1、 STD.API/PETRO PUBL TR4LL-ENGL L7b 0732290 U56458b L7 H American Petroleum Institute Health and Environmental Sciences Department Chinese Hamster Ovary (CHO)/HGPRT Mutation Assay of Tertiary Amyl Methyl Ether (TAME) DECEMBER 1996 TOXICOLOGY REPORT NUMBER 41 1 CAIS ABSTRACT NO. 43-5240 STD-API/PETRO
2、PUBL TRiLL-ENGL L77b = 0732270 05b11587 853 = Chinese Hamster Ovary (CHO)/HGPRT Mutation Assay of Tertiary Amyl Methyl Ether (TAME) Health and Environmental Sciences Department API PUBLICATION NUMBER TR411 PREPARED UNDER CONTRACT BY: RICHARD H.C. SAN, PH.D. JANE J. CLARK, B.A. 9900 BLACKWELL ROAD RO
3、CKVILLE, MARYLAND 20850 MICROBIOLOGICAL ASSOCIATES, INC., DECEMBER 1996 American Petroleum Institute STD*API/PETRO PUBL TR4LL-ENGL L77b = 0732290 05b4588 77T FOR E WORD API PUBLICATIONS NECESSARILY ADDRESS PROBLEMS OF A GENERAL NATURE. WITH RESPECT TO PARTICULAR CIRCUMSTANCES, LOCAL, STATE, AND FEDE
4、RAL LAWS AND REGULATIONS SHOULD BE REVIEWED. API IS NOT UNDERTAKING TO MEET THE DUTIES OF EMPLOYERS, MAN- UFACTURERS, OR SUPPLIERS TO WARN AND PROPERLY TRAIN AND EQUIP THEIR EMPLOYEES, AND OTHERS EXPOSED, CONCERNING HEALTH AND SAFETY RISKS AND PRECAUTIONS, NOR UNDERTAKING THEIR OBLIGATIONS UNDER LOC
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6、NSTRUED AS INSURING ANYONE AGAINST LIABILITY FOR INFRINGEMENT OF LEITERS PATENT. Copyright O 1996 American Petroleum Institute ACKNOWLEDGMENTS THE FOLLOWING PEOPLE ARE RECOGNIZED FOR THEIR CONTRIBU- TIONS OF TIME AND EXPERTISE DURING THIS STUDY AND IN THE PREPARATION OF THIS REPORT: API STAFF CONTAC
7、 T Richard Rhoden, Ph.D., Health and Environmental Sciences Department MEMBERS OF THE TAME WORKGROUP Phil Andrews, Citgo Petroleum Corporation Paul C. Bucknam, Amerada Hess Corporation Christopher Colman, Amerada Hess Corporation Wayne C. Daughtrey, Exxon Biomedical Sciences, Inc. Carol A. Fairbroth
8、er, Exxon Company, USA Barry Fulda, Citgo Petroleum Corporation Nancy Kralik, Marathon Oil Company Greg Lehman, Sun Refining and Marketing Company Craig M. Parker, Marathon Oil Company Susan A. Rodney, Texaco, Inc. Robert J. Staab, RTA Inc. Ravi Vangipuram, Texaco Refining and Marketing Russell D. W
9、hite, Chevron Research protected from exposure to light Test Article Receipt: December 26, 1995 Study Initiation: January 23, 1996 Laboratory Manager: qzz/+L Jand. Clad and ONeill et al., 1977). Exponentially growing CHO-K,-BH, cells were seeded in F12FBSS-Hx at a density of 5x16 cells125 cm2 flask
10、and were incubated at 3711C in a humidified atmosphere of 51% CO, in air for 18-24 hours. F12Fl3S5-H is Hams F12 medium without hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillitdml, 100 pg streptomycidd and 2mM L-glutamine/ml. The time of initiation of chemical treatment was desig
11、nated as day O. Treatment was carried out by refeeding the treatment flasks with 5 ml F12FBS5-HxI25 cm2 flask for the non- activated study and 4 ml F12FBS5-Hx and 1 ml S9 reaction mixture/25 cm2 flask for the S9- activated study, to which was added either 50 pl dosing solution of test or control art
12、icle in vehicle or vehicle alone. Duplicate flasks of cells were exposed to one of at least five concentrations of the test article for 5 hours at 37tl“C. After the treatment period, all media were aspirated, the cells washed with Ca“- and Mg“-free Hanks balanced salt solution (CMF-HBSS) and culture
13、d in F12FBSS-Hx for an additional 18-24 hours at 37itl“C. At this time, the cells were subcultured to assess cytotoxicity and to initiate the phenotypic expression period. EVALUATION OF CYTOTOXICITY For evaluation of cytotoxicity, the replicates from each treatment condition were detached using tryp
14、sin and subcultured independently in FFBSS-HX, in triplicate, at a density of 100 cells/60 mm dish. After 7-10 days incubation, the colonies were rinsed with HBSS, fixed 19 - STD.API/PETRO PUBL TRqLL-ENGL L77b E 0732270 05b4b07 471 c1 cells/6O mm dish. After 7-10 days of incubation, the colonies wer
15、e rinsed with HBSS, fixed with methanol, stained with 10% aqueous Giemsa and counted; cloning efficiency was then determined. EXPRESSION OF THE MUTANT PHENOTYPE For expression of the mutant phenotype, the replicates from each treatment condition were trypsinized and subcultured independently in FFBS
16、S-HX, in duplicate, at a density no greater than lo6 cells/100 mm dish. Subculturing by trypsinizing at 2-3 day intervals was employed for the 7-9 day expression period. At the end of the expression period, selection for the mutant phenotype was performed. SELECTION OF THE MUTANT PHENOTYPE For selec
17、tion of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2x16 cells/100 mm dish in F12FBSS-Hx containing 10 ph4 6-thioguanine (TG, 2-amino-6-mercaptopurine). For cloning efficiency determinations at the time of
18、selection, 100 cells/60 mm dish were plated in triplicate. After 7-10 days of incubation, the colonies were fixed, stained and counted for both cloning efficiency and mutant selection. EVALUATION OF TEST RESULTS The cytotoxic effects of each treatment condition were expressed relative to the solvent
19、- treated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2x106 cells: 10 plates at 2x105 celldplate), corrected for the cloning efficiency of cells p
20、rior to mutant selection, and is expressed as TG-resistant mutants per lo6 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses givi
21、ng I 10% relative survival are presented in the data but were not considered as valid data points. 20 - STD-API/PETRO PUBL TRqLL-ENGL 199b 0732290 05b4b00 308 .I Because spontaneous mutant frequencies are very low for the CHO/HGPRT assay, calculation of mutagenic response in terms of fold increase i
22、n mutant frequency above the background rate does not provide a reliable indication of the significance of the observed response. The wide acceptable range in spontaneous mutant frequency also suggests the need to set a minimum mutant frequency for a response to be considered positive. Hsie et af. (
23、1981) refer to a level of 50 mutants per lo6 clonable cells. In this laboratory, a more conservative approach was used which set the minimum level at AO mutants per lo6 clonable cells. The assay would be considered positive in the event of a dose-dependent increase in mutant frequencies with at leas
24、t two consecutive doses showing mutant frequencies which were elevated above 40 mutants per lo6 clonable cells. If a single point above 40 mutants per lo6 clonable cells was observed at the highest dose, the assay would be considered suspect. If no culture exhibited a mutant frequency of 40 mutants
25、per lo6 clonable cells, the test article would be considered negative. CRITERIA FOR A VALID TEST The cloning efficiency of the solvent control must be greater than 50%. The spontaneous mutant frequency in the solvent control must fall within the range of 0-25 mutants per lo6 clonable cells. The posi
26、tive control must induce a mutant frequency at least three times that of the solvent control and must exceed 40 mutants per lo6 clonable cells. ARCHIVES Upon completion of the final report, all raw data and reports are maintained by the Quality Assurance Unit of Microbiological Associates, Inc., Roc
27、kville, Maryland, in accordance with the relevant Good Laboratory Practice Regulations. RESULTS AND CONCLUSIONS SOLUBILITY TEST Ethanol was determined to be the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in ethanol
28、at a concentration of 500 mg/ml, the maximum concentration tested. 21 STD.API/PETRO PUBL TRqLL-ENGL L77b 0732290 05b4bO 244 m PRELIMINARY TOXICITY ASSAY The results of the preliminary toxicity assay are presented in Table 1. CHO cells were exposed to solvent alone and nine concentrations of test art
29、icle ranging from 0.5 to 5000 pg/ml in the absence and presence of S9 reaction mixture. No test article precipitate was observed at any dose level in treatment medium. The osmolality of the solvent control and the highest soluble dose could not be determined due to equipment malfunction. This deviat
30、ion from the protocol was determined by the Study Director to have had no adverse effect on the integrity or outcome of the study. Cloning efficiency relative to the solvent controls (RCE) was 89% at 5000 pg/ml without activation and 42% at 5000 pg/ml with S9 activation. Based on the results of the
31、toxicity test, the doses chosen for the mutagenesis assay ranged from 1000 to 5000 pg/ml for the non-activated cultures and 500 to 5000 pg/ml for the S9-activated cultures. MUTAGENESIS ASSAY The cytotoxic effects of the test article (concurrent cytotoxicity) are presented in Table 2. Mutagenicity da
32、ta are presented in Tables 3 and 4. In the non-activated system, cultures treated with concentrations of 1000,2000, 3000,4000 and 5000 pg/ml were cloned. In the S9-activated system, cultures treated with concentrations of 500, 2000, 3000, 4000 and 5000 pg/ml were cloned. No test article precipitate
33、was observed at any dose level in treatment medium. Relative cloning efficiency was 75% and 44% at the highest dose tested in the non- activated and SPactivated systems, respectively. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per lo6 clonable cells. CONCLUS
34、ION All criteria for a valid study were met as described in the protocol. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, Tertiary Amyl Methyl Ether (TAME) did not cause a positive response in the non-activated and S9-activated systems and was concluded
35、 to be negative. 22 STD-API/PETRO PUBL TRLiLL-ENGL 177b m 0712290 O5bllbLO Tbb II REFERENCES Hsie, A.W., D.A. Casciano, D.B. Couch, B.F. Krahn, J.P. ONeill, and B.L. Whitfield. 1981. The Use of Chinese Hamster Ovary Cells to Quantify Specific Locus Mutation and to Determine Mutagenicity of Chemicals
36、. A Report of the Gene-Tox Program. Mutation Research. 86: 193-214. Assay of Mutation Induction at the Hypoxanthine-guanine Phosphoribosyl Transferase Locus in Chinese Hamster Ovary Cells (CHO/HGPRT System): Development and Definition of the System. Mutation Research. 45:91-101. ONeill, J.P., P.A. B
37、rimer, R. Machanoff, G.P. Hirsch, and A.W. Hsie. 1977. A Quantitative 23 STD.API/PETRO PUBL TRLiLL-ENGL L776 0732270 0564bLL 7T2 Y d +I pc m (d Y 1 O c vi L d? o o - .- 5 Y ul o o O rf Y 5 Y s i2 o o 2 3 ul - 5 - 24 STD.API/PETRO PUBL TR4LL-ENGL L77b 0732270 05bllbL2 839 m d t- O c? O0 dm Dio wm mCu
38、 mm 8 O d SCI m 2 moo 0% t-2 g2 -p. - O O O d 6 c 4 k a + u c i 4 i c c 4 i 3 k t S .C c U I- U U a .C + .L c C C c c + i + I i c : ; r a k d d m O c? we me ov me NO mc 8 O rn io O 2 -1 m -1 00 O O O io O O mo Dt- + wn b 25 -q E c 5 E V .C c C (1 c c c C II o 5 .- g o en c E O .- - u N STD-API/PETRO
39、 PUBL TRLiLL-ENGL 177b 0732270 05b4b13 775 m I OC - O oc - oc oc - m 2 rn - .o B= U E w M M -c cis - wc wo I - Y - O E 2 ES w ro 5 r U .C c - C C c c E c cc C c c II 0 z c O o - .a 4 . m iii c m iii 26 STD.API/PETRO PUBL TRLiLL-ENGL 177b W 0732270 OSbLibL4 bOL W mR m O d - drn 00U o 5 5i V E: C C .C - c $ c E n c c II o 0 -. in CI E td Y 5 in o o - 0 - X m X G. .- g 0 on E E O o .- - X in o U E O o o 5 .- .- Y - 2 B i 1 E STD*API/PETRO PUBL TRiLL-ENGL L77b W 0732290 O5bibLS 54A = APPENDIX A HISTORICAL CONTROL DATA
