The intravenous administration of DEHP solubilized by means of a number of different detergents leads to respiratory distress and death in rats. At autopsy the lungs are grossly enlarged, edamatous, and hemorrhagic. Light and electron microscopic evaluation of the lungs indicate engorgement of the interalveolar septa with edema fluid and polymorphonuclear leucocytes, degranulation of the leukocytes, and progessive destruction of the endothelial and epithelial cells. Consistent with the conclusion that solubilized DEHP results in a syndrome of "shock lung" is the associated massive fall in arterial blood pressure and the prevention of the lung pathology by pretreatment with pharmacologic doses of an antiinflammatory steroid, methylprednisolone. Evidence is also presented that suggests that the DEHP inadvertently administered to humans during transfusions is also in a solubilized state in the plasma

Rubin, R. J.

English

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https://ntrs.nasa.gov/search.jsp?R=19750017459 2020-03-22T22:05:27+00:00Z
NiltSA CR= ^TY3
FINAL PEPO RT
TOXICOLOGIC EVALUATIO:J OF T11E MIGRATION Or A PLASTICIZER,
DI (2-ETHYLH1r:►YL) PHTFIAL:^TE (DEiIP)' FROM VINYL PLASTICS.
Submitted April 18, 1975
ATTN: Mr. Donald S. Friedman
Technology UtiliZutic^;i
Office
GSFC Code 207
Contract No. NAS-5-22071
Awarded to: Dr. Robert J. Rubin
Department of Env. Med.
Johns Iiopkins Universit-.y
School of Eiy,^iene and
Public Health
(Y 1SA-CF-143801)
	 TOXICOLOGIC FV UUATION OF
THE MIGFATION OF A PLASTICTZER, DI(2-F T dYLHFXY 1.)
 PHTHAL AT F ( Dw hP) FROM VINYL
PLASTICS Final Report (Johns Hopkins Univ.)
10 p HC $3.25	 CSCL 06C G3/51
v75-255.i1
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SUMMITRY
The intravenous administration of DMIP solubilized by
means of a number_ of different detergents leads to respiratory
distress and death in rats. At autopsy the lungs are grossly
enlarged, edamatous, and hemorrhagic. Light and electron
microscopic evaluation of the lungs indicate engorgement of
the interalveolar septa with edema fluid and polymorphonuclear
leucocytes, degranulation of the leukocytes, and progres,i.ve
destruction of the endothelial and epithelial cells. Red
blood cell structure was also seen to be altered. With the
exception of this latter observation, all of these pathologic
effects arc identical to those seen in "shock lung", a disease
state whose pathogenesis is not clearly understood at th.:, time.
Consistent with the conclusion that solubilized DEHP results
in a syndrome of "shock lung" is the associated massive fall
in arterial blood pressure and the prevention of the lung
Vpathology by pretreatment with pharm.acol.ogic doses of an anti-
inflammatory steroid, methylprednisolone. Evidence is also
presented that suggests that the DEIIP inadvertently administered
to humans during transfusions is also in a solubilized state
in the plasma.
I
'
1.
In the previous progress report of October 24, 1973 the
development of a detergent-solubilized dosage form of DEIIP
and preliminary observations of its pulmonary pathology were
described. Recent progress has dealt with an in-depth study
of this pathologic effect. Information in the following
areas has been developed:
1. Light anti electron microscopy of lung damage.
2. Pulmonary effects of DEIIP solubilized in various
detergents.
3. Elffects on blood pressure.
4. Effects on red and white blood cells.
5. Protection against lung effects by steroids.
6. Relevance of detergent-solubilized DEIIP by comparison
with kinetics of disappearance of naturally migrated
DEIIP in patients transfused with Mood products
that worn prepared and stored in vinyl plastic bags.
Some of this above information has already been submitted
for publication in th^ biomedical literature. One paper has
been accepted by the Journal of Toxicology and Applied Pharmacology
and is currently in press. A second haC been submitted to
Lancet-. Copies of both manuscripts are enclosed with this report.
i1
I	 ,
2
1. I i ht and Electron Microscornv
The intravenous administration of 250 mg/kg of DEEP
solubilize4 in 13.3% aqueous Tween 80 was found to be
approximately an LD`' d-se in Wistar albino rats. Treated
animals had obvious respiratory difficulty within minutes
after an injcz ction and within 5--15 minutes %.,ere markedly cyanotic.
Deaths were usually seen within 90-120 minutes. i+,nimals that
survived beyond 2 hours appeared grossly to h.ve recovered. from
Oic respiratory insult. At autopsy the lungs of animals that
died appeared grossly enlarged and hemor riagic. Lung weights
were rarkedly elevated. Control injections of the detergent
vehicle alone produced no symptoms of respiratory distress or
deaths. In addition, there lungs were not different in qross
appearance or weight from those of non-injected rats.
Under the light microscope (160 x) the inter-alveolar
septa of lungs from DEFIP-treated rats appeared greatly distended
and engorges: with abnormal numbers of polymorphonucicar
leukocytes (PMN's) . This abnormal pathology could be seen at
closes of solubil.ized DL'11P as low as 50 mg/kg., It was observable
as early	 15 minutes after injection, and in animals that
survived the initial dose the pulmonary histology was still
markedly abnormal 50 hours after t h,_ injection. The alveolitis
and associated ede,:,d was quite si , ..ilar in appearance to the
3.
pathologic state in man that has been described ar; "shock
lung". The cause of this type of lung pathology in man is not
currently understood but is known to be associated with the
transfusion of large num'.)ers of blood units and with endotoxin
shock.
At the electron micrk.^scopic level the picture was once again
highly suggestive of "shock lun g". Large numbers of PWI's
were found marginated on the pulmonary endothelium. Significant
numbers of these PMN's were found to be undergoing degranulation
and the endothelial -ells were seen to bc. separating and
allowing the leakage of fluid into the interstitium. In areas
the epithelial lining cells were also found to be rupturing
and allowing leakage of blool into the alveolar spaces. One
auditional feature not usually se. Ti in shock lung was the
deformation of the red blood cells from the normally appearing
biconcave disks to a more spherical, swollen shape.
2. I_> u lmonZry Effects of DF.HP Solubilized in Oth .,r Detergents
Experiments were undertaken to determine if DF11P, solubilized
in detergents other than Ti'w^en 80, also resulted in comparable
lung damage or if the effect was specific to the use of Tween 80.
Comparable lethality and lung pathology were seen when DE11P was
solubilized in two other typ<!s of Tween detergents (Tween 20
T4.
anJ Tween GG O as well as in polyethylene glycol 400 (Carnowax
400) or in a polyhydroxylated vegetable oil (Emulphor 620).
Thus, tro toxic effect of the DEHP app-g ars to be related to
its physical state as a solubilized molecule rather than to
an interaction with any specific detergent. It should also
further be emphasized that the injection of any of the detergent
vehicles alone resulted in no respiratory distress, lung
pathology or lethality.
3. Ef fect s on Aloud Pressure
The similarity of the lung pathology to previously described
"shock lung" at the light e.nd electron microscopic levels has
previously teen mentioned. Another %•:ell-described characteristic
of "shock lung" is a massive fall in mean arterla1 pressure.
In order to further characterize the DEEP eff,.. . experiments
were undertaken in the rat to determine the effect of intravenous
doses of solubilized DEHP on blood pressure. At doses near
the lethal level (00-20U mg/kg i.v.) massive falls in mean
arterial pressure were noted, but only after a time lag of 1-2
minutes; this initial. period was then followed by a precipitous
drop. This observation would tend to rule out a direct effect
of the solubilized DL11P on vascular smooth muscle and would
4
J .
suggest a time-dependent release of a potent vasoactive
material from some intracellular site. When blood pressure
fell below approximately 30 mm fig, death invariably ensued.
If the anima survived the dose, the blood pressure slowly
returned to control levels over a 20-30 minute period.
At doses of solubilized DEHP of 40 mg/kg at which no deaths
were observed, the mean fall in blood pressure was 18-27 trm Hg.
This fall in blood pressure was all the ,nore uramatic since
injection of the Tween 80 vehicle alone resulted in a hyper-
tensive response of 24-45 mm 11q. Thus, the net fall in blood
pressure with DE11P was the resultant of the hypertensive
effect of the vehicle overcome by the hypotensive effect of
the DI-I'V FEIr se.	 It is interesting to note that a second
dose of DE11P in the same animal resulted in no significant
alteration in blood pressure. Thus, although the direct
hypertensive effect of the vehicle was overcome, the hypotensive
effect of the DE11P was not sufficient, under these conditions,
to result in a net fall in blood pressure. This observation of
a decreased effect of a subsequent dose of DE11P is interpreted
to indicate that the intracollular pool of vasoactive material
that is presumably released by the DEHP is considerably
depleted by the first dose and has not had a chance to be
regenerated over the time-course of the experiment.
4. Ef fact on Red and White Blood Cell;
hnothcr well-recognized aspect of "shock lung" is an
increase in the number of circulating leukocytes in the
blood. 'Phis leukocytosis has also been confirmed in the
rats treated with solubilized DI?IIP. The %,rhite blood cell count
was observed to increase approximately 2-3 fold within 60-90
minutes at doses of 100 mg/kg. Since the studies described
above on the electron microscopic picture of the lungs of
treated animals revealed a marked accumulation of leukocytes
(PMN's) in the interal.veolar septa, experiments are currently
underway to directly measure the uptake of PMN's from tl, e blood
by the l.ung!^. However, no definitive results are available
at the time of this report.
During the experiments on white blood cell counts, an
in.travascular hemolvsis of red blood cells (RBC's) was noted.
No effect was observed with injection of the vehicle alone.
rollowinr, the solubilized DEHP no hemolysis was noted for
approximately 20-30 minutes; this was followed by an increasing
degree of hemolytis over time. This lag period for in vivo
hemolytis once again argues against a direct hemolytic effect
of the solubilized DEHP and can be interpreted to represent
the release or activation of intracellular hemolytic factors.
7.
Consistent with this interpretation is the observed lack of a
hemolytic effect of solubilized DL111' incubated with RFC's in vitro.
In fact, under i;, vi'.^ro conditions the vehicle alone resulted
in hemolysis which was prevented by the presence of DE11P.
The in vivo hemolysis was shown to result from an increased
fragility of the IBC's. Washed red cells isolated from DEHP-
treated rats exhilited markeu hemolysis during further in vitro
incubation in saline at 37°C.
These: results of increased fragility and hemolysis of RFC's
is consistent with the altered morphological appearance of
*hose cells in the electron microscope, as was described above.
Although this feature h.E s not previously been recognized a-,-
part of the "shock lung" synOrome, it may represent an additional
aspect of DL'11P toxicity.
5. Protection by Steroids
Since the pathologic alterations and lethality of "shock
lung" are known to be prevented by pretreatment with anti-
inflammatory steroids, experiments were undertaken in which
one of these potent agents, inethylprcdnisolone (--olu-mcdrol) ,
was tested for its ability to protect against the DE11P toxicity
detailed above. Methylprednisolone, at a dose of 30 tr.g/kg i.v.
given 15 minutes prior to the administration of the solubilized
8.
DUHP completely prevented the gio'ns lung pathology and reduced
the acute lethality. Lr j weights • sere not significantly
different from vehicle-injected or non-injected contrcls.
Lethality was reduced from 1C0% oC the animals given DEHP alone
(death occurring within 3G.3 + 5.9 minutes) to 33% of the
pre- treated animals (with these dying at a mean time of 61.7
minutes). Thus, once again the symptoms induced by solubilized
DEHP are entirely consistent with other models of "shock lung".
6. Relevance of Detergent- solubilized DUHP to that Occurring
by Naturil Migration from Vinyl Plastic Surfaces into
Biologic Products
DEHP is a highly water-insoluble oil. This very property
is what makes surprising the high levels of this chemical in
blood and blood products stored in vinyl plastic bags (0.25 mg/
100 ml/24 hours of storage in whole blood at 4°C to 28.0 mg1100 ml/
24 hours of storage in blood platelets at 23°C). Although the
appropriate physical/chemical tests have not yet been carried out
on UEHP-contaminated biological solutions to determine the
actual physical state of the oil, certain kinetic studies
carried out in this laboratory are consistent with it existing
in a solubilized state.
9.
Previous work in rats reviewed in an earlier progress
report, has shown that emulsified droplets of DEIIP are cleared
from the blood at a bi-exponential ra`e whereas solubilized
DEIIP is cleared at a single exponential rate which is approximately
equal to the slower of the two rates seen with the emulsified
dosage form. Additional experiments, also previously reviewed,
have allowed the conclusion that the rapid rate seen with
emulsified DEHP is lue to the uptake, of particulate droplets
of the oil by the liver. The slow rate seen with both the
emulsified and solubilized DUIP is presumably due to the
disappearance of solubilized DEHP by the natural processes of
metabolism and urinary excretion. In recent experiments
(manuscript submitted as part of report) the rate of disappearance
of DEHP has been followed in humans who had inadvertently
received large amounts of this chemical during transfusion of
platelets prepared and stored in vinyl plastic bag.. The
disappearance was found to be mono-exponential and to have a
rate approximately equal to the slow rate of disappearance
of the emulsified DEIIP and the mono-exponential rate of solubilized
DEIIP in rats. THus, the kinetic behavior of DEIIP in man
following natural migration from a vinyl plastic surface is
entirely consistent with the phthalate existing in a solubilized
state. This lends support to the over-all approach represented
ii	 ,
lU.
A n this report of being concerned with the toxicologic potential
of solubilized DEIIP.


Toxicologic evaluation of the migration of a plasticizer, DI (2-ethylhexyl) phthalate (DEHP) from vinyl plastics

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