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					                                                           NUREG/CR-3745




Biologica! Characterization
of Radiation Exposure and
Dose Estimates for Inhaled
  ranlum Milling ffluents
Annual Progress Report
April 1, 1982- March31, 1983




Project Coordinator: A. F. Eidson

Inhalation Toxicology Research Institute
Lovelace Biomedical and Environmental Research Institute


Prepared for
U.S. Nuclear Regulatory
Commission
                                              NOTICE

This report was prepared as an account of work sponsored by an agency of the United States
Government. Neither the United States Government nor any agency thereof, or any of their
employees, makesany warranty, expressed or implied, or assumesany legal liability          or re-
sponsibility for any third party’s use, or the results of such use, of any information, apparatus,
product or process disclosed in this report, or represents that its use by such third party would
not infringe privately ownedrights.




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                                                      NUREG/CR-3745
                                                      LMF-108
                                                      RH



Biological Characterization
of Radiation Exposure and
Dose Estimates for Inhaled
UraniumMilling Effluents
AnnualProgressReport
April 1, 1982- March31, 1983


ManuscriptCompleted:April 1984
Date Published: May1984

Project Coordinator
A. F. Eidson

                   Research
InhalationToxicology      Institute
Lovelace          and
         Biomedical Environmental         Institute
                                   Research
Albuquerque, NM87185

Prepared for
Division  of Health, Siting and Waste Management
Office of Nuclear Regulatory Research
U,S, Nuclear Regulatory Commission
Washington, D.C. 20555
NRC FIN AI~
                             PREVIOUS DOCUMENTS IN   SERIES


                         of
BiologicalCharacterization RadiationExposureand Dose Estimatesfor InhaledUraniumMilling
         AnnualProgressReport,March1979-March
Effluents,                                                    LMF-76.
                                            lgBO,NUREG/CR-1669,


                         of
BiologicalCharacterization RadiationExposureand Dose Estimatesfor InhaledUraniumMilling
         AnnualProgressReport,
Effluents,                                   IgBl,NUREG/CR-253g,
                              April IgBO-March                 LMF-g4.


                         of
BiologicalCharacterization RadiationExposureand Dose Estimatesfor InhaledUraniumMilling
         AnnualProgressReport,
Effluents,                                   IgB2,NUREG/CR-2832,
                              April lgBI-March                 LMF-gT.
                                           ABSTRACT


                                           of                                  exposure
    The problemsaddressedare the protection uraniummill workersfrom occupatlonal
to uraniumthrough routine bioassayprogramsand the assessmentof accidentalworker exposures.
           of
Comparisons chemicalproperties   and the biologicalbehaviorof refineduraniumore (yellowcake)
are made to identify important properties that influence uranium distributionpatterns among
organs. These studies will facilitate calculationsof organ doses for specific exposures and
associated health risk estimates and will identify important bioassay procedures to improve
          of
evaluations human exposures.
                 analytical
    A quantitative        method for yellowcakewas developedbased on the infraredabsorption
of ammonium diuranate and U308 mixtures in KBr. The method allows the fraction of ammonium
diuranate in a mixture to be determinedaccuratelywithin 7%; the U30B fraction is determined
within 13%. The method was applied to yellowcakesamples obtainedfrom six operatingmills. The
          of
composition yellowcakefrom the six mills ranged from nearly pure ammoniumdiuranateto nearly
pure U30B. The compositionof yellowcake samples taken from lots from the same mill was only
somewhatless variable.
   Becauseuraniummill workersmight be exposedto yellowcake                       of
                                                           either by contamination a wound
                a
or by inhalation, study of retention                of                       implantation
                                    and translocatlon uraniumaftersubcutaneous
in rats was done. The results showed that 49% of the implantedyellowcakecleared from the body
with a half-tlme (Tl/2) in the body of 0.3 days, and the remainder was cleared with a Tl/2
II to 30 days. Contrary to results of previous yellowcake inhalation studies using rats, the
                                                                         relatedto yellowcake
clearancefrom implantedrats was more rapid and could not be quantitatively
composition. An additional study of the effects of animal housing on response of rats to
yellowcakenephrotoxicity                                                  a
                        showed that for studiesrequiringexcretacollections, minimumof 21
                                    to         cagesbefore exposure.
days shouldbe providedfor acclimation metabolism
   Exposures of Beagle dogs by nose-only inhalationto aerosols of commercialyellowcakewere
completed. Twenty dogs exposed to a more soluble yellowcake form inhaled aerosols with
3.4 ± 0.5 ~m mass median aerodynamic diameter (MMAD) (mean ± l SE) and 1.5 ± .04 geometric
standard deviation (GSO) producing estimated Initlal lung burdens of 130 ± 9 pg U/kg body
weight. Aerosols inhaled by dogs exposed to a less soluble yellowcakeform averaged 3.0 ± 0.3
pm MMAD, with 1.7 ± O.l BSD; the estimated initial lung burden was 140 ± 7 ~g U/kg body
                 indicatorsof kidney dysfunctlon
weight.Biochemical                              that appearedin blood and urine 4 to B days
after exposure to the more soluble yellowcakeshowed significant changes in dogs, but levels
returned to normal by 16 days after exposure.No biochemicalevidenceof kidney dysfunctionwas
                                                   form.
observedin dogs exposedto the less solubleyellowcake




                                            ill
                                                                 TABLE OF CONTENTS


                                                                                                                                           Page
ABSTRACT            ..........................................                                                                             ill
LIST    OF         FIGURES              ......................................                                                             vi
LIST    OF         TABLES            .......................................                                                               vii
ACKNOWLEDGMENTS                         ......................................                                                             ix
EXECUTIVE            SUMMARY    .....................................                                                                        1
1. Infrared          Analysis of Refined                Uranium                      Ore      ........................                      3
2. Retention        of Uranium             from      Wounds Contaminated                   by Yellowcake                  ..............   11
3. Effect     of    Animal        Caging         on Nephrotoxtc                  Response to           Uranium           ...............   21
4. Two-Year Dose Pattern                 Studies          of Inhaled             Yellowcake      in the Beagle Dog .........               35
APPENDIX -         TECHNICAL PUBLICATIONS                            AND PRESENTATIONS ....................                                41
                                           LIST OF FIGURES


I.   INFRAREDANALYSISOF REFINEDURANIUMORE                                                 Page


     Figure1.1    Infraredspectra of Mill B yellowcakebefore and after heating.....
     Figure1.2    Superposed spectra of ammonium diuranate and of U308 ..........
     Figure1.3              of
                  Comparison calculated             of
                                         composition ammonium diuranateand U308
                   mixtures with their known values ....................
     Figure1.4    Spectrum of a typical commercial yellowcake sample ...... . .....
     Figure1.5    Spectraof samplestakenfrom two drums from the same lot producedby
                   Mill      E    .................................                        8


2.            OF
     RETENTION URANIUMFROM SIMULATED                 BY
                                    WOUNDSCONTAMINATED YELLOWCAKE


     Figure2.1             uraniumretention
                  Whole-body               curvesfor rats receivingsubcutaneous
                   implants       of yellowcake         from Mill A ...................   14
     Figure2.2    Uraniumcontentof kidneysin rats implantedwith Mill A yellowcake...      16
     Figure2.3    Uraniumcontentof femursin rats implantedwith Mill A yellowcake . .
                                                                               .          16
     Figure2.4              of          of
                  Comparison clearance uraniumfrom the implantsite
                   to uraniumclearancefrom lung in rats exposedto yellowcake
                   powder        by   inhalation         ..........................       18
     Figure2.5    Whole-body uraniumretentioncurvesfor rats receivingimplants of
                                    at
                   Mill A yellowcake a dose of lO mg/kg or at reduceddoses of l or
                   3    nKj/kg        ................................                    18


3.                                       RESPONSEOF RATS TO URANIUM
     EFFECTSOF ANIMAL CAGINGON NEPHROTOXIC


     Figure3.1                                                   cages beginning
                  Dose responsecurvesfor rats housed in metabolism              on
                                       implantation
                   the day of yellowcake          (NaiveRats) or for rats housed
                               cages or in metabolism
                   polycarbonate                                   21
                                                    cages beginning days before
                   yellowcake implantation (Acclimated Rats) ...............              25
     Figure9.2                                                 cagesor in
                  Mean body weightof lO rats housedin metabolism
                   polycarbonate           cages        ..........................        27
     Figure3.3                        by                          cages or in
                  Mean waterconsumption lO rats housedin metabolism
                   polycarbonate           cages        ..........................        27
     Figure3.4                        by                          cages
                  Mean food consumption lO rats housedin metabolism
                   or   in   polycarbonate          cages     .......................     28
     Figure3.5                         by
                  Mean water consumption rats implanted                       body
                                                       with lO mg yellowcake/kg
                   weight and housed in metabolism or polycarbonate cages .........       28
     Figure3.6    Volume of urinaryoutput by rats followingsubcutaneous
                                                                      implantation
                   with   yellowcake     ............................                     29
     Figure3.7              of
                  Comparison animalroom temperature               recorded
                                                   and temperatures       in
                               cages or in metabolism
                   polycarbonate                     cages during a 24-h cycle .....      30
     Figure3.8                   of
                  Gross appearance kidneysof naive rat that died 8 days after
                   implantation of yellowcake at a dose of lO mg/kg ............          30




                                                   vi
                                        LIST OF TABLES


I.                   OF
     INFRAREDANALYSIS REFINEDURANIUMORE                                                Page

     Tablel.l            of          analysisof standardmixtures ammonium
                  Results quantitative                          of
                   diuranate    and U308 ..........................                     7
     Table 1.2    Analysis of yellowcake samples from six mills .............           9
     Table 1.3    Analysis of yellowcake samples from within each mill ..........       9

2.   RETENTIONOF URANIUMFROM SIMULATED                 BY
                                      WOUNDSCONTAMINATED YELLOWCAKE


     Table2.1     Uranium         in            or
                         retention the whole-body at the wound site of rats
                   receiving           implantsof yellowcake,
                            subcutaneous                    comparedto lung
                   retention of uranium from inhaled yellowcake .............       15
     Table2.2     Uranium distributionafter subcutaneousimplantationof yellowcake...15
     Table2.3     Uraniumcontentof kidneysand femur,and dailyurinaryurinary
                   excretion of uranium in rats receiving yellowcake implants ......   17

3.          OF                         RESPONSE RATS TO URANIUM
     EFFECTS ANIMALCAGING ON NEPHROTOXIC       OF


     Table3.1                                      responseof rats to implanted
                  Effectsof cage type on nephrotoxic
                   yellowcake       ..............................                     23


4.   TWO-YEARDOSE PATTERNSTUDIESOF INHALEDYELLOWCAKE


     Table4.1                                                         powder
                  Resultsof beagledog exposuresto aerosolsof yellowcake
                   containing   I00% ammonium      dluranate   ..................      37
     Table4.2                                  to                   powder
                  Resultsof beagledog exposures aerosolsof yellowcake
                   containing     99%   U308      .........................            38




                                            vii
                                         ACKNOWLEDGMENTS


                             Personnel           to
                                      Contributln~ the Research


                                     Seniorand AssociateStaff


                 A. F. Eidson,Ph.D.                        Chemist
                 E. G. Damon,Ph.D.                         Radioblologist
                 B. B. Boecker,Ph.D.                       Radiobiologist
                 E. B. Barr,M.S.E.E.                       ResearchAssociate
                 D. H. Gray,N.S.                           Chemist
                 F. F. Hahn, D.V.M.,Ph.D.                           Pathologist
                                                           Veterinary
                                 D.V.M.,Ph.D.
                 B. A. Muggenburg,                         ResearchVeterinarian
                 J. A. Pickrell,D.V.M.,Ph.D.               ClinicalChemist
                 H. C. Redman,D.V.M.,M.P.V.M.              ResearchVeterinarian


                                         TechnicalStaff


                 E. J. Otero,B.S.                                   Technician
                                                           Laboratory
                 J. A. Romero                                       Technician
                                                           Laboratory
                 A. C. Ferris,B.A.                         ChiefResearchTechnologist



   It should be emphasized                                                 In
                          that a listingsuch as thls Is rarely comprehensive acknowledging
all the individuals who have made important contributions to the research. In the unnamed
category are the many highly skilled animal care, maintenance, shop, administrative, and
secretarialpersonnel whose efforts are essential to the continuationof a productiveresearch
project. The authors acknowledge the friendly and helpful participationof the employees and
management of the uranium mills studied to date. Research Is performed in facilities fully
accreditedby the AmericanAssociation                     of
                                    for the Accreditation LaboratoryAnimal Care. Research
is sponsoredby the U. S. NuclearRegulatoryCommissionunder an interagencyagreementvla U. S.
         of
Department Energy ContractNumber DE-ACO4-76EVOlOI3.
                                                        EXECUTIVESUMMARY


       The purpose     of this     project      is to provtde        scientific       information               to the U. S. Nuclear
Regulatory      Commission for        its    consideratlon        in determining           radiation            protection      guides       and
standards.     This will     ensure that the standards will             protect     adequately the health                    and welfare      of
mill    workers and the public        without     placing     unduly restrictive           and expensive regulations                    on the
mill operators.
       U. S. Nuclear Regulatory Commission guides for worker protection                            in urantum mills            are based on
information      derived       from accidental      human Inhalation            exposures         to slngle          chemtcal         forms of
uranium,       such as UO 2, U308, UF4 or UF6. This is                         especially    true for requirements    for
bioassay      programs at uranium mills (R. E. Alexander,                      ’Applications    of Btoassay for Uranlum,"
WASH-1251, 1974).          Recommended procedures              have since         been revised             to tnclude          more recent
information,      provided       by this     research    program and others,             that     describes         the composition           of
yellowcake     as variable mixtures of ammoniumdiuranate and U308, which vary in their solubility
properties     (U. S. Nuclear Regulatory Commission, "8ioassay at Uranium Mills," Regulatory Guide
8.22, for comment, 1978).
    Much of the information           used in the proposed procedures was derlved from studies of yellowcake
dissolution     conducted in vitro          using simulated       biological      fluids        (O. R. Kalkwarf,             NUREG/CR-0530,
1979; A. F. Etdson and 3. A. Mewhtnney, Health Phys., 39, 893, 1980; N. A. Oennis, H. M. 81auer
and 3. E. Kent, Health Phys. 42, 469, 1982).                       There ts Inadequate                  information        available         from
accidental     worker exposures to actual            yellowcake materials           to evaluate            the proposed procedures.
It remains, then, to be shown how such information                    derived from experiments conducted in vttro                             can
be used to predict       the behavior of uranlum inhaled by a mill                 worker.
       The most important       problem addressed tn this project               is the protection               of uranium mill        workers
from occupational          exposure to uranium,          specifically          through     bioassay         programs to assess the
adequacy of worker protection.                 An additional        consideration          is the assessment of accidental
exposures      of workers       and use of results           to re-evaluate         and modify            protectlon          programs,        if
necessary.
       This report    presents results        of research conducted between April                   1, 1982, and March 31, 1983,
and includes      individual      papers prepared in several            areas of research:                (1)     use of a quantitative
analytical  method to measure the variability   in ammonium diuranate  and U3O contents
                                                                                 8         of
yellowcake samples a worker might be exposed to; (2) the results of two short-term studies
rats: one designed to relate the metabolism of yelloucake    delivered via a simulated wound
contamination        for comparison to results          of earlier      yellowcake inhalation                   studies,     and the second
to investlgate the effect of caging on the toxicity of Implanted yellowcake; (3) the relationship
of the amount of ammoniumdiuranate in aerosols inhaled by Beagle dogs to biochemical indicators
of kidney toxicity.
       This format reflects        progress tn the middle stage of a five-phase                          approach to the objectives
of the project. First, ltmtted sampling during milling operations was conducted to determine the
properties of aerosols that a worker might inhale. The results were related to specific packaging
steps     and led to predictions            of appreciable        upper respiratory             tract     deposition          rates    for    the
aerosols,      tf tnhaled.     Second, laboratory        analysis     of yellowcake by infrared                   spectroscopy was used
to quantify      the range of composition           variability       of commercial yellowcake                    and to illustrate           the
use of such results        tn interpreting       the results      of animal studies or human bioassay data.
    Third,   short-term        tnhalatlon      studies     using laboratory         rats     exposed to selected       yellowcake
powders were completed.           A study designed          to investigate          the in vlvo        behavior   of yellowcake
deposited in a wound has shown that 49% of the body burden was cleared with a half-time    (T1/2)
of 0.3 days regardless of solubllity. The remainder of the more soluble yellowcake cleared with a
T1/2 : 11 days,       and the less soluble               form cleared       with    a T1/2 = 30 days. Both values            were
more rapid than the half-times   for clearance of inhaled yellowcake from lungs of rats (T1/2
~ 130 days). The retention behavior of implanted yellowcake in rats could not be quantitatively
related   to yellowcake        composition,       as could the retention            of inhaled      yellowcake.   An addltional
study on the effects of animal caging on the response of rats to nephrotoxicity   showed that for
studies requiring excreta collections, a minimum of 21 days should be provided for acclimation to
metabolism       cages before     exposure.       This should ensure that              water consumption and nephrotoxlc
response will     be similar     to that of animals housed in polycarbonate cages.
    A Z-year study of yellowcake               aerosols     from two uranium mtlls               was continued.   Aerosols were
generated from a sample of yellowcake that was 100% ammoniumdturanate                                (a more soluble    form) and
> 99~ U308 (a less soluble              form).   Twenty dogs exposed to the more soluble     yellowcake
received an estimated Initial            lung burden of 130 ± 9 ggU/kg body weight. Twenty dogs received
an estimated   initial  lung            burden of 140 ± 7 ugU/kg of an aerosol       of a less soluble
yellowcake. Biochemical indicators of kidney dysfunction that appeared in blood and urine showed
elevated levels that returned to normal within 16 days after exposure.
    In the fourth      phase, results         of the animal studies          will   be used in future         dose estimates and
hazard evaluations         of milling       effluents.     Distribution,        retention,        and excretion   data from the
2-year studies      wtll    be compared with the physical               chemistry      results      to identify   the important
yellowcake physical        properties   related      to blological         behavior.    Fifth,     data from the 2-year animal
studies   will     be compared with available              human data and incorporated                 into   improved bloassay
procedures as needed.
                              alphabetically thls project.Identifying
    Uraniummills are identified            in                       letterswere assigned
to each mill (Mills A throughF) in the order we obtainedtheir productsand do not relate to the
                               or
name of the mill, its location, the parent company.
                                  1.      INFRAREDANALYSIS OF REFINED URANIUMORE


                         In
Abstract--The varlab111ty chemlcalcoE~osltlon
and solub111tgof coBDerclalgellowcakeproducts                                    PRI#CIPALINVESTIGATOR
                             of
comp11catesthe Interpretatlon bloassay data.                                             A.F. E1dson
QuantltatlveInfraredanal~slswas used to measure
the relatlve percentages of ammonlum dluranate and %08 ~n commerclal gellowcake samples and
to estlmaCe the hounds of gellowcake composltlon varlabllltg.Rnalgsls of standard mlxtures
showed that the ammonium dluranate in a mixture could be estimated within ± 7~ of the mixture
and g308 could be estimated within 13~.                              Solutions to analgtlcal difficulties such as
overlappingspectralbands and sample varlabllltgare discussed.              for
                                                             Recommendations establlshlng
infraredanalysis         at                are
                procedures otherlaboratories given.

        Uranium ore is refined          into a commercial product known as yellowcake,                    which is packaged as a
dry powder for            storage and shipment. Previous studies             of airbone dust from yellowcake packaging
operations        (Ref.    1.1) have shown that,         if   inhaled,   the majority      of airborne        yellowcake In uranium
mills     is    likely     to deposit     in the nasopharyngea]            region.      Such particles,          if   insoluble,    are
cleared        rapidly    and excreted via the gastrointestinal              tract.      Material    deposited in the pulmonary
region is cleared by solubilization                   and excreted in urine,          or retained    in the lung, from which it
is slowly         cleared.    The two major uranium compounds in yellowcake                         are ammonium diuranate          and
U308. Because ammonium diuranate is the more soluble of the two forms (Ref. 1.2), and
absorbed and excreted in urine, chemical toxicity to kidney and deposition in bone are possible.
However, the gradual accumulation   of relatively insoluble  U308 in lung might deliver                                              an
appreciable annual radiation dose to lung. Knowledge of the relative ammonium dluranate                                             and
U308 content of yellowcake is required to predict                           the possible       target         organs and nature      of
potential health effects from tnha]ed yellowcake.
        Routine btoassay procedures are used to ensure that workers who might be exposed during their
work do not accumulate undue amounts of internally                        deposited uranium. High variability                 has been
observed         In dissolution         rates   for     yellowcake       samples from four           mills,      complicating       the
interpretation  of bioassay data (Ref.                    1.2). In vitro dissolution studles (Refs. 1.2-1.4)                         are
useful for studies of a few selected                      samples, such as a sample taken from a production                         lot
involved in an accident, but they are tmpracttca] for use in a survey of yel]owcake samples (Ref.
1.5). It is necessary to estimate the bounds of yel]owcake variability     to interpret btoassay
data, either        as part of routine      monitoring or In evaluation           of accldenta]       exposures.
    It has been shown that the more soluble percentage of a yellowcake sample can be estimated by
quantitative infrared analysis (Ref. 1.2). Yellowcake contains ammonium dturanate as a mixture
a UO3-NH3-H20 adducts and their      thermal conversion   product U308 (Ref. 1.2) along with
restdues from the mllltng process (Ref. 1.3, 1.4). Although yellowcake ts not strictly   a binary
mixture, the two oxide forms of uranium predominate. Figure 1.1 shows the infrared spectrum of a
commercial yellowcake sample before and after                     heattng at 150"C for 16 h and Illustrates                 the change
that takes place In the infrared                spectrum of yellowcake as the ammoniumdturanate ts converted to
U308 upon heating.
   The objectives            of this    study are to Illustrate           a method to determine the variability                    among
yellowcake lots,           to define the assumptions involved,             and to define the ltmtts             of application.     The
approach used quantitative               infrared      analysts    of known mtxtures         of ammonium dluranate            and U308


                                                                    3
                   2.0

               A
               B 1.6
               S
               0 1.2
               n                 UNHEATED                HEATED
               B                  U022+                  U308
               A 0.8


                   0.4
               E
                    0       I       I       I        I     I    I     I
                    1000   950    900      850    800     750   700   650   600
                                                    -1
                                                 CM


Figure 1.1 Infraredspectrumof yellowcakepowder obtalnedfrom H111B showlng the appearanceof
a U308 band and the partlal reductlon In the UO2 +2 Intenslty upon heatlng at 150°C for
15 h. Thls 111ustrates                     of
                      the theme1 converslon the UO3 In ammonlumdluranateto U30
                                                    B.



produced In the laboratory. The analysls of such standard mlxtures was used to deflne the
accuracy and preclslon of the technlque.Then It was applled to unknown commercialsamples. Two
major dlfflcultleswere encountered. First was varlab111tyIn ammonlum dluranateforms that are
producedIn operatlngm111s (and even from the same m111); and the second was overlapplngammonlum
dluranateand U308 Infraredbands (Fig. 1.1). Solutlonsto these problemsare dlscussed.


                                        MATERIALS AND METHODS


    Ammonlum dluranate was prepared In the laboratoryby dropwlseaddltlon of 1SX aqueous NH40H
to an aqueous solutlon of UO2(N03) wlth stlrrlng at pH 7.0-?.5 at room temperature. The
                                   2
resultlngyellow preclpltatewas stlrredfor 15 h, f11tered,washed wlth cold water and acetone,
and alr-drled at room temperature.Eleven known mlxtures of thls ammonlum dluranate and U308
(Natlonal Lead Company, Clnnlnatl, OH) were prepared, wlth O~ ammonlum dluranate (pure U3OB)
through I00~ ammonlum dluranate (no U308 present) In IOX Intervals. The mlxtures were ground
                                                                     Company, Chlcago, IL).
In an agate vlal uslng a W1g1-Bug shaker (Cresent Oental Manufacturlng
grlndlngtlme of 10 mln provldedmaxlmumInfraredabsorbancefor each component.Welgheda11quots
                                               spectralgrade KBr to preparemlxturesthat were
of the mlxtureswere added to 1.0 g of deslccated
0.3Z, O.SX, and 1.0~ by welght. The ammonium dluranate + U3% + KBr mlxtures were also ground
for 10 mln.
    Pellets were preparedby presslng200 mg of the ground mlxtureat 2000 psl for 5 mln uslng a
hydraullc press (Fred S. Carver, Inc., Summlt, N3). Oupllcate pellets were made for each
mlxture.Pelletsof KBr alone were made slmllarly.
    Yellowcakesamples were obtalnedfrom slx commerclalm111s (deslgnated  M111 A - M111 F), and
pellets that contalned 0.3S sample In KBr were prepared as above. There was no pretreatmentof
         samplesbefore grlndlng
yellowcake                     wlth KBr.
   Measurements were obtalned from a11 KBr pellets uslng a Perkln-E1merModel 283B Infrared
spectrophotometer equlpped wlth a mlcroprocessor-controlledunlt for quantltatlveanaiyses of
mlxtures                     law.
        uslng the Beer-Lambert



                                                 4
    Figure 1.2 shows superimposed spectra of pure ammonium dturanate and pure U308 on the same
axes. The wavelengths chosen for analysts were taken from literature  values (Ref. 1.6). The 925-
cm-1 peak and the 735-cm -1 peak were assigned            to ammonium diuranate        and U308,
respectively. The two baseline points (970 and 636 cm  -1) were chosen as the relative   minima of
the pure samples. These relative minima were also appropriate for the unknown samples measured to
date. The 852-cm-1 peak was chosen for the KBr absorption peak. An attempt was made to use the
minimum value (noted         by the crossover           of the two spectra        as the KBr absorbance).      Although this
might be preferred, the absorbance at the crossover point was occasionally less then the baseltne
determined by the 970-cm -1 and 636-cm -1 peaks. The 852-cm -1 value was chosen so that the
errors caused would probably be overestimated.
    One of the duplicate standard pellets of each known ammonium diuranate + U308 mixture was
selected randomly. The absorbances of these pellets were used to obtain the absorbtlvlty matrix

ai, J (Eq. 1.1);
                                                      AI, = al, x Cj, x b
                                                         k     j     k                                               (Eq. I.I)

where:    Ai, = absorbance wavenumber (cm
                         at         I    -1) of mixturek,
             k
                             at           I              j
          ai, = absorptivity wavenumber of components of mixturek,
             j
          Cj, = concentration
             k                                 J
                              (w%) of components of mixturek in KBr,
          b = constant                 =
                      pellet thickness 0.052 ± O.OOl cm.


    The remaining        pellets    of each pair were analyzed as unknowns. The process was then reversed
to provide   an analysis        of all    standard pellets          as If   they were unknowns. The concentrations         of
components In unknown yellowcake mixtures were then calculated                            using the absorbance spectrum and
the absorptivity       matrix as derived above.




                       0.5

                                                STANDARD MIXTURES IN KBr
                   A 0.4
                   S                     0.3%
                   S                     ADU
                   0 0.3                                                      0.3%
                   n                                                         u3~
                                         925
                   B
                   A 0.2
                   N
                                         /~                                   735

                   C o:i
                   E
                         O
                         1000      950         900       850 800            750     700      650    600
                                                                    -1
                                                               CM




Figure 1.2 Superimposed             infrared         spectra   of the 0.3% ammonium dluranate             standard   and 0.3~
U308 standard.



                                                                5
                                                    RESULTS


   Figure 1.3 shows the results of the known ammonium diuranate + U308 mixture analyses.
Individualcalculatedvalues from repeated analysesare plotted versus known values relativeto
the theoritical                                                          of
               line shown. A point above the line indicatesan overestimate the percentageof
ammonium diuranate or U308 in the mixture. The points at 0% ammonium diuranate on Graph A and
I00% U30 on Graph B represent analysis of the same pellets. Note that the method results in
        B
an underestimate for pure U30B, and the value for pure ammonium diuranate is less precise.
The accuracy and precision of the results were greatest for mixtures containing I0% to g0%
ammonium diuranate and 30% to 70% U308. Given the above accuracy and precision of results of
standard mixture analyses, results of unknown analyses showing S 20% ammonium diuranate or
U308 might indicate that the unknown mixture actually contained only ammonium diuranate or
U308¯



                       A
                120

              A
              N loo-
              A
              L
              Y eo-
              Z
              E
              0 So-

              W
              T 4o-
              X

              A
              D 2o-
              U




                                   20          40             SO         SO   1OO

                                        KNOWN WT% AMMONIUM   OIURANATE




                                                                     8   o
                           B
                 .....o

                                                                               o
                                                                               o
                                               o      0


                  4O-2o,                  ~0
                               o



                               o
                               0

                                    I           I              I          f
                                   ~o          40             8o         so   too

                                                KNOWN WT% U3OB




Figure 1.3 Standard ammonium diuranate+ U30 mixtures were analyzed as if they were unknowns.
                                           B
Total concentration of the mixtures in KBr was 0.3 wt %. Graph A shows ammonium diuranate
results,graphB shows U30 results.
                        B


                                                       6
   Table 1.1 shows a summary of analyses of the II standard mixtures where the total
            of
concentration uraniumcompoundsin KBr was 0.3%, 0.5%, and 1.0%. The data are expressedas the
         betweenthe analyzedand known amounts,such that a perfectlyaccurateand reproducable
difference
result would be 0.0 ± 0.0. The accuracy of the results shows that ammonium diuranate and
U308 were generally overestimated, but not significantlyso, when compared with the precision
of the estimates.The number of analyses(22) reflectsthe analysisof duplicatepelletsfrom
mixtures.Standarderrors expressedin this way includethe relatively large errors seen for the
extremesof the concentrationrange (Fig. 1.3). Analysesof 0.3% pellets were shown to be more
precisethan those of 0.5% pellets. The precisionof the 1.0% pellet analyses was comparableto
that of the 0.3% pellets;however,the 0.3% pelletsgave more preciseresultsin the middleof the
concentrationrange. This was assumed to be caused by decreased absorbance in the region of
spectraloverlap.
   After the 0.3% pellets were chosen for routine use in the later analysis of unknowns, the
standards were rescanned and analyzed four times during subsequentwork. The results of these
analyses were shown in Table l.l with n = B. The standard error values were greater but more
reliablefor routinework becausethey include possibleinstrumentalerror factors and possible
changes in the standard pellets with time. The results of these four seperate scans and the
        of
analyses the 0.3% pelletsare shown in Fig. 1.3.
   The spectrum shown in Fig. 1.4 illustrates a typical commercial unknown sample. Note the
broad ammonium diuranate peak that overlaps considerably with the U30B peak. The initial
analyticalapproachwas to assume that any unknownsample was a mixtureof ammoniumdiuranateand
U308. If the results suggested the yellowcake might be a pure form of either ammonium
diuranate or U30B and the spectrum appeared to be that of a pure form, the sample was
reanalyzedusing a calibrationcurve based on the pellets that containedonly 0.3%, 0.5% or 1.0%
ammonium diuranate or U30B. Analyses of suspected pure samples were based on peak areas
rather than peak maximumabsorbance.This was especiallynecessaryin the case of pure ammonium
diuranatesamples (Fig. 1.5). The spectrashown in Fig. 1.5 illustratethe variabilitypossible
among grab samples from two drums from Lot #55 produced by Mill E. Note the absence of a U30^
~-
band. The two spectral bands in the Drum #42 sample can be assigned to peaks from U02
(Ref. 1.6). These spectraillustratethe most extreme case of this type of variabilityfound
date. Clearly,analysisof this sampleshould use peak area ratherthan peak height methods.




                                          Tablel.l
                                          of
               Analysisof StandardMixtures AmmoniumDiuranateand U30 in KBr
                                                                   B

                                Deviation        %
                                         (Analyzed - KnownWt % in Mixture)
                                                   Mean ± SE (n)
          Wt % Mixture
             in KBr                  AmmoniumDluranate                  U308

              0.3                      0.43 ± 2.6 (22)             0.51 ± 6.3 (22)
              0.3                      0.07 ± 6.6 (B)              -0.03 ± 12.5 (8)
              0.5                       3.3 + 2.8 (22)             1.3 + 4.6 (22)
              1.0                       0.I ± 3.2 (22)             0.2 ± 3.8 (22)



                                             7
                    0,6 ,



                A   0.4
                S
                $   0.3
                0
                R
                 S 0.2
                A
                N
                 C 0.1
                E
                      0           I,           I    I         I      I     I         I
                      1000       950      900      850        800    750       700       650   6OO
                                                             CM -1


              Figure 1.4 Infraredspectrumof yellowcakesample obtainedfrom Mill D.




                    0.5
                                                         LOT 55
                    0.4


                ~
                    0.3    -

                ~
                    0 2        DRUM    3~.,,~/~          DRUM42




                      ot         t         I       t          I      I         I         I
                     1000       950      900       850     800       750   700       650       600
                                                             -1
                                                          CM



Figure 1.5 Infraredspectrumof yellowcakesamples taken from two drums from Lot #55 producedby
Mlll E.



   Table 1.2 summarizes analyses of samples taken from the mllls studied to date. MIll A
                                               to
samples,shown In Table 1.2, might be considered be pure ammoniumdluranateand were reanalyzed
as described. The sample from Mlll E was analyzed to contain > I00% ammonium diuranate, an
unreasonableresult. The calculated value for U308 might be artifactual when compared to the
error in the ammonium dluranate result. Analysls as a pure ammonium dluranate sample indicated
this was the case. The Mill B sample also showed an unreasonableresult for ammonium diuranate
analysis.Analysis as a pure sample dld not resolve the question,and the sample was considered
to contain 86 ± 5% ammonium dluranate, wlth the remainder U308. Analysis of the Mill C
sample suggested it might contain only pure U308; however, the spectrum showed a small


                                                         8
ammonium diuranate absorbence peak. Thus, reanalysls as a pure U308 sample was not
considered. No sample from any mill studied to date could be considered to be pure U308; each
suspectedpure sample containeda fractionof ammoniumdiuranate,  showingthat thermalconversion
to U308 was not complete.

                                            Table1.2
                                            Samplesof UnknownComposition
                        Analysisof Yellowcake
                            From Six MillsAs Mixturesand Pure Components


                                      Wt %    (Mean ± SE)
     Mill                     MixtureAnalysis          Analysisas Pure Component
                              ADU        U30
                                         B             ADU                 U308
      A                      a ± lO
                             53           a ± 2
                                          l                90 ± 20
      B                     a
                            Ill ± 4      16 ± 3            86 ± 5
      C                      a ± I
                             17          56 ± l            Not a pure spectrum
      D                      25 ± 2      71 ± 2
      E                     a
                            129 ± 5      a ± 2
                                         25                99 ± 6
      F                     49 ± 3       53 ± 4

avaluesare within the ± 20% uncertaintyfor mixtureanalysis.Sampleswere reanalyzed
                 after inspection the spectrum.
as pure components              of



     Note that samples, such as that shown for Mill C, which indicate less than 100% combined
ammonium diuranate + U30B, might occur in practice. Other species from the milling process
might represent up to 6% of the final product (Ref. 1.3, 1.4), and residual water and 3 can
also be present. A sample that might be incompletelydried or refined and not meet production
            might still be inhaledby a workerand requireanalysis.
 requirements
     Table 1.3 summarizesthe range in ammoniumdluranatecontentof differentlots from one mill.
                                     among productsfrom a single mill (Fig 1.5) Just as there
 It is clear that there is variability
among products from differentmills. The greatestvariabilitywas observedin Mill D yellowcake;
the most constantammoniumdiuranate         was in Mill F yellowcake.
                                  percentage


                                          Table 1.3
              Analysisof UnknownSamplesObtainedFrom DifferentLots From Each Nlll


                                                        Wt ~ ADU (Mean ± SE)
                  Mi11                              Mi__n_n                 Max
                    B                             77 ± 5                 lO0 ± lO
                    C                              1 ±l                   17+_ 1
                    D                              4±I                    55_+ 3
                    E                             63 ± 6                 I00 ± I0
                    F                             46 ± 3                  64 ± 4


                                             DISCUSSION

     These resultsillustratethe use of infraredanalysisto accuratelyanalyzeammoniumdiuranate
in   the presence of U30B to within ± 7% standard error of the mean. Similarly, the U308
content can be analyzed accurately to within ± 13%. These accuracy and precision values are
well within the variabilityin yellowcakecompositionobserved for lots produced by different
millsand for lots producedwithin the same mill at differenttimes.
      The followingactionsare recommendedfor those wishingto apply this techniqueto yellowcake
analysis:
    l. Standard and unknown pellets should be scanned at the same time or without turning off
                         and the instrument
           the instrument,                                   warmedup.
                                          should be thoroughly
      2.   A dried but unheatedsample of yellowcakeprecipitatefrom each facilityshould be used
                                       standard.
           as the pure ammoniumdiuranate
      3.   If experience                              lots from one mill are especially
                        shows that spectraof successive                               variable
           (see Fig. 1.5), standard mixtures of U308 and each ammonium diuranate form should
           considered.
      4.   A collectionof such standard mixtures and analyticalresults should be maintainedto
           determine                     in              of         from the facility.
                    the overallvariability the composition yellowcake


                                            REFERENCES


l.l A. F. Eidson and E. G. Damon, "Predicted DepositionRates of Uranium YellowcakeAerosols
    Sampledin UraniumMills," HealthPhysics(in press).

1.2 A. F. Eidson and 3. A. Mewhinney, "In Vitro Solubility of Yellowcake Samples From Four
    UraniumMills and the Implicationsfor BioassayInterpretation,"Health Physics39, 893-902
    (1980).
1.3 D. R. Kalkwarf, "Solubility Classification of Airborne Products From Uranium Ores and
    TailingsPiles,"NUREG/CR-0530,PNL-2BTO,1979.

1.4 N. A. Dennis,H. M. Blauer,and 3. E. Kent, "DissolutionFractionsand Half-Timesof Single
                    in
    Source Yellowcake Simulated Lung Fluids,"Health Physics42, 469-477(1982).

1.5 A. F. Eidson and W. C. Griffith,Jr., "Techniquesfor Yellowcake          StudiesIn Vitro
                                                                  Dissolution
    and TheirUse in Bioassay Interpretation,"HealthPhysics(in press).

1.6    A. M. Deane, "The Infra-red Spectra and Structure of Some Hydrated Uranium Trioxides and
       AmmoniumDiuranates,"Journalof Inorganic and NuclearChemistry21, 238 (1961).




                                               10
        2.   RETENTION OF   URANIUM FROM                               BY
                                           SIMULATED WOUNDS CONTAMINATED    YELLOWCAKE

                           and
Abstract-- The translocatlon retentionof uranium
from two dlfferentgellowcakesamplessubcutaneously               PRINCIPALINVESTIGATORS
                                        of
implantedin rats to slmulatecontamlnatlon wounds                     E.G. Damon
was assessed.    Forty-flwerats, anesthetlzedwith                    A.F. Eldson
halothane,                  implantedwlth powders
          were subcutaneously
of each of the two yellowcake                                                     in
                             samplesat a dose of 10 rag U/kg. Rats were sacrificed groups of
5 at intervals through 32 days after implantation.Selected tlssues and excreta samples were
assayed by fluorometryto determine their uranium content. Two-componentnegative exponential
functions were fitted to the uranium body burdens expressed as percentages of the inltla119
implanteduraniumbody burdens.For both yellowcakesamples,45% of the initialbody burden (IBB)
cleared with T1/2 of 0.2 days. The remaining 55~ of the IBB cleared with T1/2 of 10 days for
the first yellowcakesample and 28 days for the second sample. Resultsof prior studies of lung
         of
clearance uraniumfrom inhaledaerosolsof the two gellowcakesamplesshowed an early clearance
component(TI/2 = 1 day) that correlatedwith the ADU percentageand a late clearancecomponent
(TI/2 = 180 days), which corresponded to the U308 percentage composition of the inhaled
                                                  implantedyellowcakewas more rapid than
yellowcake.Thus uraniumclearancefrom subcutaneously
               from yellowcake
uraniumclearance                     In
                             deposited lung.


    Uranium mill workers may be exposed to uranium compounds by inhalation, ingestion,wound
             or
contamination, by absorptionfrom the eyes or mucous membranes.An earlierwork describedthe
                                of
lung retention and translocation uranium from inhaled yellowcakeaerosols (Ref. 2.1). This
report presents results of studies of whole-bodyretention and translocationof uranium after
           implantation yellowcake
subcutaneous          of                                               of
                                 powder in rats to simulatecontamination wounds.
    Althoughtoxicologicstudieson dermal applicationof uranium compoundshave been conducted
                      of        powder or uraniumcompoundsfrom contaminated
(Ref. 2.2), absorption yellowcake                                         woundshas not
been investigated.The objective of the study was to determine the rate of absorptionand the
patterns of retention,translocation,and excretion in rats exposed to one of two samples of
yellowcake powder implanted subcutaneously.The two yellowcakesamples differed widely in In
vitro solubility(Ref. 2.3). This report presentsdata on the patternof whole-bodyretention
uraniumas related to the compositionof the implantedyellowcakepowder. Data on retentionof
uranium at the site of implantationand translocationof uranium to kidneys and bone and the
       of              of
pattern urinaryexcretion uraniumare also presented.


                                    MATERIALS AND METHODS


         Powders
Yellowcake


    Two yellowcakepowderswith known solubilitypropertieswere selectedfor these studies.One
powder obtained from Mill A contained -82% of its total uranium in a soluble form, ammonium
diuranate (ADU), and ~18% U30B, a relatively insoluble compound. The second powder, from
Mill D, was composed of -25% ADU and 75% U308. These powders were chosen to provide data
             with the resultsof in vIvo studiesusing rats exposedby inhalation aerosolsof
for comparison                                                               to
the same two materials(Ref.2.1).
 Anlmals


                                                                 rats, I0-12 weeks of age, were
        Fifty male F-344, specific pathogen-free,laboratory-reared
 initiallyselectedfor each yellowcakesample used. The mean ± l S.E.M. of the body weights was
 260 ± 4 g. Rats were fed Lab Blox (Allied Mills, Chicago, IL) and watered ad libltum. Water
                                                                  in
 bottleswere changedtwo times a week. Rats were housed individually polycarbonate cages (45
 25 x 20 cm) containing a bedding of wood chips or aspen wood shavings (American Excelsior,
 Oshkosh, WI). All cages were changed weekly. Four rats implanted with each material were
 individually housed in stainless steel wire-mesh-bottom cages (18 x IB x 25 cm, Wahman’s
 Manufacturing Co., Timonlum, MD) during the time excreta were collected. After excreta
 collection, the rats were individually housed in polycarbonate cages. Animal rooms were
 maintainedon a 12-h light cycle (6 a.m. to 6 p.m.) at temperaturesof 20-23°C and a relative
 humidity 30 to 50%.
         of


          Implantations
 Yellowcake


        Anesthesiawas inducedin rats with a 5% mixtureof halothane(Halocarbon            Inc.,
                                                                             Laboratories,
Hackensack,NJ) vaporizedin 95% 02 at a flow rate of 0.6 L/min and maintainedwith 2% halothane
             by
administered a face mask. The hair was clippedfrom the dorsal thoracicarea and an incisionl
cm long was made on the dorsal midllne between the scapulae. The dorsal midline was chosen for
the implantto preventthe rat from removingthe sutures during grooming.Yellowcake(lO mg U/kg
                                          and the skin was suturedwith VETAFILBENGEN ¯ (S.
of body weight)was implantedsubcutaneously,
Jackson,Inc., Washington,DC). The incisionwas then sprayedwith Aeroplast¯ spray-on plastic
dressing (Parke-Oavisand Co., Greenwood,SC). Selectionof the dose was based on the following
assumptions:
    (1) Absorption          of a subcutaneous implant          of a soluble           uranium powder was expected to be
greater      than that of an lntraperitoneal           injection        of an aqueous uranium solution.               Haven and Hodge
(Ref. 2.4) reported an LD50 value of 86 mg U/kg after                       48 h in 200-300 g male Wtstar rats after
tntraperttoneal         injection     of a 10% aqueous solution           of uranyl nitrate.          The dose used In our study
was selected to be approximately one-tenth of the LDso/4B h"
     (2) The materials used in these studies were expected to release                                uranium more slowly than
tntraperitoneal injection of solution.
        Implantations     of either     material    at a dose of lO mg U/kg were made in 45 rats.                        Five rats were
surgically      sham-Implanted and retained           as controls.        Because rats housed in metabolism cages died
after     implantation       wtth     the more soluble       yellowcake       (Ref.      2.5),       rats     dying   as a result
nephrotoxtcity      were replaced with rats implanted at the same dose level                           or at reduced dose levels
(5 or 3 mg U/kg ).           An additional         15 rats   (tn    groups of 5) were also                   implanted      with Mill
yellowcake      at reduced doses of 3 or 1 mg U/kg and were housed In polycarbonate                                   cages. Uranium
retention     curves for these rats were compared to those for rats-implanted                               with the same yellowcake
at a dose of lO mcj U/kg to assess effects              of dose on retention           of the Mtll          A yellowcake.


                Schedule
ExcretaCollection


    Urine, feces, and cage-wash samples were collected                      from two of the rats tn each of the 16- and
32-day sacrifice         groups.    These samples were collected              daily     for   four     days after        Implantation.
Three-day composite collections   were then made weekly until sacrifice.  Excreta samples from two
of the control rats were collected on the same schedule as that of the 32-day sacrifice group.




                                                                   12
Sacrifice    Schedule


    At intervalsof 2 h, I, 2, 3, 4, 8, 16, and 32 days after implantation,rats were sacrificed
                                              injectionof 1 ml (50 mg) of sodiumpentobarbital,
in groups of five by meansof an intraperltoneal
                          by
followed by exsangulnatlon heart puncture. Rats housed in polycarbonatecages and implanted
with Mill A yellowcakeat reduced dose levels (I or 3 mg U/kg) were sacrificedat 8 or 16 days
afterimplantation.
    Rats were weighed before sacrifice, and the organs were weighed at necropsy. Fluorometric
                                        on
assayfor uranium(Ref. 2.1) was conducted the followingtissuesfrom each rat:
    I. Blood samplescollectedby heart punctureat sacrifice.
    2. Lung
    3. Kidney
    4.      Section of soft   tissue   surrounding     the site   of the implantation   (2 x 2 cm excised to the
            depth of the vertebral     column)
    5.       Liver
    6. Both femurs
    7. Remaining carcass and skin.
    A central section cut longitudinallyfrom one of the kidneys was preserved in I0% neutral
buffered                           studies.
        formalinfor histopathological


        of
Analysis RetentionData


    The uranium contents of the tissues were expressed as percentagesof the uranium implanted
         i.e.,the initialbody burden(IBB).
initially,
    Two-componentnegative exponentialfunctions (Eq.l) were fitted to the whole-bodyretention
data by a nonlinearleast squarestechnique(Ref.2.6):


                               % IBB(t) = Ale-O’693(t)/Tl
                                                        +A2e-O’693(t)/T2                                     (1)


where A1 and A2 are early and late retention components in percent, t is time after exposure
in days, and T l and T 2 are the clearance half-times for components A l and A     2,
respectively.Similar functions (Eq. l) were also fitted to the uranium retentiondata for the
soft tissue surroundingthe site where the yellowcake                         Single-component
                                                    was implanted(wound-site).
negative exponential functions (Eq. 2) were fitted to retention data for rats housed
polycarbonatecages and sacrificedat 8 or 16 days after implantationwith Mill A yellowcakeat
reduceddose levels(l or 3 mg U/kg).


                                                             -0 "6g3(t)/T
                                                     % IBB(t)= Ae                                            (2)

    Retentionfunctionsfor the two groups exposedto Mill A and Mill D yellowcakewere compared
to determine effects of composition of the implanted yellowcake powder on uranium retention.
                     of
Levels of significance differencesbetween uranium retentioncurves for the differentgroups
              by
were determined F-Tests(Ref. 2.6).
    Uranium content of kidneys, bone (femur), and the urinary excretion of uranium for rats
implanted with the two materials were compared by analyses of variance and significance of
differences                           by
           betweengroupswas determined F-Tests(Ref. 2.6).
                                             RESULTS


    The I0 mg U/kg dose was consideredlow enough that no acute biologicaleffectswere expected
                                     of
but high enoughthat:theconcentration uraniumin the tissuesof sacrificed    rats at times up to
                                                     procedures.
32 days after exposurecould be measuredby fluoromotric          However,rats exposedto the
Mill A yellowcake ~ the initial four rats housed in metabolism cages - died B days after
implantation.                  at
             Gross observations necropsyrevealed signs of uranium toxicityto the kidneys
(Ref. 2.5). Kidneysappearedpale, with mottledreddishcolorationand yellowishspeckling.None
of the rats exposedto the Mill A yellowcakeand housed in polycarbonatecages showed any acute
effects from the treatment. No acute effects were observed in rats exposed to the Mill D
yellowcake or in the sham-lmplanted control rats. The gross appearance of kidneys from the
surviving rats was normal when the rats were sacrificed 16 and 32 days after exposure. An
         to          the effectsof differences types of caging on the responseof rats to
experiment investigate                       in
                   in
uraniumis described the next paper in this report.


         Retention Uranium
Whole-Body        of


   Figure 2.1 shows the whole-bodyuraniumretentioncurves for the two yellowcakesamples,and
the parametersfor the retentionfunctionsare listed in Table 2.1. For both materials,45% IBB
cleared with a half-time of < l day, the remaining 55% IBB cleared with Tl/2 of lO days for
the Mill A yellowake and 28 days for Mill D. Whole-body retention functions for the two
yellowcake                        different < 0.005).
          sampleswere significantly         (P

                         100




                                                               MILL A


                                        I           I      !         I

                               0        10          20         30   LtO
                                   DAYS AFTER IMPLANTATION

 Figure 2.1. Whole-body uranium retention curves for rats receiving subcutaneousimplants of
 yellowcake                                         or
           from Mill A (lowercurve,data as triangles) Mill D" (uppercurve,data as circles).

                                  of
   Table 2.2 shows the distribution uraniumbetween the implantationsite (wound) and other
                kidney and bone) in rats sacrificed 8, or 32 days after implantation.
tissues(primarily                                 l,
   Figures 2.2-2.3 show comparisons of the uranium contents of kidneys and femurs of rats
sacrificed                 with the two yellowcake
          after implantation                      materials.Resultsof analysisof variance
of all of the kidney and femur data for rats sacrificed                                   with
                                                       from l to 32 days after implantation



                                               14
                                               Table 2.1
                        Uranium Retention in the Whole Body or at the Wound
                       Slte of Rats Implanted Subcutaneously with Yellowcake


                                                      a
                                                      RetentionParameters
                                     A
                                     1                 T
                                                       1            A
                                                                    2               T
                                                                                    2
Yellowcake Sample                    (~)              (days)        (~)            (days)
Mtll A:


     Whole Body                    45 + 3           0.2 ± 0.I     55 ± 3           I0 ± 1
     Wound site                    55+     3        0.2 ± 0.I     45±     3         8±   1
     b
     Lung                          70+     6        1.0 ± 0.3     30+     6        68 + 30


Mill D:


     Whole Body                    45 ± 4           0.2 ± 0.I     55 ± 4           28 ± 3
     Wound slte                    55 ± 3           0.2 ± 0.I     45 ± 3           30 ± 3
     b
     Lung                          15 ± 13          l.O ± 0.3     85 ± 13          68 ± 30



              -0.693(t)/T
                        1    -0.693(t)/T
                             2
a% IBB(t)= Ale            +A2e

bBasedon data from Ref. 2.1.
 Retentionfunctions                     samplesare significantly
                   for the two yellowcake                      differentfor both
 the whole body and the implantsite (P < 0.005).Retentionfunctionsfor lung were
             different
 significantly        from those for implantslte (p < 0.005).




                                           Table2.2
                  Uranium           After Subcutaneous
                         Distribution                           of
                                                     Implantation Yellowcake


Days After                               UraniumContent,Percentof ImplantedUranium
Exposure                         ImplantSite                                  OtherTissues
                         MILL A                 MILL D              MILL A                   MILL D
    l                   42 + 3                  48 + 5             12±2                      6±0.5
    8                   25 + 3                  38 + 3             12±2                      2±0.7
   32                    3 + 0.4                20 + 2              4±0.5                    3±0.2




                                                 16
                     10




                                     \\\~A
                                                            +
                                                            MILL D


                      0. |                              I            I
                             0       lO        20           30           ~0
                                 DAYS AFTER IMPLANTATION


Ftgure 2.2. Urantum content of kidneys of rats reclvtng tmp]ants of Mt]] A (dashed ]tne data as
triangles) or Htll D yellowcake (soltd 11ne, data as circles). Oata potnts are meanvalues
error bars tndlcate ± 1 S.E.N.




                    10




                                              MILL A




                                                            MILL D

                      O. 11.         ’        ’
                         0          lO       2O         ’30      ’ LtO

                                 DAYS AFTER IMPLANTATION



Ftgure 2.3. Uranium content of femurs In rats Implanted wtth Htll A (dashed 1the, data as
triangleS) or Mtll D yellowcake (solld 11ne, data as circles). 0ata potnts are meanvalues
error bars tndtcate± 1 S.E.N.
yellowcake are listed in Table 2.3. The mean uranium content of both kidneys and femurs was
            higher in rats Implantedwlth the Mlll A yellowcake
significantly                                                 than in those implantedwith the
Mill O material during the 32 days of the study (p < O.Ol for kidneys and p < O.O001 for
femurs).



                                              Table 2.3
                         Uranium Content of Kidneys, Femur, and Daily Urinary
                      Excretion of Uranium in Rats Receiving Yellowcake Implants


                                                    Uranium Content
                                                    Percent
                                                      of
                              Number               Implanted               Concentration,
        Yellowcake             of                   Uranium                   ~g U/g
          Sample              Samples            (Mean+ S.E.M.)            (Mean÷ S.E.M.)
        Mill A:
             Kidney             21               a
                                                 3.2 + 0.5                 34       ± a
                                                                                      5
             Femur              20               b
                                                 0.90 + 0.07                        b
                                                                                9.7 ± 0.84
             Urine              41               c
                                                 2.16 ± 0.40


        Mill D:
             Kidney             21               1.4 ± 0.2                 14       ±3
             Femur              24               0.25 ± 0.02                    2.1 ± 0.16
             Urine              25               0.65 ± 0.15

                     different
        asignificantly        from Mill O value (p < 0.005).
                     different
        bsignificantly        from Mill D value(p < O.O001).
                     different
        Csignificantly        from Mill D value(p = O.O1).




   Figure 2.4 presents a comparison of the clearance of uranium from the implant site to
clearanceof uraniumfrom the lungs of rats during the first 35 days after inhalationexposureto
the same two yellowcakesamples (Ref. 2.1) Uranium clearancefrom the wound site was more rapid
than f:’om lung for both yellowcakematerials.Retention functionsfor the curves are listed in
Table2.1.
    Figure 2.5 compares retention curves (Eq. 2) fitted to whole-body retention data for rats
                      cages and sacrificedat times from 8 to 16 days after implantationwith
housed in polycarbonate
Mill A yellowcake at a dose of lO mg U/kg with rats implanted at l or 3 mg U/kg. Whole-body
retentionfunctionsfor rats implantedwith Mill A yellowcakeat reduceddose levels (l or 3 mg
U/kg) were not significantly different (p = 0.3) from those for rats implanted with Mill
yellowcakeat a dose of lO mg U/kg. Thus, reduction in uranium dose did not significantlyalter
               rates.
uraniumclearance


                                              DISCUSSION


   Both whole-bodyand wound-siteuranium clearance half-timeswere significantlyshorter for
rats implanted with Mill A yellowcake than for those implanted with Mill D yellowcake




                                                  17
                              IMPLANT SITE                                                           LUNG

                                                                        z
                                                                        iii
        1°°°
        /                                                               a
                                                                        rr
                                                                        :3
                                                                        El
                                                                        (9
         100~
                                                                           .-I
                          o         t,_

                                                                        I-
                                                                         ra
                                                                        ii
                                                                        0
                                                                           I--
                                                                        z
                                                                        ILl
                                                                        0
                                                                        n"
                                                                        LU
          1.,         ,   i     i        i   i    i        i   i   i                 i       i   i   i       i   i   i         i   i
               0         12        24      36                                    0           12       24     36
                   DAYS AFTER IMPLANTATION                                                    DAYS AFTER
                       OF YELLOWCAKE                                                     INHALATION EXPOSURE

                                                                  of
Figure 2.4. Comparisonof clearanceof uranium from the implant-slte rats receiving
           implants yellowcake uranium
subcutaneous       of          to       clearance                     to
                                                 fromlungIn ratsexposed yellowcake
     by
powder inhalation(Ref.2.1).

               lO0-




         I
         M
         P
         L
         A                                                                                                i-i
         N                                                                                               []
         T
         E
         O

         U
         R
         A
         N
         I
         U
         M

                                     I                I                I                 I               I                I
                                    II                10               tR                14              SiS             ill

                                                 DAYS AFTER IMPLANTATION

Figure 2.5.Whole-body      retention
                     uranium         curvesfor ratsreceiving       of
                                                            implants MlllA yellowcake
                         llne)or at reduced
at a doseof 10 mg/kg(solid                dosesof l or 3 mg/kg      line).
                                                              (dashed


                                                                   18
(p < O.OOS). However, the differences                      between the retention             functions        for     the two materials
were due entlrely             to the second components (Table                 2.1).     Retention       half-times        for     the ftrst
components of the retentlon             functions      were not significantly           different     for the two materials.
        Excretlon         of urantum     In urlne       and translocatlon              of urantum        to   kidney      and bone was
slgnlftcantly            greater   (p<O.Ol) in rats Implanted with Rill                 A yellowcake than in those implanted
with Mill        O material.       Hence, retention        and translocatlon          of the Implanted uranlum depended upon
the chemical composltion   and thus the solubility   of the Implanted material.   However, the
clearance rates could not be quantitatively predlcted from the chemical composition of the two
yellowcake samples. Lung clearance of urantum tn rats exposed to aerosols of the two yellowcake
samples used in this               study were both quantitatively               and qualitatively         related       to the chemical
composltlon         and In vitro       solubility       of the yellowcake          samples In an earlier               work (Ref.         2.1).
Uranlum clearance            from the wound site         of rats Implanted            wtth elther      yellowcake sample was more
raptd than the lung clearance rates for rats exposed to these yellowcake aerosols by Inhalation.
    The difference between clearance of uranlum from wounds and from lung may be related to the
clearance         mechanisms involved.              Clearance    from a wound occurs mechanically                       by drainage         and
phagocytosis and nonmechantcally by dtssolutlon                      and translocatton.             Clearance from the resplratory
tract     occurs mechanically          (mucocillary        action   and phagocytosts by pulmonary macrophages) and
dlssolutlon.         The level      of phagocytic actlvlty          - clearlng     materlal     from a wound may or may not be
greater        in the rat than the acttvlty           of pulmonary macrophages clearing                material       from a healthy rat
lung.     It     ts not known whether the rate               of dissolution        of urantum deposited                in muscle t|ssue
dtffers        greatly    from the rate of dtssolutlon          of urantum tn lung.
        Data presented above indicate               that there Is a qualitative             correlation        between the amount of
uranlum retained  at the wound site                        and the content of insoluble   U308 In the implanted
yellowcake. However, the key questton                     is whether the Internal uptake and retention of uranium
reflect        the chemical        composition      and relative     solubillty        of the implanted             yellowcake.     Results
reported         here tndtcate       there     was greater      translocatton         of uranium to kidney and bone In rats
Implanted wtth the more soluble                 yellowcake sample. However, data on the clearance of uranium from
the wound stte could not be quantitatively      related   to the ADU and U308 composltion  of the
fmplanted yellowcake or the In vitro dissolution    data from the same yellowcake samples. Perhaps
mechanical clearance rates from the wound stte                      may have overwhelmed the effects                  of the dlfferences
tn chemical composltton and relattve solubilities of the two materials.
    Animal data presented tn thts paper tndlcate that wounds contaminated                                           with yellowcake         may
represent         a slgnlflcant        route    of entry     of uranium Into           the body. Those responsible                  for     the
protection        of the health and safety of urantum mtll              workers should be aware of thts potential                         rtsk.


                                                                 REFERENCES


2.1.      E. 6. Damon, A. F. Etdson, F. F. Hahn, W. C. 6rtfflth, 3r., and R. A. 6utlmette, "Comparison
          of Early Lung Clearance of Yellowcake Aerosols in Rats wtth In Vttro Dissolution        and
          Infrared Analysts,’ Health Phys., lg83 (in press).

2.2.      3. A. Orcutt, "The Toxicology of Compoundsof Uranium Following Application  to the Sktn,"
          The Pharmacology and Toxlcoloqv of Uranium Compounds, (C. Voegtltn and H.C. Hodge, eds.),
          pp. 377-414, NewYork (McGraw-Hill), 1949.

2.3.      A. F. Etdson and 3. A. Mewhtnney, ’In Vttro Solubility        of Yellowcake Samples from Four
          Urantum Mtlls and the Implications for Btoassay Interpretations,’     Health Phys. 3_g9:893-904,
          1980.

2.4       F. L. Haven and H. C. Hodge, ’Toxicity   Following the Parenteral Administration of Certatn
          Soluble Urantum Compounds," Pharmacology and Toxt¢oloqv of Uranium Compounds,Chapter 6, (C.
          Voegtltn and H. C. Hodge, eds.), pp. 281-308, McGraw-Hill Book Company,Inc,, NewYork, 1949.
2.5.   E. G. Damon, A. F. Eidson, and F. F. Hahn, "Acute Uranium Toxiclty         Resulting    from
       Subcutaneous Implantation   of Soluble Ye]]owcake Powder In Fischer-344 Rats," Blo]ogica]
       Characterization   of Radiation Exposure and Dose Estimates for Inhaled Uranlum Mtlltnn
       Effluents,                                                              LMF-94 Springfield,
                   Annual Progress Report, Aprll 1980-March 1981, NUREG/CR-2539,
       VA 22161, pp. 32-37, 1982.

2.6.   M. Ralston, "Oerivative-Free  Nonllnear Regresslon," BRDPStatistical   Software (W. J. Dixon
       ch.ed.), pp. 305-314, Berkley (Univ. Calif. Press), 1981.
         3.   EFFECT OF   ANIMAL CAGING ON             RESPONSE OF
                                             NEPHROTOXIC                RATS TO   URANIUM


Abstract -- Uranlummlllworkers may be exposedto
uranium compoundsby inhalation,ingestion,wound          PRINCIPALINVESTIGRTORS
             or            from eyes or mucous
contamination, by absorption                                E.G. Damon
membranes.Rats are currently being used in stu-             A.F. Eidson
                        and retentionof uranium
dies of the translocatlon                                   T.C. Marshall
from wounds contaminatedwith yellowcake(see pa-             F.F. Hahn
per no. 2, this report). Dose-response studies
were conductedto assess the effectsof two types of cages on the nephrotoxlcresponseof rats to
implanted yellowcake. The LDhO/21 days was 6 mg/kg (95% C.L. = 3-8 mg/kg) for rats housed in
metabolism cages beginning on the day of implantation(naive rats). However, rats housed
metabolismcages for 21 days before implantation(acclimatedrats) had an LDho/21 days of 360
mg/kg (95% C.L. = 220-650 mg/kg),which was the same value obtainedfor rats housed continuously
in polycarhonate cages. This significant difference (P<0.01) in response of "naive" rats
comparedto responseof "acclimated"rats was relatedto a significantlylower water consumption
by the naiverats.

     Rats are being used as an animal model to study the translocation
                                                                     and retentionof uranium
                       with yellowcake(see paper no. 2, this report).Rats are usuallyhoused
from wounds contaminated
                                                                                        cages,
in two types of cages during such studies.All rats are reared and housed in polycarbonate
but selected rats are placed in stalnless steel metabolism cages with wlre-mesh bottoms when
excreta are collected. Response of rats to uranium toxicity may be affected by environmental
factorsrelated to the type of animal caging used. For example,as noted in paper no. 2 of this
report, Fischer-344 rats reared in polycarbonate cages, then housed in metabolism cages
immediatelyafter subcutaneousimplantationof yellowcake(lO mg/kg body weight) consisting
~82% ammonium diuranate (ADU) and -18% U308, died from uranium toxicity. Identically
                      in           cages showed no overt toxic effects.This paper presents
exposedrats maintained polycarbonate
results of studies of the effects of two types of animal caging on the acute toxic response of
rats to uraniumfrom implantedyellowcake.The purposeof the study is to evaluateenvironmental
                                       responseof laboratory
factorsthat may affect the toxicological                    animalsto uranium.Such factors
                             the toxic responseof man to uranium.
may play a role in determining
     The study reported here presents a comparisonof the uranium toxicity after implantation
with yellowcake in rats housed only in polycarbonatecages (Poly Group), in metabolism cages
during a 21-day period of acclimation to these cages prior to implantation with yellowcake
(AcclimatedGroup), or housed in metabolismcages immediately                 with yellowcake
                                                           after implantation
(Naive Group). Responses of the three groups of rats to uranium toxicity are assessed for
correlationwith data on food and water consumption,changes in body weight, temperaturewithin
                                                                         with yellowcake.
the two cage types,and volumeof urinaryoutput beforeand after implantation


                                    MATERIALS AND METHODS


         Powders
Yellowcake


     Two yellowcakepowderscontalnlng the solublecompoundammoniumdiuranate(ADU) as the major
component were used in these studies, one powder obtained from Mill A contained ~82% ADU and
~18% U308 (Ref. 3.1). The second powder,from Mill B, was composedof ~I00% ADU.


                                             21
Animals


        One hundred twenty-two          male F-344, specific    pathogen-free,         laboratory-raised      rats,   10-12
weeks of age, with initial             body weights of 260 ± 4g (mean ± SEM) were used. Rats were weighed
twice      weekly     from    3 weeks before     implantation        until   death     or sacrifice        21 days after
fmplantatlon.         Rats were fed Lab Blox (Allied        Mills,      Chicago,     IL)   and water was provided        a
libitum.      Water bottles     were changed two times a week.
        All    rats   were Initially      housed individually        in polycarbonate        cages (45 x 25 x 20 cm)
containing      a bedding of aspen wood shavings (American Excelsior,                 Oshkosh, WI). Cage bedding was
changed and cages were washed weekly.              Anlmal rooms were maintained              on a 12-h light     cycle       at
temperatures of 20-22 ° C and a relative            humidity    of 40 to 60%. For details          of the animal care,
see Ref. 3.2.


Experimental Design


                          with yellowcake,
        Before implantation               rats were dividedinto three groups (Table 3.1). Group
                                     In
#1 (47 rats) were housed individually polycarbonatecages throughoutthe study. Group #2 (30
                             in                                                      Co.,
rats) were housed individually metabolismcages (18 x 18 x 25 cm, Wehman’sManufacturing
Timonium, MD) beginning on the day of implantationand until death or sacrifice 21 days after
implantation.                                          in
             Group #3 (45 rats) were housed individually metabolism cages from 21 days before
           untildeath or sacrifice days afterimplantation.
implantation                      21


                         Body Weight,
Waterand Food Consumption,           Volume of UrinaryOutput


        Water and food consumption were measured daily for I0 untreated rats in each cage type
                                     Body weights for these rats were measuredtwice per week
during a 21-day period of acclimation.
                                               the lO rats in each cage type were dividedinto
during this period.After 21 days of acclimation,
three subgroupsas follows:Four rats in each cage type were exposed to yellowcake(lO mg/kg)
           implantation,
subcutaneous                                                        as
                       two rats in each cage type were sham-implanted describedbelow, and
                                                                                     at
four rats in each cage type were retainedas cage controlsuntil 34 days of acclimation, which
time they were implanted with yellowcake at a dose of 20 mg/kg. Then, four naive rats were
                        at                                                          Water
implantedwith yellowcake a dose of lO mg/kg, and 4 naive controlswere sham-implanted.
consumptionand volume of urinaryoutput (measuredfor the rats in metabolismcages, Groups #2
#3) were measureddaily for rats In these subgroupsfrom the day of implantationuntil death or
          21
sacrifice days afterimplantation.


                        Measurements
Room and Cage Temperature

                       in                                                            measured
        The temperature the animal room and In two cages of each type was continuously
by thermistors(#4404, Omega EngineeringInc., Stanford, CT) and recorded with a five-channel
          recorder
strip-chart       (Tigraph                      Lubbock,
                          lO0, Texas Instruments,       TX).


         Implantations
Yellowcake


                                  implantedwlth Mill A or Mill B yellowcakepowder at doses listed
           Rats were subcutaneously
                                             These sham-implanted
in Table 3.1 or were surgicallysham-lmplanted.                  controlswere subjectedto
the anesthesiaand surgicalprocedures,but no yellowcakewas implanted.Anesthesiawas induced
in rats with a 4% mixtureof halothane(Halocarbon            Inc., Hackensack,
                                                Laboratories,                NJ) vaporized



                                                           22
                                            Table 3.1.
           Effects of Cage Type on Nephrotoxic Response of Rats to Implanted Yellowcake


                                             Group#I
                                                         Cages:
                                  47 Rats in Polycarbonate
                                                                              Mortality
                    Powder
           Yellowcake
                  Dose, mg Yellowcake                              No. Dead/      Per-    Time to
                                                                  No. Implanted cent       Death
 Source            per kg Body Weight
Controls                  None                                         0/2      0%
                           lO                                         0/4         0%
MILL A
                           14                                         O/l         0%
                            20                                        0/3         0%
                            36                                        O/l          0%
MILL B
                            46                                        O/l          0%
                            55                                        0/I          0%
                            65                                        0/I          0%
                            75                                        I/4         25%     II days

                            85                                        0/I         0%
                            89                                        0/1          0%
                           lOl                                        0/4          0%

                           120                                        0/4          0%
                           180                                        O/l          0%

                           270                                        O/1          0%
                           411                                        2/5         40%     18, 20 days

                           622                                        4/4        100%      4 - 1S days

                           760                                        4/4         100%     S days

                           876                                         2/2        100%     5 - 7 days

                         1,349                                         I/l        100%     5 days
                                              Group#2
                                              Cages the day of Implantation
                    30 Rats Placedin Metabolism
                          None                                    0/2       0%
Controls
                             3                                    0/4       0%
MILL A
                                                                       2/3         67%    ? days
                             5
                                                                      lO/ll        91%    7 - 8 days
                            lO
                            lO                                         5/7         71% 8 - 12 days
MILL B
                            2g                                         1/1        100% 8 days
                            98                                         I/I        100%    7 days

                            413                                        I/l        100%    6 days
                                                Table 3.1. (Continued)


                                                       Group #3
                    45 Rats Housed in Metabolism Cages More Than 21 Days Before Implantation

                            Powder
                   Yellowcake
                                                                                           Mortality
                          Dose, mg Yellowcake                                     No. Dead/    Per- Time to
   Source                 per kq Body Welqht
                                                                                ~0. Implanted    cent       Death
  Controls                         None                                              0/2         0%
  MILL A                            lO                                               0/4         0%
                                    20                                              0/4          0%
 MILL B                             25                                              0/4           0%
                                    28                                              1/4          25%    13 days
                                    46                                              O/1          0%
                                    52                                              1/4          25%    lO days
                                    66                                              O/1          0%
                                    75                                              0/1          0%
                                   121                                              0/I          0%
                                   411                                              O/l          22%    II - 14 days
                                   620                                              2/9          50%    5 - 7 days
                                   760                                              4/4         100%    6 - II days
                                   881                                              1/1         100%    6 days
                               1,3SO                                                1/1         100%    4 days




in 95% 02 at a flow rate of 0.6 L/mln and then maintained with 2% halothane via a face mask.
The hair was clippedfrom the dorsal thoracicarea and a l-cm long incisionwas made on the dorsal
midllne between the scapulae. The dorsal midllne was chosen for the implant to prevent the rat
                                                                            implanted,and
from removingthe suturesduring grooming.The yellowcakedose was subcutaneously
the skin was sutured with VETAFIL BENGENe (S. Jackson, Inc., Washington,DC). The incision was
                     spray-on
sprayedwith Aeroplaste                                 and Co., 6reenwood,
                             plasticdressing(Parke-Davls                 SC).
       Rats were observed daily for morbidity or mortality through 21 days after implantation.
Surviving rats were sacrificedby an Intraperitonealinjection of l ml of euthanasiasolution
(T-61, National LaboratoriesCorp., Somerville, N3) administered21 days after Implantatlon.
Necropsieswere performedon all rats, thoracicand abdominalviscerawere examinedgrossly, and
photographswere taken of the kidneys. Kidneyswere assayedfor uranium content by fluorometrlc
procedures (Ref. 3.3). A section of kidney from each rat was fixed In I0%, neutral-buffered
formalin, embedded in paraffin, sectloned at 6vm, stained with hematoxylin and eosln, and
        by              for hlstopathologlcal
examined light microscope                  alterations.


Analysis of Data


       Dose-response data for the three groups of rats (Table 3.1) were analyzed by probtt                  analysis
of the lethality      data (Ref.   3.4).   The following    probtt   equation    was used for    analysts    of the
        data:
mortality


                                               y=a +b log     x ,                                                 (1)


                                                       24
where y = mortality        at 21 days expressed in probit        units,   a = the intercept       constant,   b = the
slope constant,        and x = the Implanted dose of yellowcake (mg yellowcake/kg               of body weight).    The
LOG0/21 days and associated 95% confidence limits  (CL) were derived from the probtt regressions
and 95% ftduclals (Ref. 3.4).
      Food consumption, water consumption, and changes in body weight of rats in the three groups
(Table 3.1) were compared by analyses of variance,           and significance      of differences     between groups
was determined by F-tests        or by the Student’s    !-test    (Ref.   3.5).   Differences     were considered
be significant     if p<O.O5.


                                                       RESULTS


Mortality


                                                               cages (Group #1), naive rats
       Table 3.1 presentsmortalitydata for rats in polycarbonate
metabolism cages (Group #2), or acclimated rats in metabolism cages (Group #3). The LDso/2
l
days (6 mg/kg with 95% CL of 3-8 mg/kg) for naive rats housed in metabolism cages was
significantlylower than for rats housed in polycarbonatecages (LD50=342 mg/kg with 95% CL of
192-635 mg/kg) or for acclimatedrats housed in metabolismcages (LD50=444mg/kg with 95% CL
lgg-2047 mg/kg). The LD50/21 days value for rats housed in polycarbonate cages was not
significantlydifferent from the LD50/21 days for acclimatedrats housed in metabolism cages.
Therefore, data for these two groups of rats were combined to obtain the dose response curve
                 rats" for comparison the curve for "naive rats’shown in Figure3.1.
labeled"acclimated                  to




             98-




                   i                                                                                          ,


                                       10            1OO        1,OOO                                   10,OOO
                                              mg/kg BODY WEIGHT




Figure 3.1 Dose-response curves for rats housed in metabolism cages beginning on the day of
yellowcakeimplantation  (Naive Rats) or for rats housed in polycarbonatecages or in metabolism
cages beginning 21 days before yellowcakeimplantation(AcclimatedRats). LDGO values (and
confidencelimits) are for 21-day mortality.


                                                        25
Body Weight, Water, and Food Consumption


      Figure 3.2 summarizesthe body weight data for untreated rats housed in metabolismcages
                                       cages. The figure shows changesin mean body weight for
comparedto those housed in polycarbonate
lO rats in each group through 21 days after the rats were first placed in metabolismcages. At
the end of 21 days, six rats from each group were removedfrom this phase of the study for use in
          of               phase of the study. Body weight data shown in the figure for days
initiation the dose-response
21-34 are for four rats in each group. Rats placed in metabolismcages initiallylost weight,and
                                                               lower than for those housed in
the ultimategain in body weight for these rats was significantly
            cages throughout
polycarbonate              the period of observations.
                                       for the two groups of untreatedrats. Rats in metabolism
      Figure 3.3 shows water consumption
cages drank significantlyless water than those housed in polycarbonatecages throughout the
observations.Because of the increasing differencein body weight for the two groups of rats
                             was normalized body weight.When this was done, the normalized
(Figure3.2), water consumption             to
                of                                   different
water consumption the two groups was not significantly        after day 5.
      Food consumptionfor rats housed in metabolismcages initiallywas less than that of rats
housed in polycarbonate cages, but by day 3 food consumption for the two groups was not
significantlydifferent.However, even though food consumptionwas equal for the two groups of
rats from day 3 throughout the period of observations,rats in metabolismcages did not gain
weight as fast as those housed in polycarbonate cages. Figure 3.4 shows food consumption
         to
normalized body weight(g of food/kgof body weight)for the two groups of rats.
                          the water consumption
      Figure 3.5 summarizes                   data for four rats in each group through21 days
after implantationwith yellowcakeat a dose of lO mg/kg. The naive rats housed in metabolism
                        less water than acclimatedrats or rats housed in polycarbonate
cages drank significantly                                                             cages
                              Two of these four rats died with signs of uraniumnephrotoxicity
until day B after implantation.
8 or lO days after implantation.Data beyond day lO in this group are for the two surviving
rats. None of the rats in the other two groups implantedat this dose level (lO mg/kg) died. All
three groups of rats drank more water after implantationwith yellowcake.Water consumptionin
the surviving            a
             rats reached peak at about day 8 or 9 afterimplantation.
      Figure 3.6 shows the volume of urine excreted by surviving rats during the 21-day period
after implantation for four naive rats, and four acclimated rats implanted with lO mg of
yellowcake/kgand their sham-4mplantedcontrols. This figure also shows the volume of urinary
output for fbur acclimated                                         Urinary output by all of
                          rats implantedwith 20 mg of yellowcake/kg.
the treated rats increased significantly(P<O.OOl) above that of the sham-implantedcontrols
during the first 2 weeks after implantation.Urinary output by acclimatedrats implanted with
yellowcakeat a dose of lO mg.kg was significantly  greater than that of naive rats implantedat
this same dose level. However,the volume of urine excretedby naive rats survivinglonger than 8
days rose above that of the acclimatedrats during the lO- to I8-day period after implantation.
Urinary output of acclimated rats implanted with yellowcake at a dose of 20 mg/kg was not
            different
significantly                                   at
                     from that of rats implanted a dose of lO mg/kg.
                                                                                  cages was
      Reducedtoleranceto uraniumtoxicityexhibitedby naive rats housed in metabolism
related to reduced water consumptionby these rats (Figure 3.5) during the first 4 days after
yellowcakeimplantation,coincidentwith the peak nephrotoxiceffect of the implanted uranium.
Rats housed in polycarbonatecages or rats acclimated to metabolismcages for 21 days before
yellowcakeimplantationconsumed significantlymore water during this time than the naive rats
(Figure3.5).




                                              26
                 290


                 280

           E

           V
                260
           I
           W


           a 240
           0
           m



                220                                  I                        I      I
                      0          8             16   24                       32     36
                                       DAYS AFTER START


Figure 3.2 Mean body weight of rats housed in metabolism cages (triangles)        or in polycarbonate
cages (circles).Error bars represent± 1 SEM.




                 120-




          c~.
          F,, 90
          n-
          0
          LUm
          I--
                     6c
           ELU
               13.


                                  I             I             I               I        J
                     300         8          16       24                      32       36
                                       DAYS AFTER START


Figure 3.3 Mean water consumption (ml/kg body weight) for rats housed in metabolism cages
           or
(triangles) in polycarbonate                Error bars represent± l
                             cages (clrcles).



                                               27
                  90


           ,~    80

           ,,=,~
           8 6o
           ,o~
           ,,o
          ~4o
                       t
                 20                  I          I            I             I        I
                      0              8      16       24                    32 36
                                         DAYS AFTER START


Ftgure 3.4 Mean food consumption (g/kg body wetght) for rats housed In metabolism          cages
(triangles) or tn polycarbonate cages (circles). Error bars represent ± 1SEH.




                400
                I
          0                ¯ Poly
                           ¯ Metab




                      I
                 o
                 -2        0         4      8         12     16    20
                                                                               I
                                                                               24
           E
                          DAYS AFTER YELLOWCAKE IMPLANTATION



Figure 3.5 Mean water consumption by rats Implanted wtth 10 mg yellowcake/kg     body wetght. The
11ne wtth data potnts as squares ts for naive rats housed tn metabolism cages, the 11ne wtth data
potnts as triangles  ts for rats =accllmted"   to metabolism cages, and the 11ne wtth potnts as
ctrcles ts for rats housed tn polycarbonate cages. Error bars represent ± 1 SEN.


                                                28
               40-




                 30
                                                                   Accl     20 mg/kg




                         Accl      10 mglkg




                 1°
                 I
                                                                                   Naiv    10 mglkg




                   /                                                                               -         ~ - 2J2
                 02            0   2          6      10      14                                  18
                                           DAYS AFTER IMPLANTATION

      Figure 3.6 Volume of urinary               output by rats after        subcutaneous implantation           with yellowcake.



Uranium Concentration tn Kidney


        The concentration              of uranium in the kidneys (?2 ± 18 pg/g) of four naive rats                        that died
days after       Implantation          wlth yellowcake at a dose of lO mg/kg was signlflcantly                     higher than that
of five      surviving          rats (16 ± 4 pg/g) housed in polycarbonate                 cages and sacrificed         8 days after
implantation with yel]owcake at this same dose level                         (the latter     were rats in the wound retention
study described in paper no. 2 of thts report).


An(mal RoomTemperature and Cage Temperature


         The mean room temperature                (during    24 days) and mean temperature             in each of two metabolism
cages and two polycarbonate                    cages are plotted        at 4-h Intervals      through a 24-h cycle            in Figure
3.7.    Temperature            In the polycarbonate         cages was significantly           lower than the temperature                in
metabolism cages throughout the 24-h cycle.


Htstopathologlcal              Observations


      Kidneys of naive rats that died 8 days after implantation  with yellowcake at a dose of 10
mg/kg appeared pale, with mettled reddish coloration    and yellowish   speckling (Figure 3.8).
Widespread massive necrosis                of tubular       eptheltal     cells   was present,     and it    involved     essentially
all    tubules    and the proxtml              and distal    portton     of each Individual        tubule.       Nearly all     tubular
epithelial       cells         were necrotic     and sloughed.     Massive casts of necrotic            cells,    calcified     debris,
and protein           filled       the tubules.       The glomerult        were relatively        spared.        Rats in the      wound
retention      study (See paper no. 2 in this                 report)     that were killed       at 16 days after       implantation



                                                                    29
                              CAGE AND ROOM TEMPERATURE                   (°C)
                     29.3 -
                                      Metab.   ~4 Temp.--~l~
                                                                           Temp.


                     22.8
             ~O
             O

             U..I
             ¢¢

             ~- 22.3                                           ÷6 Temp.
             ,<                     ~//f
              n"                l                  Also
             LU
              ft..
             I..U
                                                                          #’7 Temp.
                     21.8
                                           Room Temp.




                     21.3       i           I    I    ~         I                  J
                              10am         2pm 6pm 10pm        2am   6am
                                               TIME OF DAY


Figure 3.7 Comparison of animal room temperature and temperaturesrecorded In polycarbonate
                     cages duringa 24-h cycle.
cages or in metabolism




Figure 3.8 6ross appearance of kidneys of naive rat that died 8 days after implantation of
          at                                to
yellowcake a dose of lO mg/kg(left) compared kidneysof normal rat (right).
(10 mg/kg) also had tubular            necrosis          but of a less widespread,             severe nature.            In addition,       many
tubules     were ltned     by small        flattened         basophilic       immature epithelial          cells.        It    appeared that
these rats had undergone a period of acute tubular                           necrosis,      and repair    of tubules had begun. The
histopathological         changes noted above were generally                      similar     to those reported               by Barnett     and
Metcalf in Voegtlin        and Hodge (Ref. 3.6) in their                     description     of the pathological               anatomy of the
kidney following        uranium poisoning.


                                                                  DISCUSSION


         The histopathologtcal         observations              noted for rats         in these studies          that    died eight        days
after    implantation      with yellowcake were generally                     similar     to those reported          by others for rats
after oral or parenteral administration   of uranyl nitrate (Refs. 3.6 - 3.12). In earlier studies
of toxicity  from parenteral administration    of soluble uranium compounds to animals, rats were
usually     housed in wire cages in groups of five                           or fewer (Refs.       3.6,     3.13).        Haven and Hodge
reported LDso/48 hr or L050/21 days values of 86 mg/kg or 2.5 mg/kg, respectively,   for male
Wlstar rats housed in wire cages after a single intraperitoneal injection  of uranyl nitrate
hexahydrate (Ref. 3.13). The LDso/21 days value (6 mg/kg with 95% confidence limits     of
mg/kg) reported here for naive F-344 rats housed in metabolism cages after implantation    with
yellowcake powder was not significantly                   different       from the 21-day LD50 value cited above.
     Orcutt   reported    an LDso value of 1 g/kg for aqueous solutlons                                                        of UO2F2 or
UO2(N03) 2 applied   to the shaved skin of rats (Ref. 3.14). This is about                                                    103 times the
LDso for a single        tntraperltoneal           injection      of uranyl nitrate          solution     and an equal amount above
the LDso value reported he6e for naive rats housed in metabolism cages after                                                    subcutaneous
implantation of dry yellowcake with ammoniumdluranate as the major ingredient.
         Results presented here indicate                 that rats housed in metabolism cages beginning immediately
after    subcutaneous implantation             with yellowcake            were more susceptible           to uranium toxicity               than
rats housed in polycarbonate                cages or rats acclimated                  in metabolism cages for                 21 days before
yellowcake      implantation.         Difference           in response          of the two groups of rats                     was related     to
differences in water consumption during the first                        8 days after yellowcake implantation.
        One might expect the water consumption                           by rats housed in metabolism cages where the
temperatures     were higher         to be greater           than for those housed in polycarbonate                       cages where the
temperatures were lower. However, water consumption by naive rats Placed in metabolism cages was
initially  lower than that of rats continually  housed tn polycarbonate cages, even though the
temperature within        the metabolism cages was higher than the temperature within                                    the polycarbonate
cages.
         The greater     tolerance     to uranium toxicity                exhibited       by rats housed in polycarbonate               cages
compared to naive rats            housed tn metabolism cages was related                         to the lower concentration                   of
uranium in the kidneys of these rats than in the naive rats housed in metabolism cages. The
difference in uranium concentration in kidney of the two groups of rats was related to differences
in water consumption          by rats        in these two groups.                Water consumption           by rats           acclimated     to
metabolism cages was equal to that of rats housed in polycarbonate cages, and the response of
these two groups of rats to uranium toxicity was similar.  These findings are similar to those
reported      by Ryan et al (Ref.                 3.7)    that     increase       in fluid      intake      and urinary            output
saline-loaded     rats was correlated              wtth reduction          in uranyl nitrate-induced               acute renal failure.
These investigators         reported       that     saline     loading       (provision     of 1% saline          as the sole source of
drinking    water) afforded protection              in rats against the development of acute renal failure                             induced
by uranyl     nitrate     (Ref.    3.7).     Saline-loaded            rats     exhibited      greater     fluid     intake       and urinary
output than rats drinking            water at 24 or 48 hrs after                  intravenous     injection         with uranyl       nttrate
solution        at a dose of 10 mg/kg. Saline loading ameliorated            the azotemta but not the renal tubular
necrosis or tubular dysfunction that are characteristic of uranium nephrotoxlcity.(Refs. 3.7
3.9). However, Avasthl et al. (Ref. 3.10) reported that saline loading protected rats against
alterations  tn both renal function and endothellal                    cell morphology (as assessed by electron
microscopy), whereas sodium depletion In rats after                   administration of uranyl nitrate (15 mg/kg)
resulted        In development of a marked reduction            tn the glomerular     filtration      rate and significant
alterations    tn endothelial          cell morphology.         In saline loaded rats, a lower concentration   of
uranyl nitrate    as a result         of increased fluid         intake and urinary output may have prevented the
cellular     injury.
        We conclude that rats         housed tn polycarbonate         cages or in metabolism cages after              a 21-day
period of acclimation  exhibited  a significantly lower nephrotoxlc  response to uranium from
Implanted yellowcake than dtd halve rats housed In metabolism cages. Greater toxlc response of
the naive rats to implanted yellowcake was related                 to reduction     In water consumption by these rats
compared to those housed In polycarbonate                       cages or rats     housed in metabolism           cages after
accllmatlon        to this    cage type.   Difference     tn water consumption of the two groups of rats may be
due to differences             In the behavior       patterns      between the naive        rats     and those     housed in
polycarbonate          cages. The differences    In water consumption could not be related               to differences       in
temperature within the two types of cages.
        It      has generally     been recognized        that    a period   of accllmatton          must be provided         for
laboratory animals placed In a new environment before undertaking toxicity studies. However, the
ttme period required   for acclimation   of rats to metabolism cages has not been determined
previously.        Data presented here tndlcate          that mtntmum periods       of 3 days or 5 days are required
for acclimation         in terms of food consumption or water consumption, respectlvely.                  However, rate of
change of body weight for rats housed In metabolism cages was less than that of rats housed tn
polycarbonate          cages throughout    a 34-day perlod        of observation.     Therefore,       further     studies    of
accllmatton         of rats     to metabollsm     cages are required        to determlne           the minimum period         Of
acclimation       needed for studies Involving          measurements In changes of body wetght of rats housed in
these two cage types.
        Figure 3.3 shows that water consumption of rats housed tn metabolism cages was less than for
rats housed tn polycarbonate           cages until      16 days of acclimation       (alt~ough      the difference     was not
statistically       slgnlftcant    beyond day 5). Therefore,         we recommendthat tn future          studies     requiring
excreta collections,          a minimum 21-day acclimation          to metabolism cages should be provtded before
exposure of rats to nephrotoxtc test substances. Thts should provtde a measure of assurance that
water consumption and nephrotoxtc responses of rats tn the two cage types wtll be similar.


                                                           REFERENCES

3.1     A. F. Eldson and 3. A. Mewhtnney, "In Vttro Solubility        of Yellowcake Samples from Four
        Uranium Mtlls and the Implications for Bloassay Interpretations,"   Health Phystcs 39: 893-902.

3.2     H. C. Redman, C. H. Hobbs, and A. H. Rebar, "Survival       Distribution  of Syrian Hamsters
        (Mesocrtcetus auratus, Sch:SYR) Used During 1972-19~7,’ Progress tn Experimental Tumor
        Research, Vol. 24, (F. Homburger, ed.), pp. 108-117, Karger, Basel, 1979.

3.3     E. 6. Damon, A. F. Etdson, F. F. Hahn, W. C. Grtfftth, 3r., and R. A. 6utlmette, "Comparison
        6f Early Lung Clearance of Yellowcake Aerosols tn Rats wlth In Vitro Dissolution        and
        Infrared Analysis," Health Physics 1983, (In press).

3.4 O. 3. Ftnney, Probtt Analysts,            3rd ed. Cambridge Untv. Press, Cambridge, England, 1971.

3.5     M. Ralston, "Derivative-Free  Nonlinear Regression," 8MOPStatistical                       Software (W. J. Dixon,
        ch. ed.), pp. 305-314, Berkeley (Univ. Caltf. Press), 1981.
3.6    C. Voegtltn and H. C. Hodge (eds.): The Pharmacology and Toxicology of Urantum Compounds,
       Natlonal Nuclear Energy Series, D1vlslon IV, Vol. 1 and Olv. Vt, Vol. 1, pp. 207-236,
       McGraw-Hill Book Co., Inc., New York, 1949 and 1951.

3.7    R. Ryan, 3. S. McNetl, W. Flamenbaum, and R. Nagle (Introduced by Robert 3. T. 3oy), ’Uranyl
       Nltrate   Induced Acute Renal Failure      tn the Rat: Effect of Varying Doses and Saline
       Loading," Proc. Soc. Exp. Biol. Med. 14__33: 289-296, 1973.

3.8    D. P. Haley, "Acute Renal Failure Induced by Uranyl Nitrate:       Structural  and Functional
       Response of Water and Saline Drinking Rats,’ Doctoral Dissertation,    The University of Texas
       Health Science Center at San Antonio, 1980.

3.9    O. P. Haley, "Morphologic Changes In Uranyl Nitrate-Induced     Acute Renal Failure   in Saline-
       and Water-Drlnklng Rats,’ Lab. Invest. 46: 196-208, 1982.

3.10   P. S. Ataschi, A. P. Evan, and O. Hay, "61omerular Endothelial        Cells in Uranyl Nitrate-
       Induced Acute Renal Failure In Rats," 3. Olin. Invest. 6_55: 121-127, 1980.

3.11   K. A. 6oel, V. K. Garg, and Veena Garg, "Histopathology     of Kidney of Albtno Rat Poisoned
       with Uranyl Nitrate," Bull. Envlron. Contam. Toxicol. 24: 9-12, 1980.

3.12   P. W. Ourbtn and M. E. Wrenn, "Metabollsm and Effects of Uranium in Animals," Occupational
       Health Experience with Urantum, (M. E. grenn, ed.), ERDA93, pp. 68-129, Arlington,     VA,
       1975. Available from National Technical Information Service, Sprlngfield, VA 22161.

3.13   F. L. Haven and H. C. Hodge, "Toxicity   Following the Parenteral Administration of Certain
       Soluble Uranium Compounds,’ Pharmacology and Toxicology of Uranium Compounds,Chapter 6, (C.
       Voegtlin and H. C. Hodge, eds.) pp. 281-308, McGraw-Hill book Company,Inc., NewYork, 1949.

3.14   m A. Orcutt, "The Toxicology of Compoundsof Uranium Following Application
       3.                                                                          to the Skin,
       The Pharmacology-and Toxicology of Urantum Compounds, (Voegtlin C. and Hodge H.C., eds.),
       pp. 377-414, New York (McGraw-Hill), 1949.




                                                33
           4.   TWO-YEAR DOSE PATTERN STUDIES OF INHALED YELLOWCAKE IN THE BEAGLE DOG


Abstract --Fortg-fourBeagle dogs were exposed
to aerosolsgenerated              samplesoh-
                    from yellowcake                       PRIWCIPALIWVESTIGATORS
talned from two uranlu~mllls. The two materlals                A.F. E1dson
          two extremesIn yellowcake
represented                       composltlon                  E.G. Damon
that occur In Industry:one was 100~ ammonlu~dl-
uranate; the other was > 99~ U308. The aerosols of the 100~ ammonlum dluranate form Inhaled
by 20 dogs averaged 3.4 ± 0.5 mlcrons (mean ± 1 SE) mass medlan aerodynamlcdlameter (MMAD),
and 1.5 ± 0.04 geometrlc standard devlatlon (GSD). The average estlmated Inltlal lung burden
was 130 ± 9 pg U/kg body welght. Exposure aerosols of the > 99~ U308 form Inhaled by
the second group of 20 dogs averaged 3.0 ± 0.3 ~um MMAD and 1.7 ± O.l GSD. The estlmated
Inltlal lung burden for the second group was 140 ± 7 pg U/kg body welght. Sacrlf~ces are
                                                          and analyslsof tlssue and excreta
completedthrough64 days after exposurefor both experlments,
samples                  Is
       for uraniumcontent In progress.


                    of             patternsof retentionand excretionof inhaleduraniumin rats
        Investigation the short-term
exposed to yellowcake aerosols from two uranium mills has shown that clearance of inhaled
         from the lung depends, part, on the yeIIowcake
yellowcake                     in                              in
                                                      solubility body fluids (Ref.4.1).
                to
The translocation other tissues(e.g., bone) and excretionthroughthe kidney also appears
            related.
be solubility
    Experimentswere designedto providedata on the long-termpatternof clearanceof uraniumin
                                of                      a
beagle dogs exposedby inhalation two types of yellowcake, more solubleform and a less sol-
uble form. Aerosols of the two yellowcake forms obtained from operating mills were generated
directlyfrom powdersfor nose-onlyinhalationexposures.The resultsof this study in dogs will
be comparedwith the availabledata on human exposureto U308, U02, and to UO3 (Ref. 4.2, 4.3).
                 of
    The objectives this studyare:
    (1) to ~ssess the patternsof retentionand excretionin dogs of two chemicalforms of ura-
nium commonlypresentin yellowcake aerosols;
    (2) to relate the above metabolismof uraniumfrom inhaledyellowcakeaerosolsto their phy-
                           and
sicaland chemicalproperties;
        (3) to relate the observed behavior of yellowcaketo humans and suggest possible bioassay
schemes.


                                       MATERIALS AND METHODS


                           were chosen,based on infraredanalysisto represent
    Two samplesof yellowcake                                                the extremesof
compositionobservedat uranium mills: one materialwas 100% ammoniumdiuranate (a more soluble
form); the other was < I% ammonium diuranate and > 99% U30B (a relatively insoluble form;
see Paper l of this report).
        Forty-fourbeagle dogs, includingan equal number of males and females,2 to 6 years of age
were selected. Twenty dogs were exposed to each material, and four were retained as unexposed
controlsto be used as quality controlsfor the uranium fluorometryanalyses.Exposure aerosols
Were generatedfrom the dry yellowcakepowdersusing a DeVilbissModel 125 powder generator(Ref.
4.4).


                                                35
        Aerosol concentration             was monitored during exposure by a Model RAM-Snephelometer (GCA Corp.,
Bedford,      MA) callbrated          with aerosols         generated from the same yellowcake                  powder. Calibration
aerosols were generated using constant alr                        flow rates and were sampled simultaneously                     using the
nephelometer and membraneftlters.                       The uranium deposited on the membrane filters                    was determined
by reflectance fluorometry (Ref. 4.5), and the aerosol concentration                                  was calculated      using the flow
rate through the fllter  and the sampling duration.
    The nephelometer was used to monitor the exposure aerosol concentration   contlnuously during
each exposure and to allow a more accurate estimate of the amount inha]ed by the dog. The breath-
ing frequency        and ttdal            volume of the dog were monitored                 during     exposure using a whole-body
plethysmograph (Ref.              4.6).     Estimates of the lnlttal            lung burden were made using the cumulative
volume of alr       Inhaled,       the average aerosol concentration,                 and deposition      efficiency     of 20% for the
pulmonary compartment of the lung. The aerosol particle                              size distribution      was determined by anal-
ysls     of the yellowcake           deposited      on each stage of cascade Impactors                    and fitting      a lognormal
distribution   function to the data.
      Because renal toxicity  might be caused by Inhalatlon                           of uranium, blood and urine samples were
collected     to monitor renal function.                  Blood and urine samples were also collected                     from all       dogs
before     exposure and at sacrifice.                   Additional        samples were collected           at 8 and 16 days after
exposure and at 90-day intervals                   thereafter.        Blood serum was analyzed for blood urea nitrogen,
creatlnlne,     total     protein,        albumln, calclum, and" Inorganlc phosphate content using a Multistat                            III
mlcrocentrlfugal          analyzer         (Instrumentation          Laboratories       Co., Lexington,       MA). Whole blood was
analyzed for        hematocrtt,           hemoglobln,      red and white cell           counts,     and mean cell       volume using a
Coulter Node1 2BI Counter (Coulter Electronics                       Co., Hlaleah, FL) and a Coulter hemogloblnometer.
    Comparative differential   cell counts and platelet estimation tests were Included. Standard
chemical analyses of urine include: protein, glucose, ketones, urobiltnogen, blltrubtn, blood and
hemoglobin content,          specific        gravity,     and pH. Sediments were analyzed mlcroscoplcally                       for cells,
casts, and crystals.
     Urine, feces, and cage wash water were collected                           daily    from 2 days before exposure until                   16
days after exposure, then three daily collections per week were made for 2 weeks. Subsequently,
three daily collections  per month were made every other month until 180 days after exposure.
After     180 days, three consecutive               daily     collections       of excreta        were made at 3-month intervals
until     sacrtf!ce.
        Dogs are scheduled for sacrifice                 at 0.08, 2, 4, 8, 32, 64, and 180 days and at l,                         1.5,   and
2 years after       exposure. Four exposed dogs in each study were not assigned to speclftc                                      sacrifice
groups, but are reserved as replacements in case of deaths during the term of the study. One of
the control     dogs in each study was sacrificed                    at 2 days and one will           be sacrificed       at 2 years to
provide quality         control     data on fluorometrtc          analyses during the study. Selected tissues taken at
necropsy      for   uranium        analysis      Include:        blood,     skull,      turbtnates,      trachea,       lung,     kidney,
gastrointestinal      tract (including  esophagus and stomach), liver, spleen, tracheobronchial  lymph
nodes, femur,       and lumbar vertebrae.  Tissues taken for htstopathologtcal    examination include
samples of kidney, femur, liver,                 spleen, lymph nodes, lung, and any lesions                  observed, Tissues and
excreta are analyzed for uranium content by reflectance                         fluorometry.


                                                        RESULTSAND DISCUSSION


    Scheduled sacrifices are complete through one year after exposure; all other #ogs are alive.
Results describing the exposure aerosol characteristics and estimated achieved tntt!al lung burden
for the animals that tnhaled one of the two yel]owcake forms are shown in Tables ~.l                                    and 4.2.




                                                                       3e
                                             Table 4.1.
 Results of Beagle Dog Exposures to Aerosols of Yellowcake Powder Containing 100~ AmmonlumDluranate



                          Mass Median
                          Aerodynamic                           Estimated Achieved    Indication
Exposure    Animal          Diameter      Geometric Standard    Initial Lung Burden    of Kidney
 Number     Number         + SE (um)        Deviation + SE           (ug U/kg)        Dysfunction
3120-01      1229A          3.6 + 0.01        1.34 + 0.03               130               No
3120-02      1176V          2.4 + 0.01        1.07 + 0.02               110               Yes
3241-03      11798          Control                -                     -                No
3242-04      1143U          3.4 +_ 0.2        1.55 +_ 0.07               95               No
3242-05      1182A          3.2 +_ 0.8        1.62 +- 0.07              150               Yes
3242-06      1240T         3.3 + O. 1         1.56 + O. 04              160               No
3243-07      1182B         3.9 + 0.2          1.54 + 0.05               250               Yes
3243-08      1241S         2.7 + 0.2          1.16 + 0.03               150               Yes
3244-09      1213B         3.3 + 0.2          1.38 + 0.07               130               Yes
3244-10      1242W         4.0 + 0.4          1.9 + 0.2                 140               Yes
3245-11      1224B         3.6 + 0.2          1.26 + 0.05               210               Yes
3245-12      1243S         3.0 +_ 0.1         1.5 + 0.03                140               Yes
3246-13      1226A         3.5 + O. 1         1.54 + O. 05              120              Yes
3246-14      1243U         3.3 + 0.2          1.59 + 0.07               100              No
3246-15      1226E         3.1 + 0.1          1.58 + 0.04               120               Yes
3246-16      1244S         3.2 + 0.1          1.66 + 0.06               130               Yes
3247-17      1244T          Control                -                     -               No
3248-18      1227D         3.4 + 0.2          1.63 + 0.06               140              No
3248-19      1181B         3.8 + 0.2          1.41 + 0.06               100              No
3248-20      1245H         3.8 + 0.3          1.6 + 0.1                 110              No
3248-21      12328         4.8 + 0.2          1.52 + 0.06                66              No
3249-22      1247S         2.9 + 0.2          1.5 + 0.1                  80              No
Mean+ SE (n - 20)          3.4 + 0.5          1.50 + 0.04             130 + 9
                                               Table 4.2.
           Results of Beagle Dog Exposures to Aerosols of Yellowcake Powder Containing 99% U308

                           Mass Median
                          ~, Aerodynamic                                 Achieved
                                                                 Estimated             Indication
Exposure     Animal          Diameter      GeometricStandard     InitialLung Burden     of Kidney
 Number      Number         +_ SE (~m)                ±
                                             Deviation SE             (uq U/kg)        Dysfunction
3121-23        1145S        2.6 ± 0.2          1.7+_ O.l                 160              No
3121-24        1178G        3.1 ± O.l          1.5 ± O.l                 130               No
3121-25        1148S         Control               -                      -                No
3292-26        118l A       1.9+_ O.l          l.S ± O.l                 130               No
3292-27        1220C        1.5+- O.l          1.5+_ O.1                 IgO               No
3292-28        1174T        2.2+_ 0.6          1.9 ± 0.3                 120               No
3292-29        1176T        2.7+- O.l          1.4+- O.i                 110               No
3293-30        12218        2.7 _+ 0.1         1.7 + O.1                 110               No
3293-31        1240S        3.0 ± 0.1          1.5 ± 0.04                160               No
3294-32        1242T        2.3 + 0.1          1.6 ± 0.1                 140               No
3294-33        1222B        3.3 ± 0.2          1.5 ± 0.1                 130               No
3294-34        1222D        2.9 +_ 0.2         1.6 +_ O.1                120               No
3294-35        1245T        2.9_+ 0.3          1.7 + 0.I                 150               No
3295-36       1247T         2.4+_ O.1          1.8_+ O.l                 190               No
3295-37        1235A        2.7+- 0.2          1.8+_ O.l                 160               No
3295-38       1236A         2.6 + 0.2          1.7 ± 0.1                 200               No
3295-39       1236B          Control               -                      -                No
3295-40       1248S         7.7 ± 2.3          3.2 ± 0.7                 100              No
3296-41       1248T         3.2 ± 0.I          1.6 +_ 0.04               II0              No
3296-42       1223A         3.6+- 0.2          1.5 ± O.l                 llO              No
3297-43       1237A         3.2 ± 0.2          1.6+- O.l                 120              No
3297-44       1250T         3.0 ± 0.3          1.8+_ O.l                 180              No
Mean ± SE (n = 20)          3.0 +- 0.3         1.7 +- O.1              140 +- 7
       Use of filter           sampling data to calibrate                   the nephelometer            assumes that        the parttcle      size
distribution          of the aerosol               remains constant             durtng      sampling.      Thts assumption might not be
warranted         in some cases. However, the nephelometer                               was used to monitor          the exposure aerosol
concentration         and overcome a major limitatfon                      of filter        sampling,      the time required      to determine
the amount of uranium on the filter                         by reflectance       fluorometry      (a minimum of 24 h).
       Inittal      lung burdens estimated using breathing                        parameters and aerosol concentrations                 measured
during exposure were approximately                          65% of the desired value and approximately                      7% of the LDso/30
day dose for dogs given a stngle injection     of UO2(N03)2 solution  (Ref. 4.7). More refined
estimates of the initial lung burden for each animal will also be made when the results of tissue
and excreta          analyses          for    uranium content            become available           for    Individual       animals.     The low
intensity         of gammaradiation                    from natural       uranium precludes             the use of external         whole-body
counting in this study.
       Eleven dogs exposed to the more soluble                           yellowcake (containing            100% ammoniumdiuranate)            have
shown changes in biochemical                       indicators        of renal dysfunction              (Table 4.1).     No such biochemical
changes have been observed for dogs exposed to the less soluble form (containing    > 99% U308,
Table 4.2). Dog 1213B (Table 4.1), sacrificed on schedule at 4 days after exposure, had elevated
glucose levels             and albumtn in urine above pre-exposure                           values.      Dog 1241S had elevated           glucose
levels      and albumin in urine at 8 days after                         exposure. Dogs identified                as having possible        kidney
dysfunction         had elevated glucose levels                    (250-3000 mg/dl) and protetn              (lO0-1000 mg/dl) in urine
8 days after exposure. Control antmals had normal levels of 0-0.25 mg/dl for these indicators.
In addition, elevated levels of 30-50 mg%(control value = 1S mg%)blood urea nltrogen and 2.2-4.3
mg%creatinine               (control         value      = 0.8-1.1      mg%) were measured at 8 days after                    exposure.       These
elevated         levels     returned         to normal at 16 days after                exposure.       Histopathologlcal       examination      of
kidney tissues             from dogs that              had biochemical          evidence of kidney dysfunctlon                is in progress;
however, the dysfunction                     was possibly          caused by acute tubular             necrosis    in the proximal         tubules
(Ref. 4.1) that was repaired by approximately 16 days after exposure.
    These results show clearly   that only the dogs exposed to the more soluble                                               yellowcake      form
showed evidence               of kidney         toxicity,          indicating        that    the content       of ammonium dluranate            in
yellowcake aerosols is important for health protection purposes.
     The occurrence of kidney dysfunction cannot be quantitatively                                      related    to the estimated initial
lung burden Mow. The dogs that experienced kidney dysfunction                                      (Table 4.1) had estimated initial
lung      burdens         of 150 ± 13 ~g U/kg body weight                          (mean ± SE),           and dogs with        normal      kidney
function          had estimated              initial        lung    burdens      of 106 ± 11 ~g U/kg.               These values        are not
significantly             different      at the 95% confidence              level.       A more quantitative         treatment    of the dose-
response relationshlps                   between tnhaled             yellowcake        and kidney toxicity           will   be possible       when
results      of analyses for uranium content in tissues                           and excreta become available              during the coming
year.


                                                                        REFERENCES


4.1     E. G. Damon, A. F. Etdson, F. F. Hahn, W. C. Grtfftth, 3r., and R. A. Gutlmette, "Comparison
        of Early Lung Clearance of Yellowcake Aerosols tn Rats with In Vitro Dissolution and Infrared
        Analysis,’ Health Physics (in press).

4.2     R. E. Alexander, "Applications of Btoassay for Uranium," Directorate of Regulatory Standards,
        U.S. Atomic Energy Commission, WASH-12S1, Superintendent      of Documents, U. S. Government
        Printing Office, Washington, OC, 1974.
4.3   Occupational Health Experience wtth Uranium, M. E. Wrenn, ed., ERDA-93*, 1975.

4.4   A. F. Etdson, "An Improved Technique for Aerosoltzatlon  of Dry Powders of Industrial Uranium
      and Plutonium Mixed-Oxlde Nuclear Fuel Materials,"      Radiation Dose Estimate and Hazard
      Evaluations for Inhaled Airborne Radionuclldes, Annual Progress Report, 3uly l, 1978-June 30,
      1979, 3. A. Mewhlnney, Project Coordinator, NUREG/CR-1458, LF-71, pp. 5-10, 1980".

4.5   R. A. Gullmette, "Analytlcal Methods for the Quantttatlve   Determination of Uranium In Bio-
      logical and Nonblologtcal Material," Biological  Characterization  of Radiation Exposure and
      Dose Estimates for Inhaled Uranium Mtlltng      Effluents,   Annual Progress Report, March
                                            pp.
      197g-March 1980, LMF-76, NUREG/CR-1669, 23-26, 1980".

4.6   B. B. Boecker, F. L. Agullar,   and T. T. Mercer, "A Canine Inhalation     Exposure Apparatus
      gtlllztng a Whole-Body Plethysmograph," Health Physics 10, 1077-1089 (1964).

4.7   P. W. Ourbln and M. E. Wrenn, "Metabolism and Effects of Uranium in Animals," Occupational
      Health Experience with Uranium, Wrenn, M. E., ed., pp. 68-129, ERDA-93, VA, 1975".


*Available   from the National Technical Information   Service,   Springfield,   VA 22161.




                                                  4O
                                                               APPENDIX


                                       Technical Publications          and Presentations


 Publications
 1.  A. F. Eldson and O. A. Hewhlnney, "In Vltro                  Solubility      of Yellowcake Samples From Four Uranium
      MIlls   and the Implications      for Bloassay Interpretatlons,"                 Health Phystcs 39, 893-902 (1980).
 2.   A. F. Etdson and N. C. Grlfftth,              3r.,    "Techniques for Yellowcake Dissolution             Studies In Vftro
      and Thetr Use tn Bloassay Interpretation,"                Health Phystcs (In press).
 3.   A. F. Eldson and E. G. Damon, "Characteristics                    of Yellowcake Aerosols Sampled In Operatlng
      Uranlum Mtlls,"     Health Physlcs (tn press).
4.    E. G. Oamon, A. F. Eldson, F. F. Hahn, W. C. Grtfflth                     Jr.,    and R. A. Gutlmette,      "Comparison of
      Early Lung Clearance of Yellowcake Aerosols In Rats wtth In Vitro                            Otssolutlon      and Infrared
      Analysts," Health Physics (In press).
5.    A. F. Eldson, "In Vttro 0tssolutton   of Commercial Yellowcake and Comparisons With Available
      HumanData," International          Conference, Radlatlon Hazards In Mtnlng: Control,                     Measurement, and
      Medical Aspects, M. Gomez, Ed., Amertcan Instltute                        of Mtnlng,    Metallurgical,      and Petroleum
      Engineers, Inc., pp. 1073-1078, 1981.


 Presentations
1. A. F. Etdson and 3. A. Mewhtnney, "In Vitro                    Dissolution          of Uranlum Product Samples from Four
      Urantum Mtlls,"      24th Annual Meettng of the Health Physics Soctety,                        Philadelphia,      PA, 3uly
      8-13, 1979.
2.    A. F. E1dson, "In       Vttro    Solubility          of Yellowcake       Samples From Four Urantum Ntlls           and the
      Implications     for Bloassay Interpretation,"             25th Annual Conference on Bloassay, Environmental
      and Analytical Chemistry, Las Vegas, Nevada, October 31-November 1, 1979.
3.    A. F. Etdson, "Comparlson of Techniques for In Vttro Yellowcake Dissolution                                Studies,"     25th
      Annual Meettng of the Health Physics Soctety, Seattle,                    WA, 3uly 20-25, 1980.
4.    A. F. Etdson,      "Characteristics       of Urantum Ht111ng Aerosols,"                 Meettng of Radiation           Safety
      Officers     and Membersof the Hyomtng Htntng Association,                 Casper, WY, August 29, 1980.
5.    A. F. Etdson, "Characteristics           of Urantum Yellowcake Aerosols                 Sampled In Operating       Urantum
      Ntlls," 26th Annual Meettng of the Health Phystcs Soctety, Louisville, KY, 3une 21-25, 1981.
6.    A. F. Eldson, "In Vttro Dissolution  of Commarctal Yellowcake and Comparison wtth Available
      HumanData,"      International     Conference on Radiation               Hazards In Mtntng: Control,         Measurement,
      and Medtcal Aspects, Golden, CO, October 4-9, 1981.
7.    E. G. Damonand A. F. Etdson, "Six-Month Studtes of the Translocatton and Retention of Urantum
      In Rats Exposed by Inhalation of Aerosols of Yellowcake Samples From Two Urantum Mtlls,"  Rto
      Grande Chapter, Amortcan Industrial              Hygtene Association         and the Health Phystcs Soctety 3otnt
      Neettng, Albuquerque, NN, October 5-6, lg81.
8.    E. 6. Damon and A. F. Eldson,            "Translocatlon         and Retention         of Urantum tn Rats Exposed by
      Inbalatton     of Aerosols of Yellowcake Samples from Two Urantum Ntlls,’                      27th Annual Meettng of
      the Health Phystcs Soctety,       Las Vegas, NV, June 26-July 1, 1982.




                                                                41
Abstracts
1.  A. F. Eldson and 3. A. Mewhlnney, "In Vitro Dissolution of Uranium Product Samples from Four
    Uranium Hills, H Health Physics 37, 821 (1979).
2. A. F. Eldson, "Comparlso~s of Techniques for In Vitro Yellowcake Dissolution Studies," Health
     Physics 39, 1050 (1980).
3.   A. F. Etdson and E. G. Damon, "Characterlstlcs       of Uranium Yellowcake   Aerosols   Sampled In
     Operating Uranium Mtlls,"   Health Physics 41, 847 (1981).
4.   E. G. Damon and A. F. Etdson,     "Translocatlon   and Retention   of Uranium in Rats Exposed by
     Inhalation   of Aerosols of Yellowcake Samples from Two Uranium Mills,"   Health Physics 43, 143
     (1982).
 NRC FORM 335
  q11-81)                  U.S.   NUCLEARREGULATORYCOMMISSION
                    BIBLIOGRAPHIC DATASHEET                    LMF-108
                                                 Characteri-2, (Le~e blenk/
 4. TITLE ANDSUBTITLE ~ddvommoN~.,f~p,~,,,JBiological
 zationof Radiation     Exposureand Dose Estimatesfor Inhaled
 UraniumMillingEffluents     (AnnualProgress              l,
                                              Report:April 3. RECIPIENT’S ACCESSION NO.
 1982- March31~ 1983)
 7.     AUTHOR(S)                                                                                  5. DATE REPORTCOMPLETED
 A.F. Eidson,ProjectCoordinator                                                                          MONTH                  [ YE AR
                                                                                                     April                        1984
 9.     PERFORMING ORGANIZATION    NAME AND MAILING     ADDRE~ flnclu~     Z,D    Code)                  DATE REPORTISSUED
 Inhalation         Research
           Toxicology      Institute
 Lovelace         and
         Biomedical EnvironmentalResearchInstitute
 P.O.Box 5890                                                                                      6. (Le,we INdmk/
            NM
 Albuquerque, 87185
                                                                                                   8. (Leawe blamk)
 12.     SPONSORING ORGANIZATION     NAME AND MAILING     ADDRE~ flnc/u~     Z~ Co~)
 Division of Health, Siting and Waste Management                                                   10.    PROJECT/TASK/WORK UNIT NO.

 Office of Nuclear Regulatory Research
                                                                                                   11. FIN NO.
 U.S. Nuclear Regulatory Commission
 Washington, D.C. 20555                                                                                  A 1222
 13.    TYPE OF REPORT                                                           PERIOD   COVERED (Inclusive dates)


 Technical                                                                   Aprill. 1932 - March31. 1983
 15. SUPPLEMENTARY       NOTES                                                                     14. (Lewe ola~k)


16. ABSTRACT ~OOwordsor~ssJA             infrared
                              quantitative       absorption method for yellowcakeallowedthe
          of
fraction ammonium                 in
                        diuranate a mixtureto be determined    accurately  within7% and the
UR08 fraction                              of
               within13%.Thecomposition yellowcake       from six operating millsranged
fFom nearlypure ammonium              to
                             diuranate nearlypure U^08.A studyof retention      and trans-
          of
location uraniumaftersubcutaneous                     ~n
                                         implantation rats was done.The resultsshowed
that 49% of the implanted    yellowcake                                       in
                                       clearedfrom the body with a half-time the body of
0.3 days,and the remainder                                 of
                               was clearedwitha half-time II to 30 days.Twentydogs
         to
exposed a more soluble       yellowcakeform inhaledaerosols            an
                                                             producing estimated    initial
                                 of               of
lung burdenof 130 micrograms U per kilogram body weight.Aerosols            inhaledby dogs
         to
exposed a less solubleyellowcake                     an
                                       form averaged estimated    initial lung burdenof
                 of
140 micrograms U per kilogramof body weight.Biochemical                     of
                                                                indicators kidney
dysfunction                  in                                           to
             that appeared bloodand urine4 to 8 days afterexposure the more
soluble  yellowcake  showedsignificant         in                            to
                                        changes dogs,but levelsreturned normal
                              No
by 16 days after exposure. biochemical               of                     was
                                            evidence kidneydysfunction observed
                 to
in dogsexposed the lesssolubleyellowcake        form.



  7.    KEY WORDS AND DOCUMENT ANALYSIS                                     17a.    DESCRIPTORS



~ranium,aerosol,yellowcake                                                       rats, dogs, uranium
~eposition, inhalation, exposure                                                 fuel cycle, yellowcake
~ose, solubility,   mill, health
bioassay, rats, dogs, wounds,
infrared  analysis,  fuel cycle
  7b.    IDENTIFIE   RS/OPEN-EN DED TERMS



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