Trinitrofluorenone (TNF) is a major component of a toning formulation that at one time was used widely in certain photocopy processes. Because the principal route of exposure of humans to TNF probably would be dermal, studies were conducted to compare chemical absorption, distribution, excretion, and tissue retention, as well as toxicity in 14-day studies, by oral and dermal routes of exposure. Further, 13-week toxicity studies were carried out with TNF incorporated into the feed of rats and mice of both sexes. In genetic toxicity evaluations, TNF was found to be mutagenic in Salmonella typhimurium, with and without metabolic activation.
In disposition and metabolism studies, excretion patterns following oral administration of radiolabeled TNF (in doses ranging between 1 and 100 mg/kg body weight) were similar; 20% and 70% of the administered dose appeared in urine and feces, respectively, during the first 72 hours. Residual radiolabel in tissues comprised less than 1% of the dose. The appearance in the feces of 60-70% of a 1 mg/kg i.v. dose provided evidence for substantial biliary excretion of TNF; studies of the radiolabeled materials extracted from urine and feces suggested that glucuronidation is a major biotransfomation of TNF and its metabolites.
In the dermal exposure studies, groups of 5 F344/N rats and 5 B6C3F1 mice of each sex were administered TNF in acetone by topical application once a day, 5 days per week, for 14 days. Doses were 0, 7.5, 15, 30, 60, or 120 mg/kg body weight for rats and 0, 12.5, 25, 50, 100, or 200 mg/kg for mice. There were no deaths, no adverse clinical signs, and no gross or microscopic changes related to treatment in either species, except for discoloration of skin at the site of application. Disposition studies with female rats showed that less than 10% of a dermal dose of 47 mg and less than 3% of a dermal dose of 400 mg were available systemically.
In contrast, toxicity was observed in the 14-day feeding studies with TNF. Groups of 5 rats and 5 mice of each sex were fed diets containing TNF at concentrations of 0, 500, 1600, 5000, 16000, or 50000 ppm. There were no deaths of rats or mice, but body weight gains of rats receiving 50000 ppm were reduced by as much as 45%. Animals receiving diets with 5000 ppm or higher TNF had a black discoloration of the skin and hair and enlarged and/or dark thyroid glands. Mild follicular cell hypertrophy and pigmentation of the epithelium and colloid were noted in the thyroid gland. Among mice, the brain and gallbladder were dark; the spleen of females was dark and also enlarged by hematopoiesis. Thymic lymphoid depletion and atrophy of the seminal vesicles were present in top-dose male rats (50000 ppm).
In 13-week studies, groups of 10 animals of each sex received diets containing TNF at concentrations of 0, 1000, 2000, 4000, 8000, or 16000 ppm for rats, and 0, 3125, 6250, 12500, 25000, or 50000 ppm for mice. No rats died, but the deaths of several mice in the 50000 ppm groups indicated a possible relation to TNF ingestion. Body weight gains of dosed rats were lower than controls and were dose-related. Top-dose male mice gained markedly less weight than controls. A mild macrocytic anemia and increase in methemoglobin was present in dosed rats at the end of the study. In both species, there was a widespread occurrence of a dark brown pigment in dosed animals, with little evidence of toxicity related to the pigment accumulation. Other treatment-related effects in male rats included mesenteric vascular inflammation, renal inflammation, testicular degeneration with reduced sperm count and motility, splenic hematopoiesis, and oval cell hyperplasia, cytoplasmic alteration, and mixed cell foci in the liver. Top-dose female rats had centrilobular hepatocyte cytoplasmic alteration and splenic hematopoiesis. Dosed mice of both sexes showed cystic degeneration of the thyroid gland, liver hypertrophy, and splenic hematopoiesis.
In summary, TNF caused a variety of lesions in oral feeding studies. The no-observed-adverse-effect-level (NOAEL) for microscopic changes other than pigment accumulation was 1000 ppm for rats. A NOAEL could not be determined for mice from this study. Limited dermal absorption likely would prevent significant systemic toxicity resulting from contact of TNF with the skin.