Document 0g1X4pZDgvNex9v7LapRxQZ6J

1*10 PRONTAOC ROAD NOdTHIIOOK, lUINOIt *0069 REPORT TO MONSANTO COMPANY MUTAGENIC STUDY WITH AROCIjOR 1260 ' " IN ALBINO MICE JANUARY 13, 1972 Mr. Elmer P. Wheeler Manager, Environmental Health Monsanto Company 800 N. Lindbergh Boulevard St. Louis, Missouri 63166 Dear Mr. Wheeler: * Re: IBT No. E623 - Mutagenic Study with Aroclor 1260 in Albino Mice We are submitting herewith our laboratory report dated January 13, 1972, prepared in connection with the above study. Very truly yours, J. C. Calami ra President HONS 068868 4 REPORT TO MONSANTO COMPANY MUTAGENIC STUDY WITH AROCI-OR 1260 IN ALBINO MICE JANUARY 13, 1972 IB T NO. .62 3 I. Introduction A mutation is a change in the character of a gene such that morpho logic, physiologic, and/or biochemical alterations are produced. If this change in gene character occurs in the germinal cell, the alteration can be transmitted to succeeding generations. Changes of this nature can be artificially induced (irradiation, chemical exposure, etc. ) or they may be spontaneous. A dominant lethal mutation, occurring in the male germinal cell, may lead to the inability of the affected cell to fertilize an egg or, once having fertilized, to the failure of development beyond the blastocyst stage (implantation). Male mice, treated with the lest compound, are mated with untreated females. The numbers of pre-implantation losses and early resorptions in female mice, dissected at mid-gestation, arc used to calcu late the mutation rate based on the induction of dominant lethal mutations, KONS 068869 a- II, Summary 't,V-r A dominant Uthal mutagenic atudy waa conducted uting albino mica. -m Male* were treated with a tingle Intraperitoneal Injection of Aroclor 1260 iff levels of either 500 (T-l) or 1,000 (T-II) mg/kg of body weight. Another group of male* received methyl mcthaneaulfonate (MMS) at 100 mg /kg and was used a* a positive control. Mating indices for Aroclor 1260 treated animals were unaffected. Two positive control males and 1 T-I male died during the course of the study. Data for treated animals regarding implantation sites, resorption sites, and embryos did not differ from the control data. Mutation rates were not unusual for the Aroclor treated animals. MMS treated animals showed a positive response for Ihe first 2 weeks following treatment. Therefore, treating male mice with Aroclor 1260 via an intraperitoneal injection of 500 or 1,000 mg/kg did not cause a dominant lethal response. Respectfully submitted, INDUSTRIAL DIO-TEST LABORATORIES, INC. Report prepared byif:; Dennis Arnold, B.S. Group Leader Mutagenic Studies ' Report approved by: Gerald Kcnne<ly7'B. S. Senior Group Leader Metabolic and Mutagenic Studies HONS 068870 111. Procedure A. Outline of Expcrlmcr.t The teat materiel was Aroclor 1260. Methyl metheneaul/onete wte used as a positive control. Charles River strain albino mice were received et this laboratory at 60 to 70 lays of age for use in the study. The organi sation of groups is presented in Tabic 1. Croup C PC T-I T-II TABLE 1 TEST MATERIAL: Aroclor 1260 Mutagenic Study - Albino Mice Organization of Croups Dose Level* (ms /kg) 100 500 1, 000 Number of Males Treated 12 12 17 12 * Aroclor 1260 was administered as a 10 percent solution in corn oil. Control males received th vehicle in amounts equivalent to those received by the T-II males. Positive control males received methyl methancsulfonate in a 1 percent corn oil solution. HONS 068871 4 B. Dosage Levels The treatment level* were selected by intraperitoneal admlnii" v (ration of tingle graded doses of the test material to male mice. The maximum tolerated dose was determined and dose levels employed in the main study were based on these findings. The test material was adminis tered intrapcritoneally in a corn oil solution to male mice. Control animals received the vehicle in volumes equivalent to those giver the high teat i group and positive control males received methyl methanesulfonatc. C. Mating Schedule Each group consisted of 12 male mice, each of which was placed in a cage with 3 untreated virgin femalcs-immcdiately after dose adminis tration. At the end of 1 week, the females were removed from the cage and replaced by another group of 3 females. This procedure continued for 6 consecutive weeks, a period of time required for maturation of the male mouse germ cells from the spermatocyte to the mature spermatozoon. Experiments at this laboratory have shown that the reference mutagenic compound* affects germinal cells in the meiotic and posl-mciotic stages prior to or during the spermatid stage. Recovery from the effects of MMS is generally seen 3 weeks post-treatment. Following the sixth week of mating, all males were sacrificed. D. Female Sacrifice The females were sacrificed approximately 1 week after re moval from the breeding cage, at which time most females that had The reference mutagen $ methyl methaneaulfonate (MMS). ; MOMS 06SBIZ 4 mated were at mid-pregnancy. All animals ware sacrificed by carbon dioxide asphyxiation. The numbers of implantation sites, resorption sites, and embryos were recorded. Females were judged to be pregnant if corpora lutca were present in the ovaries. Resorption sites were divided into 2 groups, early deaths (deciduomata) and late deaths. Late deaths refer to embryos w lich develop to a relatively advanced stage prior to death, so that the placenta and fetal membranes or remnants thereof arc visible. The frequency of late deaths is apparently unaffected by mutagens and, therefore, would appear to be non-gcnctic in this test system. Dcciduomata occur it the sites of implantsd blastocysts which fail to develop following implantation. The mutagenicity of the chemical can be measured by the proportions of all implantations which are deeiduomata. Mutagenicity can also be measured by comparing the mean num ber of viable embryos in the test group to the number obtained in the con trol group. Calculation of the mutation rale using this criterion assumes that pro-implantation losses among non-affccted test animals will be es sentially the same as losses observed among control animals. MOMS 068873 4 IV. Rcsulta A. Treatment levels ^^ Doaei were administered to groups of 4 male mice each to determine the levels for the main study. Exposures of 30, 100, 300, or 1,000 mg of Aroclor 1260 per kg of body weight failed to elicit any abnormal reactions and no deaths were recorded during the M-day observation period ^ following treatment. B. Mortality and Reactions Two positive control males (weeks 1 and 3) and 1 T-I male (week 3) died during the study. No other deaths occurred. C. Mating Performance . Mating indices, defined as the number of animals pregnant divided by the number of females mated times 100, and the number of aurviving males for each post-treatment week are shown in Table II. All mating indices for the Aroclor 1260 treated animals seen in the experiment arc considered to be normal for the mouse strain employed. Positive control animals had a low mating index for the first week post treatment. MOMS 066874 TABLE II TEST MATERIAL; Aroclor 1260 Mutagenic Study - Albino Mice Mating Performance Test Number of Week Surviving Number of Animals Pregnant Croup Number__Males________Number of f emales Mated Mating Index (Percent! 1 12 2 12 3 12 4 12 5 12 6 12 25/36 28/36 31/36 29/36 28/36 29/36 69. 4 77.8 86. 1 80.6 77. 8 80.6 1 11 2 11 3 11 4 10 5 10 6 10 14/33 19/33 23/33 26/30 23/30 24/30 42,4 57.6 69.7 86. 7 76. 7 80.0 D. Sacrifice Data Data pertaining to the numbers of corpora lutca, implantation lies, resorption sites (early and late differentiated), and embryos are ^ presented in Table III. laboratory data have shown that the untreated female mouse lias a mean of 13.5 corpora lutca. Therefore, the num ber of pregnant animals is multiplied by 13. 5 to obtain the total number of corpora lutca. The corpora lutea for females in control and T-l (at week 3) that did not have implantations arc included in the results. These data show no significant differences between test and control* animals with regard to the numbers of implantations,. resorptions, and embryos.* Effects were noted for the first 2 weeks following treatment of the positive control animals. Group C Test Week Number , 2 3 4 5 6 PC 1 2 3 4 5 6 TABLE III TEST MATERIAL: Aroclor 1260 Mutagenic Study - Albino Mice Summary of Sacrifice Data Pregnant Animals Examined 25 28 31* 29 28 29 14 19 23 26 23 24 Calculated Corpora Lutea Implantation Sites 338 378 417 392 378 392 293 (11. 7) 355 (12. 7) 342 (11. 0) 357 (12. 3) 341 (12.2) 349 (12.0) 189 256 310 351 310 324 134 ( 9.6) 178 ( 9.4) 294 (12.8) 334 (12.8) 291 (12.6) 292 (12.2) Resorption Sites Early Late 5 (0.2) 9 (0. 3) 14 (0.4) 15 (0.5) 13 (0.5) 12 (0.4) 1 (0.1) 0 (0.0) 5 (0.2) 5 (0.2) 3 (0. 1) 3 (0. 1) 4 1 (2.9) 47 (2.5) 13 (0.6) 16 (0.6) 14 (0.6) 17 (0.7) 2 (0. 1) 0 (0.0) 3 (0. 1) 4 (0.2) 5 (0.2) 2 (0. 1) Embryos 287 (11.5) 346 (12.4) 323 (10.4) 337 (11.6) 325 (11.6) 334 (11.5) 91 ( 6.5) 131 ( 6.9) 278 <12. 1) 314 (12. 1) 272 (11.8) 273 (11.4) Note: Numbers in parentheses are the means per female. Includes 1 pseudo-pregnar? female (corpora lutea only). 8 9 B 9 0 SNOW Li:,, Group Test Week Number T-I 1 2 3 4 5 6 T-u i 2 3 4 5 6 TABLE III continued TEST MATERIAL: Aroc lor 1260 Mutagenic Study - Albino Mice Summary of Sacrifice Data Pregnant Animals Examined 31 31 26 28 29 32 Calculated Corpora Lutea Implantation Sites 418 418 349 378 392 432 339 (10.9) 384 (12.4) 314 (12.1) 335 (12.0) 381 (13. 1) 398 (12.4) Resorption Sites Early Late 13 (0.4) 7 (0. 2) 7 (0. 3) 11 (0.4) 14 (0. 5) 14 (0.4) 1 (0. 1) 2 (0. 1) 2 (0. 1) 5 (0.2) 1 (0. 1) 4 (0. 1) 30 405 342 (11.4) 13 (0.4) 1 (0.1) 30 405 371 (12.4) IS (0.5) 2 (0. 1) 25 338 312 (12.5) 15 (0.6) 4 (0.2) 31 418 376 (12. 1) 16 (0.5) 3 (0. 1) 27 364 330 (12.2) 18 (0. 7) 1 (0.1) 28 378 349 (12.5) 13 (0.5) 1 (0.1) Embryos 325 (10.5) 375 (12. 1) 305 (11.7) 319 (11.4) 366 (12.6) 380 (11.9) 328 (10. 9) 354 (11.8) 293 (11.7) 357 (11.5) 311 (11.5) 335 (12.0) Note: Numbers in patentheses are the means per female. * Includes 1 pseudo-pregnant female (corpora lutea only). E. MuUcenk Dsts A lumimry of results for the mutagenic study is presented in Table IV. Pre-Implantation Ls>s* is defined as: Number of Corpora Lutea - Number of Implantation Sites x jqq Number of Corpora Laitea The mutation rate may be calculated by comparing the number of early resorptions (dcciduomata) to the total number of implantations for each group. Number of Early Resorption Sites x jqO Number of Implantation Sites table) Another way of expressing the mutation rate, which takes into account pre-implantation losses, is by comparing the mean number of normal embryos in each test group to the mean number of embryos in the control group. ,_ . Embryos Test Croup/Female ,, 100 " 1 Embryos Control Group/Fcmalo x 100 1 J#% , , , <B ,n taMc> Values presented were obtained by comparing each group to the contem porary control group (a) and to cumulative control data (b). Values with a minus (-) sign indicate that the mean number of embryos for that group and week wss greater than that of controls. The deviation from aero should not exceed 25 percent in the positive direction, IJrr MO 066880 3 animals compared favorably to those of controls. The mutation rates for the positive control animals indicated a response for the first 2 weeks post-treatment. & ... .  Croup C PC TABLE IV TEST MATERIAL! Aroclor 1260 Mutagenic Study - Albino Mice Summary of Mutagenic Data Test Week Number Pre-Implantation Ijoss and Mutation Rates Mutation Rates Pre-Implantation Loss B (Percent) Aa b 1 13. 3 1.7 - 0.9 .2 6. 1 2. 5 -10.7 .3 78.0 4. 1 10.4 .4 8.9 4. 2 - 0.9 .5 9.8 3.8 0.9 6 11.0 3.4 - 1.7 1 89. 1 30.6 43. 5 43.0 2 30.5 26.4 44.4 a. 4 3 5.2 4.4 16.3 - 4.3 4 4. 8 4. 8 - 4. 3 - 5.2 5 6. 1 4. 8 - 1. 7 - 0.8 6 9. 9 5.8 0.9 2.6 MGNS 068382 Croup TABLE IV continued TEST MATERIAL: Aroclor 1260 Mutagenic Study - Albino Mice Summary of Mutagenic Data Test Week Number Pre-Implantation Ix>ss and Mutation Rates Mutat ion Rates Prc-Implanlaiion Loss (Percent! A B ab 1 18. 9 3.8 8. 7 7. 9 2 8. 1 1. 8 2. 4 -fl. 0 3 10. 0 2. 2 -12.5 -0. 9 4 11.4 3. 3 1, 7 0, 9 5 2.8 3. 7 - 8.6 -7. 7 6 7. 9 3. 5 - 3. 5 -i:7 l 15. 6 3. 8 5.2 4.4 2 8.4 4. 0 4.8 -5.4 3 7. 7 4. 8 -11.5 -0.9 4 10. 0 4. 2 0.9 0.0 5 9. 3 5. 4 0.9 1.7 6 7. 7 3. 7 - 4. 3 -2. 5 MGNS 068883 *fr