Urethane (ethyl carbamate) is produced commercially for use in the preparation and modification of amino resins and as a cosolvent during the manufacture of pesticides, fumigants, and cosmetics. It has also been used as a chemical intermediate in the textile industry to impart wash and wear properties to fabrics, as a cosolvent with drugs, and, for a brief period, as an antineoplastic agent for the treatment of chronic leukemia and multiple myeloma. The major source of human exposure to urethane is from fermented foods and beverages. Urethane in combination with ethanol was nominated by the U.S. Food and Drug Administration for in-depth toxicological evaluation by the NTP because of the widespread exposure to urethane in alcoholic beverages and a lack of adequate dose-response carcinogenicity data to conduct meaningful risk assessments. Male and female B6C3F 1 mice were exposed to urethane (99% pure) and/or ethanol (92.6% ethanol, 7.4% water) in drinking water for 4 weeks or 2 years.
Four-week mechanistic study
Groups of four male and four female mice were exposed to 0, 10, 30, or 90 ppm urethane in the presence of 0%, 2.5%, or 5% ethanol in drinking water ad libitum for 4 weeks. Concentrat ions of 10, 30, and 90 ppm urethane resulted in average daily consumption of approximately 35, 110, and 315 mg urethane for males and 30, 80, and 245 mg for females. Concentrations of 2.5% and 5% ethanol resulted in average daily consumption of approximately 85 and 170 mg ethanol for males and 70 and 130 mg for females. Liver and lung samples were collected for cell proliferation and apoptosis analyses. Additional groups of four male and four female mice were sacrificed after 4 weeks of exposure; liver and lung samples were collected for induction of glutathione, cytochromes P450 and P450 2E1, and DNA adduct formation, and blood was collected for measurement of urethane and ethanol serum concentrations.
Terminal group mean body weights were not affected by either urethane or ethanol. Increasing the urethane concentration had no effect on water consumption by mice. Increasing the ethanol concentration caused a significant decrease in water consumption by males. Increasing the ethanol concentration caused a significant exposure-related decrease in feed consumption by males.
Urethane was detected in the serum of mice exposed to 30 or 90 ppm urethane and 5% ethanol; ethanol was not detected in any of the samples. The percentage of hepatocytes in the G 0 phase was decreased and the percentage in the G1 phase was increased in females exposed to 30 or 90 ppm urethane; this effect was independent of the ethanol concentration. The percentage of PCNA-labeling was decreased in the lung of mice exposed to 30 or 90 ppm urethane, and the effect was independent of the ethanol concentration. Increasing the concentration of ethanol caused an exposure-related increase in cytochrome P450 2E1 activity and an exposure-related decrease in glutathione content in the liver of females; these parameters in females exposed to 2.5% or 5% ethanol were significantly greater or less than those in the controls; the changes were independent of the urethane concentration. Etheno-dA adduct concentrations in hepatic DNA were significantly increased by exposure to urethane and decreased by exposure to ethanol.
Two-year study
Groups of 48 male and 48 female mice were exposed to 0, 10, 30, or 90 ppm urethane in the presence of 0%, 2.5%, or 5% ethanol in drinking water ad libitum for 2 years. Concentrations of 10, 30, and 90 ppm urethane resulted in average daily consumption of approximately 40, 115, and 360 µg urethane for males and 35, 105, and 325 µg for females. Concentrations of 2.5% and 5% ethanol resulted in average daily consumption of approximately 100 and 180 mg ethanol for males and 80 and 155 mg for females.
Survival, body weights, and water consumption
Urethane caused an exposure-related decrease in survival of mice. Ethanol caused a marginal exposure-related increase in survival of males, but had no effect on survival of females. Mean body weights of mice exposed to increasing concentrations of urethane and 0%, 2.5%, or 5% ethanol showed evidence of urethane-induced reductions in body weights, especially in female mice. Mean body weights of mice exposed to 90 ppm urethane and 0%, 2.5%, or 5% ethanol were generally decreased during the last 24 weeks of the study; in addition, females exposed to 10 or 30 ppm urethane and 2.5% ethanol or 0, 10, or 30 ppm urethane and 5% ethanol had generally reduced body weights during this time period. Water consumption by mice exposed to increasing concentrations of urethane and 0%, 2.5%, or 5% ethanol was unchanged throughout the study; water consumption by mice exposed to increasing concentrations of ethanol and 0, 10, 30, or 90 ppm urethane was generally decreased throughout the studies. This ethanol-induced reduction in water consumption was more marked in males than in females.
Pathology findings
Exposure to increasing concentrations of ethanol and 0, 10, 30, or 90 ppm urethane caused an exposure-related decrease in liver weights of males. The incidences of hemangiosarcoma of the liver in mice exposed to 90 ppm urethane and 0%, 2.5%, or 5% ethanol were significantly increased. The incidences of hepatocellular neoplasms were significantly increased in males exposed to 30 or 90 ppm urethane and 0% ethanol or 90 ppm urethane and 2.5% ethanol. In female mice, an increase in the concentration of urethane in the presence of 0%, 2.5%, or 5% ethanol caused an exposure-related increase in the incidences of hepatocellular neoplasms. The incidences of hepatocellular neoplasms were higher in males exposed to greater concentrations of ethanol and 0 ppm urethane (hepatocellular adenoma: 0% ethanol, 7/46; 2.5% ethanol, 12/47; 5% ethanol, 19/48; hepatocellular adenoma or carcinoma: 12/46, 16/47, 25/48). Nonneoplastic liver lesions related to urethane exposure occurred in male and female mice.
Incidences of alveolar/bronchiolar neoplasms were higher in mice exposed to greater concentrations of urethane and 0%, 2.5%, or 5% ethanol. The incidences of alveolar/bronchiolar adenoma or carcinoma (combined) increased in females exposed to increasing concentrations of ethanol and 10 ppm urethane, and the incidence was significantly increased in females exposed to 5% ethanol. Incidences of alveolar/bronchiolar adenoma decreased in males exposed to ethanol and 10 or 30 ppm urethane.
Incidences of harderian gland neoplasms were higher in mice exposed to greater concentrations of urethane and 0%, 2.5%, or 5% ethanol. The incidences of harderian gland adenoma or carcinoma (combined) were significantly increased in all urethane-exposed groups except in females exposed to 10 ppm urethane and 2.5% ethanol. The incidences of harderian gland neoplasms were decreased in males exposed to ethanol and 30 ppm urethane.
Incidences of adenoacanthoma, adenocarcinoma, and adenoacanthoma or adenocarcinoma (combined) of the mammary gland were higher in females exposed to greater concentrations of urethane and 0%, 2.5%, or 5% ethanol. The incidences of adenocarcinoma were significantly increased at 90 ppm urethane and 0%, 2.5%, or 5% ethanol, and at 30 ppm urethane and 2.5% ethanol; the incidences of adenoacanthoma were significantly increased at 90 ppm urethane and 0% or 5% ethanol.
The incidences of hemangiosarcoma of the heart were significantly higher in males exposed to 90 ppm urethane and 0%, 2.5%, or 5% ethanol and in females exposed to 90 ppm urethane and 5% ethanol; the incidence of this neoplasm in females exposed to 90 ppm urethane and 2.5% ethanol was also increased. Increasing the ethanol concentration in the 90 ppm female group caused an exposure-related increase in the incidence of this neoplasm. Incidences of endothelial hyperplasia and angiectasis of the heart increased in mice exposed to increasing concentrations of urethane and 0%, 2.5%, or 5% ethanol.
The incidences of granulosa cell tumor (benign or malignant) of the ovary were significantly higher in females exposed to 90 ppm urethane and 0% ethanol, and the incidences of benign and benign or malignant (combined) granulosa cell tumor were significantly increased in females exposed to 30 ppm urethane and 5% ethanol. The incidences of angiectasis and thrombosis of the uterus in females exposed to 30 or 90 ppm urethane and 0% or 2.5% ethanol were significantly increased.
The incidences of squamous cell papilloma or carcinoma (combined) of the forestomach were higher in males exposed to greater concentrations of urethane and 0% ethanol, and the incidence in the 90 ppm group was significantly increased.
The incidences of squamous cell papilloma or carcinoma (combined) of the skin were higher in males exposed to greater concentrations of urethane and 0%, 2.5%, or 5% ethanol. The incidence was significantly increased in the 30 and 90 ppm groups in the presence of 0% and 2.5% ethanol and in the 90 ppm group in the presence of 5% ethanol.
Incidences of hemangiosarcoma of the spleen was higher in males exposed to greater concentrations of urethane and 2.5% ethanol and in females exposed to increasing concentrations of urethane and 0% or 2.5% ethanol. The incidence of this neoplasm in females exposed to 90 ppm urethane and 0% ethanol was significantly increased. Urethane also caused slight increases in the incidences of hemangiosarcoma of the uterus and skin in females.
Conclusions
Under the conditions of this 2-year drinking water study, there was clear evidence of carcinogenic activity of urethane in male B6C3F 1 mice based on increased incidences of liver, lung, harderian gland, skin and forestomach neoplasms and of hemangiosarcoma, primarily of the liver and heart. There was clear evidence of carcinogenic activity of urethane in female B6C3F 1 mice based on increased incidences of liver, lung, harderian gland, mammary gland, and ovarian neoplasms and of hemangiosarcoma, primarily of the liver and spleen. The occurrences of hemangiosarcoma of the spleen in males and of the uterus and skin in females may have been exposure related.
Exposure to urethane resulted in increased incidences of nonneoplastic lesions of the liver and heart in males and females and of the uterus in females.
The design of this 2-year drinking water study was inadequate to determine the carcinogenic activity of ethanol in male and female B6C3F 1 mice.
Overall, there was weak evidence of an interaction of ethanol in the carcinogenicity of urethane in B6C3F 1 mice. In males, increasing the ethanol concentration may have decreased the alveolar/bronchiolar and harderian gland adenoma or carcinoma responses to urethane. In females, increasing the ethanol concentration may have increased the incidence of hemangiosarcoma of the heart and alveolar/bronchiolar adenoma or carcinoma responses to urethane.