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 ------- 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. ------- 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 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 ------- |