o-Nitrotoluene and o-toluidine hydrochloride are structurally related chemicals that are suspected and demonstrated animal carcinogens, respectively. The metabolic potential of the gastrointestinal flora is considered an important factor in o-nitrotoluene-induced toxicity and involves the reduction of the nitro group to the corresponding amine (forming o-toluidine). These studies were designed to 1) compare the target organ toxicities of o-nitrotoluene and o-toluidine hydrochloride administered in feed at approximately equimolar doses (5,000 ppm) to male F344/N rats for 13 or 26 weeks, 2) determine the potential progression or reversibility of toxic or proliferative lesions following chemical withdrawal (stop-exposure) for 13 weeks after 13 weeks of exposure, and 3) examine the effect of antibiotic-altered gastrointestinal flora on the toxicity and/or carcinogenicity of o-nitrotoluene.
o-Nitrotoluene and o-toluidine hydrochloride caused mesothelial hyperplasia and mesothelioma in male rats after 13 or 26 weeks of dietary exposure. The incidence of mesothelioma was greater and the latency was less in rats administered o-nitrotoluene than in rats administered o-toluidine hydrochloride. Additionally, o-nitrotoluene caused testicular degeneration in rats. Effects of o-nitrotoluene administration in the liver included progressive, irreversible increases in liver weight and irreversible increases in the incidences of cytoplasmic vacuolization and oval-cell hyperplasia. Placental glutathione S-transferase (PGST)-positive foci of cellular alteration occurred in the liver after 13 weeks of o-nitrotoluene exposure, and the number and size (as reflected by the volume fraction) of foci were increased after 26 weeks of continuous exposure. During the recovery period, the number of PGST-positive foci in rats in the stop-exposure group decreased slightly, but the size of the foci continued to increase. After 26 weeks, cholangiocarcinoma occurred in 2 of 20 rats in the stop-exposure group and 1 of 20 rats in the continuous-exposure group administered o-nitrotoluene. In contrast, liver effects in rats administered o-toluidine hydrochloride consisted of minimal hemosiderin accumulation in Kupffer cells; the incidence of this lesion in the stop-exposure group decreased during the recovery period. o-Toluidine hydrochloride caused fewer and much smaller PGST-positive foci than those caused by o-nitrotoluene.
o-Nitrotoluene caused an accumulation of hyaline droplets in the renal tubule epithelium; this accumulation did not increase in severity with continued exposure and completely regressed during the recovery period. Exposure to o-toluidine hydrochloride did not cause hyaline droplet accumulation but did cause an accumulation of hemosiderin pigment in renal tubule epithelium. This change progressed in severity during the 26-week continuous-exposure study but decreased in severity in the stop-exposure study during the recovery period.
Exposure to o-nitrotoluene or o-toluidine hydrochloride caused increased incidences of hematopoiesis, hemosiderin accumulation, and capsular fibrosis in the spleen. In rats administered o-toluidine, spleen effects were much more prominent and were also reflected by congestion and markedly increased spleen weights. During the recovery period for the stop-exposure groups administered either compound, incidences of hemosiderin accumulation and hematopoiesis were decreased, but the capsular fibrosis did not resolve. Hyperplasia of the transitional epithelium in the urinary bladder was observed only in rats administered o-toluidine hydrochloride; this lesion did not increase in severity after 26 weeks of continuous exposure and completely regressed in the stop-exposure group during the recovery period. Alteration of the gastrointestinal flora by daily gavage administration of antibiotics did not affect the pattern or severity of toxicity at any site or the development of mesothelioma in rats exposed to o-nitrotoluene, although cholangiocarcinomas that occurred in three rats with the normal flora did not occur in groups with the altered intestinal flora. Subsequent studies determined that the antibiotic regimen used was effective only in reducing the gut population of aerobic microorganisms and had little effect on obligate anaerobes, which are thought to play a mayor role in nitro group reduction.
In summary, these studies confirmed the target organs and compared the relative toxicity for o-nitrotoluene and o-toluidine hydrochloride administered to male F344/N rats at equimolar concentrations in feed. With the exception of the spleen toxicity observed with each chemical, but more prominently with o-toluidine hydrochloride, morphologic effects of exposure to each of these chemicals in the testis/epididymis, liver, kidney, and urinary bladder were different. The results of these studies demonstrate the somewhat greater relative carcinogenic potential of o-nitrotoluene compared to o-toluidine hydrochloride after 13 or 26 weeks of administration based on the occurrence of mesothelioma and cholangiocarcinoma. The apparently lower potency of o-toluidine hydrochloride relative to o-nitrotoluene in the induction of mesothelioma suggests that simple intestinal reduction of the nitro group may not be sufficient for carcinogenic activity in the mesothelium.