Document NEG2wL46MDKy6znNmZzwg7jXR

AR226-2788 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HASKELL LABORATORY REPORT NUMBER 441-84 Copies Company Sanitized. Does not contain TSCA CBI FOR DU PONT USE ONLY E. I. DU PONT DE NEMOURS & CO., INC. HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT NEWARK, DELAWARE 19714 C-8 ALTERNATES1: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) Dates: Initiation (breeding date) - July 13, 1981 Completion (sacrifice date) - August 19, 1981 Noteboo Date Written: September 26, 1984 Date Issued: October 2, 1984 No. pages in this report: 72 Compaq Sanitized- Does not contain TSCA < 0 8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINA8X STUDY, BY GAVAGE) HLR 441-84 Staff Teratologist Haskell Laboratory RES:jy:MR4130.2 -2- not contai" TSCACB1 Company S a n i m i - Dcoo C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY. BY GAVAGE) X. i HASKELL LABORATORY REPORT NUMBER 441-1U TABLE OF CONTENTS Title Page. . . . Signature Page . . Table of Contents . I. SUMMARY II. INTRODUCTION A. Background . . . B. Protocol. . . . H I . MATERIALS AND METHODS. A. Test Chemicals............................... B. Animals and H u s b a n d r y ............ ... . . C. Dose Selection............................... D. Animal Breeding and Distribution. . . , . E. Test Chemical Administration ............... F. Maternal and Fetal Examination and Sacrifice. G. Statistical Tests..................... IV. RESULTS A. Eye Examination of Prospective Parents............ ... , B. Maternal Clinical Signs Observed......................... C. Maternal Feed Consumption............... ... D. Maternal Body Weight G a i n ...................... ! E. Gross Examination of Maternal Organs at Sacrifice . . . F. Reproductive Effects and Fetal Body Weight ............ G. Fetal Alterations...................... V. DISCUSSION VI. CONCLUSION . . VII. ACKNOWLEDGEMENTS VIII. REFERENCES Page 1 2 3 7 8 9 10 10 11 12 12 12 12 13 14 14 14 14 15- Company Sanitized. Dees notcontain TSCA CBl -3- DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARjL|STUDY. BY GAVAGE) o HLR 441-84 TABLE OF CONTENTS (CONT.) TABLES Page I. Feed Consumption in Rats Given Test Chemicals by Gavage from Days 6-15 of G e s t a t i o n ..................................... 18 II. Reproduction and Fetal Development in Rats Given Test Chemicals by Gavage from Days 6-15 of G e s t a t i o n...................... III. Fetal Malformations in Rats Given Test Chemicals by Gavage from Days 6-15 of G e s t a t i o n ..................................... IV. Fetal Variations in Rats Given Test Chemicals by Gavage from Days 6-15 of G e s t a t i o n ..................................... 19 21 22 APPENDIX Protocol for Maternal and Developmental Toxicity Study in Rats after Administration by Gavage with C-8 Alternates* . . . Amendment to the Protocol 28 39 ATTACHMENTS 1. *Letter, from J. R. Barnes to R. E. Staples, 3 / 2 0 / 8 1 ........... 2. Letter, from J. M. Clinton to R. E. Staples, 7 / 7 / 8 1 ............ 3. Memorandum, from W. D. Kerns to R. E. Staples, 8/19/81 . . . . 4. ' Memorandum, from W. D. Kerns to R. E. Staple's, 1/29/82 . . . . 42 54 55 56 * Du Pont classified information * not con!" C A C Com?a'ny Samfad DO' -4- C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT ,(PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 TABLE OF CONTENTS (CONT.) ATTACHMENTS (CONT.) 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...................... 6. Memorandum, from W. D. Kerns to R. E. Staples, 12/18/81. . . . 7. Memorandum, from R. E. Staples to Teratology Section File, 1 0 / 3 0 / 8 1 ..................................................... 8. Tabulation of Results of Dose Selection Based Upon Comparison of LD50 or ALD V a l u e s ........................................... Page 57 67 71 72 -5- Company Sanitized. Doss not contain TSCA CBI C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT .(PRELIMINARY. STUDY, BY GAVAGE) HLR 441-84 SUMMARY ^ ^^ Lm p t p administered to rats by gavage at 100, 500, 2 5 ( ^ o ^ 5 0 ( M n g / k g D o d y weight/day, respectively, from Days 6 through 15 of gestation to determine the d e g r e ^ ^ ^ hazard to the developing conceptus. The dosage J ^ m ^ r i v e n was lethal to six of the seven rats tested, an^CTie rat " n i ^ ^ s ^ ^ i v e d 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 Pont obtains ammonium perfluorooctanoate (C-8) from 3M Company for use in the manufacture of a variety of fluoropolymer dispersions, including some of Du Pont's Teflon products. A study of the -- ^embryotoxicity and teratogenic potential of C-8 was requested by B f l Q W H B g l Polymer Products Department, and o n e m ^ ^ ^ ^ ^ i d Pigments Department, at a meeting 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. not contain TSCA CBI Sanitized. Does C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT .(PRELIMINA R Y 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 onduct a maternal and _developmental toxicity study was received from larch 23, 1981 (Attachment Drallvrequested that the study for comparison to C-8. also pilot B. Protocol (Appendix A) An MR request was sent to PPD on July 17, 1981; it was authorized on July 28, 1981. The protocol was issued on July 23, 1981, and it was amended on December 3, 1981. III. MATERIALS AND METHODS Test Chemicals* C-8 is a white powder which sublimes at 110^ weight is 431, and its structural formula The purity of the sample used was! contaminants present were! \\Its molecular and Number is and it was assigned Haskell Number 14,045. The sample was received from the Polymer Products Department. !. a white solid with greater t h a ^ ^ ^ ^ s o ^ b i l i t y in water. Its structural' formula 7ts molecular weight is 463, its CAS Registry Number is anc* t*ie Haskell_Number assigned_is 14,075. The samp^^fas received f r o m P P & R , Washington Works. * Du Pont classified information 7ijjnpany. tSCA CBl C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 A. Test Chemicals (Cont.) 3. ( M s a w^ite solid, with greater than 10% solubility in water. Its structural formula isl"^ Its molecular weight is 525 and the Haskell Number assigned is 14053. The sample was received from| PP&R, Washington Works, with a purity ofl lis an off-white powder with a density of 2.0, pH 7 - 9 a n ^ ^ ^ ^ ^ ^ s o ^ j b ^ ^ ^ y i j ^ 7 a t e r . - Its structural formula a molecular weight of 550. Its assigned.Haskell Number is 14,068 and the sample was received f r o m P o l y m e r Products, E353/318 Experimental Station. ____ lis a beige powder with a density of about 2, pH 4-5 in water, and 1-10% solubility in water. I t s _ ^ structurual formula Its molecular weight is 540 and its assigned Haskell Number is 14,008. The sample was the The sample was received f r o m ' 5SL 269/306. PPD. 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 170+0.7 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. Company Sanitized. Does not contain TSCA CBI -8- 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 3% (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 dead on the morning of the fifth day by which time it 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. - 9- (inTSCACBl Sanitized. Does nol contain Company 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-8. The calculated dosages were 100, 5 0 0 ^ 5 0 0 ^ a n d 1500 mg/kg/day respectively. Due t o w i t h wo 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 delivered 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 1 CAS Registry Number 8001-30-7; Item 13266, Lot No. DCo4m-p4a5ny Sanitized. Does not contain TSCAC - 10 - C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT .(PRELIMINARY STUDY, BY GAVAGE) u 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'. 11 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT 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<0.05. 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. 3 not contain TSCACBI Company Sanitizsd. Dos 12 C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT IPRELIMINAM 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. I *No deaths or clinical signs were^hseyrr\ved in the ;roup, Similarly, no deaths occurred in t h e ^ H f e 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. In t h e g r o u p 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 stairringofthe uterus with ammonium sulfide. Hence, fetal data for the I ^ ^ ^ ^ ^ H M g r o u p are not available for tabulation. *-n ^ h e g r o u p five o ^ J ^ ^ a m ^ h a d clinical signs similar to those seen in the C-8 and f U H R g r o u p s . 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 iatrogenic pulmonary injury, hence the deaths were not found to be related to improper gavaging (Attachment 3). C . Maternal Feed Consumption ' ^ ^ ^ ^ ^ r i n g the dosing period, the groups given C-8,1 _ _ _ _ _ _ _ _ _ ^ ^ ^ ^ I ^ ^ R c o n s u m e d significantly less feed than the control group (Table IT". F e e d consumption was similar to the control value in the post-exposure period. Insufficient data were available for thefll^V jroup to permit meaningful statistical comparison. ~* - 13 not contain t s c a c b i SaniUzed. Doe CompaDX 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 group (Table II). However, from Days 6-15G, the group receiving C-8 gained about one-third less (p<0.05) 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<0.05) the control value. During the dosing period, the body weight gain of the||^J3group was not significantly different from that of the control group, but during the postdosing period, the^^^Bgroup gained significantly more than did the control group. The ^ B g a n d M H j j H I H H l g r o u p s gained little during the dosing period, but, t h e r e a ^ e ^ ^ t n e W H j g r o u p gained significantly more body weight than the control- group. 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 ^ W J g r o u p 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 f e t a l b o d y w e i g h t were not adversely affected by administration of C-8, the dosages tested (Table II). G. Fetal Alterations No more than one fetus was malformed in each of the groups given test chemicals (Table III). 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 above the control value in both t! [groups (Table IV). The increase for the 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. Doss not contain TSCA CBJ C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 G. Fetal Alterations (Cont.) Stereoscopic examination of the bisected eyes from two fetal heads per litter (Boilin's fixed) from each group did not reveal malformations or variations (Tables III and IV). The only eye alteration noted 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 thejm^^group 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 groups extra unmated female rats from dosages fej^g^ag^t^ian were used in t h i ^ s t u d y ( W g r o u p ) . Administration mg/kg o r f l j j j ^ m | H M & a t 150 mg/kg for a period oT ten days still resulted l ^ ^ ^ ^ m u c n b o d y 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 toxicity testing be undertaken. ^ ^ ^ < 3 - 8 was previously evaluated in Haskell Laboratory Report No. ^ H U a n d will not be repeated here; the data on C-8 are presented in this report for comparative purposes only. { j j ^ w a s 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 f ^ u s e s were demonstrated to be adversely affected by exposure to 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 that was not significantly increased above the control value 05). In view of the concurrent toxicity present among the damSjjSSKtfas not demonstrated to represent a unique hazard to the conceptus. rot contain TSCACB Company Sanitized. Doe*n C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT 0(PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 V. DISCUSSION (CONT.) briilso 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 w h i c h ^ i m m ^ w a s given was too high to permit evaluation of toxicity to the dam or their concepti. Subsequent range-finding studies in non-pregnant rats indicated that the dosage level should not exceed 35 mg/kg body weight, if given daily for 10 days, by gavage, in corn oil. T h e a l s o 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^idua^varLations were not statistically significant (p>0.05). | ^ | ^ | | | | m ^ ^ d i d not pose a unique hazard to the rat conceptus at the toxic level tested. VI. CONCLUSION not demonstrated to represent a unique hazard to the conceptus after administration by gavage to rats ^ n ^ ^ ^ ^ p e r i o d of major organogenesis at dosages toxic to the dam. given at a dose level that was too toxic to the dam to assess its potential hazard to 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 Sect! on j Has k.sli Lsboiratoiry - 16 Company Sanitized. Doss not contain TSCA CBJ C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) HLR 441-84 v i n . REFERENCES 1. Unpublished Du Pont Data, Haskell Laboratory: 2. Unpublished Du Pont Data, Haskell Laboratory: 3. Unpublished Du Pont 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, 12:165-172 (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., 18_: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). Santtized Doeassnoi coniataTSCACB4 - 17 - c o m p l y C HLR 4 41 -8 4 TABLE I C -8 ALTERNATES: MATERNAL AND DEVELOPMENTAL T O X IC IT Y IN THE RAT (PRELIM IN ARY STUDY, BY GAVAGE) FEED CONSUMPTION3 IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6 -1 5 OF GESTATION (G ) I 00 I Oo 3 rs Cal) if} ) s N<D CL Days 1-5G D a ys 6-1 5G Days 16-20G Number o f dams In clu d e d Corn o l 1 C-8 (5 mL/kq/day) (100 mq/kq/dav) 21.0+0.59 20.6+0. 32 21.9+0.48 17. 2+0.37^ 28. 1+0.58 2 9 .0 + 0 . 52 24 22 C ) (100 mq/kq/dav) 2l.8_+0. 79 18. 3+0. 25*^ 30. 5+J.04 7 a 4> 9 ra m s/ ra t/ d a y + S .E .M .; v a lu e s f o r n o n -p re g n a n t fe m a le s were e xclu d e d s i g n i f i c a n t l y d if f e r e n t from c o n tro l va lu e by D u n n e t t 's t e s t (p <0 .0 5) (500 mq/kq/dav) 2 1 .3j+0.52 13.7+0.40^ 2 7.9 ^ 0. 52 6 Cl (250 mq/kq/dav) (500 mq/kq/day) 19.3+0.0 I9.6+J.67 11.7+0.0 13. 3_+1.90*^ 18. 5+0. 0 26.7+J.78 17 Does not contain TSCA CB. HLR 4 4 1 -8 4 TABLE I I C - 8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TO XIC ITY IN THE RAT (PRELIM INARY STUDY, BY GAVAGE) R E PRODUCTION AND FETAL DEVELOPMENT IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6 -1 5 OF GESTATON Corn o I C-8 (5 mL/kq/day) (100 mq/kg/day) (100 mg/kq/day) CAO (500 mq/kq/day) FemaI es .. a 1 No. p regnant /no. mated 1-- vO No. deaths 1 No. 1Itte rs Mean no. c o rp o ra lu te a e> 3 Mean no. im p la n ts S u Mean 1 Iv e r w eight (g )^ vi \ Mean w e igh t g a ln ( g ) ^ i 3 M <a0. n= Days 6-15 a o Days 16-21 ww 3 Days 6-21 O oo D ays 6 - 2 1C^ 3 . 3 -(F e ta l Death U f~ ^ M e a n no. resorptlons/dam jjgoNo. l i t t e r s w ith t o t a l re s o rp t io n 25/25 0 24 16. H O . 55 13. 6_+0. 57 1 5 .4 t0 .3 4 33.' 2_+1. 1 2 5 6 .7 ^ 2 .3 4 72.6_t1.31 1 2 9 .2 t2 .6 8 57.8 +2. 35 0 . 7_+0. 16 1 22/25 3b 22 16.7 t o . 87 13. 8_+0.64 1 6 .2 t0 .4 4 30. 0+1 . 09^ 3 8 .3_t2.89^ 84. 5+3.00^ 122.8jM .26 4 9 .8 + 2 .7 5 ^ 0 . 6 J O . 13 0 7/7 0 7 15 .3+ J.19 14. 6_+0. 65 1 7 .5 t0 .6 8 3 5 .4j+2.08 4 4 .4 + 5 .2 5 8 6 .6 + 4 .6 9 Y 131.0t_7.78 5 6 .4 t5 .6 9 6/7 0 6 15. 3+0. 67 15. 2+0. 54 22. 9+1.20^ 32 . 1_+2. 75 -1 .5 + 8 .9 5 ^ 9 2 . 6_+5.99^ 9 1 .0 + 4 .2 9 ^ 2 1 . 8_+2. 72*^ 1 .0 + 0 .3 8 0 0. 8jtO. 17 0 0/7 6C 0 -- -- -- -- -- ___ -- -- 0 3/7 3d 3 17 .3 + 2 . 8 1 4 .7 + 0 .8 8 1 4 .9 + J . 12 3 4 .6 + 2 .0 7 12. 5_+1.87^* 7 7 .0 + 6 .5 1 8 9 .5 + 7 .7 9 ^ 20. 7j+3.78^ 1.3JKJ.33 0 HLR 4 41 -8 4 TABLE I I (CONT. ) C -8 ALTERNATES: MATERNAL AND DEVELOPMENTAL T O X IC IT Y IN THE RAT (PRELIM INARY STUDY, BY GAVAGE) REPRODUCTION AND FETAL DEVELOPMENT JN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6 -1 5 OF GESTATON Company Sanitized. Doss not contain TSCA CBl Corn o iI C-8 (5 mL/kg/day) (100 mq/kq/day) life! (100 mq/kg/day) i Fetuses 0 No. liv e 1 322 292 95 86 40 Mean no. liv e 13.4+0.32 13.3+0.67 13.6+0.95 14.3+0.61 13.3+0.88 No. stunted 00 0 0 0 Mean w eight (g ) 3.8+0.08 4.0+0.05 3 .9 + 0 . 14 3.3+0.09 3.6+0.10 d b a lI female s had v i s i b l e s ig n of pregnancy e vid e n t a t autopsy c one was no+ed a s b e in g dead on Day 11G, and two more on Day 12G r a t s were found dead on D ays 10, 11, 12, 13, and 14G and one was s a c r i f i c e d d t h a t su rv iv e d had r e s o r p t io n s detected by s t a in o n ly In extre m is on Day 12G; th e r a t e ne ra + Was found dead on DaV ,3G ; +he o t h e r s were s a c r i f i c e d In e x tr e m is on D ays 12 and 14G f - ne fem ale had o n ly two e a r ly r e s o r p t io n s In u te ro on Dav 21G x+ S.E.M . 9-- h fem ales w ithout a l i t t e r were excluded 4 " '9h, f *xeiu<,,"s ,h* s i g n i f i c a n t l y d if f e r e n t from c o n tro l va lu e by D u n n e t t 's t e s t <p0.05) * ^ n TABLE III Company Sanitized. Dees not contain TSC CB1 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 GESTATION External Malformations3 N o . examined Visceral Malformations N o . examined Great vessels Innominate - none Kidneys - none Kidneys - small Spleen - small Brain - ventricles enlarged Corn oil C-8 (5 mL/kg/day) (100 mg/kg/dav) 322/24b 292/22 171/24 c l/ld l/ld X/1H l/ld 155/22 Head Malformations3 No. examined . Skeletal Malformations N o . examined Sternebrae - fused Total with Malformations Avg. % Malformed Fetuses per Litter (+S.E.M.) 221/24 322/24, l/ld 2/1 0.7+0.70 198/22 292/22 Cm S (100 mg/kg/day) 95/7 49/7 1/1 1/1 65/7 95/7 1/1 1.1+1.10 c (500 mg/kg/day) 86/6 45/6 60/6 86/6 40/3 22/3 1/1 27/3 40/3 1/1 2.6+2.56 k no malformations were noted fetuses/litters d blanks represent zero incidence occurred in a single fetus HLR 1-84 TABLE-- IV 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 GESTATION Company Sanitized. .Does no!contain TSCACffl Developmental Variations Corn oil C-8 (5 mL/kg/day) (100 mg/kg/day) (100 mg/kg/day) i External ISJ No. examined Hematoma I Petechia Eye - red 322/24 15/10 60/19 c 292/22 10/8 34-/1..Q5 1/1 95/7 6/3 22/6 Visceral No. examined Renal papilla -reduced Renal pelvis -large Pulmonary arteries -common trunk 171/24 l/le 1/1 3/2 155/22 2/Is 9/6 49/7 3/2 Head1 N o . examined 221/24 198/22 65/7 86/6 6/3 16/6 2/2d 45/6 3/2 60/6 40/3 1/1 10/3 22/3 1/1 5/3 27/3 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 GESTATION Company Sanitized. Doss not contain TSCACBfc Corn oil C-8 t(5 mL/kg/day) (100 mg/kg/day) Developmental Variations (Cont ) I ro Skeletal LO I No. Examined Sternebrae -misaligned( 1)'1. -misaligned(2+)J -bipartite 322/24 14/11 7/7 1/1 292/23 6/6 9/8 Centrum -bipartite 3/3 -dumbbelled 2/2 4/4 Rib extra ossification center -rudimentary -extra -thickened -calloused -wavy * 45/16 2/2 1/1 1/1 76/16 11/5 1/1 1/1 . 1/1J h(100 mg/kg/day) 95/7 2/2 2/2 1/1 2/2 30/16 1/1 (500 mg/kg/day) 86/6 5/4 3/3 14/5 1/1 2/1 4/1 2/1 (500 mg/kg/day 40/3 1/1 5/2 1/1 8/3 fl HLR 441-84 TABLE IV (CONT.) C-8 ALTERNATES: MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETA.L VARIATIONSa IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF GESTATION Developmental Variations (Cont.) Corn oil C-8 (5 mL/kg/day) O O P mg/kg/day) (100 i ^ ^ d a y ) (SOO^TTgTdav) ^ 5 0 ^ / S d ^ i N> P- I oo 3 o &r3 o> o N<0 O. o o Subtotal Developmental Variations --No. Affected -Avg. % Affected Fetuses/Litter (+S.E.M.) Variations Due to Retarded Development Visceral Renal papilla -slightly reduced Skeletal Sternebra -partiall ossified -unossified Rib -partially ossified Ischium -partially ossified Centrum -partially ossified -unossified 119/22 36.3+3.92 > 51/13 10/6 1/1 128/21 43.2+4.53 l/lg 22/8 1/1 l/iJ 51/7 53.7+9.41* 40/6 46.7+6.87 2/2 6/4 2/1 2/1 1/1 1/1 22/3 55.7+5.85** 13/3 i/i r-> CM CM not contain TSCAC^ HLR 441-84 TABLE IV (CONT.) C-8 ALTERNATES : MATERNAL AND DEVELOPMENTAL TOXICITY IN THE RAT (PRELIMINARY STUDY, BY GAVAGE) FETAL VARIATIONSa IN RATS GIVEN TEST CHEMICALS BY GAVAGE FROM DAYS 6-15 OF GESTATION Company SanUized.ofiS noUonlain Variations Due to Retarded Development (Cont.) Corn oil c-8 (5 mL/kg/day) (100 mg/kg/day) Skeletal (Cont.) Pubis -partially ossified No. examined with heads Skull bones partially ossified -parietal -interparietal -supraoccipital -squamosal -frontal -zygoma Maxilla -partially ossified H Hyoid O -partially ossified ^ -unossified i.* 2/2 104/24 1/1 4/3 1/1 1/1 3/3 5/4 2/2k 94/22 2/2k 5/5k 5/5k 2/2k 1/lJ 2/2K l/lk 2/2, 4/3k , COD (100 mg/kg/day) 30/7 26/6 3/1 1/1 1/1 1/1 2/2 HLR 441-84 TABLE IV (CONT.) 0 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 GESTATION Variations Due to Retarded Development i (Cont.) K3 ^ Subtotal - Variations Due to Retarded Development Oo 3 -No. Affected T3 6> 3 -Avg. % Affected V) Fetuses per Litter n> 3 (+S.E.M.) N TOTAL No. Fetuses with Variations Avg. % Fetuses with Variations per Litter (+S.E.M.) Corn oil C-8 (5 mL/kg/day) (100 mg/kg/day) (100 mg/kg/day) (500 mg/kg/dav) (5QoT!g7kg7da 70/14 20.6+4.61 35/12 10.9+3.08 154/23 46.5+5.18 145/21 48.5+4.35 4/7 4.9+2.82 53/7 56.0+8.80 14/6 16.5+4.36 41/6 47.9+6.84 14/3 36.2+21.04 29/3 73.5+11.27* Doss not contain TSCACBt HLR 441-84 TABLE IV (CONT.) 0 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 GESTATION a- malformedlnClUde thSe PreSent malformed fetuses as these ^ t u s e s were previously tabulated as being b - fetuses/litters c - blanks represent zero incidence d e - tSffeturn^H anterlor chamber of the eye at histomorphologic examination NJ f - l u l l weighed 2.89 g; hydroureter was not detected at visceral examination ,, _ 3 `58 g ' hydroureter as not detected at visceral examiantion 8 a S, ^ ' 0 2 ' Z ) with left kidney affected had associated hydroureter; the other a littermate ( A 21 r S tk sid:yS aft'" ed " lthUt aSSCl8Ced * - i W reduce^papilla'wa^presen^on^^' h - one fetus (3.47 g); both renal pelves slightly enlarged 1 _ et!i8 /o ^ith develoPmentai variations of head were detected k - one fei-,io rR r u r mre sternebrae were misaligned, respectively one *-etas (3.26 g) had each of these variations ** I S^a^iS^ Ca^ y siSnificant difference by one-tailed Mann-Whitney U test (p<0.05) statistically significant difference detected by two-tailed Mann-Whitney test (p<0.05) Company Sanitized. Doss 3 o oo 3 o 3* co O > O CD r- U ^ C ^ 7 Z ^ l ^ a ^ E S - 575 REV- 1-75 cc: AFi'lNUl A % ESTABUSHED1802 E. I. d u P o n t d e N e m o u r s & C o m p a n y INCORPORATED W i l m i n g t o n , D e l a w a r e 19898 CENTRAL RESEARCH & DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOG Y AND IN D U STR IA L M EDICINE July 23, 1981 M E M O R A N D UM TO: POLYMER PRODUCTS DEPARTMENT CUSTOMS HOUSE SQUARE 314 CHEMICALS AND PI OIENTS DEPARTMENT BRANDYWINE BUILDING 6210 ) FROM: R. E. STAPLES SUBJECT: C-8 AND PROSPECTIVE ALTERNATES: PROTOCOL FOR TERATOGENICITY TESTING The protocol for the teratogenicity testing of C-8 and its alternates is attached if you have questions concerning this s t u a ^ ^ p ^ a s ^ ^ l e t me know (366-5302). RES /mie Attachment (1) gompany Sanitized. Does not contain TSCA CB1 - 28 - BETTER THINGS FOR BETTER LIVING . . . THROUGH CHEMISTRY E. I. DU PONT 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 I. BACKGROUND .A ^request for a proposal to conduct a, teratogenicity study on ^ lwas received fromC" " (Haskell Coordinator on March 23, 198 is request was made response to a TSCA 8(e) notice from the 3M Company On .Tune 11. 1Q81. E. D. Champney orally requested ft so he tested in to a second TSCA 8(e) notice trom the 3M Company for The oral LD50 and ALD estimates for the rat were 1981. by the Acute Section at Haskell Laboratory on June 12, test the teratogenicity ' in the rat after ther 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. ~ Company sa n iferf. c o w nolconlan TSCA CB! 29 - o?2^0C0L F0R teratogenicity study in ^ T S ^ ^ e^DMINISTRAT-ion by GAVAGE c PAGE 2 Test Chemical Products The following Department and chemicals were received from assigned Haskell Numbers: the Polymer B. Animals , Crl:CD(SD)BR femalp rate weighing between 170-180 q will hp ulliparous) 50-60 days old Breeding Laboratories nr m S or?ered frm Charles River The rats will be quarantined f ^ a t ^ i " 11? ^ 011' Massachusetts. by air-conditioned truck nrv-m _iaast one week after arrival unique identific^ion nieberPthatarwnialA ^ w U l be assi^ ' a and by a combination of ear Dunrhpc p !! written on a cage card will be mated by overnight c?hab i t L ? n?n ^Cllps` The females strain and mating will be v e r i f L k Jo mature males of the same the vaginal lavage each morning (Day^ J f ^ e s S t i o n f ? 6TM ^ 0203 in a positive teratogenic Response ^to C - ^ i ^ t V because 3M rePrted C - 8 f r | ^ j ^ ^ i ^ ^ ^ n d i n g Also, the degr S T o f ^ x i c i t j of Hask e f l L a b o ^ S ^ ^ ! h ^ f f c D f S D ! :RRmiie d 'in this species at extensive background teratdgenfc t? S t f iain,W&S ChSen because Laboratory on this rat Strain' 6Xlsts 3t Haske11 including cataracts" Z r t T e TM * 1 in^ ence f alterations (according to^nsultinq' in CD rats is 3% prospective parents f o A h i s s t u d A i n A S Clinton) a11 alterations before breedina n ^ be screened for these both e y e s will be examined nd a n ^ J ^ . f ' l ^ ^ ^ . P ^ c e d u r e , eliminated. "* 11 aiLectea rats will be - 30 - es noi contain TSCA C. Company Sanitized. Do c * Route of Administration , . Admin istration of the test chemical; 11 ka u S M M Ii 2 TM ^ V - *" * tls? m s^tiTnis r / aS^ " ^ * ^ ^ n^ "wbsio" to calculate the doieW^ 9bi rfcently corded will be used samples of each solution w i l ? ^ " ? ac^ Sat* Periodically, of concentration a ^ Uniformitf of fr Possible analysis group in each exDen'monk ^ . lxture. a vehicle control body weight for the same oeriod^f^ f I* striPPed corn oil/kg oxidation during the doL^! d .i R a t i o n . To minimize 9 wtiolpls. be stored It ^ C ^lIn ?reeddbboottttllees?tshtJopCpOerrned? Uwitfhrmgrooupnednedglacsasns D * Dose Levels The recommended dosage levels are Experiment I irtn ra-i/^9 100 mg/kg 100 mg/kg 500 mg/kg 250 mg/kg 500 mg/kg Stripped corn oil C-8 Experiment 5 ml/kg Stripped corn oil 100 mg/kg C-8 The C-8 level is recommended because it i<? t-ho level that can be tolerate " se lt: 1S the maximum by range-findinq testino- i h .damf by Sava9e as determined alternate chemicals was'determl^d s l m U ? y . f0r-eaCh of the E ` Animal Distribution randomly If the dis^finiil aSSlgned to will be selected 1 Eastman Kodak, Rochester, NY, not contain TSCACfc. Company Sanitized. Does - 31 PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTER ADMINISTRATION BY GAVAGE WITH 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 rreecceeiivviinngg*^C-8. Each of <trhentrroe1mai9nrionugP *tnedst togrotuhpes teisnt tghirsoupP mated assign<rd about 7 mated females. About 12 mated females will be assigned to each test group for Experiment F Husbandry w i n ho h UP2 arr*val ab Haskell Laboratory, the female rats will be housed two/cage m suspended wire-mesh steel cages Purina Certified Rodent Chow #5002, Checkers, and wate? from libitum!"00 Suburban Water Corporation (WSWC) will be provided ad The Ptential effects of dietary contaminants were a"d ' ?n the basis of the manufacturer's data contaminant levels are believed to be within acceptable ranqes f L 2 ther conta"11^ ants reasonably anticipated to be present in the feed are expected to interfere with the results of this studv The potential effects of water contaminants reported by WSWC were considered and appear to be within acceptable Liges. 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. Records Maintenance _ . . Records for parameters in the protocol will be finita^ ed fr 6aCh animal* when the study is completed and the Tnf raPrt 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 . ... A11 Personnel will wear flock-lined latex gloves2 when ndling test compounds or mixtures. Kevlar gloves will be wnm ntae^ordnu ?r i nang" aault?opasrye. hanCdolnetdamiannadteldatw^asteexlmmaitner!ia*l Sgillolvesbe,, t n FlbarPaks ^ incineration at Goioen Thumb Glove Company, Glove #L-61, 18 mils thick - 32 - Company sanitized, Does not contain TSCA O il PROTOCOL FOR TERATOGENICITY STUDY IN j A T S j j j g ^ ^ ^ g ^ T I O N BY GAVAn F IOR ITS PAGE 5 Parameters to be Studied Experiment I - Teratogenicity Study 1 Dams a. --* 7 wex9nea on tne day of arrival, before breeding, and on the morning of Days 1, or 9r 13, 16, and 21 of gestation Feed consumption - the average amount of feed consumed daily per rat for each group will be determined for the pre-exposure, exposure, and S i n "hP? SSre Periods. The measurements will be taken at the same time each morning. Clinical signs - observed upon arrival, at breeding, and daily at least from Days 5 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 W uig- the intact and empty uterus of eacn dam having one or more fetuses will be weighed to permit calculation of actual maternal body weight gain during gestation Corpora lutea - counted and recorded for each ovary 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 earlv resorptions . * l Resorptions - counted and recorded for each rat (not those detected by stain only) 2 Fetuses Number, location, and condition recorded for each fetus in each litter ~ n3l coniato TSCAOBI Sanitized. Dess Compaq - 33 - PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTER ADMINISTRATION RV c a v a c p WITH fa ----- ^ 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. tissue alterations - detected3and 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 heads'* of 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 a n tetuses (including "Dead" fetuses); the fetai heads that were fixed in Bouin's solution will be excluded All grups will be coded from just before sacrifice until all -raw data are collected. " j staples, R. E., Teratology, 9;A3/-A38 /Iqrtq).--------- ~ ffzaz?' v''* M` and " J- Taylr- J. Morph., 127:291-306 HC> 5n?a;n TSCa CBI J now: - 34 - R A T S a ^ 5 0R TERATGENICITY STUDY IN WITh BY GAVA GR Experiment U - Extended Teratogenicity Study 1 Dams PAGE 7 r -rival, ^ i, 7 , 14, L i r a a ; s 6^ r p a2i t gestation Clinical signs - observed upon arrival, at allt6lng' daily frm Da^s 6 t r ough 15 of partum10nL and n lf 7' l4' and 22 days post ?fLtU: i u A^ 9nrS eIeCtS bserved at " y th.r Date of delivery - noted for each dam Reproductive indices For each test group: o Fertility index (% matings resulting in pregnancy) y in ? i i f M ? ? h s ? deX <S ",atingS resultin9 in For each litter; survivprf^zi index (% animals born that survived 4 days or more) jactation index (% animals alive at 4 days ' that survived to 22 days post partum) 2. Offspring The number of live pups, per litter and the number of dead or cannibalized pups per litter post p L S S ? ^ ^ n X ' 4' 14' and 22 da^s ~.recorded foe each litter on the deltvery- 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, Itffplrtum3 ' W9lgh9d 11 4' 7' 14' 22 days ainTSCACBK .Sant!a * . * * * * * * ;omPan'- - 35 - PROTOCOL FOR TERATOGENICITY STUDY IN RATS AFTER ADMINISTRATION BY GAVAGE WITH " ---- ------ IOK ITS ALTERS PAGE 8 Clinical signs - observed 1, 4, 7, 14 and 22 days post partum. Pups with adverse signs noted will be marked for subsequent identification within the litter. e * External alterations - detected and recorded for all live pups Soft tissue and skeletal alterations - pups will not be examined for these types of alterations unless otherwise indicated 9 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 exsangu j.ua uru 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. 1 III. STATISTICS Experiments I and II The litter will be used as the experimental unit. The Fisher s exact test will be used to determine the significant differences in the incidence of pregnancy, and mortallfcy- Jonckheere's test will be used to determine the presence of a dose response. Dunnett's test will be used for testing the significance of d i f f e ^ c e s 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 . diferenCes between the control group and CA^ iUiental groups. The level of significance will be p < 0.05. In addition, the reproductive indices given earlier will be calculated. ... od npesnci contain TSCACBl Company_Samteed.-ocsn - 36 - d ?SJ0C0L f o r t e r a t o g e n i c i t y s t u d y in ^ T S y ^ E ^ D M iNISTRATION BY GAVAftR Iv* CRITICAL DATES Starting Date (breeding): Completion: July 1 3 , 1981 January 18, 1982 PAGE 9 - 37 - not contain TSCACBl CpmpanySani^26*3' Poes PROTOCOL FOR TERATOGENICITY STUDY IN J^SSILa m i n i strati on by gavage IOR ITS ALTEl P AGE 10 Prepared by: L L^montia -- b iologist Teratology Section Toxicology and Pathology Study Director Teratology Section Toxicology and Pathology - 38 ized.Does ,,otcontaiC*CBI Company San CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE December 3, 1981 M E M. O R A N D U M TO: QUALITY ASSURANCE COMMITTEE - C. M. BARBA FROM: R. E. STAPLES 9y " SUBJECT: PROTOCOL AMENDMENT FOR TERATOGENICITY STUDY- C-8 AND PROSPECTIVE ALTERNATES r"----- * treessttiinnag o C-8 and its alternatPersotoicso1attfaocrhetdh.e teratogenicity RES /mle Attachment (1) - 39 - ,,,,t contain TSCACW Sanitized. Does Company E. I. DU PONT DE NEMOURS & CO., INC. CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ELKTON ROAD NEWARK, DELAWARE /u'iniMuiyuJMT TO THE PROTOCOL FOR TERATOGENICITY 'ESTING IN RATS AFTER ADMINISTRATION BY GAVAGEJWITH "" " OR ITS of the study^ following chan9es were made after initiation Page 8 II. MATERIALS AND METHODS I* Parameters to be Studied Experiment II - Extended Teratogenicity Study 2. Offspring <? Eye examinations Because no compound-related effects were detected grossly or microscopically m the fetal eyes from Experiment I, the eyes from all offspring in Experiment II were fixed, identified, and retained but none were processed or examined microscopi cally by a pathologist. J* Retention of Specimens All skeletal, head, and selected visceral speci mens, as well as histologic preparations, will be e t a m e d till issuance of the final report. There after, they will be retained for as long as the quality of the material affords proper evaluation. - 40 - n0l contain TSCACa :Sd.Does Company Sanii1, AMENDMENT TO THE PROTOCOL FOR TERATO- PAGE 2 PREPARED BY: C. L. Lamontia - Biologist Teratology Section Toxicology and Pathology Staples'y S Staff Teratologist Teratology Section Toxicology and Pathology APPROVED BY J. Aftosmis - Associate Director Toxicology and Pathology CLL/RES/mle 12/3/81 - 41 t s c ^ cbi . noes not coni*" Sanifesd-D ~ Compaq CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ATTACHMENT 1 M A R 2 3 1981 March 20, 1981 MEMORANDUM TO R. E. Staples FROM: J. R. Barnes i f f TERATOGENIC STUDY WITH Polymer Products Department would like Haskell Laboratory to pre pare a protocol and cost estimate for an oral teratology study with The purpose of the study is to determine whether [produces effects similar to those reported by the 3M Company forI H | M ? - n 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 t o ^ m ^ H ^ ^ PPD, Customs House Square-314. JRB:mjh Attach. 42 - , not colala C o m p a n S j ^ 00' POLYMER PRODUCTS DEPARTM ENT February 11, 1981 TO: E. D. CHAMPNEY D-11070 FROM: J. B. ARMITAGE CHS-314./vs/i TSCA SECTION 8(e) REPORT FROM 3M ^ ^ Under the Freedom of Information Act, we obtained ^ ^ ^ ^ t ^ ^ E P ^ ^ ^ ^ ^ ^ ^ t a c h e ^ T S C A 8 ^ ) notice on the was noted in my December 12, T380 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 JB M S a wi&fcC-d ,, C0>`a*n ll0Y - 43 There's a world of things we're doing something about REC'D UNITED STATES ENVIRONMENTAL PROTECTION AGENCY W A S H IN G T O N , D .C . 20 46 0 J A N s Q 1931 .K-tii HC Mr. J. B. Armitage E.I. duPont de Nemours & Company, Inc. Patents and Regulatory Affairs Division Wilmington, Delaware 1989S O FFIC E O F PESTIC ID ES AND TO XIC SUBSTANCES Dear Mr. Armitage: Re: Freedom of Information Request Act (A-101) RIN-247-81 ' In response to your letter dated January 23, 1981, enclosed is a nonconfidential co p y of substantial risk notification 8EHQ-11800374S, 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. West of my staff at (202) 755-8050. Please use the reference number RIN-247-81 when inquiring about your request. Sincerely Enclosure Edward J. Cull, Chief Information Control Branch 44 - nTSCACei X 0 0 `0 5 ' / ^ o C-dneral O/ficas/CM ' Certified P a :1 - " e - u r - =?:=-- -- 2WCanter St. Paul. Minnesota 55101 612/733 1110 N o v e m b e r 1 9 , 1980 ^ rr% Document Control Officer Chemical Information Division Office of Toxic Substances (WH-557)- ' Environmental Protection Agency 4 0 1 M S t r e e t , S. W. W a s h i n g t o n , D. C. 2 0 H 6 0 B H l /1 $ 0 - 0 - 3 7 o Gentlemen: % -^ D o l^ -- ~a '^2> S u b j e c t : S e c t i o n 0 ( e ) T o x i c S u b s t a n c e s C o n t r o l A c C 3 TJ Please Potassium Salt find attached the of Perfluoroalkyl Sulfonates no i % f o l l o w i n g i n f o r m a t i o n r e l a t i n g to the subject chemicals: (1 ) (2) (3) P r o t o c o l s for O r a l T e r a t o l o g y Study, in Rats. Preliminary report, 3M 12, 1980. memo, dated November 3M Technical Report entitled "Analysis of Selected Decatur Employee Serum for Sulfonic and Carboxylic F l u o r o c h e m i c a l s ". Preliminary information from the ongoing teratology study cited in (2 ) above indicates that the subject chemicals are t eratogenic in rats. This information and c h e f i n d i n g s d e s c r l b e d ' ~ i h "(3 7 ""a b o v e , i n d i c a t i n g t h e p r e s e n c e o f f l u o r o c h e m i c a l s i n t h e b l o o d o f s o m e o f o u r p l a n t * ...... > I s a a s us to s u b m i t this, i n f o r m a t T o r T p u r s u a n t to S e c t i o n 8 (e) of the T o x i c S u b s t a n c e s C o n t r o l Act and ' E P A 's s t a t e m e n t o f i n t e r p r e t a t i o n p u b l i s h e d i n t h e F E D E R A L R E G I S T E R , M a r c h 16, 1978. As n o t e d on page 584 of an article publi s h e d in the October i960 American Industrial Hygiene J o u r n a l t entitled "Health Status of Plant Workers Exposed to Fluorochemicals-- A P r e l i m i n a r y R e p o r t " , 'this p u b l i c a t i o n a n d the a t t a c h e d i n f o r m a t i o n r e f l e c t , in p art, 3M*s t e s t i n g and m o n i t o r i n g p r o g r a m designed to "...evaluate the overall impact of exposure of fluorochemicals on the health of workers". A l t h o u g h cnis p r e l i m i n a r y i n f o r m a t i o n c i t e d in (2 ) i n d i cates tret the subject chemicals are most probably animal t e r a t o g e n s , o ur employee records and the epidernioiog^IT''d a t a d e s c r i b e d in the a f o r e m e n t i o n e d publica.^{ajnIfi^i;?Sce t n a t t o d a t e n o h u m a n h e a l t h p r q &jUaasS. B f f i y e o e e n o b s e r v e d n o r d i s e a s e p a t t e r n s detecG<S'8r>a5fiia.ch a r e a t t r i b u t a ' o T a ^ o g ^ Environmental Protection Page Two N o v e m b e r 19, 1980 Agency related to iTuorochemical exposure. This publication also described the industrial hygiene measures undertaken in further reducing employee exposure, s^Ll t s ,, oi* p e r f l u r o a l k y l s u l f o n a t e s " i s a g e n e r i c cheraicarl n a m e "Tor "a l f i O i ' m i r r ^ E T i O T o l o g s l ^ S I c h c a n b e e x p r e s s e d b y t h e g e n e r a l f o r m u l a C n F 2 n + 1 SC>3 K . T h e s e h o m o l o g were reported on the TSCA inventory __ Homologous mixture, o r uhe c o r r e s p o n d i n g a m m o n i u m s alts, is c u r r e n t l y " sold" as"" varioiis p r o d u c t s c o n t a i n i n g fr o m 100 p e r c e n t "solids" to 0 5 ..Percent o f t h e m i x t u r e . T h e s e ' p r o d u c t s "' a n d " t h e i r "" customer uses""are"described as follows: FLUORAD1 Brand Etching Bath Additive FC-93 (correponding ammonium salt of the subject chemical) Electronic Manufacturing FLUGRAD^ Brand Fluoroch'emical Surfactant, FC-95 ... Chrome Plating FLUORAD^ Brand Fluorochemical Surfactant, FC-99 (corresponding amine salt of subject chemical) Chrome Plating LIGHT WATER^ Brand Aqueous Film Forming Foam FC-203 Fire Suppression L I G H T WATER** B r a n d A q u e o u s F i l m Forming Foam FC-203A Fire Suppression L I G H T W A T E R 1* B r a n d A q u e o u s F i l m Forming Foam, FC-206A Fire Suppression L I G H T WATER** B r a n d Aqueous F i l m Forming Foam Alcohol Type Concentrate, FC-600 Fire Suppression A F F F 6% C o n c e n t r a t e , F C - 7 8 0 B rare suppression Approximately ^ 0f perfluoroalkyl sulfonates are pro- ced p e r year domestically at our Decatur production facilit' l o c a t e d a t P . 0 . B o x 2 2 0 6 , D e c a t u r , A l a b a m a 3 5 6 0 2 , w i t h '18 ` r h l T JQt S -D t ^ n t l a l l y e x p o s e d o n a n i n t e r m i t t e n t b a s i s . Chemical reaction occurs in a closed system. Approximately t are P rocessed at our Chemolite p r o d uction s i t e l o c a t e d at H i g h w a y 6l & W a s h i n g t o n C o u n t y Rd. 19, a u ,, , M i n n e s o t a 5 5 1 3 3 , w i t h 3 7 e m p l o y e e s p o t e n t 4 a < M <581 exposed on an intermittent basis. , n a V c o ^ 9 1^ - 46 - c o m p a s Sanllte^ - Environmental Protection Agency Page Three N o v e m b e r 1 9 , 1980 c o _ o r m , by m id-December, all those customers and 3H employees who^haye a potential, through, certain uses anci/or processing, 0 1 .significant e x p o s u r e to the subject c h e m i c a l s . A t t h a t t i m e w e w i l l s u m m a r i z e t h e s e **!"* n e s SSotMpfilRe" Ur reP o m m e n d ^ i o n a for h a n d l i n g and us ini thes p r o d u c t s . w e a r e b y c o p y o f t h i s l e t t e r a d v i s i n g n t 'o s H of these new but preliminary teratogenic findings, "when - s _ g n i i . . c a n t n e w i n f o r m a t i o n b e c o m e s a v a i l a b l e t o ys V/e to advise these customers and employees accordingly.' '" ^^7*^ o u r o n g o i n g t e s t i n g a n d m o n i t o r i n g u r o g r a m in fluorochemicals, this is to a d vise the A g e n c y * o f the following work planned for Initiation in the near future: (1) A second teratogenic study designed to further evaluate these findings in both rats ana 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^Drovidino- a d ? ? i t i 2 e d v e r s i o n O f t h i s r e p o r t for t t e public n e ?n deleted from the confidential submission i n c o n s e q u e n t i a l i n f o r m a t i o n s u c h a s t h e n a m e s o f 3jyr employees for the purpose of protecting their privacy. FW C*- 9o 5S, " tSh:e 1 m iPx t/u rteUi5orf. " p oft? ar sTMs i1u mt C s ah letrse i no f lps e rbfalsueodr ooanl kfyele d i n g sulfonates described as a b o v e . When ( L f is complete, a final written report will be provided to EPA. In the interim, should additional correspondence' be necessary on this matter, please c o n t a c t : * Larry Magill . Manager, Regulatory Affairs Department Commercial Chemicals Division 3M 3M Center, 223-68-04 Saint Paul, MN 55144 Telephone: 612/733-7062 . Yours truly, V / > . / j v , L. D. D e S i m o n e Group Vice President cea Attachments - 47 c: Anthony. R o b b i n s , a . Director, NIOSH Park Lawn Building 5600 Fishers Lane Rockville, MD 20855 Company Sanitise Does cot contain TSCA C3[ November 12, 1980 ' cc: TO: FROM: SUBJECT: Teratogenicity of Two Fluorochemical Compounds in Rats (Study numbers 680TR0008 and 680TRQ010) Preliminary results from rat teratology studies on FC-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 appearances observed under the dissecting microscope varied from a slight dis coloration on the lens near _the anterior margin to a dark colored oval area, orten 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. - 48 lot contain TSCACBl .. Company Sambts- Doa3! TITLE : Pr otocol fo r Oral Teratoloqy study of Number 0680TR0008). ^ in (Experiment ("Guideline* fo r Stoerodnctic * * January' 1966 for Huaan Use"). Good Laboratory stody w ill b * ^ Saity Svaiuai:ion o f Drugs according to the 1973 Standard Operating P rc c e d u ^ ^ ^ ^ *"* ^ *ty 3vaiuaic" Laboratory's SPONSOR: 3* C cw srcial Chenical Division. S t . Paul, TESTING FACILITY: Safety Evaluation Laboratory St. Paul, Minnesota. STUDY DIRECTOR: . STAKT O P DOSISK. W d J u ly , 1980. test system. E l9hty-l9iit s.aually aatu r. ti- --a - fteeeeaaliee rraattea frca Charliee Rive'r tBiareesdaintegdLSapbroargauteo"ryCaww leilyl wderived , in hanging stainless gteei cacea ,,ith V % WiU *** houSPd In a temperature and h u e id i^ ? , i " eh floors and fronCs .m b H L . ^ f0? - Tllia B tral- f f e U , are raadUy . a i l ^ S p L f ! ? ,r i . dat* and * * mated ^ x r^ r rarsrs ^ test system " 2 TM , . ^ ^ 1 E ^ a - d hat be "" a ^ X ^ r S i . r W l9 M d CONTROL ARTICLE: Corn o i l ; TEST ARTICLE: FC-95. -- - - a conputer-qttn.ratiid ICAI" SPEd i w 2 " TM SbV S e tha start 0/ ^ " ^ co"positlon OTd Puriy ill h.. 2.T,," ' ^" DiXSAGE LEVELS AND EXPERIMENT DESIGN Th ' . Oil daily. The artJcle Wil 1 b<? upended in corn administered by oral int h *.su*pen8-on ""d control article will be qestation aeco ^6 ' '* <*' - 49 - inot contain TSCA CBl piiPanXSanvli-sc.Doe Dose Group High Mid Low Control Doso LeveI JO rog/Jcg/day 5 isg/kg/day 1 agAg/day 0 agAg/day Group Size 22 ? 22 ? 22 ? 22 ? The oral route of administration will be used because of tahoiin c o n t l l i n f r 13 raf io la b *la d 7C"9S * 11 absorbed. No contaminants are kncxm to interfere with tit* teat article. ^ WiiL f a cb3 rV9d ^ l y 2rca day 3 through day 20 of gestation for abnormal c lin ic a l aigns. Body weights w ill be ^Se^da cccocordrdiningolly 3u' si?n'g a cUo#ns1t5an*t"d*os2e0 v0oi luPar9onafc5yalnAdg thoef braodtsy Iittss contents will be examine00d *to* da2t0aamnidne: nOuVsabreireao'fctoorruposraandlutea oo.fZimipllaLntiatliolnZ sVit^es, puapndwerig?ht-anndu=bgreoroaofabnoraalitiea Aonr-o^Ii-e; T^ l t l Z l 'T r Z ^ L ^ V U * >- -- 1 am vi^rJ *il3" technique to determine any visceral abnoraalitiea using a diaaocting microscope. The aPPrXl2ataly two-thirds of the pupa wiU bTfixed in ethyl .!, Z letal aitar cl*arl" - data ANALYSIS AND PINAL REPORT: The proposed statistical methods to be used for analysis of the data are, Dunnetfs t test for dan and pun weights s^Tand f.TM*Ptinn n,ber of implantation t o T T / f i iea*' Chl 8qUarS f r ^ rc- t abnormalities, p u p e x a S n lt io n h ^ 1980) V i , , a l reP rt 18 2" 2 aonth8 aiter detailed completed (approxiaately fourth quarter. Date I kllklu Date Date y J z /c ''Date - 50 Company Sanitized. Does not contain TSCA CBI Report Number: Date; October 4, 1979 T IT LE ; Analysis o f Selected Decatur Employee Serum fo r Sulfonic and Carboxylic Fluorocheaicals . AUTHOR: ABSTRACT: The precenca ox -,,1 ^-' , using bobn uluctnon _ = 1U Microwave plasma dTe?te*ct?or methods - 51 - Report No. Data_____ _ V4V. _____ Subject: Characterization of Decatur RF Values R eq u esto r:-------- *------------------ Dept. Name Coaaercial .Chemicalsp -o j. No. P- 2 qu est D a ted -p cto b er 1979 Report: . . . 'Hie s e r u m o f 1 0 s e l e c t e d 3 M D e c a t u r e m p l o y e e s w a s s u b m i t t e d (A72754) i a J u n e 197 for analysis of organic fluorida levels and the total organic fluoride values ver reported in Anal y t i c a l Report 7194. Subsequent to that work, it became of latere co f u r t h e r c h a r a c t e r i s e t h e f l u o r i d e s e r u m v a l u e a n d a t t e m p t to i d e n t i f y the pres of 3M fluorochemicals. w Initial analysis (7/18/79) by the microwave plasma detector (MPD) established the presence of the expected perfluoro sulfonate and perfluorooctanoate anions in the s e r u m e x t r a c t o f .-- 25 and . -22. H o w e v e r , t h e o b s e r v a t i o n of p e r f l u o r o - s u l f o n a t e an i o n in che above extracts was unexpected and it therefore becam< n e c e s s a r y co s p e n d a d d i t i o n a l a n a l y t i c a l d e v e l o p m e n t t i m e ia o r d e r to m e a s u r e the: 3 fluorochemical3 in the same serum extract sample. D u r i n g this time, the method was evaluated on the electron capture (SC) gas chromatograph. The EC detector would be expected to have greater sensitivity than the MPD for these fluorochemics but with less specificity since the MPD can be operated on the fluorine channel eliminating any confusion by other EC active compounds. We selected che 5 highest fluorine c o n taining Decatur samples and analyzed the serum extract for perfluoro sulfonate, perfluoro sulfonate, and perfluor occanoace anion. W e used both che M P D and EC d e t e c t i o n systems and report the following values: Person -22 -23 -24 -2^ -26 RF (ppm) 10.1 5.7 9.4 11.8 4.1 Perfluorosulfonate 52 15% 5% 5% 10% % FOUNtP Perfluorosulfonate 60% 70% 80% 55% 65% - Perfluocooctanoate Tn r 30% 20% '15% 20% 25% 95% 105% *' ' 100% so% 100% coaduc!:ed oa a d e t e r m i n a t i o n (1 m l o f serum) d u e to limit, availability of sample and time and represents our interpretation of che average - e -C and MPD values. No internal standard was used; expect 10-20% accuracy A R 6962, 7028, 7194 P r e v i o u s l y identified as - 2 1 (Z) d u e to m i s - l a b e l i n g b y m e d i c a l p e r s o n n e l . Company Sanitized. Dees not contain TSCA C% 52 L ..o. 7230 October A, 1979 Pass 2 The above resulcs are necessarily somewhat approximate due co Che lack of aa iacer standard and c o n t i n u a l ref i n e ments in the deri v a c i x a t i o n and GC of the correspond! methyl ester. Sufficient sample remains for additional analysis. Further improve in the a n a l y s i s a r e snd w i l l c o n t i n u e to be m a d e aa time permius. tr>cujht -- best to report chase value now, especially since we identified the presence of perfluoro s u l f o n a t e i n h u m a n s e r u m e x t r a c t a n d n o t o x i c o l o g y s t u d i e s to dace have been completed on this fluorocheaical. Three years ago (10-13-- 76) w e reported that Decatur employee . aarum extract contained perfluoro sulfonate (baaed on and a minor fluorine containin component n o w identified as perfluorooctanoate. T h e refore, the presence of per fluoro sulfonate in this individual haa been known for scae time. Now, with improved analytical methods, w e have greater sensitivity and can determine tha presence of perfluoro sulfonate in addition to perfluoro sulfonate anion / 53 - con inollco n * O A C B ' od- '4A / > D IP LO M A T E : AMERICAN C O LLE C E O F VETERINARY OPH THALM O LO G ISTS ATTACHMENT 2 JA M E S M. C L IN T O N . V. M. D. ANIMAL EYE CLINIC AT SOUTH JERSEY ANIMAL HOSPITAL ROUTE SA 1 ABOVE CHURCH ROAD - P. O. BOX 115 MEDFORD. NEW JERSEY 0 6 0 5 5 T elepho ne (6 0 9 ) 6 5 4 -0 3 0 4 d l STAPLES .IUL 2 2 1981 RECEIVED Haskell Laboratories SAlternates Exam Date: 7 July 1981 Robert Stapels, Ph.D. Initial Ophthalmoscopic Examination sol, 3n S v f l r 1 s 7 o n s *7ere i d e n t i f i e d i n f e m a l e r a t s 3 0 6 5 1 4 , 3 0 6 5 1 8 3 0 6 4 4 0 a n d l n mf l s r a t s 3 0 5 2 7 5 , 3 0 5 2 8 9 , 3 0 5 1 8 4 a n d 3 0 4 8 4 6 . T h e y T - e - T' o T * l e n b u r g ? ra " " * * * " d * " * " " * * b y t e c h n o l o g i s t s S. V i v e n d i . fSxdthcofingrstud" e " ^ " P ^ y nonnal and J a m e s M. Clinton, V.M.D. - 54 - Company San Sized. Doss noi contain TSCA CB U iN I U U j iS J L fiA O n ALNU UEj v E i L i V r r u u * u l j m v i r u j i ' i x HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ATTACHMENT 3 E.STAPLES AUo 2 i1981 August 19, 1981 MEMORANDUM TO : R. E. Staple /< FROM: W. D. Kerns : l 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 55 - ATTACHMENT 4 R. E. STAPLES JAM 2 9 *to02 m c E iv m DATE: TO: FROM: SUBJECT: January 29, 1982 R. E. Staples y' J J ^ D ^ K e r n s / t } lb 1\ ----- Three fetuses were examined microscopically (12-12, 13-6, 30-6) for a histomorphological 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 Company Sanitized. Doss not contain TSCACBI - 56 - ES-3606 HASKELL LABORATORY ATTACHMENT 5 cc: October 5, 1981 MEMORANDUM TO: R. E. Staple.? FROM: 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 anterior lens capsule in some cases. In others, the lens material in this area had been fractured by the microtome. Six additional fetuses 'ere evaluated for the presence of microscopic lesions. Five imals 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. WDK:1jm - 57 - Company Sanitized. Does not contain TSCA CBJ TABLE 1 Dam Code Fetus . Number 2 1, 5, 9 4 3, 5, 9 5 3/ 4, 7 6 1, 8, 13 7 If .5, 11 8 1, 5, 11 9 3, 7, 13 10 . 4, 7, 12 14 5, 8, 11 15 3, 5, 15 ^ / 5,. 9, 15 18 4, 9, 15 19 .1/ 8, 13. .21 3, 5, 7 22 I# 4, 11 23 1, 5, 9 24 4, 7, 11 25 1, 7, 11 26 5/ 9, 13 29 4 r 9, 15 30 If Ilf 15 32 5, 7, 9 33 3, 8, 13 . Dam Code Fetus Number 34 3, 7, 12 37 3, 1, 9 38 1/ 8, 11 39 , If 8, 11 40 5, 11, 15 41 3, 8, 17 42 1, 7, 12 43 5, 11, 15 45 4, 13, 15 46 3 49 1, 9, 12 50 4, 9, 11 51 If 7, 13 53 5, 9, 11 55 1, 5, 12 57 I, 9, 13 59 3, 9, 13 60 5, 9, 10 61 . 4, 9, 15 ' 62 3, 11, 13 64 3, 7, 13 65 If 7, 11 66 4, 7, 11 - 58 - Company Sanitized. Doss not contain TSCA CB1 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 - gom panySaniiastl- Goes not contain TSCA C 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 refractile particulates Unilateral alteration (artifact) with brownish-green refractile particulates 60 - Conipany SanUjzed. Doss not contain TSCA CE1 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE cc : October 1, 1 9 8 1 MEMORANDUM TO: N. C. CHROMEY FROM: C. L. LAMONTIA SUBJECT: HISTOLOGIC PREPARATION AND C-8 TERATOGENICITY STUDIES: Please have these two groups of fetal specimens (attachment) embedded, sectioned, and examined. Embed onehalf 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/mle Attachments (2) . Company Sanitized. Does no! contain TSCA CB* 61 SPECIMENS TO BE PREPARED F O R E V A L U A T I O N BY W. D. K E R N S The following fetuses had no eye alterations detected during external examination: Dam Code 68 54 58 58 Fetus Number 1 7 9 11 Observations during Head Examination Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration CLL/as 9/30/81 - 62 - Company San;; scL Do-33 not contain TSCA CBJ SPECIMENS TO BE PREPARED F O R E V A L U A T I O N B Y W. D. K E R N S The following fetuses had no eye alterations detected during external examination: Dam Code Fetus Number Observations during Head Examination 35 1 Faint brown area in anterior half of lens of left eye ^0.5x0.4mm. 35 3 No alterations detected 35 4 Faint brown area in anterior half of lens of left eye ^0.6x0.4mm 35 7 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 '''0.4x0.4mm, 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. v 0 .4x0.4mm, right 'vO.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 63 company. , c o n t a i n ^ 0" Dess*10' SPECIMENS TO BE PREPARED FOR E V A L U A T I O N B Y W. D. K E R N S (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 'vO.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 'vO.Smm 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 browncvery light brownclight brown CLL/as 9/30/81 - 64 :d. Ocas not contain TSCA CJ. Company Sami R. 6. STAPLES 1881 C-8 AND ALTERNATES: CODE FOR ROUTINE TERATOGENICITY TEST IN RATS 3Y GAVAGE yen Corn Oil (5 ml /kg) 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 C-8 (100 mg/kg) Dam Animal Code Number 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 /tw- CLL/mle 8/17/81 a Females without dam codes died or were sacrificed before Dav 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 notconiato TSCAOBI Company Saniiis- Ocas 65 - C-8 AND ALTERNATES: CODE FOR ROUTINE TERATOGENICITY TEST IN RATS BY GAVAGE C-9 (100 mg/kg) Dam Animal Code Number 1 306340 11 306420 20 306439 31 306516 36 306367 48 306412 56 306421 Cadi Dam Animal Code Number 3 306355 12 306484 13 306503 27 306342 44 306390 47 306334 58 306353 250 mg/kg) Dam Animal Code Number 54 . 306373 -- a 306354 -- 306486 -- 306469 -- 306346 -- 306551 -- 306338 1 (50 0 mg /kg Dam Animal Code 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/mle 8/13/81 66 Company Ssntiized. Doss not contain TSCACB (am t HASKELL LABORATORY MEMORANDUM ATTACHMENT 6 December 18, 1981 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. WDKrwfd 67 - Company Sanitized. Does not contain TSCA CBt SPECIMENS TO BE PREPARED F O R E V A L U A T I O N BY W. D. K E R N S The following fetuses had no eye alterations detected during external examination: Dam Code 68 54 58 58 Fetus Number 1 7 9 11 Observations during Head Examination Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration Not sufficient discoloration to be considered an alteration CLL/as 9/30/81 - 68 :2Cd. Does not contain TSCA CBI Company Sar SPECIMENS TO BE PREPARED F O R E V A L U A T I O N B Y W. D. K E R N S The following fetuses had no eye alterations detected during external examination: Dam Code Fetus Number Observations during Head Examination 35 1 35 3 35 4 35 7 Faint brown area in anterior half of lens of left eye ,v0.5x0.4mm. No alterations detected Faint brown area in anterior half of lens of left eye ^*0.6x0.4mm 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 35 11 52 1 52 3 52 7 Light brown area in anterior half of lens of right eye ^0.6x0.2mm Very light brown area in anterior half of lens of both eyes; Left ^0.4x0.4mm, right ^0.4x0.4mm. Light brown area in anterior half of lens of both eyes; Left ^0.8x0.5mm, right ^0.6x0.4mm Light brown area in anterior half of lens of both eyes; Left ^0.5x0.3mm, right ^0.5x0.3mm Light brown area in anterior half of lens of both eyes; Left. ^0.4x0.4mm, right 'x-O.5x0.3mm ' 52 8 Very faint brown area in anterior half of' lens of both eyes; Left ^0.4x 0.4mm, right M).5x0.4mm Company Sar 69 - Sd. Does not contain t sc a c b i SPECIMENS TO BE PREPARED F O R E V A L U A T I O N B Y W. D. K E R N S (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 'vO.6x0.4 m m , right ^0.6x0.4mm No alterations detected Light brown area in anterior half of lens of both eyes? Left ^0.3x0.3mm, right v 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 v 0 .5x0.5mm . Note - Alterations listed by severity: Very faint brown<faint brown<very light brown<light brown CLL/as 9/30/81 70 - CoW? -<&V.s_?n ea**`T * * e* , r - 3snl E S -3253 REV. 6-62 DOIV'T SAY I T --W R ITE IT TERATOLOGY SECTION FILE - MEMO T O __________________________________________________ A T ___ PROM R. E. STAPLES ATTACHMENT 7 DATE 1 0 / 3 0 / 8 1 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. He also aske_d about the findings on the alternates and in particular, I 1slaved that the dose levels selected forboti^Tne' were too h i q h a n ^ a s a result, no fetuses were obtamec from the n H H H H U n d only three litters were obtained from the m H ^ j f t e v e n though exposure was stopped before the comple^or^oi^tne 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/mle Company Sanitized. Does not contain TSCA CBi - 71 TH E JOB YO U SAVE M A Y BE YOUR OW N-- P.I.P. 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 C-8 435 691 300 550 m 2250 1500 3000 1500 7500 4000 Therefore, on the basis of demonstrated toxicity, the relative ratios were about as follows: Toxicity Ratios 11 55 15 Initial dose levels tested ' 150* (2 rats/group) 150* 750* 750* 2250** 2nd Pre-test (2 rats/group) 100 100 500 500** 1000** Used for the main study (7/23/81) 200 100 500 250** 500** 1st Additional test (4 rats/group), Dosed for 5 days 125* 250* 2nd Additional test (4 rats/group), Dosed for 10 days 501 1501 Estimated maximum tolerated dose levels for 10 days of dosing 35 100 ** severe toxicity including deaths * decreased body weight gain and adverse clinical signs body weight gain still too low but np adverse clinical signs 72 - Company 5a; contain TSC ACBt Doss net