Document 2ZBM0NV1GOgzM9Da82RDVvog

XENOS / nay 24, 1931 consultation in drug safety assessment nr. C.UJ. Olsan Group Compliance flanager ninnesota nining and Hanufacturing Company 3n Center, Bldg. 230-GL-04 St. Paul, nN 5514-1000 Re: F n -3 9 2 4 - Response to FDA Questions of December 10, 1990 CFoad Additive Petition Nos. 0B4197 and 0842063 Dear Craig: The Division of Toxicology in the Center Far 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 C0IET3 TOXICITY / CARCINOGENICITY STUDY OF FLUDRQCHEHICAL F n -3 9 2 4 IN RATS CRIKER Exp No 0281CR0012, April 1981/nay 19833. 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 informa tion on; post mortem procedures, grass/microscopic comparisons, com puter generated tissue comparisons and laboratory generated histor ical rat control data on hepatocellular tumors. Each question has been addressed and a response documented based on a review of; the original report, the NTP reference CToxicol. Pathol. 14(23:263-273, 19863, existing data files at RIKER and XEN0S, and direct communication with the original reviewing pathologist, Dr. R. Geil. The documentation resulting from this review is enclosed. It has been passible 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 pre sented in the final report by Dr. Geil. The FDA request to provide a separate (computer generated3 gross/microscapic comparison is not passible, but also not really necessary. Dr. Geil has presented bath grass and microscopic findings on each individual animal histopathology sheet in his original report. In addition, Dr. Geil has also in cluded summary incidence tables for bath macroscopic and microscopic findings. P .0 . Box 5008 .V orth Branch, New Jersey 08876 ooaoss 201/526-2910 fir. C.W. Olson May 22, 1331 Page 2 Please critically review the enclosed material and if any of the'res ponses are not clear, let me know and corrections can be made very quickly . With best personal regards, I remain, Enclosures Ph.D. 003089 RESPONSE TO FDA LETTER OF DECEMBER 10, 1930 Food Additive Petition Nos. 0B4197 AND 0B4206 Toxicology Questions 1. Additional examination/information on the occurrence of the liver lesions in this study particularly in light of the recent national Toxicology Program's classification scheme of liver lesions in rats <Maronpot et al., national Toxicology Program, nomenclature for Hepatoproliferative Lesions in Rats, Toxicologic Pathology, Dol. 4. No. 2 (1986),_ pages 263-273. Please provide a discussion of the pathogenesis of that of hyperplasia ("hyperplastic nodule") and other non-neoplastic lesions, in relation to the neoplastic changes. Your submission should also include the basis/support 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-3924 CN-Ethyl FOSE. Technical Grade, 86*) The absorption of N-Ethyl F0SE-14C C2-N-Ethylperfluoroactanesulfanamido ethanol, 96*. CFM-3422H) administered to rats as a single dose in the diet, was 60* Analytical CNMR) results obtained from extract ed rat liver two days post-dose, contained perfluorooctanesulfonate at 4.6* of the original single dose of N-Ethyl F0SE-14C. However, approximately 10* of the original carbon-14 administered was still present in the liver 32 days post-dose. Elimination of the carbonl4 was primarily via the feces; i.e., 62* at 32 days. C3M Internal Report, J.D. Johnson, FC-Experiment 11, 1981) Multiple dosing by the same route of administration for 7 days was performed with N-Ethyl F0SE-14C. Feces and urine were collected far an additional 21 days at which time, the rats were sacrificed and tissues collected. One metabolite, 2-N-ethylperfluoroactanesulfonamida acetic acid, was extracted and identified in the feces pooled from the 24-48 post-dose collection period. C3M Internal Report, J.D. Johnson, FC-Experiment 12, 1981) These experiments imply that there is entero-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. ^03090 3f1 FDA Petition Nas. 0B4197 & 034206 PAGE 2 Toxicology Questions In a 90 day rat toxicity study, N-Ethyl FOSE CFh-3422) given in the diet established the liver as the primary target organ. At concen trations of 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 of the study. At 100 and 300 ppm, terminal male liver weights were significantly increased in both absolute and rela tive 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. Histopathologically, hepatocytic hypertrophy Cmegalacytosis) was seen in all of the treated groups, however hyperplasia was seen only in the treated groups terminated early. Hepatocellular cytoplasmic lipid containing vacuoles, chronic inflammatory cells, pigment in both hepatacytes and Kupffer cells 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. CFH-3422 90 Day Subacute Rat Toxicity Study, IRDC, Proj . 137-086, 11/10/78) A corresponding 90 day oral Cgavage) toxicity study in rhesus monkeys did not produce any evidence of hepatocellular damage. All of the above described studies were also performed with ammonium perfluarooctanoate. All of the rodent hepatocellular findings were essentially identical to those reported for N-Ethyl FOSE. Likewise, the rhesus monkey 90 day results were also similar; i.e., there was no evidence of hepatocellular damage. CGriffith, F.D. and Long, J.E., Am. Ind. H y g . Assoc. J., 41:576-583, 1980) Having summarized the hepatic changes seen in the shorter term tox icity studies and comparing these findings directly with those repor ted in the 2 year oral feeding of FH-3924 CN-ethy1-2-perfluoroactylsulfonamida ethanol; N-Ethyl FOSE, Technical), it becomes apparent that they are essentially identical. Furthermore, based on the ab sorption, distribution, metabolism and excretion data derived from N-Ethyl 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 hepatocellular alterations are species Crodent) specific, since the rhesus monkey did not develop similar lesions in 2 separate studies utilizing the same test sub stances . Therefore, the pathogenesis of the hepatocellular proliferative lesions in rats treated with FM-3924 can be described as follows. The absorption of the test article is achieved through the gastro intestinal 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 xenabiotic induces a chronic cellular stimulation, exceeding the limits of normal homeostasis, resulting in the following reactive morphological changes: hepato cellular hypertrophy Cmegalacytosis), hepatocellular vacuolation 003091 an FDP Petition Noe. QB4137 a 084206 PAGE 3 Toxicology Questions Cfatty infiltration) and hepatocellular degeneration. This repre*sents a typical first phase of the livers reaction to exogenous chem ical stress. Host importantly, the cellular hypertrophy seen at this time is considered to be a manifestation of exaggerated intracellular metabolic activity, hence causing each 'turned o n ' 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* hepatocytes wear out, the number of degenerated or atrophied hepatocytes usually increase in a focal pattern of distribution. Either single cell necrosis and/or apoptosis begins to became apparent. Also at this time, other focal sites of increased *intracellular ac tivity take on a difference histological appearance due to their re action to the hematoxylin (blue) and eosin Cred) stain used to visu alize the cells for microscopic examination: e.g., foci of cellular alteration - basophilic, eosinophilic or clear cell reactions. As more of the worn out cells shrink (atrophy) and die Cnecrasis), the rat liver Cin contrast to other species of animals, including the human) replaces the empty spaces with new hepatocytes Cregenerative hyperplasia) and to very limited extent, fibrous connective tissue Cfibrosis) . Several other cellular events may also be observed at this phase. Cystic degeneration can develop producing the visual ap pearance of a clear-spaced sponge, hence the term spongiosis hepatis. The etiology of this change may be attributed to alteration in the perisinusoidal fat-staring cells. Pigment deposits in metabolically induced rat livers is mast commonly endogenous and represents accumu lations of normal degratory products in the form of hematin or lipofuscin. Finally, vascular ectasia (telangiectasis) or focal dilation and congestion of hepatic sinusoids, may occur as a result of venous flaw restriction due to local sinusoidal compression by hypertrophied hepatocytes. The last phase of the pathogenesis involves the interpretation of the significance of increased hepatocytic proliferation in rat toxicity assays. Hepatocytic 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 differen tiate hyperplasia from neoplasia, an International Symposium entitled ''Rodent Liver Nodules: Significance to Human Cancer Risk" was convened by the Society of Toxicologic Pathologist in 1S82 to speci fically address this issue. As a result of this Symposium, the term neoplastic nodule was eliminated as an acceptable diagnosis and a clear distinction between hyperplastic and neoplastic nomenclature was recommended. The proceedings of this Symposium CToxicol. Pathol. 10: 1-227, 1SS2) provide excellent references on this subject. The consensus derived from the Symposium provided the scientific opinion from which the NTP nomenclature guideline were later promulgated. CToxicol. Pathol. 14: 263-273, 1S86) Pictorial representations of both the nan-neoplastic and neoplastic liver lesions described above 003092 3n FDA Petition N a s . GE4197 S QB4206 PAGE 4 Toxicology Questions are admirably presented in a text entitled "Current Histopathalagy, Ual.13, Atlas of Experimental Toxicological Pathology. C. Gapinath, D.E. Prentice and O.J. Lewis, flTP Press, Norwell, HA, 1987. Within the context of this experiment, hepatocellular 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 from a central care, hence farming 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 nan-neoplastic. However, if in the Judgement of the reviewing pathologist the lesions exceed that defined far 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 for accepting the interpretive statements above are based an the fact that the non-neoplastic proliferative hepatocytic reactions observed in rats after 2 years of continuous dietary feed ing of Ffl-3924, 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 finding of continuous hepatocytic prolifer ation through the geriatric period of the rodents life-span. When any cell population is induced to divide abnormally over an extended period of time, particularly in old aged rats, the probability of producing a neoplastic clone is greatly enhanced. In this study, a statistically non-significant increase in benign hepatocellular adenomas were found in 4/50 C8k) high dose female rats, but not in any of the remaining treated and/or control animals. However, the historical control values for this finding ranged from 0-5?i. The incidence of the malignant form of the tumor, hepatocellular carcin oma, 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 FH-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 ta display both degenerative and chronic nan-neoplastic regenerative cellular changes with no signif icant evidence for the induction of hepatocellular neoplasia. 2. A glossary/description of the histopathologic teras used in describing liver lesions in rats. In Table 1, a listing of the original hepatocellular histopathologic terms used in the final report for the FM-3924 2 year rat study are compared to the nomenclature proposed by the National Toxicology Pro gram CToxical. Pathol. 14C23: 2S3-273, 19963. 003093 an FDA Petition Nos. 084137 & 084206 PAGE 5 Toxicology Questions The paper by Maronpat, et al., clearly defines and describes all of the lesions reported by Dr. Geil in the 2 year study. The terms used by Dr. Geil are not identical with those proposed by the NTP. However, from a diagnostic and interpretative paint of view, they are identical. 3. Your submission should include the following inform ation concerning the examination of the livers! a. The scope of the gross examination (external and cut surfaces). b. Method of sampling the livers from all animals in the study. c. Information on any additional sections taken from gross lesions observed at the time of necropsy. d. Number of liver sections examined microscopically from each animal. e. Correlation of gross findings with the microscopic lesions (for each animal in the study). f. An explanation for those cases in hich the gross findings are not accounted for by the microscopic findings. g. If the testing laboratory's computer system is able to provide a correlation report (gross findings versus microscopic results), the information should be made available for further assessment of 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 atten ding pathologist, recorded and if none were seen, a recording of NUL far 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 labs 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 CsuspeCted tumors? were recorded in cen timeters giving either the diameter or its outside dimensions. 003094 3M FDA Petition Nas. CIB4197 & 0B420B PAGE 5 Toxicology Questions c. All information on the relatively few supplementary tissues taken from gross lesions, are contained in the individual pathology re port 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 identifying a simi lar lesion in other sections from the same organ, the final inci dence 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 con cert. 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 estab lished for the study. Uariations from this procedure are listed in Table 2 as total liver sections histopathalogically 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. f. There were no recognized instances noted were it would be neces sary to question the absence of a microscopic liver lesion to correspond to a recorded gross finding. A major reason far the efficiency of 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 on hepatocellular tumors (adenoma, car cinoma). hyperplasia, hyperplastic nodules and other hepatocellular proliferative lesions in rat of the strain used in the present fluorochemical study, col lected at the sponsor's laboratory over the past five years. Provide the dates during which each study mas conducted as well as the age of the animals when sacrificed. No additional 2 year carcinogenic studies have been performed in the same facilities since the completion of the FM-3924 assay. However, two similar two year tests were completed prior to the start of the current study. 60J095 an FDA Petition Nos. 084197 & 0B4206 PAGE Toxicology Questions same source and were housed in the same facility on the 3M campush The studies were initiated in 1977 and 1979, respectively, 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 N o . 277CR001 Necropsy 1979 hales Female Hepatocellular adenoma Hepatocellular carcinoma 0/50 1/50 2/50 0/50 Study No. 279CR022 Hepatocellular adenoma Hepatocellular carcinoma Hepatocellular hyperplastic nodule Necropsy 19B1 Males Female 3/50 0/50 1/50 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. 003096 TABLE 1 TWO YEAR ORAL CDIET3 TOXICITY/CARCINOGENICITY STUDY OF FLUOROCHEMICAL Fd-BSEH IN RATS April 1981 / day 1983 Slossaru of Rat Liver Histopathologic Terms Original vs NTP Nomenclature NON-NEOPLASTIC TERMS ORIGINAL DIAGNOSIS Atrophy Cystoid degeneration Fibrosis Hematopoiesis, extramedullary Hepatocyte alteration, basophilic Hepatocyte alteration, eosinophilic Hepatocyte alteration, vacuolated Hepatocyte vacuolation Hyperplastic nodule Megalocytosis Mitotic activity, increased Necrosis Pigment Pigment, brottn Pigment, hepatocyte Pigment, Kupfer cell Portal bile duct proliferation Portal mononuclear cell infiltrate Telangiectasis NTP* N0MEHCLATURE/DESCR1PT10H * Hepatocellular degeneration * Cystic degeneration, spongiosis hepatis Increase in fibrous connective tissue Red blood cell production in the liver * Foci of cellular alteration, basophilic * Foci of cellular alteration, eosinophilic * Foci of cellular alteration, clear cell * Cytoplasmic vacuolation * Hepatocellular hyperplasia * Hepatocellular hypertrophy Increased cell division * Necrosis Nondescript pigment Broun pigmentf e.g., hemosiderin Pigment in liver cell(s) Pigment in phagocytic cell(s) * Bile duct proliferation Aggregates of chronic inflammatory cells * Localized vascular ectasia NEOPLASTIC TERMS Hepatocellular adenoma Hepatocellular carcinoma Hemolymphoreticular neoplasm * Hepatocellular adenoma * Hepatocellular carcinoma Disseminated 1ymphoreticular cell tumor 003037 TABLE E TWO YEAR ORAL CDIET) TOXICITY/CARCINOGENICITY STUDY OF FLUQRQCHEMI CAL Fn-3384 IN RATS k Number of Liver Sections Examined .,Pag_JSr-OUP Controls High Dose Hid Dose Low Dose One Year Interim Sacrifice CiS/sex/grp) Hales Females 30 3S 30 3E ---- Two Year Terminal Sacrifice CSO/sex/grp) Hales 100 Females 106 111 119 103 99 104 101 Number of Liver Sections. oer Animal C<S or >E) Sections/Rat 1 Grauo Control Animal Numbers DlS Females 3540 Hid 4848 LObi 384S 4875, 4915 3 Control 3534 46E9, 4640 4595, 4596 4603, 4619 High 3707, 3703 3738, 3739 3745, 3750 3753 4775, 47BS 4771, 477S 4781, 4784 4807, 4813 4819 Hid 3770, 380S 3808 Lauj 4879, 4891 4909 4 Control 46ES High 37E4, 3743 4 7 6 S ,4804 4809, 48E0 Law 3831 5 Low 3B1S 6 High G0093 480S