Urethane, a byproduct of fermentation found in alcoholic beverages, iscarcinogenic in rodents and is classified by the International Agency forResearch on Cancer as a possible human carcinogen. The United States Food andDrug Administration nominated urethane for study because of the widespreadexposure of humans through the consumption of fermented foods and beverages andbecause of a lack of adequate dose-response data about the carcinogenicity ofurethane with and without the coadministration of ethanol. Comparative studies ofurethane in drinking water and in 5% ethanol were conducted to investigatepossible effects of ethanol on urethane toxicity. Toxicokinetic studies ofurethane in drinking water and in 5% ethanol and genetic toxicity studies ofurethane in vivo and in invitro were also conducted.
Groups of 10 male and 10 female F344/N rats and B6C3F1 mice, 6 weeks of age, received 0, 110, 330, 1,100, 3,300,or 10,000 ppm urethane in drinking water or in 5% ethanol for 13 weeks.Toxicokinetic evaluations were performed for urethane in the plasma of male miceafter 13 weeks of administration in drinking water or 5% ethanol. Themutagenicity of urethane in Salmonella typhimuriumstrains TA97, TA98, TA100, TA1535, and TA1537 with and without S9 was tested atdoses up to 16,666 ug/plate; urethane was also tested for induction of sisterchromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovarycells and sex-linked recessive lethal mutations and chromosomal reciprocaltranslocations in Drosophila melanogaster. Thefrequency of micronucleated erythrocytes induced in peripheral blood and bonemarrow cells of mice by urethane in drinking water and in 5% ethanol was alsoevaluated.
In rats that received urethane in drinking water, seven males and fourfemales administered 10,000 ppm and one female administered 3,300 ppm died beforethe end of the study; body weight gains were reduced at these concentrations. Twomales and all females given 10,000 ppm urethane in 5% ethanol died during thestudy, and the body weight gains of males and females that received 3,300 ppmwere lower than those of the controls. Relative right kidney, liver, and lungweights of males and females and relative right testis weights of malesadministered 1,100 ppm or greater were generally higher than those of thecontrols in each study. Leukopenia and lymphopenia were observed in ratsreceiving urethane in either drinking water or ethanol and occurred in malesreceiving 330 ppm or greater and females receiving 110 ppm or greater. Otherdifferences in hematology and clinical chemistry variables were not considered tobe biologically significant.
Lymphoid depletion of the spleen, lymph nodes, and thymus was observed inmale and female rats receiving 1,100, 3,300, or 10,000 ppm urethane in drinkingwater. Cellular depletion of the bone marrow occurred in males and females in the10,000 ppm groups. Hepatocellular fatty changes and clear cell foci of alterationwere noted in the liver of males and females that received 3,300 or 10,000 ppm.The incidences of nephropathy were significantly increased in female rats thatreceived 1,100 ppm or greater; the severity of this lesion in exposed males andfemales was greater than that in the controls. Females that received 330 ppm orgreater had higher incidences of cardiomyopathy than the controls; the severityof this lesion was greater in males in the 10,000 ppm group and females in the3,300 and 10,000 ppm groups than in the controls.
In rats that received urethane in 5% ethanol, lymphoid depletion occurred inmales and females in the 3,300 and 10,000 ppm groups. Cellular depletion of thebone marrow was observed in males and females in the 10,000 ppm groups. Onlymales in the 10,000 ppm group had hepatocellular fatty change (8/10) and clearcell foci (1/10); the incidence and severity of nephropathy in males and femalesand cardiomyopathy in males were similar to those in rats administered urethanein drinking water; however, no cardiomyopathy was observed in females receivingurethane in ethanol. The estrous cycle length of females receiving urethane inethanol appeared to be longer than that of females receiving urethane in drinkingwater. Because cycle length was longer in the 10,000 ppm groups than in thecontrols in both the drinking water and ethanol vehicle studies, this differencemay represent an exacerbation of the toxicity of urethane. A longer estrous cyclemay be a sign of reproductive impairment and correlates with a decrease in femalefecundity.
All mice administered 10,000 ppm urethane in either vehicle died. All mice thatreceived 3,300 ppm urethane in drinking water died, while only one male and fourfemales receiving 3,300 ppm urethane in 5% ethanol died. Body weight gains ofmales and females in all 1,100 ppm groups were less than those of the respectivecontrols, but the weight gains of mice receiving 1,100 ppm urethane in 5% ethanolwere greater than those of mice receiving urethane in drinking water. The meanbody weights of the lower exposure groups were similar to those of the respectivecontrols, and there were no other differences between the body weights of micereceiving urethane in drinking water and those receiving urethane in 5% ethanol.Fluid consumption, and therefore total urethane intake, appeared lower in micereceiving the 5% ethanol vehicle than in those receiving the water vehicle. Therelative right kidney, liver, and lung weights of males and females administeredurethane in drinking water or ethanol were generally greater than those of thecontrols. Clearance of urethane from the plasma of male mice was complete within2 hours after urethane was administered in water, but urethane was not cleared 12hours after administration in 5% ethanol. At the end of 13 weeks of urethaneadministration, the plasma urethane elimination half-life was 0.8 hours; thekinetics were similar for concentrations of 110, 330, and 1,100 ppm urethane inwater and in ethanol. However, at each exposure level, the plasma urethaneconcentration was four times greater for urethane administered in 5% ethanol thanfor urethane administered in drinking water, indicating a possible inhibition ofurethane metabolism by ethanol. Kinetic measurements for elimination by femalemice could not be obtained from the data collected.
In mice administered urethane in drinking water, lung inflammation occurredin males and females that received 1,100 ppm or greater. Alveolar epithelialhyperplasia occurred in the lungs of males in the 330 and 1,100 ppm groups andfemales in the 1,100 ppm group; one male mouse in the 330 ppm group had analveolar/bronchiolar adenoma (see the following summary table). Mice receivingurethane in 5% ethanol had lower incidences and severity of lung inflammation butgenerally greater incidences and severity of alveolar epithelial hyperplasia thanmice receiving the same concentrations of urethane in drinking water.Alveolar/bronchiolar adenomas occurred in four males and one female administeredurethane in ethanol.
Incidence and Severity of Lung Lesions in B6C3F 1 Mice in the 13-Week Studies
of Urethane in Drinking Water and Urethane in 5% Ethanol1
|Alveolar epithelial hyperplasia||0||0||3 (2.0)||1 (4.0)||0||0|
|Alveolar epithelial hyperplasia||0||0||0||4 (1.0)||0||0|
|Alveolar epithelial hyperplasia||0||1 (2.0)||2 (4.0)||3 (2.0)||5* (1.8)||0|
|Alveolar epithelial hyperplasia||0||0||2 (2.0)||7** (1.1)||2 (2.5)||0|
1Ten mice per group wereexamined. In the urethane in drinking water study, all males administered 3,300ppm died by Week 4, and all females administered 3,300 ppm died by Week 5. Allmice administered 10,000 ppm urethane in either vehicle died by Week 3. Averageseverity (in parentheses) is based on the number of animals with lesions:
1=minimal, 2=mild, 3=moderate, and 4=marked
*Significantly different (P<0.05) fromthe control group by the Fisher exact test.
**Significantly different (P<0.01 ) from the controlgroup by the Fisher exact test.
Nephropathy was observed in males and females that received urethane in eithervehicle, and the lesions in female mice were more severe than those in male mice;ethanol did not appear to increase the incidence or severity of nephropathy.Cardiomyopathy occurred in males and females that received 1,100 or 3,300 ppmurethane in drinking water and in females that received 3,300 ppm urethane inethanol. Lymphoid depletion occurred in mice that received 3,300 or 10,000 ppmurethane; 5% ethanol did not appear to enhance these effects. However, urethanein 5% ethanol induced ovarian atrophy; the incidence of this lesion was lower infemales receiving urethane in drinking water. A concentration of 1,100 ppmurethane in either drinking water or ethanol effectively stopped estrouscycling.
Urethane is clearly genotoxic in vitro and <spanclass="italic"> in vivo. In vitro, urethaneinduced mutations in Salmonella <spanclass="italic"> typhimurium strain TA1535 in the presence of liver S9enzymes. Sister chromatid exchanges were induced in cultured Chinese hamsterovary (CHO) cells with and without S9. However, no induction of chromosomalaberrations was observed in CHO cells treated with urethane, with or without S9.In vivo, urethane induced sex-linked recessivelethal mutations and reciprocal translocations in germ cells of adult male <spanclass="italic"> Drosophila melanogaster fed urethane. Significantlyincreased frequencies of micronucleated erythrocytes were observed in peripheralblood obtained from male and female mice after 45 days of exposure and in bonemarrow and peripheral blood obtained after 13 weeks of exposure to urethane indrinking water. There appeared to be no significant difference in the magnitudeof the response in the peripheral blood micronucleus test between miceadministered urethane in drinking water and mice administered urethane in 5%ethanol.
In summary, concentrations of 1,100 ppm urethane or greater in drinking watercaused lymphoid and bone marrow cell depletion and hepatocellular lesions andincreased the severity of nephropathy and cardiomyopathy in male and female rats.The lethal effects of 10,000 ppm urethane were slightly exacerbated by 5% ethanolin female rats. Urethane administered in drinking water induced lunginflammation, alveolar and bronchiolar hyperplasia, alveolar/bronchiolaradenomas, nephropathy, cardiomyopathy, lymphoid and bone marrow cell depletion,seminiferous tubule degeneration, and ovarian atrophy and follicular degenerationin mice. In female mice, 5% ethanol appeared to exacerbate ovarian atrophy. Miceadministered urethane in 5% ethanol consumed less fluid, and therefore lessurethane, than mice receiving urethane in drinking water. Coadministration ofurethane and ethanol inhibited the clearance of urethane from plasma. Incidencesand severity of alveolar epithelial hyperplasia and alveolar/bronchiolar adenomaappeared to be slightly enhanced in mice receiving urethane in 5% ethanolcompared to mice receiving urethane in drinking water. However, theadministration of urethane in 5% ethanol for 13 weeks did not enhance thefrequency of micronucleated erythrocytes in the peripheral blood of male orfemale mice.