, 13 5, 9, 10 4, 9, 15 3, 11, 13 3/ 7, 13 I/ 7, 11 4, 7, 11 58 - Company Sanitized. Doss not contain TSCACB1 TABLE 2 Dam Code 52 35 55 266842 253340 "6" Fetus Number 11 12 11 2 1 "6" Observations During Head Examination "Pale bro-wn area in lens; right lens ~ 1 mm, left lens ~ 0.5 mm" "Light brown area in center of lens of right eye ~ 0.5 mm" No alterations detected Slightly dark area in center of left lens. Currently, this is the slightest degree that will be noted as an effect. Dark area in center of both lenses Very small dark area in the right lens between "y" suture and epithelium - 59 - no.snotcontainTSCACBl Sompanx ,Sam...te^s^ G02-" "alw TABLE 3 MICROSCOPIC OBSERVATIONS D52F11 D35F12 D55F11 D266842F2 D253340F1 D"6"F"6" Unilateral alteration (artifact) with brownish-green refractile particulates Bilateral alteration (artifact) with brownish-green refractile particulates No significant lesions observed (NSLO) NSLO Unilateral alteration (artifact) with brownish-green refraetile particulates Unilateral alteration (artifact) with brownish-green refractile particulates ConipanySanifaed. Does nol contain TSCA CEi 60 - /L"--."^^^^^?^^.c?&^^ CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE MEMORANDUM TO: FROM: N. C. CHROMEY C. L. LAMONTIA ^ SUBJECT: HISTOLOGIC PREPARATION AND E C"8 TERATOGENICITY -STUDIES JJATION OF FETAL EYES: Please have these two groups of fetal specimens (attachment) embedded, sectioned, and examined. Embed one- half with the cut surface of the lens up. Since evaluation of the lenses of these fetuses is particularly important, each specimen should be sectioned so as to obtain cross-sections which include the largest diameter of each lens. Stain selected sections from both groups with hematoxylin and eosin and send . them to W. D. Kerns for evaluation. Please contact W. D. Kerns when the specimens are sectioned. Also, the slides should remain separated by groups. Please send any information concerning these specimens directly to R. E. Staples /(Study Director) or me. CLL/mIe Attachments (.2) Company Sa^Iized. Does not contain TSCA CK. 61 SPECIMENS TO BE PREPARED FOR EVALUATION BY W. D. KERNS The following fetuses had no eye alterations detected during external examination: Dam Code 68 54 58 58 Fetus Number 7 9 11 Observations during Head Examination Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration CLL/as 9/30/81 Sani^Do-nolconlamTSCACB? 62 - Company SPECIMENS TO BE PREPARED FOR EVALUATION BY W. D. KERNS The following fetuses had no eye alterations detected during external examination: Dam Code Fetus Number Observations during Head Examination 35 Faint brown area in anterior half of lens of left eye 'v0.5x0.4mm. 35 No alterations detected 35 Faint brown area in anterior half of lens of left eye '\0. 6x0. 4mm 35 Light brown area in anterior half of lens of left eye ^0.4x0.3mm; Very faint brown area in anterior half of lens of right eye 'v-0. 4x0. 2mm. 35 9 Light brown area in anterior half of lens of right eye ^0.6x0.2mm 35 11 Very light brown area in anterior half of lens of both eyes; Left ^0.4x0.4mm, right ^0.4x0.4mm. 52 Light brown area in anterior half of lens of both eyes; Left ^0.8x0.5mm, right ^0 . 6x0 . 4mm 52 Light brown area in anterior half of lens of both eyes; Left ^0.5x0.3mm, right M). 5x0. 3mm 52 Light brown area in anterior half of lens of both eyes; Left. ^0.4x0.4mm, right ^0. 5x0. 3mm 52 Very faint brown area in anterior half of lens of both eyes; Left ^0.4x 0.4mm, right ^0.5x0.4mro intain' ^--ed.Dcssnotco' 63 Company. ,S^"-00 SPECIMENS TO BE PREPARED FOR EVALUATION BY W. D. KERNS (cont'd) Dam Code 52 52 52 35 52 52 Fetus Number 9 13 15 5 4 5 Observations during Head Examination Light brown area in anterior half of lens of both eyes; Left ^0.6x0.4mm, right 'v.0 . 6x0 . 4mm No alterations detected Light brown area in anterior half of lens of both eyes; Left '\/0. 3x0. 3mm, right ^0.5x0.4mm Light brown area in center of lens of right eye ^0.5mm No alterations detected Light brown area in anterior half of lens of both eyes; Left ^0.6x0.5mm, right ^0.5x0.5mm Note - Alterations listed by severity: Very faint brown<faint brown<very light brown<light brown CLL/as 9/30/81 64 - cowpanxS2"1^, -0p0.,,-not contain - TSCACI^. """ R. E. STAPLES ^E'P 30 W C-8 AND ALTERNATES: CODE FOR ROUTINE TERATOGENICITY TEST IN RATS 3Y GAVAGE Corn Oil C -8 f./^iA^^^-f^ ^ ^r^. ^^ (5 ml/kg) (100 mgYkg) Dam Animal Code Number Dam Animal Code Number 2 306376 5 306385 6 306541 7 306571 9 306523 10 306443 17 306501 19 306475 22 306482 24 306471 26 306368 28 306458 32 306447 34 306397 38 306352 40 306556 42 306438 45 306391 49 306433 50 306370 55 306384 59 306563 60 306382 64 306406 66 306538 4 306365 8 306426 14 306465 15 306558 18 306494 21 306341 23 306539 25 306464 29 306517 30 306472 33 306414 37 306547 39 306557 41 306399 43 306416 46 306425 51 306532 53 306374 57 306481 61 306540 62 306375 65 306524 .,, 306409 -- 306498 306477 CLL/mIe 8/17/81 Females without dam codes died or were sacrificed before Day 21 Division by Breeding Lots Dam Code Lot A Lot B Lot C Lot D 1 through 5 6 through 24 25 through 44 45 through 66 65 - Qowa^.y.S^^ ^'w^wcw C-8 AND ALTERNATES: CODE FOR ROUTINE TERATOGENICITY TEST IN RATS BY GAVAGE I:-9 (100 mg/kg) Dam Animal Code Number 1 306340 11 306420 20 306439 31 306516 36 306367 48 306412 56 306421 I . (500 mg/kg) Dam Animal Code Number 3 306355 12 306484 13 306503 27 306342 44 306390 47 306334 58 306353 T250 Dam Code mg/kg) Animal Number 54 . 306373 --a 306354 -- 306486 -- 306469 -- 306346 -- 306551 306338 ^ (500 mg/k^j Dam Code Animal Number 16 306470 35 306473 52 306336 63 306396 -- 306363 -- 306519 306508 Females that were not assigned dam codes died or were sacrificed before the usual sacrifice period. Division by Breeding Lots Dam Code Lot A Lot B Lot C Lot D 1 through 5 6 through 24 25 through 44 45 through 66 CLL/mIe 8/13/81 66 Company SanW. DO.. no> contain TSCACK ATTACHMENT 6 HASKELL LABORATORY December 18, 1981 MEMORANDUM TO: FROM: SUBJECT: R. E. Staples W. D. Kems All animals listed in the attached tables were evaluated microscopically for the presence of histomorphologic lesions in the fetal lens. There were no microscopic lesions detected in any of the sections that were evaluated. Frequently, lenses contained arti facts that were attributed to trimming and processing. WDK:wfd - 67 - Company S^^seS. Dcss not contain TSCA CB! SPECIMENS TO BE PREPARED FOR EVALUATION BY W. D. KERNS The following fetuses had no eye alterations detected during external examination: Dam Code 68 54 58 58 Fetus Number 11 Observations during Head Examination Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration CLL/as 9/30/81 - 68 - not contain TSCACB1 CompanyS.^.Docs SPECIMENS TO BE PREPARED FOR EVALUATION BY W. D. KERNS The following fetuses had no eye alterations detected during external examination: Dam Code Fetus Number Observations during Head Examination 35 1 35 3 Faint brown area in anterior half of lens of left eye '\-0. 5x0. 4mm. No alterations detected 35 4 35 7 Faint brown area in anterior half of lens of left eye ^.6x0.4mm Light brown area in anterior half of lens of left eye ^0.4x0.3mm; Very faint brown area in anterior half of lens of right eye ^0.4x0.2mm. 35 9 Light brown area in anterior half of lens of right eye ^0.6x0.2mm 35 11 Very light brown area in anterior half of lens of both eyes; Left ^0.4x0.4xan, right ^0.4x0.4mm. 52 1 Light brown area in anterior half of lens of both eyes; Left ^0.8x0.5mm, right ^0.6x0.4mm 52 3 Light brown area in anterior half of lens of both eyes; Left ^0. 5x0. 3mm, right ^0.5x0.3mm 52 7 Light brown area in anterior half of lens of both eyes; Left. ~0.4x0._4mm, right ^0.5x0.3mm 52 8 Very faint brown area in anterior half of' lens of both eyes; Left ^0.4x 0.4mm, right ^0.5x0.4mm -...^ Doc-snotconlainTSCACB. Company San--- " - 69 - SPECIMENS TO BE PREPARED FOR EVALUATION BY W. D. KERNS (cont'd) Dam Code 52 52 52 35 52 52 Fetus Number 9 13 15 5 4 5 Observations during Head Examination Light brown area in anterior half of lens of both eyes; Left ^0.6x0.4mm, right ^0.6x0.4mm No alterations detected Light brown area in anterior half of lens of both eyes; Left ^0.3x0.3mm, right ^0. 5x0 . 4mm Light brown area in center of lens of right eye 'Y'0.5mm No alterations detected Light brown area in anterior half of lens of both eyes; Left ^0.6x0.5mm, right ^-0 . 5x0. 5mm Note - Alterations listed by severity: Very faint brown<faint brown<very light brown<light brown CLL/as 9/30/81 - 70 - . tSCi^1 noiool^"1 ^^ossno CoW?^51" ES-3253 REV. 6-62 IT-WRITE IT DON'T SAY TERATOLOGY SECTION FILE TO. MEMO AT. PROM. R. E. STAPLES ATTACHMENT 7 DATE 10/30/81 SUBJECT: C-8 ALTERNATES - TELEPHONE CALL FROM D. P. CORDS, 10/30/81_____________ I received a telephone call from Phil Cords (C&P) this morning and responded to several questions asked concerning the report on C-8 given to representatives of several departments yesterday morning. ^Ull He also asked about the findings on the alternates and in particular, UBB^BBMBP I relayed that the dose levels selected for^lotntne^^HHHHBBHIIIIBIIIBBlk were from too the f.hlfiegHh Iand a result'7 no~fetuses were obta-med^ ,nd only three litters were obtained ~ from the even though exposure was stopped before the completion intended treatment period. I relayed also that the lenses of the eyes of most of the fetuses of two of the three litters had apparent discoloration of the lenses as seen under the dissecting scope. Subsequent processing of the fetuses of these two litters for his- tologic examination revealed only changes seen in the control groups. Phil Cords asked that he be notified of any meetings scheduled to consider further teratogenicity studies con ducted on the alternates and that a notation be included in the file on this point. This memorandum is intended to comply with his request. RES/mIe Company SanEfcsd. Doss not contain TSCA CBI - 71 - THE JOB YOU SAVE MAY BE YOUR OWN--P.I.P. HLR 441-84 ATTACHMENT 8 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) TABULATION OF RESULTS OF DOSE SELECTION BASED UPON COMPARISON OF LD50 OR ALD VALUES Data available at the time this study was started indicated that the approximate LD50 and ALD values in the male rat were as follows: LD50 ALD W 0 4 C-8 435 691 2250 3000 7500 300 550 1500 1500 4000 Therefore, on the basis of demonstrated toxicity, the relative ratios were about as follows: Toxicity Ratios Initial dose levels (2 rats/group) tested 1150* 1150* 7550* 5750* 15 2250** 2nd Pre-test (2 rats/group) 100 100 500 500** 1000** Used for the main study 200 100 500 250** (7/23/81) 500** 1st Additional test (4 rats/group). Dosed for 5 days 2nd Additional test (4 rats/group). Dosed for 10 days Estimated maximum tolerated dose levels for 10 days of dosing 125* 50 35 250* 1501 100 ** severe toxicity including deaths * decreased body weight gain and adverse clinical signs body weight gain still too low but no adverse clinical signs - 72 - TSCACB1 Company ,Sa.'.".^^-"D"OSS not contain