Document wgd4Rgp5qv2eXNvMqkooY13aQ

SnSu&faaL 5IO-TiST JlaiMa&yusl. Sue. mo HONTAOf OAO NOKTHiKOOK. ILLINOIS 6002 REPORT TO MONSANTO COMPANY MUTAGENIC STUDY WITH AROCLOR 1260 IN ALBINO MICE JANUARY 13, 1972 IBT NO. E62 3 # >-1. Dear Mr. Wheeler: R: 1ST No. 623 - Mutagenic Study with Aroclor 1260 in Albino Mica We are submitting herewith our laboratory report dated January 13, 1972, prepared la connection with the above study. Very truly yours, & ts f J. C. Calandra President . i* j .. JCC/kjl V4| -X* ,, *. '"rTjL? ,tM0NS 0684,9 ."'I- --vi-J . -- - - REPORT TO MONSANTO COMPANY MUTAGENIC STUDY WITH AROCLOR 1260 IN ALBINO MICE JANUARY 13, 1972 IBT NO. E623 I. Introduction A mutation it a change in tha character of a gone euch 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 fertilise an egg or. once having fertilised, to the failure of development beyond the blastocyst stage (implantation). Male mice, treated with the test compound, are mated with untreated femalea. 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. MGhS 060490 II. Summary . _ .j'. ., A dominant lethal mutagenic study was conducted using albino mice-. - Males were treated with a single intraperitoneal injection of Aroclor 1260 at levels of either S00 (T-I) or 1,000 (T-1I) mg /kg of body weight. Another group of males received methyl methanesulfonate (MMS) at 100 mg/Vg and was used as 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 the 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. Report prepared by: INDUSTRIAL BIO-TEST LABORATORIES, INC. J1 Dennis Arnold, B.S. ' Croup Leader Mutagenic Studies Report approved by: Gerald Kennedy, Hb. S. Senior Group Leader Metabolic and Mutagenic Studies January 13, 1972 - Manager, Toxicology HOMS 066491 ' W ' .', \V" v; 13' '.if:-,-. .. m. Procedure - o*7"' V\>7^':^'^ ' , ` . . ~'V '}v . ^,^:3 * * ^ ?' ?f% A. Outline of Experiment The test materiel was Aroclor 1260. Methyl methanesulfonate wme used as a positive control. Charles River strain albino mice were received at this liboratory at 60 to 70 days of age for use in the study. The organi zation of groups is presented in Table I. Grouo C PC T-[ T-U TABLE 1 TEST MATERIAL: Aroclor 1260 Mutagenic Study Albino Mice ~ Organization of Groups Do.e Level* (mg/kg) 100 $00 1. 000 Number of Melee Tre.ted 12 12 12 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-ll males. Positive control males received methyl methane sulfonate in a 1 percent corn oil solution. AOAS 06649^ -vt-.T.v-'V **&. V 'V.; *-.&jpg** f? . .: - * .** * y" 9 3nJ*itual BIO T E S T JI*Utftnd0i, 3m vr B, Dosage Levels '' The treatment levels were selected by intraperitoneal admtnis(ration o/ single 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 intraperitoneally in a corn oil solution to male mice. Control animals received the vehicle in volumes equivalent to those giver the high test group and positive control males received methyl methane sulfonate. C. Mating Schedule Each group consisted of 12 male mice, each of which was placed in a cage with 3 untreated virgin females immediately sfter 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 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 in methyl methanesulfonate (MhlSV* .' k i-'-'i*S*"::- -*y . ' .kiTTr"-.-.J*'--1V-- ' (J3'I *" VV* aT. ' MOWS 060493 MuUtial Bno.TEST JiiM4 A?- :?\j^-y -'- ."-C-^S--; r*\ mated were at mid-pregnancy. AH animals were sacrificed by carbon . dioxide asphyxiation. The numbers of Implantation sites, resorption rites, 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 (dec!duomata) and late deaths. Lnte 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 bo 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 rate using this criterion assumes that pre-implantation losses among non-affected test animals will be es sentially the same ae losses observed among control animals. w.* , r HONS 068494 BIO-TES? Jmi***U*iM, . **? IV. Results . A. Treatment Levels \\ ' ^< Doses 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 14-day observation period following treatment. B. Mortality and Reaction# 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 ammalt pregnant divided by the number of females mated times 100, and the number of surviving males for each poet-treatment week are shown in Table IT. All mating indices for the Aroclor 1260 treated animals seen in the experiment are considered to bo normal for the mouse strain employed. Positive control animals had a low mating index for the first week post treatment. ' :r: ^ HONS 060495 m Mating Performance To it Number of Week Surviving Crouo Number Melee Number of Animals Precnant Number of Females Mated Meting Index (Percent! C1 2 3 4 5 & 12 12 12 12 12 12 25/36 26/36 31/36 29/36 26/36 29/36 69.4 77.8 86. 1 80.6 77.8 80.6 PC i 2 3_ 4 5 6 11 11 tl 10 10 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 1 -! 81 o T E S T JaUtalttlM. im. ' .;~r TABLE II continued TEST MATERIAL: Aroclor 1260 Mutagenic Study - Albino Mice Mating Performance Teat Number of Week Surviving Group Number Males Number of Animals Pregnant Number of Females Mated Mating Index (Percent) T-I i 2 3 4 5 6 12 12 12 11 11 11 31/36 31/36 26/36 28/33 29/33 32/33 86. 1 86. 1 72.2 84.8 87.9 97.0 T-II 1 2 J 4 3 6 12 12 12 12 . 12 12 30/36 30/36 23/36 31/36 27/36 28/36 83.3 83.3 69.4 86. 1 75.0 77.8 HONS 068497 1 Vi I i' . 'l hi m. ( '*.1 :: .VjjV - Group Test Week Number CI 2 3 4 S 6 PC 1 2 3 4 5 6 TABLE III TEST MATERIAL: Aroclor 1260 Mutagenic Study - Albino Mice Pregnant Animals Examined 25 28 31* 29 28 29 14 19 23 26 23 24 Summary of Sacrifice Data --t----------------------- Calculated Implantation Corpora Lutea Sites Resorption Sites Early Late 338 378 417 392 378 392 293 (11.7) 355 (12.7) 342 (11.0) 357 (12. 3) 341 (12.2) 349 (U.0) 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) 189 256 3 10 351 310 324 134 (9.6) 178 ( 9.4) 294 (12.8) 334 (12.8) 291(12.6) 292 (12.2) 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 (It.6) 325 (11.6) 334 (11.5) 91 ( 6.51 131 ( 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-pregnant female (corpora lutea only). s-i HONS 0 6 8 4 9 9 1 ru Group Tt W.k Number T-l 1 2 3 4 5 6 T-II 1 2 3 4 5 6 TABLE III continued TEST MATERIAL: Aroclor 1260 Mutagenic Study - Albino Mice Summary of Sacrifice Data Pregnant Animals Examined 31 31 26* 28 29 32 30 30 25 31 27 28 Calculated Implantation Corpora Lutea 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) 405 342 (11.4) 405 371 (12.4) 338 312 (12. 5) 418 376 (12. 1) 364 330 (12.2) 378 349 (12.5) 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) 7 (0. 1) 2 (0. 1) 5 (0. 2) 1 (0. 1) 4 (0. 1) 13 (0.4) IS (0. 5) 15 (0.6) 16 (0.5) 18 (0. 7) 13 (0. 5) 1 (0. 1) 2 (0. 1) 4 (0.2) 3 (0. 1) 1 (0. 1) 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) Notes Numbers In parentheses are the means per female. * Includes 1 pseudo-pregnant female (corpora lutea only). E. Mutagenic Data *:Ki A summary of results for the mutagenic study is presented in Table IV. PreImplantatinn Loss is defined ast Number of Corpora Lutes Number of Implantation Sites K jqq 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. Numbor of Early Resorption Sites x jqq ^ Number of Implantation Sites table) Another way of expressing the mutation rate, which takes into account prs-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. inn / Embryos Test Croup/Female * ' Embryos Control Group/Female x ' (B in table) Values prtsented 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 embryoa for that group and week was greater than that of controls. The deviation from aero should not txeeed 25 percent in the positive direction. HONS 068501 MONS 06fl502 D Group c PC TABLE IV TEST MATERIAL* Aroclor 1260 Mutagenic Study - Albino Mice Summary of Mutagenic Data Tet Wek Number 1 2 3 4 5 6 1 2 3 4 5 6 Pre-Implantation Loaa and Mutation Rate a Mutation Ratee Pre-Implantation Loaa B (Percent) 13. 3 Aa b .1.7 - 0.9 6. 1 78.0 . .2.5 -10.7 4. 1 10.4 8.9 4.2 - 0,9 9.8 3.8 0.9 11.0 3.4 - 1.7 29. 1 10.5 5.2 4.8 6. 1 9.9 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 HONS 068503 fUuUiml BIO-TEST ?/SS.;*-X Croup T-I T-II TABLE IV continued TEST MATERIAL: ArocJor 1260 Mutagenic Study - Albino Mice Summary of Mutagenic Data Teet Week Number Pre-Implantation Ijoas and Mutation Rates Pre-Implantation Loaa (Percent) A Mutation Rate* B ab 1 18.9 3.8 8.7 7.9 2 8. 1 1.8 2.4 8.0 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 -1.7 1 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 mm HONS 068504 m