in ICR Mice - Defense Technical Information Center Intravenous Toxicity Study of Hypertonic...

Institute Report No. 333N

Acute Intravenous Toxicity Study of:1 Hypertonic Saline/Dextran 70® and Constituents

in ICR Mice

Denzil F. Frost, MS, DVM, CPT, VCGary M. Zaucha, DVM, CPT, VCG. David Young, DVM, MA, VC

andDon W. Korte, Jr., PhD, MAJI, MSC

DTICSELECTE

MAMMALIAN TOXICOLOGY BRANCH PR 2DIVISION OF TOXICOLOGY

December, 1988 Toxicology Series: 245

LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

: " " ' . - -' ' " ' . _ _ ' -- ' '

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Acute Intravenous Toxicity Study of Hypertonic Saline/Dextran 70® and Constituents in ICRMice (Toxicology Series 245)--Frost et al

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In conducting the research described in this report, the investigation adhered tothe "Guide for the Care and Use of Laboratory Animals," as promulgated by theCommittee on Revision of the Guide for Laboratory Animal Facilities and Care,Institute of Laboratory Animal Resources, National Research Council.

This material has been reviewed by Letterman Army Institute ofResearch and there is no objection to its presentation and/orpublication. The opinions or assertions contained herein are theprivate views of the author(s) and are not to be construed as officialor as reflecting the views of the Department of the Army or theDepartment of Defense, (AR 360-5)

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Institute Report No.: 333

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Mammalian Toxicology (If applicable) Letterman Army Institute of Research

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I1 TITLE (include Security Clasificaiion)

(U) Acut-e Intravenous Toxicity Study of Hypertonic Saline/Dextran 70® andur.it.uterits in ICR Mice.

12. PERSO',AL AUTHOR(S)

DF Frost, GM Zaucha, GD Young, and DW Korte, Jr.13-). C REPORT 13b. TIME COVERED 14. DATE OF REPORT (Year, Month, Day) 15. PAGE COUNT

It t 2:e I FROM25MAY88TO28JUN8 December 1988 15916. )oY'LEMENTARY NOTATION

r.L)xJ: olo.gy Series No. 24517 COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)

FIELD GROUP SUB-GROUP"I Acute Toxicity, Intravenous Injection, MaximumTolerated Dose, Hypertonic Saline/Dextran 70®,

...... HvD tonic Saline. Dextran 70®, Ringer's Lactate.19, ABSTRACT (Continue on reverse if necessary and identify by block number)

The acute toxicity following intravenous administration of a proposed new1r S.sciation fluid, hypertonic saline/ Dextran 700 (HSD), was evaluated inmale ard female ICR mice. Animals were administered a single intravenous dosez HSD, 28 ml/kg over a 5-minute period, in an attempt to define its maximum

tolerated dose. Equal volumes of each HSD component, 7.5% hypertonic saline(HS) and 6% Dextran 700 (D70) in normal saline, and Ringer's lactate (RL)were :42o evaluated. Observations were made at 1, 2, and 4 hours afteradrinistration on the day of dosing and twice daily for the remainder of thestudy. The animals in each group were divided into two subgroups based onwhiether they were submitted to necropsy on Day 3 or Day 14 after dosing. HSDrrcdu~i death within 12 minutes of the injection in 2 of 20 mice, pale eyes,and tail sloughing, findings similar to those observed with HS. Major signs

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18. SPECIFIC TERMS (cont.)

Resuscitation Fluid, ICR Mice

19. ABSTRACT (cont.)

observed in all groups were hunched posture, rough coat, hyperactivity,inactivity, and increased rate and/or depth of respiration. Body weights wereunaffected by the dosing and no gross or microscopic lesions could beattributed to HSD or its constituents. These data suggest that the maximumtolerated dose of HSD following acute intravenous administration is less than28 ml/kg, and that the toxicity associated with HSD is consistent with theadministration of large volumes of hypertonic saline. Since the proposedtheiapeutic dose of HSD is only 4 ml/kg, these findings indicate that therewill be minimal adverse effects associated with the therapeutic administrationof HSD.

ABSTRACT

The acute toxicity following intravenous administrationof a proposed new resuscitation fluid, hypertonic saline/Dextran 70® (HSD), was evaluated in male and female ICR mice.Animals were administered a single intravenous dose of HSD,28 ml/kg over a 5-minute period, in an attempt to define itsmaximum tolerated dose. Equal volumes of each HSD component,7.5% hypertonic saline (HS) and 6% Dextran 700 (D70) innormal saline, and Ringer's lactate (RL) were also evaluated.,Observations were made at 1, 2, and 4 hours afteradministration on the day of dosing and twice daily for theremainder of the study. The animals in each group weredivided into two subgroups based on whether they weresubmitted to necropsy on Day 3 or Day 14 after dosing. HSDproduced death within 12 minutes of the injection in 2 of 20mi.e, pale eyes, and tail sloughing, findings similar tothose observed with HS. -Major signs observed in all groupswere hunched posture, rough coat, hyperactivity, inactivity,and increased rate and/or depth of respiration. Body weightswere unaffected by the dosing and no gross or microscopiclesions could be attributed to HSD or its constituents.These data suggest that the maximum tolerated dose of HSDfollowing acute intravenous administration is less than 28ml/kg, and that the toxicity associated with HSD isconsistent with the administration of large volumes ofhypertonic saline. Since the proposed therapeutic dose ofHSD is only 4 ml/kg, these findings indicate that there willbe minimal adverse effects associated with the therapeuticadministration of HSD.

Key Words: Acute Toxicity, Intravenous Administration,Maximum Tolerated Dose, Hypertonic Saline/Dextran704, Hypertonic Saline, Dextran 700, Ringer'sLactate, Resuscitation Fluid, ICR Mice

AcceSSiOn For

NTIS GRA&IDTIC TAB

justification------

Distrlbutlo /Avaiability CoesA--- - A - .o io -- !-Di t. pe u I

PREFACE

TYPE REPORT: Acute Toxicity GLP Study Report

TESTING FACILITY:US Army Medical Research and Development CommandLetterman Army Institute of ResearchPresidio of San Francisco, CA 94129-6800

SPONSOR:US Army Medical Research and Development CommandLetterman Army Institute of ResearchPresidio of San Francisco, CA 94129-6800

PROJECT DIRECTOR: Charles Wade, PhD

PROJECT/WORK UNIT/APC: 3S463807D836/087/TLRO

GLP STUDY NUMBER: 88001

STUDY DIRECTOR: Don W. Korte, Jr., PhD, MAJ, MSCDiplomate, American Board of Toxicology

PRINCIPAL INVESTIGATOR: Denzil F. Frost, MS, DVM, CPT, VCDiplomate, American College ofVeterinary Preventive Medicine

CO-PRINCIPAL INVESTIGATOR: Gary M. Zaucha, DVM, CPT, VC

PATHOLOGIST: G. David Young, DVM, MAJ, VC

DATA MANAGER: Yvonne C. LeTellier, BS

REPORT AND DATA MANAGEMENT: A copy of the final report,study protocols, retired SOPs,raw data, analytical andstability data, and an aliquotof the test compound will beretained in the LAIR Archives.

TEST SUBSTANCE: Hypertonic Saline/Dextran 700

INCLUSIVE STUDY DATES: 25 May 88 - 28 Jun 88

OBJECTIVE: The objective of this study was to determinethe acute toxicity of hypertonic saline/Dextran700 following intravenous administration in maleand female ICR mice.

iii

ACKNOWLEDGMENTS

SPC Dean K. Magnuson, BS, SPC Eric Dahlberg, BS, and SPC, ilmar 0. L. Villa provided assistance in dose preparation

and administration; SGT Tammie Heineman, SGT Barbara D.3reen, and Richard Katona provided data collection, animalare, and facility management.

iv

SIGNATURES OF PRINCIPAL SCIENTISTS AND MANAGERSINVOLVED IN THE STUDY

We, the undersigned, declare that GLP study number 88001 wasperformed under our supervision, according to the proceduresdescribed herein, and that this report is an accurate recordof the results obtained.

DO W. KO TE, &-, PhD/DATE Y . LeTELLIER, BS/DATEMAJ, MSC DACStudy Director Data Manager

DENZIL F. FROST, MS, DVM/DATE GARY M' ZAUCHA, DVM/DATECPT, VC CPT, VCPrincipal Investigator Co-Principal Investigator

CHARLES B. CLIFORD, DVM, PhD/DATEMAJ, VCSenior Pathologist

v

DEPARTMENT OF THE ARMY

LETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 941294800REPLY TO

ATTENTION OF:

SGRD-ULZ-QA 19 December 1988

MEMORANDUM FOR RECORD

SUBJECT: GLP Compliance for GLP Study 88001

1. This is to certify that in relation to LAIR GLP Study 88001,the following inspections were made:

27 January 1988 - Protocol Review01 June 1988 - Animal Receipt/Room Inspection09 June 1988 - Randomization13 June 1988 - Dosing13 June 1988 - Observations15 June 1988 - Test Chemical Log16 June 1988 - Interim Sacrifice20 June 1988 - Weighing27 June 1988 - Final Sacrifice14 July 1988 - Histology

2. The institute report entitled "Acute Intravenous ToxicityStudy of Hypertonic Saline/Dextran 700 and Its Constituents inICR Mice," Toxicology Series 245, was audited on 9 December 1988.

CAROLYN M. LEWIS, MSDiplomate, American Board of ToxicologyChief, Quality Assurance

vi

TABLE OF CONTENTS

Page

Abstract.....................................................1

Preface....................................................1iii

Acknowledgments............................................. iv

Signatures of Principal Scientists .......................... v

Report of Quality Assurance Unit ............................vi

Table of Contents.......................................... vii

BODY OF REPORT

INTRODUCTION................................................. 1

Objective of Study ......................................2

MATERIALS.................................................... 2

Test Substance.......................................... 2Test Substance Constituents .............................2Control................................................. 3Animal Data............................................. 3Husbandry............................................... 3

=EHODS...................................................... 4

Group Assignment/Acclimation ............................4Dose Levels, Preparation, and Analysis ................. 5Tect Procedures......................................... 5Observations............................................ 5Necropsy................................................ 5Statistical Analysis.................................... 6Duration of Study .......................................6Changes/Deviations...................................... 6Storage of Raw Data and Final Report ................... 7

RESULTS...................................................... 7

Clinical Observations ...................................7Body Weights............................................ 8Necropsy Findings .......................................8

vii

TABLE OF CONTENTS (continued)

Page

DISCUSSION..................................................8

CONCLUSION .................................................. 12

: ""-ENCES. .................................................. 13

A-E ENDICES ................................................. 15

Appendix A. Chemical Data............................... 16Appendix B. Animal Data .............................. 20

Appendix C. Historical Listing of Events ............. 21Appendix D. Individual Animal Histories .............. 22Appendix E. Individual Body Weights .................. 68Appendix F. Pathology Report ......................... 72

OFFICIAL LIST OF DISTRIBUTION ............................. 159

viii

Acute Intravenous Toxicity Study of HypertonicSaline/Dextran 70® and Its Constituents in ICR Mice --Frost et al

INTRODUCTION

During conventional land warfare it has been estimatedthat 90% of the deaths occur either in the field or en routeto a fixed medical treatment facility and that 50% of thosedeaths occur due to hemorrhage (1). Conventional treatmentof hemorrhage has involved infusion of isotonic resuscitationfluids at volumes equivalent to two or three times the volumeof blood lost (2). Supplying this volume of resuscitationfluid on the battlefield for treatment of multiple casualties4s not feasible.

Hypertonic crystalloid solutions have been used for thepast 70 years in the treatment of hemorrhage (3). However,h.e consensus has been that unless followed by replacement of

the lost blood volume, the beneficial effects of treatmentwith hyperosmotic solutions are transient (4). Recently, theaddition of a hyperoncotic colloid, 6% Dextran 70®, to thehypertonic crystalloid, 7.5% saline, hnas significantlyextended 96-hour survival rates compared with those obtainedwith normal saline or 7.5% saline (5). If the effectivenessor this hypertonic saline/Dextran therapy is verified byclinical trial, it would be a significant advance in thetreatment of blood loss due to traumatic injuries.

As with any new treatment regimen, there are potentialrisks. Low molecular weight dextran may cause bleedingabnormalities and phlebitis or interfere with the cross-matching of blood (6). Hypertonic solutions may causeneurologic abnormalities induced by the rapid increases inosmolalities (7-9) or cardiac arrhythmias induced by thehypokalemia associated with the rapid expansion ofextracellular space (2, 10). Consequently, the Division ofToxicology, Letterman Army Institute of Research, was taskedto provide an acute and subchronic toxicity profile of the7.5% hypertonic saline/6% Dextran 70® resuscitation fluid(HSD). This report describes the results of an acutetoxicity study of hypertonic saline/Dextran 70® followingintravenous administration of the maximum tolerated dose inmale and female ICR mice.

Frost et al--2

Objective of Study

The objective of this study was to determine the acutetoxicity of hypertonic saline/Dextran 70® followingintravenous administration in male and female ICR mice.

MATERIALS

Test Substance

Name: Hypertonic saline/Dextran 70® (HSD)

LAIR Compound Code: TP96

Lot Number: NC 54845

Expiration Date: 31 December 1988

Composition per 100 ml: Dextran 70® 6 gsodium chloride 7.5 gwater for injection to 100 ml

Source: Pharmacia LEO PharmaceuticalsUppsala, Sweden

Test Substance Constituents

Name: Hypertonic (7.5%) saline (HS)


Lot Number: I 318712


Composition per 100 ml: sodium chloride 7.5 gwater for injection to 100 ml


Name: Dextran 70® (D70)


Lot Number: NE 54941

Expiration Date: 31 May 1989

Frost et al--3

Composition per 100 ml: Dextran 70® 6 gsodium chloride 0.9 gwater for injection to 100 ml

Source: Pharmacia LEO Pharmaceuticals

Uppsala, Sweden

Control

Name: Ringer's lactate (RL)


Lot Number: NC 54847


Composition per 100 ml: sodium chloride 600 mgpotassium chloride 30 mgcalcium chloridedihydrate 20 mgsodium lactate 310 mgwater for injection to 100 ml


Other test substance information is presented in

Appendix A.

Animal Data

Forty-two male and 42 female ICR mice (Harlan Sprague-Dawley, Inc., P.O. Box 29176, Indianapolis, IN 46229) wereassigned to this study. They were identified individuallywith neck tags. Two male and 2 female mice were utilized forquality control necropsy. The animal weights on receipt (25May 88) ranged from 16.5 to 26.9 g. Additional animal dataare presented in Appendix B.

Husbandry

Study animals were individually housed in stainlesssteel battery-type cages with screen bottoms, in racksequipped with automatically flushing dump tanks. The diet,fed ad libitum, consisted of Purina Rodent Laboratory Chow®5001 (Ralston Purina Company, St. Louis, MO); water, purifiedby reverse osmosis, was provided by continuous drip from acentral line. The animal room temperature and humidity weremonitored continuously by hygrothermograph. The temperature

Frost et al--4

was maintained in a range from 23.3'C to 30.0°C. Therelative humidity was maintained in a rango of 29% .o 47%with occasional spikes to as high as 63% during roomcleaning. The photoperiod was 12 hours of light per dayi0600 - 1800 hours).

METHODS

This study was conducted in accordance with FDAguidelines (11) and LAIR SOP-OP-STX-113, "Acute IntravenousToxicity Study" (12).

group Assignment/Acclimation

The animals were randomized into four groups of 10 malesand 10 females each. Since animals were to be terminated attwo different time intervals following dosing, each group wasfurther divided into 2 subgroups of 5 males and 5 femaleseach (Table 1). Allocation was accomplished using acomputer-based, stratified, weight-biased method. The XYBIONPath/Tox AESLCT Animal Allocation Program was used inconjunction with a VAX 750 Computer.

Table 1

DOSE GROUPS

Group/Subgroup Solution Sacrifice Day

1/1 hypertonic saline/Dextran 70® 3

1/2 hypertonic saline/Dextran 70® 14

2/1 hypertonic saline 3

2/2 hypertonic saline 14

3/1 Dextran 70/normal saline 3

3/2 Dextran 70/normal saline 14

4/1 (controls) Ringer's lactate 3

4/2 (controls) Ringer's lactate 14

Frost et al--5

The male and female study animals were acclimated for 18and 19 days, respectively, before the day of dosing. Duringthis period they were checked daily for signs of illness, andweighed weekly.

Dose Levels. Preparation. and Analysis

The maximum tolerated intravenous dose of HSDadministered over a 5-minute period was determined to be 28ml/kg in preliminary pilot studies. Equal volumes of HS,D70, and RL were administered to animals in the correspondingdose groups. Solutions were used as supplied by themanufacturer, Pharmacia LEO Pharmaceuticals. Analysis of thedosing solutions was provided by Pharmacia LEOPlarmaceuticals. Additional chemical data are presented inAppendix A.

Test Procedures

The acute intravenous toxicity of hypertonicsaline/Dextran 700 (HSD) was evaluated in parallel withsolutions of its major constituents, hypertonic (7.5%) saline(HS) and Dextran 70® (6%) in normal saline (D70), withRinger's lactate (RL) serving as the control. The dosingsolutions were administered via the tail veins. Injectionswere made using tuberculin syringes (Becton Dickinson & Co.,Rutherford, NJ 07070, Lot No. 7H355) and 26 gauge, 1/2 inchneedles (Becton Dickinson & Co., Rutherford, NJ 07070, LotNo. 1D017). A 12-inch length of PE-20 polyethylene tubing(Clay Adams, Division of Becton Dickinson & Co., Parsippany,NJ 07054, Lot No. 91198, exp. date 8/92) was used to attach asecond, hubless needle to the needle on the syringe tofacilitate completion of the injections. Immediatelyfollowing the injection, digital pressure was applied tocontrol any bleeding from the injection site.

Observations

Clinical observations were accomplished 1, 2, and 4hours after dosing on Day 0, and then at least twice dailyfor the remainder of the observation period. Body weightswere recorded upon receipt of the animals, weekly during thequarantine and observation periods, before dosing, and atnecropsy.

Animals that died spontaneously after dosing wereimmediately submitted for necropsy. Subgroups 1 and 2animals were submitted for necropsy on Days 3 and 14,

Frost et al--6

respectively, immediately after sacrifice by barbiturateoverdose. Histopathology was performed on the heart, Liver,kidney, brain, lung, spleen, and all gross lesions.

Statistical Analysis

Clinical signs were tabulated by groups. The means andstandard errors for the body weights for each group werecalculated. For Day 0, the equality of the variances for thegroups was tested by the Levene test. If equal, the standardone-way analysis of variance was performed. If unequal, theWelch's one-way analysis of variance was performed. For Days3, 7, and 14, an analysis of covariance was performed withDay 0 body weights as the covariate. If the F-statistic wassignificant for an analysis of variance or covariance for aparticular measurement, differences among the groups wereevaluated using the Newman-Keuls test (13). Gross andmicroscopic pathology findings were described for each animaland tabulated by groups.

Duration of Study

Appendix C is a complete listing of historical events.

Changes/Deviations

The diet consisted of Purina Rodent Laboratory Chow®#5001 instead of Certified Rodent Chow® #5002.

The animal room lights were found on at 2148 hours, 11Jun 88.

Animals 88C00-01, -08, and -38 of Group 1 were misdosedand received 40 ml/kg HSD instead of 28 ml/kg. Since 88C0008tolerated the dose, it was not deleted from the datatabulation. Animals 88C00-01 and-38 died from the overdoseand were replaced by 88C00-48 and -42, respectively. Due tothe nature of XYBION programming, it was not possible tochange animal numbers. As a result, the pathology data forthese two animals appear under the number of the animal eachreplaced.

Some tissues were not available for histologicalevaluation because they were lost or not processed correctly.The following tissues were not available for animal 88C0094:brain, liver, heart, spleen and kidneys.

It is believed that these changes had no adverse effectson the results of this study.

Frost et al--7

Storage of Raw Data and Final Report

A copy of the final report, study protocol, raw data,retired SOPs, and an aliquot of the test compound will beretained in the Letterman Army Institute of ResearchArchives.

RESULTS

Clinical Observations

The clinical signs observed were grouped into thefollowing categories: general health, respiratory,

b&rfavioral, ocular, reflexive, and miscellaneous.

The most frequently observed signs were in the generalhealth category. Hunched posture (65 of 80 animals) was

observed on Day 0 (day of dosing) and was observed in femalesand males through Days 3 and 4, respectively. Rough coat (36of 80) was observed on Day 0 in both sexes but predominantlyin the males. Rough coat was present through Days 3 and 7for females and males, respectively. Bruising of the tailwas observed in eleven animals (seven males and four females)from both the HSD- and HS-treated groups. Eventual sloughingof the tail distal to the injection site occurred in two HSDand three HS animals. Two HSD animals and one HS animal diedwithin 12 minutes of the start of the infusion.

The second major category of clinical signs observed inthe study were respiratory. Increased respiratory rate wasobserved in 65 of 80 animals on Day 0. The increasedrespiratory rate was observed until Day 3 in the females andin a majority of male animals; however, there were isolatedoccurrences of increased respiratory rate in some malesobserved through Day 7. The increase in respiratory depthwas observed in 12 of 80 animals. The increase inrespiratory depth occurred primarily on Day 0 in animalsreceiving HS or D70.

Ocular signs were observed in 23 of 80 animals. Theeyes of these animals became pale or a lighter red in color.The pale eyes were observed only on Day 0 and were seen withincreased incidence in the groups receiving D70, HSD, and HScompared with the controls.

Behavioral, reflexive, and miscellaneous signs wereequally distributed among all groups. Behavioral signsobserved included inactivity, hyperactivity, irritability,apprehension, circling, tremors, disorientation, and ataxia.

Frost et al--8

Only inactivity (16 of 80) and hyperactivity (13 of 80)occurred in an appreciable number of anixals. Th-. otherbehavioral signs were isolated and occurred sporadicallyamong the different groups. Increased startle reflex(reflexive) and nasal staining (miscellaneous) were eachobserved in one animal.

Table 2 contains a summary of clinical observations.Appendix D contains individual animal histories.

Body Weights

Animal body weights were not significantly (p < 0.05)affected by dosing. Table 3 presents the mean body weightsby group. Appendix E contains individual weight tables.

:ecropsy Findings

No gross or microscopic pathological lesionsattributable to the test compound or its constituents werereported. The Veterinary Pathologist's report is presented4n Appendix F.

DISCUSSION

The acute intravenous toxicity of HSD in mice wasevaluated by dosing the animals with an apparent maximumtolerated dose via the tail vein. This dose was establishedin preliminary studies as 28 ml/kg administered over 5minutes. This dose, 28 ml/kg, is seven times the proposedtherapeutic dose of 4 ml/kg (2). When administered at a rateof 28 ml/kg over 5 minutes, HSD caused death in 1 of 10 malesand 1 of 10 females. This suggests that the maximumtolerated dose is less than 28 ml/kg. In addition to dosingwith HSD, groups of mice were dosed with equal volumes of HS,D70, or RL. The HS also caused a death in 1 of 10 malesdosed which suggests that the HS component is responsible forthe lethality of the HSD mixture. The deaths were very rapidoccurring within 1 - 12 minutes of the initiation of theinfusion. The rapidity of onset and the fact that death wasobserved only in those groups receiving hypertonic salinesuggest that the deaths were due to cardiac dysrhythmiasarising from the putative hypokalemia produced by the salineinfusions. This is consistent with the observation thatdextran is well tolerated when administered intravenously atdoses up to 40 ml/kg in various animal models (G. Jonsson,Pharmacia Pharmaceuticals AB, personnel communication).

Frost et al--9

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Frost et al--10

Table 3

GROUP MEAN BODY WEIGHT (GMS)

DAY OF STUDYGROUP -18 -13 -7 0 3t 7 14

Males

1 23.0" 25.6 29.5 31.8 31.7 29.5 33.0±1.79 3.78 2.97 4.91 1.76 7.61 3.67(10) (10) (10) (10) (4) (4) (5)

2 23.2 24.5 29.8 32.7 31.9 32.4 34.5±2.29 5.20 2.73 2.32 2.58 1.39 1.91(10) (10) (10) (10) (5) (4) (4)

3 23.7 26.4 29.8 32.2 31.9 31.2 28.8±1.80 2.08 2.87 2.62 2.80 3.04 6.76(10) (10) (10) (10) (5) (5) (5)

4 23.7 26.2 29.1 31.2 31.4 30.7 30.4±1.97 2.20 2.67 4.13 3.10 3.43 5.32(10) (10) (10) (10) (5) (5) (5)

Females*

1 20.0 21.4 24.8 26.7 27.2 27.2 29.2±2.38 3.32 1.80 2.45 1.58 2.57 3.70(10) (10) (10) (10) (4) (5) (5)

2 20.7 21.9 24.6 27.1 25.8 27.2 30.4±2.08 2.54 1.67 1.95 1.62 1.85 0.89(10) (10) (10) (10) (5) (5) (5)

3 21.5 22.9 24.6 26.9 28.9 26.7 28.6±1.27 1.81 1.60 2.30 1.57 2.40 2.51(10) (10) (10) (10) (5) (5) (5)

4 20.8 21.7 24.7 25.4 26.9 25.3 28.4±1.43 2.92 1.73 2.21 2.37 2.04 2.07(10) (10) (10) (10) (5) (5) (5)

t Day 3 body weights include only interim sacrifice animals.

" Data presented as mean ± standard deviation with the numberof animals, n, in parenthesis.* Days for females = -19, -14, -8, 0, 3, 6, 14.

Frost et al--ll

An equal volume of RL was administered as a control.Since the RL is an isotonic solution it would only permitcomparison of the effects of the volume administered.Therefore, differences between the RL group and the HSD, HS,and D70 groups would be attributable to the volume expansioncapabilities of the latter groups. The majority of clinicalsigns whether general health, behavioral, respirative,reflexive, or miscellaneous in nature, were observed at anequal rate of occurrence in each of the four groups. The twoobservations, other than death, associated primarily with thevolume expanders were pale eyes and tail sloughing. Inalbino mice, the reddish color of the eyes is associated withthe concentration of erythrocytes in the highly vascularretina; thus, the "pale eye" observation would suggest a-eduction (or dilution) in the erythrocyte concentration.The increased incidence of pale eyes in the HSD, HS, and D70qroups compared with the LR group is attributed to thehyperosmotic action of these solutions to draw additionalLiuid intu Lhe vascular compartment resulting in a greaterdilution of erythrocytes as compared with the LR group. Thepale eyes were observed only within the first 24 hours offluid administration suggesting that fluid balance hadreturned to normal within this time.

The tail sloughing occurred in two animals receiving HSDand three animals receiving HS. This was attributed tounavoidable extravasation of injection fluid around the siteof injection. Assuming that the injection technique was thesame for all animals, extravasation occurred in all dosegroups. Tail sloughing in HSD and HS, but not in D70 treatedanimals, suggests that extravascular hypertonic saline causestissue necrosis by a direct effect rather than indirectlythrough an increase in extravascular fluid pressure occludingperfusion. The resulting sloughing of the tail distal to theinjection site was a consequence of the local necrosis. Thisfinding was unexpected as HSD was not irritating followingintravenous, intra-arterial, paravenous, subcutaneous, andintramuscular administration when evaluated in a standardirritation test system (14).

No significant findings associated with any treatmentregimen were observed during necropsy at 3 or 14 days afterinjection of the dosing fluids nor in the histologicalexamination of selected tissues. This suggests thatmorphological changes, if, in fact, there were any due to theadministration of the volume expanders, were transient.

These data suggest that the maximum tolerated dose of1I3D when administered intravenously over a 5-minute period isless than 28 ml/kg. The toxicity observed (death in 2 of 20animals, pale eyes, and tail sloughing) was attributable to

Frost et al--12

the HS component of the HSD resuscitation fluid and is anexpected physiological response to large volumes cfhypertonic saline. Since the proposed therapeutic dose ofHSD is only 4 ml/kg, these findings indicate that there willbe minimal adverse effects associated with the therapeuticadministration of HSD.

CONCLUSION

The maximum tolerated dose of HSD following acuteintravenous administration is less than 28 ml/kg, and thetoxicity associated with the HSD administration is consistentwith the administration of large volumes of hypertonicsaline. Since the proposed therapeutic dose of HSD is only 4ml/kg, these findings indicate that there will be minimaladverse effects associated with the therapeuticadministration of HSD.

Frost et al--13

REFERENCES

1. Bellamy RF. The causes of death in conventional landwarfare: Implications for combat casualty care research.Milit Med 1984; 149: 55-62.

2. Kramer GC, Perron PR, Lindsey DC. Small-volumeresuscitation with hypertonic saline dextran solution.Surgery 1986; 100(2): 239-246.

3. Penfield WG. The treatment of severe and progressivehemorrhage by intravenous injection. Am J Physiol 1919; 48:121-128.

'. Velasco IT, Pontieri V, Rocha e Silva M. HyperosmoticNaC1 and severe hemorrhagic shock. Am J Physiol 1980; 239:HC54-H673.

5. Maningas PA, DeGuzman LR, Tillman FJ, Hinson CS,Priegnitz KJ, Volk KA, Bellamy RF. Small-volume infusion of7.5% NaCI in 6% Dextran 70® for the treatment of severehemorrhagic shock in swine. Ann Emerg Med 1986; 15(10):1131-1137.

6. Holcroft JW, Vassar MJ, Turner JE, Derlet RW, Kramer GC.3' NaCI and 7.5% NaCl/dextran 70 in the resuscitation ofseverely injured patients. Ann Surg 1987; 206(3): 279-288.

7. Norengerg MD, Leslie KO, Robertson AS. Associationbetween rise in serum sodium and central pontinemyelinolysis. Ann Neurol 1982; 11(2): 128-135.

2. Laureno R. Central pontine myelinolysis following rapidcorrection of hyponatremia. Ann Neurol 1983; 13(3): 232-243.

9. Sterns RH, Riggs JE, Schochet SS. Osmotic demyelinationsyndrome following correction of hyponatremia. N Engl J Med1986; 314(2): 1535-1541.

10. Shackford SR, Fortlage DA, Peters RM. Serum osmolar andelectrolyte changes associated with large infusions ofnypertonic 6odium lactate for intravascular volume expansionof patients undergoing aortic reconstruction. Surg GynecolObstet 1987; 164(2): 127-136.

11. FDA, Nonclinical Laboratory Studies (21 CFR 58) FinalRule, 22 Dec 78 (43 FR 59986-60025).

Frost et al--14

REFERENCES (cont.)

12. Acute Intravenous Toxicity Study. LAIR StandardOperating Procedure OP-STX-113, Letterman Army Institute ofResearch, Presidio of San Francisco, CA, 8 June 1988.

13. Dixon DJ, ed. BMDP statistical software. Berkeley:University of California Press, 1981: 555-573.

14. Zaucha GM, Frost DF, Young GD, Korte DW. Localirritation toxicity study of hypertonic saline/Dextran 70® inNew Zealand White rabbits. Toxicology Series 244. InstituteReport No. 332. Presidio of San Francisco, CA: LettermanArmy Institute of Research, 1988.

Frost et al--15

APPENDICES Page

Appendix A. Chemical Data ............................ 16Appendix B. Animal Data .............................. 20Appendix C. Historical Listing of Events ............. 21Appendix D. Individual Animal Histories .............. 22Appendix E. Individual Body Weights .................. 68Appendix F. Pathology Report ......................... 72

Frost et al--16

Appendix A: CHEMICAL DATA

Pharmacia CERTIFICATE OF ANALYSIS 1988-12-19

6 % DEXTRAN 70 ZN 7,5 %SODIUM CHLORIDE INJECTION

ChaurNo. NC 54845

Inherent viscosity 27 m/g

Absorbance (375 nm, 10 mm) 0,006

pH 4,6

Heavy metals < 5 ppm

Sodium chloride 74,7 g/1000 ml

Dextran 70 60 g/1000 ml

Particulate matter passed test

Sterility passed test

Pyrogens passed test

Released for clinical trials.

Pharmei ABAnalytical Chemistry Department

S-T5 1 02 u e Nut 016S 1630 00 M ro027 M o 6.1541 Ia Sweden Iml +46 Is 16 30 Wm Int +4618 164139

Frost et al--17

Appendix A (cont.): CHEMICAL DATA

~)Pharmac la CERTIFICATE OF ANALYSIS 1988-12-19

7,5 % SODIUM CHLORIDE

Charg No. I 318712

Identification passed test

Absorbance 0.000

pH 6.0


Sodium chloride 75,0 g/1000 ml




Released for biological trials.

Pharmaeca ABAnlytical ChemIstry Department

. ........E'l~sset Frss~on .. ....

SPhwnWGABWm 8 U.,m. Nlt 01-48000 -pei. 70027 kt018-154136

Int+46 is is 30 00 l eoI phlaiot Im +46181S 4%

Frost et al--18


tJ Pharmacia Certificate of analysis

Name: MACRODEX 60 mg/mI in Normal so:ine

ftm No.: 10.4510-0 'oI No: NE 54941

Te Result Tolerance limit Method

Inherent viscosity mI/g 26 25-28 03700Colour 0.01 Max. 0,04 03811pH 4.9 4,0-7,0 USP XX p. 968Heay metals ppm <5 Max. 5 USP XX p. 909Assay for-sodium chloride g/1000ml 8,88 8,10-9.90 02355-dextran 70 g/1000ml 59 54-66 02356Sterility Passed test To pass test 02885Pyrogens Passed test To pass test 02983

The Identity is assured through strict adherence to established GMP rules throughoutthe manufacturing procedures.

Released for sale: 1987-05-25

Pharmacla ABQuality Control Deportment

Mats Wberg M09

PhOMocla A TePelh"on Telw Telefto8.75182 UPPSALA Not 018- 163=O 76027 phomus Not 018- 154135

&V~eInt +46 IS 163000 Ilt +46 18 154135

Frost et al--19


e Pharmacla CERTIFICATE OF ANALYSIS 1988-12-19Pr~dUc

LACTATED RINGER'S INJECTION

Cherge No. NC 54847

pH 5,8


Sodium 127 mmol/1000 ml

Potassium 3,91 mmol/1000 ml

Calcium 1,30 mmol/1000 ml

Chloride 105,5 mmol/1000 ml

Lactate 27,2 mmol/1000 ml




Released for clinical trials.

Pharmacia ABAnalytical Chemistry Department

P= dins TO*a'o Cabm ile T60469Wll'ffr"* As

75.2 UPPOe a NUOI163000 w 760M NMo010-16413Im +46 18 103000 vpam p mu. Int +48 4 10S 4136

Frost et al--20

Appendix B: ANIMAL DATA

Species: Mus musculus

Strain: ICR

Source: Harlan Sprague-Dawley, Inc.P.O.Box 29176Indianapolis, IN 46229

Se :: Male and Female

Date of birth: 22 Apr 88

Method of randomization: Weight bias, stratified animala.iocation (XYBION Medical Systems PATH/TOX AESLCT AnimalAllocation program)

Animals in each group: 10 Males and 10 Females

Condition of animals at start of study: Normal

Bo-dy weight range at dosing: Males 18.8 - 36.7 gFemales 21.9 - 30.9 g

Identification procedures: Neck tag

Pretest conditioning: Quarantine/acclimation 26 May 1988 to

12/13 June 1988 (males/females)

Justification: The laboratory mouse is a standard laboratorymodel for acute toxicity studies and is accepted by allregulatory agencies.

Frost et al--21

Appendix C: HISTORICAL LISTING OF STUDY EVENTS

Date Event

25 May 88 Animals arrived at LAIR. They weresexed, observed for illness, andcaged in the GLP Suite. Eightyanimals were assigned to the study.

27 May 88 Animals were tagged.

26, 31 May 88 Animals were weighed.6, 13, 14, 16(a)17(a), 20(b)27 (b), 28 (b)Tun 88

31 May 88 Quality control animals weresubmitted for necropsy.

26 May 88- Animals were observed twice daily.27 Jun 88

9 Jun 88 Remaining animals were randomizedinto groups.

13, 14 Jun 88 Animals were dosed at approximately0900 hours. Observations wereconducted one, two, and four hoursafter dosing.

16(a), 17(a) Food was removed by 0600 hours.27(b), 28(b) Animals were observed for clinicalJun 88 signs at approximately 0730 hours.

Animals were delivered to theNecropsy Suite for euthanasia andnecropsy.

Frost et al--22

Appendix D: INDIVIDUAL ANIMAL HISTORIES

The protocol schedule refers to the day of dosing as Day0. Since XYBION programming refers to the day of dosing asDay 1, a one-day dicrepancy exists between the actual studyday and the day listed in the Individual Animal Histories.The following table lists the actual study day and thecorresponding day which appears in Appendix D under "STUDYDAY/TIME DATA WERE TAKEN".

STUDY DAY XYBION

Dosing 0 11 2£3

interim Sacrifice 3 44 55 66 77 88 99 10

10 1111 1212 1313 14

Terminal Sacrifice 14 15

Frost et al--23

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Frost et al--56

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7'rost et al--68

Appendix E: BODr WEIGHTS (GMS) - MALES

DAY OF STUDYA*NIMAL GROUP -18 -13 -7 0 3 7 14

sco- ___

08 1/1 22.7 25.9 29.7 31.5 31.327 1/1 24.5 27.7 31.0 35.6 33.635 1/1 22.3 25.1 28.9 31.6 died42 1/1 23.7 26.2 30.3 32.1 3.48 1/1 19.2 26.8 30.0 32.1 29.503 1/2 24 .7 29.3 33.3 36.7 3.3 3611 1/2 25.1 27.7 32.1 34.7 33.9 3612 1/2 21.3 15.5 22.3 18.8 18.2 2723 1/2 22.5 24.8 29.7 32.6 31.4 3343 1/2 23.7 26.6 27.9 32.5 NR* 33

Mean 23.0 25.6 29.5 31.8 31.7 29.5 33.0Std Dev 1.79 3.78 2.97 4.91 1.76 7.61 3.67SEM 0.56 1.19 0.94 1.55 0.88 3.80 1.64

14 2/1 26.2 30.3 33.2 35.7 34.520 2/1 24.0 17.9 29.9 32.9 34.229 2/1 23.8 25.8 27.8 28.2 28.237 2/1 19.3 13.3 25.2 29.6 31.146 2/1 25.7 28.5 31.9 33.4 31.409 2/2 23.9 26.8 29.0 32.7 30.7 3716 2/2 21.6 26.5 32.5 34.4 died22 2/2 22.4 26.2 31.4 34.5 34.1 3541 2/2 24.7 27.1 30.9 33.9 32.3 3345 2/2 20.1 22.5 26.0 31.4 32. 3 33

%'ean 23.2 24.5 29.8 32.7 31.9 32. 34.5Std Dev 2.29 5.20 2.73 2.32 2.58 1.39 1.91_EM 0.72 1.64 0.86 0.74 1.15 0 69 0.96

* Not recorded

Frost et al--69

Appendix E (cont.): BODY WEIGHTS (GMS) - MALES

DAY OF STUDYANIMAL GROUP -18 -13 -7 0 3 7 1488C00-

04 3/1 23.1 24.7 27.0 30.1 31.005 3/1 24.1 25.9 29.0 32.1 27.821 3/1 24.8 27.7 30.8 31.8 31.736 3/1 26.0 28.2 33.0 34.8 35.244 3/1 24.2 27.1 29.9 33.9 33.602 3/2 21.2 24.4 27.5 31.1 30.9 2218 3/2 24.2 26.5 29.3 30.5 29.2 2219 3/2 23.5 28.9 34.9 36.3 33.7 3626 3/2 20.1 22.3 25.2 27.5 27.5 2930 3/2 25.3 28.2 31.1 34.3 34.8 35

Mean 23.7 26.4 29.8 32.2 31.9 31.2 28.8Std Dev 1.80 2.08 2.87 2.62 2.80 3.04 6.76SEM 0.57 0.66 0.91 0.83 1.25 1.36 3.02

13 4/1 25.5 26.9 30.0 34.0 33.517 4/1 23.0 25.1 29.1 31.4 31.128 4/1 24.4 27.6 27.7 33.8 32.732 4/1 20.5 22.3 24.8 26.1 26.234 4/1 24.6 27.8 30.9 34.0 33.706 4/2 26.9 28.5 33.1 35.1 34.9 3607 4/2 24.4 27.2 30.5 31.4 30.1 2415 4/2 24.7 28.3 31.8 34.2 33.4 3525 4/2 21.6 23.1 25.5 22.3 26.4 2633 4/2 21.8 24.8 27.9 30.0 28.9 31

Mean 23.7 26.2 29.1 31.2 31.4 30.7 30.4Std Dev 1.97 2.20 2.67 4.13 3.10 3.43 5.32SEM 0.62 0.70 0.84 1.31 1.39 1.53 2.38

Frost et al--70

Appendix E (con.) BODY WS:GHTS (GMS) - FEMALES

DAY OF STUD)ANIMAL GROUP -19 -14 -8 0 3 6 14

71 1/1 19.9 21.3 23.7 25.7 24.9-' I/i 19.i 22.3 24.0 22.8 27.5S1 1/1 20.6 22.8 25,3 27.9 27.686 1/1 22.8 21.1 22.7 28.5 29.

1/1 19.2 21.5 224 24.0 died50 1/2 16.5 21.3 26.0 26.3 24.2 2458 1/2 19.2 12.8 26.8 30.1 30.2 3463 1/2 25.0 25.5 28.0 30.0 29.2 3119 1/2 19.3 21.1 23.7 24.9 25.1 28S7 1/2 18.3 23.7 24.9 26.6 27.4 29

Mean 20.0 21.4 24.8 26.7 27.2 27.2 29.2Std Dev 2.38 3.32 1.80 2.45 1.58 2.57 3.70SEM 0.75 1.05 0.57 0.77 0.79 1.15 1.66

57 2/1 21.1 21.3 23.4 26.3 25.773 2/1 21.8 22.1 24.4 26.4 26.2- 2/1 16.9 16.3 24.9 28.1 28.184 2/1 19.6 22.2 23.3 26.3 25.496 2/1 19.9 40.5 24.0 24.2 23,659 2/2 19.3 23.6 26.9 30.9 L5.7 316<2 2/2 24.1 25.3 27.5 26.9 25.5 3164 2/2 19.3 23.6 24.7 27.8 27.8 2975 2/2 22.6 23.8 25.4 28.7 27.7 3183 2/2 22.2 20.1 21.9 25.1 25.2 30

New 20.7 21.9 24.6 27.1 25.8 27.2 30.4Std Dev 2.08 2.54 1.67 1.95 1.62 1.85 0.89SEM 0.66 0.80 0.53 0.62 0.72 0.83 0.40

Frost et al--71

Appendix E (cont.): BODY WEIGHTS (GMS)FEMALES

DAY OF STUDYANIMAL GROUP -19 -14 -8 0 3 6 1488C00-

49 3/1 20.5 21.8 23.7 27.4 28.954 3/1 21.3 22.1 24.2 27.0 27.355 3/1 22.9 24.8 27.1 29.0 28.283 3/1 21.9 24.7 24.9 28.5 28.693 3/1 22.3 25.2 26.3 28.7 31.561 3/2 23.7 23.8 25.7 26.4 28.7 2970 3/2 21.9 19.5 25.3 29.1 29.1 3282 3/2 19.4 21.3 21.6 22.7 23.1 2585 3/2 21.0 23.3 23.7 27.2 26.2 2994 3/2 20.5 22.2 23.4 23.1 26.4 28

Mean 21.5 22.9 24.6 26.9 28.9 26.7 28.6Std Dev 1.27 1.81 1.60 2.30 1.57 2.40 2.51SEM 0.40 0.57 0.51 0.73 0.70 1.07 1.12

52 4/1 21.2 22.2 23.6 24.0 26.560 4/1 20.4 21.9 24.1 24.5 25.566 4/1 21.4 23.2 24.7 26.0 26.474 4/. 23.8 24.7 28.3 29.7 31.077 4/1 19.2 19.7 22.9 25.0 25.151 4/2 20.8 21.3 25.1 21.9 24.9 2753 4/2 19.2 20.9 22.5 24.9 24.6 2867 4/2 22.1 24.7 26.3 26.6 27.1 3068 4/2 19.4 14.7 25.7 27.8 27.5 3190 4/2 20.8 23.3 23.9 23.8 22.5 26

Mean 20.8 21.7 24.7 25.4 26.9 25.3 28.4Std Dev 1.43 2.92 1.73 2.21 2.37 2.04 2.07SEM 0.45 0.92 0.55 0.70 1.06 0.91 0.93

Frost et al--72

Appendix F: PATHOLOGY REPORT

GLP Study 88001

Principal Investigator: CPT Denzil FrostCo-Principal Investigator: CPT Gary ZauchaPathologist: MAJ David Young

INTRODUCTION:

Study: Acute mouse intravenous toxicity test.Test Compound: Hypertonic Saline/Dextran (HSD)-Animal: Mouse (Mus musculus), ICR, 20-25 grams, male

and female.

3=a~e Groups:

Group 1: (HSD) 28 ml/kg*roup 2: (Hypertonic Saline) 28 ml/kg,group 3: (Dextran 70) 28 ml/kgGroup 4: (Lactated Ringer's) 28 ml/kg

I. S TJARY OF PROCEDURES:

Euthanasia: Sodium pentobarbital, IP (One subgroup at72 hrs; one subgroup at 14 days)

Fixative: 10% Neutral Buffered FormalinHistopathology: Routine.

GROSS FINDINGS:

Table 1 lists animals from all groups with recordedcss lesions. No gross lesions were considered to be

-"--tied to the test compounds.

* . MICROSCOPIC FINDINGS:

-issues saved for microscopic examination from all~--_s include brain, heart, liver, lung, spleen and kidney.

Table 2 lists the incidence summary (with percentages)all microscopic observations. The most frequent lesions

%e present in the lungs (congestion) -nd liver (subacutei:il ~'a1hepatitis). Multiple findings of lower incidence::-.,uJe hepatic sinusoidal congestion and renal cortical

., often with an associated minimal subacute interstitial.hitis. Single cases of cardiac myofiber mineralization,

a:. ic necrosis and renal pelvis mononuclear infiltrate were

Frost et al--73

Appendix F (cont.): PATHOLOGY REPORT

The incidence summary for microscopic observations ofnonneoplastic graded diagnoses are listed on Table 3.Analysis of these findings using the Kolmogorov-Smirnov two--ailed test revealed no significant differences at the 0.05level. Annex A lists the individual animal report of grossand microscopic diagnoses.

V. SUMMARY COMMENTS:

The etiology of the single case of mild hepatic necrosis,,as not determined. The multiple cases of minimal and oftenfocal hepatitis were most consistent with liver lesionscaused by mouse hepatitis virus. The pulmonary and hepaticsinusoidal congestion indicated local variation in vascularperfusion and was most likely a consequence of exsanguinaticnwhile under barbiturate anesthesia. Other lesions observedin these mice were interpreted as incidental findings oflittle to no clinical significance. No lesions wereconsidered related to the test compound or procedure.

~G. DAVID YOUNGMAJ, VCDivision of Pathology

15 August 1988i'dbj

:-rest et ai--74

Appendix F (coit. 1 1ATVJ. .EPORT

_. ineral depcs-:ion - Cardiac mofibers are -- laced by-e ,ui and variable amountos of mineral, with an absence of

:n-6.'matory resoornOe. A few aduent myofibers a:eJ.e--ener ..tive. Coded as piLsent.

-.?erivasculitis/vasculitis - Arterioles are surrounded bySe ::roohils and few lymphocytes. The neutrophils are present.:.J TargincLed within the lumen of the vessel and are

4 nsnigrating through the vascular wall. A mirimal amountSyaline necrosis is present within the vessel wail.

euerity grades are as follows:--ace = Small, widely scattered foci not to exceed

three foci per lung section.Mild Four to six small foci of inflammatory ce"1ic-,.2.Ierate Seven ro ten small foci or presence of three

more foci with extension of infiammation intothe pulmonary parenchyma (alveolar regions).

Marked = Moro than ten smalL foci or more titan fourlarge fooi.

2. Congestion - Vascular spaces are distended with large.:.uns of blood. Coded as present when the congestion

- utes a promir .en hisLologic characteristic.

.... dal congestio !- See lungs.

Z. rosis - Discrete areas of hepotocellu!ar architecturalSotn contain cells with nuclear and cytoplasmic changes

retention of th, <r cellular outlines. Cytologic changespyknosis and loss of nuclei with an eosinophilic

. of the -ytop'asm. Few inflammatory cells,nc. of ne':troh Js and lymphocytes are present.

,-Ver t~y grade ,iteria for this lesion are as follows:,Troe Necrotic area is no larger than a 40x

microsccoic field.Combined areas of focal or multifocal necrotic

ES1ions are less than 10% of the liver section.orate = combined areas of focal or multifocal necrotic

lesions are 10% -- 20% of the liver section.

Frost et al--75

Appendix F (cont.): PATHOLOGY REPORT

Marked = Combined areas of focal or multifocal necroticlesions are 20% - 40% of the liver section.

Severe = Combined areas of focal or multifocal necroticlesions are greater than 40% of the liversection.

3. Hepatitis - Subacute inflammatory lesions in the liverconsist of small focal accumulations of neutrophils,lymphocytes and macrophages within the hepatic parenchyma.Individual cell necrosis or degeneration of hepdtocyteswithin these inflammatory foci are occasionally observed.Severity grade criteria are based on the number ofnflammatory cell foci present:Trace = One to three foci.Mild = Four to ten foci.Moderate = Ten to twenty foci.Marked = More than twenty foci.

KIDNEYS

i. Cortical cysts - One or more cystic spaces are presentwithin the renal cortex. The cysts are lined by flattenedepithelium and may be empty or filled with pale homogenouseosinophilic fluid. Coded as present.

2. Interstitial nephritis - Interstitial accumulation oflymphocytes, macrophages and few neutrophils are primarilyand almost exclusively associated with and abutting thecortical cysts. Severity grade criteria are as follows:

Trace Small foci not to exceed three per kidneysection.

Mild = Four to six small foci.Moderate = Seven to ten small foci or the presence of

three or more foci with extension into andobliteration of renal tubules and glomeruli.

Marked = More than ten small foci or more than fourlarge foci.

3. Mononuclear cell infiltrate - Focal infiltration ofmacrophages and lymphocytes are immediately subjacent to theurothelium of the renal pelvis. Severity grade criteria areas follows:

Trace = One to two small foci.Mild = T.-iree to five small foci.Moderate= Six to ten small foci or one to three larger

foci not exceeding a 40x microscopic field.Marked = More than ten small foci or more than four

large foci.

Frost et aI--76

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