[CANCER RESEARCH 34, 255-258, Januarv 1974] Brief Communication Reaction of Drugs with Nitrous Acid as a Source of Carcinogenic Nitrosamines1 William Lijinsky Carcinogenesis Program, Biology Division, Oak Ridge National Laboratory. Oak Ridge, Tennessee 37830 SUMMARY tischistosomiasis), cyclizine (for motion sickness), disul- firam (antialcoholic), and methadone (narcotic). Twelve common drugs that are tertiary amines react with nitrite in aqueous solution at ph 3 to 4 to form dialkylni- trosamines that are known carcinogens. Aminopyrine gave MATERIALS AND METHODS dimethylnitrosamine in 30% yield or higher at all concentra tions down to 50 ppm (with 25 ppm nitrite); the other Aminopyrine (dimethylaminoantipyrine) was from Al- product of this reaction was the nitrite salt of 4-hydroxyan- drich Chemical Co. (Milwaukee, Wis.), quinacrine hydro- tipyrine. The other drugs, when present at 0.01 Mwith 0.04 chloride and methapyrilene hydrochloride were from Sigma Mnitrite, formed nitrosamines in yields ranging from 0.03% Chemical Co. (St. Louis, Mo.), and methadone hydrochlo from dextropropoxyphene to 2.4% from lucanthorre in 4 hr ride was from Mallinckrodt Chemical Works (St. Louis, at 37°. Mo.). The remaining compounds were kindly supplied gratis by the manufacturers as follows: chlorpheniramine (Schering Corp., Bloomfield, N. J.), chlorpromazine (Smith, Kline and French, Philadelphia, Pa.), dextro INTRODUCTION propoxyphene (Eli Lilly and Co., Indianapolis, Ind.), lucan thone (Sterling-Winthrop Pharmaceutical Co., Rensselaer, Some preliminary studies of the interaction of tertiary N. Y.), tolazamide (Upjohn Co., Kalamazoo, Mich.), amines with nitrous acid have indicated that formation of disulfiram (Ayerst Laboratories, New York, N. Y.), cycli dialkylnitrosamines by nitrosative dealkylation is a reaction zine (Burroughs-Wellcome, Research Triangle Park, N. usually undergone by tertiary amines at moderately acid pH C.), and oxytetracycline hydrochloride (Pfizer Pharmaceuti (6). Such reactions taking place in the stomach of man cals, Groton, Conn.). might be a source of carcinogenic nitrosamines (3), since Reactions were carried out in 20- to 40-ml volumes of nitrite is commonly used as a curing and preserving agent in aqueous solution containing the drug, 0.5 to 1.0 ml acetic meat and fish and can be formed by reduction of nitrate. A acid (as a convenient acidifying agent), and sodium nitrite (4 significant exposure of man to tertiary amines is in the form to 8 moles/mole of amine). The pH of the solution was of the many drugs commonly taken (a large proportion of taken at the beginning and end of the reaction. Reactions which are tertiary amines). A short report of reactions of a were carried out at 90°under reflux or at 37°in corked few drugs with nitrous acid has been made (4), and a flasks. At the end of the allotted time the reactions were biological study of the reaction of one tertiary amine, stopped by addition of a few pellets of sodium hydroxide aminopyrine, has demonstrated the formation of large (this made the solutions alkaline), and the aqueous solutions amounts of dimethylnitrosamine in this way (5). were extracted twice with double volumes of méthylène Here are reported further studies on the reactions of chloride; this was adequate to extract almost completely all aminopyrine and other commonly used drugs with nitrous of the nitrosamine present (the partition coefficient for acid, with special regard to the extent of interaction at dimethylnitrosamine between méthylènechloride and rather low concentrations, to offer some indication of the water, or dilute alkali, is 3.5:1, and higher than this for other magnitude of the hazard to man from such interactions. nitrosamines). The méthylènechlorideextracts were back- The drugs examined were aminopyrine (analgesic), chlor- extracted with a small volume of 5 N hydrochloric acid and pheniramine and methapyrilene (antihistaminics), chlorpro- were evaporated to a small volume at room temperature in a mazine and dextropropoxyphene (tranquilizers), tolazamide stream of nitrogen. The residual solutions were made to 5 or (hypoglycémie),quinacrine (antimalarial), lucanthone (an- 10 ml in volumetric flasks, and the nitrosamines were 1Research jointly sponsored by the National Cancer Institute and the estimated by gas-liquid chromatography on 8.4% diethyl- United States Atomic Energy Commission under contract with Union eneglycol succinate on Chromosorb (analyses were carried Carbide Corporation. out by the Analytical Chemistry Division, Oak Ridge Received June 21, 1973; accepted September 24, 1973. National Laboratory, and by Dr. George Singer of the JANUARY 1974 255 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1974 American Association for Cancer Research. William Lijinsky Biology Division). Identification of the product as the drug to nitrite of 1:1 or 1:2 are given in Table 1; the nitrosamine was based on the retention time in a gas-liquid concentration in the reaction mixture and the fraction of the chromatogram and was in most cases confirmed by mass theoretical yield are given. In all cases the expected volatile spectrometry carried out on the products of the reactions on nitrosamine was formed from the tertiary amine. No a larger scale, as for cyclizine (Table 1) and in Ref. 4. A attempt was made to identify the other products of the Coulson nitrogen-specific detector was used for the estima reactions, among which must have been the other possible tion of concentration; this eliminated possible over-estima nitrosamine formed by cleavage of one of the small alkyl tion of nitrosamine because of the presence of by-products groups. having the same retention time, among which is acetic acid In Table 2 are the results of similar reactions of the drugs with the same retention time as dimethylnitrosamine. The with nitrite, but under identical conditions of concentration yields given are minimal because of inevitable losses in the of amine and of nitrite, each solution being 0.01 Min amine extraction and evaporation procedures. Recovery of the and 0.04 Min nitrite. The acid was acetic acid, and the pH's most easily lost nitrosamine, dimethylnitrosamine, was 75 of the solutions were all 3.3 to 3.4. Again the yields of to 80%. volatile nitrosamine are given in /ug/ml of solution and as a percentage of theoretical. Because the reaction of aminopyrine with nitrous acid RESULTS was so much more vigorous than that of any other tertiary amine we have examined, this reaction was explored more The yields of nitrosamine by reaction of the several drugs thoroughly. Table 3 shows the yield of dimethylnitrosamine with nitrite at arbitrary concentrations and with a ratio of at quite low concentrations of aminopyrine with various Table 1 Nitrosamines formed by interaction of drugs with nitrite in acetic acid nitrosamineTemper of tion of drug tion of DrugAminopyrineOxytetracyclineChlorpromazineDextropropoxypheneChlorpheniramineMethadoneMethapyrileneDisulfiramQuinacrineLucanthoneTolazamideCyclizineConcentra(mg/ml)0.2581555555555555555540ConcentraNaNO2 ature37°37373790379037903790379037379037903790Time(hr)24241444441412444434PH3.23.03.23.43.43.33.53.53.53.33.33.43.43.63.53.53.63.63.13.7Mg/ml33200.5103821712522691754.51.512104070155DMN"DMNDMNDMNDMNDMNDMNDMNDMNDMNDMNDMNDMNDiethylnitros-amineDiethylnitros-amineDiethylnitros-amineDiethylnitros-amineDiethylnitros-amineNHMIDNP%of (mg/ml)0.251611010101010101010101010101010101036Yield theoretical40150.30.883.30.161.50.071.80.182.40.7140.30.151.20.73.03.48.2 'DMN, dimethylnitrosamine; NHMI, nitrosohexamethyleneimine; DNP. dinitrosopiperazine. 256 CANCER RESEARCH VOL. 34 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1974 American Association for Cancer Research. Nitrosamines from Drugs and Nitrous Acid Table 2 Formation of nitrosamines from drugs at 0.01 M amine and 0.04 M nitrite Yield of nitrosamine" DrugChlorpromazineChlorpheniramineDextropropoxypheneMethadoneMethapyrileneQuinacrineLucanthoneDisuinramTolazamideTrimethylamineTemperature90°37903790379037903790379037373737Time(hr)14141414141414334pH3.43.43.43.43.33.33.43.43.43.33.33.33.33.33.33.theoretical0.060.050.70.20.40.030.40.040.70.080.15O.I1.92.40.080.61.4 -33.3Jig/ml0.50.3551.630.230.350.61.5119240.8810.5DMNDMNDMNDMNDMNDMNDMNDMNDMNDMNDiethylnitros-amineDiethylnitros-amineDiethylnitros-amineDiethylnitros-amineDiethylnitros-amineNHMIDMN%of " DMN, dimethylnitrosamine; NHMI, nitrosohexamethyleneimine. Table 3 corresponds quite well with the composition 52.59% C, Reaction of aminopyrine with nitrite in acetic acid at 37°in I hr 5.21% H, 16.72% N for CuHnNaO«. It is reasonable to deduce that the yellow product of reaction of aminopyrine Concentrationof aminopyrinemg/ml0.0.0.0.0.0.050.050.05mM0.430.430.430.430.430.220.220.22ConcentrationofNaNOmg/mlO.I0.0750.050.0250.0120.050.0250.012imM1.45I.I0.70.350.170.70.350.17Dimethylnitrosawith nitrous acid is the nitrite salt of the compound shown mineyield(% below (4-hydroxyantipyrine), formed by oxidativedeamina- of theoretical)5554482613523522tion of aminopyrine (Chart 1). A yellow compound was precipitated by addition of sodium nitrite to an acid solution of 4-hydroxyantipyrine. This had a UV absorption spectrum similar to that of the compound derived from aminopyrine and had the same melting point, a mixture of the 2 products showed no depression of melting point, and the mass spectra of the 2 products were the same. The 2 products seem identical. concentrations of nitrite. At high concentrations of