Document 3xbYjdeMn7d4gz4BeM2vB6zO

X EN0 S Ammm~ E**@ consultationdnrugsafetayssessment May 24, lSS1 CIAI/ Mr. C.W. Olson Group Compliance Manager Minnesota Mining and Manufacturing Company 3M Center, Bldg. 230-GL-04 St. Paul, MN SS14-1000 Re: FM-3924 - Response to FDA Questions of December 10, 1330 (Food Additive Petition Nos. OB4lS7 and OB4206) Dear Craig: The Division of Toxicology in the Center for Food Safety and Applied Nutrition at the Food and Drug Administration has requested further information relating to a RIKER Laboratories toxicity study entitled TWO YEAR ORAL (DIET) TOXICITY / CARCINOGENICITY STUDY OF FLUOROCHEMICAL FM-3324 IN RATS CRIKER Exp No 0281CROO12, April 1SB1/MaW 1SS3). The litany of questions raised by the FDA are directed toward gaining further information on rat liver changes observed during the course of the study and coordinating the original histopathologic results with the diagnostic criteria published in 1986 under the auspices of the National Toxicology Program, National Institute of Environmental Health Sciences. In addition, the FDA also requested more information on; post mortem procedures, gross/microscoPic comparisons, computer generated tissue comparisons and laboratory generated historical rat control data on hepatacellular tumors. Each question has been addressed and a response documented based on a review of; the original report, the NTP reference (Tcxical - Pathol 14C2):263-273, 1966), existing data files at RIKER and XENOS, and direct communication with the original reviewing pathologist, Dr. R. Geil. The documentation resulting from this review is enclosed. It has been possible to compile essentially all of the information requested by the FDA and the results do not change in any way, the original diagnosis and interpretation of the liver findings as presented in the final report by Dr. Gail. The FDA request to provide a separate Ccomputer generated) gross/microscopic comparison is not possible, but also not really necessary. Dr. Gail has presented both gross and microscopic findings an each individual animal histapatholcgW sheet in his original report. In addition, Dr. Gail has also included summary incidence tables for both macroscopic and microscopic Findings. P.O.Bo.-.c5008 VorthBranchN,ew Jerse0y8876 2011526-2910 Mr. C.W. Olson May 22, lSS1 Page 2 Please critically review the enclosed material and if any of the responses are not clear, let me know and corrections can be made very quickly. With best personal regards, I remain, Sincere ours, Conrad 0. King, D.U.M., Ph.D. Consultant to 3M Enclosures 3M RESFONSE TO FDA LETTER OF DECEMBER 10, 1990 Food Additive Fetition Nos. OEqlS7 AND OBq2OS Toxicology Questions 1. Additional examinationlinformation on the occurrence of the liver lesions in this study particularly in light of the recent Hational Toxicology Program's classification scheme of liver lesions in rats (Maronpot et al., Hational Toxicology Program, Homenclature for Hepatoppoliferative Lesions in Rats, Toxicologic Pathology, V-ol. 4. Ho. 2 (1986), pages 263-273. Pi-ease prov ide a discussion of the pathogenesis of that of hyperplasia (ohyperplastic nodule') and other non-neoplastic lesions, in relation to the neoplastic changes. Your submission should also include the basislsupport for the overall interpretation of the liver data in the study. The pathogenesis of the hepatocellular proliferative lesions in the long term rat study under review is best understood when placed into perspective with metabolic and toxicologic profiles of this class of compounds. The test article in the two year carcinogenic assay was FM-392Lt CN-Ethyl FOSE, Technical Grade, 66%) The absorption of N-Ethyl FOSE-lLfC:C2-N-EthylperFlucrooctanesulfonamido ethanol, 96%, CFM-3Li223) administered to rats as a single dose in the diet, was 60% Analytical CNNR) results obtained From extracted rat liver two days post-dose, contained perfluccoactanesulfonate at Li.E% cE the original single dose of N-Ethyl FOSE-lLiC. However, approximately 10% of the original carbon-llt administered was still present in the liver 32 days post-dose. Elimination of the carbanILi was primarily via the Feces; i.e. , 62% at 32 days. (3M Internal Report, J.D. Johnson, Fl:-Experiment 11, 1381) Multiple dosing by the same route of administration for 7 days was performed with N-Ethyl FOSE-14C. Feces and urine were collected For an additional 21 days at which time, the rats were sacrificed and tissues collected. One metabolite, 2-N-ethylperFluoroactanesulfanamida acetic acid, was extracted and identified in the Feces pooled from the 2Lt-LfEp3ost-dose collection period. C3N Internal Report, J-13. Johnson, FC:-Experiment 12, 1961) These experiments imply that there is enteco-hepatic recirculation of test substance and combined with morphologic evidence produced in the multiple dose shorter term toxicity studies, suggest that the liver is actively involved in the detoxification process. 3m FDA Fatition Nos. OB'il97 & 081-iEO6 FAGE a Toxicology Questions In a 30 day cat toxicity study, N-Ethyl FOSE CFM-3422) given in the diet established the liver as the primary target organ. At concentrations cE 30, 100 and 300 ppm, there was a dose dependent decrease in mean body weight gains. Higher dosage groups of 1,000, 3,000 and 10,000 ppm were too toxic and these groups were terminated prior to the termination cE the study. At 100 and 300 ppm, terminal male liver weights were significantly increased in both absolute and relative values while the Female values were increased in the relative comparison only at 300 ppm. At necropsy, there were gross liver lesions at 100 and 300 ppm, but not in the 30 ppm group. Histapathologically, hepatocytic hypertrophy (megalacwtosis) was seen in all of the treated groups, however hyperplasia was seen only in the treated groups terminated early. Hepatocellular cytoplasmic lipid containing vacuales, chronic inflammatory cells, pigment in both hepatocytes and KupfEer calls and hepatocellular necrosis was observed in dosage groups at 100 ppm and higher. In the 300 ppm group, renal tubular nephrosis was a prominent Finding. Based on these findings, the dietary dosage concentrations selected For the rat 2 year carcinogenic study were 0, 10, 30 and 100 ppm in the diet. CFM-3422 90 Day Subacute Rat Toxicity Study, IRDC, Fraj. 137-086, 11/10/78) A corresponding 90 day oral (gavage) toxicity study in rhesus monkeys did not produce any evidence of hepatocellular damage. All of the above described studies were also performed with ammonium pereluarcoctanoate. All cE the rodent hepatocellular findings were essentially identical to those reported For N-Ethyl FOSE. Likewise, the rhasus monkey 90 day results were also similar; i.e., there was no evidence of hapatocellular damage. (Griffith, F.D. and Long, J.E., Am. Ind. Hyg. Assoc. J., ltl:S76-583, 1SE30) Having summarized the hepatic changes seen in the shorter term toxicity studies and comparing these findings directly with those reported in the 2 Wear oral Feeding cE FM-3924 CN-athyl-2-parEluorooctyl- sulfonamido ethanol; N-Ethyl FOSE, Technical), it becomes apparent that they are essentially identical. Furthermore, based an the absorption, distribution, metabolism and excretion data derived from N-EthUl FOSE rat studies, it is also clear that the compound has an affinity For the rat liver with a relatively long retention time. Finally, it may be assumed that the hopatacellular alterations are species (rodent) specific, since the chasus monkey did not develop similar lesions in 2 separate studies utilizing the same test sub- stances. Therefore, the pathogenesis of the hepatocallular proliferative lesions in rats treated with FM-3324 can be described as follows.. The absorption of the test article is achieved through the gastrointestinal tract via the portal vascular system into the liver. Hepatocellular detoxification processes are activated stimulating intracellular protein synthesis. Continued exposure of the liver to the xenobictic induce5 a chronic cellular stimulation, exceeding the limits of normal homeostasis, resulting in the Following reactive morphological changes: hapatocellular hWpectrophW Cmegalacytosis), hapatacellular vacualation 3M FDA Petition Nos. OB4197 & OB4206 PAGE 3 Toxicology Questions (fatty infiltration) and hepatocellular degeneration. This represents a typical First phase of the livers reaction to exagenous chemical stress. Most importantly, the cellular hgpertrophy seen at this time is considered to be a manifestation cE exaggerated intracallular metabolic activity, hence causing each 'turned an' liver cell to swell and look larger than an adjacent less active cell. In a great majority of these cases, the swelling is due to an increase in smooth endoplasmic reticulum associated with microsomal enzyme induction. The second phase which Follows continued hepatocellular activation, can be described morphologically as extensions of the above noted cellular alterations. As the metabolically overworked or 'turned on' hepatacytes wear out, the number aE degenerated or atrophied hepatocytes usually increase in a focal pattern of distribution. Either single call necrosis and/or apaptosis begins to become apparent. Also at this time, other focal sites of increased -intracal-lularactivity take on a difference histological appearance due to their reaction to the hematoxwlin (blue) and ecsin Cred) stain used to visualize the cells for microscopic examination: e.g., foci of cellular alteration - basophilic, ecsinaphilic or clear call reactions. As more of the worn out cells shrink (atrophw) and die (necrosis), the rat liver (in contrast to other species cE animals, including the human) replaces the empty spaces with new hepatocgtes (regenerative hyperplasia) and to very limited extent, fibrous connective tissue CEibrosis). Several other cellular events may also be observed at this phase. Cystic degeneration can develop producing the visual appearance cE a clear-spaced sponge, hence the term spongiosis hapatis. The etiology cE this change may be attributed to alteration in the perisinusaidal Cat-staring cells. Pigment deposits in metabolically induced rat livers is most commonly endogenous and represents accumulations of normal degratory products in the Form cE hematin or lipoFuscin. Finally, vascular ectasia (telangiectasis) or focal dilation and congestion cE hepatic sinuscid5, may occur as a result aE venous Flow restriction due to local sinusoidal compression by hgpertraphied hepatocytes. The last phase of the pathogenesis involves the interpretation of the significance of increased hepatocytic proliferation in rat toxicity assays. HepatocWtic proliferation is a very predictable reaction in the rat and mouse liver to a nonlethal hepatatoxic insult. Since this finding represents a major interpretative problem to differentiate hgperplasia from neoplasia, an International Symposium entitled "Rodent Liver Nodules: Significance to Human Cancer Risk" was convened by the Society of Toxicologic Pathologist in 1982 to specifically address this issue. As a result of this Symposium, the term necolastic nodule was eliminated as an acceptable diagnosis and a clear distinction between hwperplastic and neoplastic nomenclature was recommended. The proceedings of this Symposium CTaxicol. Pathal10: 1-227, 1362) provide excellent references an this subject. The consensus derived from the Symposium provided the scientific opinion from which the NTP nomenclature guideline were later promulgated. CToxicol. Pathal. lLi:263-273, 1966) Pictorial representations of both the non-neoplastic and nacplastic liver lesions described above 3M FDA Petition Nos. 084197 & OBLi2CE PAGE 14 Toxicology Questions are admirably presented in a text entitled "Current Histapatholo@N, Ual.13, Atlas of Experimental Toxicological Pathology. C. Gopinath, D.E. Prentice and D.J. Lewis, MTF Press, Norwell, MA, 1987.@ Within the context of this experiment, hepatacellular hyperplasia should be considered as a reparative, regenerative response that is a sequela of the replacement process of worn-out or necrotic hepatocytes. When a single foci of hepatocytic regeneration is established, there is a tendency for the hyperplastic reaction to enlarge Erom a central core, hence forming what has been referred to as hyperplastic nodules. IF this hyperplastic growth does not exceed the defined morphologic limits imposed by generally accepted practice, i.e., NTP guidelines, then it is considered to be non-neoplastic. However, if in the judgement of the reviewing pathologist the lesions exceed that defined Ear hyperplasia, then it will be classified as either a benign or malignant neoplasm. Likewise, the interpretation of bile duct hyperplasia is classified in a similar manner. The rationale Ear accepting the interpretive statements above are based on the fact that the non-neoplastic proliferative hepatocytic reactions observed in rats after 2 years cE continuous dietary Eeeding of FM-392Li, were essentially no different than those seen in rats treated in a similar manner For just 90 days. The incidence of liver neoplasms observed in treated rats From the 2 year Feeding study is considered to be extremely low. This would not be expected based on the Einding of continuous hepatacytic proliferation through the geriatric period cE the rodents liee-span. When any cell population is induced to divide abnormally over an extended period of time, particularly in old aged rats, the probabilitg of producing a neoplastic clone is greatly enhanced. In this study, a statistically non-significant increase in benign hepatocellular adenomas were Eound in Li/50 (8%) high dose Female rats, but not in any cE the remaining treated and/or control animals. However, the historical control values for this finding ranged from 0-6%. The incidence of the malignant form of the tumor, hepatocellular carcinoma, was found in both control and treated groups, also without any statistically significant differences. In summary and based on the information presented above, it can be stated that FM-3924 administered orally to rats for a period of 2 years and at the dosage levels given, has the potential to metabolically stimulate rat hepatacytes to display both degenerative and chronic non-neoplastic regenerative cellular changes with no signieicant evidence Ear the induction of hepatocellular neoplasia. 2. A glossarvldescriptioonf the histopethologicterms used in describingliverlesionsin rats. In Table 1, a listing of the original hepatocellular histapathologic terms used in the final report for the FM-3324 2 year rat study are compared to the nomenclature proposed by the National Toxicology Pro- gram CTaxical. Fathal. 14C2): 263-273, 1986). 3M FDA Petition Nos. OB4197 & OB4206 FACIE s Toxicology Questions The paper by Maranpot, et al., clearly defines and describes all of the lesions reported by Dr. Geil in the 2 year study. The terms used by Dr. Beil are not identical with those proposed by the NTP. However, from a diagnostic and interpretative point of view, they are identical. 3. Your submissionshould include the followinginformation concerningthe examinationof the livers: a. The scope of the gross examinationfexternaland cut surfaces). b. Method of samplingthe livers fro& al-1a.nimalsin the study. c. Informationon any additionalsectionstaken from gross lesions observedat the time of necrapsy. d. Husber of liver sectionsexaminedmicroscopically fro& each animal. e. Correlationof gross findingswith the microscopic lesions (for each animal in the study). f. An explanationfor those cases in which the gross findingsare not accountedfor by the microscopic findings. 9. If the testing laboratory'scomputersystem is able to provide a correlationreport (gross findings versus microscopicresults),the informationshould be made availablefor furtherassessmentof the liver lesions. a. For each animal necropsied, a complete external and internal gross examination was performed under the supervision of the attending pathologist. All abnormalities found were confirmed by the attending pathologist, recorded and if none were seen, a recording of NUL for that organ and/or tissue was made. b. Livers were First examined in situ, removed intact and examined, at least two lobes were examined an cut surface, two sections one from the right lateral lobe and one from the medial lobe were taken For- histopathologic examination. In addition, under the direction of the attending pathologist, one or more sections were taken from all gross lesions noted in the gross observations. The size of tissue masses (suspected tumors) were recorded in cen- timeters giving either the diameter or its outside dimensions. 3M FDA Petition Nos. 084197 & OB420S PAGE Toxicology Questions c. All information on the relatively Few supplementary tissues taken from gross lesions, are containedin the individual pathology report sheets. Since gross and microscopic findings are correlated on these same sheets, any aberrant Finding specifically related to the gross lesion can be identified. Since each microscopic lesion is counted once for each animal, regardless of identiEUing a similar lesion in other sections From the same organ, the Final incidence table will identify unusual or aberrant Findings. A current review of the individual animal pathology sheets allowed for the correlation of two or more liver lesions which may appear in concert. The review did not reveal any remarkable correlations which were different From those documented in the pathology incidence tables. d. Two liver sections per animal was the standard procedure established For the study. Variations from this procedure are listed in Table 2 as total liver sections histopathologicallu examined by group as well as the identification of animals were the number of liver tissues varied from the standard protocol. e. The Final study report contains the correlation of gross findings with the microscopic lesions For- each animal in the study. This comparison is presented on the individual pathology sheets. E. There were no recognized instances n.oted were it would be necessary to question the absence of a microscopic liver lesion to correspond to a recorded gross finding. A major reason for the efficiency aE the gross/microscopic correlation process in this study, was that a qualified pathologist was present at the time the gross observations were made. g. It would appear that the reason to have a computer generated gross to microscopic correlation report would be in the absence of such a comparison. Since this comparison exists in the report for each animal, it would appear that any additional documentation would be redundant. 4. Information should be provided on the historical control data an hepatocellulartumors (adenona, carcinoma). hyperplasia,hyperplasticnodules and other hepatocellular proliferativelesions in rat of the strain used in the present fluorochemicalstudy, col- lected at the sponsor's laboratoryover the past five years. Provide the dates during which each study was conducted as well as the age of the animals when sac- rificed. No additional 2 same Facilities two similar two current study. Wear carcinogenic studies have been performed in the since the completion of the FM-3924 assay. However, year tests were completed prior to the start of the 3M FDA Petition Nos. OB4197 & 094206 PAGE 7 Toxicology Questions same source and were housed in the same facility on the 3M campu@.. The studies were initiated in 1977 and 1979, respectivelw, and the rats were approximately 6 weeks of age when placed on test. The control data for liver proliferative lesions from these two studies are as follows: Study No. 277CRO01 Necropsy 1975 Males Female Hepatocallular adenoma Hapatocellular carcinoma 0/50 1/SO 2/SO 0/50 Study No. 27SCRO22 Hepatocallular Hepatocellular Hepatocallular adenama carcinoma huperplastic nodule NecrapsW malf3s 3/50 O/SO 1/SO 1981 Female 1/50 2/50 0/50 This will complete the response to the toxicologic questions, however if any of these answers are unclear or-if any of the above questions need further explanation, we will be available at your convenience, to discuss these or any additional experimental or interpretive issues which may be relevant to your review. Conrad D. King, O.V.M , Ph. May 24, 19 TABLE 1 TWO YEAR STUDY ORAL COIET) TOXICITY/CARCINOGENICITY OF FLUOROCHEMICAL FM-3324 IN RATS April lgel / May 1563 !."71assarouf Rat liver HistoDatholonic Terms Orininal vs NTP Nomenclature HON-HEOPLASTICTERMS ORZSZHAL DZASNOSZS atrophy Cystaid degeneration Fibrosis Hematopoiesis,extrazedullary Hepatocyte alteration, basophilic Hepatacyte alteration,easinaphilic Hepatocyte alteration, vacuolated Hepatocyte vocuolation Hyperplasticnodule Kegalocvtosis Mitotic activity, increased Necrosis Pigment Pigment, brown Piasent, hepatocyte Pigeent, Kupfer cell Portal bile duct proliferation Portal sonanuclear cell infiltrate Telanciectasis NTP* NOMENCLATUREIDESCRZPTZON # Hepatacellulardegeneration # Cystic degenerationospongiosis hepatis Zncreasein fibrous connective tissue Red blood cell production in the liver 0 Foci of cellular alteration?basophilic 0 Foci of cellular alterationfeasinophilic # Foci of cellular alterationpclear cell # cvtoplasnicvacualation # Hepatocellularhvperplasia # Hepatacellularhypertraphy Zncreasedcell division # Necrosis Nondescriptpigment Brown pignent; e.g., hemosiderin Pigment in liver cell(s) Pigment in phagocvticcell(s) # Bile duct proliferation Plgregatesof chronic inflammatorycells # Localizedvascular ectasia HEOPLASTZC TERMS Hepatocellalaradenona Hepatocellularcarcinoma Hemolysphoreticularneoplasm f Hepatocellularadenoza # Hepatocellularcarcinoma Disseminatedlysphoreticularcell tumor TABLE 2 TWO YEAR ORAL (DIET) TOXICITY/CARCINOGENICITY STUDY OF FLUOROCHEMICAL FM-3SEq IN RATS Number of Liver Sections_Examined Controls High Dose Mid Dose Low Dose One Year Interim Sacrifice ClS/sax/grp) Males Females 30 32 30 32 - - - Pmr- (3roUI3 Two Year Terminal Sacrifice (SO/sax/grp) Males 100 ill 103 lolk Females 106 lis 99 101 Number mE Liver Sections per Animal (<2 or >2) Sections/Rat 1 3 Group Control Mid Low Control Animal Numbers Mal Females 3SLiO 4848 3BLi2 3534 467S, 4915 4629, 4595, 4603, 4s4o 4sss 4619 High 3707, 3738, 3745, 37S3 3709 3739 3750 4775, 4771, 4781, 4B07, 4Bls 4782 4772 4784 4813 Mid Low Control High 3770, 3802 3808 3724, 3749 4879, 4BS1 LLSOS Lt622 4762,4804 4809, 4B2o Low 5 Low 6 High 3831 3815 ItBO2