C.I. Pigment Red 3, a yellowish red solid, is widely used for coloring paints, inks, plastics, and rubber, and in textile printing. It is used in a wide range of consumer items such as wallpaper, typewriter ribbons, carbon paper, and art materials. Toxicology and carcinogenicity studies were conducted by feeding groups of F344/N rats and B6C3F1 mice of each sex diets containing C.I. Pigment Red 3 (97% pure) for 2 weeks, 13 weeks, and 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and cultured Chinese hamster ovary cells.
Groups of five rats and five mice of each sex were given feed containing 0, 6,000, 12,500, 25,000, 50,000, or 100,000 ppm C.I. Pigment Red 3 for 2 weeks. No chemical-related deaths occurred in rats or mice. Final mean body weights of exposed rats and male mice were lower than controls; female mice that received 6,000 and 50,000 ppm had significantly increased final mean body weights compared to that of the controls. The feed consumption of treated rats and mice was slightly greater than that of the controls, suggesting that C.I. Pigment Red 3 had no adverse effects on the feed palatability. Dose-related decreases in erythrocyte counts and hematocrit values and an increase in reticulocyte counts were observed in rats. Changes in these parameters were observed in mice, but there were no clear, dose-related trends.
Groups of ten rats and ten mice of each sex were given feed containing 0, 3,000, 6,000, 12,500, 25,000, or 50,000 ppm C.I. Pigment Red 3 for 13 weeks. No chemical-related deaths were observed in rats or mice. The final mean body weights of exposed female rats were significantly lower than that of the controls; the final mean body weights of exposed male rats and exposed mice were similar to controls. There were significant increases in relative liver and kidney weights of exposed male rats. Increases in the relative liver weights in mice did not occur with a dose-related trend and thus they were not considered related to chemical administration. Sites for the toxicity of C.I. Pigment Red 3 were the bone marrow, kidney, liver, and spleen in rats. Lesions observed in rats included bone marrow hyperplasia, congestion and hematopoietic cell proliferation of the spleen, and iron-positive pigmentation of the spleen, kidney, and liver. Sites for the toxicity of C.I. Pigment Red 3 in mice were the liver, kidney, and spleen in males and the liver and spleen in females. Lesions noted among mice in the spleen were hematopoietic cell proliferation and iron-positive pigmentation. In the liver, there was hematopoietic cell proliferation in male and female mice. Cytomegaly occurred in the renal tubule epithelium of the male mouse kidney.
Doses selected for the 2-year feed studies were 0, 6,000, 12,500, and 25,000 ppm for rats and 0, 12,500, 25,000, and 50,000 ppm for mice. The dose selection for rats was based on body weight changes observed for females that received 50,000 ppm; the dose selection for mice was based on the lack of body weight depression or death at the doses tested during the 13-week studies. Concentrations higher than 50,000 ppm in the feed were not used because higher levels might have adversely affected the nutritional value of the diet during the 2-year studies.
Body weight, feed consumption, clinical findings, and survival
Final mean body weights for male rats that received 25,000 ppm, female rats that received 12,500 and 25,000 ppm, and male and female mice that received 50,000 ppm were more than 10% lower than those of the controls. Feed consumption of exposed rats and mice was similar to that of the controls. No clinical findings indicative of toxicity were observed in rats or mice. The survival of low-dose male rats was greater than that of the controls (0 ppm, 28/50; 6,000 ppm, 40/50; 12,500 ppm, 28/50; 25,000 ppm, 20/50). Survival of exposed female rats and exposed male mice was similar to the controls; the survival of high-dose female mice was significantly decreased compared to that of the controls (39/50, 37/50, 31/50, 25/50). The reduced survival in this dose group may have been due to the increased incidence of ovarian abscesses.
Neoplasms and nonneoplastic lesions
Benign adrenal pheochromocytomas were significantly increased in the 12,500 and 25,000 ppm groups of male rats compared to the controls (22/50, 29/50, 35/50, 34/50). However, malignant neoplasms were not increased in incidence (6/50, 7/50, 10/50, 4/50). The incidence of adrenal pheochromocytomas in dosed groups exceeded the range for NTP historical controls for feed studies (22%-48%), and the increased incidence of this neoplasm was attributed to C.I. Pigment Red 3 administration.
Squamous cell papillomas of the skin occurred with a positive trend in male rats (0/50, 4/50, 2/50, 6/50), and the incidence in the high-dose group was significantly greater than that of the controls. A poorly differentiated squamous cell carcinoma (diagnosed as carcinoma) was observed in a control male. The historical control rate for squamous cell papillomas in NTP feed studies is low (16/800 or 2%, range 0%-4%), and the higher incidence of this tumor in male rats may have been caused by the administration of C.I. Pigment Red 3.
Hepatocellular adenomas occurred with a positive trend in female rats, with a significantly greater incidence in the high-dose group than in the control group (0/50, 0/50, 1/50, 10/50). This neoplasm has occurred in only one historical control group in NTP feed studies (3/800, range 0%-6%), and the increase in hepatocellular adenomas in female rats was attributed to chemical administration.
Chemical-related nonneoplastic lesions observed in the livers of male and female rats included eosinophilic or mixed type foci of cellular alteration. Foci were often accompanied by angiectasis and cystic degeneration in males and by granulomas and cholesterol pigmentation in females. Chronic nephropathy occurred with increased severity in exposed male and female rats. The lesions were more severe in males than in females. Other lesions considered secondary to renal disease included parathyroid gland hyperplasia, fibrous osteodystrophy of the bone, and mineralization of various organs (stomach, intestine, heart, and blood vessels). The increased incidence of hyperplasia of the transitional epithelium of the renal papilla observed in treated rats was considered to be part of the chronic nephropathy.
Zymbal's gland carcinoma incidences were marginally increased in the mid- and high-dose male rats (0/50, 0/50, 2/50, 3/50). The incidence in the high-dose group was outside the NTP historical control range (0%-4%), and the Zymbal's gland carcinomas may have been related to C.I. Pigment Red 3 administration.
Mononuclear cell leukemias, mammary gland fibroadenomas, and preputial gland/clitoral gland adenomas occurred at lower incidences in exposed male and female rats. The decrease in mononuclear cell leukemia was attributed to the direct effect of C.I. Pigment Red 3 or its metabolites on the mechanism responsible for inducing leukemias in aging rats, while the decreased incidence of mammary gland fibroadenomas might be attributed to decreased body weights in female rats. The cause of the decreased incidences of preputial and clitoral gland tumors is unknown.
Tubule adenomas of the renal cortex occurred at a significantly higher incidence in high-dose male mice than in controls (0 ppm, 0/50; 12,500 ppm, 0/50; 25,000 ppm, 0/50; 50,000 ppm, 6/50). Because this tumor occurred only in exposed males and was outside the range for NTP historical controls in feed studies (0%-2%), renal cortical tubule adenomas in male mice were considered to be related to the administration of C.I. Pigment Red 3.
Follicular cell adenoma of the thyroid gland occurred with a positive trend in male mice (0/50, 0/49, 1/50, 5/50). The incidence in the high-dose group was significantly greater than that in the controls. This chemical-related effect is supported by the increased incidence of follicular cell hyperplasia. Because the incidence of this tumor exceeded the range of the historical controls from NTP feed studies (0%-4%), the increase of follicular cell adenoma was attributed to chemical administration. Female mice receiving C.I. Pigment Red 3 had a significant increase in follicular cell hyperplasia but showed no increase in tumor incidence at this site.
Focal renal tubule hyperplasia and cystic hyperplasia occurred in exposed male mice but not in the controls. Cytomegaly (karyomegaly) of the renal tubule epithelium was seen in all treated male mice. The severity of the accompanying chronic nephropathy was increased in both male and female mice.
C.I. Pigment Red 3 was mutagenic in Salmonella typhimurium strains TA100 and TA98 in the presence of exogenous metabolic activation (S9); no increases in gene mutation were observed in strains TA1535 and TA1537, with or without S9. C.I. Pigment Red 3 did not induce sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells in either the presence or the absence of S9.
Under the conditions of these 2-year feed studies, there was some evidence of carcinogenic activity of C.I. Pigment Red 3 in male F344/N rats as exhibited by increased incidences of benign pheochromocytomas of the adrenal gland. The marginal increase in the incidences of squamous cell papillomas of the skin and Zymbal's gland carcinomas may have been related to C.I. Pigment Red 3 administration. There was some evidence of carcinogenic activity of C.I. Pigment Red 3 in female F344/N rats as indicated by the increased incidence of hepatocellular adenomas. There was some evidence of carcinogenic activity of C.I. Pigment Red 3 in male B6C3F1 mice as exhibited by the increased incidences of tubule adenomas of the renal cortex and follicular cell adenomas of the thyroid gland. There was no evidence of carcinogenic activity of C.I. Pigment Red 3 in female B6C3F1 mice that received 12,500, 25,000, or 50,000 ppm.
The incidences of mononuclear cell leukemia and preputial gland tumors in male rats and mononuclear cell leukemia, mammary gland fibroadenoma, and clitoral gland tumors in female rats were lower in the exposed groups. The incidences of liver foci were markedly increased in exposed male and female rats. The severity of chronic nephropathy was increased in male rats and to a lesser extent in female rats given C.I. Pigment Red 3. An increase in the severity of nephropathy was observed in male and female mice; cytomegaly (karyomegaly) of renal tubule epithelium was observed in male mice. Thyroid follicular cell hyperplasia occurred with an increased incidence in male and female mice receiving C.I. Pigment Red 3.