title: Toxicology and carcinogenesis studies of diisopropylcarbodiimide (Cas No. 693-13-0) in F344/N rats and B6C3F1 mice (dermal studies).
abstract: Diisopropylcarbodiimide is used as a reagent for peptide syntheses and as a chemical intermediate. The National Cancer Institute nominated diisopropylcarbodiimide for study as a representative chemical in the alkylcarbodiimide class because of its acute toxicity; its use in chemical, pharmaceutical, and recombinant DNA industries; and the absence of data on potential health effects. Male and female F344/N rats and B6C3F1 mice were administered diisopropylcarbodiimide (greater than 99% pure) dermally for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 2-WEEK STUDY IN RATS: Groups of five male and five female F344/N rats were dermally administered 0.3 mL ethanol containing 0, 3, 9, 27, or 81 mg diisopropylcarbodiimide or 0.3 mL of the neat chemical containing 242 mg per animal, 5 days a week for 2 weeks. All rats in the 27, 81, and 242 mg groups died before the end of the study. Of the surviving groups, final body weights were similar to those of the vehicle controls. Clinical findings included convulsions/seizures, nasal/eye discharge, tremors, and comatose conditions in 81 and 242 mg rats and lethargy, ataxia, and abnormal breathing in 27 mg rats. The incidences of epidermal hyperplasia at the site of application in 9 and 27 mg males and 27 mg females were significantly greater than those in the vehicle controls; the incidences of hyperkeratosis in 3 and 9 mg males and 9 mg females were also significantly increased. 2-WEEK STUDY IN MICE: Groups of five male and five female B6C3F1 mice were dermally administered 0.1 mL ethanol containing 0, 1, 3, 9, or 27 mg diisopropylcarbodiimide or 0.1 mL of the neat chemical containing 81 mg per animal, 5 days a week for 2 weeks. All 9, 27, and 81 mg mice died before the end of the study. Final body weights of the surviving groups were similar to those of the vehicle controls. Clinical findings in 9, 27, and 81 mg mice included comatose conditions, convulsions/seizures, tremors, abnormal breathing, nasal/eye discharge, lethargy, and irritation at the site of application. Incidences of chronic active inflammation at the site of application in 9 mg males and females were significantly greater than those in the vehicle control groups. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female core study F344/N rats were dermally administered 0, 10, 20, 40, 80, or 160 mg diisopropylcarbodiimide/kg body weight in ethanol, 5 days per week for 3 months. Groups of 10 male and 10 female clinical pathology rats were administered the same doses for 22 days. All 160 mg/kg core study rats were sacrificed moribund or died within the first week of the study. All 80 mg/kg rats died or were found moribund by day 59. Significant decreases in body weight gain occurred in 40 mg/kg males and females, and a significant decrease in final mean body weight occurred in 40 mg/kg females. Clinical findings in groups administered 40 mg/kg or more generally included irritation of the skin at the site of application, seizures, ataxia, abnormal breathing, ruffled fur, thinness, and lethargy. Significantly increased incidences of skin lesions at the site of application included epidermal hyperplasia in all dosed groups of males (except 160 mg/kg) and 40 mg/kg or greater females, epidermal necrosis in 160 mg/kg males and females, and chronic active inflammation in 80 and 160 mg/kg males and females. Significantly increased incidences of nonneoplastic lesions occurred in the brain, lung, and liver (males only) of rats administered 80 or 160 mg/kg. 3-MONTH STUDY IN MICE Groups of 10 male and 10 female B6C3F1 mice were dermally administered 0, 17.5, 35, 70, 140, or 280 mg/kg diisopropylcarbodiimide in ethanol, 5 days per week for 3 months. All mice in the 280 mg/kg group and nine males and nine females in the 140 mg/kg group died before the end of the study. The final mean body weight gain of 70 mg/kg males was significantly less than that of the vehicle control group. Clinical findings observed in 140 and 280 mg/kg mice included abnormal breathing, ataxia, comatose conditions, convulsions/seizures, irritation at the site of application, lethargy, ruffled fur, and thinness. Significant increases in kidney weights occurred in 17.5 and 35 mg/kg males. Significant decreases in total spermatid heads per testis and average spermatid count occurred in 17.5 mg/kg males. At the site of application, the incidences of epidermal hyperplasia in males and females administered 70 mg/kg or greater, chronic inflammation in 140 and 280 mg/kg males and 70 mg/kg or greater females, and sebaceous gland hyperplasia in 140 mg/kg males were significantly increased. Thymic atrophy was significantly increased in 140 and 280 mg/kg males and females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were dermally administered 0, 10, 20, or 40 mg/kg diisopropylcarbodiimide in anhydrous ethanol 5 days per week for 2 years. Survival of 20 mg/kg males was significantly greater than that of the vehicle controls; survival of all dosed groups of females was similar to that of the vehicle controls. Body weights of 40 mg/kg rats were generally less than those of the vehicle controls after week 13. Clinical findings frequently observed in 40 mg/kg males included ataxia, excitability, impaired gait, low muscle tone, abnormal breathing, lethargy, vocalization, and seizures. Because of severe neurological signs exhibited by the 40 mg/kg males, a neuropathological review of these animals was performed. The principal pathological findings of the brain included neuronal necrosis, hemorrhage, and/or fibrinoid arteriole necrosis. Incidences of hemorrhage in the lung of 40 mg/kg males, chronic lung inflammation in 10 and 20 mg/kg females, and alveolar epithelium hyperplasia in 20 mg/kg females were significantly greater than those of the vehicle controls. At the site of application, the incidences of epidermal hyperplasia in all dosed groups of males and 20 and 40 mg/kg females and chronic inflammation in all dosed groups of males and 40 mg/kg females were significantly increased. There was no increased incidences of neoplasms related to diisopropylcarbodiimide administration. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were dermally administered 0, 10, 20, or 40 mg/kg diisopropylcarbodiimide in anhydrous ethanol, 5 days per week for 2 years. Survival of all dosed groups was similar to that of the vehicle control groups. Mean body weights of dosed groups of mice were generally similar to those of the vehicle control groups throughout the study. There were no increased incidences of neoplasms that were attributed to the administration of diisopropylcarbodiimide. Significantly increased incidences of epidermal hyperplasia and focal dermal inflammation of the skin at the site of application occurred in 20 mg/kg male mice.
  
  
  RESULTS
  Diisopropylcarbodiimide was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535 with or without liver S9 activation enzymes. In vivo, the frequency of micronucleated normochromatic erythrocytes was significantly increased in male and female mice after 3 months of dermal exposure to diisopropylcarbodiimide. In addition, significantly elevated frequencies of micronucleated polychromatic erythrocytes (reticulocytes) and micronucleated normochromatic erythrocytes were seen in male mice during a 4-month dermal exposure to diisopropylcarbodiimide. Negative results were obtained, however, in an acute three-injection rat bone marrow micronucleus study. A three-treatment acute micronucleus test in male mice also showed no increase in micronucleated erythrocytes, but results of a single injection micronucleus test in male mice were concluded to be equivocal, due to an increase in micronucleated erythrocytes seen in peripheral blood but not in bone marrow preparations.
  
  
  CONCLUSIONS
  Under the conditions of these 2-year dermal studies, there was no evidence of carcinogenic activity of diisopropylcarbodiimide in male or female F344/N rats or B6C3F1 mice administered 10, 20, or 40 mg/kg. Clinical and histological signs of neurotoxicity in male rats were associated with diisopropylcarbodiimide administration.
mesh_terms: D000279:Administration, Cutaneous; D000818:Animals; D001835:Body Weight / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000473:pathology; D001927:Brain Diseases / Q000139:chemically induced* / Q000473:pathology; D002234:Carbodiimides / Q000008:administration & dosage / Q000633:toxicity*; D005260:Female; D006470:Hemorrhage / Q000139:chemically induced / Q000473:pathology; D006965:Hyperplasia; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced; D009153:Mutagens / Q000008:administration & dosage / Q000633:toxicity*; D016273:Occupational Exposure; D051381:Rats; D011916:Rats, Inbred F344; D012640:Seizures / Q000139:chemically induced / Q000503:physiopathology; D012871:Skin Diseases / Q000139:chemically induced / Q000473:pathology; D018675:Toxicity Tests; D015262:Xenobiotics / Q000008:administration & dosage / Q000633:toxicity*
chemical_list: D002234:Carbodiimides; D009153:Mutagens; D015262:Xenobiotics; C081611:1,3-diisopropylcarbodiimide