Document KR8jo4NDOdp1p4gnz7aMmxnEr

m*+ -- /O' 7 project no: . Rt'PORT FILE, _ i r 9w9uMaJjqI B10 - T E s T Jkkrtafoiied', 9m. . 1810 FRONTAGE ROAD NORTHBROOK. ILLINOIS 60062 REPORT TO MONSANTO COMPANY MUTAGENIC STUDY WITH AROC LOR 1242 IN ALBINO MICE JANUARY 13, 1972 IBT NO. E621 ( It ( OSW 036793 /- STLCOPCB4020617 1 - v fi ( Sn&uAtAidl Bid-TEST 1810 FRONTAGE ROAD NORTHBROOK, ILLINOIS 60062 Sm. 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. E621 - Mutagenic Study with Aroclor 1242 in Albino Mice We are submitting herewith our laboratory report dated January 13, 1972, prepared in connection with the above study. Very truly yours. JCC/kjl J. C. Calandra President t DSW 036794 STLCOPCB4020618 REPORT TO MONSANTO COMPANY MUTAGENIC STUDY WITH AROCLOR 1242 IN ALBINO MICE JANUARY 13, 1972 IBT NO. E621 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 ger.e 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 maybe 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 test compound, are mated with untreated females. The numbers of pre-implantation losses and early resorptions in female mice, dissected at mid-gestation, are used to calcu late the mutation rate based on the induction of dominant lethal mutations. OSW 036795 STLCOPCB4020619 ' v .. , Unduibial BIO T E S t Ja&MaiviUl, Dm. 't II, Sumnury A dominant lethal study was conducted in albino mice treated with either 500 jT-I) or 1,000 (T-II) mg Aroclor 1242/kg of body weight, A group of males was treated with methyl methanesulfonate (MMS) at 100 mg. kg to serve as positive controls. Several deaths, 2 PC, 1 T-I, and 2 T-II males, occurred during the study. The ability of the treated males to mate with and fertilize the un treated females was unaffected. Autopsies of the females that had mated with the treated males did not reveal any changes indicative of dominant lethality nor did the mutation indices, calculated from the data obtained at these sacrifices, show any positive results. ,, MMS treated animals showed increased mutation rates for the first 2 weeks following treatment. DSs 036796 STLCOPCB4020620 S'ndusiUal BIO-TEST labtoafotteAv 9tt&. It can be concluded that treating male mice with a single intraperitoneal injection of 500 or 1, 000 mg Aroclor 1242/kg of body weight does not induce dominant lethal mutations in the germinal cells. Respectfully submitted, INDUSTRIAL BIO-TEST LABORATORIES, INC. Report prepared by: <C, .. Dennis Arnold, B.S. Group Leader Mutagenic Studies Report approved by: Gerald Kennedy, js>. S. Senior Group Leader Metabolic and Mutagenic Studies January 13, 1972 M. L. Keplingo^, Ph Manager, Toxicology trm:psh DSW 036797 STLCOPCB4020621 DmLuAIaIoI BIO-TEST JakyudvUei, Unc. .4 4 III. Procedure A. Outline of Experiment The test material was Aroclor 1242. Methyl methanesulfonate (MMS) was used as a positive control. Charles River strain albino mice were received at this laboratory at 60 to 70 days of age for use in the study. The organization of groups is presented in Table I. Group C PC T-I T-II TABLE I TEST MATERIAL: -Aroclor 1242 Mutagenic Study - Albino Mice Organization of Groups Dose Level* (mg/kg) . 100 500 1, 000 Number of Males Treated 12 12 12 12 Aroclor 1242 was administered as a 10 percent solution in corn oil. Control males received the vehicle in amounts equivalent to those received by the T-II males. Positive control males received MMS in a 1 percent corn oil solution. DSM 036798 STLCOPCB4020622 /,,.(/)icl B I O - T E S T Id&iaiMiM, 9hc. 5 B. Dosage Levels ; The treatment levels were selected by intraperitoneal adminis tration of single graded doses of the test material to male mice. The i! maximum tolerated dose was determined and dose levels employed in the main study were based on these findings. The test material was adminis tered intraperitoneally as a corn oil solution to male mice. Control animals received the vehicle in volumes equivalent to those given the high test group and positive control males received methyl methane sulfonate (MMS). . C. Mating Schedule Each group consisted of 12 male mice, each of which was placed in a cage with 3 untreated virgin females immediately 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 post-meiotic stages prior to or during the spermatid_stagp._. 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 is methyl methanesulfonate (MMS). 036799 STLCOPCB4020623 i BIO-TEST lahyvd&tlel, $hc. 6 mated were at mid-pregnancy. All animals were sacrificed by carbon dioxide asphyxiation. The numbers of implantation sites, resorption sites, and embryos were recorded. Females were judged to be pregnant if corpora lutea were present in the ovaries. Resorption sites were divided into 2 groups, early deaths (deciduomata) and late deaths. Late deaths refer to embryos which develop to a relatively advanced stage prior to death, so that the placenta and fetal membranes or remnants thereof are visible. The frequency of late deaths is apparently unaffected by mutagens and, therefore, would appear to be non-genetic in this test system. Deciduomata occur at the sites of implanted blastocysts which fail to develop following implantation. The mutagenicity of the chemical can be measured by the proportions of all implantations which are deciduomata. 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 fate using this criterion assumes that pre-implantation losses among non-affected test animals will be es sentially the same as losses observed among control animals. DSW 036800 jW'uiUk& B i O TEST Jedo^aU^Ui-, 9*6. ' ' ' ' i '' ' IV. Results A. Treatment Levels Mice, groups of 4 males, were each given a single intrapcri- toneal injection of 30, 100, 300, or 1,000 mg of Aroclor 1242 per kg of body weight. There were no deaths or reactions observed among ' any of the animals treated. The levels chosen for the study were 500 (T-I) and 1,000 (T-II) mg/kg of body weight. B. Mortality and Reactions Two positive control males died, 1 on the day of treatment and 1 during week 3. One T-I and 2 T-II males died within 24 hours posttreatmer.t. No other deaths occurred and no unusual reactions were noted. 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 surviving males for each post-treatment week are shown in Table II. The mating indices of treated animals compared favorably to those of the control animals. The index at week 1 for the MMS treated animals was low. CSW 036d0i STLCOPCB4020625 JW.,thud BIO-T EST JaiMa&U&L, 9h&. 8 TABLE II TEST MATERIAL: Aroclor 1242 Mutagenic Study - Albino Mice Mating Performance Group Test Week Number Number of Surviving Males Number of Animals Pregnant Number of Females Mated Mating Index (Percent) C1 2 3 4 5 6 12 12 12 12 12 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 PC 1 2 3 4 5 6 11 11 11 10 10 10 14/33 . 19/33 23/33 26/30 v 23/30 24/30 42.4 57.6 69.7 86. 7 ' 76.7 80.0 DSW 036802 STLCOPCB4020626 yndiftitual B I O - T E S T Xalxyuit&Uel, $*c. 9 TABLE II continued TEST MATERIAL: Aroclor 1242 Mutagenic Study - Albino Mice Mating Performance Group Test Week Number Number of Surviving Males Number of Animals Pregnant Number of Females Mated Mating Index (Percent) T-1 T -1*. 1 2 3 4 5 6 1 2 3 4 5 6 11 11 11 11 11 11 10 10 10 10 10 10 22/33 27/33 30/33 28/33 33/33 27/33 19/30 26 /30 27/30 ' 23/30 22/30 25/30 66. 7 81. 8 90. 9 84. 8 100. 0 81. 8 . 63. 3 86. 7 90. 0 ' 76.7 73. 3 83. 3 CSW 036803 STLCOPCB4020627 hubiibual B I O - T E S T loluyicit&a&L, Hue. 10 D. Sacrifice Data Data pertaining to the numbers of corpora lutea, implantation sites, resorption sites (early and late differentiated), and embryos are presented in Table III. Laboratory data have shown that the untreated female mouse has a mean of 13. 5 corpora lutea. Therefore, the num ber of pregnant animals is multiplied by 13. 5 to obtain the total number of corpora lutea. The corpora lutea for the pseudo-pregnant females in control, T-I, and T-II (at week 3) that did not have implantations are 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. Positive control animals showed a marked effect (increase in early resorptions) at weeks 1 and 2. OSW 036804 STLCOPCB4020628 Group C PC TABLE HI TEST MATERIAL: Aroclor 1242 Mutagenic Study - Albino Mice . Summary of Sacrifice Data Test Week Number Pregnant Animals Examined Calculated Corpora Luton 1 2 3 4 5 6 '1 2 3 4 5 6 25 : 28 31* 29 28 . - 29 r'. . , 14 19 23 . : ' 26 23 24 .. 338 378 417 ' 392 , 278 : 392 ' ; 189 : v ; 256 ' 310 : 351 : 310 324 Implantation Sites 293 (11.7) 355 (12.7) 342 (11.0) 357 (12.3) 341 (12.2) 349 (12.0) 134 ( 9.6) 178 ( 9.4) 294 (12.8) 334 (12.8) 291 (12.6) 29 2 (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) 41 (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 (H.6) 325 (11.6) 334 (11.5) 91 ( 6.5) 131 ( 6. 9) 278 (12. 1) 314 (12. 1) 272 (11.8) 273 (H.4) DSW 0 3 6 8 0 5 Note: Numbers in parentheses are the means per female. * Includes 1 pseudo-pregnant female (corpora lutea only). 0B l - T E S T 2c STLCOPCB4020630 i .1 1 ' Group T-I T-n TABLE III continued TEST MATERIAL: Aroclor 1242 Mutagenic Study - Albino Mice _t Summary of Sacrifice Data Test Week Number . Pregnant Animals . Examined . Calculated Corpora Lutea Implantation Sites Resorption Sites Early Late 1 22 2 21 3 : 30* 4 28 5 33 6 27 1 19 2 ; 26 3' 27* 4 23 .5 22 6 25 297 . 364 . 408 378 446 364 268 (12. 2) 334 (12.4) 375 (12.5) 340 (12. 1) 424 (12.8) 337 (12.5) 4 (0. 2) 7 (0.2) 12 (0.4) 12 (0.4) 17 (0. 5) 10 (0.4) 0 (0.0) 1 (0. 1) 1 (0.1) 3 (0. 1) 3 (0. 1) 5 (0.2) 256 " 235 (12.4) 5 (0. 3) 1 (0.1) . 351 324 (12.5) 5 (0.2) 1 (0. 1) . / 370 343 (12. 7) 8 (0.3) 2 (0. 1) ' 310 292 (12.7) 8 (0. 3) 2 (0. 1) 297 268 (12.2) 5 (0. 2) 0 (0.0) ' 338 308 (12.3) 14 (0.6) 2 (0. 1) Embryos 264 (12. 0) 326 (12. 1) 362 (12. 1) 325 (11.6) 404 (12. 2) 322 (11. 9) 229 (12. 0) 318 (12. 2) 333 (12. 3) 282 (12. 3) 263 (12. 0) 292 (11. 7) o </; Note: Numbers in parentheses are the means per female. o * Includes 1 pseudo-pregnant female (corpora lutea only). OJ O' CD o Cr i tv Sttduihial B 1 O - T E S T Jed&taiori&i; Due. i1 13 E. Mutagenic Data A summary of results for the mutagenic study is presented in Table IV. Pre-Implantation Loss is defined as: Number of Corpora Lutea - Number of Implantation Sites x jqO Number of Corpora Lutea The mutation rate may be calculated by comparing the number of early resorptions (deciduomata) to the total number of implantations for each group. Number cf Early Resorption Sites v jqq ^ in tabie) Number of Implantation Sites 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. 100 - ( Embryos Test Group/Female x'100)-. (B in table) ............................... Embryos Control Group/Female -. Values presented were obtained by comparing each group to the contemporary 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 was greater than that of controls. The deviation from zero should not exceed 25 percent in the positive direction. OSW 036807 STLCOPCB4020631 Duduihkd B I O - T E S T Ja&MalvdeJ., 9m. 14 Mutation rates and pre-implantation losses among treated animals are essentially the same as those of the control animals. OSW 036808 STLCOPCB4020632 SnduitolaL BIO-TEST ^abyiai&U&i, 9no. 15 Group C PC TABLE IV TEST MATERIAL: Aroclor 1242 Mutagenic Study - Albino Mice Summary of Mutagenic Data T o ft t Week Number 1 2 3 4 5> 6 1 3 4 5, o/ Pre-Implantation Loss and Mutation Rates Mutation Rates Pre-Implantation Loss B (Percent) A ab 13. 3 6. 1 18.0 8.9 9.8 11. 0 1.7 2. 5 4. 1 4. 2 3. 8 3.4 - - 0.9 - -10. 7 - 10.4 - - 0.9 - 0.9 - 1. 7 29. 1 30. 5 5.2 4. 8 6. 1 9. 9 v 30.6 26.4 4.4 4.8 4.8 5. 8 43. 5 44.4 .-16. 3 -4.3' .- 1.7 0.9 43. 0 38.4 - 4.3 - 5.2 - 0. 8 2.6 DSW 036809 STLCOPCB4020633 OtvtuiUial B I O - T E S T 2al*Mai&U&l, Dtto. 16 Group T-1 T-II TABLE IV continued TEST MATERIAL: Aroclor 1242 Mutagenic Study - Albino Mice Summary of Mutagenic Data Test Week Number Pre-Implantation Loss and Mutation Rates' Mutation Rates Pre-Implantation Loss B (Percent) Aab 1 9.8 1. 5 - 4.3 - 5.3 2 8. 2 2. 1 2.4 - 8. 0 3 8. 1 3. 2 -16. 3 - 4.3 4 10. 0 3. 5 0. 0 - 0.9 5 4.9 4.0 - 5.2 - 4.2 6 7.4 3. 0 - 3.5 - 1.7 1 8. 2 2. 1 - 4. 3. - 5.3 2 7. 7 1. 5 1.6 - 8.9 3 7. 3 2. 3 -18. 3 -6.0 4 5. 8 " 2.7 - 6.0 ' - 7. 0 5 9. 8 1. 9 - 3.4 - 2.6 6 8. 9 4. 5 - 1.7 0. 0 DSW 036810 STLCOPCB4020634