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Histopathological re-examination of brain sections from the NTP (1996) subchronic and chronic rat studies and the Pozzani (1959) monkey study were conducted by an independent consulting pathologist, Dr. Robert H. Garman, DVM.  No changes were found that were attributed to acetonitrile exposure.

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Pozzani Monkey Study (1959). A study on the toxicity of acetonitrile in rhesus monkeys performed at Mellon Institute in the late 1950s (completed on 12/24/58) had reported gross hemorrhages within the dura or along the superior sagittal sinus of the brains of some of the monkeys that had died on study (Pozzani et al; J. Occup. Med., Vol I, No. 12, December 1959). Although microscopic examination ofthe monkey brain sections had not revealed any evidence of brain hemorrhage to the original Study Pathologist, an additional histopathologic examination was performed by Dr. Garman to be certain that there were no treatment-related neuropathologic alterations. This slide review indicated that the reported foci of gross "hemorrhage" actually represented a process of agonal or postmortem congestion with associated diapedesis (i.e. the postmortem migration of red blood cells out of vessels as a result of separation of the endothelial cell "tight junctions"). Dr. Garman concluded that critical evaluation of the tissue sections from the monkeys on this study is compromised by concurrent infection and parasitism, as well as by postmortem alteration and tissue handling artifact. Nevertheless, there is no evidence of any neuropathologic effects of the acetonitrile exposures on the rhesus monkey nervous system at the light microscopic level under the conditions of this study. The gross finding of hemorrhage of the superior sagittal sinus could not be substantiated by the microscopic examination (as it also was not substantiated by the original pathologist). It is Dr. Garman’s opinion that this gross finding should not, therefore, have been reported as a manifestation of toxicity in the publication by Pozzani et al (J. Occup. Med., Vol 1, No. 12, December 1959).

Because brain hemorrhages had also been reported in some of the rats in the highest concentration (1600 ppm) group of the 13-week subchronic inhalation study on acetonitrile performed by the National Toxicology Program (NTP, 1996), it was deemed prudent to also examine the brain sections from these rats to look for the presence of any treatment related neuropathologic alterations.

NTP Subchronic Rat Study. The NTP subchronic study on the toxicity of acetonitrile in rats had six concentration levels (control, 100 ,200, 400, 800, and 1600 ppm), with there being 10 rats/sex/group. A gross observation of "red foci" had been recorded for the brains of some of the rats in the 1600 ppm group that had died early in the study. Furthermore, the Study Pathologist had reported brain hemorrhages in 3/10 of the 1600 ppm male group and in 1/10 of the 1600 ppm females. In the EPL Quality Assessment Report on this study, the Original Reviewing Pathologist (Dr. H. Roger Brown) stated (pg. 15 of the EPL Quality Assurance Report) that he believed all these foci of "hemorrhage" to either represent congestion or to be secondary to congestion. He indicates further that he found blood outside of vessels in many sections of brains from control rats. Dr. Brown states in his report: "In the opinion of the reviewer, this change was associated with agonal congestion primarily although hemorrhage that occurs as a result of agonal hypoxia may have also occurred." Dr. Robert Garman found the foci of extravascular red blood cells to primarily be meningeal or submeningeal in location, although a few very small-sized foci of peracute/agonal intraparenchymal hemorrhage were also found. Such foci are frequently seen in the brains of rats that have died from a variety of causes and are not exsanguinated. Dr. Garman classified only one of the rats on the subchronic study as actually having hemorrhage. This particular rat (male Rat No. 1002) had extensive peracute intraventricular hemorrhage but no evidence of hemorrhage within the parenchyma of the brain. Furthermore, there was no evidence within the brain sections from this rat of any neuropathologic alteration. Although the Original Reviewing Pathologist commented that the hemorrhage in this rat's brain might have been secondary to congestion, Dr. Garman’s opinion is that a more likely pathogenesis of this lesion may have been one of acute head trauma. While this degree of intraventricular hemorrhage is unusual in this pathologist's experience, it was limited to a single rat on this study and was not considered to represent a toxicologic lesion. In Dr. Brown's Quality Assessment Report (p. 25), he states: "Agonal congestion was also confined in several organs, but hemorrhage in the brain which was reported by the original pathologist was considered in this review to be agonal or secondary to congestion."

NTP Chronic Rat Study.Dr. Garman examined all the brain sections from the top concentration group (400 ppm) male and female rats on the chronic study that had been reported to have histopathologic changes of any type within the brain. Some of these rats had also been reported to have brain hemorrhages. Others had brain tumors or "hydrocephalus." None of these changes were significantly elevated in frequency over control group levels or were considered to be treatment related in the chronic study. The foci of reported hemorrhage in all but one rat represented the same type of peracute/agonal hemorrhage or of postmortem diapedesis (secondary to congestion) that had been seen in the rats on the subchronic study. Furthermore, this type of hemorrhage/erythrocyte extravasation is also frequently seen in rats that die spontaneously and are not exsanguinated. One small-sized focus of peracute meningeal hemorrhage (in Rat No. 741, a 400 ppm female) had an associated intrameningeal mononuclear lelikemic cell infiltrate that had not been documented by the Study Pathologist. Another rat (No. 614, a 400 ppm group male) did have some foci of brain hemorrhage, but these hemorrhages were present within the center of a glioma. Dr. Garman does not believe that this focus of hemorrhage should have been documented as a primary diagnosis on that rat. (Note: the most common cause of frank hemorrhage within the brains of rats, in this pathologist's experience, is that of brain neoplasia -- usually either mononuclear cell leukemia or gliomas). All of the rats on the chronic study reported to have "hydrocephalus" by the Study Pathologist actually represented cases of ventral brain compression by pituitary adenomas.

 

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No classification is proposed based on available animal data.