Document nm5rXzVKdKd5JQgY0wVKL6X8R

HAZLV;OCCN WASHINGTON S200 LEESBURG PIKE VIENNA. VA. 22lE32-169S MUTAGENICITY TEST ON T-5710 IN AN IN VIVO RAT MICRONUCLEUS ASSAY DATA REQUIREMENT U.S. EPA FIFRA Guideline 84-2 FINAL REPORT AUTHOR Hemalatha Murli, Ph.D. PERFORMING LABOR.ATORY Hazleton Washington, Inc. 9200 Leesburg Pike Vienna, Virginia 22182 LABORATORY PROJECT ID HWA Study No.: 15516-0-454 SUBMITTED TO 3M Corporation 3M Center St. Paul, Minnesota 55144 STUDY COMPLETION DATE April 23, 1993 , I AP@R 2 8 IS93 3 1%4 k 15516-0-454 1 of 25 -"'*HAZLEN:C:N WASHINGTON STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS No claim of confidentialityis made for any informationcontained in this study on the basis of its fallingwithin the scope of FIFRA 10(d)(1)(A), (B),or (C). Company: Company Agent: (Typed Name) (Title) Signature Date 2 15516-0-454 HAZLE'C(MN WASHINGTON STATEMENT OF DATA CONFIDENTIALITY CLAIMS Information claimed confidential on the basis of its falling within the scope of FIFRA 10(d)(1)(A),(B), or (C) has been removed to a confidential appendix, and is cited by cross-reference number in the body of the study. Company: Company Agent: (Typed Name) (Title) Signature Date 2 15516-0-454 HAZLF.-CC:N WASHINGTON GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with the EPA FIFRA Good Laboratory Practice Standards as set forth in Title 40 of the'U.S. Code of Federal Regulations Part 160. Any significant deviations from the protocol and/or GLP are attached as an appendix to this report. There were no deviations from the aforementioned regulation which affected the quality or integrity of the study or the interpretation of the results in this report. Study Director: al.l.fi2tz - 9),-, f@14- Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Molecular and Cellular Toxicology Study Monitor: Applicant/Submitter: 15516-0-454 Dite Date Date 3 ' HAZLENCON k.,W@ WASHINGTON THIS PAGE INTENTIONALLY LEFT BLANK 15516-0-454 @4 w HAZLETON WASHINGTON THIS PAGE INTENTIONALLY LEFT BLANK 5 15516-0-454 HAZLCOCC:N WASHINGTON QUALITY ASSURANCE STATEMENT PROJECT TITLE: IN VIVO RAT MICRONUCLEUS ASSAY PROJECT NO.: 20996 ASSAY NO.: 15516 PROTOCOL NO.: 454 EDITION NO.: 5 QualityAssurance inspections of the study and/or review of the final report of the above referenced project were conducted according to the Standard Operating Procedures of the Quality Assurance Unit and according to the general requir ements of the appropriate Good Laboratory Practice regulations. Findings from 'the inspections and final report review were reported to management and to the study director on the following dates: Insi)ection/Date Harvest-3/411993 Draft Report review/4/1411993 Final Report review/4/23/1993 Findings Reported 3/04/1993 4/14/1993 4/23/1993 Auditor B. Mullett B. Mullett M. Murphy 15516-0-454 QuaN-ty @,ssurance untt Date Released 6 '"HAZLENCON WASHINGTON STUDY COMPLIANCE AND CERTIFICATION The described study was conducted in compliance with the Good Laboratory Practice regulations as set forth in the Code of Federal Regulations (21 CFR 58, 40 CFR 792, and 40 CFR 160); the Organization for Economic Cooperation and Development Principles of Good Laboratory Practice C(81)30 (Final) Annex 2, issued 1979-1980 (effective 1981). To the best of the signers' knowledge, there were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrity of the study or the interpretation of the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation of the test article as presented herein represents an appropriate conclusion within the context of the study design and evaluation criteria. All test and control results in this report are supported by an experimental data record and this record has been reviewed by the Study Director. All raw data, documentation, records, protocols, and a copy of the final report generated as a result of this study will be archived in the storage facilities of Hazleton Washington, Inc., for at least one year following submission of the final report to the Sponsor. After the one year period, t-heSponsor may elect to have the aforementioned materials retained in the storage facilities of Hazleton Washington, Inc., for an additional period of time, or sent to a storage facility designated by the Sponsor. SUBMITTED BY: Ji5s6Arriaga,t.S Research Associate Study Director: Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Molecular and Cellular Toxicology 15516-0-454 tate' -?,3 3 Study Completion Date 7 HAZLV;ECC:N WASHINGTON TABLE OF CONTENTS SUMMARY 1. III. Page No. . . . . .. . . . . . . .. . . . .. . . . . . .. . . . . . 10 SPONSOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 MATERIAL (TEST ARTICLE) . . . . . . . . . . . . . . . . . . . . . 11 A. Client's Identification B. Dates Received C. Physical Description D. Genetics Assay No. TYPE OF ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . 11 IV. PROTOCOL NO . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 V. STUDY DATES . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 A. Initiation Date B. Experimental Start Date C. Experimental Termination Date Vi. SUPERVISORY PERSONNEL . ... . . . . . . . . . . . . . . . . . . . 11 A. Study Director B. Laboratory Supervisor vii. OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Viii. MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 IX. SOLUBILITY AND STABILITY . . . . . . . . . . . . . . . . . . . . 12 A. Solubility and Stability B. Dose Selection C. Micronucleus Assay Dosing Information X. BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS . . . . . . . 18 Xi. EVALUATION CRITERIA . . . . . . . . . . . . . . . . . . . . . . . 19 A. General B. Data Presentation and Interpretation Xii. RESULTS AND INTERPRETATION . . . . . . . . . . . . . . . . ... . 20 xiii. CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 XIV. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 xv. DEVIATION FROM THE SIGNED PROTOCOL . . . . . . . . . ... . . . . 20 15516-0-454 8 w' HAZLEICCDN WASHINGTON I TABLE OF CONTENTS (Continued) XVI. EXPERIMENT DATA TABLES . . . . . . . . . . . . . . . . . . . . . 21 15516-0-454 9 HAZLE-CC:N WASHINGTON SUMMARY Mutagenicity Test on T-5710 in an i-n vivo Rat Micronucleus Assay The objective of this in vivo assay was to evaluate the ability of the test article, T-5710, to induce micronuclei in bone marrow polychromatic erythrocytes of Sprague-Dawley rats. The test article was suspended in corn oil. At the request of the Sponsor, Trial I of the micronucleus study was conducted dosing T-5710 by oral gavage at 1250, 2500, and 5000 mg/kg. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups euthanatized approximately 24 hours after dosing were included in the assay. The animals were dosed with the test article and were euthanatized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. Due to the excessive toxicity/mortality in the animals at all dose levels, especially at the 72 hour harvest, this assay was repeated. After consultation with the Sponsor. Trial Il of the micronucleus study was conducted dosing T-5710 by oral gavage at 312.5, 625, and 1250 mg/kg. Again, ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups euthanatized approximately 24 hours after dosing were included in the assay. The animals were dosed with the test article and were euthanatized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. The test material, T-5710, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the rat bone marrow micronucleus test. 15516-0-454 10 '"-'@'HAZLETON WASHINGTON Mutagenicity Test on T-5710 in an i-nvivo Micronucleus Assay I. SPONSOR: 3M Corporation Il. MATERIAL (TEST ARTICLE): A. Client's Identification: L-10059 (T-5710) B. Dates Received: February 24, 1993; March 11, 1993 C. Physical Description: Cream-colored granular material D. Genetics Assay No.: 15516 III. TYPE OF ASSAY: In Vivo Rat Micronucleus Assay IV. PROTOCOL NO.: 454, Edition 5 V. STUDY DATES: A. InitiationDate: February 24, 1993 B. Experimental Start Date: March 2, 1993 C. Experimental Termination Date: March 30, 1993 vi. SUPERVISORY PERSONNEL: A. Study Director: Hemalatha Murli, Ph.D. B. Laboratory Supervisor: Jos6 Arriaga, B.S. VII. OBJECTIVE: The objective of this in vivo assay was to evaluate the ability of the test article, T-5710, to induce micronuclei in bone marrow polychromatic erythrocytes of Sprague-Dawley rats. This study was conducted using modifications of the procedures suggested by Heddle et al. (1983). 15516-0-454 11 HAZLC'CC:N WASHINGTON VIII. MATERIALS: Adult male and female rats, strain Sprague-Dawley, were purchased from Charles River Laboratories, Raleigh, NC. This healthy, random bred strain was selected to maximize genetic heterogeneity and at the same time assure access to a common source. Animals were isolated by sex. Animals were housed two per cage during quarantine, and housed individually at randomization. The temperature was maintained at 71.8 to 80.20F for Trial I and at 7260F for Trial II and humidity was maintained at 5020Z. A 12-hour light/12-hour dark cycle was maintained. A commercial diet (Purina'*Certified Laboratory, ChowO 15002) and water were available ad libitum for the duration of the study. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed on a retrospective basis for specified microorganisms, pesticides, alkalinity, heavy metals, and halogens. Sanitary, stainless steel, hanging wire cages were used. Personnel handling animals or working within the animal facilities were required to wear suitable protective garments and equipment. Animals were quarantined for seven days before being placed on study. Animals were randomly assigned to study groups and were individually weighed prior to dosing. All animals were dosed based upon the individual body vteights. Animals were uniquely identified by ear tag. Dose or.treatment groups were identified by cage card. At the termination of the study all surviving animals were euthanatized by C02 inhalation, followed by penetration of the thorax. Any extra animals not used for the study were euthanatized by C02 inhalation, followed by penetration of the thorax. IX. SOLUBILITY AND STABILITY: A. Solubility and Stability The test article, T-5710, was supplied as a cream-colored granular material. The solubility of the test article was evaluated in corn oil and 0.52 high viscosity carboxymethylcellulose (CMC). Unsuitable suspensions were obtained in CMC. An opaque, viscous suspension was obtained when 0.66 ml of corn oil was added to 589.2 mg of T-5710, resulting in a final volume of about 2.3 ml, and a final concentration of about 256.2 mg/ml that passed easily through an 18G gavage needle. The vehicle used to suspend the test article for the bone marrow micronucleus assay was corn oil. The stability of the test article under the preparation and dosing conditions of the assay is the responsibility of the sponsor. 15516-0-454 12 HAZLEEC(ON WASHINGTON B. Dose Selection The dose levels used in Trial I of this assay were selected by the Sponsor. The doses selected for this assay were 1250, 2500, and 5000 mg/kg body weight, and were administered by oral gavage. C. Micronucleus Assay Dosing Information TRIAL I The animals used in Trial I of the micronucleus assay were dosed on Tuesday, March 2, 1993. Cyclophosphamide (CP, Sigma, Lot 470HO948), the positive control, was solubilized in sterile deionized water (Lotll3, prepared at HWA) and was administered by oral gavage at 60 mg/kg. The vehicle control consisted of corn oil (Duke's corn oil, Lot 2B 25 17:59) and was administered concurrently with the test article at a volume of 20 ml/kg. The weight range of the animals used in the micronucleus assay was 251.1 - 302.7 grams and 169.7 - 210.4 grams for the males and females, respectively. The dosing solutions for the assay were prepared by making a 250 mg/ml stock for the high dose (5000 mg/kg). An opaque, cream-colored, slightly viscous suspension was obtained when 112.5 ml of corn oil was added 87.5001 grams of T-5710, resulting in a final volume of 350.0 ml and a final concentration of 250 mg/ml. This suspension was homogenized in a Tissuemizerg) for about 5 minutes to ensure an uniform suspension. Dilutions of this stock were prepared for the 2500 and 1250 mg/kg dose levels. A second group of animals (designated Secondary Dose Group) was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group. The test article dosed animals were euthanatized approximately 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized approximately 24 hours after the administration of the control articles. An outline of the dosing scheme is found in the following table: 15516-0-454 13 HAZLEGE(ON WASHINGTON ... Dosing Scheme for Trial I of the Micronucleus Assay A total of 126 animals was used in this assay. Number of Animals Assigned Primary Dose Groups 24 Hr 48 Hr 72 Hr Secondary Dose Groups' Treatment M F M F M F Male Female T-5710 1250 mglkg 2500 mg/kg 5000 mg/kg 55 55 55 55 55 55 - - 5 5 5 5 55 8 8 Vehicle Control, Corn oil 20 ml/kg 5 5 -- -- - - Positive Control, Cyclophosphamide 60 mg/kg 5 5 -- -- The animals assigned to the secondary dose groups were dosed and were only used to replace animals which died in the primary dose group at the high dose level. All extra animals not used as replacementswere euthanatizedat the completion of the trial. The age of the animals at the time of dosing was -8 weeks. Volumes dosed were 20 ml/kg (except for the positive controls) and were based upon individual animal weights. The positive control animals were dosed at 10 ml/kg. Animal Observations All animals were observed immediately after dosing and periodicallythroughoutthe duration of the assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriateharvest times. All test article dosed groups appeared normal immediately after and approximately 4 hours after dosing. 15516-0-454 14 w HAZLV;@CCN WASHINGTON Approximately 23 hours after dosing, all animals from the 1250, 2500, and 5000 mg/kg dose groups appeared ataxic with diarrhea. One male (16216, 2500 mg/kg dose and 72 hour harvest group) and 1 female (16273, 5000 mg/kg and 48 hour harvest group) were also experiencing lacrimination of the eyes. Approximately 47 hours after dosing, 2 males (I's 6250, 6238,, 72 hour harvest group) and 2 females (16273, 48 hour harvest group; 16241, secondary dose group) from the 5000 mg/kg dose group, and 2 males (16234, 48 hour harvest group; #6216, 72 hour harvest group) from the 2500 mg/kg dose group, and 1 female (16198) from the 1250 mg/kg dose and 48 hour harvest group were found dead. All remaining animals from the 5000, 2500, and 1250 mg/kg dose groups appeared ataxic with diarrhea. Approximately 49 hours after dosing, 2 males (I's 6235, 6287, secondary dose group) and I female (16246, secondary dose group) were found dead. All remaining animals from the 5000, 2500, and 1250 mg/kg dose groups appeared ataxic with diarrhea. Approximately 70 hours after dosing, 3 males (1 6217, 72 hour harvest group; I's 6205,6240, secondary dose group) and 3 females (I's6l9l, 6259, 72 hour harvest group; 16299, secondary dose group) from the 5000 mg/kg dose group, 3 males (I's6210, 6310, 6281, 72 hour harvest group) and 2 females (I's6309, 6204, 72 hour harvest group) from the 2500 mg/kg dose group, and 1 male (16303, 72 hour harvest group) and 1 female (16242, 72 hour harvest group) from the 1250 mglkg dose group were found dead. All remaining animals from the 5000, 2500, and 1250 mg/kg dose groups appeared ataxic with diarrhea, and red crusts around the nose. Approximately 71 hours after dosing, 1 female (1 6290, 72 hour harvest group) from the 5000 mg/kg dose group and 1 female (16257, 72 hour harvest group) from the 2500 mg/kg dose group were found dead. All remaining animals from the 5000, 2500, and 1250 mg/kg dose groups remained unchanged from the earlier set of observations. There were too few animals surviving in this Trial for a valid analysis. Thus, after consultation with the Sponsor, a Trial II was conducted testing dose levels of 312.5, 625, and 1250 mglkg body weight. 15516-0-454 15 '"-"*HAZLENCON WASHINGTON TRIAL II The animals used in the micronucleus assay were dosed on Tuesday, March 23, 1993. Cyclophosphamide (CP, Sigma, Lot 170HO948), the positive control, was solubilized in sterile deionized water (Lotil3, prepared at HWA) and was administered by oral gavage at 60 mg/kg. The vehicle control consisted of corn oil (Duke's corn oil, Lot 2B 25 17:59) and was administered concurrently with the test article at a volume of 10 mllkg. The weight range of the animals used in the micronucleus assay was 224.4 - 275.0 grams and 165.0 - 213.0 grams for the males and females, respectively. The dosing solutions for the assay were prepared by making a 125 mg/ml stock for the high dose (1250 mg/kg). An opaque, pale yellow suspension was obtained when 179.2 ml of corn oil was added 23.7500 grams of T75710, resulting in a final volume of 190.0 ml and a final concentration of 125 mg/ml. This suspension was homogenized in a Tissuemizere for about 5 minutes to ensure an uniform suspension. Dilutions of this stock were prepared for the 625.0 and 312.5 mg/kg dose levels. A second group of animals (designated Secondary Dose Group) was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group. The test article dosed animals were euthanatized approximately 24, 48 and 72 hours after administration of the test article. The positive and vehicle control animals were euthanatized approximately 24 hours after the administration of the control articles. An outline of the dosing scheme is found in the following table: 15516-0-454 16 '"-**HAZLC'CC:N WASHINGTON Dosing Scheme for Trial II of the Hicronucleus Assay A total of 130 animals was used in this assay. Number of Animals Assigned Treatment Primary Dose Groups 24 Hr 48 Hr 72 Hr Secondary Dose Groups' MF MF MF Male Female T-5710 312.5 mg/kg 625.0 mg/kg 55 55 55 55 55 55 - - 1250 mglkg 55 55 55 10 10 Vehicle Control, Corn oil 10 ml/kg 55 -- -- - - Positive Control, Cyclophosphamide 60 mg/kg 55 -- -- The animals assigned to the secondary dose groups were dosed and were only used to replace animals which died in the primary dose group at the high dose level. All extra animals not used as replacements were euthanatized at the completion of the trial. The age of the animals at the time of dosing was -8 weeks. Volumes dosed were 10 ml/kg and were based upon individual animal weights. Animal Observations All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately after dosing. 15516-0-454 17 HAZLC@TC:N WASHINGTON Approximately 5 hours 625.0, and 1250 mg/kg group were ataxic. after dosing, all animals dose groups including the from the 312.5, secondary dose Approximately 23 hours after dosing, 1 male (16557) from the 1250 mg/kg dose and 24 hour harvest group appeared languid. All remaining animals from the 1250 mg/kg dose group including the secondary dose group appeared ataxic. All animals from the 312.5 and 625.0 mg/kg dose groups appeared normal at this time. Approximately 47 hours after dosing, all animals from the 1250 mg/kg dose group including the secondary dose group appeared ataxic. All animals from the 312.5 and 625.0 mg/kg dose groups appeared normal at this time. Approximately 71 hours after dosing, 2 males (16481, 1250 mg/kg dose and 72 hour harvest group; #6454 from the secondary dose group) were found dead. All remaining animals from the 1250 mg/kg dose group including the secondary dose group appeared ataxic. All animals from the 312.5 and 625.0 mg/kg dose groups appeared normal at this time. X. BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS: At the appropriate harvest time, the animals were euthanatized with C02 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was aspirated from the bone and mixed in a syringe with -0.1 ml fetal calf serum to form a suspension. The cells were then placed on slides and air-dried, fixed in methanol, and stained in May-Grunwald solution followed by Giemsa (Schmid, 1975). The air-dried slides were coverslipped using Depexl mounting medium. The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this rat strain is about 0.0-0.4z. The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring the first 1000 erythrocytes. 15516-0-454 is HAZLV;T.CDN WASHINGTON Xi. EVALUATION CRITERIA: A. General: The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively). B. Data Presentation and Interpretation Data are summarized by sex and dose groups for the different time points. Individual animal data are also presented. The analysis of the data was performed using an Analysis of Variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons (Sokal and Rohlf, 1981) was used at each harvest time to determine which dose groups, if any, were significantly different (p<0.05) from the vehicle control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined. The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCES, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment. 15516-0-454 19 HAZLV;'CCN WASHINGTON XII. RESULTS AND INTERPRETATION: The test article, T-5710, induced no significantincreases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 4.64Z t 0.64Z and 3.12Z 0.70Z for the males and females, respectively. The data summarized by dose group are presented in Table 1 and individual animal data are found in Tables 2 through 4. XIII. CONCLUSION: The test material, T-5710, did not induce a significantincrease in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the rat bone marrow micronucleus test. XIV. REFERENCES: Heddle, J.A., Hite, M., Kirkhart, B., Larsen, K., MacGregor, J.T., Newell, G.W., and Salamone, M.F.: -The induction of micronuclei as a measure of genotaxicity. Mutation Res., 123:61-118, 1983. Schmid, W.: The micronucleus test. Mutation Res., 31:9-15, 1975. Schmid, W.: The micronucleus test for cytogenetic analysis. In, Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4 (A. Hollaender, ed.). Plenum, pp. 31-53. 1976. Sokal, R.R. and Rohlf, J.J.: Biometry, Freeman, 1981. xv. DEVIATION FROM THE SIGNED PROTOCOL: The following deviation was made from the protocol. The temperaturewas maintained at 71.8 to 80.20F for Trial I of the micronucleus assay and not maintained at 72*60F. This was due to technicaldifficultiesat the animal facility. This slightdeviation did not affect the animals in any way. 15516-0-454 20 . . - 'w HAZLE-CC)N WASHINGTON I XVI. EXPERIMENT DATA TABLES 15516-0-454 21 Ln LA H SPONSOR: 3M Corporation ASSAY: 15516 TABLE I MICRONUCLEUS DATA SUMMARY TABLE TEST ARTIC TRFAR04T HARVEST DDSE TIME (HR) Z @ffCRONLICLEATED PCEs WAN OF 1000 PER ANDIAL S.E. MALES FEMALES 10TAL RATIO PCI WAN S KALES VEHICLE CONTROL OORN OIL 10 id/kg POSITIVE CDNTROL 60 irg/kg CYCLOPHOSPHAMME 24 0.12 0.05 24 4.64 0.64* 0.12 0.04 3.12 0.70* 0.12 0.03 3.88 0.51* 0.82 0.09 0.70 0.08 TEST ARTICLE T-5710 24 0.12 0.04 312.5 ng/kg 48 0.06 0.02 72 0.14 0.04 0.12 0.04 0.10 0.04 0.12 0.04 0.12 0.02 0.08 0.02 0.13 0.03 0.71 0.07 0.63 0.11 0.74 0.08 24 0.08 0.05 625.0 ug/kg 48 0.16 0.09 72 0.16 0.07 0.14 0.07 0.08 0.06 0.12 0.05 0.11 0.04 0.12 0.05 0.14 0.04 0.66 0.06 i 0.54 0.10 0.74 0.09 1250 ag/kg 24 0.14 0.05 48 0.04 0.02 72 0.22 0.02 0.12 0.04 0.04 0.02 0.04 0.02 0.13 0.03 0.04 0.02 0.13 0.03 0.58 0.05 0.56 0.18 0.53 0.04 Significantly different from the corresponding vehicle control, p<O. HAZLENCC:N WASHINGTON TABLE 2 MICRONUCLEUS,TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-5710 ASSAY NO.: 15516 MALES FEMALES TREATMENT ANIMAL NUMBER NO. MN PCEs (1000) RATIO PCE:NCE ANIMAL NUMBER NO. MN PCEs (1000) -RATIO PCE:NCE Vehicle Control (24 hour harvest) Positive Control (24 hour harvest) Test Article (24 hour harvest) 312.5 mg/kg 625.0 mg/kg 1250 mg/kg 6477 6482 6508 6509 6523 6502 6517 6524 6554 6582 6526 6528 6541 6561 6569 6462 6464 6473 6491 6571 6460 6556 6557 6570 6579 1 0.78 1 0.66 3 0.99 0 1.04 1 0.62 6476 1 6487 2 6497 0 6522 1 6539 2 36 0.69 47 0.72 29 0.48 65 0.68 55 0.95 6467 20 6472 15 6479 51 6527 26 6578 44 0 0.94 2 0.69 1 0.48 2 0.71 1 0.73 6488 2 6512 2 6536 1 6572 1 6584 0 0 0.78 0 0.64 2 0.79 0 0.45 2 0.64 6500 3 6531 0 6567 0 6568 3 6583 1 1 0.69 3 0.58 1 0.39 0 0.62 2 0.62 6458 1 6558 2 6575 0 6577 2 6580 1 MN Micronucleus PCE Polychromatic erythrocyte No. MN PCEs - Micronucleated PCEs NCE - Normochromatic erythrocyte 0.58 0.32 0.42 0.31 0.31 0.38 0.49 0.24 0.33 0.68 0.42 0.65 0.40 0.51 0.67 0.12 0.41 0.62 0.39 0.94 0.98 0.25 0.69 0.57 0.21 15516-0-454 23 HAZLF;NC(ON WASHINGTON TABLE 3 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-5710 ASSAY NO.: 15516 MALES FEMALES TREATMENT ANIMAL NUMBER NO. MN PCEs RATIO (1000) PCE:NCE ANIMAL NUMBER NO. MN PCEs RATIO (1000) PCE:NCE Test Article 6461 1 0.60 (48 hour harvest) 6484 0 0.23 312.5 mg/kg 6486 0 0.70 6559 1 0.92 6573 1 0.69 6493 0 0.47 6511 2 0.40 6546 0 0.67 6550 2 0.66 6551 1 0.80 625.0 mg/kg 6457 1 0.40 6470 0 0.33 6480 5 0.80 6499 0 0.76 6566 2 0.39 6449 0 0.36 6483 0 0.24 6532 3 0.67 6544 1 0.68 6552 0 0.58 1250 mg/kg 6451 6469 6496 6518 6543 1 0.68 0 0.10 0 0.20 0 1.02 1 0.81 6459 0 0.22 6505 1 0.18 6540 0 0.31 6542 0 0.99 6562 1 0.68 MN Micronucleus PCE Polychromatic erythrocyte NO. MN PCES - Micronucleated PCEs NCE Normochromatic erythrocyte .15516-0-454 24 HAZLCMCON WASHINGTON TABLE 4 MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M Corporation TEST ARTICLE: T-5710 ASSAY'NO.: 15516 MALES FEM&LES TREATMENT ANIMAL NUMBER NO. MN PCEs RATIO (1000) PCE:NCE ANIMAL NUMBER NO. MN PCEs RATIO (1000) PCE:NCE Test Article 6456 2 0.71 (72hour harvest) 6503 1 0.66 312.5 mg/kg 6534 0.50 6563 2 0.88 6581 0 0.94 6463 1 0.67 6489 2 0.31 6490 2 0.35 6507 1 0.37 6565 0 0.73 625.0 mg/kg 6465 2 0.58 6468 4 1.00 6513 1 0.58 6545 1 0.65 6574 0 0.89 6455 1 0.83 6485 3 0.29 6498 1 0.43 6514 0 0.22 6533 1 0.27 1250 mg/kg 6448 2 0.46 6452 2 0.44 6492 2 0.54 6529 3 0.53 6564 2 0.68 6495 0 6519 0 6548 0 6555 1 6585 1 MN Micronucleus PCE Polychromatic erythrocyte No. MN PCEs = Micronucleated PCEs NCE - Normochromatic erythrocyte 1.00 0.40 0.20 0.55 0.43 15516-0-454 25