EPA-600/1-77-039
July 1977
Environmental Health Effects Research Series
SE TO PLATINUM
COMPLEXES •
NO-EFFECT LEVEL
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Triangle Park, North Carolina 27711
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
EPA-600/1-77-039
July 1977
ALLERGIC RESPONSE TO PLATINUM AND PALLADIUM COMPLEXES
DETERMINATION OF NO-EFFECT LEVEL
by
James Taubler
Department of Biology
St. Vincent College
Latrobe, Pennsylvania 15650
Grant No. 803036
Project Officer
Robert M. Bruce
Clinical Pathology Branch
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
-------
DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
ii
-------
FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-ma'de environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park, .
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
The relationship of the following report to the overall objectives of
the Health Effects Research Laboratory is based on the health risks
associated with the potential release of noble metal attrition products
into the atmosphere from the use and operation of catalytic converters.
Principle components among these suspected attrition products include
platinum and palladium compounds. Hypersensitivity has been a recognized
occupational hygiene problem among workers in platinum refineries as well
as a concern with the possible therapeutic use of organoplatinum compounds
as anticancer agents.
inn H. Knelson, M.D.
Director,
Health Effects Research Laboratory
•ill
-------
ABSTRACT
Section A
Rabbits, guinea pigs and mice were subcutaneously injected with PtSO4 (with
and without NH4C1) and PdSO4 (with an without NH4 Cl) in an attempt to sensi-
tize the animals to platinum or palladium. No allergic induction was found.
Levels of platinum or palladium in the serum, urine and spleens of animals were
monitored by AAS (atomic absorption spectrophotometer) . Significant levels were
found primarily in the urine of guinea pigs.
Section B
No allergic induction to platinum or palladium was found in rabbits, guinea
pigs or mice when these animals were injected intravenously with platinum or
palladium. Dermal contact with platinum was also tested on rabbits and guinea
pigs but failed to induce an allergic state.
Platinum and palladium levels in the sera, urine and spleens of the animals,
as monitored by AAS, were not significant.
Section C
Rabbits, guinea pigs and mice were intravenously or subcutaneously injected
with a platinum-egg albumin complex or a palladium-egg albumin complex. Skin
tests or footpad tests were performed 10-14 days after the last intravenous or sub-
cutaneous injection. Only guinea pigs injected subcutaneously with palladium-egg
albumin complex developed a hyper sensitivity. This hypersensitivity to palladium
was of the delayed type and was passively transferred to normal recipient guinea
pigs via spleen cells from sensitive donors.
Section D
A delayed type allergy was induced in guinea pigs subcutaneously injected
with palladium complexed to egg albumin. A minimum palladium concentration
of 3.7 mg/ml is needed for induction. When guinea pig albumin is complexed to
palladium a higher (5.0 mg/ml) palladium concentration is necessary.
iv
-------
CONTENTS
Foreword . . , iii
Abstract . iv
Introduction . . ........ 1
Conclusions 3
Section A - Subcutaneous Injections 5
Section B - Intravenous and Dermal Contact 13
Section C - Metal Albumin Complex ... 17
Section D - Albumin Complex 24
References 31
Tables 32-81
-------
INTRODUCTION
The immunological phenomena of man and many other lower animals that have
evolved during thousands of centuries can be arbitrarily categorized in two
groups, i.e., those that are protective and those that are harmful. At man's
present level of knowledge the protective immunological phenomena far outnumber
the harmful, however, this may be due to the subtle obscure nature of the immu-
nological phenomena involved in producing harmful effects. Hypersensitivity or
allergy is the term used to denote an immunological reaction that is harmful to the
host within which it occurs. Hypersensitivity can be classified into two types,
immediate and delayed. The essential difference between the two is the type of
immune response, i.e., the immediate is mediated thru a humoral (antibody)
immune response, the delayed thru a cellular immune response. Delayed hyper-
sensitivity or delayed allergy also requires a longer time to develop ranging from
6 - 214 hours after challenge whereas the immediate allergy is usually manifested
within the first hour. .
Immediate Allergy:
Immediate allergy or hypersensitivity involves the sensitization of the host
by an allergen (antigen) . The allergens of hay fever can be obtained from a wide
variety of material such as pollen, grasses (hay, ragweed, golden rod, lawn
grasses, etc.) and animal dander. The antigen involved in the production of
hives can be obtained from almost any food but in particular foods such as fish,
milk, chocolate and fruit. Asthma and anaphylaxis are also examples of
immediate allergy; anaphylaxis, being the most severe of all allergic reactions,
often can be fatal.
Allergens that by themselves can induce the allergic state are usually complex
macromolecules. However, low molecular weight substances such as penicillin,
certain chemical compounds and indeed even elements can complex with large
molecular weight host proteins and induce the hypersensitive state. Platinum is
an example of an element that can induce immediate type allergies. Little is
known about platinum allergies but from the few reports concerning platinum
allergies(l - 8), it appears that platinum can induce an asthma and a dermatolo-
gical(rash) type reaction. It should be added that simple chemicals are usually
themselves not allergens but act as haptenes. A haptene can combine with large
molecules (usually proteins) and induce allergies specific for the haptene.
-------
Delayed allergy:
Delayed hypersensitivity or delayed allergy is also known as infection allergy
since for many years it could only be demonstrated subsequent to infection (i.e.,
certain viral diseases and intracellular bacterial diseases such as tuberculosis
and brucellosis) .
Immune stimulation by antigens (usually virons, bacterial cells or chemicals)
is required to induce delayed allergy. Upon reexposure to the antigen the sensi-
tized lymphoid cells are injured. The mechanism of injury is ill defined; never-
theless, delayed allergy can be demonstrated by means of various in vivo or in
vitro tests. Smallpox vaccination, the various tuberculin tests and brucellin
tests are all manifestations of delayed allergy. Additionally poison ivy rash and
the dermatological reactions to various chemicals, some of which are carcino-
gens are all forms of allergy.
Whether simple elements such as platinum or palladium can induce delayed
allergy is unknown. However, simple haptenes such as penicillin can induce
delayed allergy thus, delayed allergy to metals such as platinum and palladium
may indeed be probable. Apparently a prime criterion for antigenicity of a small
molecular weight substance (i.e., mercury or platinum) is the ability of the metal
to form complexes with skin or serum protein, most likely by reacting with free
amino or sulphydryl groups on the protein to form stable complexes.
Immediate and delayed allergy, which include numerous autoimmune diseases
'are but two manifestation of the immunological capabilities of man spanning such
diverse phenomena as general immunity, tumor immunity and transplantation
immunity. Basic research on the possible allergic reactions of metals such as
platinum and palladium will not only provide environmental benefits but also aid
in understanding the complex nature of man's immunological capacity.
-------
CONCLUSIONS
Section A
Rabbits, guinea pigs and mice which were subcutaneously injected three
times a week for 4 weeks with various concentrations of platinum and palladium
salts showed:
1) no induction of an allergic state to platinum or palladium
2) significant palladium levels in the urine of rabbits and guinea pigs
3) significant platinum levels in the splenic tissue of rabbits and guinea pigs.
Section B
Rabbits, guinea pigs and mice which were intravenously injected three
times a week for 4 weeks with various concentrations of platinum and palladium
salts showed:
1) no induction of an allergic state to platinum or palladium
2) no significant platinum or palladium levels in the urine, serum or splenic
tissue.
Also, rabbits and guinea pigs which were exposed to platinum paste (dermal
contact with platinum paste) for 5 weeks showed:
1) no allergic induction
2) no significant levels of platinum in the urine, serum or splenic tissue.
Section C
Rabbits, guinea pigs and mice which were intravenously injected three
times a week for 3 weeks with various concentrations of palladium - egg albumin
complex showed:
1) no allergic induction
2) no significant palladium levels in the serum
3) significant palladium levels in the urine of guinea pigs
4) no significant palladium levels in the splenic tissues .
-------
Guinea pigs and mice which were subcutaneously injected three times a week for
3 weeks with various concentrations of a palladium-egg albumin complex showed:
1) a delayed allergy to palladium in guinea pigs
2) transfer of the palladium delayed allergy via spleen cells
3) no significant palladium levels in serum of guinea pigs
4) significant palladium levels in the urine of guinea pigs
5) no induced allergy in mice.
Rabbits and guinea pigs which were subcutaneously injected three times a
week for 3 weeks with various concentrations of platinum-egg albumin complex
showed:
1) ' no induced allergy
2) significant platinum levels in the serum of rabbits
3) significant platinum levels in both the urine and serum of guinea pigs.
Section D
Guinea pigs which were subcutaneously injected three times a week for 4
weeks with various concentrations of PtCl > or Pt -albumin (guinea pig or egg)
complex showed no allergic induction.
Guinea pigs which were subcutaneously injected three times a week for 3
weeks with various concentrations of PdCl. complexed to guinea pig or egg
albumin showed:
1) induction of a delayed allergy
2) that 3.7 mg/ml of palladium complexed to egg albumin is the minimum
concentration of palladium necessary for allergic induction
3) that higher palladium levels when complexed to guinea pig albumin will
induce a delayed allergy
4) no immediate type allergy.
Although platinum allergies of the immediate type have been reported (1-8) ,
no such allergic induction (immediate or delayed) has been demonstrated under
the experimental conditions of this report. However, palladium when complexed
to guinea pig or egg albumin will induce a delayed type allergy. As of this
time, no report of palladium allergy is known except that demonstrated in this
report.
-------
SECTION A
SUBCUTANEOUS INJECTIONS
The following is a series of experiments (preliminary) to determine dose, the
route of exposure and animal species needed to develop a test system for the
allergic induction of platinum and palladium.
MATERIALS AND METHODS (GENERAL)
1) Animals .
a) Rabbits - all experimental rabbits used were 5 Ib. albino
females.
b) Guinea pigs - all experimental guinea pigs used were 300 -
350 gram albino females.
c) Mice - all experimental mice used were 28 .- 30 gram white
Swiss females.
2) Determination of platinum and palladium concentration
a) Standardization of AAS (atomic absorption spectro-
photometer) for platinum or palladium in sera
A known serum sample containing 1.0 ml serum (to
avoid matrix interference) , 2.0 ml LaCL and 7.0 ml
platinum or palladium solution (various amounts) for
a total of 10.0 ml was assayed against a serum blank
containing 1.0 ml serum, 2.0 ml LaCl, and 7.0 ml H, 0
to avoid serum adsorption. Solutions containing 1 ppm .
or more recovery were 99.7% for platinum and 96% for
palladium, based upon 140 determinations.
b) Standardization of AAS for platinum or palladium in
urine
A known urine sample containing 1.0 ml urine
2.0 ml LaCl, and 7.0 ml of platinum or palladium
(various amounts) for a 10.0 ml total was assayed
against a urine blank containing 1.0 ml urine,
20 ml LaCl -3 and 7.0 ml H-, 0. In solutions containing
1 ppm or more, recovery was 90% for platinum and
95% for palladium based upon 140 determinations.
-------
c) Determination of palladium and glatinum in urine
and serum
Serum and urine samples were assayed by AAS against
known serum and urine samples.
d) Determination of palladium and platinum in spleen
Spleens were removed and weighed and cell extracts
were assayed by AAS for platinum or palladium con-
centrations . The concentrations were recorded as
mg metal/spleen mass.
Determination of allergic induction
a) Skin test - guinea' pigs and rabbits
The flanks of the animals were shaved and the hair
removed by a depilatory agent two days prior to the
skin testing.
All skin tests were carried out 10-14 days
after the last exposure to platinum or palladium.
0.1 ml of the platinum or palladium solution was
injected intradermally. Test sites were examined
for erythema and/or induration \, 1, 2, 9, 12, 24
and 48 hours later.
b) Passive transfer
A spleen cell suspension in Hank's solution was
used for transfer. A minimum of 5 x 10 cells
were used for transfer. Cell counts were made
with a hemocytometer. Twenty-four hours after
transfer the recipient animals were skin tested.
c) Footpad test in mice
Mice were footpad tested with 0.1 ml of the various
concentrations of PtSO^or PdSO^. Saline was in-
jected in the opposite foot as a control. Caliper
reading was used to determine if a positive footpad
test did occur. The net swelling in the test foot at
24 hr (difference in measurement between 24 hr
reading and pre-injection reading) is compared to
the net swelling in the control foot at 24 hr (difference
in measurement between 24 hr reading and pre-injection
reading) . This net swelling is due to the metal (PtSO^
or PdSO 4. Normal mice were footpad tested using the
same procedure.
-------
EXPERIMENTS AND RESULTS'
Experiment L
Fifteen rabbits and 15 guinear pigs were separated into 3 groups" (5 animals/
group:) and subcutaneousljr injected; with 1.0 ml of PdSO* in-Q,3, 0.1,
0.0 !j mg/ml concentrations three times weekly for four weeks. The palladium:
solution also, contained. Q^Zr OLftT and. Q;JEL g/ml o£ NH^CL (the: result-of.
attempts, to- increase, the. solubility" of FdSGX ) „
Results
1> SfcmrTest
In arrt^KfrnriL tn j-hf "% reme-Htntntiemcr at 'PtftKd -,r alt s^mrrofrtg arrttpyT'cr ixr&r-t*
injected: intradermaliy with. ft. 1 mL o£.QL21.g/ml of Nff^l as a. control.
ATT sitesL-sfeesBeds- a. . J£ to 1.5. cnt ck&xmetvxK swsettE&g, at. 31(L maiv andL I_ &E:~ TBei ..
sweEEing" vwasi ^uun. atrZ lax* snA, at necratic: area; w-itlt erytfaenra otcucred: at
alL :dtes_afe Z
Z) F'alladianK levels .in. sera: and. urine of guiaea pigs and, rabbitsr
Tabtes T aisdr Z'TTstep^ITig«^TiTrv T'tyggfern' trrrgi.g--a.TMt sgrtiTn: erf^ itt.tllu-itlTi'aT'
pig£t injeGtedr subcutaneously" with: various f rt^tfrgrtt p^trmrs- ryf • PdSO'A. Tables
artdr 4; Bigg: 'fef^ p jVT'a-t.Trnm. Tiggt»Tg fn. t?I»«* TTTrfrrt^ amf«$ saj rt.irrtK
TIT gr»
Nbi palladiufli was. detected, in. the spleens of guinea pigs; or rabbits:.
Disc.ussion of ResuBs
tf
of s Q'.iing/nilPjdSC^ soiutionr.shosyedrsign€6can£le^eikaf paBkdia^
frame one aniTpgl . En pigs- recervingp Q-.T. mg/ml dose of EidSCi^ the palladrum; con-
centration ITL the: urine- steadily- rose: to a. high point- at, 18-25 days after: which a£L
o£ tire? animafe diedr. Pigs? reeewitt^ fee; feKesfe: dbse? oi- E3S&£ C®"- ®5 mg/m:i:>
QGCtEEred"
AIL the animals (except; one.} died: fromr the mjectfcms^ w^Mch vecas: unexpected^
This; unexpected, result: deserves; the follawin g camments-. There was marked;
and extensile necrosis at the injectioit site (.hip')i artrf after. 4^6- days- paralysis of
the land? limb' was- observed. Death.oecurred1 within 5 days with the- highes dose.
The highest dose of PdSG-g was 3 mg/ml which also contained 0.21 g/ml of NH^CI.
(Ther NH^d was there because of attempts to increase the solubility of PdSO^ .' )
This.Mghlsa;lt-(NH,;GlXconceiitration: mostrliifcel.y" wooidrcauseiQsnBatic: effects: (ceil
danut-ge and permeability changes) which could: damage or cause malfunction of
the sciatic nerve.
-------
Similar conditions were observed in pigs injected with 0.1 mg/ml PdSCh in 0.07
g/ml NH4C1 and 0.05 mg/ml PdSO4 in 0.035 g/ml NH4 Cl, although paralysis and
death were delayed. Was death due to the PdSC>4 or the NH 40! or both? To obtain
some insight, five pigs were injected with 1.0 ml of a 0.2 g/ml Nffy Cl. solution.
The results were strikingly similar to those observed in pigs receiving both
NH 4C1 and PdSO 4. Paralysis and death occurred.within.5 days .- Thus it would •
appear that the high salt concentration is toxic enough to cause, death in pigs. It
should be noted that the larger (by weight) the pig the longer, it survived; thus .
body weight - dose relationship appears to be involved. This is supported by
the fact that no rabbits (much higher weight - dose relationship) died due to the
injection. However, necrosis at the injection site was observed.
With extensive tissue damage (evident by large necrotic area) it would seem
reasonable that the palladium would enter the blood stream and be filtered by
the kidney which would explain the palladium levels in the urine. However, :
from Table 2 it is obvious that no significant level of palladium in the serum
occurred. How palladium levels occur in the urine and not in the blood is unre-
solved at present.
2) Rabbits ;
Examination of Table 3 shows that the highest palladium level in the urine was
found in rabbits injected with the high dose (0.3 mg/ml) . The lower doses, :
0.1 and 0.05 mg/ml, showed lower palladium urine levels. In all animals, how-
ever, the palladium levels increased up to the 18th day of the experiment.
Table 4 demonstrates no significant palladium serum levels throughout the experi-
ment. ',
Although there was necrosis at the injection site (hip) it was less extensive than
that observed in the pigs. Also, no rabbits died from the injection; the rabbit
deaths that did occur were the result of blood sampling (cardiac puncture) .
3) Skin Tests , .. - ; _ •; '<
All tests were negative.
4) Palladium levels in splenic tissue . - . .
No palladium levels were found. : .,
EXPERIMENT II '. - .,'.'
Fifteen rabbits, guinea pigs and mice were separated into three groups (5 animals/
group) and subcutaneously injected with 1.0 ml of PtSO, in the following concen-
trations: 0.35, 0.10 and 0.05 mg/ml three times weekly Tor four weeks.
Results •
1) Skin test
No edema or erythema occurred. .
2) Footpad test in mice
All were negative.
-------
3) Platinum levels in urine and sera of guinea pigs and rabbits
Tables 5 and 6 list platinum levels in the urine and serum of individual guinea
pigs injected subcutaneously with various concentrations of PtSC>4 .
Tables 7 and 8 list platinum levels in the urine and serum of individual rabbits
injected subcutaneously with various concentrations of PtSC>4 .
4) Platinum concentration in splenic tissues
Table 9 shows the average of PtSC>4 concentration in splenic tissue of guinea
pigs, rabbits and mice subcutaneously injected with various PtSC>4 concen-
trations.
Discussion of Results
1) PtSO. in urine of guinea pigs and rabbits
Significant levels of PtSO. were found in the urine of guinea pigs at only one
point; i.e., in guinea pigs injected with the highest concentration of PtSO. (0.35
mg/ml), the level of PtSO4 in the urine at 21 days reached 0.002 ing/ml. This is
quite low, but significant. No significant levels of PtSO. were found in the urine
of rabbits.
2) PtSO 4 in serum of guinea pigs and rabbits .
No significant levels of PtSO4 were found in the serum of guinea pigs or rabbits.
3) Skin Test
A small area of necrosis occurred at the skin test site with the highest concen-
tration of PtSO4 (0.1 ml of a 0.35 mg/ml cone). This proved to be a toxic reaction
and not an allergic one, since normal animals also show a similar response.
Apparently a 0.1 ml of a 0.35 mg/ml PtSQj solution injected intradermally is toxic
to the dermis.
4) Uptake of Pt5O4 by the spleen in guinea pigs, rabbits and mice
Table 9 lists the amounts of PtSO 4 concentrated in the spleens of the 3 species of
animals. The amounts are comparable since the PtSO4 concentrations are given
in mg/gm spleen weight. In animals injected with the high cone (0.35 mg/ml) .
of PtSO4 , rabbit spleens picked up approximately 0.016 mg/gm, guinea pig
spleens 0.050 mg/gm and mouse spleens 0.141 mg/gm. This suggests roughly a
3-fold increase from rabbits to guinea pigs to mice; i.e., rabbit spleens pick up
X, guinea pigs 3X and mice 10X. The large PtSO4 cone in mouse spleens as
compared to the rabbit spleens is most likely due to the relative size of the spleens
of each species, the rabbit spleen being approximately 6-7 times as large (by
weight) as that of the mouse. The guinea pig spleen is approximately midway
(regarding weight) between that of the rabbit and the mouse. The significance
of PtSO4 concentration in the spleens of various species is questionable at this
point, but may be critical in the over-all immunology and physiology of a species
if a certain concentration would be shown to have toxic effects.
5) Mouse footpad tests
Test animals (animals injected with PtSO4 ) and normal animals gave similar
footpad test reactions.
-------
CONCLUSIONS
1) Generally no significant levels of PtSO4 were found in the urine of guinea
pigs and rabbits.
2) No significant levels of PtSO4 were found in the serum of guinea pigs and
rabbits.
3) No immediate or delayed type allergy was induced in guinea pigs, rabbits
or mice.
4) All skin tests in guinea pigs and rabbits and all footpad tests in mice were
negative.
5) Considerable levels of PtSO4 were found in the spleens of the three species.
The largest amounts of PtSO4 were found in the mouse spleens, the least in
the rabbit spleens.
COMPARISON OF EXPERIMENTS I AND II
Urine levels of palladium and platinum in rabbits and guinea pigs
1) Injected with 0.30 mg/ml PdSO4 and 0.35 mg/ml PtSO4
Rabbits - Significant levels of palladium were found in the urine of rabbits
while no significant levels of platinum were found.
Maximum levels of palladium occurred at 18 days and reached 0.0047 mg/ml,
while the maximum level of platinum occurred at 7 days being 0.0012 mg/ml,
a reading of doubtful significance.
Guinea pigs - Significant levels of palladium were found in the urine of
guinea pigs, maximum levels occurring at 4 days and reading 0.021 mg/ml.
A low but significant level of platinum occurred at 21 days, reading only
0.002 mg/ml.
2) Injected with 0.1 mg/ml PdSO4 and 0.1 mg/ml PtSO4
Rabbits - Significant levels of palladium were found in the urine of rabbits
while no significant level of platinum was found. Maximum levels of pal-
ladium occurred at 4 days (0.0015 mg/ml) .
Guinea pigs - Significant levels of palladium were found throughout the
experimental period, reaching a maximum level of -.0117 mg/ml at 25 days.
No significant levels of platinum were found in the urine of guinea pigs.
3) Injected with 0.05 mg/ml PdSO4 and 0.05 mg/ml PtSO4
Rabbits - Significant levels of palladium were found in the urine. Maximum
level was 0.0015 mg/ml at 4 days. No significant levels of platinum were
found in the urine of rabbits.
10
-------
Guinea pigs - Significant levels of palladium were found throughout the
experimental period, reaching a maximum level of 0.0084 mg/ml at 25 days.
No significant levels of platinum were found.
4) Spleen levels of palladium and platinum in rabbits and guinea pigs
No significant levels of palladium were found. High levels of platinum were
found, which suggests that platinum has a much greater affinity for splenic
tissue than palladium.
Table 10 lists maximum levels of palladium and platinum in the urine of
guinea pigs and rabbits. .
The following statements regarding palladium and platinum levels in the urine of
rabbits and guinea pigs can be made.
1) Much more palladium enters the urine of rabbits and guinea pigs than
platinum.
2) Much more palladium enters the urine of guinea pigs than rabbits.
3) The levels of platinum are below significance for both rabbits and
guinea pigs.
EXPERIMENT III
The purpose of this experiment is to determine the effect of NH4C1 on platinum
levels in urine, sera and spleens. Nine rabbits and guinea pigs were separated
into 3 groups (3 animals/group) and subcutaneously injected with 1.0 ml of 0.35
mg PtSO4/ml and 0.2 g NH4Cl/ml, 0.1 mg PtSO4/ml and 0.07 g NH4/ml, and
0.05 mg PtSO4/ml and 0.03 g NH4Cl/ml three times weekly for four weeks.
Results
1) Platinum levels in urine and sera of guinea pigs and rabbits
Tables 11 and 12 list the platinum levels in serum and urine of individual
rabbits injected subcutaneously with various concentrations of PtSO4
andNH4Cl.
Tables 13 and 14 list the platinum levels in serum and urine of individual
guinea pigs injected subcutaneously with various concentrations of PtSO4
and NH4C1.
2) Platinum concentration in splenic tissues
No platinum was detected in the spleens of the guinea pigs and rabbits.
EXPERIMENT IV
Fifteen rabbits and guinea pigs were separated into three groups (5 animals/
group) and subcutaneously injected with .1.0 ml of 0.3 mg, 0.1 mg and
0.05 mg/ml PdSO^ three times weekly for four weeks.
11
-------
Results
Tables 15, 16, 17 and 18 show no significant levels of palladium in the urine
or sera of guinea pigs or rabbits.
All skin tests were negative.
Discussion
Comparison of the sub cutaneous ly injected PdSO^ and PtSO^ (with and without
•NH d) experiments
Tables 19 and 20 give the comparative palladium and platinum levels in the
sera and urine of rabbits and guinea pigs . The following statements can be
made regarding palladium and platinum in urine and sera of rabbits and
guinea pigs.
1) Sera
Rabbits: Minimal significant levels of PtSO4 occurred only when
NH^Cl was injected with the PtSO4. No significant levels of PdSO4
were detected .
Guinea pigs: No significant levels of PtSO4 or PdSO4 were found.
All pigs died, however, with NH^Cl experiment.
. 2) Urine
Rabbits: Significant levels of palladium occurred in urine and higher
levels. when NH 4C1 is used. No significant levels of platinum were found
Guinea pigs: Significant levels of palladium occurred in urine, higher
when NH^jCl is used. No significant levels of platinum were found.
Note: It does appear that NH 4C1 caused higher levels of palladium to be
found in the urine.
3) Spleen
Significant levels of platinum were found in the spleens of rabbits
and guinea pigs only when PtSC>4 was injected alone. In experiments
where PtSO 4 plus NH 4C1 were injected no significant platinum levels
could be found . Thus it appears that platinum can be taken up in the
spleens of rabbits and guinea pigs and NH4C1 can inhibit this uptake.
Palladium was never found in the spleens of rabbits or guinea pigs.
12
-------
SECTION B
INTRAVENOUS AND DERMAL CONTACT
The following experiments (preliminary) were used to determine if the
intravenous or dermal contact with platinum or palladium would induce an
allergic response.
MATERIALS AND METHODS
See Section A . .
EXPERIMENTS AND RESULTS
.EXPERIMENT V
Fifteen rabbits were separated into three groups (5 animals/group) and
injected intravenously (marginal ear vein) three times a week for three weeks
with 1.0 ml of a 0. 35 mg, 0.10 mg and 0. 05 mg/ml PtSO4 solution.
Forty-five mice were separated into three groups (15 mice/group) and
injected intravenously (tail vein) three times a week for three weeks with 0.25
ml of a 0.35 mg, 0.10 mg and 0.05 mg/ml PtSO4 .
Rabbit skin testing was done 14 days after the last intravenous injection by
injecting 0.1 ml of the 3 PtSO4 concentrations intradermally. Mice were footpad
tested 14 days after the last intravenous injection by injecting 0.05 ml of 3 PtSO4
coneentrations into the hind footpads.
Results
All rabbit skin tests and mouse footpad tests were negative. .Tables 21 and
22 list the PtSO4 levels in the urine and serum of individual rabbits injected
intravenously with various concentrations of PtSO 4. No significant levels were
found. Note: Mouse serum and urine were not used since sufficient quantities
cannot be obtained.
13
-------
Conclusions
1) No allergic induction in rabbits or mice injected intravenously with PtSO4 .
2) No significant levels of PtSC>4 in the urine or sera of rabbits. (Mouse serum
and urine were not used since sufficient quantities cannot be obtained.
SXPI
fteen rabbits (5 rabbits/group), 15 guinea pigs (5 guinea pigs/group)
and 45 mice (15 mice/group) were separated into 3 groups and injected intra-
venously. Rabbits and guinea pigs received 1.0 ml of 0.35 mg, 0.10 mg and
0.05 mg/ml PdSO4 three times a week for 3 weeks. Mice received 0.25 ml instead
of 1.0 ml. The intravenous injections in rabbits were via the marginal ear vein.
In guinea pigs the dorsal sapheous vein and in mice the ventral tail vein were
injected.
Skin and footpad tests were done 12 days after the last intravenous injection.
Results
1) All skin and footpad tests were negative. Note: In the case of the guinea
pig, the dorsal sapheous vein collapses and an extreme hematoma occurs,
making subsequent intravenous injection impossible. The hematoma most
likely is due to the toxic nature of PdSC>4 . (This toxic effect is also seen in
skin test sites where necrosis of the dermis is seen.) Thus guinea pigs re-
-ceived only 5.of the scheduled 9 injeetions. Nevertheless, they were skin
tested along with the rabbits and all sites were negative.
2) There were no detectable palladium levels in the animal spleens.
3) There were no detectable levels of PdSO4 in the urine or sera of rabbits or
guinea pigs.
Conclusions
1) There was no induction of allergy in rabbits, guinea pigs or mice.
2) No PdSO^ was found in urine, sera, spleen or kidney in any of the three
species.
3) The results of this experiment are similar to those of the PtSCK (i.v.)
experiment, i.e. , no metal was found in any of the tissues or urine examined .
4) The guinea pig does not appear to be a good experimental animal for intra-
venous injections of platinum or palladium since these metals are toxic,
causing necrosis in the leg area (injection site) .
14
-------
EXPERIMENT VII
Platinum Paste Experiment
Fourteen rabbits and 15 guinea pigs were exposed to platinum (7 rabbits
exposed.to 0.25 gm/application and 7 exposed to 0.1 gm/application; 8 guinea
pigs exposed to 0.25 gm/application and 7 exposed to 0.1 gm/application) via a
paste (H2O arid PtSC>4) . The paste was applied to the flank of each animal and
held in position by gauze and tape. (Prior to the paste application the hair of
each animal was removed so that the bare skin was in direct contact with the
metal.) Fresh applications were made each week for 5 weeks. Fourteen days
after the last application of platinum paste the animals were skin tested with
various concentrations of PtSO 4.
Results
1) Skin test - All surviving* animals were skin tested 14 days after last
application of platinum paste.
Skin test procedure: 0.1 ml of a 0. 35 mg/ml, 0.1 mg/ml or 0.05 mg/ml PtSC>4
solution was injected intradermally. As a control 0.1 ml of a saline solution
was intradermally injected. Sites were examined 30 min, 1 and 2 hrs, and 1
and 2 days later. All guinea pigs gave a negative skin test. Six rabbits (2
exposed to 0.25 gm/application and 4 exposed to 0.1 gm/application) produced
a questionable skin reaction upon skin testing. A description of the skin
test reactions in these rabbits follows: No reaction at 30 min or 1 hr. At 24
hrs some swelling with mild erythema. Less swelling at 48 hrs with faint
erythema. At 3-4 days necrosis at the platinum site.
Normal rabbits produce the same reactions, suggesting that the reaction was
not allergically mediated, but due to toxic effects of platinum. Nevertheless
2 rabbits (those giving the best reaction on skin testing) were skin tested
again with a 1/50 dilution of the 0.05 mg/ml PtSC>4 solution. The rationale
for injecting the 1/50 dilution of the 0.05 mg/ml PtSO4 was to determine
whether the reaction was due to toxicity or allergy. Again a small area of
necrosis occurred at the test site in these and normal rabbits. Thus it was
concluded that the skin test results were negative. Additionally, serum and
spleen cells from these 2 rabbits were used in an attempted passive transfer
for immediate and delayed allergy. The results for both were negative.
*Deaths in animals before sacrificing were due to bleeding and not due to
exposure to platinum paste (cardiac puncture) .
2) After skin testing all animals were sacrificed (except those used in attempted
passive transfer) and the PtSO 4 levels in the splenic tissue assessed. No
significant levels of PtSO 4 in the spleens of guinea pigs or rabbits exposed
to platinum paste were found.
15
-------
3) Concentration of PtSO4 in urine and serum of rabbits and guinea pigs
exposed to the platinum paste - Samples were taken once a week for 5 weeks
and no significant levels of PtSO^ were found. (Tables 23 - 26)
Conclusions
1) No induction of immediate or delayed allergy by exposure of guinea pigs or
rabbits to platinum paste was observed.
2) No significant levels of PtSO 4 in urine or serum of guinea pigs or rabbits
exposed to platinum paste were noted.
3) No significant levels of PtSO^ were found in the spleens of rabbits or guinea
pigs exposed to platinum paste.
16
-------
SECTION C
METAL ALBUMIN COMPLEX
Since the subcutaneous, intravenous and dermal exposure routes to palladium
arid platinum appear not to induce an allergic state, an alternate approach was
taken. Allergies to metals do exist and in many cases are induced by the metal
complexing to skin or blood proteins. Based on this rationale, experiments were
designed to determine the ability of a palladium or a platinum complex to induce
the allergic state.
MATERIALS AND METHODS
The materials and methods are basically the same as in Sections A and B, with
the following addition.
Procedure for complexing palladium to albumin
A stock solution of palladium-albumin complex is prepared in the following
manner.
One gram of Na2PdCl4 is dissolved in 5 ml phosphate buffer pH7.0
and allowed to set for 18 hours. This solution is autoclaved, and
then 5 ml of a 1 g/50 ml solution of albumin is added (total amount
of albumin added is 100 mg) and allowed to set overnight. The pH
is adjusted to 7.0 with 2.0 M NaOH and the palladium content de-
termined by AAS. This Pd~alb complex solution is centrifuged and
the supernatant is checked for the presence of chlorine by adding
AgNO3 (0.1 g AgNO3 and 5 ml H^O) . A white precipitate forms and
the solution centrifuged. The supernatant is discarded and the pre-
cipitate washed with Ho O. H20 and concentrated NH 4 OH are then added
to the precipitate which dissolves, indicating the presence of chloride.
The presence of the protein was confirmed by the nitrous acid test.
Working solutions of the palladium-albumin complex were prepared by
dilutions of the stock solutions.
EXPERIMENT VIII
Palladium/Egg Albumin Complex (Intravenous Route)
Fifteen rabbits and 15 guinea pigs were separated into three groups(5 animals/
group) and injected intravenously with 1.0 ml of 10 mg Pd and 1.5 mg egg
albumin/ml, 1 mg Pd and 0.15 mg egg albumin/ml and 0.3 mg Pd and 0.05 mg
egg albumin/ml three times per week for 3 weeks. Rabbits and guinea pigs
were skin tested 10 days after the last intravenous injection by an intradermal
17
-------
injection of 0.1 ml of the three palladium/albumin complexed solutions. (Note:
after 4 intravenous injections to guinea pigs, it was necessary to change to
the subcutaneous route due to the collapse of the sapheous vein. Thus guinea
pigs had 4 intravenous and 5 subcutaneous injections. Forty-five mice were
separated into 3 groups (15 mice/group) and injected intravenously with 0.25
ml of each of the 3 palladium/egg albumin complex solutions. Mice were footpad
tested 10 days after the last intravenous injection.
Results
1) Palladium Urine Levels
Tables 27 and 28 list the urine levels of palladium in rabbits and guinea
pigs. Significant levels were attained only in guinea pigs receiving 10 mg
palladium. All serum readings were zero for both species. In rabbits all
skin tests were negative.
2) Skin Test ;
Skin test measurements in guinea pigs are shown in Table 29. Positive
skin reactions were found in guinea pigs receiving all 3 doses. However,
passive transfer was not successful.
3) Footpad Test
Thirty of the 45 mice died before being footpad tested. The concentration
of palladium used apparently is toxic. Breakdown of deaths is as follows:
13/15 died when injected with high concentration
(2.5 mg Pd and 0.4 mg albumin)
10/15 died when injected with middle concentration
(0.25 mg Pd and 0.04 mg albumin)
7/15 died when injected with low concentration
(0.08 mg Pd and 0.01 mg albumin)
The surviving mice (15) were footpad tested, but
demonstrated no allergic induction.
4) Palladium Spleen Level
Palladium was not found in the spleens of any of the species.
CONCLUSIONS
1) There was no allergic induction in rabbits, guinea pigs or mice injected
intravenously with various concentrations of palladium complexed to
albumin three times per week for 3 weeks.
Note: The intravenous injections were administered to guinea pigs only 4
times (8 days), followed by subcutaneous injections. When these pigs
were skin tested they had developed a delayed reaction (delayed hypersensi-
tivity, Table 29). However, this "sensitivity" could not be transferred to
normal recipients and therefore could not be considered a bona fide allergic
18
-------
reaction. The pigs that experienced the apparent delayed reaction were injected
subcutaneously for less than 2 weeks. Thus, they may not have had enough time
to develop a full hypersensitivity. From the results of the second palladium-
albumin complex experiment (2nd part, of this report) , this appears to be the
explanation.
2) Palladium levels in urine and sera of animals
(a) Rabbits - no significant palladium levels in either sera or urine of
rabbits injected intravenously with palladium-albumin complex
(b) Guinea pigs - no significant palladium levels in the sera of guinea
pigs injected intravenously (4 times) and subcutaneously (5 times)
with palladium-albumin complex
(c) Guinea pigs - significant palladium levels found in urine of pigs
injected (4 times i.v. and 5 times s.c.) with 10 mg Pd-1.5 mg
albumin complex •
3) Palladium levels in spleen, kidney and liver of the three species
No significant palladium levels were found in these tissues when the3 species
were injected intravenously with the palladium-albumin complex.
EXPERIMENT IX
Palladium/Egg Albumin Complex (Intravenously in Rabbits and Subcutaneously
in Guinea Pigs)
This experiment was designed to recheck the negative results (no allergic
induction) obtained when rabbits were intravenously injected with the palladium/
egg albumin complex. Also, it was performed to check on the induction of an
allergic state by egg albumin alone, using this immunizing schedule. Since
questionable results were obtained in guinea pigs when part of the injection was
subcutaneous, the subcutaneous route was chosen here for guinea pigs.
Procedure:
' Guinea pigs were injected subcutaneously with 1.0 ml 3 times a week
for 2 weeks with the following solutions:
2 rabbits received 10 mg Pd and 1.5 mg alb/ml
2 rabbits received 1 mg Pd and 0.15 mg alb/ml
1 rabbit received 0.3 mg Pd and 0.05 mg alb/ml
1 rabbit received 1.5 mg alb.ml
1 rabbit received 0.15 mg alb/ml
Guinea pigs were injected subcutaneously with 1.0 ml 3 times a week for
2 weeks with the following solutions:
2 guinea pigs received 10 mg Pd and 1.5 mg alb/ml
2 guinea pigs received 1 mg Pd and 0.. 15 mg alb/ml
19
-------
2 guinea pigs received 0.3 mg Pd and 0.05 mg alb/ml
4 guinea pigs received 1. 5 mg alb
3 guinea pigs received 0.15 mg alb
Mice were injected intravenously with 0.25 ml 3 times per week for
3 weeks with the following solutions:
9 mice received 1.5 mg alb
9 mice received 0.15 mg alb
9 mice received 10 mg Pd and 1.5 mg alb
9 mice received 1 mg Pd and 0.15 mg alb
9 mice received 0.3 mg Pd and 0.05 mg alb
Results
1) Allergic induction to palladium or albumin occurred.
2) There were no significant levels of palladium in sera or urine of rabbits.
See Table 30. . .
3) No significant levels of palladium were found in the liver, kidney or spleens
of rabbits.
4) Skin test results for individual guinea pigs are listed in Table 31 which
shows a delayed hypersensitivity to the palladium-albumin complex de-
veloped in guinea pigs subcutaneously injected with the complex. Passive
transfer was effected via spleen cells as seen from Table 32.
' c^
5) An immediate hypersensitivity to albumin developed in guinea pigs sub-
cutaneously injected with albumin as seen from Table 31 (guinea pigs 1-7) .
Table 32 also shows that passive transfer was effected via serum.
6) All readings of palladium levels in sera of guinea pigs were zero.
7) Palladium levels in urine of guinea pigs showed significance only in pigs
receiving 10 mg Pd - 1.5 mg alb complex (Table 33).
8) No significant levels of palladium were found in the liver, kidney or spleen
of guinea pigs.
9) 14 of 18 mice in the first two groups (those receiving 1.5 and 0.15 mg alb)
died by the fifth injection. This may have been due to the large amount of
foreign protein per body weight or due to an allergic reaction (anaphylaxis)
or a combination of both.
10) 7 of 9 mice in the third group (those receiving 10 mg Pd and 1.5 mg alb) died
before footpad testing. It appears that the Pd-alb complex may inhibit any
anaphylactic reaction since the survival rates are increased. However, the
high concentration of palladium may in itself be toxic.
Only 2 of 9 mice in the fourth group died. Apparently, this concentration of
palladium is better tolerated (1 mg Pd and 0.15 mg alb) . None of the mice
receiving 0.3 mg Pd and 0.05 mg alb died.
20
-------
11) All surviving mice were footpad tested. No allergic induction was demon-
strated by the footpad test; all tests were negative.
12) No significant palladium levels were found in the spleen, liver or kidney
of mice.
CONCLUSIONS . . '
1) There was no allergic induction to palladium by intravenous injection of
Pd-alb complex into rabbits. This confirms the first Pd-alb experiment
and also demonstrated an unexpected result. There was no development
of immediate allergy to the albumin alone. The development of immediate
allergy to albumin under these conditions would be expected, thus the
rabbit appears not to be a good species.
2) Regarding the palladium levels in the sera and urine of rabbits injected
intravenously with Pd-alb complex, the results are quite similar to the
first Pd-alb complex experiment.
3) A delayed hypersensitivity (tuberculin type) was induced in guinea pigs
by the subcutaneous injection of the Pd-alb complex. This .hypersensitivity
can be transferred to normal recipients by spleen cells but not by serum.
Apparently, the palladium acts as a haptenic group on the albumin and
prevents an immediate type allergy to the albumin from developing. Note
that albumin alone injected subcutaneously into a guinea pig does cause
an immediate allergy which can be transferred by serum. This guinea
pig palladium or platinum/albumin complex system should provide a suitable
model by which the immunological response to palladium or platinum can be
studied.
4) No palladium levels were found in sera of guinea pigs subcutaneously
injected with Pd-alb complex. This is similar to the first palladium-
albumin complex experiment.
Significant palladium levels were found in the urine of guinea pigs
subcutaneously injected with 10 mg Pd-1.5 mg alb complex only.
Significant palladium levels were not found with lower Pd-alb com-
plex concentrations. Again this is similar to the results of the first
palladium-albumin complex experiment.
5) No allergic induction to palladium by intravenous injection of a Pd-alb
complex in mice. Mice receiving high concentrations of albumin alone
may have died due to anaphylaxis, but this is only a theoretical expla-
nation and this result is not germaine to this project.
6) No significant palladium levels were found in the spleen, liver or
kidney of the 3 species of animals when injected intravenously or in
guinea pigs when injected subcutaneously, with a Pd-alb complex.
7) Finally, it appears that the guinea pig is the animal of choice for induction
of an allergic state to palladium by the subcutaneous injection of a Pd-alb
complex.
21
-------
EXPERIMENT X
Pt-egg albumin complex in rabbits and guinea pigs
Rabbits and guinea pigs were grouped and subcutaneously injected with various
concentrations of Pt-egg albumin complex 3 times a week for 3 weeks according
to the following schedule:
a) Rabbits received three 1.0 ml subcutaneous injections per week
for 3 weeks.
4 rabbits received 11.2 mg Pt/ml and 7 mg alb/ml
1 rabbit received 7 mg alb/ml
4 rabbits received 3 mg Pt/ml and 1.4 mg alb/ml
1 rabbit received 1.4 mg alb/ml
4 rabbits received 0.02 mg Pt/ml and 0.004 mg alb/ml
1 rabbit received 0.004 mg alb/ml
b) Guinea pigs received three 1.0 ml subcutaneous injections per week for
three weeks.
4 guinea pigs received 11.2 mg Pt/ml and 7 mg alb/ml
1 guinea pig received 7 mg alb/ml
4 guinea pigs received 3 mg Pt/ml and 1.4 mg alb/ml
1 guinea pig received 1.4 mg alb/ml
4 guinea pigs received 0.02 mg Pt/ml*and 0.004 mg alb/ml
1 guinea pig received 0.004 mg alb/ml
Results
1) All skin tests were negative in surviving animals.
2) Serum and urine levels of platinum in rabbits and guinea pigs are shown
in Tables 34- 37.
a) In rabbits, significant levels of platinum were found only
in the serum. Also, with the high concentrations (11.2 mg
Pt), all rabbits died by one week (3rd injection.) Fewer
deaths occurred with the two lower platinum concentrations.
b) In guinea pigs, significant levels of platinum were found in
both serum and urine with all pigs receiving the high concen-
tration (11.2 mg Pt) dying by the first week.
CONCLUSION
A dose of 11.2 mg Pt/ml subcutaneously 3 times appears to be toxic for both
guinea pigs and rabbits; even the lower dose of 3 mg killed 80% of the animals.
In the surviving animals, no allergic induction occurred.
22
-------
EXPERIMENT XI
Rabbits and guinea pigs were grouped and subcutaneously injected with various
concentration of Pt-egg albumin complex 3 times a week for 3 weeks according to
the following schedule:
a) Rabbits received three 1.0 ml subcutaneous injections per week
for 3 weeks
6 rabbits received 2.48 mg Pt and 0.02 mg alb/ml
1 rabbit received 0.02 mg alb/ml
6 rabbits received 0.25 mg Pt and 0.002 mg alb/ml
1 rabbit received 0.002 mg alb/ml
b) Guinea pigs received three 1.0 ml subcutaneous injections per week
for 3 weeks
6 guinea pigs received 2.52 mg Pt and 0.02 mg alb/ml
1 guinea pig received 0.02 mg alb/ml
6 guinea pigs received 0.26 mg Pt and 0.002 mg alb/ml
1 guinea pig received 0.002 mg alb/ml
All animals were skin tested 14 days after the last subcutaneous injection.
Results
All skin tests were negative and no platinum was found in the sera or urine of
either species.
CONCLUSION
Platinum alone or complexed to albumin at concentrations of 4; 5 mg/ml or less
would have minimal toxicity. However, platinum does not appear to induce an
allergic state.
The guinea pig and the subcutaneous route of injection appear to be the best for
allergic induction based upon experimental results using palladium complexed
with egg albumin.
23
-------
SECTION D
ALBUMIN COMPLEX
It has been established from Sections A, B and C that the guinea pig is the best
of the three species (rabbit, guinea pig and mouse) used to induce an allergic
state to platinum or palladium. The subcutaneous route is the best for exposure
of the metal. Pt^+ or Pd^+ alone will not induce an allergic state. If, however,
Pd2+ is complexed to egg albumin, the complex will sensitize (induce an allergic
state in) the guinea pig. A Pt^+ - egg albumin complex will not induce an allergic
state.
The effect of valances on the induction of the allergic state was studied by the
following series of experiments.
EXPERIMENT XII
Guinea pigs were divided into ten groups and injected subcutaneously three
times a week for four weeks with 1.0 ml of the solutions according to the following
schedule:
Number of pigs Solutions
5 PtCl4 -4.5 mg/ml
5 PtCl4 - 1.5 mg/ml
5 PtCl4 complexed to guinea pig albumin
4.5 mg/ml PtCl4 and 0.070 mg/ml albumin
5 PtCl4 complexed to guinea pig albumin
1.5 mg/ml PtCl4 and 0.025 mg/ml albumin
5 PtCl4 complexed to egg albumin
4.5 mg/ml PtCl4 and 0.070 mg/ml albumin
5 PtCl4 complexed to egg albumin
1.5 mg/ml PtCl4 and 0.025 mg/ml albumin
4 Guinea pig albumin - 0.70 mg/ml
4 Guinea pig albumin - 0.025 mg/ml
4 Egg albumin - 0.70 mg/ml
4 Egg albumin - 0.025 mg/ml
Fourteen days after the last subcutaneous injection, all the guinea pigs were skin
tested (intradermally) with 0.1 ml of the 10 solutions plus a buffer solution used
as a diluent in the above 10 solutions. Skin reactions were checked at 30 min,
and at 1, 9, 12, 24 and 48 hours.
Results
All skin test reactions were negative.
24
-------
Comments
The failure of a platinum-albumin complex to induce an allergic reaction in guinea
pigs while a similar palladium-albumin complex experiment has been successful
in allergic induction, prompted the following observations:
1) The concentration of platinum per injection was lower (4.5 mg/ml) than that
of the palladium (10 mg/ml). However, platinum concentrations higher than
4.5 mg/ml per injection were toxic. Also, delayed allergic response to pal-
ladium was induced with metals as low as 1.5 mg/ml per injection.
2) The albumin concentration was less in the present experiment (with platinum)
than the concentration used in the palladium experiment. Thus the failure
to induce an allergic response to platinum complexed to albumin and also to
albumin itself suggests that a higher concentration of albumin may be neces-
sary. Note: the reason a low albumin concentration was used was to de-
termine the minimum albumin concentration needed to complex to the metal
(platinum) for allergic induction.
EXPERIMENT XIII
To determine if the concentration of albumin is critical, a higher concentration was
used here. Guinea pigs were divided into ten groups and injected subcutaneously
three times a week for four weeks with 1.0 ml according to the following schedule:
Number of pigs Solutions
5 . PtCl - 4.5 mg/ml
5 PtClJj - 1.5 mg/ml
5 PtCl4 complexed to guinea pig albumin
4.5 mg/ml PtCl4 and 7 mg/ml albumin
5 PtCl4 complexed to guinea pig albumin
1.5 mg/ml PtCl4 and 2.3 mg/ml albumin
5 PtCl^complexed to egg albumin
4.5 mg/ml PtCl^ and 7 mg/ml albumin
5 PtCl4 complexed to egg albumin
1.5 mg/ml PtC^ and 2.3 mg/ml albumin
4 Guinea pig albumin - 7 mg/ml
4 Guinea pig albumin -2.3 mg/ml
4 Egg albumin - 7 mg/ml
4 Egg albumin - 2.3 mg/ml
Fourteen days after the last subcutaneous injection, all the guinea pigs were skin
tested (intradermally) with 0.1 ml of the 10 solutions plus a buffer solution used
as a diluent in the above 10 solutions. Skin reactions were checked at 30 min,
and at 1, 9, 12, 24, and 48 hours.
Results
1) No positive skin tests in guinea pigs sensitized to PtCl^ alone.
25
-------
2) No positive skin tests in guinea pigs sensitized to PtCL complexed to guinea
pig albumin.
3) Guinea pigs sensitized to PtCl^ complexed to egg albumin gave positive
(delayed) skin tests only to guinea pig or egg albumin.
4) Guinea pigs sensitized to guinea pig albumin gave a positive delayed skin
test reaction only to guinea pig albumin .
5) Guinea pigs sensitized to egg albumin gave a positive delayed skin test
reaction to both guinea pig and egg albumin .
CONCLUSIONS
1) PtCl^ injected subcutaneously (dose 1.4-5 mg/ml) does not induce an
allergic response in guinea pigs.
2) PtCl4 - guinea pig albumin or PtCl4 -egg albumin complex injected subcutane-
ously does not induce an allergic response to platinum in guinea pigs . How-
ever, a delayed type allergy is induced to the albumin. This result differs
from the palladium-egg albumin complex experiment. In that experiment a
delayed type allergy to palladium was induced with no albumin allergy
induction .
3) Thus far it appears that palladium complexed to egg albumin will induce an
allergic state, while platinum complexed to egg albumin will not when injected
subcutaneously into guinea pigs.
EXPERIMENT XIV
Guinea pigs were divided into six groups and were injected subcutaneously three
times a week for three weeks with 1.0 ml according to the following schedule:
Number of pigs Solutions
4.5 mg/ml PdCl4 complexed to egg albumin
4.5 mg/ml PdCl4 complexed to guinea pig albumin
3.8 mg/ml PtCl^ complexed to egg albumin
3.8 mg/ml PtC^ complexed to guinea pig albumin
4 7.0 mg/ml egg albumin
5 7.0 mg/ml guinea pig albumin
Fourteen days after the last subcutaneous injection, all the guinea pigs were skin
tested (intradermally) with a 0.1 ml of the 6 solutions plus a buffer used as a
diluent in the above 6 solutions. Skin reactions were checked at i, 1, 9, 12, 24
and 48 hours.
Results
1) Skin test reactions in guinea pigs injected with 4.5 PdQL complexed to egg
albumin. See Table 38.
2) Skin test reactions in guinea pigs injected with 4.5 PdCl. complexed to
26
-------
guinea pig albumin. See Table 39.
3) Skin test reactions in guinea pigs injected with PtCl, complexed to egg
albumin. All sites negative.
4) Skin test reactions in guinea pigs injected with PtCl^ complexed to guinea pig
albumin. All sites negative.
5) Skin test reactions in guinea pigs injected with egg albumin. Slight reaction
in 2 of 4 pigs when skin tested with egg albumin or the palladium-egg albumin
or the palladium-guinea pig albumin complex.
6) Skin test reactions in guinea pigs injected with guinea pig albumin. All sites
negative.
7) Passive transfer - Skin test reaction in guinea pigs injected with spleen cells
from palladium-egg albumin complex sensitive pigs. See Table 40.
8) Passive transfer - Skin test reactions in guinea pigs injected with spleen cells
from palladium-guinea pig albumin complex sensitive pigs. See Table 41.
CONCLUSION AND COMMENT
1) Guinea pigs sensitized to palladium-egg albumin complex (4.5 mg/ml Pd and
7.0 mg/ml egg albumin) showed a delayed allergy when skin tested with pal-
ladium-egg albumin or palladium-guinea pig albumin complexes. No allergy
developed to egg albumin or guinea pig albumin alone. It appears that in the
palladium-egg albumin complex the palladium acts as an haptene, thus de-
terming the antigenicity of the complex.
Confirmation of the induction of delayed allergy to palladium-egg albumin
complex was obtained via passive transfer.
Spleen cells from palladium-egg albumin sensitive pigs, when injected intra-
peritoneally into normal recipients will confer a delayed type allergy.
2) Guinea pigs sensitized to palladium-guinea pig albumin complex (4.5 mg/ml
Pd and 7.0 mg/ml guinea pig albumin) showed a delayed type allergy when
skin tested with palladium-guinea pig albumin or palladium-egg albumin
complexes.
Passive transfer was also effected by the intraperitoneal injection into normal
recipients spleen cells from guinea pigs sensitized to palladium-guinea pig
albumin complex.
3) Guinea pigs injected with platinum-egg albumin or platinum-guinea pig
albumin complexes (3.8 mg/ml platinum and 7.0 mg/ml of egg or guinea pig
albumin) failed to induce an allergic response.
It thus appears that platinum alone or in the complexed state (complexed with egg
or guinea pig albumin) will not induce any type of allergy. However, palladium
when complexed to egg or guinea pig albumin will induce a delayed allergy which
27
-------
can be transferred to normal recipients via spleen cells from sensitive
donors.
Two additional experiments were designed to determine the minimal amount of
palladium (in the complexed state) needed to induce the allergy.
EXPERIMENTS XV AND XVI
Guinea pigs were separated into 8 groups and injected subcutaneously three
times a week for three weeks with 1.0 ml of various concentrations of pal-
ladium-albumin complex,according to the following schedule:
Number of pigs Solutions
7 2.0 mg/ml Pd complexed to 3.5 mg/ml guinea pig albumin
7 2.0 mg/ml Pd complexed to 3.5 mg/ml egg albumin
7 1.0 mg/ml Pd complexed to 1.7 mg/ml guinea pig albumin
7 1.0 mg/ml Pd complexed to 1.7 mg/ml egg albumin
7 0.88 mg/ml Pd complexed to 1.4 mg/ml guinea pig albumin
7 0.88 mg/ml Pd complexed to 1,4 mg/ml egg albumin
7 0.22 mg/ml Pd complexed to 0.75 mg/ml guinea pig albumin
7 0.22 mg/ml Pd complexed to 0.75 mg/ml egg albumin
Seven days after the last subcutaneous injection all guinea pigs were skin tested.
Results
All skin tests were negative.
CONCLUSIONS
Palladium concentrations of 2.0 mg/ml or less fail to induce an allergic state.
EXPERIMENT XVn
Guinea pigs were "separated into 8 groups and injected subcutaneously three times
a week for 3 weeks with 1.0 ml of various concentrations of palladium-albumin
complex solution according to the following schedule:
Number of pigs Solutions
7 3.7 mg/ml Pd complexed to 5.75 mg/ml egg albumin
7 3.2 mg/ml Pd complexed to 5.0 mg/ml egg albumin
7 2.7 mg/ml Pd complexed to 4.2 mg/ml egg albumin
7 3.7 mg/ml Pd complexed to 5.75 mg/ml guinea pig albumin
7 3.2 mg/ml Pd complexed to 5.2 mg/ml guinea pig albumin
7 2.7 mg/ml Pd complexed to 4.2 mg/ml guinea pig albumin
4 5.75 mg/ml egg albumin
4 5.75 mg/ml guinea pig albumin
Ten days after the last subcutaneous injection all guinea pigs were skin tested
intradermally with 0.1 ml of each of the palladium-albumin (egg or guinea pig)
28
-------
solutions plus buffer solution containing the three concentrations of palladium
(without albumin), buffer solution containing egg or guinea pig albumin, and
buffer alone for a total of 12 skin test solutions.
Results
Only guinea pigs injected with 3.7 mg/ml palladium complexed to 5.75 mg/ml-
egg albumin gave a positive skin test when tested with 0.1 ml of the 3.7 mg/ml
Pd complexed to 5.75 mg/ml egg albumin. Table 42 lists the individual reaction
areas. Passive transfer of the allergy was effected in four normal guinea pigs
by intradermally injecting 5 X 10° spleen cells from 4 of the 6 sensitized guinea
pigs . Table 43 lists the reaction areas in the recipient pigs. When skin tested
24 hr after receiving sensitized spleen cells.
CONCLUSIONS
A palladium concentration of 3.7 mg/ml appears to be the minimum concentration
of palladium that will induce an allergic response when the palladium is
complexed to egg albumin. If guinea pig albumin is used no allergic induction
occurs.
EXPERIMENT XVIII
Guinea pigs were separated into 6 groups and injected subcutaneously three
times a week for three weeks with 1.0 ml of various concentrations of a palladium-
albumin complex solution according to the following schedule:
Number of pigs Solutions
8 5.0 mg/ml Pd complexed to 7.0 mg/ml egg albumin
8 3.75 mg/ml Pd complexed to 7.0 mg/ml egg albumin
8 5.0 mg/ml Pd complexed to 7.0 mg/ml guinea pig albumin
8 3.75 mg/ml Pd complexed to 7.0 mg/ml egg albumin
2 7.0 mg/ml egg albumin
2 7.0 mg/ml guinea pig albumin
Ten days after the last subcutaneous injection all guinea pigs were skin tested
intradermally with 0.1 ml of each of the palladium-albumin (egg or guinea pig)
solutions plus buffer solution containing the 2 concentrations of palladium (with-
out albumin), buffer solution containing egg or guinea pig albumin, and buffer
alone for a total of 9 skin test solutions.
Results
Only guinea pigs injected with palladium complexed to egg albumin showed re-
actions upon skin testing. The only solutions used for skin testing that produced
a positive reaction were 5.0 mg and 3.75 mg palladium complexed to egg albumin.
Those reaction areas are listed in Table 44. Passive transfer of the sensitivity
was successful and the skin test reactions in recipient guinea pigs are listed in
Table 45.
29
-------
CONCLUSIONS
1) Delayed allergy is induced with palladium complexed to egg albumin, mini-
mum concentration 3.7 mg/ml.
2) Palladium concentrations higher than 5.0 mg/ml complexed to guinea pig
albumin will induce a delayed allergy.
30
-------
REFERENCES
1. Freedman, S.O. and J. Krupey. Respiratory allergy caused by platinum
salts. J. Allergy 43:233. (1968).
2. Levene, G.M. and C. D. Calnan. Platinum hypersensitivity: treatment by
specific hyposensitization. Clin. Allergy. 1:75. (1971).
3. Roberts, A. Platinosis - a five year study of the effects of soluble platinum
salts on employees in a platinum laboratory and refinery. Arch. Ind. Hyg.
4:549. (1951).
4. Hunter, D. The diseases of occupations. The English University Press .
LTD. London, Fourth Ed. p. 480. (1969).
5. Parrot, J. L., R. Herbert., A. Saindelle, and F. Ruff. Platinum and platin-
osis. Arch. Environ. Health 19:685. (1969).
6. Levene, G.M. Platinum sensitivity. Br. J. Derm. 85: 590. (1971).
7. Saindelle, A. andF. Ruff. Histamine release by sodium chloroplatinate.
Br. J. Pharmac. 35:313. (1969).
8. Hunter, D. , R. Milton and K. M. A. Perry. Asthma caused by the complex
salt of platinum. Brit. J. Indust. Med. 2: 92 . (1945).
31
-------
TABLE 1. Pd Levels at Various Times in Urine of Guinea Pigs Injected S.C,
3 Times a Week for 4 Weeks with Various
Concentrations of
(Significant to 0.001 mg/ml)
Cone.
Inj.
mg/ml
0.3
0.1
0.05
Guinea
Pig
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
*
0.0000
0.0001
0.0001
0.0001
0.0000
0.0000
0.0000
0.0000
*
Day of Experiment
4 11 18
mg/ml
0.0022
0.0101
0.0175
*
0.0570
*
0.0071
0.0015
0.0015
0.0073
0.0019
0.0069
0.0017
0.0059
*
mg/ml
0.0018
*
*
*
*
*
*
0.0024
0.0052
0.0017
0.0000
0.0001
0.0004
0.0057
*
mg/ml
0.1055
*
*
*
*
*
*.
0.0108
0.0104
0.0097
0.0008
0.0029
0.0003
0.0049
*
25
mg/ml
*
*
*
*
*
*
*
0.0090
0.0143
*
0.0090
*
0.0100
0.0062
*
32
mg/ml
*
*
*
*
*
*
*
*
*
*
*
*.
0.0002
*.
*
*No results due to death of animal.
32
-------
TABLE 2. Pd Levels at Various Times in Serum of Guinea Pigs Injected S.C.
3 Times a Week for 4 Weeks with Various
Concentrations of PdSC>4 •
(Significant to 0.0005 mg/ml)
Day of Experiment
Coric .
Inj.
mg/ml
0.3
0.1
0..05
Guinea
Pig
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
mg/ml
0.0005
0.0004
0.0005
0.0004
0.0001
*
.0.0001
0.0000
0.0001
0.0000
0.0002
0.0000
0.0002
0.0008
*
9
mg/ml
0.0000
*
*
*
*
*
0.0000
0.0000
0.0000
0.0002
0.0000
0.0002
0.0000
0.0000
*
16
mg/ml
0.0000
*
*
*
*
*
*
0.0000
0.0002
0.0000
0.0000
0.0000
0.0000
0.0000
*
23
mg/ml
0.0002
*
*
*
*
*
*
0.0001
0.0001
*
0.0000
*
0,0000
0.0007
*
30
mg/ml
*
*
*
*
*
*
*
*
*
*
*
*
0.0000
0.0000
*
*No results due to death of animal.
33
-------
TABLE 3. Pd Levels at Various Times in Urine of Rabbits Injected S.C,
3 Times a Week for 4 Weeks with Various
Concentrations of PdSO/j.
(Significant to 0.001 mg/ml)
Day of Experiment
Cone.
Inj.
mg/ml
0.3
0.1
0.05
Rabbit
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
*
0.0000
0.0000
0.0000
0.0002
0.0000
0.0006
0.0000
0.0003
0.0000
0.0000
*
4
mg/ml
0.0065
0.0026
0.0023
*
0.0000
0.0042
0.0000
0.0050
0.0004
0.0006
0.0013
0.0022
0.0018
0.0009
*
11
mg/ml
0.0020
0.0045
0.0000
*
*
0.0010
0.0005
0.0006
0.0003
0.0008
0.0003
0.0001
0.0014
0.0004
*
18
mg/ml
0.0033
0.0000
0.0050
*
*
0.0060
0.0009
0.0011
0.0010
0.0005
0.0005-
0.0005
0.0012
0.0005
*
25
mg/ml
0.0010
*
0.0010
*
*
0.0020
0.0001
0.0001
0.0000
0.0001
0.0001
0.0001
0.0001
*
*
32
mg/ml
0.0005
*
0.0001
*
*
0.0001
0.0000
0.0000
*
0.0000
0.0000
0.0000
0.0000
*
*
*No results due to death of animal.
34
-------
TABLE 4. Pd Levels at Various Times in Serum of Rabbits Injected S.C.
3 Times a Week for 4 Weeks with Various
Concentrations of
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
•Inj,
mg/ml Rabbit
0.3 1
2
3
4
5
6
0.1 7
8
9
10
0.05 11
12
13
14
15
0
mg/ml
0.0000
0,0000
0.0005
*
0.0000
0.0003
0.0000
0.0002
0.0000
0.0006
0.0000
0.0003
0.0000
0.0000
*
9
mg/ml
0.0000
0.0004
0.0001
*
0.0008
0.0001
0.0000
0.0000
0.0000
0.0000
0.0001
0.0000
0.0000
0.0000
*
16
mg/ml
0,0002
*
0.0000
*
*
0.0002
0.0000
0.0002
0.0000
0.0006
0.0000
0.0000
0.0000
0.0002
*
23
mg/ml
0.0003
*
0.0000
*
• *
0.0002
0.0000
0.0000
0.0001
0.0007
0.0001
0.0001
0.0000
0.0001
*
30
mg/ml
0.0002
*
0.0001
*
*
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000.
0.0001
*
*
*No results due to death of animal.
35
-------
TABLE 5. Pt Levels at Various Times in Urine of Guinea Pigs Injected S. C,
3 Times a Week for 4 Weeks with Various
Concentrations of PTSO4.
(Significant to 0.001 mg/ml)
Day of Experiment
Cone.
Inj . Guinea
mg/ml Pig
0.35 1
2
3
4
5
0.1 6
7
8
9
10
0.05 11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0010
0.0005
0.0005
0.0005
0.0010
0.0005
0.0005
0.0000
0.0000
0.0000
7
mg/ml
0.0020
0.0030
0.0010
0.0005
0.0000
0.0000
0.0000
0.0020
0.0015
0.0000
0.0010
0.0005
0.0015
0.0005
0.0000
14
mg/ml
0.0006
0.0006
0.0000
0.0006
0.0000
0.0000
0.0000
0.0000
0.0006
0.0000
0.0012
0.0000
0.0006
0.0000
0.0000
21
mg/ml
0.0015
0.0005
0.0025
0.0040
0.0025
0.0000
0.0005
0.0010
0.0005
0.0000
0.0010
d.oooo
0.0005
0.0000
0.0005
36
mg/ml
0.0013
0.0000
0.0007
0.0000
0.0000
*
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*No results due to death of animal.
36
-------
TABLE 6. Pt Levels at Various Times in Serum of Guinea Pigs Injected S .C
3 Times a Week for 4 Weeks with Various
Concentrations of PtSO A .
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
Inj . Guinea
mg/ml Pig
0.35 1
2
3
4
5
0..1 6
7
8
9
10
0,,05 11
12
13
14
15
0
mg/ml
0.
0.
0.
0.
0.
•o.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0000
0000
0000
0000
0005
0000
0000
0000
0000
0000
0000
oooo
0005
0000
9
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0004
0004
0000
0000
0000
0004
0000
0004
0004
0004
0000
0000
0004
0000
0004
16
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0,
0.
0.
0.
0.
0.
0004
0000
0000
0000
0000
0000
0000
0000
0000
0000
0004
0000
0000
0000
0000
21
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0006
0006
0000
0006
0006
0000
0006
0000
0006
0000
0000
0000
0006
0000
0006
35
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
•o.
0.
0.
0000
0000
0000
0000
0000
*
0000
0000
0000
0000
0000
0000
0000 ,
0000
0000
*No results due to death of animal.
37
-------
TABLE 7. Pt Levels at Various Times in Urine of Rabbits Injected S .C .
3 Times a Week for 4 Weeks with Various
Concentrations of PtSO A.
(Significant to 0.001 mg/ml)
Cone.
Inj.
mg/ml Rabbit
0.35 1
2
3
4
5
0.1 6
7
8
9
10
0.05 11
12
13
14
15
Day of Experiment
07 14
mg/ml
0.0000
0.0000
0.0000
0.0000
0,0000
0.0000
0.0010
0.0000
0.0000
0.0000
0.0005
0.0000
0.0000
0.0000
0.0000
mg/ml
0.0015
0.0015
0.0005
0.0005
0.0020
0.0005
0.0015
0.0010
0.0005
0.0010
0.0005
0.0005
0.0005
0.0010
0.0010
mg/ml
0.0006
0.0006
0.0006
0.0012
0.0006
0.0006
0.0006
0.0006
0.0000
0.0006
0.0000
0.0000
0.0006
0.0000
0.0000
21
mg/ml
0.0010
0.0010
*
0.0000
0.0000
0.0005
0.0000
0.0000
0.0010
0.0000
0.0000
0.0005
0.0000
0.0005
0.0000
36
mg/ml .
0.0013
0 . 0000
*
0.0000
0,0000
*
0.0013
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0013
*
*No results due to death of animal.
38
-------
TABLE 8. Pt Levels at Various Times in Serum of Rabbits Injected S .C.
3 Times a Week for 4 Weeks with Various
Concentrations of PtSO ..
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
Inj. .
mg/ml Rabbit
0.35 1
2
3
4
5
0.1 6
7
8
9
10
0.05 11
12
13
14
15
0
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0000
0000
0000
0005
0000
0000
0000
0000
0000
0000
0000
0000
0000
0005
9
mg/ml
0.
0.
0.
0.
0.
0.
o.
0.
0.
0.
0.
0.
0.
0.
0.
0004
0004
0000
0000
0004
0004
0004
0000
0004
0004
0000
0000
0000
0000
0004
16
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
21
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0006
0000
*
0000
0000
0006
0000
0000
0000
0000
0006
0006
0000
0000
*
35
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0000
*
0000
0000
*
0000
0000
0000
0000
0000
0000
0000
0000
*
*No results due to death of animal.
39
-------
TABLE 9. Average concentration of PtSO, in splenic tissue of guinea pigs,
rabbits and mice injected with various concentrations of PtSO,
3 times a week for 4 weeks.
Guinea Pigs Rabbits Mice
Inj. no. of avg. PtSO4no. of avg. PtSO4 no. of avg. PtSO4
Dose animals in spleen animals in spleen animals in spleen
mg/ml mg/gm mg/gm
0.35 5 0.0503 5 0.0164 4 0.1414
0.10 5 0.0296 5 0 5 0.2865
0.05 5 0.0288 5 0.0118 5 0
40
-------
TABLE 10. Levels of PdSO4 and PtSO4 in
the urine of rabbits and guinea pigs.
Rabbits Guinea Pigs
Irij. maximum levels of metal in urine maximum levels of metal in urine
dose mg/ml mg/ml
mg/ml PdSO4 PtSO4 PdSO4 PtSO4
0,35 0.0047 0.0012 0.0210 0.0012
0.10 0.0015 0.0009 0.0117 0.0007
0.05 0.0015 0.0007 0.0084 0.0007
41
-------
TABLE 11. Pt Levels at Various Times in Serum of Rabbit Injected S .C.
3 Times a Week for 4 Weeks with Various
Concentrations of PtSO 4 + NH 4<31.
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
Inj.
0 . 3 mg PtSO 4/ml
and
0.2 g NH4Cl/ml
0 . 1 mg PtSO 4/ml
and
0.07 g NH 4Cl/ml
0.05 mg PtSO4/ml
and
* 0.03 g NH4Cl/ml
Rabbit
1
2
3
4
5
6
. 7
8
9
0
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0000
0000
0000
0000
0000
0008
0000
0008
5
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0000
0000
0010
0010
0010
0000
0010
0000
12
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0000
0000
0000
0007
0000
0007
0007
0000
19
mg/ml
0
0
0
0
0
0
0
0
.0008
.0000
.0008
.0000
.0000
.0000
*
.0008
.0000
26
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0010
0000
0000
0000
0010
0000
*
0000
0000
*No results due to death of animal,
42
-------
TABLE 12, Pt Levels at Various Times in Urine of Rabbits Injected S.C.
3 Times a Week for 4 Weeks with Various
Concentrations of PtSC>4+ NP^Cl.
(Significant to 0.001 mg/ml)
Day of Experiment
Cone .
Inj.
Rabbit
0
mg/ml
7
mg/ml
13
mg/ml
20
mg/ml
27
mg/ml
0.3 mg PtSO4/ml 1
and 2
0.2 g NH Cl/ml 3
0.1 mg PtSO /ml 4
and 4 5
0.07 g NH4Cl/ml 6
0.05 mg PtSOVml. 7
and 8
0.03 g NH4Cl/ml 9
0.0000 0.0000 0.0010 0.0004 0.0004
0.0000 0.0000 0.0005 0.0000 0.0000
0.0000 0.0000 0.0000 0.0000 *
0.0005 0.0000 0.0000 0.0000 0.0011
0.0000 0.0005 0.0015 0.0004 0.0004
0.0000 0.0000 0.0010 0.0004 0.0004
0.0000 0.0005 0.0005 * . ' *
0.0000 0.0000 0.0000 0.0000 0.0000
0.0000 0.000 0.0000 0.0000 0.0007
*No result due to death of animal.
43
-------
TABLE 13. Pt Levels at Various Times in Serum of Guinea Pigs Injected S.C,
3 Times a Week for 4 Weeks with Various
Concentrations of PtSO 4 + NH 4 Cl.
(Significant to 0.0005 mg/ml)
Cone . Guinea
Inj.
0.3 mg PTSO /ml
and 4
0.2 g NH4 Cl/ml
0 . 1 mg PtSO 4 /ml
and
0.07 g NH4 Cl/ml
0.05 mg PtSO4 /ml
and
0.03 g NH4 Cl/ml
Pig
1
2
3
4
5
7
9
10
11
Day of Experiment
0 5
mg/ml
0
0
0
0
0
0
0
0
0
.0008
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
mg/ml
0
0
0
0
0
0
0
.0010
*
*
.0010
.0000
.0010
.0000
.0000
.0010
12
mg/ml
0
0
0
0
0
*
*
*
.0000
*
.0000
.0007
.0000
.0000
19
mg/ml
0
0
0
0
0
*
*
*
.0008
*
.0008
.0000
.0000
.0000
26
mg/ml
0
0
0
0
0
*
*
*
.0020
*
.0010
.0010
.0010
.0000
*No results due to death of animal.
44
-------
TABLE 14. Pt Levels at Various Times in Urine of Guinea Pigs Injected S.C,
3 Times a Week for 4 Weeks with Various
Concentrations of PtSO4 + NH 4 Cl.
(Significant to 0.001 mg/ml)
Day of Experiment
Guinea
Cone . Inj .
0.3 mg PtSO4 /ml
and
0.2 g NH4 Cl/ml
0.3. mg PtSO4 /ml
and
0.07 g NH4 Cl/ml
0.05 mg PtSO4 /ml
and
0.03 g NH4 Cl/ml
Pig
1
2
3
4
5
7
9
10
11
0
mg/ml
0.0000
0.0005
0.0000
0.0005
0.0000
0.0005
**
0.0000
0.0000
7
mg/ml
*
*
*
0.0000
*
0.0000
0.0000
0.0000
0.0000
13
mg/ml
*
*
*
0.0005
*
0.0000
0.0005
0.0000
0.0000
20
mg/ml
*
*
*
0.0000
*
0.0004
0.0000
0.0000
0.0004
27
mg/ml
*
*
*
0.0000
*
0.0004
0.0004
0.0000
0.0000
*No results due to death of animal
**No urine collected.
45
-------
TABLE 15. Pd Levels at Various Times in Urine of Guinea Pigs Injected S.C,
3 Times a Week for 4 Weeks with Various
Concentrations of PdSO. .
(Significant to 0.001 mg/ml)
Day of Experiment
Cone.
Inj.
mg/ml
0.3
0.1
0.05
Guinea
Pig
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
4
mg/ml
0.0006
0.0009
0.0003
0.0005
0.0006
0.0005
0.0002
0.0002
0.0001
0.0001
0.0000
0.0000
0.0000
0.0000
0.0000
11
mg/ml
0.0000
0.0000
0.0003
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
0.0000
0.0000
0.0000
0.0000
18
mg/ml
0.0006
0.0007
0.0003
0.0006
0.0009
0.0000
*
*
0.0000
*
*
0.0005
0.0000
0.0001
0.0000
25
mg/ml
0.0009
0.0004
0.0006
0.0005
0.0003
0.0005
*
*
0.0004
. *
*
0.0000
0.0000
0.0001
0.0003
*No results due to death of animal.
46
-------
TABLE 16. Pd Levels at Various Times in Serum of Guinea Pigs Injected S.C,
3 Times a Week for 4 Weeks with Various
Concentrations of PdSO *.
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
Inj.
mg/ml
0.3
0.1
0.05
Guinea
Pig
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
2
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
9
mg/ml
0.0000
0.0002
0.0002
0.0000
0.0000
0.0000
0.0000
0.0000
0.0002
0.0000
*
0.0000
0.0000
0.0002
0.0002
16
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
0.0000
0.0000
0.0000
0.0000
23
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
.*
*
0.0000
*
*
0.0000
0.0000
0.0000
0.0000
*No results due to death of animal.
***No blood was obtained.
47
-------
TABLE 17. Pd Levels at Various Times in Urine of Rabbits Injected S .C,
3 Times a Week for 4 Weeks with Various
Concentrations of PdSO4 .
(Significant to 0.001 mg/ml)
Day of Experiment
Cone.
Inj.
mg/ml
0.3
0.1
0.05
Rabbit
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
o.oobo
0.0000
0.0000
0.0000
*
4
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0002
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
11
mg/ml
0.0000
Q.OOOO
0.0000
0.0000
0.0000
0.0000
0.0000
0.0002
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
18
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0002
0.0000
0.0000
0.0002
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
25
mg/ml
*
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
*No results due to death of animal.
48
-------
TABLE 18. Pd Levels at Various Times in Serum of Rabbits Injected S.C,
3 Times a Week for 4 Weeks with Various
Concentrations of PdSO 4 .
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
Inj.
mg/ml Rabbit
0.3 1
2
3
4
5
0, 1 6
7
8
9
10
0.05 11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
2
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
9
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0002
0.0000
0.0000
0.0000
0.0000
0.0000
*
16
mg/ml
0.0000
0.0000
0.0000
0 . 0002
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
23
mg/ml
0.0000
0 . 0000
O.OQOO
0 . 0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0002
0.0000
*
*No results due to death of animal.
49
-------
TABLE 19. Average Maximum Levels of Metal in the Urine of Animals
in mg/ml. (Significant to 0.001 mg/ml)
Ui
o
Injection
Dose
mg/ml
0.35
0.10
0.05
Pd S04
NH4 Cl
0.0047
0.0015
0.0015
RABBITS
Injected with
Pd S04
0.0000
0.0000
o.oooo
Pt SO4 Pt SO4
NH4C1
0.0005 0.0012
0.0008 0.0009
0.0003 0.0007
GUINEA PIGS
Injected with
Pd SO4 Pd S04
NJ| Cf
0.0210 0.0006
0.0117 0.0005
0.0084 0.0003
Pt S04
NH4 Cl
0.0005
0.0005
0.0005
Pt S04
0.0020
0.0007
0.0007
-------
TABLE 20. Average Maximum Levels of Metal in the Serum of Animals
in mg/ml (Significant to 0.0005 mg/ml)
Injection
Dose
mg/ml
0.35
0.10
0.05
Pd SO4
NH4 Cl
0.
0.
0.
0001
0000
0000
RABBITS
Injected with
Pd SO4
0.0000
0.0000
0.0000
Pt SO4 Pt SO4
NH 4 Cl
0,0005 0.0002
0.0003 0.0003
0.0005 0.0003
Pd SO4
NH4 Cl
All died
0.0000
0.0000
GUINEA PIGS
Injected with
Pd SO4
0.0000
0.0000
0.0000
Pt SO4
NPtj Cl
0.0010
0.0015
0.0007
Pt S04
0.0004
0.0003
0.0002
-------
TABLE 21. Pt Levels at Various Times in Urine of Rabbits Injected I'.V,
3 Times a Week for 3 Weeks with Various
Concentrations of PtSO 4 .
(Significant to 0.001 mg/ml)
Day of Experiment
Cone .
In j .
mg/ml Rabbit
0.3 1
2
3
4
5
0.1 6
7
8
9
10
0.05 11
12
13
14
15
0
mg/ml
0.0004
0.0000
**
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
0.0000
4
mg/ml
0.0005
0.0015
0.0000
0.0015
0.0000
0.0000
0.0000
0.0005
0.0000
0.0000
0.0005
0.0000
0.0000
*
0.0000
11
mg/ml
0.0006
0.0000
0.0000
0.0006
0.0000
0.0000
0.0000
0.0000
0.0000
o.oooo
0.0000
0.0000
0.0000
*
0.0000
18
mg/ml
0.0007
0.0000
0.0000
0.0000
0.0007
0.0000
0.0000
0.0013
0.0000
0.0000
0.0000
0.0000
0.0000
*
0.0000
*No results due to death of animal.
**No urine.
52
-------
TABLE 22 . Pt Levels at Various Times in Serum of Rabbits Injected I'. V.
3 Times a Week for 3 Weeks with Various
Concentrations of PtSO 4.
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
Inj.
mg/ml Rabbit
0.3 1
2
3
4
5
0.1 6
7
8
9
10
0.05 11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0008
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
0.0008
2
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0,0000
0.0000
0.0000
0.0000
*
0.0000
9
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0020
0.0000
0.0000
0.0000
*
0.0000
16
mg/ml
0.0010
0.0000
0.0000
0.0010
0.0010
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
*
0.0000
*No results due to death of animal.
53
-------
TABLE 23. Pt Levels at Various Times in Urine of Rabbits Exposed
Once a Week for 5 Weeks to Various Concentrations
of PtSO4 Paste.
(Significant to 0. 001 mg/ml)
Cone . Rabbit
g
0.25 1
2
3
13
5
6
14
0.10 7
8
9
10
11
12
15
0
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0006
0011
0006
0017
0017
0006
0006
0000
0000
0011
0000
0011
0000
Day of Experiment
3 10
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0006
0000
0000
0000
0011
0011
0000
0006
0000
0000
0000
0006
0000
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0004
0004
0000
0004
0009
0009
0013
0000
00.00
0000
0000
0009
0004
0004
17
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0015
0015
0015
0005
0010
0010
0015
0005
0000
0183*
0005
0000
0000
0015
24
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0011
0011
0000
0000
0000
0000
0000
0000
0000
0000
0000
0006
0006
0000
31
mg/ml
0.0007
0.0007
0.0000
0.0000
0.0007
0.0013
0.0027
0.0000
0.0007
0.0007
0.0000
0.0020
0.0040
0.0000
*Rabbit removed patch and PtSO * fell into urine collection pan,
54
-------
TABLE 24. Pt Levels at Various Times in Serum of Rabbits Exposed
Once a Week for 5 Weeks to Various Concentrations
of PtSO4 Paste.
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone . Rabbit
g
0.25 1
2
3
13
5
6
14
0.10 7
8
9
10
11
12
15
0
mg/ml
0.0005
0.0000
0.0000
0.0000
0.0000
0.0000
0.0005
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0005
4
mg/ml
0.0000
0.0005
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0005
0.0005
0.0000
0.0005
11
mg/ml
0.0000
0.0005
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
18
mg/ml
0.0006
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
25
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
o.odoo
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
32
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0 . 0000
0.0000
55
-------
TABLE 25. Pt Levels at Various Times in Urine of Guinea Pigs Exposed
Once a Week for 5 Weeks to Various Concentrations
of PtSO 4 Paste.
(Significant to 0.001 mg/ml)
Cone . Guinea
g Pig
0.25 1
2
3
4
5
6
7
8
0.10 9
10
11
12
13
14
15
0
mg/ml
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.0000
.0005
.0005
.0000
.0000
.0000
.0000
.0011
.0006
.0000
.0000
.0006
.0006
.0011
.0006
Day of Experiment
3 10 17
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
0005
0005
0000
0000
0000
0000
0005
0000
0000
0000
0000
0006
0000
0000
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0009
0437**
0027
0000
*
0009
0004
0022
0000
0000
0009
0004
0004
0004
0007
mg/ml
0.0000
0.0040
0.0035
0.0000
*
0.0000
0.0000
0.0010
0.0010
0.0000
*
0.0005
0.0000
0.0010
0.0010
24
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0006
*
0000
0000
*
0000
0017
0017
0000
0006
*
0000
0000
0000
0000
31
mg/ml
0
0
0
N
0
0
0
0
0
0
0
0
.0000
*
.0020
.0007
*
.0040
.0353**
.0007
.0000
***
*
.0007
.0000
.0020
.0007
*No results due to death of animal.
**Unusual high reading due to the pigs removal of the patch and its
falling into the urine collection pan.
***No urine collected.
56
-------
TABLE 26. Pt Levels at Various Times in Serum of Guinea Pigs Exposed
Once a Week for 5 Weeks to Various Concentrations
ofPtSO, Paste.
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
i
•0.-25
0.10
Guinea
Pig
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
mg/ml
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.0000
.0000
.0000
.0000
.0005
.0005
.0005
.0000
.0005
.0000
.0000
.0000
.0000
.0005
.0000
4
mg/ml
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.0000
.0005
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
11
mg/ml
0.0005
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0005
0.0000
18
mg/ml
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.0005
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
.0000
*
.0000
.0000
. 0000
.0000
25
mg/ml
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0000
*
0000
0000
0000
0000
0000
0000
0000
0000
*
0000
0000
0000
0000
32
mg/ml
0.0000
*
0.0000
0.0000
*
0.0000
0.0000
0.0000
0.0000
0.0000
*
0.0000
0.0000
*
0.0000
*No results due to death of animal,
57
-------
TABLE 27. Pd Levels at Various Times in Urine of Guinea Pigs Injected I.V.
3 Times a Week for 3 Weeks with Various
Concentrations of Pd-Albumin Complex. *
(Significant to 0.001 rag/ml)
Day of Experiment
Cone.
Inj . Guinea
mg/ml Pig
10.0 1
2
3
4
5
1.0 6
7
8
9
10
0.3 11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0.000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
4
mg/ml
0.0000
0.0005
**
0.0023
**
0.0000
0.0001
0.0002
0.0005
0.0001
0.0001
0.0002
0.0002
0.0001
0.0003
11*
mg/ml
0.0029
**
**
0.0020
**
0.0000
0.0001
0. 0000
0.0001
0.0000 -
0.0001 *
0.0001
0.0001
0.0000
**
. 18
mg/ml
0:0023
**
**
0.0025
**
0.0001
0.0005
0.0000
0.0002
0.0001
0.0002
0.0002
0.0002
0.0000
**
*After 4 injections animal received complex S .C.
**No Results due to death of animal.
58
-------
TABLE 28. Pd Levels at Various Times in Urine of Rabbits Injected I.V.
3 Times a Week for 3 Weeks with Various
Concentrations of Pd-Albumin Complex.
(Significant to 0.001 mg/ml)
Day of Experiment
Cone .
Inj.
mg/ml Rabbit
10.0 1
2
3
4
5
1.0 6
7
8
9
10
0.3 11
12
13
14
15
0
mg/ml
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
4
mg/ml
0.0002
0.0003
0.0001
0.0004
*
0.0001
0.0003
0.0001
0.0001
0.0000
0.0001
0.0001
0.0001
0.0002
0.0003
11
mg/ml
0.0003
. 0.0004
0.0006
0.0007
*
0.0001
0.0001
0.0001
0.0002
0.0000
0 . 0001
0.0001
0.0000
0.0002
0.0000
18
mg/ml
0.0000
0.0002
0.0000
0.0004
*
0.0001
0.0000
0.0002
0.0002
0.0001
0.0004
0.0001
0.0001
0.0000
0.0001
*No results due to death of animal.
59
-------
TABLE 29. Skin test reactions in guinea pigs in mm^ (erythema and induration)
Guinea Pig
Sensitive
to
01 mg Pd
1 . 5 mg alb
10 mg Pd
1 . 5 mg alb
1 mg Pd
0.15 mg alb
1 mg Pd
0.15 mg alb
0.3 mg Pd
0.05 mg alb
Hour
4
1
2
24
30
48
4
1
2
24
30
48
i
1
2
24
30
48
4
1
2
24
30
48
4
1
2
24
30
48
1.5 mg
alb
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.15 mg
alb
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
10 mg Pd
1 . 5 mg alb
0
0
0
151
64
49
0
0
0
80
56
64
0
0
0
24
25
20
0
0
0
77
77
70
0
0
0
180
56
64
1 mg Pd
0.15 mg
0
0
0
30
75
25
0
0
0
20
9
15
0
0
0
20
16
15
0
0
0
20
20
25
0
0
0
35
6
9
0.3 mg Pd
alb 0.005 mg alb
0
0
0
9
9
9
0
0
0
25
16
25
0
0
0
16
16
9
0
0
0
16
20
25
0
0
0
12
8
9
Buffer
0
0
0
9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
TABLE 30. Rabbit urine palladium levels in mg/ml
(Significant to 0.001 mg/ml)
Injection
10 mg Pd
1/5 mg alb
10 mg Pd
1 . 5 mg alb
1 mg Pd
0.15 mg alb
1 mg Pd
0.15 mg alb
0.3 mg Pd
0 . 05 mg alb
Pre-Injection
0.00006
0
0
0.00006
0
Day 7
0.0001
0,0006
0.00007
0
0
Day 14
0.00016
0.00065
0.00008
0
0.00008
Day 21
0
0.0003
0
0
Q
Note: All sera readings were zero,
61
-------
TABLE 31. Skin test reactions in guinea pigs in mm^ (erythema and induration)
Skin tested with 0.1 ml intradermal injection of
Guinea Pig
Sensitive
to
G.P.#1
1 . 5 mg alb
G.P. #2
1 . 5 mg alb
G.P. #3
1 . 5 mg alb
G. P. #4
1.5 mg alb
Hour
i
1
2
5.5
24
48
4
1
2
5.5
24
48
4
1
2
5.5
24
48
\
1
2
5.5
24
48
1 . 5 mg
alb
121
144
180
240
0
0
121
169
196
484
0
0
120
120
255
320
0
0
143
110
195
625
0
0
O.lSmg 10 mg Pd 1 mg Pd 0.3mgPd
alb 1.5 mg alb 0.15mgalb O.OSmgalb
132 -
100 -
120 - -
210 . -
0 - -
0 - -
100
121
130
182 - -
0 - -
0 - -
120 - - -
100 - - -
120 - -
210
0 - -
0 - -
0 - -
0 - -
0
255 - -
0 - -
0
Buffer
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
TABLE 31 - page 2
G. P. #5
0.15 mg alb
G. P, #6
0.15 mg alb
G. P. #7
0.15 mg alb
G. P. #8
10 mg Pd
1.5 mg
alb
G. P. #9
10 mg Pd
1.5 mg
alb
i
i
2
5.5
24
48
"I
1
2
5.5
24
48
1
2
5.5
24
48
i
1
2
5.5
24
27
48
1
2
5.5
24
27
48
143
156
165
210
0
0
72
80
168
255
0
0
182
156
306
120
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
110
120
100
132
0
0
130
100
144
180
0
0
72
110
168
81
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-
-
—
~
-
-
-
~
0
0
0
0
80
60
36
0
0
0
0
28
42
0
-
-
""~
.
-
-
—
0
0
0
0
64
30
20
0
0
0
0
6
0
0
-
-
~
s _
-
'
-
~
0
0
0
0
20
16
12
0
0
0
0
20
9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
. 0
0
0
0
0
0
0
0
0
0
0
0
0
-------
TABLE 31 - page 3
G. P. #10
1 mg Pd
0.15 mg
alb
G. P. #11
1 mg Pd
0.15 mg
alb
G. P. #12
0.3 mg Pd
0.05 mg alb
G. P. #13
0.3 mg Pd
0.05 mg
alb
4
1
2
5.5
24
27
48
4
1
2
5.5
24 .
27
48
4
1
2
5.5
24
27
48
4
1
2
5.5
24
27
48
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
121
90
72
0
0
0
0
210
196
132
0
0
0
0
42
72
16
0
0
0
0
130
144
25
0
0
0
0
12
25
9
0
0
0
4
49
72
9
0
0
0
12
16
30
9
0
0
0
0
36
25
36
0
0
0
16
25
16
9
0
0
0
25
16
16
9
0
0
0
20
49
30
9 •
0
0
0
0
16
9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
2
TABLE 32. Skin test reaction in mm (erythema and induration) in normal guinea pigs
receiving ceils or sera from sensitized pigs and skin tested with O.I ml intradermaliy.
1.5mg 0.15mg 1.5 ing alb O.lSmgalb O.OOSmgalb
Hour alb alb 10 mg Pd 1 mg Pd 0.3mgPd Buffer
G. P. #1
received
spleen
cells (107 )
from alb
sen .
G.P. #2
received
spleen
cells (10 7 )
from Pd-
alb . sen .
G.P. #3
received
spleen
cells (107 )
from Pd-
alb sen .
G. P. #4
received
serum
from alb
sen.
i
1
2
6
24
48
i
1
2
6
24
48
i
1
2
6
24
48
1
2
6
24
48
0
0
0
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
0
99
100
0
Q
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
0
0
0
0
0
70
70
0
o
0
-
—
0
0
0
50
84
40
0
0
0
25
56
25
: -
-
-
-
* ' ~
'
—
0
0
0
35
35
9
0
0
0
9
6
0
—
-
' -.
-
-
-
~ • •
0
.0
.0
9
12
0
0
0
0
9
4
o
—
.
'
-
-
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-------
TABLE 33. Guinea pig urine palladium levels in mg/ml
(Significant to 0.001 mg/ml)
Injection
10 mg Pd
1.5 mg alb
complex
1 mg Pd
0.15 mg alb
complex
0.3mgPd
0.05 mg alb
complex
Guinea
Pig
1
2
3
4
5
6
Pre-
Injection
0
0
0.00006
0
0
0
Day 7
0.0029
0.0017
0.0001
0.0004
0.00007
0.0001
Day 14
0.0018
0.0022
0.0005
0.0004
0.0003
0.0002
66
-------
TABLE 34. Pt Levels at Various Times in Serum of Rabbits Injected S.C.
3 Times a Week for 3 Weeks with Various
Concentrations of Pt-Albumin Complex.
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
Inj,
mg/ml Rabbit
11.7 1
2
3
4
2.6 5
6
7
8
0.02 9
10
11
12
0
mg/ml
0.0000
0.0008
0.0008
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
5
mg/ml
0.0016
0.0016
0.0016
0.0008
0.0000
0.0000
0.0008
0.0000
0.0008
0.0000
0.0000
0.0000
12
mg/ml
*'
*
*
*
0.0008
0.0000
*
0.0000
0.0000
0.0008
0.0000
0.0000
19
mg/ml
*
*
*
*
0.0000
0.0008
*
0.0008
0.0008
*
0.0000
0.0000
*No results due to death of animal.
67
-------
TABLE 35. Pt Levels at Various Times in Urine of Rabbits Injected S .C.
3 Times a Week for 3 Weeks with Various
Concentrations of Pt-Albumin Complex.
(Significant to 0.001 mg/ml)
Day of Experiment
Cone .
Inj.
mg/ml Rabbit
11.7 1
2
3
4
2.6 5.
6
7
8
0.02 9
10.
11
12
0
mg/ml
0.0006
0.0006
0.0006
0.0006
0.0006
0.0009
0.0006
0.0006
0.0006
0.0003
0.0006
0.0003
7
mg/ml
0.0009
0.0006
*
*
0.0014
0.0009
0.0000
0.0003
0.0009
0.0000
0.0006
0.0009
14
mg/ml
*
*
*
*
0.0009
0.0029
*
0.0026
0.0006
0.0009
0.0011
0.0011
19
mg/ml
*
*
*
*
0.0017
0.0011
*
0.0023
0.0009
*
0.0011
0.0009
*No results due to death of animal.
68
-------
TABLE 36. Pt Levels at Various Times in Serum of Guinea Pigs Injected S .C,
3 Times a Week for 3 Weeks with Various
Concentrations of Pt-Albumin Complex.
(Significant to 0.0005 mg/ml)
Day of Experiment
Cone .
Inj -
mg/ml
11.7
2.6
0.02
Guinea
Pig
1
2
3
4
5
6
7
8
9
10
11
12
0
mg/ml
0.0000
**
**
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
5
mg/ml
*
*
*
0.0024
0.0008
0.0008
0.0008
0.0008
0.0000
0.0000
0.0000
0.0000
12
mg/ml
*
*
*
*
0.0024
0.0024
0.0016
*
0.0000
0.0000
0.0000
0,0000
19
mg/ml
*
*
*
*
* .
0.0024
0.0032
*
0.0000
0.0000
0.0000
0.0000
*No results due to death of animal,
**No serum.
69
-------
TABLE 37. Pt Levels at Various Times in Urine of Guinea Pigs Injected S .C ,
3 Times a Week for 3 Weeks with Various
Concentrations of Pt-Albumin Complex.
(Significant to 0.001 mg/ml)
Day of Experiment
Cone .
Inj . Guinea
mg/ml Pig
11.7 1
2
3
4
2.6 5
6
7
8
0.02 9
10
11
12
0
mg/ml
\
0.0011
0.0003
0.0006
0.0003
0.0000
0.0011
• 0.0003
0.0003
0.0006
0.0009
0.0006
0.0003
7
mg/ml
*
*
*
0.0020
0.0017
0.0051
0.0049
0.0029
0.0023
0.0006
0.0000
0.0009
14
mg/ml
*
*
*
*
0.0037
0.0023
0.0020
*
0.0003
0.0000
0.0009
0.0009
19
mg/ml
*
* .
*
*
*
0.0017
0.0011
*
0.0006
0.0000
0.0006
0.0003
*No results due to death of animal.
70
-------
TABLE 38. Skin test reactions in guinea pigs in mm^ (erythema and induration)
Skin tested with 0.1 ml (cone = me/ml) intradermal injection of
Guinea
Pig
sensitive
to
4.5 mg Pd
7.0 mg egg
albumin
4.5 mg Pd
7.0 mg egg
albumin
4.5 mg Pd
7.0 mg egg
albumin
4.5 mg Pd
7.0 mg egg
albumin
Hour
4
1
9
12
24
48
4 .
1
9
12
24
48
4
1
9
12
24
48
4
1
9
12
24
48
Buffer
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3.8PtCl4 3.8PtCl4 4.5PdCl4
7.0mg 7.0mg 7.0 mg 7.0 mg 7.0mg
egg g • Pig g • Pig egg g . pig
albumin albumin albumin albumin albumin
_ • _ _ _ -
- - -
201
- - 201
- - - - 226
- - - - 78
_ _ _ - _ -
_ _ _
- - - 154
154
• _ 254
- - 64
_ _ _ . _ _
_
- : - - 452
• _ 452
452
- - - - 78
_ _ _ _ _
• _ ' _ _ _
452
452
452
- 176
4 . 5 PdCl4
7.9 mg
egg
albumin
-
-
176
176
346
132
_
-
346
225
346
78
-
-
346
346
380
113
-
-
380
380
380
176
-------
TABLE 39. Skin test reactions in guinea pigs in mm (erythema and inducation)
Skin tested with 0.1 ml (cone = mg/ml) intradermal injection of
Guinea
Pig
Sensitive
to
4.5mg Pd
7.0 mg
guinea pig
albumin
4.5 mg Pd
7.0 mg
guinea pig
albumin
4.5 mg Pd
7.0 mg
guinea pig
albumin
4.5 mg Pd
7.0 mg
guinea pig
albumin
Hour
4
1
9
12
24
. 48
4
1
9
12
24
48
4
1
9
12
24
48
•4
1
9
12
24
48
Buffer
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4.6PtCl4 3.8PtCI4 4.5PdCl4
7.0mg 7.0mg 7.0mg 7.0mg 7.0mg
egg g- pig g- Pig egg g. pig
albumin albumin albumin albumin albumin
_ _ _ _ _
- - - -
380
380
176
- . - 176
_ _ _ _
- - - -
- - 346
346
314
154
_ ' _ _ _ _
_
. - - - - 415
415
452
132
_ _ _ • _ _
_
- - 132
132
113
-
4 . 5 PdCL
7.0 mg
egg
albumin
_
-
380
380
176
176
_
-
154
154
201
154
_
-
254
254
314
113
_
-
28
28
28
-
-------
TABLE 39 - page 2
4.5 tng Pd i 0 - - - - - -
10---- - -
7.0 mg 9 0 - - - - 50 314
guinea pig 12 0 - - - - 50 314
albumin 24 0 - - - - 113 314
48 0 - _ _ _ 113 153
U)
-------
TABLE 40. Skin test reactions in guinea pigs, which received intraperitoneally
5 X 10 8 spleen cells from Pd-egg albumin sensitive pigs, in mm
(erythema and induration)
Skin tested with 0.1 ml (cone = mg/ml) intradermal injection of
G. pig
received
5 X 10 8
• spleen
cells sen-
sitized to
Pd-egg alb.
G. pig
received
5 X 10 8
spleen
cells sen-
sitized to
Pd-egg alb.
Hour
i
1
9
12
24
48
i
1
9
12
24
48
Buffer
0
0
0
0
0
0
0
0
0
0
0
0
4.5 PdCl4
7,0 mg
egg
albumin
_
-
314
314
283
283
_
-
314
346
346
283
4.5 PdCl4 3.8 PtCl4 3.8 PtCl4
7.0 mg 7.0 mg 7.0mg 7.0mg 7.0mg
g. pig egg g. pig egg g. pig
albumin albumin albumin albumin albumin
-
— — — — —
283 - - - -
314 - - -
314 - - - • -
226-
- - ' -
• — — — — . —
346 -
380 - - - -
380 ~
314 - - -
-------
TABLE 41. Skin test reactions in guinea pigs; which received intraperitoneally
5 X 10 8 spleen cells from Pd-g. pig albumin sensitive pigs, in mm2
(erythema and induration)
Skin tested with 0.1 ml (cone = mg/ml) intradermal injection of
G. pig
received
5 X 10 8
j spleen
cells sen-
sitized to
Pd-g. pig
albumin
G. pig
received
5 X 10 8
spleen
cells sen-
sitized to
Pd-g. pig
albumin
Hour
i
1
0
12
24
48
i
1
9
12
24
48
Buffer
0
o
0
0
0
0
0
0
0
0
0
0
4.5PdCl4
7.0mg
egg
albumin
-
-
314
380
380
314
»
- -
314
314
314
314
4.5PdCl4 3.8PtCl4 3.8PtCl4
7.0 mg 7.0 mg 7.0 mg
g. pig egg g. pig
albumin albumin albumin
' - -
— — —
314
346
314 - -
283
-
- - -
284
314 - -
346
285 -
7.0 mg 7.0 mg
egg g- pig
albumin albumin
.
_ • —
— —
— —
— • ~~
— . ~
'
_ —
- -
— —
— —
— —
-------
TABLE 42. Skin test reactions (mm erythema and induration)
in guinea pigs sensitized with 3.7 mg/ml palladium
complexed to 5.7 mg/ml egg albumin.
Guinea pigs
sensitized
3.7 mg/ml Pd-
5.7 mg/ml egg
albumin
Skin tested with 0.1 ml (cone = mg/ml)
Hour intradermal injection of 3.7 Pd and
5.7 egg albumin
#1 \
1
9
12
24
#2 ' 4 '
1
9
12
24
#3 \
1
9
12
24
#4 ' 4
1
9
12
24
#5 4
1
9
12
24
0
0
176
143
132
0
0
284
176
132
0
0
113
177
132
0
0
214
283
226
o
0
113
132
122
76
-------
TABLE 42 - page 2
#6 i 0
1 0
9 71
12 113
24 104
#7 4 ' 0
1 0
9 0
12 0
24 0
77
-------
TABLE 43. Skin test reactions (mm erythema and induration)
in normal guinea pigs receiving 5 X 10 spleen cells
intraperitoneally from sensitive donors.
Recipient
guinea pig
sensitized
with cells
from
G. pig #1
Table 41
G. pig #2
Table 41
G. pig- #3
Table 41
G. pig #4
Table 41
Hour
i
1
9
12
24
i
1
9
12
24
4
1
9
12
24
4
1
9
12
24
Skin tested with 0. 1 ml (cone = mg/ml)
intradermal injection of 3.7 mg Pd and
5.7 egg albumin
0
0
240
226
;103
0
0
240
213
200
0
0
226
200
113
0
0
314
268
113
All other sites and times - readings were zero.
78
-------
TABLE 44. Skin test reaction (mm^ erythema and induration)
in guinea pigs sensitized with 5.0 mg/ml Pd
complexed to 7.0 mg/ml egg albumin. Skin tested
with 0.1 ml (cone = mg/ml)
Sensitized to 3.7 Pd Sensitized to 5.0 Pd
Guinea Hour
Pig
#1 4
1
9
12
24
#2 4
1
9
12
24
#3 \
1
9
12
24
#4 4 ' ' '
1
9
12
24
#5 4
1
9
12
24
3.7 Pd
7.0. egg
alb
0
0
226
240
240
0
0
254
254
254
0
0
254
200
200
0
0
70
86
86
0
0
186
188
176
5.0 Pd
7.0 egg
alb
0
0
346
416
379
0
0
362
362
362
0
0
200
226
200
0
0
314
314
314
0
0
268
329
329
3.7Pd
7.0 egg
.alb
0
0
268
283
246
0
0
268
283
254
0
0
240
240
226
0
0
177
177
177
0
0
213
226
200
5.0Pd
7.0 egg
alb
0
0
490
490
397
0
0
314
314
314
0
0
490
314
314
0
0
188
188
188
0
0
346
380
314
79
-------
TABLE 44 - page 2
#6 i
1
9
12
24
0
0
346
346
240
0
0
213
213
213
0
0
213
213
176 .
0
0
. 324
314
314
#7 i
1
9
12
24
0
0
176
153
153
0
0
433
530
490
0
0
200
200
176
0
0
0
0
0
#8 i 00
1 00
9 0 240
12 0 254
24 0 240
80
-------
TABLE 45. Skin test reaction (mm^ erythema and induration)
in normal guinea pigs receiving 5 X 10 ° spleen
cells intraperitoneally from sensitized donors.
Recipient
guinea pig
sensitized
with cells
from
5.0 Pd-7.0
egg alb sen-
sitized pig
5.0 Pd-7.0
egg alb sen-
sitized pig
3.7 Pd-7.0
egg alb sen-
sitized pig
3.7 Pd-7.0
egg alb sen-
sitized pig
Hour
i
1
9
12
24
i
1
9
12
24
i
1
9
12
24
i
1
9
12
24
Skin tested with 0.1 ml
intradermal injection of
3.7 Pd-7.0 egg alb 5.0
0
0
200
213
165
0
0
0
0
0
o
0
94
94
94
0
0
165
165
165
(cone = mg/ml)
Pd-7.0 egg alb
0 .
0 .
254
254
254
0
0
0
0.
0
0
0
200
200
188
0
0
213
254
254
81
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-77-039
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Allergic Response to Platinum and Palladium Complexes -
Determination of No-Effect Level
5. REPORT DATE
July 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
James Taubler
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biology
St. Vincent College
Latrobe , Pennsylvania 15650
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
Grant No. 803036
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
RTP/NC
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Section A - Rabbits, guinea pigs and mice were subcutaneously injected with PtSO^ (with
and without NH^Cl) and PdS04 (with and without KH4C1) in an attempt to sensitize the
animals to platinum or palladium. No allergic induction was found. Levels of
platinum or palladium in the serum, urine and spleens of animals were monitored by
AAS (Atomic absorption spectrophotometer). Significant levels were found primarily in
the urine of guinea pigs.
Section B - No allergic induction to platinum or palladium was found in rabbits, guinea
pigs or mice when these animals were injected intravenously with platinum or palladium.
Dermal contact with platinum was also tested on rabbits and guinea pigs but failed to
induce an allergic state. Platinum and palladium levels, in the sera, urine and spleens
of the animals, as monitored by AAS, were not significant.
Section C - Rabbits, guinea pigs and mice were intravenously or subcutaneously injectec
with a platinum-egg albumin complex or a palladium-egg albumin complex. Skin tests or
footpad tests were performed 10-14 days after the last intravenous or subcutaneous
injection. Only guinea pigs injected subcutaneously with palladium-egg albumin complex
developed a hypersensitivity of the delayed type.
Section D - A delayed type allergy was induced in guinea pigs subcutaneously injected
with palladium complexed to egg albumin.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
platinum
allergic diseases
palladium
06 T
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
85
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
82
------- |