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UNCk92 2s MUTEN-DNY SUUNmCUIC OA o et ?OXICTY T4-NITROPHENYL MONOCI4LOROME (U) LETTERMAN ARMY INST OFRESEARCH PESIDIO OF SAN FRANCISCO CA
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Institute Report No. 255
NFourteen-Day Subchronic Oral Toxicity Study of 4-
IN Nitrophenyl Monochloromethyl (Phenyl) Phosphinate inam) Male Rats
S-Carolyn M. Lewis, M
Craig W. White, DVK CPT CICThomas P. Kxelner, BA, SP5
Paul P. Waring, BSJohn C. Turnier, DVM, M&J VC
andJohn T. Fruin, PhD, COL VC
Mammalian Toxicology Branch DTIC
Division of Toxicology TELE! E
MR2 21988?
February 1988 Toxicology Series: 74
LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129
D.STR.. .TA7,
Approve for pjutbc rejea 8 821 vO t 14 "7
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Fourteen Day Subchronic Oral Toxicity Study of 4-NitrophenylMonochloromethyl (Phenyl) Phosphinate in Male Rats (Toxicology Series 74)--
Lewis, White, Kellner, Waring, Turnier, and Fruin
Reproduction of this document in whole or in part is prohibited except with the permission of theCommander, Letterman Army Institute of Research, Presidio of San Francisco, California 94129.However, the Defense Technical Information Center is authorized to reproduce the document forUnited States Government purposes.
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Citation of trade names in this report does not constitute an official endorsement or approval of theuse of such items.
In conducting the research described in this report, the investigation adhered to the "Guide for theCare and Use of Laboratory Animals," as promulgated by the Committee on Revision of the Guidefor Laboratory Animal Facilities and Care, Institute of Laboratory Animal Resources, NationalResearch Council.
This material has been reviewed by Letterman Army Instituteof Research and there is no objection to its presentation and/or publication. The opinions or assertions contained hereinare the private views of the author(s) and are not to be con-strued as official or as reflecting the views of the Departmentof the Army or the Department of Defense. (AR 360-5)
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11. TITLE (Include Security Classification)
Fourteen Day Subchronic Oral Toxicity Study of 4-Nitrophenyl Monochloromethyl(Phenyl) Phosphinate in Male Rats
12. PERSONAL AUTHOR(S) Carolyn M. Lewis, MS, Craig W. White, DVM, CPT VC, Thomas P.Kellner, BS, SP5 r Paul P. Warina. BS. John C. Turnier. DVM. MAJ VC. and
13a. TYPE OF REPORT 113b. TIME COVERED 14. DATE OF REPORT (Year.MonthOay) 15. PAGE COUNT
Final IFROMI7Nov 2TCLEj 82 1988 February 7416. SUPPLEMENTARYNOTATION (Item 12 continued): John T. Fruin, PhD, COL VC
17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)
FIELD GROUP SUB-GROUP Subchronic Oral Toxicity, 4-Nitrophenyl Monochlorcmethyl(Phenyl) Phosphinate, Phosphinates, Rat
19, ABSTRACT (Continue on reverse if necessary and identify by block number)The 14-day subchronic oral toxicity of 4-nitrophenyl monochloromethyl (phenylphosphinate (MCP) was evaluated in male rats. MCP was administered by gavageat dose levels of 0, 12.5, 25, 50 and 100 mg/kg/day for 14 days. At necropsyblood samples were obtained for hematological and serum clinical analyses. Acomplete histological examination was performed on all animals. In addition,plasma, red blood cell, and brain acetylcholinesterase and butyrylcholines-terase activities were determined. Although MCP was lethal to one rat inboth the 50 and 100 mg/kg dose groups, no definitive pattern of clinicalchemical, hematological or histopathological alterations was found. Thissuggests that the deaths observed could be due to a transient toxic responseassociated with cholinesterase inhibition
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ABSTRACT
The 14-day subchronic oral toxicity of 4-nitrophenylmonochloromethyl (phenyl) phosphinate (MCP) was evaluated inmale rats. MCP was administered by gavage at dose levels of0, 12.5, 25, 50 and 100 mg/kg/day for 14 days. At necropsy,blood samples were obtained for hematological and serumclinical analyses. A complete histological examination wasperformed on all animals. In addition, plasma, red bloodcell, and brain acetyicholinesterase and butyrylcholinesteraseactivities were determined. Although MCP was lethal to onerat in both the 50 and 100 mg/kg dose groups, no definitivepattern of clinical chemical, hematological orhistopathological alterations was found. This suggests thatthe deaths observed could be due to a transient toxic responseassociated with cholinesterase inhibition.
Key Words: Subchronic Oral Toxicity, 4-NitrophenylMonochloromethyl (Phenyl) Phosphinate,Phosphinates, Rat
Aooession For
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PREFACE
TYPE REPORT: Fourteen-Day Subchronic Oral Toxicity GLP Study Report
TESTING FACILITY: US Army Medical Research and Development Command
Letterman Army Institute of ResearchPresidio of San Francisco, CA 94129-6800
SPONSOR: US Army Medical Research and Development Command
US Army Medical Research Institute of Chemical DefenseAberdeen Proving Ground, MD 21010-5425
PROJECT/WORK UNIT/APC: 35162772A875 Defense Against Chemical Agents,WU 304, Toxicity Testing of PhosphinateCompounds, APC TL04
GLP STUDY NUMBER: 82034
STUDY DIRECTOR: COL John T. Fruin, DVM, PhD, VC,Diplomate, American College ofVeterinary Preventive Medicine
PRINCIPAL INVESTIGATORS: CPT Craig W. White, DVM, VC
Carolyn M. Lewis, MSSP5 Thomas P. Kellner, BA
PATHOLOGIST: John C. Turnier, DVM, MAJ, VCDiplomate, American College ofVeterinary Pathologists
SREPORT AND DATA MANAGEMENT: A copy of the final report, studyprotocol, retired SOPs, raw data,analytical, stability, and puritydata of the test compound, tissues,and an aliquot of the test compoundwill be retained in the LAIR Archives.
TEST SUBSTANCE: 4-Nitrophenyl Monochloromethyl (Phenyl) Phosphinate
INCLUSIVE STUDY DATES: 17 November - 16 December 1982
OBJECTIVE: The objective of this study was to determine the
subchronic toxicity of 4-nitrophenyl monochloromethyl(phenyl) pnosphinate (MCP) in male rats.
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ACKNOWLEDGMENTS
SP5 L. Sauers, MS; SP5 L. Mullen, BS; SP5 J. Rodriguez,
BS; and SP5 E. Zimmerman assisted with daily dosing andobservations. CPT(P) G. Makovec, DVM; CPT(P) M. Langford,DVM; SSG C. Beckett; SP5 M. McKinley, BA; SP5 F. McKinley,BA; SP5 T. Loughead; SP4 C. Dumlao, BS; SP4 M. Kostrna; L.Cote and T. Hironaga contributed in the collection,preparation and histological examination of tissues and inperforming the hematology and urinalysis. M. Lyons and J.Knudsen, BS, perfcrmed the various biochemical analyses.Claire N. Lieske, US Army Research Institute of ChemicalDefense, provided the compound, advice, and support.
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SIGNATURES OF PRINCIPAL SCIENTISTS AND MAINAGERS INVOLVED IN TE STUDY:
We, the undersigned, declare that GLP study number 82034 wasperformed under our supervision, according to the procedures describedherein, and that this report is an accurate record of the resultsobtained.
17JOHN T. FRUIN / DATE THOMAS P. KELL9ER, BS / DATECOL, VC SP5, USAStudy Director Principal Investigator
".. iJOBI-C. TURNIEI D ATE CAROLI M. LEWIS, MS / DATE
J CPT, VC DAC
Pathologist Principal Investigator
IG W. WHITE /LJYATE VIRGIff L. GILDENGORIN, P DATECPT, VC DACPrincipal Investigator Statistician
PAUL P. WARING, B9 DATEDAC
Chemist
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DEPARTMENT OF THE ARMY
LETTERMAN ARMY INSTITUTE OF RESEARCH
PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129-6800
ATTENTION OF
SGRD-ULZ-QA (70-In) 13 January 1988
MEMORANDUM FOR RECORD
SUBJECT: Report of GLP Compliance for Study 82034
1. I hereby certify that in relation to LAIR GLP Study 82034, thefollowing inspections were made:
02 November 1982 - Protocol Review03 December 1982 - Dose Preparation03 December 1982 - Dosing14 December 1982 - Observations15 December 1992 - Necropsy15 December 1982 - Tissue Processing15 December 1982 - Clinical Chemistry
2. The report and raw data for this study were audited on 26 May1987.
A2RY .DUTCHER
Princi al AdvisorQuality Assurance Section
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S" II p-S22 KIz;Z
TABLE of CONTENTS
Abstract ............................
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . .1i
Acknowledgments .. . . . . . . . . . . .. . . . . . . . . . . iv
Signature of Principal Scientists............................... v
Report of Quality Assurance Unit............................... vi
Table of Contents............................................. vii
BODY OF REPORT
INTRODUCTION................................................1I
Objective of Study ...................... .......... 1
0 MATERIALS
Test Substance .................... ............... 1*Vehicle................................. .. .. . .2
Animals ........................... *.......... . ... .2Husbandry.............................. ... . .......... 2
METHODS
Group Assignment/Acclimation.......... ........... 2
5.Dose Levels............................ ......... 3
Compound Preparation ....................... ..... 3
Test Procedures .............................. 3Statistics....................... ...... ..... 4
RESULTS
Mortalities.................................. .... 4Clinical Signs ...... ... .................
Animal Weights.................................... 5Clinical Chemistry ........... .................
Pathology/Hematology .. . .. .. ............. . ......... 6
DISCUSSION....................... ............... 7
-RECOMMENDATIONS ...... .. .. .. . ............. .. ........ .8
REFERENCES..................... ... ................... 9
vii
Table of Cc ts (continued)
APPENDICES
V.Appendix A, Chemical Data................................... 11Appendix B, Tables........................13Appendix C, Historical Listn Of Study Events............. 21Appendix D, Procedures for Analytical Chemistry............ 23Appendix E, Deviations from the Original Protocol.......... 24Appendix F-I, Individual Clinical Signs.................... 25Appendix F-2, Individual Body Weights....................... 29Appendix F-3, Individual Clinical Chemistry................ 31
Appendix G-1, Pathology Report.............................. 37% Appendix G-2, Individual Hematology Values................. 72
9..OFFICIAL DISTRIBUTIONi LIST...................................... 74
.pviii
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Lewis--i
FOURTEEN-DAY SUBCHRONIC ORAL TOXICITY STUDY OF 4-NITROPHENYLMONOCHLOROMETHYL (PHENYL) PHOSPHINATE IN MALE RATS--Lewis et al
[ One mission of the US Army Medical Research and
- ./Development Command is to develop a prophylactic regimen. against organophosphate intoxication. The organophosphinate
~compounds offer an effective strategy of prophylaxis. Thestrategy requires protecting a critical percentage of theavailable acetylcholinesterase from irreversible bindingduring chemical agent poisoning. This is accomplished byreversible binding with a compound, such as 4-nitrophenylmonochloromethyl (phenyl) phosphinate, from which the enzymemay be reactivated using standard antidotal therapy (1-4).
'-..Objective of the Study
-' The objective of this study was to determine the[. subchronic toxicity of 4-nitrophenyl monochloromethyl (phenyl)_ phosphinate (MCP) in male rats.
a.-•
; MATERIALS
;£ Test Substance
• Chemical name: 4-Nitrophenyl Monochloromethyl (Phenyl)Phosphinate
..J LAIR Code: TA009
' Code name: MCP, CMP
e.
Chemical Abstract Service Registry Number: None
-[. Empirical formula: CI3HICINO4P
The test compound was received from the US Army MedicalInstitute of Chemical Defense, Aberdeen Proving Ground, MD21010 on 23 June 1982. The test chemical was stored at 4Cuntil the time of compounding with the vehicle before dosing.Detailed chemical data on the test compound are given in
o p
may b reativaed usnrtnadatdtlteay(-)
Lewis--2
Appendix A.
Vehicle
The vehicle contained 20% Tween 80- (Fisher ScientificCompany, Fairlawn, NJ), 10% ethanol and 70% citrate buffer (pH3.0). This vehicle was selected because it significantlyretarded phosphinate hydrolysis.
Animals
Sixty-eight male albino Sprague-Dawley rats were receivedfrom Bantin-Kingman Breeding Laboratories, Fremont, CA for usein this study. Ear tags, numbers 82D00974 to 82D01041,without exclusions, were used to identify each animalindividually. Two animals were sacrificed for quality controlnecropsies, six extra animals were eliminated duringrandomization as extras, and two other animals were removedfrom the study after being misdosed. The rats' weights (17November 1982) ranged from 145 to 177 g.
Husbandry
The animals in this study were housed individually instainless steel mesh drawer rack cages. No bedding was usedin any of the cages.
Diet consisted of Certified Purina Rodent Chow No. 5002(Ralston Purina, Checkerboard Square, St. Louis, MO, Lot No.OCT14822F and SEPT09822K) ad libitum. Water was provided byautomatic Lixit dispenser.
The temperature range maintained throughout this study was20-26 0 C with a relative humidity of 40-55% with occasionalspikes up to 72% during room cleaning. The photoperiod was 15hours of light daily (0500-2000 hours).
METHODS
Group Assignment/Acclimation
The animals were acclimated for 13-14 days from receipt tothe day of dosing. During the acclimation period the animals
* were observed daily for signs of illness.
Ten male animals were assigned to each of six dose groups.Allocation was accomplished using a computer-based stratified,randomization method (LAIR SOP-OP-STX-78).
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4
Lewis--3
Dose Levels
The dose for each animal was based on the body weight andthe assigned dose group. Doses were calculated by a programon a Hewlett-Packard 98A calculator (LAIR SOP OP-ISG-8). Theanimals were weighed twice a week and doses were adjustedaccordingly. The volume administered ranged from 0.21 to 2.7ml depending on dosage and animal weights.
Four dose levels were given to male rats (10 animals/doselevel) at 1/16, 1/8, 1/4, and 1/2 of the acute LD for MCP(200 mg/kg) each day. Table 1 in Appendix B show the dosing
. scheme. Each dose group was further divided into twosubgroups. One subgroup (a) was dosed beginning on 1 Dec 82and the other subgroup (b) on 2 Dec 82. This procedurereduced the number of animals sacrificed on one day to amanageable level.
Compound Preparation
The solutions for the vehicle control group were preparedvjust before the study started. The MCP dosing solutions were
prepared daily according to LAIR SOP OP-STX-48, "Preparation- -[ of Phosphinate Compounds for Oral Toxicity Studies", except
that the concentrations of Tween 80", ethanol, water, andcitrate buffer were changed to minimize hydrolysis. Thedosing solutions were analyzed for hydrolysis (stability)immediately after preparation and within 20 minutes afterdosing was completed. The results from these analyses are
given in Appendix A.
Test Procedures
.A All animals were dosed daily between 0830 and 1030 hoursfor 14 days. The animals were not fasted. An 18-gauge, 3-inch gastric gavage needle (Popper and Sons, Inc., New Hyde
A Park, NY 11040) was used to administer the compound by gastricintubation. This procedure was performed withoutadministering sedatives or anesthesia to the animals.
One hour after each dosing the animals were observed formortality and signs of toxicity. Animals were observedundisturbed in cages, outside of cages, and after return tocages. If an animal exhibited severe signs of toxicity, itwas observed more frequently. Moribund animals wereeuthanized and submitted for necropsy. Body weights wererecorded twice weekly and on the day of sacrifice. Appendix Ccontains a listing of the historical events.
All animals assigned to this study were subjected tocomplete necropsy procedures. All tissues itemized in SOP OP-
Lewis--4
STX-52 were examined microscopically in the cage control,vehicle control, and high dose groups. The other three dosegroups had histopathology performed only on the liver, kidney,heart, and those organs with gross lesions. Hematology andblood chemistry analyses were also performed. A list of LAIRSOPs used for the blood chemistry is in Appendix D.
Changes to the original protocol are discussed in AppendixE.
St3tistics
The animal weights and the results from hematology andblood chemistry analyses were analyzed statistically withpackaged programs available on BMDP software (5). Theequality of the variances of the groups was tested using theLevene's Test. If the variances were equal, the vehiclecontrol group and the dose groups were compared by thestandard one-way analysis of variance (ANOVA). Otherwise, theWelch one-way ANOVA, which is not based on the assumption thatthe variances are equal, was performed. If the F-statisticwas significant in either case, the Dunnett's test wasperformed to determine whether or not the vehicle controlgroup was significantly different from any of the dose groups.The Student's t-test was used to compare all values of thecage and vehicle control groups except total bilirubin. Ifthe variances of the two control groups were not equal by theLevene's test, the t-statistic was calculated with thevariance of each group estimated separately; otherwise, it wascalculated with the variances pooled (averaged). Totalbilirubin values were nonparametric data which were analyzedby using the Kruskal-Wallis one-way ANOVA. The totalbilirubin levels in the two control groups were compared byusing the Mann-Whitney test.
RESULTS
Mortalities
Four deaths were observed during the study; however, twoof the four mortalities were attributed to misdosing. Animals82D01009 (12.5 mg/kg group) and 82D01019 (100 mg/kg group)were removed from the study based on the pathology report.The other two deaths, one at 50 mg/kg and one at 100 mg/kg,were compound-related (Table 1, Appendix B).
Clinical Signs
MCP produced dose-dependent increases in the incidencerate of some signs. These signs included sluggishness orinactivity, excitation, decreased respiratory rate, rough
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Lewis--5
coat, excessive salivation (clear or yellow material aroundthe mouth and on the front legs), yellow stain/material aroundthe perianal and ventral areas (presumably urine), and redstain/material around the mouth, nose, head and neck(presumably harderian gland secretions).
A few signs were seen less frequently, but did occurprimarily in the higher dose groups suggesting that they weremore severe signs of toxicity. These included aggressiveness,loss of equilibrium, increased respiratory rate, increased ordecreased respiratory depth, wheezing, hunched posture, orangeor clear stain perianal, and brown urine.
Individual clinical signs appear in Appendix F-1.
Animal Weights
The mean body weights and standard error of the mean foreach group are given in Table 2, Appendix B. The body weightsfor the vehicle control and test groups were not significantlydifferent when compared by ANOVA. When the control groupswere compared, the vehicle control group had significantlylower weights than the cage control group on the last threeweighings.
Individual body weights appear in Appendix F-2.
Clinical Chemistry
The effect of MCP on the level of several electrolytes,various biochemical components, and the activity of severalenzymes in serum was examined. In addition,acetylcholinesterase and butyrylcholinesterase activity wereanalyzed in plasma, red blood cells, and brain tissue. Themean and standard error of the mean for each dose group forthese measurements are shown in Tables 3 through 6, AppendixB.
When the vehicle control and dose groups were compared byANOVA, significant differences were found with the levels ofblood urea nitrogen, creatine phosphokinase, and alkalinephosphatase in serum and acetylcholinesterase in brain.However, when the Dunnett's test was performed, no significant
0. differences were found except with alkaline phosphataselevels. The high dose group (100 mg/kg/day) had significantlylower alkaline phosphatase levels than the vehicle controlgroup. When the vehicle and cage control groups were comparedusing the Student's t-test, no differences were found in anyclinical chemistry values.
0I
4: Lewis--6
Individual clinical chemistry values appear in Appendix F-3.
Pathology/Hematology
Gross necropsies of the two rats whose deaths wereattributed to the compound revealed signs of gastricirritation. Gross necropsy findings in terminally sacrificed
": rats include dilated renal pelvis in one vehicle control and~ "* one 100 mg/kg rat, thickening of the splenic capsule in one
12.5 mg/kg rat, a focal skin abrasion in another 12.5 mg/kgrat, and yellow-brown and red-brown pulmonary foci in one 25mg/kg and one 50 mg/kg rat, respectively.
The histopathological lesions found in terminallysacrificed rats included peritracheal hemorrhage in one 100mg/kg rat, periesophagitis in two 100 mg/kg rats, interstitialpneumonitis in two vehicle control, one 50 mg/kg and two 100mg/kg rats, hemorrhage and/or erythrophagocytosis in themesenteric lymph nodes of four 100 mg/kg rats, portallyoriented subacute hepatitis in two 50 mg/kg and three 100mg/kg rats, and renal tubular mineralization in one cagecontrol rat, one vehicle control rat, six 12.5 mg/kg rats, six25 mg/kg rats, three 50 mg/kg rats and three 100 mg/kg rats.The histopathological findings in the two rats that died fromthe compound included renal tubular mineralization in the 100mg/kg rat, periportal subacute hepatitis in both rats, hepaticnecrosis in the 50 mg/kg rat, hemorrhage and/orerythrophagocytosis in the mesenteric lymph node of the 100mg/kg rat, acute gastric inflammation in the 50 mg/kg rat,gastric hemorrhage in both rats, slight intestinal hemorrhagein the 100 mg/kg rat and slight necrosis of the stomach andintestines in the 50 mg/kg rat.
The effect of MCP on various hematological measurements
was examined. The mean and standard error of the mean foreach group are shown in Table 7, Appendix B. When the control
S groups were compared by the Student's t-test, no significantdifferences were found in any of the measurements. When thedose groups and the vehicle control group were compared byANOVA, a few significant differences were found. The 12.5mg/kg group had significantly higher hematocrits than thevehicle control group. The mean corpuscular hemoglobin valueswere significantly lower in the 50 mg/kg dose group than thevehicle control group. In addition, the mean corpuscularhemoglobin concentration values in the 100 mg/kg dose groupwere significantly lower than the vehicle control group.
The pathology report appears in Appendix G-1. Individualhematology values appear in Appendix G-2.
J, 0
Lewis--7
DISCUSSION
The types of clinical signs observed in the 14-daysubchronic study of MCP were similar to those reported in theacute study (6), although the frequency and the severity wereusually lower. Nearly all the signs observed could beattributed to effects of MCP on the nervous system. The mostfrequent signs were sluggishness or inactivity, excitation,loss of equilibrium, changes in respiration, excessivesalivation (often yellow presumably from hydrolysis of thecompound), excessive urination, excessive harderian glandsecretions and piloerection.
Although the body weights for the vehicle control groupwere not significantly different from those of the test groupsat any time during the study, they were significantly lowerthan the cage control group after the first week of dosing.There are several possible explanations for their lowerweights. The animals may have been traumatized by the dosing
*" which affected their appetite, or the vehicle itself may haveaffected their appetite. The vehicle could have also affectedthe absorption or transit time so that less food was absorbed.At this point we cannot be certain which, if any, of thesefactors contributed to the weight differences observed in thisstudy.
A few statistically significant differences were seen inthe clinical chemistry data. Of these few differences, noneappeared to be compound-related. Alkaline phosphatase levelswere significantly lower in the highest dose group whencompared to the vehicle control group. In general, one isconcerned about elevated levels of alkaline phosphatase, notdecreased levels. This difference was considered incidental.
The difference in the creatine phosphokinase levelsbetween groups was significant when the Welch one-way ANOVAwas performed. However, none of the treatment groups were
Jr significantly different from the vehicle control group by theDunnett's test. The difference found with the ANOVA was dueprimarily to elevated levels in two animals in the 50mg/kg/day dose group. Creatine phosphokinase is particularlysensitive to skeletal muscle damage. Even exercise,intramuscular injections, and psychotic reactions can resultin elevated levels (7). Since MCP is known to cause tremors,
convulsions, and fasciculation, elevated levels in a fewanimals are not surprising. One of these animals had slightto moderate signs of toxicity the day before sacrifice;however, the other animal never exhibited any signs oftoxicity during the study period. The pathology report andother clinical chemistry results were examined for these twoanimals, but no other evidence supporting the possibility of
t
Lewis--8
muscle damage was found.
Pathological examinations of the rats that died and thosethat survived the 14-day dosing period revealed few distinctcompound-related effects. Two rats that appeared to have diedfrom the compound exhibited signs of gastrointestinalirritation. Gross necropsy findings in the rats that survivedwere regarded as minimal and considered unrelated to thecompound administration. Microscopic examination of theserats revealed hemorrhages within the lymph nodes, portallyoriented hepatic inflammation and renal tubular mineralizationin several animals in some of the dose groups. However, thesefindings were considered of dubious significance.
Only a few statistically significant differences werefound in the hematology data. The hematocrits in the 12.5mg/kg dose group were significantly greater than in thevehicle control group. Since the hematocrits in the otherdose groups were not significantly higher, this differencedoes not appear to be compound-related. When compared to thevehicle control group the mean corpuscular hemoglobin and meancorpuscular hemoglobin concentration values were significantlylower in the 50 mg/kg and 100 mg/kg dose groups, respectively.The lack of any other significant changes in the otherhematological measurements makes these findings difficult toexplain.
CONCLUSIONS
Although MCP caused a few deaths, no definitive clinicalchemical, hematological or histological alterations werefound. This suggests that death could be due to a transienttoxic response associated with cholinesterase inhibition.
RECOMMENDATIONS
Metabolic and pharmacokinetic studies correlating dose andcholinesterase inhibition would aid in the interpretation ofdata and in the design of dosage regimens for future studies.
Lewis--9
REFERENCES
1. Lieske CN, Clark JH, Meyer HG, Lowe JR, Lawson MA, Lennox
WJ. The concept and chemistry of organophosphinates aspr )phylactic agents in organophosphate intoxication.Toxicology Research Projects Directory 1981: 7.
2. Lieske CN, Meyer HG, Clark JH, Lowe JR, King JW.Spontaneous reactivation of bovine erythrocyteacetyicholinesterase inhibited by five organophosphinates.Aberdeen Proving Ground, MD: U.S. Army Medical BioengineeringResearch and Development Laboratory, 1979.
3. Horton GL, Lieske CN, Lowe JR. Phosphinate inhibitionstudies of cholinesterases. Pestic Sci 1978; 9:135-138.
4. Hodgson E, Guthrie FE, eds. Introduction to biochemicaltoxicity. New York: Elsevier Science Publishing Co., Inc.,1980: 193-223.
5. Dixon WJ, ed. BMDP statistical software. Berkeley:* - University of California Press, 1981:555-573.
6. White CW, Rodriguez J, Kellner, TP. Acute oral toxicity* (LD ) of 4-nitrophenyl monochloromethyl (phenyl) phosphinate
(TA88 9) in male rats. Toxicology Series No. 55. Presidio ofSan Francisco, CA: Letterman Army Institute of Research.Institute Report No. 191. October 1984.
7. Zimmerman HJ, Henry JB. Serum enzyme determinations as anaid to diagnosis. In: Davidsohn I, Henry JB, eds. Todd-Sanford clinical diagnosis by laboratory methods.Philadelphia: W.B. Saunders Co., 1974:837-869.
I.
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Lewis-- 10
".
V.
Page
Appendix A, Chemical Data ................................. 11Appendix B, Tables ........................................ 13Appendix C, Historical Listing of Study Events ........... 21Appendix D, Procedures for Analytical Chemistry ........... 23Appendix E, Deviations from the Original Protocol ......... 24Appendix F-i, Individual Clinical Signs ................... 25Appendix F-2, Individual Body Weights ...................... 29Appendix F-3, Individual Clinical Chemistry ............... 31Appendix G-1, Pathology Report ............................ 37Appendix G-2, Individual Hematology Values ................ 71
APPENDICES
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Lewis--il
CHEMICAL DATA
-1.emical name: 4-Nitrcphenyl monochloromethyl (phenyl)phosphinate (MCP)SLot Xuncer: L-90
LAIR Code: -.A009
3trucural Formula:
0
P N0
H2 CI/
Molecular Formula: C13HllClNO4 P
Physical State: White crystalline solid
Melting Point: 77-78.50 C
Source: Dr. Clair LieskeUS Army Medical Institute of Chemical DefenseAberdeen Proving Ground, MD 21005
Analytical Data:Stability: The dosing solutions were assayed for intact
and hydrolyzed phosphinate immediately after preparation anddosing. p-Nitrophenol, a product of phosphinate hydrolysis,
- was quantitated spectrophotometrically at 400 nm using avalue of 18,300 for the molar extinction coefficient. Absor-bance was measured in accordance with LAIR SOP-OP-STX-49,
A. "Spectrophotometric measurement of p-nitrophenol for phos-phinate determination". The concentration of unhydrolyzedphosphinate in the dosing solution was determined from thedifference in p-nitrophenol concentraton before and afterNaOH hydrolysis. The initial hydrolyzed phosphinate wasdivided by the total hydrolyzed phosphinate to obtain thepercent hydrolysis for each solution. The percent hydrolysisbefore and after dosing is shown in Table 1.
Concentration: The same analysis described understability provided information regarding the concentration ofthe dosing solutions. These results are summarized in Table2.
0O' APPENDIX A
16 A. -1 -6A-VA
Lewis--12
TABLE _e hydrolysis of MCP in the dosing vehicle.
Percent HydrolysisBefore After Average
1 lec 82 7.20 7 .36 7.282 :ec 82 6.86 6.50 6.683 Dec 82 6.86 7.40 7.134 Dec 82 5.59 5.78 5.695 Dec 82 6.63 7 .05 6.846 Dec 82 5.88 6.20 6.047 Dec 82 6.46 7.13 6.80
Ni 8 Dec 82 7 .42 7.36 7.399 Dec 82 6.42 6.77 6.60
10 Dec 82 5.32 7.00 6.1611 Dec 82 6.08 6.68 6.3812 Dec 82 5 .94 6.38 6.1613 Dec 82 5.98 6.89 6.4414 Dec 82 6.17 6.88 6.5315 Dec 82 6.24 6.79 6.52
TABLE 2. Actual concentration of MCP in dosingsolutions.
Intact MCP (mg/ml)Date Before After Average i Target
1 Dec 82 13.2 12.0 12.6 90
2 Dec 82 12.3 11.9 12.1 863 Dec 82 13.0 12.5 12.8 91
. 4 Dec 82 12.0 12.4 12.2 875 Dec 82 12.5 13.0 12.8 916 Dec 82 13.9 11.8 12.9 927 Dec 82 12.3 13.5 12.9 92
- 8 Dec 82 12.6 12.0 12.3 889 Dec 82 12.8 13.5 13.2 94
10 Dec 82 11.6 11.5 11.6 8311 Dec 82 13.1 12.1 12.6 9012 Dec 82 12.8 12.3 12.6 9013 Dec 82 12.4 12.5 12.5 8914 Dec 82 11.5 11.5 11.5 8215 Dec 82 14.2 14.2 14.2 101
-
APPENDIX A luded)
0'% ~ ihV * - ... %'S. . % ~ - ~ - 5 ~.v N
Lewis--13
-'"p
LIST OF TABLESPage
Table 1 - Dosing Scheme and Related Deaths by GroupSfor 14-Day Subchronic Toxicity of MCP ............... 14
Table 2 - Mean Body Weights 14-Day SubchronicToxicity of MCP ..... #................................ 15
Table 3 - Effects of MCP on Electrolyte Levelsin Serum .................................. *.......... 16
Table 4 - The Effect of MCP on Biochemical Constituents
of Serum .......................... *..............*.... 17
Table 5 - Effects of MCP on Serum Enzyme Activity ............. 18
".
Table 6 - Effect of MCP on Cholinesterase Activity inPlasma, Red Blood Cell and Brain .................... 19
Table 7 - Effect of MCP on Hematological Parameters ........... 20EII
"pPPNDX
Lewis--1 4
TABLE 1
Dosing Scheme and Related Deaths Dy Groupfor
14-Day Subchronic Toxicity of MCP*
Concentration Group Deaths/(mg/kg/day) No. Group Totals
Cage control (0) 1 0/10
Vehicle control (0) 2 0/10
12.5 3 0/9t
25 4 0/10
50 5 1/10
100 6 1/9t
.JMCP=4-Nitrophenyl Monocnloromethyl (Phenyl) Phosphinate
one animal misdosed.
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Lewis--15
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Lewis--17
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Lewis-- 18
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Lewis--23
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Lewis--21
-~ HISTORICAL LISTING OF STUDY EVENTS
Date Events
17 Nov 82 Animals arrived at LAIR. They were observed forillness, eartagged, weighed, and caged in GLPSuite. Two animals were submitted to the LAIRPathology Group for quality control necropsy.
18-30 Nov 82 Animals were checked daily.
19,22,26,29 Nov 82 All animals weighed.
30 Nov 82 Animals removed from quarantine status and dosagelevel calculated for Groups 2(a) - 6(a).
1 Dec 82 Groups 2(a) - 6(a) dosed. Observations conductedat 1000 hours throughout the study period. Dosagefor groups 2(b) - 6(b) calculated.
*2 Dec 82 Groups 2-6 (a + b) weighed, dosed and observed.Group 1 weighed and observed. Dose levelscalculated for Groups 2-6.
3- e 2Gop - oe n bevd ru bevd
3- Dec 82 Groups 2-6 wid dosed and observed. Group 1vd
weighed and observed. Dose levels calculated forp. Groups 2-6.
7 Dec 82 Groups 2-6 dosed with newly calculated doselevel and observed. Group 1 observed.
8-9 Dec 82 Groups 2-6 dosed and observed. Group 1 observed.
-. 10 Dec 82 All animals weighed. Dose levels for Groups 2 -
6 recalculated.
*10-12 Dec 82 Groups 2-6 dosed with newly calculated doselevel and observed. Group 1 observed.
- ~ 13 Dec 82 Groups 2-6 dosed and observed. Group 1 observed.All animals weighed.
14 Dec 82 Groups 2-6 dosed and observed. Group 1 observed.Food removed from Groups 1(a) - 6(a) at 1630 hours.Twelve animals transferred to metabolic cages.
15 Dec 82 Groups l(a) - 6(a) observed and weighed at 0730.4, necropsy Groups 1(a) - 6(a). Blood and tissue
samples taken for the measurements specified.Groups 2(b) - 6(b) weighed, dosed, and observed.Group lb observed. Food removed from Groups1(b) - 6(b) at 1630 hours.
* APPENDIX C
b'I -pr o
% % % %
Lewis--22
Date Events
16 Dec 82 Animals observed and weighed at 0730 hours.Groups l(b) - 6(b) submitted for necropsy. Bloodand tissue samples taken for the measurementsspecified.
I
I
A(
Lewis--23
PROCEDURES FOR ANALYTICAL CHEMISTRY
The following are LAIR GLP SOPs for the Analytical Chemistryperformed for the study.
A'
1. Calcium - OP-ACH-17
2. Sodium and Potassium - OP-ACH-19
3. Chloride - OP-ACH-20
4. Magnesium - OP-ACH-50
5. Phosphorus - OP-ACH-18
6. Glucose - OP-ACH-7
7. Cholesterol - OP-ACH-11
8. Triglycerides - OP-ACH-9
"-. 9. Creatinine - OP-ACH-15
".'. 10. Blood Urea Nitrogen - OP-ACH-16
- 11. Uric Acid - OP-ACH-14
12. Albumin - OP-ACH-12
13. Total Protein - OP-ACH-13
14. Total Bilirubin - OP-ACH-8
15. Serum Iron - OP-ACH-22
16. Aspartate Amino-Transferase - OP-ACH-4
17. Alanine Amino-Transferase - OP-ACH-3
18. Lactate Dehydrogenase - OP-ACH-5
19. Creatine Phosphokinase - OP-ACH-6
20. Alkaline Phosphatase - OP-ACH-10
21. Acetyl Cholinesterase - OP-ACH-30 and OP-ACH-46
22. Butyryl Cholinesterase - OP-ACH-52
Globulin values were calculated by subtracting the albumin valuesfrom the total protein values.
APPENDIX D
Lewis---
DEVIATIONS FROM THE ORIGINAL PROTOCOL
1. On 10 Dec 82 dose volumes were recalculated based on theweights taken that day. Normally, the new volumes were not useduntil the following day. However, this time the new volumes were
4~. given the same day they were calculated.
2. On 3 Dec 82 the cage control animals were overlooked whenobservations were performed.
3. According to the original protocol the vhcewas to be 21.5%Tween 80', 18.5% ethanol, 37.5% 50mM citrate buffer (pH 3.2), and22.5% water. The test compound was more susceptible to hydrolysisthan previous phosphinate compounds tested, so the vehicle waschanged to 20% Tween 80 ,10% ethanol, and 70% 50mM citrate buffer
(pH 3.0) which increased the stability of the test compound.
APEDI6
Lewis--25
Coding for Clinical Signs
- Normal* Observation not performed
A AggressiveB Brown UrineC Rough CoatD DiarrheaE ExcitedF Decreased Respiratory RateG Increased Respiratory RateH Hunched PostureI inactive or SluggishJ Decreased Respiratory DepthK Increased Respiratory DepthL Loss of EquilibriumM Clear Stain PerianalN Toe Nail Bleeding0 Orange Stain PerianalP Piloerection
-.' 0 IrritableR Red Stain/Material Head/NeckS Yellow/Clear Stain/Material Mouth/Front Legs or SalivationT Hair LossU ScabW Sound ProductionX DeadY Yellow Stain/Material Perianal/Ventral
O."
4,
J APPENDIX F-1
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Lewis--26
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Lewis--37
Pathology Report
Fourteen Day Sub-chronic Toxicity Study of 4-NitrophenylMonochloromethyl(phenyl)phosphinate in Male Albino Sprague-Dawley Rats,
Study 82-034
1. Introduction.
The objective of this study was to determine the sub-chroniceffects of 4-Nitrophenyl Monochloromethyl(phenyl)phosphinate whenadministered daily for 14 days (oral gavage) in male Sprague-Dawleyrats. Each animal was randomly assigned to one of 6 dose groups of 10animals each (5 in each subgroup).
Cage controls - groups 1A & IB
Vehicle* controls - groups 2A & 2B
12.5 mg/kg/day - groups 3A & 3B25 mg/kg/day groups 4A & 4B50 mg/kg/day - groups 5A & 5B
100 mg/kg/day - groups 6A & 6B
After 14 days on test, the rats were submitted for necropsy.Following anesthesia with pentobarbitol sodium, administered byintraperitoneal injection, blood was collected from the right ventricle
* ~. of each rat and submitted for hematologic examination [red blood cellcount (RBC), hemoglobin concentration (Hb), hematocrit (Hct), meancorpuscular volume (MCV), mean corpuscular hemoglobin (MCH), meancorpuscular hemoglobin concentration (MCHC), white blood cell count(WBC), WBC differential and blood cell morphology, platelet count, andreticulocyte count]. Additional blood was submitted to AnalyticalChemistry Services Group, Division of Research Support, for chemicalanalyses. All rats were killed by exsanguination and gross necropsyexaminations were performed. Portions of anterior cerebrum (unfixed)
-. were submitted to Analytical Chemistry Services Group, Division ofResearch Support, for cholinesterase determinations. Tissue specimensfrom major organs and systems were fixed in 10% neutral bufferedformalin (except the eyes which were fixed in Karnovsky's solution) for
* subsequent microscopic examination. Tissues were embedded in paraffin,sectioned at approximately 6 microns thickness and stained withhematoxylin and eosin. All tissues itemized in SOP OP-PSG-12 wereexamined microscopically in the cage controls, vehicle controls, and
S.. the 100 mg/kg dosage level. In the 50 mg/kg, 25 mg/kg, and 12.5 mg/kgdosage levels, only hearts, livers, and kidneys were examined
*. microscopically. In addition, organs with gross lesions were examinedmicroscopically.
*Vehicle: 20% Polysorbate 80 (Tween 80), 10% Ethanol, 70% 50 mMCitrate Buffer.
,. APPENDIX G-1
0N*
Lewis--38
2. Results, interpretation, and discussion.
The gross and/or microscopic findings are itemized in IncidenceTables 1 - 3.
a. Table 1 tabulates the incidence and severity of lesions
observed grossly or microscopically in each rat.
b. Table 2 tabulates group gross necropsy observations.
c. Table 3 tabulates the group histopathologic observations.
Hematology: One way analysis of variance followed by Dunnett'stest if applicable, was performed on white cell differentials, MCV's,MCH's and MCHC's, hematocrits, RBC, WBC, reticulocyte, and plateletcounts to determine if there were any differences among the vehiclecontrol and each of 12.5, 25, 50, and 100 mg/kg dose groups. The mean
* hematocrit was significantly greater in the 12.5 mg/kg rats. The meancorpuscular hemoglobin was significantly lower in the 50 mg/kg rats.The mean corpuscular hemoglobin concentration was significantly lowerin the 100 mg/kg animals.
d. Gross necropsy:
There were four spontaneous deaths during the course of the study;one of which was a group 3, 12.5 mg/kg rat #33084 on day 10. Atnecropsy, the presence of oily, reddish-tinged staining around themuzzle of this rat suggested that it had aspirated the test material.A group 5, 50 mg/kg rat #33062 was found dead on day 7. The only grossfinding in this animal was a diffusely reddened glandular stomachmucosa. Two group 6, 100 mg/kg rats (#33068 and #33093) died on days 2and 12 respectively. Rat #33068 when necropsied had a soft brain and aslightly distended mucoid filled small intestine suggesting some degreeof autolysis. This rat's stomach's glandular mucosa was also reddened,however. Rat #33093 had several gross necropsy findings suggestive ofaspiration (red oily material around the muzzle, firm dark noncollapsedlung lobes) as well as esophageal rupture and intrathoracicinstallation of test material (dark red subserosal esophageal focus,oily material in the thorax).
Necropsy findings of spontaneously dying rats would therefore
suggest that two of the rats died as a direct result of a dosing-. accident (*33084, #33093) while the other two rats (#33062 and #33068)
whose mode of death is speculative may have exhibited signs of mildgastric irritation.
Of the sixty animals in the study; 10 of 10 cage controls, 10 of 10.* vehicle controls, 9 of 10 12.5 mg/kg rats, 10 of 10 25 mg/kg rats, 9 of
10 50 mg/kg rats, and 8 of 10 100 mg/kg rats survived to study
2* APPENDIX G-1 (cont.)
%,
Lewis--39
termination at which time they were necropsied. Gross necropsyobservations were minimal and considered unrelated to compoundadministration. They consisted (see tables I & 2) of: dilated renalpelvises in one vehicle control and one 100 mg/kg rat; thickening ofthe splenic capsule in one 12.5 mg/kg rat; a focal skin abrasion in
another 12.5 mg/kg rat; as well as yellow-brown and red-brown pulmonaryfoci in one 25 mg/kg and one 50 mg/kg rat respectively.
There were no gross findings in terminally sacrificed rats thatmight indicate any degree of gastro-intestinal irritation.
e. Microscopic findings:
The majority of histopathologic lesions observed in tissues from
animals surviving to terminal sacrifice were considered unrelated totreatment due to frequency of occurance, distribution among dosegroups, and incidence rates in normal healthy Sprague Dawley rats.
Peritracheal hemorrhage in one of 6 100 mg/kg tracheas as well asesophagitis and periesophagitis noted in two of seven 100 mg/kg ratswere most probably related to the gavage procedure.
Interstitial pneumonitis in 2 of 10 vehicle controls, the onehistologically examined 50 mg/kg rat lung and 2 of 8 examined 100 mg/kgrat lungs may well have been related to the gavage procedure withassociated aspiration of small quantities of test material and/orconcurrent disease.
Hemorrhage and/or erythrophagocytosis was observed in 4 of 8histologcally examined mesenteric lymph nodes in the 100 mg/kg group.
Portally oriented subacute hepatitis was present in the livers of 2of 9 and 3 of 8 (50 and 100 mg/kg respectively) histologically examinedrats.
There was an increase, although not dose related, in renal tubularmineralization in all four treatment groups.
Rats that died spontaneously had a few of the above-noted lesions.Renal tubular mineralization was present in high dose (100 mg/kg) inrat #33068. Periportal subacute hepatitis was present in group 6 (100mg/kg) rat #33068 and group 5 (50 mg/kg) rat #33062. Hepatic necrosiswas seen in 50 mg/kg rat #33062. Hemorrhage and/or erythrophagocytosisin the mesenteric lymph node was present in rat #33068 (100 mg/kg).
Although gastro-intestinal lesions were not foundhistopathologically in sacrificed rats, necrosis, hemorrhage, and acuteinflammation were observed in stomach of rat #33062 (50 mg/kg), a ratpreviously noted as having a reddened glandular stomach. This rat also
3 APPENDIX G-1 (cont.)
% % % % % %- -- *4~--- . -*.d~ ~ V * ~ f d% .. * .. . -1
*. Lewis--40
had slight intestinal epithelial necrosis. Rat *33068 (100 mg/kg) had
slight mucosal hemorrhages in both the stomach and small intestine.
3. Summary.
a. The low numbers of deaths reflect the relative innocuous nature- .- of 4-Nitrophenyl Monochloromethyl (phenyl) phosphinate when given at• . the dose levels of 12.5, 25, 50, and 100 mg/kg by gavage in a Tween 80-" .based vehicle for fourteen days.
b. The deaths of one 12.5 and one 100 mg/kg rat could beattributed to the gavage procedure and hence were not directly compoundrelated. The unscheduled deaths of the other two rats (50 and 100mg/kg), however, may have been due to the toxic effects of the compoundand, in these cases, were specifically manifested by gross andmicroscopic gastro-intestinal irritation.
c. Gross necropsy observations in sacrificed animals from alltreatment groups revealed no compound related effects. Similarly therewere no distinct compound related histopathologic tissue alterations inthese animals. Portally oriented hepatic inflammation, renal tubular
- mineralizations, and hemorrhages within lymph nodes were considered tobe of dubious significance.
d. The mean corpuscular hemoglobin and mean corpuscular hemoglobinconcentration values were decreased in the 50 and 100 mg/kg rats,respectively. Although statistically significant, these differenceswere not accompanied by other statistically significant hematologicalterations and are as yet unexplained.
</ JOHN C. TURNIER, DVMDiplomate, A.C.V.P.MAJ, VCDivision of Pathology
13 November 1987
/-X c
S.
'P
Lewis--41
-APPENDICES
I. Appendix A - Supplementary Guide to Interpretation of
'J Histopathologic Observations
II. Appendix B - Key to Tables 1, 2 & 3
- Tables 1, 2 & 3
III. Appendix C - Statistical Analysis of Hematologic Values, Study#82-034
5 A D
S
;'U.
,
' 5 APPENDIX G-l (cont.)
Lewis--42
APPENDIX A
Supplementary Guide to Interpretation of Histopathologic Observations
The following observations were not coded as they occur withconsiderable frequency in normal male Sprague Dawley Rats.
1. Interstitial, paraductular lymphoid aggregates in the pancreas andsalivary glands.
2. Plasmacytosis and lymphoid hyperplasia of very slight degrees inthe submandibular lymph node.
3. Very slight to slight hemosiderin deposition in the spleen.
4. Very slight degrees of sinus ectasia, sinus histiocytosis, andlymphoid hyperplasia in the mesenteric lymph node. Greater degreeswere coded.
5. Submucosal lymphoid aggregates in nasal cavity. Acute inflammationin paired vomeronasal organs. Flocculent eosinophilic material +/-
artifactually induced hemorrhage within lumens of sinuses.
6. Very slight lymphoid aggregates in seminal vesicles or prostate.
7. Tiny inconspicuous foci of mineralization in gastric glandularepithelium. Very slight aggregates of neutrophils, lymphocytes, andother inflammatory cells in the submucosa and lamina propria of thestomach.
8. Artifactual vacuolation of neurons and white matter of brain and/orspinal cord.
9. Slight amounts of flocculent eosinophilic material within themiddle ear.
Very slight progressive nephropathy diagnosed in the kidney when therewas evidence of early glomerular alterations (capsular basementthickening + synechia and hypercellularity) + tubular epithelialhyperplasia and the variable presence of local inflammatory cellinfiltrates.
Subacute hepatitis was used to describe foci of lymphoid cellsaccompanied by cellular degeneration and/or necrosis and the variablepresence of neutrophils and/or macrophages.
6
* APPENDIX G (cont.)
~W,
Lewis--43
APPENDIX B
Fourteen Day Sub-chronic Toxicity Study of 4-NitrophenylMonochlorcmethyl(phenyl)phosphinate in "Iale Albino Sprague-Dawley Rats,
Study 82034
Fey to "'icroscopic Findings (-ables 1 - 3):
I. (-) = Tissue or organ present, no significant lesions were observedunless recorded as present (P) or graded as to severity (1-5).
2. (-) = Tissue or organ not present.
3. (P) = Lesion recorded as present and not graded as to severity.
4. Grading for severity of lesion is as follows:
1 =minimal
2 = mild
3 = moderate
4 = marked
5 = severe.
5. ( [] ) = Gross lesions observed during necropsy.
6. (*) = No gross lesions.
7. Died (x)/Moribund (m) = Rats that died during the study or uerekilled when moribund.
7
S APPENDIX G-1 (cont.)
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DEPARTMENT OF THE ARMY Lewis--71
-=-;IA ARMY NS qEZ ESEAPC-.
=RES 0 OF SAN FRANC SCO. CA- CRN A 94129
RP~y '0A'-ENTICN AF-
SGRD-ULZ-I 6 June 1985
MEMORANDUM FOR RECORD
SUBJECT: Statistical Analysis/Study 4r82-034
I. A computer package, BMDP on the Data General MV8000 computer, wasutilized to analyze the hematology data of study i82-034.
2. Student's t-tests were performed to compare the measurements of thecage control group with the vehicle control group. No significantdifferences between the groups were found for red blood cell count,hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular
" hemoglobin concentration, mean corpuscular hemoglobin, reticulocytes,platelets, white blood cell count, neutrophils, lymphocytes, eosinophils
and monocytes values.
3. One-way analysis of variance was used to test for differences
among the vehicle control, 12.5 mg/kg, 25 mg/kg, 50 mg/kg, and 100 mg/kgdose groups. When a.significant F-value for a group effect was found,
a posteriori multiple comparisons were used to test for differencesamong means for the vehicle control group with a one-sided Dunnett'stest.
4. No differences were found for the following: red blood cell count,
hemoglobin, mean corpuscular volume, reticulocytes, platelets, white
blood cell count, neutrophils, lymphocytes, eosinophils and monocytes.
5. The dose group, 12.5 mg/kg, was found to have a significantly
greater mean for the hematocrit than the vehicle control. The meancorpuscular hemoglobin mean value was found to be significantly lowerfor the 50 mg/kg group than the vehicle control group. In addition,the 100 mg/kg group had a significantly lower mean for the meancorpuscular hemoglobin concentration values than the vehicle control
O. group.
., 6. The 0.05 level of significance was used with all statistical tests.
VIRGINIA L. GILDENGORIN, PhD
Chief, Biometric Team, ISG
APPENDIX G-1 (concluded)
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OFFICIAL DISTRIBUTION LIST
Commander CommanderUS Army Medical Research US Army Medical Bioengineeringand Development Command Research & Development Laboratory
A ATTN: SGRD-R\MS, Mrs. Madigan ATTN: SGRD-UBG-MFort Detrick. MD 21701-5012 Fort Detrick. Bldg 568
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. ATTN: DASG-TLO Kansas StreetWashington, DC 20310 Natick, MA 01760-5007
HQ DA (DASG-ZXA) CommanderWASH DC 20310-2300 US Army Institute of Surgical Research
Fort Sam Houston, TX 78234-6200Commandant
Academy of Health Sciences CommanderUS Army US Army Research Institute" -' ATTN: HSHA-CDM of Chemical DefenseFort Sam Houston, TX 78234-6100 ATTN: SGRD-UV-AJ
Aberdeen Proving Ground, MD 21010-5425l Uniformed Services University
of Health Sciences CommanderOffice of Grants Management US Army Aeromedical Research Laboratory4301 Jones Bridge Road Fort Rucker, AL 36362-5000Bethesda, MD 20814-4799
AIR FORCE Office of ScientificUS Army Research Office Research (NL)ATTN: Chemical and Biological Building 410, Room A217
Sciences Division Boiling Air Force Base, DC 20332-6448PO Box 12211Research Triangle Park, NC 27709-2211 Commander
USAFSAM/TSZDirector Brooks Air Force Base, TX 78235.5000ATTN: SGRD-UWZ-LWalter Reed Army Institute Head, Biological Sciences Division
of Research OFFICE OF NAVAL RESEARCHWashington, DC 20307-5100 800 North Quincy StreetArlington, VA 22217.5000Commander
US Army Medical Research Instituteof Infectious Diseases
ATTN: SGRD-ULZ-AFort Detrick, MD 21701-5011
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