Document J3LKnQbyByJRJGer4rVVgMzQX

AFOT-OiasAttachments to Letter to C. Auer dated May 4, 2000 Perfluoroctane Sulfonate Studies Genotoxicity 1) Mutagenicity Evaluation of T-2014 CoC in the Ames Salmonella/Microsome Plate Test Final Report, Litton Bionetics Project No. 20838, Protocol No. DMT-100, February 20, 1978. 2) Memorandum Report from S. R. Rohfing to A. N. Welter, dated March 31 1977, on Results of the Ames Spot Test for Mutagenicity screening of various FCs, including Sample 12-583 which is FC-95, Notebook Reference 45867-24, 25. 3) Mutagenicity Test on T-6295 in an in vivo Mouse Micronucleus Assay, Final Report, Corning Hazleton , Inc. (CHV), CHI Study No. 17403-0-455, May 23, 1996, and protocol and amended protocol. 4) Final Report, Chromosomal Aberrations in Human Whole Blood Lymphocytes with PFOS, Covance Laboratories, Inc., Covance Study No. 20784-0-449, 3M Reference No. T-6295.18, October 25, 1999. 5) Final Report, Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures with PFOS, Covance Laboratories, Inc., Covance Study No. 20784-0-447, 3M Reference No. T-6295.19, November 9, 1999, and protocol. 6) Final Report, Salmonella-Escherichia coh'/Mammalian-Microsome Reverse Mutation Assay with PFOS, Covance Laboratories, Inc., Covance Study No. 20784-0-409, 3M Reference No. T-6295.17, November 5, 1999, and protocol. 7) Final Report, In Vitro Microbiological Mutagenicity Assays of 3M Company Compounds T-2247 CoC and T-2248 CoC, SRI International, SRI Project No. LSC-4442-016, 3M Refemece No. T-2247.1 (FC-99 Old Formula, L-4299 which is 50 % of the diethanolamine salt of perfluorooctanesulfonate in water), September 5, 1978. 8) Prof. Nicola Loprieno, "Evaluation of Mutagenicity Studies Developed on (PFOS) Perfluorooctane Sulfonate," prepared at the request of John L. Butenhoff, Ph.D., 3M Corporate Toxicology, January, 2000. 9) Final Report - Bacterial Reverse Mutation Assay of x-1, Hita Research Laboratories, Chemical Biotesting Center, Study Code K01-1802, 3M Reference No. T-6667.1 (FC-98, Potassium PerfluoroethyIcyclohexyl Sulfonate), September, 1996. 001794 i Q-! MUTAGENICITY EVALUATION OF T-2014 CoC IN THE AMES SALMOREUS/MICROSOME ^La t e TEST FINAL REPORT SUBMITTED TO: 3M COMPANY 3M CENTER SAINT PAUL, MINNESOTA 55101 m BIONETCS Litton SUBMITTED BY: LITTON BIONETCS, INC. 5516 NICHOLSON LANE KENSINGTON, MARYLAND 20795 LBI PROJECT NO. 20838 FEBRUARY 1978 001795 I. SPONSOR: 3M Company II. MATERIAL* A. Identification: T-2014 CoC B. Date Received: December 20, 1977 C. Physical Description: White powder II. TYPE OF ASSAY: Ames Sal monel 1a/Mi crosome Plate test IV. PROTOCOL NO.: DMT-100 V. RESULTS The results of this assay are presented in Table 1. VI. INTERPRETATION OF RESULTS AND CONCLUSIONS The test compound was examined for mutagenic activity in a series of in vitro microbial assays employing Salmonella and Saccharomyces indicator organisms. The compound was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats. The compound was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effect at the highest dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this compound was from 0.1 pg to 500 pg per plate. The results of the tests conducted on the compound in the absence of a metabolic activation system were all negative. The test with TA-100 was repeated at 100, 500 and 1000 pg's because of increased revertants observed at 500 pg dose level in the initial test. The repeat test was negative. The results of the tests conducted on the compound in the presence of a rat liver activation system were all negative. The test with TA-100 was repeated at 500, 1000 and 2000 pg dose per plate, because of increased revertants observed at 500 pg dose level in the initial test. The repeat test was negative. Information was supplied by the sponsor. If information was not indicated by the sponsor, N.I. was entered. CBB IO N E T 1C S Litton 1 C01736 VI. INTERPRETATION OF RESULTS AND CONCLUSIONS (Continued) The test compound, T-2014 Coc did not demonstrate genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions. m BIONET1C S Litton Submitted by: Study Director D. R. Jagannath, Ph.D. Section Chief Submammalian Genetics Department of Genetics and Cell Biology -7? Date Reviewed by: and Cell Biology 2 V. RFSlILTS TABLE 1 A. NAME OR CODE DESIGNATION OF t h e t e s t COMPOUND: R. SOLVENT: DMSO C. TFST INITIATION DATE: DEC. 28. 1977 NOTE: CONCENTRATIONS APE GIVEN IN MICROLITERS <UL> T-2014 COC OR MICROGRAMS (UG) PER PLATE. R E V E R T A N T S P ER P L A T E >1H TEST SPECIES TISSUE TA-1535 TA-1537 TA-1538 TA-98 100 NONACTIVATION 12 12 12 12 12 DA 1 SOLVFNT CONTROL POSITIVE CONTROL TFST COMPOUND 0.100000 UG UG1 . 0 0 0 0 0 0 10.000000 UG 100.000000 UG soo.oooooo UG 1000.000000 UG ACTIVATION --- --- -- -- -- ... -- -- -- ---- --- 19 AIO 1A 1R 11 13 18 10 19 A3 180 198 87 52A 1555 1198 1258 8A0 1A 2A A6 212 111 10 2A Al 193 87 11 20 31 210 93 8 1A 35 223 176 92 10 18 37 275 1Al 98 92 SOLVENT CONTROL POS1TJVF CONTROL* TEST COMPOUND 0.100000 UG 1.000000 UG 10.000000 IIG 100.000000 UG soo.oooooo UG 1000.000000 UG 2000.000000 UG RAT LIVER RAT LIVER RAT LIVER RAT LIVER RAT LIVER RAT LIVER RAT LIVER RAT ' LIVER RAT LIVER * TRY* CONVERTANTS PER PLATE TA-1*35 TA-1S37 TA-1538 TA-98 TA-100 04 solvent FMS DM NF NF FMS EMS DMSO 10 UL/PLATE 10 UG/PLATE 10 u g /p l a t e 10 UG/PLATE 10 UL/PLATE 10 UL/PLATE 50 u l /p l a t e 20 12 2A A6 282 233 963 872 20 13 27 A6 30 16 20 39 2A 22 23 A2 19 1A 2A A9 15 13 27 3A ** Ta -1535 Ta -1537 TA-1538 Ta -98 Ta -100 04 SOLVENT ANTH ANTM ANTH ANTH ANTH OMNA DMSO 2.5 UG/PLATE 2.5 UG/PLATE 2.S UG/PLATE 2.5 UG/PLATE 2.5 UG/PLATE too MICROMOLF5/PLATE 50 UL/PLATE 277 167 138 1A26 189 23A 167 2A8 138 288 1A7 271 1A8 291 157 16A 79 78 01798 CO PROTOCOL 1. PURPOSE The purpose of this study was to evaluate the test material for genetic activity in a microbial assay with and without the addition of mamma lian metabolic activation preparations. 2. MATERIALS A. Indicator Microorganisms A description of strain verification is given in Standard Operat ing Procedure on page 10. Salmonella typhimuri um TA-1535 TA-1537 TA-1538 TA-98 TA-100 Saccharomyces cerevisiae D4 B. Activation System 1. Reaction mixture ________ Component Final Concentration/ml TPN (sodium sait) Glucose-6-phosphate Sodium phosphate (dibasic) MgCl2 KC1 Homogenate S9 fraction 4 pmol 5 pmol 100 pmol 8 pmol 33 pmol 0.1 .05 ml 2. S9 homogenate A 9,000 x supernatant was prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254 five days prior to kill according to the procedure of Ames et aT. (1975). S9 samples were coded by lot number and assayed for milli grams protein per milliliter and relative P448/P450 activity by methods described in LBI Technical Data on Rat Liver S9 Product. m BIONETICS Litton 4 001799 2 . MATERIALS (Continued) C. Positive Control Chemicals The chemicals used for positive controls in the nonactivation and activation assays are given in Table 1 of Section V. Results. D. Solvent Either deionized water or dimethyl sulfoxide (DMSO) was used to prepare stock solutions of solid materials. All dilutions of test materials were made in either deionized water or DMSO. The sol vent employed and its concentration are recorded in Table 1 of Section V. Results. 3. EXPERIMENTAL DESIGN A. Plate Test (Agar Incorporation)* Approximately 108 cells from an overnight culture of each indica tor strain were added to separate test tubes containing 2.0 ml molten agar supplemented with biotin and a trace of histidine. For nonactivation tests, at least 4 dose levels of the test com pound were added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates. In activation tests, at least 4 dose levels of the test chemical were added to the appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 ml containing the 9,000 x 3 liver homogenate) was added to each of the activation overlay tubes, which were then mixed, and the contents poured over the surface of a minimal agar plate and allowed to solidify. The plates were incubated for 48 hr at 37C and scored for the number of colonies growing on each plate. D4 yeast plates were incubated at 30C (nonactivation) and 37C (activation) for 3-5 days and then scored. The concentrations of all chemicals are given in Table 1 of Section V. Results. Positive and solvent controls using both directly active positive chemicals and those that require meta bolic activation were run with each assay. * Certain classes of chemicals known to be mutagens and carcinogens do not produce detectable responses using the standard Ames agar incorporation method. Some dialkyl nitrosamines and certain substituted hydrazines are mutagenic in suspension assays, but not in the plate assay. Chemicals of these classes should be screened in a suspension assay. m BIONETICS Litton , 5 001300 3. EXPERIMENTAL DESIGN (Continued) B. Recording and Presenting Data The numbers of colonies on each plate were counted and recorded on printed forms. These raw data were analyzed in a computer program and reported on a printout. The results are presented as revertants (or convertants for D4) per plate for each indicator strain employed in the assay. The positive and solvent controls are pro vided as reference points. Other relevant data are provided on the computer printout. 4. EVALUATION CRITERIA Plate test data consist of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semisolid overlay. Because the test chemical and the cells are incubated in the overlay for 2 days, and a few cell divisions occur during the incubation period, the test is semiguantitative in nature. Although these features of the assay reduce the quantitation of results, they provide certain advantages not contained in a quantitative suspension test: The small number of cell divisions permits potential mutagens to act on replicating DNA, which is often more sensitive than nonreplicating DNA. The combined incubation of the compound and the cells in the overlay permits constant exposure of the indicator cells for 2 days. A. Surviving Populations Plate test procedures do not permit exact quantitation of the number of cells surviving chemical treatment. At low concentra tions of the test chemical, the surviving population on the treat ment plates is essentially the same as that on the negative control plate. At high concentrations, the surviving population is usually reduced by some fraction. Our protocol normally employs several doses ranging over 2 or 3 log concentrations, the highest of these doses being selected to show slight toxicity as determined by subjective criteria. B. Dose-Response Phenomena The demonstration of dose-related increases in mutant counts is an important criterion in establishing mutagenicity. A factor that might modify dose-response results for a mutagen would be the CBBIONET1C S Litton OOlSOl 4. EVALUATION CRITERIA (Continued) B. Dose-Response Phenomena selection of doses that are too low (usually mutagenicity and tox icity are related). If the highest dose is far lower than a toxic concentration, no increases may be observed over the dose range selected. Conversely, if the lowest dose employed is highly cyto toxic, the test chemical may kill any mutants that are induced, and the compound will not appear to be mutagenic. . C. Control Tests Positive and negative control assays are conducted with each experiment and consist of direct-acting mutagens for nonactivation assays and mutagens that require metabolic biotransformation in activation assays. Negative controls consist of the test compound solvent in the overlay agar together with the other essential com ponents. The negative control plate for each strain gives a reference point to which the test data are compared. The positive control assay is conducted to demonstrate that the test systems are functional with known mutagens. D. Evaluation Criteria for Ames Assay Because the procedures used to evaluate the mutagenicity of the test chemical are semi quantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical data base. Most data sets are evaluated using the following criteria: 1. Strains TA-1535, TA-1537, and TA-1538 If the solvent control value is within the normal range, a chemical that produces a positive .dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic. 2. Strains TA-98, TA-100, and D4 If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA-100 and 2-3 times the solvent control value for strains TA-98 and D4 is considered to be mutagenic. For these strains, the dose-response increase should start at approximately the solvent control value. C H BIONET1C S Litton 7 001802 4. EVALUATION CRITERIA (Continued) 0. Evaluation Criteria for Ames Assay 3. Pattern Because TA-1535 and TA-100 are both derived from the same parental strain (G-46) and because TA-1538 and TA-98 are both derived from the same parental strain (D3052), there is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain, e.g., TA-1537, responds to a mutagen in nonactivation tests, it will generally do so in activation tests (The converse of this relationship is not expected.). While similar response patterns are not required for all mutagens, they can be used to enhance the reliability of an evaluation decision. 4. Reproducibility If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data lose significance. The preceding criteria are not absolute, and other extenuating factors may enter into a final evaluation decision. However, these criteria are applied to the majority of situations and are presented to aid those individuals not familiar with this pro cedure. As the data base is increased, the criteria for evalu ation can be more firmly established. E. Relationship between Mutagenicity and Carcinogenicity It must be emphasized that the Ames Salmonel 1a/Microsome Plate Test is not a definitive test for chemical carcinogens. It is recognized, however, that correlative and functional relationships have been demonstrated between these two endpoints. The results of comparative tests on 300 chemicals by McCann et al_. (1975) show an extremely good correlation between results of microbial mutagenesis tests and i_n vivo rodent carcinogenesis assays. All evaluations and interpretation of the data presented in this report are based only on the demonstration, or lack, of mutagenic activity. OE BIONET1C S Litton 001S03 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome muta genicity test. Mutation Res. 31, 347-354. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. 72, 5135-5139. C H BIONETICS Litton 9 001S04 5. STANDARD OPERATING PROCEDUPE All data will be entered ui ink (no pencil). All changes or corrections in entries will be made with a single line through the change, and an explanation for the change must be written. All calculations (weights, dilutions, dose calculations, etc.) will be shown on data records. All data entries will be dated and initialed. All laboratory operations will be written out in standard protocol manuals. These manuals will be present in each laboratory area. Deviations from any established protocol will be described and justi fied. Data will be stored in bound form (notebooks or binders). These bound data books will be reviewed by the appropriate Section Heads. Chemicals submitted for testing will have date of receipt and initials of entering person. Lot numbers for all reference mutagens, solvent, or other materials used in assays will be recorded. Animal orders, receipts, and identification will be recorded and main tained such that each animal can be traced to the supplier and ship ment. All animals on study will be properly identified. A copy of the final report plus all raw data and support documents will be permanently stored in the archival system of Litton Bionetics, Inc. Current curricula vitae and job descriptions will be maintained on all personnel involved in the study. Salmonella strains will be routinely checked for the his, uvrB, rfa, and pKM 101 phenotypes. Only appropriately screened stock cultures will be used in chemical evaluations. BIONETICS Litton