Background:

Dicyclohexylcarbodiimide is used as a stabilizing agent in elastics and fibers and for the chemical synthesis of peptides. We tested if dicyclohexylcarbodiimide could cause cancer in two different strains of genetically modified mice.

Methods:

We applied solutions containing dicyclohexylcarbodiimide dissolved in ethanol to the backs of female Tg.AC hemizygous mice for 20 weeks and to female p53 haploinsufficient mice for 27 weeks. The daily doses were 0.75, 1.5, 3, 6, or 12 milligrams of dicyclohexylcarbodiimide per kilogram of body weight. Besides the skin where the solutions were applied, tissues from over 15 organs were examined for every animal.

Results:

Exposure to dicyclohexylcarbodiimide caused inflammation and hyperplasia of the skin at the site of application in both strains of genetically modified mice. Female Tg.AC hemizygous mice given dicyclohexylcarbodiimide developed squamous cell papillomas of the skin at the site the chemical was applied. Female p53 haploinsufficient mice receiving dicyclohexylcarbodiimide did not have any increase in tumors related to the chemical.

Conclusions:

We conclude that dicyclohexylcarbodiimide caused cancer of the skin in female Tg.AC hemizygous mice but not in female p53 haploinsufficient mice.

3-Week Study in F344/N Rats:

Groups of five male and five female rats were dermally administered 0.3 mL ethanol containing 0, 0.6, 1.8, 5.1, 15, or 45 mg dicyclohexylcarbodiimide, 5 days per week for 3 weeks. All males and females in the 15 and 45 mg groups, four 5.1 mg males, and all 5.1 mg females died before the end of the study. Of the surviving groups, final mean body weights were similar to those of the vehicle controls, although the one surviving 5.1 mg male rat lost weight during the study. Histopathologic examination of rats dosed with 5.1 mg dicyclohexylcarbodiimide or less revealed treatment-related lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, or chronic active inflammation in the dermis.

3-Week Study in B6C3F₁ Mice:

Groups of five male and five female mice were dermally administered 0.1 mL of ethanol containing 0, 0.2, 0.6, 1.7, 5, or 15 mg dicyclohexylcarbodiimide, 5 days per week for 3 weeks. One 0.6 mg female mouse and all mice in the 1.7, 5, and 15 mg groups died before the end of the study. Final mean body weights of the 0.6 mg groups were significantly less than those of the vehicle controls, and animals in these groups generally lost weight during the study. Histopathologic examination of mice dosed with 1.7 mg dicyclohexylcarbodiimide or less revealed treatment-related lesions of the skin at the site of application including epidermal hyperplasia, epidermal necrosis, and acute or chronic active dermal inflammation.

13-Week Study in F344/N Rats:

Groups of 10 male and 10 female core study rats were dermally administered 0, 0.75, 1.5, 3, 6, or 12 mg dicyclohexylcarbodiimide/kg body weight in ethanol, 5 days per week for 13 weeks; groups of 10 male and 10 female clinical pathology study rats were administered the same doses for 22 days. All 12 mg/kg male and female core study rats died or were found moribund and sacrificed prior to day 45. Final mean body weight and body weight gain of 6 mg/kg males were significantly less than those of the vehicle controls. The predominant clinical pathology changes suggest a secondary, treatment-related inflammatory leukogram and minimal decreased erythron of chronic inflammation that would be consistent with necrosis and chronic active inflammation of the skin. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in 3 mg/kg or greater males and 1.5 mg/kg or greater females, chronic active inflammation in 6 and 12 mg/kg males and 1.5 mg/kg or greater females, and epidermal necrosis in 12 mg/kg males. The incidences and severities of epidermal hyperplasia increased in a dose-related manner in both sexes of rats.

13-Week Study in B6C3F₁ Mice:

Groups of 10 male and 10 female mice were dermally administered 0, 1.5, 3, 6, 12, or 24 mg dicyclohexylcarbodiimide/kg body weight in ethanol, 5 days per week for 13 weeks. All 24 mg/kg male and female mice died or were found moribund and sacrificed prior to day 16. Final mean body weights of 6 and 12 mg/kg males and mean body weight gains of 6 and 12 mg/kg males and females were significantly less than those of the vehicle controls. The predominant clinical pathology changes suggest a secondary, treatment-related inflammatory leukogram and minimal decreased erythron of chronic inflammation that would be consistent with necrosis and chronic active inflammation of the skin. Dermal administration of dicyclohexylcarbodiimide significantly decreased the weight of the epididymis in 6 and 12 mg/kg males and significantly decreased epididymal spermatozoal motility in 6 mg/kg males. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in all dosed groups except those administered 24 mg/kg, chronic active inflammation in all dosed groups except 1.5 mg/kg females, and epidermal necrosis in 24 mg/kg males and females.

20-Week Study in Female Tg.AC Hemizygous Mice:

Groups of 10 female Tg.AC hemizygous mice were dermally administered 0, 0.75, 1.5, 3, 6, or 12 mg dicyclohexylcarbodiimide/kg body weight in ethanol, 5 days per week for up to 20 weeks. Due to the severity of skin lesions observed in 12 mg/kg animals, the application of dicyclohexylcarbodiimide was discontinued after eight dermal applications in this group. There were no deaths considered related to dicyclohexylcarbodiimide administration, although 13 animals died or were sacrificed moribund prior to the end of the study: three each from the vehicle control and 0.75 mg/kg groups, four from the 3 mg/kg group, two from the 6 mg/kg group, and one from the 12 mg/kg group. Overall, the survival was within the range known for the Tg.AC hemizygous mouse. Mean body weights of dosed groups of mice were similar to those of the vehicle controls. At the site of application, the incidences of squamous cell papilloma were increased in a dose-related manner. The incidences of chronic active inflammation of the dermis and epidermal hyperplasia were significantly increased in mice administered 3 or 6 mg/kg.

27-Week Study in Female p53 Haploinsufficient Mice:

Groups of 15 female mice were dermally administered 0, 0.75, 1.5, 3, 6, or 12 mg dicyclohexylcarbodiimide/kg body weight in ethanol, 5 days per week for up to 27 weeks. Dosing of the 6 and 12 mg/kg groups was discontinued after 11 and 8 days, respectively, because of the severity of skin lesions at the site of application. Twelve animals died or were sacrificed moribund prior to the end of the study: three from the 3 mg/kg group, one from the 6 mg/kg group, and eight from the 12 mg/kg group. Mean body weights of dosed groups of mice were similar to those of the vehicle controls. No neoplasms were attributed to administration of dicyclohexylcarbodiimide. At the site of application, the incidences of focal epidermal hyperplasia were significantly increased in 1.5, 3, and 12 mg/kg mice, the incidences of focal chronic active inflammation of the dermis were increased in groups administered 3 or 12 mg/kg, and the incidences of focal ulcer and focal chronic active inflammation of the subcutaneous tissue were increased in the 12 mg/kg group.

Genetic Toxicology:

Dicyclohexylcarbodiimide was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535, with or without rat or hamster liver S9 activation enzymes. In vivo, there was a small but significant increase in the frequency of micronucleated normochromatic erythrocytes in male and female B6C3F₁ mice after 13 weeks of dermal exposure to dicyclohexylcarbodiimide. Negative results were obtained, however, in an acute three-injection micronucleus study in bone marrow of male F344/N rats.

Conclusions:

Under the conditions of this 27-week dermal study, there was no evidence of carcinogenic activity^* of dicyclohexylcarbodiimide in female p53 haploinsufficient mice administered 0.75, 1.5, 3, 6, or 12 mg/kg in ethanol.

Female Tg.AC hemizygous mice dermally dosed with dicyclohexylcarbodiimide for 20 weeks had significantly increased incidences of squamous cell papilloma of the skin at the site of application.

Nonneoplastic lesions noted at the site of application included chronic active inflammation and epidermal hyperplasia in female p53 haploinsufficient mice and female Tg.AC hemizygous mice.

Footnotes

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Explanation of Levels of Evidence of Carcinogenic Activity is on page 9. A summary of the Technical Reports Review Subcommittee comments and the public discussion on this Report appears on page 11.