1、 STD.API/PETRO PUBL TR4LO-ENGL L79b 0732290 5b4260 7b m American Petroleum Institute Health and Environmental Sciences Department Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells Exposed to Tertiary Amyl Methyl Ether (TAME) DECEMBER 1996 TOXICOLOGY REPORT NUMBER 410 CAIS ABSTRACT NO. 43-5
2、239 Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells Exposed to Tertiary Amyl Methyl Ether (TAME) Health and Environmental Sciences Department API PUBLICATION NUMBER TR410 PREPARED UNDER CONTRACT BY: PATRICK T. CURRY, PH.D 9900 BLACKWELL ROAD ROCKVILLE, MARYLAND 20850 MICROBIOLOGICAL ASSO
3、CIATES, INC., DECEMBER 1996 American Petroleum Institute STD-API/PETRO PUBL TR41O-ENGL 197b 0732270 05b1i2b2 730 FOREWORD API PUBLICATIONS NECESSARILY ADDRESS PROBLEMS OF A GENERAL NATURE. WiTH RESPECT TO PARTICULAR CIRCUMSTANCES, LOCAL, STATE, AND FEDERAL LAWS AND REGULATIONS SHOULD BE REVIEWED. AP
4、I 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 LOCAL, STATE, OR FEDERAL LAWS. NOTHING CONTAINED I
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6、ENT OF LETTERS PAEN“. Copyright O 1996 American Petroleum Institute 1 - STD.API/PETRO PUBL TRLiLO-ENGL 177b W 0732270 05b42b3 b77 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 CON
7、TACT Richard Rhoden, Ph.D., Health and Environmental Sciences Department 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. Fairbrother, Exxon Company, USA Barry
8、 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. Saab, RTA Inc. Ravi Vangipuram, Texaco Refining and Marketing Russell D. White, Chevron Research howeve
9、r, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner with one or more 23 STD*API/PETRO PUBL TRYLO-ENGL 199b 0732290 05b11285 238 concentrations being statis
10、tically elevated relative to the solvent control group (10.05). A significant increase at the high dose only with no dose response was considered suspect. A significant increase at one dose level other than the high dose with no dose response was considered equivocal. Test articles that did not demo
11、nstrate a statistically significant increase in aberrations were concluded to be negative. CRITERIA FOR A VALID TEST The frequency of cells with structural chromosome aberrations in either the untreated or solvent control must be no greater than 6%. The percentage of cells with chromosome aberration
12、s in the positive control must be statistically increased (10.05, Fishers exact test) relative to the solvent control or to the untreated control if a solvent other than water was used. ARCHIVES Upon completion of the final report, all raw data, reports, and stained and coded slides are maintained i
13、n the archives of Microbiological Associates, Inc., located in Rockville, Maryland. 24 STD.API/PETRO PUBL TRqLO-ENGL 177b 0732270 05b428b 179 RESULTS AND DISCUSSION SOLUBILITY TEST Ethanol was determined to be the solvent of choice based on solubility of the test article and compatibility with the t
14、arget cells. The test article was soluble in ethanol at a maximum concentration of approximately 500 mg/m and was soluble in the treatment medium at a concentration of 5000 pg/ml. PRELIMINARY TOXICITY ASSAY Dose levels and post-treatment cell harvest times for the chromosome aberration assay were se
15、lected following a preliminary toxicity test based upon a reduction of cell growth (cell growth inhibition) and cell cycle delay after treatment relative to the solvent control. The results of the evaluation of cell growth inhibition are presented in Tables 1 and 2 and the results of the evaluation
16、of cell cycle delay are presented in Tables 3 and 4. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 0.5 pg/ml to 5000 pg/ml in the absence and presence of an S9 reaction mixture. The test article was soluble in solvent at a stock concentration of 500
17、mg/ml, and soluble in treatment medium at all concentrations tested. The osmolality in treatment medium of the highest concentration tested (5000 pg/ml) was 294 mOsm/kg. The osmolality of the solvent was 282 mOsm/kg. The pH of the highest concentration of test article in treatment medium was approxi
18、mately 7. Cell growth inhibition relative to the solvent control was 66% at 5000 pg/ml, the highest concentration tested in the non-activated test system, and 64% at 5000 pg/ml, the highest concentration tested in the SPactivated test system. Based on these findings, the doses chosen for the chromos
19、ome aberration assay ranged from 313 to 5000 pg/ml for both the non- activated and the S9-activated systems. No cell cycle delay was apparent at any of the doses tested in the non-activated study. The cell cycle was delayed from approximately 12 hours to 23.8 hours at the highest dose tested (5000 p
20、g/ml) in the S9-activated study. Based on the cell cycle kinetics observed in the preliminary toxicity assay, the cell harvest times for the chromosome aberration assay were 12 hours for the non-activated study and 20 hours for the 25 STD-API/PETRO PUBL TRqLO-ENGL L99b 0732290 05b4287 O00 = S9-activ
21、ated study. The selected harvest times were used to ensure microscopic evaluation of first-division metaphase cells. CHROMOSOME ABERRATION ASSAY The activity of Tertiary Amyl Methyl Ether (TAME) in the induction of chromosome aberrations in CHO cells when treated in the absence of SPactivation is pr
22、esented by treatment flask in Table 6 and summarized by group in Table 9. The test article was soluble in solvent at a stock concentration of 500 mg/ml, and soluble in treatment medium at all concentrations tested. Toxicity (cell growth inhibition relative to the solvent control) was approximately 4
23、3% at 5000 pg/ml, the highest test concentration evaluated for structural chromosome aberrations (Table 5). An additional concentration of 313 pg/ml was tested as a safeguard against excessive toxicity at higher concentrations but was not required for microscopic examination. The percentage of cells
24、 with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control only at 2500 pg/ml (10.05, Fishers exact test). However, the response seen at this dose level was within the range of the historical control and was not concluded to be biolo
25、gically significant. The percentage of damaged cells in the MMC group was 9.5% (pS0.01, Fishers exact test). The activity of Tertiary Amyl Methyl Ether (TAME) in the induction of chromosome aberrations in CHO cells when treated in the presence of an S9 reaction mixture is presented by treatment flas
26、k in Table 8 and summarized by group in Table 9. Due to contamination of the cultures, this portion of the assay was repeated. Only data collected from the repeat assay are contained in this report. The test article was soluble in solvent at stock concentration (500 mg/ml) and was soluble in treatme
27、nt medium at all concentrations tested. Toxicity (cell growth inhibition relative to the solvent control) was approximately 72% at 5000 pg/ml, the highest test concentration evaluated for structural chromosome aberrations (Table 7). An additional concentration of 3 13 pg/d was tested as a safeguard
28、against excessive toxicity at higher concentrations but was not required for microscopic examination. The percentage of cells with structural aberrations in the test article-treated groups was statistically increased above that of the solvent control at the 1250, 2500 and 5000 pg/ml dose levels (50.
29、05, 26 - STD.API/PETRO PUBL TRVLO-ENGL L77b 0732270 05b4288 T97 Fishers exact test). The Cochran-Armitage test was also positive for a dose response (50.05). The percentage of damaged cells in the CP group was 32.5% (10.01, Fishers exact test). 27 STD-API/PETRO PUBL TRqLO-ENGL L97b 0732270 05b4287 783 CONCLUSION The positive and negative controls fulfilled the requirements for a valid test. Under the conditions of the assay described in this report, Tertiary Amyl Methyl Ether (TAME) was concluded to be positive in the chromosome aberration assay using Chinese hamster ovary (CHO) cells. 28
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