title: Bioassays of nitrilotriacetic acid (NTA) and nitrilotriacetic acid, trisodium salt, monohydrate (Na3-NTA-H2O) for possible carcinogenicity.
abstract: Bioassays for the carcinogenicity of nitrilotriacetic acid, trisodium salt, monohydrate (Na3-NTA-H2O) were conducted at Stanford Research Institute (SRI), using Fischer 344 rats and at Litton Bionetics, Inc. (LBI), using both Fischer 344 rats, and B6C3F1 mice. Similar bioassays using rats and mice, were conducted at LBI on the free acid, nitrilotriacetic acid (NTA). Each chemical was mixed in respective diets and administered ad libitum. The Na3-NTA-H2O was tested in rats at SRI at 200, 2,000, and 20,000 ppm for a 24-month period. It was also tested in rats at LBI at 7,500 and 15,000 ppm and in mice at 2,500 and 5,000 ppm using 18-month feeding periods for both species. The NTA was tested in rats and mice at LBI at 7,500 and 15,000 ppm for the 18-month period. The numbers of animals used in tests at SRI were 24 of each sex for each dose group and for the controls; at LBI, 50 of each sex for each dose group and 20 of each sex for the controls. Since equimolar quantities of Na3-NTA-H2O and NTA were not used, given concentrations of Na3-NTA-H2O represented 30% less NTA than did equal concentrations of the free acid. Average weights attained by high-dose groups of rats and mice were consistently lower than those of control groups. Less difference was observed with the low-dose groups. Survival, however, was not decreased by the compounds administered, except in rats given 20,000 ppm Na3-NTA-H2O. Lesions of the urinary tract were found in most treated groups of both rats and mice. They were characterized, especially in the high-dose groups, by primary tumors of epithelial origin. These tumors were particularly significant since they were not found in the urinary tract of the control mice and only rarely occur spontaneously in the strains of animals on test. Lesions of the urinary tract were also characterized by hydronephrosis and/or nephritis in high-dose rats and by nephritis in both high- and low-dose mice. Statistical evidence of the carcinogenicity of Na3-NTA-H2O and NTA was provided by incidences of tumors at different sites in the urinary tract. For example, among animals given 20,000 ppm Na3-NTA-H2O at SRI, tumors of the kidney occurred in male (treated, 9/24; untreated, 0/24; P=0.001) and female (treated, 4/24; untreated, 0/24; P=0.054) rats; tumors of the ureter in male (treated, 8/24; untreated, 0/24; P=0.002) and female (treated, 6/24; untreated, 0/24; P=0.011) rats; and tumors of the bladder, in female rats (treated, 5/24; untreated, 0/22; P=0.031). Similarly, among animals given 15,000 ppm NTA at LBI, tumors of the bladder occurred in female rats (treated, 12/48; untreated, 0/18; P=0.014) and tumors of the kidney occurred in male mice (treated, 24/44; untreated, 0/20; P<0.001). Additional tests at LBI, using 15,000 and 7,500 ppm Na3-NTA-H2O and 7,500 ppm NTA in male and female rats, 15,000 ppm NTA in female mice, and 7,500 ppm NTA in male mice, also induced tumors of the urinary tract, but in numbers too low to be statistically significant. Metastatic tumors, appearing to have arisen from primary tumors of the urinary trac the urinary tract, were found in 5/24 male and 5/24 female rats given 20,000 ppm Na3-NTA-H2O at SRI and in one male rat given 15,000 ppm NTA at LBI; none were found in rats given lower doses or in mice. Thus, NTA and Na3-NTA-H2O were shown to be carcinogenic to the urinary tracts of both rats and mice at the higher doses tested. Lower doses, as delineated in this report, did not induce significant numbers of such lesions.