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Abstract for TR-494

Toxicology and Carcinogenesis Studies of Anthraquinone in F344/N Rats and B6C3F1 Mice (Feed Studies)

CASRN: 84-65-1
Chemical Formula: C14H8O2
Molecular Weight: 208.22
Synonyms/Common Names: 9,10-Anthracenedione; anthradione; 9,10-anthraquinone; 9,10-dioxoanthracene; 9,10-dihydro-9,10-dioxoanthracene
Report Date: September 2005

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Abstract

Anthraquinone is used as an intermediate in the manufacture of dyes and pigments, an additive in the kraft pulping process in the paper industry, a catalyst in the isomerization of vegetable oils, an accelerator in nickel electroplating, and as a bird repellant. The National Toxicology Program is conducting a class study of naturally occurring quinones containing the anthraquinone ring; anthraquinone is the parent compound of this class. Male and female F344/N rats and B6C3F1 mice were exposed to anthraquinone (approximately 99.8% pure by gas chromatography and liquid chromatography) in feed for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, mouse bone marrow cells, and mouse peripheral blood erythrocytes.

14-Week Study in Rats

Groups of 10 male and 10 female F344/N rats were fed diets containing 0, 1,875, 3,750, 7,500, 15,000, or 30,000 ppm anthraquinone (equivalent to average daily doses of approximately 135, 275, 555, 1,130, or 2,350 mg anthraquinone/kg body weight) for 14 weeks. All rats survived until the end of the study. Mean body weights of females were significantly less in the exposed groups than in the control group. Feed consumption by the exposed and control groups was similar at the end of the study. Liver and kidney weights of exposed groups were greater than those of the controls, as were testis weights of males exposed to 7,500 ppm or greater. A minimal, responsive anemia was apparent in groups of male and female rats exposed to 3,750 ppm or greater by day 26 of the study. The anemia persisted and involved all exposed groups of rats at the end of the study. Renal function was also affected by anthraquinone exposure as demonstrated by increases in urine protein and glucose concentrations and aspartate aminotransferase and N-acetyl-ß-D-glucosaminidase activities. Estrous cycles were longer in 15,000 and 30,000 ppm females than in the controls.

Groups of exposed rats had liver hypertrophy; eosinophilic hyaline droplets in the kidney; congestion, hematopoietic cell proliferation, and pigmentation of the spleen; and bone marrow hyperplasia. The incidences of nephropathy in 15,000 and 30,000 ppm females were significantly greater than that in the controls, and the severities of nephropathy were increased in exposed groups of males and in 30,000 ppm females. The concentrations of α2u-globulin in the kidneys were significantly greater in all exposed groups of males. Thyroid gland follicular cell hypertrophy was present in all males and females exposed to 3,750 ppm or greater. Incidences of inflammation and transitional cell hyperplasia in the urinary bladder of 30,000 ppm females were greater than those in the controls.

14-Week Study in Mice

Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 0, 1,875, 3,750, 7,500, 15,000, or 30,000 ppm anthraquinone (equivalent to average daily doses of approximately 250, 500, 1,050, 2,150, or 4,300 mg/kg to males and 300, 640, 1,260, 2,600, or 5,300 mg/kg to females) for 14 weeks. All mice survived until the end of the study. Mean body weights and feed consumption were similar among exposed and control groups. A responsive anemia occurred in exposed mice at week 14. Liver weights of exposed groups of mice were significantly greater than those of the control groups.

The incidences of centrilobular hypertrophy in the liver of mice exposed to 3,750 ppm or greater were significantly greater than those in the controls, and the severities increased with increasing exposure concentration. Cytoplasmic alteration of the urinary bladder was observed in all exposed mice, and the severities increased with increasing exposure concentration. The incidences of hematopoietic cell proliferation were increased in all exposed groups of males and females, and pigmentation was observed in the spleen of all exposed mice (except one male and one female in the 30,000 ppm groups).

2-Year Study in Rats

Groups of 50 male and 50 female F344/N rats were fed diets containing 469, 938, or 1,875 ppm anthraquinone for 105 weeks. Groups of 60 male and 60 female F344/N rats received 0 or 3,750 ppm anthraquinone for the same period. Five males and five females receiving 0 or 3,750 ppm were evaluated for histopathology and α2u-globulin concentrations at 3 months and for organ weights and histopathology at 12 months. These dietary anthraquinone concentrations resulted in average daily doses of approximately 20, 45, 90, and 180 mg/kg to males and 25, 50, 100, and 200 mg/kg to females. Additional groups of 18 males given 469, 938, 1,875, or 3,750 ppm for 8 days and 10 males and 10 females given 469, 938, or 1,875 ppm for 3, 6, 12, or 18 months were designated for toxicokinetic studies.

Survival, Body Weights, and Feed Consumption

Survival of all groups of males was similar, and survival of exposed groups of females was greater than that of the controls. Mean body weights of exposed groups of males during the latter part of the study and mean body weights of exposed females throughout most of the study were less than those of the controls. Feed consumption by exposed groups was similar to that by the controls.
 

Pathology Findings

The incidences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) occurred with positive trends and were increased in all exposed groups of female rats. The incidences of renal tubule adenoma in all exposed groups of male rats exceeded the historical control range, and the incidence was significantly increased in the 938 ppm group. Increased incidences of nonneoplastic lesions of the kidney associated with anthraquinone exposure included hyaline droplet accumulation, pigmentation, and mineralization in the renal medulla and transitional epithelial hyperplasia in males and females and renal tubule hyperplasia in females. Incidences of nephropathy were increased in females, and severities of nephropathy were increased in males. At 3 months, the concentration of α2u-globulin in the kidney of 3,750 ppm males was greater than that in the control group.

The incidence of urinary bladder transitional epithelial papilloma was significanty greater in 1,875 ppm males than in the control group, and the incidences in groups of males exposed to 938 ppm or greater exceeded the historical control range. There were positive trends in the incidences of transitional epithelial hyperplasia and papilloma or carcinoma (combined) of the urinary bladder in females.
 

The incidences of hepatocellular adenoma or carcinoma (combined) were slightly increased in exposed males and females; the incidences in groups of females exposed to 938 ppm or greater exceeded the historical control range. The incidences of several nonneoplastic liver lesions of minimal severity were also increased. The incidences of congestion, pigmentation, and hematopoietic cell proliferation of the spleen were greater in exposed males and females than in the controls. The incidences of bone marrow hyperplasia were increased in most groups of exposed rats, and the incidences of bone marrow atrophy were increased in exposed females.

The incidences of mononuclear cell leukemia were significantly less in all exposed groups than in the controls at 2 years, and the incidences were less than the historical control ranges.

2-Year Study in Mice

Groups of 50 male and 50 female B6C3F1 mice were fed diets containing 0, 833, 2,500, or 7,500 ppm anthraquinone (equivalent to average daily doses of approximately 90, 265, or 825 mg/kg to males and 80, 235, or 745 mg/kg to females) for 105 weeks. Additional groups of 36 males given 833, 2,500, or 7,500 ppm for 8 days and 10 males and 10 females given 833, 2,500, or 7,500 ppm for 12 months were designated for toxicokinetic studies.

Survival, Body Weights, and Feed Consumption 

Survival was less for 7,500 ppm males than for the control group. Mean body weights of 7,500 ppm males during the last 6 months of the study and mean body weights of 7,500 ppm females at the end of the study were less than those of the control groups. Feed consumption was similar in all groups of males and females.
 

Pathology Findings 

Incidences of hepatocellular neoplasms (including multiple neoplasms) increased with a positive trend in male and female mice, and the incidences were increased in all exposed groups. Incidences of hepatoblastoma were significantly increased in males exposed to 2,500 or 7,500 ppm. The incidences of several nonneoplastic lesions of the liver were increased in exposed mice. There was a marginal increase in the incidences of neoplasms of thyroid gland follicular cells in males and females. Incidences of intracytoplasmic inclusion body of the urinary bladder and hematopoietic cell proliferation of the spleen in males and females and thyroid gland follicular cell hyperplasia and kidney pigmentation in males were greater in exposed groups than in the controls.

Genetic Toxicology

Anthraquinone (97% pure) was mutagenic in S. typhimurium strains TA98 and TA100, with and without rat and hamster S9 metabolic activation enzymes. A 100% pure anthraquinone sample showed no mutagenic activity in strains TA98, TA100, or TA102, with or without rat liver S9 enzymes. Sample A07496, the compound used in the 2-year studies (99.8% pure), was negative in TA98, TA100, and TA1537, with and without rat S9. Samples A65343 (Diels-Alder process) and A54984 (Friedel-Crafts process) were negative in TA98 and TA100, with and without rat S9. Sample A40147 (Diels-Alder process) was mutagenic in TA98 and TA100, with and without rat S9.

Several substituted anthraquinones were also tested in Salmonella, and results showed significant mutagenic activity for 2-hydroxyanthraquinone and 1-, 2-, and 9-nitroanthracene, with and without S9. 1-Hydroxyanthraquinone was not mutagenic in Salmonella, with or without S9.

Significant increases in the frequencies of micronucleated normochromatic erythrocytes were observed in peripheral blood samples from male and female mice exposed to anthraquinone (99.8% pure) in feed for 14 weeks. However, results of an acute exposure mouse bone marrow micronucleus test, with anthraquinone administered by intraperitoneal injection, were negative.

Physiologically based Pharmacokinetic Model

A physiologically based pharmacokinetic model was developed to characterize tissue concentrations of anthraquinone resulting from oral exposure in rats. Data used to create the model were obtained from the literature or from the current studies. The physiologically based pharmacokinetic model indicates that anthraquinone is slowly and incompletely absorbed, slowly distributed to tissues by a diffusion-limited transport process, stored in fatty tissues, and slowly metabolized. Model-based plasma anthraquinone concentrations may serve as a surrogate dosimeter for evaluating neoplasm exposure concentration-response data.
 

Conclusions

Under the conditions of these 2-year feed studies, there was some evidence of carcinogenic activity of anthraquinone in male F344/N rats based on increased incidences of renal tubule adenoma and of transitional epithelial papillomas of the kidney and urinary bladder. Hepatocellular neoplasms may have been related to exposure to anthraquinone. There was clear evidence of carcinogenic activity of anthraquinone in female F344/N rats based on increased incidences of renal tubule neoplasms. Increases in the incidences of urinary bladder transitional epithelial papilloma or carcinoma (combined) and of hepatocellular adenoma in female rats were also related to anthraquinone exposure. There was clear evidence of carcinogenic activity in male and female B6C3F1 mice based on increased incidences of liver neoplasms. Thyroid gland follicular cell neoplasms in male and female mice may have been related to anthraquinone exposure.

Exposure to anthraquinone for 2 years caused increases in the incidences of nonneoplastic lesions of the kidney, liver, spleen, and bone marrow in male and female rats, the liver, urinary bladder, and spleen in male and female mice, and the thyroid gland and kidney in male mice.

Decreased incidences of mononuclear cell leukemia in male and female rats were attributed to exposure to anthraquinone.

Studies

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Anthraquinone

 


 
Male
F344/N Rats
Female
F344/N Rats
Male
B6C3F1 Mice
Female
B6C3F1 Mice
Concentrations
in feed
0, 469, 938, 1,875, or 3,750 ppm 0, 469, 938, 1,875, or 3,750 0, 833, 2,500, or 7,500 ppm 0, 833, 2,500, or 7,500 ppm
Body weights Exposed groups less than control group Exposed groups less than control group 7,500 ppm group less than control group 7,500 ppm group slightly less than control group
Survival rates 22/50, 23/50, 22/50, 26/50, 22/50 23/50, 40/50, 35/50, 37/50, 40/50 45/50, 41/50, 43/50, 23/50 35/50, 42/50, 35/50, 42/49
Nonneoplastic effects Kidney: hyaline droplet accumulation (3/50, 14/50, 10/50, 16/50, 16/50); severity of nephropathy (2.2, 3.1, 3.1, 3.0, 3.0); pigmentation (25/50, 31/50, 36/50, 38/50, 33/50); medulla, mineralization (30/50, 42/50, 46/50, 47/50, 49/50); transitional epithelium, hyperplasia (28/50, 45/50, 44/50, 48/50, 48/50)

 

Liver: centrilobular hypertrophy (0/50, 4/50, 21/50, 13/50, 29/50); cystic degeneration (9/50, 31/50, 36/50, 28/50, 29/50); inflammation (13/50, 30/50, 28/50, 30/50, 27/50); eosinophilic focus (9/50, 22/50, 30/50, 29/50, 20/50); mixed cell focus (4/50, 12/50, 15/50, 13/50, 10/50); cytoplasmic vacuolization (5/50, 18/50, 23/50, 17/50, 23/50)

Spleen: congestion (6/50, 35/50, 37/50, 30/50, 31/50); pigmentation (12/50, 36/50, 38/50, 33/50, 28/50); hematopoietic cell proliferation (37/50, 45/50, 44/50, 43/50, 39/50)

Bone Marrow: hyperplasia (25/50, 28/50, 37/50, 36/50, 33/50)

Kidney: hyaline droplet accumulation (33/50, 48/50, 45/50, 44/50, 44/49); nephropathy (39/50, 49/50, 47/50, 49/50, 49/49); pigmentation (27/50, 50/50, 48/50, 50/50, 47/49); medulla, mineralization (17/50, 25/50, 27/50, 28/50, 20/49); renal tubule, hyperplasia (0/50, 12/50, 13/50, 15/50, 11/49); transitional epithelium, hyperplasia (0/50, 5/50, 12/50, 3/50, 10/49)

Liver: centrilobular hypertrophy (0/50, 18/50, 23/50, 19/50, 26/49); cystic degeneration (0/50, 5/50, 10/50, 10/50, 6/49); inflammation (25/50, 46/50, 44/50, 38/50, 46/49); eosinophilic focus (8/50, 32/50, 34/50, 39/50, 34/49); mixed cell focus (3/50, 30/50, 20/50, 23/50, 13/49); angiectasis (3/50, 15/50, 18/50, 15/50, 21/49)

Spleen: congestion (1/50, 46/50, 42/50, 44/50, 45/49); pigmentation (33/50, 45/50, 48/50, 48/50, 47/49); hematopoietic cell proliferation (39/50, 50/50, 47/50, 47/50, 46/49)

Bone Marrow: hyperplasia (19/50, 31/50, 28/50, 19/50, 23/50); atrophy (4/50, 13/50, 13/50, 11/50, 13/50)

Liver: centrilobular, hypertrophy (24/50, 34/50, 41/50, 33/49); degeneration, fatty, focal (0/50, 7/50, 6/50, 0/49); hepatocyte, erythrophagocytosis (1/50, 9/50, 13/50, 6/49); eosinophilic focus (14/50, 17/50, 24/50, 20/49); focal necrosis (2/50, 3/50, 3/50, 8/49)

Urinary Bladder: intracytoplasmic inclusion body (0/50, 46/49, 46/49, 42/45)

Thyroid Gland: follicular cell hyperplasia (7/50, 10/50, 15/49, 21/46)

Spleen: hematopoietic cell proliferation (12/50, 14/50, 12/49, 30/42)

Kidney: pigmentation (0/50, 2/50, 2/50, 18/47)

Liver: centrilobular hypertrophy (1/49, 27/50, 22/50, 39/49); degeneration, fatty, focal (2/49, 3/50, 1/50, 9/49); eosinophilic focus (6/49, 15/50, 11/50, 22/49)

Urinary Bladder: intracytoplasmic inclusion body (0/44, 40/48, 43/46, 46/48)

Spleen: hematopoietic cell proliferation (9/45, 17/49, 17/48, 26/48)

Neoplastic effects

Kidney: renal tubule adenoma (1/50, 3/50, 9/50, 5/50, 3/50); transitional epithelial papilloma (0/50, 0/50, 2/50, 0/50, 1/50)

Urinary Bladder: transitional epithelial papilloma (0/50, 1/50, 3/50, 7/50, 3/49)

Kidney: renal tubule adenoma (0/50, 4/50, 9/50, 7/50, 12/49); renal tubule adenoma or carcinoma (0/50, 6/50, 9/50, 8/50, 14/49)

Urinary Bladder: transitional epithelial papilloma or carcinoma (0/49, 0/49, 0/49, 1/50, 2/49)

Liver: hepatocellular adenoma (0/50, 2/50, 6/50, 4/50, 3/50)

Liver: hepatocellular adenoma (21/50, 32/50, 38/50, 41/49); hepatocellular carcinoma (8/50, 13/50, 17/50, 21/49); hepatoblastoma (1/50, 6/50, 11/50, 37/49); hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma (26/50, 35/50, 43/50, 48/49)

Liver: hepatocellular adenoma (6/49, 28/50, 27/50, 40/49); hepatocellular carcinoma (2/49, 3/50, 8/50, 8/49); hepatocellular adenoma or carcinoma (6/49, 30/50, 30/50, 41/49)

Equivocal findings

Liver: hepatocellular adenoma or carcinoma (1/50, 3/50, 4/50, 5/50, 3/50)

None

Thyroid Gland: follicular cell adenoma (0/50, 0/50, 2/49, 2/46)

Thyroid Gland: follicular cell adenoma (1/45, 1/48, 2/48, 2/48); follicular cell carcinoma (0/45, 0/48, 0/48, 2/48); follicular cell adenoma or carcinoma (1/45, 1/48, 2/48, 4/48)

Decreased incidences

Mononuclear Cell Leukemia: (25/50, 2/50, 1/50, 5/50, 7/50)

Mononuclear Cell Leukemia: (18/50, 1/50, 1/50, 2/50, 0/50)

None None
Level of evidence of carcinogenic activity Some evidence Clear evidence Clear evidence Clear evidence
Genetic Toxicology of Cumene
Assay Test System Results
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
Anthraquinone (97% pure)
Positive in strains TA98 and TA100 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
Anthraquinone (100% pure)
Negative in strains TA98, TA100, and TA102 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
Anthraquinone (A07496, 99.8% pure)
Negative in strains TA98, TA100, and TA1537 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
Anthraquinone (A65343, Diels-Alder)
Negative in strains TA98 and TA100 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
Anthraquinone (A54984, Friedel-Crafts
Negative in strains TA98 and TA100 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
Anthraquinone (A40147, Diels-Alder, 99.4% pure)
Positive in TA98 and TA100 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
1-Hydroxyanthraquinone
Negative in strains TA98, TA100, and TA102 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
2-Hydroxyanthraquinone
Positive in strain TA98 without S9, negative in strain TA98 with S9, and negative in strain TA100 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
1-Nitroanthracene
Positive in strains TA98 and TA100 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
2-Nitroanthracene
Positive in strains TA98 and TA100 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations:
9-Nitroanthracene
Positive in strains TA98 and TA100 with and without S9
Micronucleated erythrocytes Mouse bone marrow in vivo: Negative
Micronucleated erythrocytes Mouse peripheral blood in vivo
(99.8% pure anthraquinone):
Positive