United States
                    Environmental Protection
                    Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
                    Research and Development
EPA-600/S1-83-002  Apr. 1983
4MEPA          Project  Summary
                    Development  of a  Model of
                   Toluene  Blood Level  Following
                    Subcutaneous Injection  of
                   Toluene  in  the Rat

                   Vernon A. Benignus, Keith E. Muller, and Curtis N. Barton
                     A  model of toluene level in blood
                    following subcutaneous (SC) injection
                    of toluene mixed with polyoxyethylated
                    vegetable oil vehicle  was developed.
                    The purpose was to provide a means of
                    predicting dose received, for subse-
                    quent toxicologic studies for any time
                    and dose combination. The pharmaco-
                    kinetics were of secondary interest.
                    With the use of data from 111 rats, a
                    4-parameter equation was devised to
                    predict  the course of toluene blood
                    levels from 20-480 minutes, for doses
                    of 50-1000 mg/kg. Blood concentra-
                    tions rose at a rate that was independent
                    of dose level. Maximum blood levels
                    were uniquely determined by dose level.
                    Blood  levels fell  at differing  rates,
                    depending upon dose level. When com-
                    pared to inhalation, injection exposure
                    has the advantages of low expense, low
                    equipment requirements, and simplic-
                    ity. Its disadvantage for some experi-
                    ments is poor temporal simulation of
                    the normal route of exposure, inhala-
                    tion.
                     This Project Summary was developed
                    by EPA's Health Effects Research Lab-
                    oratory, Research Triangle Park, NC, to
                    announce key findings of the research
                   project that is fully documented in a
                    separate report of the same title /see
                    Project Report ordering information at
                    back).

                    Introduction
                     Toluene is a commonly occurring sub-
                    stance, widely used as a solvent in paints,
glue, and rotogravure printing, to name
but a few applications. Threshold concen-
trations for effects on neural and be-
havioral functions range from 100ppm in
ambient air to about 1000 ppm, depending
upon the dependent variables.
  Humans are normally exposed to tol-
uene via inhalation.  Injection has been
used by many investigators as a conven-
ient and inexpensive alternative to the
more complex technology of inhalation
exposure. Despite the common use of this
method, only one study was found in
which tissue levels of toluene after injec-
tion were reported (Sato and Nakajima,
1979). Using intraperitoneal (IP)  injec-
tions of 30, 115, and 460 mg/kg, the
researchers measured blood levels at 2,
4, 8,  12, and 24 hours in rats. Although
most of the rising  limb  of the curve
occurred before the  first  blood sample
was  taken, it appeared  that toluene
reached its peak blood value later for
larger doses. Since statistical tests were
not performed, it was not clear whether
the elimination rate  was  a function of
dose level. Sampling  rate for blood level
was also quite low. Due to infrequent and
late sampling and lack of statistical testing
of the curve fits, it is unclear whether the
curves which these  investigators used
adequately described the time course of
blood levels.
  Toluene has been injected both IP and
SC by various investigators, but  blood
levels over time were not reported. For
many experiments, it is desirable for blood
toluene to be maintained at a stable level

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for a long period of time rather than to
reach a peak and to be eliminated rapidly.
Drugs delivered by SC injection are more
slowly  and evenly absorbed  over time
than are those given by IP injection. No
data were found, however, on toluene
blood levels after SC injection.
  This study was  performed to discover
and describe the time course of toluene in
blood after injection, because the design
of subsequent toxicologic studies would
be greatly simplified if such information
were available.  The  pharmacokinetics
were of secondary interest. SC injection
was used because a long, slow absorption
is often desirable. If the time course of
toluene blood level after injection were
known,  preliminary study could be con-
ducted using injections rather than  the
more expensive and complex  inhalation
procedure.
Materials and Methods
  The work in this report was an alter-
nating series of exploratory and confirma-
tory studies. The process of devising the
injection technique and the  model for
predicting results is described in detail in
the "Results" section of the full publica-
tion.
  All  254  subjects  were Long-Evans
hooded rats of similar age. All rats were
kept in a colony on a night-dark schedule
for a minimum of 30 days before use.
  Injections were administered SC after
the rat was grasped with the non-dextrous
hand and the skin of its  back was pushed
upward  to form a "hump." A  22-gauge,
one-inch needle on a plastic syringe was
inserted intotheskin hump, in an anterior
direction parallel to the back of the rat.
After  the injection was complete, the
needle was withdrawn  and the injection
site was rubbed in a posterior to anterior
direction 10  times  in order to  avoid
leakage  from the site.
  Toluene was mixed  in  various ratios
with  polyoxyethylated vegetable oil
(Emulphor, EL610)toachievean injection
volume  of  1.6 ml/kg  of body  weight.
Dosages were 50,  100, 120,  150/500,
and 1000 mg/kg.
  Rats were injected in the morning and
then housed  in individual cages  in  a
lighted room without food or water until
they were sacrificed. Rats were rendered
unconscious by cervical dislocation, and
35 ml  of blood was  drawn from the
anterior vena cava  as described  in
Benignus ef at. (1981). Rats were sacri-
ficed at 20-minute intervals after injection
for up to 8 hours.  Gas chromatography
was used to analyze the blood samples
(Benignus et a/., 1981).
  Previous data have shown that varia nee
of blood levels is much greater for higher
doses than for lower doses. Exploratory
analyses in this study confirmed  this.
Consequently, all data were analyzed in
log  concentrations. The models of most
interest are inherently nonlinear. Meth-
ods used for fitting inherently nonlinear
equations were programmed in Statistical
Analysis System. When parameter esti-
mates for different nonlinear equations
were to be compared, the technique of
seemingly unrelated nonlinear regression
was used.

Conclusions
  The time course of toluene in blood
following SC injection was observed (see
Figure 1).  Rise rates, maximum blood
level, and declmeof toluene in blood were
all functions of dose level. The following
4-parameter  equation was devised  to
predict blood level  as a function of time
after injection and dose level:
logC, = (log 1.58) (log D,)
     + log[1-exp(.0106t,)]
          -t, (.00838-.0012 log D,).
                       (D
Here C, = blood toluene (ppm) for rat i, D =
dose of toluene in mg/kg, and t = time
after injection. The purpose was to enable
predictions of dose received  in  subse-
quent toxicologic research without inves-
tigations of the pharmacokinetics.
  The SC injection technique appears to
have  utility  as  a method of toluene
exposure.  Its advantages  are  its low
expense and equipment requirements, as
well as  its simplicity. Its disadvantage is
its poor temporal simulation of inhalation
exposure for certain  kinds of studies.
Perhaps it would be most useful  in con-
ducting preliminary work on the effects of
toluene. Effects would have to be verified
with inhalation exposures  in  order  to
generalize the results to the usual route
of exposure, inhalation.

References
Sato, A and T. Nakajima. Dose-dependent
  metabolic interaction between benzene
  and toluene in vivo and in vitro. Toxicol.
  Appl. Pharmacol. 48:249-256,  1979.
Benignus, V., K. Muller, C. Barton, and J.
  BittiKofer. Toluene levels in blood and
  brain of rat during and after respiratory
  exposure.   Toxicol. Appl.  Pharmacol.
  61:326-334, 1981.
  -7.0JL
             60      120      ISO     240      300
                              Time After Injection, min
                                           360
                                                   420
                                                           480
 Figure 1.
Lines of best fit for the 4-parameter model. The crosshatched bars show the times
during which the toluene concentration in blood is estimated to be at least 90% of
max/mum (raw data) for each dose.

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     The EPA author Vernon A. Benignus (also the EPA Project Officer, see below) is
      with the Health Effects Research Laboratory, Research TrianglePark, NC27711;
      Keith E. Muller and Curtis N. Barton are with the University of North Carolina,
      Chapel Hill. NC 27514.
     The complete report, entitled "Development of a Model of Toluene Blood Level
      Following Subcutaneous Injection of Toluene in the Rat." (Order No. PB83-172
      494; Cost: $10.00, subject to change) will be available only from:
            National Technical Information Service
            5285 Port Royal Road
            Springfield, VA 22161
            Telephone:  703-487-4650
     The EPA Project Officer can be contacted at:
            Health Effects Research Laboratory
            U.S. Environmental Protection Agency
            Research Triangle Park. NC 27711
              Government  Printing Office:    1983-659-017/7056
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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Fees Paid
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Agency
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Penalty for Private Use $300

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