The Nitrosation of Hexetidine and Hexedine: Characterization of the

Richard N. Loeppky, Sukhjeet P. Singh, Saleh Elomari, Riley Hastings, and Thomas E. Theiss. Journal of the American Chemical Society 1998 120 (21), 51...
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Chem. Res. Toxicol. 1994, 7, 868-876

868

The Nitrosation of Hexetidine and Hexedine: Characterization of the Major Nitrosamine from Common Antimicrobial Agents J.-Y. Bae,? P. Mende,$ G. Shevlin,? B. Spiegelhalder,$ R. Preussmann,*>$ and R. N. Loeppky*?’ Department of Chemistry, University of Missouri, Columbia, Missouri 65211, and The German Cancer Research Center, Heidelberg, Germany Received June 8, 1994@

The acidic nitrosation of hexetidine and hexedine, common antimicrobial agents and drug constituents, leads t o a mixture of nitrosamines. The major nitrosamine product, “HEXNO”, forms rapidly in yields a s high a s 60% over the pH range 1-4.8 at incubation times of 1h a t 37 “C with 40 mM NOa- and 10 mM hexetidine. On the basis of extensive spectroscopic characterization and independent synthesis HEXNO has been assigned the structure of 142ethylhexyl)-3-nitroso-4-methyl-4-[[N-(2-ethylhexyl)-N-nitrosoaminolmethyllimidazolidine (7). The synthesis of HEXNO involves the novel interception by potassium nitrite in ether/l& crown-6 of a n imminium ion produced from the reaction of hexedine with benzyl chloroformate. Collapse of the a-amino nitrous ester produced by this reaction yields the nitrosamine containing carbamate 8 , which yields HEXNO after removal of the carbamate with trimethylsilyl iodide and subsequent nitrosation. The rapid formation of HEXNO from hexetidine and hexedine supports the hypothesis that tertiary geminal diamines will produce nitrosamines rapidly by a mechanism which involves the cleavage of a nitrosammonium ion with the assistance of the neighboring nitrogen atom. This process is deemed to be of possible importance in the endogenous production of potentially carcinogenic nitrosamines because of its low nitrite requirement and high nitrosation rate. The available data suggest the probable formation of HEXNO and other nitrosamines from hexetidine under conditions of its use.

Introduction Hexetidine (1) is a common “formaldehyde releasing” antimicrobial agent used in mouth washes and numerous other commercial products. In this publication we report on the characterization and identity of the major nitrosamine product which arises from the astonishingly rapid nitrosation of both 1 and hexedine (2), a related antimicrobial substance. This report is the collaborative culmination of two independent investigations of the nitrosation of hexetidine. While the overall objective of both investigations is the reduction of human exposure to possible carcinogens, the starting points of each study were somewhat different. Most common tertiary amines do not nitrosate rapidly enough to be major precursors of nitrosamines in the human environment (1-3). The nitrosation of hexetidine and related model compounds was undertaken t o test the hypothesis that geminal diamines would be susceptible to rapid nitrosation with ensuing generation of nitrosamines. This publication and the accompanying paper ( 4 ) , which reports the results of a related model study, clearly support this hypothesis.

\I

CH/’NH, 1

R=

~

R

W

CH3, NH, RNHCH&H,NHR

2

3

CH,CH ( C2H51 CH,CH,CH,CH,

In a preliminary account Loeppky et aZ. reported that the nitrosation of hexetidine resulted in the rapid pro+ @

University of Missouri-Columbia. The German Cancer Research Center, Heidelberg. Abstract published in Advance ACS Abstracts, October 15, 1994.

duction of at least eight different nitrosamines (3). Model studies by these workers revealed the identity of two nitrosamines which arise from a substrate lacking the NH2 group of hexetidine (eq 1). The work in Germany

arose from a different perspective. German federal health guidelines require that new nitrogen containing compounds or older compounds proposed for new commercial uses be tested for their ability to produce nitrosamines by the WHO-NAP test (5). Application of this test to hexetidine revealed rapid nitrosamine formation with limited quantities of acidic nitrite. Mende et al. have characterized the products from the nitrosation of N1,N3-bis(2-ethylhexyl)-2-methyl-l,2,3-triaminopropane (3),the hydrolysis product of hexetidine (6). These products (Scheme 1)also result from the nitrosation of hexetidine, but the structure of the major product of that nitrosation reaction has not been determined and is the subject of this paper.

Experimental Procedures Caution:Since many nitrosamines are known to be carcinogenic, nitrosation mixtures and specific nitrosamines should be handled with extreme care.

0893-228x/94I2707-086~~04.50l00 1994 American Chemical Society

The Nitrosation of Hexetidine

Scheme 1 CH\

,NH2

RNHCH/%H,NHR

3

+ NO 0

HNO,

I

R HO CH,

Materials and Methods. Hexetidine, as the free base, was obtained either from Angus Chemie (Heidelberg, Germany) or Aldrich Chemicals (Milwaukee, WI) and was stored at -20 "C. Its purity was verified by GC-MS. Commercial hexetidine preparations were obtained from pharmacies in the Heidelberg, FRG, area. Reagents were obtained from commercial suppliers (reagent grades) and used as received unless otherwise specified. Solvents were distilled according to conventional procedures shortly before use. Flash chromatography was carried out on E. Merck silica gel 60 (230-400 mesh), and medium pressure liquid chromatography (MPLCY on E. Merck (supplier) silica gel 60 (25-310 mesh). Thin layer chromatography (TLC) was performed on precoated silica gel 60 F-254 plates (20 cm x 20 cm, layer thickness 0.2 mm) manufactured by E. Merck. TLC plates were visualized by W irradiation (254 nm) followed by development with the Griess reagent for N-nitroso compounds. High pressure liquid chromatography (HPLC) was performed on a Waters chromatograph equipped with a M-510 solvent delivery system, a n automatic sampler (WISP Model 710B), a multiwavelength detector, and a MAXIMA 820 data system. Reverse phase columns were used: a Zorbax analytical column (ODS, 25 cm x 4.6 mm i d . ) and an Alltech (Deerfield, IL) semipreparative column (ODS, 25 cm x 10 mm id.). GC-MS was run on a Hewlett-Packard Model 5890 gas chromatograph equipped with a Supelco (Bellefonte, PA) SPB-1 fused silica capillary column (30 m x 0.25 mm id.) connected to a HewlettPackard 5970 mass selective detector. GC-FID was performed on a Hewlett-Packard Model 5890-11 with a Supelco SPB-1 column (30 m x 0.25 mm id.). GC-TEA was run on a HewlettPackard Model 5880A gas chromatograph equipped with an Alltech SE-30 fused silica capillary column (30 m x 0.54 mm i.d.1 and a TEA analyzer (Model 502). FT-IR spectra were recorded on a Nicolet 20 DXB spectrophotometer. lH and 13C NMR spectra were recorded on a Joel FX-SOQ, a Nicolet NT300, or Bruker AMX-500 spectrometers. COSY and HETCOR experiments were run on a Bruker AMX-500 spectrometer, and DEPT was measured on a Nicolet NT-300 spectrometer. Deuterated chloroform was used as a solvent. High resolution mass spectra (HRMS) were determined either a t the Midwest Center for Mass Spectrometry, Lincoln, NE, or in Heidelberg on a Finnigan MAT 711 MS, 70 eV, direct probe a t 80 "C. Elemental analyses were obtained from Desert Analytics (Tucson, AZ). Nitrosation of Hexetidine. Hexetidine (54.3 mg) was dissolved in 10 mL of acetic acid (16 mM, pH 4-5) and nitrosated with 1,2, 4, and 8 equiv of aqueous NaNOz (from a 3 M stock solution). Each mixture was stirred for 1h at room temperature and then neutralized with saturated aqueous Naz-

Chem. Res. Toxicol., Vol. 7, No. 6, 1994 869 c o s . The pH was adjusted to 11with additional Na~C03.The products were extracted into benzene, and the organic layer was washed with brine, dried (MgS041, and concentrated to 15 mL. Each analytical sample was subjected to analysis by GC (FID, MS, and TEA) and HPLC. In order to isolate and characterize the major N-nitroso product, a scaled up nitrosation procedure was utilized. An icecold aqueous solution (40 mL) of 8-fold excess of sodium nitrite (4.9 g) was added in one portion to 3 g of hexetidine dissolved in 30 mL of acetic acid. Within 5 min a water-insoluble yellow oil separated. After 1h the mixture was made alkaline to pH 11with aqueous NaOH at 0 "C. A typical workup gave 3.4 g of product mixture. More than 8 spots were visualized by UV irradiation on TLC (1:l hexane/EtOAc), most of which showed positive response to the Griess reagent. The crude mixture was separated by flash chromatography (5% EtOAdhexane). The most nonpolar product (Rf= 0.56,30% EtOAc in hexane) from the mixture turned out to be the major N-nitroso product HEXNO [1-(2-ethylhexyl)-3-~tros~4-methyl-4-[[N-~2-ethy~e~l)N-nitrosoaminolmethyllimidazolidine(711 as determined by GCTEA. This fraction was concentrated and rechromatographed on a MPLC column with gradient solvent system (hexane to 5% EtOAc in hexane). After removal of solvent in uucuo, 840 mg of a yellow oil was isolated. Analytical HPLC showed four peaks. Further purification of the product was achieved by preparative HPLC [methanovwater (90:10), flow 2.0 mumin, 30 min run time, W detection a t 250 nml. The pure product (yellow oil) shows 2 peaks on analytical HPLC. IR (neat, cm-l): 2960 (s), 2930 (s), 2873 (m), 2859 (m), 1459 (s), 1298 (m), 1091 (m). lH NMR (500 MHz, CDC13): 6 4.58-2.19 (m, lOH), 1.68 and 1.58 (29, 1CH3), 1.70-1.50 (m, 2CH), 1.39-1.00 (br m, 16H), 0.98-0.75 (br m, 12H). 13C NMR (125 MHz, CDC13): 6 68.1,68.0,67.9,66.1,66.0,65.7,62.9,62.8,62.4,62.3,62.2,58.7, 57.3,57.2,56.4,56.3,48.9,48.8,48.3,48.2,37.6,37.5,37.2,37.1, 37.0,36.2,31.1,31.0,30.9,30.5,30.4,30.2,29.9,28.7,28.6,28.3, 28.2,28.1,24.6,24.5,24.4,24.3,24.2,24.1,23.9,23.8,23.7,23.6, 23.5,23.1,23.0,22.9,22.8,14.0, 13.9, 10.5, 10.4, 10.3, 10.2,9.9, 9.8. HRMS for C21H43N502: calcd 397.3417, found 397.3410. Anal. Calcd for C Z I H ~ ~ N ~ C, O Z63.42; : H, 10.91; N, 17.62. Found: C, 63.49; H, 10.94; N,17.24. Nitrosation of Hexetidine and Hexetidine Drug Formulations under Standard Conditions. Hexetidine was nitrosated under the standardized conditions for drug nitrosation [ l o mM drug, 40 mM nitrite (pH 3-41, incubation 1 and 4 h a t 37 "Cl according to the method of Coulston and Dunne (5). The nitrosation reactions were stopped with ammonium sulfamate and the mixtures made alkaline with NaOH. The nitrosation products were extracted into dichloromethane, dried, and stripped of solvent in uucuo. The residues were taken up in 5 mL of methanol and analyzed by HPLC. The nitrosamine content of each sample was quantified by comparison with external standards made from purified nitrosamines. This procedure was also used for determinations of nitrosamine formation from hexetidine in commercially available drug formulations. Since the drugs contained 0.1% (3.2 mM) of hexetidine, a nitrite concentration of 13 mM was employed rather than 40 mM. Incubations and workups were performed as described above.

Determination of Total Nitroso Compounds with the Griess Reagent. The nitrosamine content of the fraction

isolated from the nitrosation of hexetidine was determined by denitrosation with anhydrous HBr in acetic acid followed by photometry detection of HNOZequivalents released using the Griess reagent (7).Sodium nitrite and dinitrosopiperazine were used as reference compounds. Nitrosation of Hexedine. Hexedine (0.53 g, 1.5 mmol) was dissolved in 5 mL of acetic acid. To this solution was added 0.4 Abbreviations: FID, flame ionization detector; TEA, thermal g of NaNOz (5.8 mmol, 4-fold excess) in 3 mL of water. After energy analyzer; COSY, NMR pulse sequence for determination of stirring at room temperature for 1 h, the reaction mixture was coupled nuclei; HETCOR, NMR pulse sequence for determination of brought to pH 11using saturated aqueous NazC03 solution. The which carbon signals are correlated with which proton signals; DEPT, NMR pulse sequence for determining the number of protons attached products were extracted into ether. After drying over anhydrous to each carbon; HRMS, high resolution mass spectrometry; HEXNO, sodium sulfate, the solution was filtered and concentrated. The l-(2-ethylhexyl)-3-nitroso-4-methyl-4-[[~-(2-ethylhe~l)-~~nitrosoamiresulting residue (3 spots on TLC) was subjected to flash nolmethyllimidazolidine;TMSI, trimethylsilyl iodide; TMSOMe, trichromatography (10%EtOAc in hexane) to give 209 mg of yellow methylsilyl methoxide; MPLC, medium pressure liquid chromatograviscous liquid as a major product (from the most nonpolar phy.

870 Chem. Res. Toxicol., Vol. 7, No. 6, 1994

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I

fraction). The spectroscopic data for this isolated product were identical to those for the major product (HEXNO) from the nitrosation of hexetidine. Synthesisof 3-Carbobenzoxy1-(2-ethylhexyl)-4-methyl-

7b

4-[[N-(2-ethylhexyl)-N-nitrosoaminolmethyllimidazolidine (8). Hexedine (2) (3.6 g, 10.3 mmol) and anhydrous NazC03 (0.11 g, 1.0 mmol) were mixed in 20 mL of dry diethyl ether. To this heterogeneous solution was added 4.4 g of KNOz (51.7 mmol, &fold excess) premixed with 16.4 g of 18-crown-6 (62.0 mmol, &fold excess) in 30 mL of diethyl ether. The mixture was vigorously stirred a t room temperature for 30 min, followed by addition of 1.6 mL of benzyl chloroformate (95% purity, 10.3 mmol). Vigorous stirring was continued a t room temperature for 12 h, and the reaction was stopped by adding 20 mL of water. The ether layer was separated and then washed repeatedly with saturated aqueous KC1 solution to remove excess 18-crown-6. The organic phase was dried over anhydrous NazS04, filtered, and concentrated. The resulting residue was applied to flash chromatography (10% EtOAc in hexane) to give 1.7 g of a light yellow viscous oil (33%). IR (neat, cm-'): 2960-2850 (m), 1705 (s), 1455 (m), 1412 (m). 'H NMR (500 MHz, CDC13): 6 7.30-7.18 (m, 5H), 5.14-4.95 (m, 2H), 4.64-3.65 (m, 3CHz), 3.39-2.01 (m, 2CH2), 1.78-1.49 (m, 2CH), 1.44-1.33 (4s, IC&), 1.29-1.00 (m, 8CH2), 0.99-0.62 (m, 4CH3). 13C NMR (125 MHz, CDC13): 6 157.4, 156.4, 153.4, 152.4, 152.3, 136.7, 136.6, 136.3, 136.0, 135.7, 128.5, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 70.0, 69.2, 69.1, 69.0, 67.3,67.6,66.9,66.4,66.1,63.7,63.5,63.2,62.8,57.4,57.0,56.9, 56.8,56.3,56.2,55.3,48.6,44.9,37.6,37.5,37.4,37.3,36.1,31.0,

c (6)

3 4

1

2

retention time

(min)

Figure 1. 1. HPLC RP (reverse phase) chromatogram of hexetidine nitrosation products.

Results

Isolation and Purification of the Major Nitrosation Product. The acidic nitrosation of hexetidine produces a complex mixture of products. Analysis of the reaction mixture by several different methods reveals the presence of nitrosamines. Use of gas chromatography coupled to the N-nitroso compound-selective detector (TEA) reveals at least eight major peaks and several 30.9,30.8,30.5,30.4,30.1,29.9,28.7,28.6,28.5,28.4,28.2,27.9, minor components. TLC of the mixture followed by UV 24.3,24.2,23.9,23.6,23.2,23.0,22.9,22.8,22.5,22.1,14.0,13.9, irradiation and spraying with the nitrosamine-detecting 10.4, 10.3, 10.1, 9.7. HRMS for CzgH50N302 (M - NO): calcd Griess reagent gave similar results. Hexetidine contains 472.3903, found 472.3919 (Mf not observed). Anal. Calcd for no W absorbing functionality, but HPLC of the reaction Cz9H50N403: C, 69.27; H, 10.03; N, 11.15. Found: C, 69.46; H, mixture using W detection reveals the presence of a t 10.18; N, 11.36. least seven peaks (Figure 1). The NNO functionality has Synthesis of 1-(2-Ethylhexyl)-4-methy1-4-[[N-(2-ethyla strong W absorption a t 240 nm. Comparison of hexyl)-N-nitrosoaminoJmethyl]imidazolidine (9). The carretention volumes and spiking experiments clearly showed bamate 8 (660 mg, 1.3 mmol) and 0.23 mL of iodotrimethylsilane peaks 1-4 to be the same as those obtained from the (97% purity, 1.6 mmol) were mixed in 15 mL of dry chloroform. nitrosation of the related propanetriamine 3 (6). The The mixture was heated at reflux for 24 h, and the reaction was quenched by adding small amounts of methanol (1mL) a t presence of two additional nitrosamines (Figure 1, peaks room temperature. Volatile solvents (methanol and chloroform) 5 and 6), arising from the nitrosation of 3, in the and the excess of TMSI and TMSOMe (byproduct) were removed hexetidine nitrosation mixture, is likely, but their idenunder vacuum. The resulting residue was dissolved in methtity could not be confirmed by HPLC because of the ylene chloride, and the organic phase was washed with brine overlap of their peak positions by the major componentsolution, dried over anhydrous magnesium sulfate, filtered, and (s) of the hexetidine nitrosation (peaks 7a and 7b). The concentrated. Flash chromatography (10% EtOAc in CHZC12) major component(s1 of the nitrosation mixture, peaks 7a gave 140 mg of a light-yellow oil (29%). IR (neat, cm-l): 2960and 7b, were isolated by silica gel chromatography 2850 (s), 1458 (m). *H NMR (500 MHz, CDCl3): 6 4.30-2.07 followed by preparative HPLC or MPLC. HPLC of the (m, 5CHz), 1.77-1.67 (m, lCH), 1.37-0.94 (br m, 8CHz and isolated peaks 7a or 7b showed that they interconverted, lCH), 1.16 and 1.04 (2s, IC&), 0.87-0.77 (m, 4CH3). 13CNMR (125 MHz, CDC13): 6 70.8, 70.7,63.5,63.4,63.1,62.8,62.1,59.8, a phenomenon common for nitrosamines, resulting from 59.7,57.5,57.4,56.4,49.2,49.1,48.6,48.5,38.3,38.2,37.2,36.9, synlanti isomerization of the NNO group. Except for the 36.3,36.2, 31.3,31.2,30.6,30.2,28.8,28.7,28.4,28.3,28.2,25.9, interconversion of 7a and 7b, this fraction was homoge25.8,25.6,24.5,24.3,23.9,23.6,23.0,22.8,14.0,13.9,10.6,10.5, neous by analytical HPLC. In the discussion which 10.4, 10.3,lO.l. HRMS for C21Hd3 (M - NO): calcd 338.3535, follows we will refer to this fraction as HEXNO. found 338.3542 (M+ not observed). LR-FAB (NaI matrix) for Structural Characterization of the Major NitroC21H44N40 (M+): calcd 368.3, found 368.3. Anal. Calcd for sation Product (HEXNO). Elemental analysis of HEXCz1H44N40: C, 68.41; H, 12.04; N, 15.21. Found: C, 68.68; H, NO, a viscous yellow oil, provided a formula of Cd&N502. 12.16; N, 14.92. Synthesis of 1-(2-Ethylhexyl)-3-nitroso-4-methyl-4-[[N- The high resolution mass spectrum (Figure 2) shows a molecular ion a t mlz = 397.3417 which yielded the same (2-ethylhexyl)-N-nitrosoaminolmethyllimi (HEXmolecular formula. HEXNO was assayed for "tetal NO). The synthetic intermediate 9 (10 mg, 0.03 mmol) was dissolved in 2 mL of acetic acid, and then 5 mg of NaNOz (0.07 N-nitroso content" by measuring the quantity of HNOz mmol) in 1mL of water was slowly added a t 0 "C. The mixture liberated upon HBr/HOAc treatment (7). The value of was stirred a t room temperature for half an hour and then 1.5 mol of HNOdmol of HEXNO is consistent with the neutralized by adding saturated aqueous Na2C03 solution. The presence of two nitrosamine functional groups per molpH was adjusted to 11,and the product was extracted into ether. ecule as suggested by the molecular formula. The IR The organic phase was dried over anhydrous sodium sulfate, spectrum of HEXNO shows no OH, NH, or C=O but does filtered, and concentrated. The resulting residue was subjected reveal strong bands a t 1450 cm-l characteristic- of the to flash chromatography (10% EtOAc in hexane) to give 3 mg NNO group. Both the 13C and lH NMR spectra of of a yellow viscous oil (25%). NMR data for this product were HEXNO are complicated by a large number of lines identical to those for the major product from the nitrosation of emanating from the presence of stereoisomers. This hexetidine (HEXNO).

Chem. Res. Toxicol., Vol. 7, No. 6, 1994 871

The Nitrosation of Hexetidine

Scheme 2

1

NO

R=

201

I

I

I

I

I

m/Z

Figure 2. 2. High resolution mass spectrum of HEXNO. Table 1. C atomn a b C

d e f g

h 2 (8)c 4,6 5 Me

13C Chemical

hexetidineb 14.05 24.55-24.58 31.32-31.36 28.68-28.88 36.40-36.56 65.95-66.16 23.05-23.11 10.60-10.76 77.06-77.30 59.34 47.94 25.44

Shift Assignments for Related Amines hexedine 14.08 24.51 31.37 28.88 38.11 65.12 23.08 10.17 77.55 56.66 68.74 27.22

2-ethyl-1-hexanamine 14.03 23.77 30.70 28.97 42.33 44.72 23.02 10.86

Carbon atoms are labeled consecutively from the methyl (a) end of the hexyl chain to the N-CH2 (0. "he CH3 of the ethyl is (h) and the CH2 (g). Ring carbons are numbered according to their position by conventional standards. Several lines are present for each atom due to stereoisomerism, and the range is reported. Ring C-8 of hexedine.

complication is also manifest in the NMR spectra of hexetidine itself (81, which is synthesized from racemic 2-ethyl-1-hexanamine. As a result of the pseudochirality of the quaternary carbon, three diastereoisomers are present in the mixture. The 13C NMR spectral assignments for hexetidine are given in Table 1. Three lines can be observed for many of the carbons in the molecule. The complete N M R assignment of HEXNO and related compounds is presented below. The data and rationale which led to the structural hypothesis are presented here. Through the use of the DEPT pulse sequence it is possible to clearly identify those lines in the 13C NMR spectrum of HEXNO which arise from CH3, CH, and C carbons. At least eight peaks are observed for the CH (6 36.337.2) and CH3 group associated with the ethyl side chain (6 10.21-10.57), respectively. HEXNO contains two 2-ethylhexyl chains, and these groups are in magnetically distinct regions. Subtraction of the formulas (C8H17) of the two 2-ethylhexyl groups from the molecular formula of HEXNO leaves a formula for the molecular core of C5HgN502. The two nitrosamine functional groups account for N402, and the NMR clearly indicates the presence of one quaternary carbon and a CH3. The remaining carbons constitute three CH2 groups appearing between 6 45 and 68, indicating their proximity to electronegative functionality. The CH3 not associated with the 2-ethylhexyl chains appears at 6 24.5 which is close to its position in hexetidine (6 25.44). Although the complexity of the lH spectrum limits its value, two

- CH,CH

(

C,H,

No

) CH,CH,CH,CH3

singlets are evident a t 6 1.66 and 1.56, suggesting that this CH3 in HEXNO is also attached to the quaternary carbon. A series of three closely spaced peaks between 6 67.89 and 68.09 arising from a CH2 are the most downfield peaks in the I3C spectrum of HEXNO. To facilitate the structural assignment of HEXNO, the N,N-dimethyl analog of hexetidine was nitrosated ( 4 ) and found to give a n N-nitrosooxazolidine as its major product. The 13C NMR spectrum of this N-nitrosooxazolidine exhibits its most downfield peak a t 6 80.3 (-OCHzNNO-) (4). The absence of a peak near this value means that hexetidine does not produce a N-nitrosooxazolidine as its major nitrosation product. The CH2 between the two nitrogen atoms in hexetidine is found a t 6 77.06-77.30. The relative shielding of the most downfield resonance in the I3C of HEXNO suggests that it may be syn to a N-N=O oxygen because it is well-known that CH2 groups syn to the nitrosamine oxygen are shielded to the extent of 5-10 ppm from their anti counterparts. The chemical shift of this CH2 is consistent with its placement between two nitrogen atoms, one of which is an amine and the other a nitrosamine. The quaternary carbon in HEXNO appears a t 6 66.05-66.72 (three lines). This carbon is deshielded by 18 ppm from its position in hexetidine and suggests its attachment to a nitrosamine nitrogen. On the other hand, the close spacing of the lines from the three isomers indicates that syn-anti isomerism is not being observed for this N-nitroso group. This is to be expected when the nitrosamine functional group is adjacent to a quaternary carbon because the relative amounts of syn and anti isomers are very dependent upon the steric bulk of the attached groups, the anti position to the large group being preferred. This interpretation suggests the presence of a fragment within HEXNO consisting of RNCHzC(CH3)NN=O, where the R group is a 2-ethylhexyl moiety and the other two groups attached to the quaternary carbon are probably CH2s. Hexetidine disproportionates in acidic solution to give hexedine (2) and 3. NMR and chromatographic monitoring of hexetidine when placed in acetic acid revealed that hexedine is formed rapidly ( < 5 min a t 25 " C ) . The nitrosation of pure hexedine (2) gives HEXNO as the major nitrosamine product. These data and those discussed in the preceding paragraphs permit a structural hypothesis for HEXNO. Initial reaction of hexedine with the nitrosating agent would give the nitrosammonium ion 4 (Scheme 2), which can be expected to rapidly open to give 5. Rapid nitrosation of the secondary amine 6 would give 7. We propose that HEXNO can be assigned the structure of 7. The high resolution mass spectrum of HEXNO (Figure 2) also supports the assigned structure. The major fragmentation pathways are given in Scheme 3. Like many nitrosamines, the molecular ion of HEXNO has a relatively low abundance. A peak resulting from the loss

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Bae et al.

C

D

Scheme 4 WCHZPh

7 HEXNO

of a single NO is found a t 367, and peaks resulting from the subsequent loss of another NO or NOH, mass spectral fragmentations characteristic of nitrosamines, are seen a t mlz 337 and 336, respectively. The most abundant high mass peak (196) is a doublet. The major ion (196.20652) has the formula C ~ ~ H and, Z~N as shown in path A of Scheme 3, arises from a series of steps involving (1) the loss of NO, (2) the interaction of the other NO oxygen with the nitrogen cation, and (3) the coupled loss of RNO and Nz. Although precise mass measurements for the other peak constituting the 196 doublet were difficult, the data suggest the formula C ~ ~ H Zfor ~ Nthis Z ion, and its origin and further fragmentation are shown in path B of Scheme 3. Although the other fragmentation paths are generally typical of those observed for nitrosamines, the structural assignment is particularly supported by paths A and C, which involve an interaction of the two nitrosamine centers. Synthesis of HEXNO. HEXNO has been synthesized by the route shown in Scheme 4. Preliminary experiments showed that reaction of hexedine with benzyl chloroformate resulted in the replacement of one of the ring methylenes with the carbonyl of the chloroformate. This cyclic urea was too unreactive to be of further synthetic use, but its formation suggested attack a t the central ring nitrogen followed by ring opening as shown in Scheme 5. There is good evidence supporting the intermediacy of imminium ions in some nitrosation reactions (9). In these transformations the nitrite ion has

Scheme 5 &H,Ph

been proposed to react with the imminium ion to give a n a-amino nitrous ester, which then decomposes to formaldehyde and a nitrosamine. The intermediate shown in Scheme 5, from the reaction of hexedine and benzyl chloroformate, is a n imminium ion, and we devised a strategy to trap it with ionic nitrite to yield a n intermediate which we presumed to lead to the desired nitrosamine. This hypothesis was experimentally verified. Reaction of hexedine with benzyl chloroformate in ether containing the 18-crown-6 complex of KNOz led to the formation of the nitrosamino carbamate 8. Cleavage of the carbamate was accomplished easily with iodotrimethylsilane, and the amine 9, resulting from basification, was carefully characterized as described below. The nitrosation of 9 gave a compound which was spectroscopically identical to HEXNO in all respects. NMR Assignment. The 13C and lH NMR spectra of 9 are complicated but less complex than that of either

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The Nitrosation of Hexetidine

65

DPm

50

55

50

i5

57.28

e n t i

40

i5

30

I

2t

15

IO

5

0

Figure 3. 3. 13C NMR spectrum of HEXNO. 1 a i - 2 4 5

in

3. 2n.n

.

1

1

57.5

70n

9.14

0

13 9

.2.

5

30 1

9-E 28 3 21 3 24 5

I

2n

e

I

I 7 'P

E . HEXNO 57 21 '4

2 2 0 21 9

8

8 0 07 ,

l3 8 1

22 0 2 3 9 28 0

24 3 2 4 5

58

2 5 e5

.

37 0

9-2

Z-HEXNO

Figure 4. 4. 13C NMR chemical shift assignments for the predominant diastereoisomers of 2 and E 9 and HEXNO. HEXNO (Figure 3) or 8 , and for this reason, and because of the greater availability of this compound, its spectra were used to arrive a t a complete spectral assignment for it and HEXNO. These assignments required 2D N M R data utilizing the DEPT, COSY, and HETCOR pulse sequence methods for both 9 and HEXNO. The complete spectral assignment for the 2 and E isomers of 9 is given in Figure 4. The following method was used to make the assignment. The 13Cchemical shifts of the butyl portions of the 2-ethylhexyl groups can be expected to vary little from their positions in hexetidine and related compounds and are assigned accordingly. The 13Csignals for the CH3 group of the ethyl portion of this chain are the most upfield peaks in the spectra, and six lines can be seen, indicating a greater chemical shift dispersion for those atoms closest to the centers responsible for the stereoisomerism. The positions of the methylenes attached to these methyls can be discerned from the combined use of COSY and HETCOR spectra. The critical task in making the NMR assignments for 9, and subsequently HEXNO, is the identification of the resonances emanating from the N-bound CH2 groups a t the termini of the 2-ethylhexyl chain and the other N-bound CH2 groups. The 13CDEPT spectra permit the identification of the CH groups of the 2-ethylhexyl chain, and the use of the HETCOR connects them to their corresponding lH signals. The COSY spectrum then permits connection of these resonances to those of the

N-bound CH2 groups of the two chains. The reasonable assumption is made that the most downfield CH signals (6 1.75 and 1.82) come from the chain which is attached to the nitrosamine, and the COSY clearly shows the connection (coupling)of these protons in the range 6 3.54.3,chemical shifts expected of CHzs attached to NN=O. The other CH (6 1.2) is connected to protons in the range 6 0.85-2.2 and is assigned to the chain attached to the tertiary amino N. This analysis permits the assignment of the 13C and lH peaks of the N-bound CH2 groups a t the termini of the 2-ethylhexyl chain. The syn CH2 groups attached to the nitrosamine N are a t higher field than their anti counterparts, as is well-known for nitrosamines. The assignment of the lH and 13C peaks arising from the CH2 groups attached to quaternary carbon relied on the long range proton coupling of the quaternary carbonbound CH3 to the CH2 groups. This coupling could be located in the COSY spectrum for one diastereotopic CH2 proton of each synlanti isomer. Because of the diastereotopic nature of CH2 protons, the HETCOR spectrum reveals 13C C-H coupling of one carbon to two different protons. Since every CH2 in the molecule possesses, in principle, diastereotopic hydrogens, this phenomenon is exhibited by many of the carbon atoms in the molecule. We have made the assumption that the more downfield methyl resonance arises from the E nitrosamine. We have also assumed that the exocyclic CH2 group (in comparison with the endocyclic CH2) will exhibit considerable chemical shift dispersion because of its attachment to the nitrosamine N. The distinction of which of these CH2 groups is attached to the nitrosamine relies on the more downfield (6 3.2-3.9) lH chemical shift for the CH3connected protons compared to the endocyclic CH2 (6 2.02.6). The quaternary carbon assignment is easily made from the absence of these peaks in the DEPT I3C spectrum. The remaining carbon is the ring CH2 located between the two nitrogen atoms and has the most downfield chemical shift. The protons attached to this carbon are easily located in the HETCOR spectrum. A comparison of the chemical shift assignments for 9 with those from HEXNO shows relatively minor changes (see Figure 4). The quaternary carbons (6 65.7 and 66.0) are shifted to lower field as is anticipated because of the nitrosation of the attached N atom. Similarly, those due to the isolated ring CH2 appear a t slightly higher field (6 68.1) due to the effect of the syn N-nitroso group. The shifts of the other carbons are not altered by more than 1 ppm from their position in the amine precursor 9.

874 Chem. Res. Toxicol., Vol. 7, No. 6,1994

Bae et al.

other nitrosamines at approximately the same level, 3680 and 10-21 ,ug/mL, respectively. The yields, however, were lower than that of the hexetidine control by factors of 3-10. Since all of the drug formulations contained up to 5%ethanol, a known competitive inhibitor of amine nitrosation ( l o ) ,we also compared the nitrosatability of the hexetidine control mixed with ethanol. As expected, the nitrosamine content produced from the nitrosation of this control was in the same range as that obtained for the drug samples. The chromatographic profile of nitrosamines produced from these nitrosations was identical to that produced from hexetidine alone (Figure 1).

Discussion

PH

Figure 6. 5. pH profile for the formation of HEXNO ( 0 )and other nitrosamines (0). Table 2. Nitrosamine Formation from Hexetidine Drug Formulationsa sample product A product B product C product C (stored 1yr) hexetidine control hexetidine control with 5% ethanol (v/v)

nitrosamines NA1-NAG, total +g/mL) 14 21 10 10 79 12

HEXNO OldmL) 55 80 42

36 330 78

a Incubation time 1 h of samples containing 13 mM NOz- and 1 mg/mL (3.2 mM) hexetidine at pH 3.

The structure assignment for HEXNO, then, is well supported by both the interpretation of its NMR and mass spectra and its synthesis from hexedine by a reasonable, if not completely unambiguous route. The ambiguity in the synthesis is introduced by the novel interception of the imminium ion by nitrite and the subsequent transformation of that species to the nitrosamine 9. Nitrosation Experiments. HEXNO forms rapidly from hexetidine even when limited quantities of nitrous acid are employed. Preliminary experiments showed that nitrosamine formation could be detected immediately after the introduction of 0.5 equiv of sodium nitrite to an acetic acid solution of hexetidine maintained a t 25 “C. A 60% yield of HEXNO is obtained a t pH 1 while the combined yield of the other nitrosamine products is only 15% a t their optimal pH for formation. A pH-yield profile for HEXNO and the other nitrosamines arising from the nitrosation of hexetidine under the “NAPtest” conditions of Coulston and Dunne ( 5 ) is presented in Figure 5 (see the Experimental Procedures for the delineation of these conditions). These data show that HEXNO is, by far, the major nitrosamine product a t all pH values and that its yield decreases with increasing pH. This phenomenon is somewhat unusual, a s most amine nitrosations display a definite pH maximum for production near pH 3.8. This phenomenon may be linked to the acid catalyzed conversion of hexetidine to hexedine (see below). Three drug formulations containing hexetidine, commercially available on the German market, were also tested for nitrosatability (Table 2). Each drug preparation consisted of a 0.1% acidic (pH 4.3-4.7) aqueous hexetidine solution. Pure hexetidine was used for comparison a t the same concentration. The nitrosations were conducted according to the method of Coulston and Dunne ( 5 )but employed lower drughitrite ratios of 1:4. Each of these drug preparations formed HEXNO and the

Reactivity and Mechanism of Nitrosation. The data of Table 2 show that HEXNO and other nitrosamines form rapidly from hexetidine a t low concentrations a t 37 “C. Most tertiary amines would produce immeasurable quantities of nitrosamines under these conditions (1-3). The differences in reactivity probably arise from significantly different mechanisms of reaction and the lower basicity of the geminal diamine functionality present in these molecules. The results generated in the standardized nitrosation assay show that hexetidine gives a total nitrosamine yield of 70%. This places hexetidine near the top of a comparative scale of the nitrosatability of drugs constructed by Gillatt et al. (11). Hexetidine is known to disproportionate in aqueous acidic solution to hexedine (2) and the propanetriamine 3 (8). While the structure of HEXNO suggests its formation from hexedine, several lines of evidence suggest its rapid formation directly from hexetidine as well. If the disproportionation route leading first to 2 and 3 were the main pathway, then one would expect nitrosamine yields from 3 to equal those of HEXNO, assuming ample reaction times and nitrite quantities. While we have insufficient data a t this time to ascertain that latter conditions are met for all of the nitrosation reactions reported here, the yield of HEXNO is larger than the maximum 50% permitted by the disproportionation of hexetidine to 2 and 3. Both the data of Table 2 and the pH-yield profile of Figure 5 show that the yields of the nitrosamines produced from 3 are much lower than the yield of HEXNO. Additionally, it is remarkable that the product pattern of nitrosamines obtained from all the hexetidine drugs was identical to those produced from a freshly prepared control sample. These data suggest that 2 and 3 do not accumulate during storage in the acidic drug preparations and that HEXNO arises, a t least in part if not totally, directly from hexetidine (conditions of Table 2). On the other hand, the nitrosamine chromatographic profile resulting from the nitrosation of 2 (hexedine) is simpler than that generated from hexetidine showing the probable intervention of 3 in the nitrosation of the latter. Thus, HEXNO appears to be produced by three competing pathways, two of which are shown in Scheme 6 and the third (from hexedine 2) in Scheme 2. The route moving across the top of Scheme 6 involves the direct nitrosation of-hexetidine by assisted ionization of the nitrosammonium ion followed by rapid ring closure of the primary amino group on the resulting imminium ion. The reversible acid catalyzed ring opening of hexetidine followed by ring closure to the primary amino group either preceding or following nitrosation provides a second pathway. . This same intermediate (center left of Scheme 6) also leads to the propanetriamine 3 upon hydrolysis, or hexedine (2) upon reaction with formalde-

Chem. Res. Toxicol., Vol. 7, No. 6, 1994 876

The Nitrosation of Hexetidine

Scheme 6 HNO,

___, CHVH,

"+

wR CH,X

H2

I C

H

kW

R=- CH,CH (C,H, 1 C,H9

hyde. These pathways cannot be distinguished on the basis of our present data. The lower yields of the products derived from 3 argue against the prominence of the latter routes, although the higher basicity of the triamine (8)may retard its competitive nitrosation in the presence of limited nitrite. On the other hand, the possible importance of the second pathway is supported by studies showing that the reversible ring opening of perhydropyrimidines, similar to hexetidine, becomes more important a s the size of the nitrogen substituents increases (12). The key to the rapid nitrosation of these molecules is located in the geminal arrangement of the amino groups. Although each molecule contains three basic nitrogen atoms, single pKa values reported (8) for 1, 2, and 3 of 8.3, 6, and 9.1, respectively, show the effect of structure on basicity. In hexedine (2) the geminal arrangement of nitrogen atoms results in reduced basicity due to the electron withdrawing character of the proximal nitrogen atom. The pKa value reported for hexetidine is obviously influenced by the presence of the more basic primary amine, but the ring nitrogens probably have pK, values close to what is reported for hexedine. The triamine shows a pKa value typical for an aliphatic amine. Once the nitrosammonium ion forms, a process facilitated by the lower basicity of the tertiary nitrogens, nitrosamine formation can occur by assisted ionization involving the other nitrogen atom. This process involves a single mole of nitrosating agent per mole of nitrosamine produced, in contrast to the classical mechanism of nitrosamine formation from tertiary amines which requires one mole of nitrite to prqduce an imminium ion through NOH elimination and-a second mole of nitrite to generate the nitrosamine from the resulting secondary amine (11. The concept of nitrosamine formation through assisted displacement has been demonstrated for amidines (13) and electron-rich benzylic (3,141 and heterobenzylic tertiary amines (15, 16). While detailed kinetic studies of hexetidine formation have not yet been done, the HEXNO yield-pH profile is unusual for amine nitrosation. Most amine nitrosation reactions possess a bell shaped pH-rate profile with highest reactivity a t about pH 3.8 (17).The formation of the active nitrosating agent is enhanced a t lower pH values, but the protonation of the amine is greater a t lower pHs and reactivity is reduced by lower concentrations of free amine. The yield of HEXNO is highest a t

the lowest pH examined (pH = 1, see Figure 5 ) . This unusual reactivity could result from the probable availability of one free tertiary amine nitrogen even a t relatively low pHs. Nitrosation of the other ring nitrogen results in a dication, and proton loss is likely to be facile. The freed nitrogen atom can then assist the fragmentation of the nitrosammonium ion to generate the nitrosamine as shown in Scheme 6. Hexetidine contains two tertiary amine groups and one primary amine while all of the nitrogens in hexedine are tertiary amines. Yet, both compounds rapidly react to give HEXNO. It is remarkable that there does not appear to be significant nitrosation and deamination of the primary amine in hexetidine. This pathway was found to be the major pathway for the nitrosation of a hexetidine model compound where the 2-ethylhexyl groups were replaced with methyl groups (4). While the reasons for the differences in reactivity are still under active investigation, a simple rationalization for the reactivity is related to the probable differences in basicity of the primary and tertiary amines in hexetidine, as discussed above. The protonation of the NH2 will inhibit its nitrosation. On the other hand, the statistical preponderance of the two tertiary amine nitrogens and their lower basicity favors nitrosation at these positions. Once the nitrosammonium ion is formed a t one of the tertiary nitrogens, its ring opening may also be facilitated by the large N-alkyl group substitutents as has been observed for the acid catalyzed ring opening of perhydropyrimidines (12). This process may be faster than primary amine diazotization, in contrast to the case ( 4 )where the N-substitutents are the smaller methyl groups. The larger alkyl groups could also facilitate the conversion of hexetidine to hexedine, a process which requires ring opening. This process converts the NH2 into a tertiary amino group and prevents diazonium ion formation. Obviously, definitive mechanism determination for these nitrosation reactions awaits careful rate and expanded product studies. Possible Health Effects. Because of the difficulties associated with the structural assignment and the synthesis of HEXNO we have not yet assayed commercial preparations containing hexetidine or hexedine to determine whether HEXNO or the other nitrosamines are present in these mixtures. We also can provide no definitive data on the possible toxic effects of HEXNO and the other nitrosamines arising from the nitrosation of hexetidine. It is likely, however, that HEXNO possesses genotoxicity. A comparison of its structure with compounds tested for carcinogenicity by Druckrey et al. (18) reveals structural similarities. All of the nitrosamines formed from hexetidine have free methylene groups adjacent to the nitrosamine functional groups. A common mode of carcinogenic activation of nitrosamines involves the a-hydroxylation of this CH2 to give a n unstable a-hydroxynitrosamine which generates alkylating agents (19). Regardless of whether HEXNO is found in commercial preparations, there is a significant probability that hexetidine, given its high proclivity toward nitrosation, will be easily nitrosated in vivo in humans. Although hexetidine is applied locally for prophylaxis and therapy for microbial infections in the oral cavity, formation of nitrosamines may occur if the drug is swallowed during application. Conditions in the stomach are well suited for endogenous nitrosation (20)because of the acidity and the generation of nitrite in the oral cavity through microbial nitrate reduction (21,22). The acidity of many

876 Chem. Res. Toxicol., Vol. 7, No. 6, 1994

of the drug preparations also makes nitrosation in the oral cavity a real possibility since it is known that nitrite concentrations in the saliva can be quite high. It is also now well established that bacteria can catalyze nitrosamine formation from readily nitrosatable substrates and nitrate (23). This process is particularly likely in the oral cavity where the drug may be retained. While the nitrite and amine concentrations used in the assay are low, they are still higher than probable in vivo concentrations. Work to date suggests that other pathways may be important in the nitrosation of hexetidine a t even lower concentrations, and further investigation is underway.

Conclusion The major compound, HEXNO, produced from the nitrosation of hexetidine and hexedine has been characterized. The structure elucidation was significantly complicated by the chirality of the 2-ethylhexyl chains and the number of diastereoisomers generated in the major product. The structure assignment is made firm both by the synthesis of HEXNO and by the extensive spectroscopic characterization. This research demonstrates the usefulness of two different approaches for predicting and evaluating the formation of trace quantities of possible deleterious nitrosamines from common chemical ingredients. Because sensitive methods are available for detecting the presence of N-nitroso compounds in mixtures, the WHONAP test (5),a simple chemical procedure, easily showed the formation of nitrosamines from hexetidine (6). On the other hand, a recognition of molecular structural features which make compounds vulnerable to rapid N-nitrosation led to the prediction that hexetidine and hexedine would produce nitrosamines much more rapidly than most tertiary amines (3,24). The use of structurereactivity concepts permits a visual evaluation of the N-nitrosation propensity of a large number of structures. Compounds whose high reactivity toward N-nitrosation is suspected can then be tested in the laboratory for nitrosamine formation using the WHO-NAP test or a similar procedure. This procedure should be employed with all new nitrogen containing compounds because of human in vivo nitrosation capacity. The possible occurrence of HEXNO and other nitrosamines in products containing hexetidine and hexedine should be investigated. An assessment of the biological activity of HEXNO and related compounds should be made because of the high propensity of its precursors toward nitrosation and the probability of its or related N-nitroso compound formation in vivo in humans.

Acknowledgment. The support of this research by a grant from the National Cancer Institute, R37 CA 26914, is gratefully acknowledged. We also thank Mrs. G. Walter of Heidelberg for her assistance in the nitrosation experiments. References (1) Smith, P. A. S., and Loeppky, R. N. (1967)Nitrosative cleavage of tertiary amines. J . Am. Chem. SOC.89, 1147-1157.

Bae et al. Gowenlock, B., Hutcheson, R. J., Little, J., and Pfab, J . (1979) Nitrosative dealkylation of some symmetrical tertiary amines. J. Chem. Soc., Perkin Trans. 2,1110-1114. Loeppky, R. N., Shevlin, G., and Yu, L. (1990)Rapid nitrosamine formation from tertiary nitrogen compounds: An overview. In Significance of N-Nitrosation of Drugs (Eisenbrand, G., Bolzer, G., and Nicolai, H. v, Eds.) Drug Development and Evaluation, Vol. 16,pp 253-266, Gustav Fischer Verlag, New York. Loeppky, R. N., and Bae, J.-Y. (1994)An aziridinium ion intermediate in the nitrosation of a hexetidine model. Chem. Res. Toxicol. (preceding paper in this issue). Coulston, F., and Dunne, J. F., Eds. (1978) The Potential Carcinogenicity of Nitrosatable Drugs, WHO Symposium, Ablex Publishing Corp., Norwood, NJ. Mende, P., Wacker, C.-D., Preussmann, R., and Spiegelhalder, B. (1993) Nitrosation of the antimicrobial drug hexetidine: Nitrosamines derived from a triamine decomposition product. Food Chem. Toxicol. 31, 53-58. (7)Eisenbrand, G., and Preussmann, R. (1970)A new method for the colorometric determination of nitrosamines by cleavage of the N-nitroso group with hydrogen bromide in acetic acid. Arzneim. Forsch. 20,1513-1517. Satzinger, G., Hermann, W., and Zimmermann, F. (1975)Analytical profile of pure hexetidine. Drug Res. 25, 1849-1853. Keefer, L. K., and Roller, P. P. (1973)N-Nitrosation by nitrite ion in neutral and basic medium. Science 181,1245-1247. Williams, D. L. H., and Aldred, S. E. (1982) Inhibition of nitrosation of amines by thiols, alcohols, and carbohydrates. Food Chem. Toxicol. 20,79-81. Gillatt, P. N., Hart, R. J., Walters, C. L., and Reed, P. I. (1984) Susceptibilities of drugs to nitrosation under standardized chemical conditions. Food Chem. Toxicol. 22,269-274. Evans, R. F.(1967)Hydropyrimidines. Aust. J . Chem. 20,16431661. Loeppky, R. N., and Yu, L. (1990)Nitrosamines, N-nitrosoamides, and diazonium ions from tri-N-substituted amidines. Tetrahedron Lett. 31, 3263-3266. Schlemmer, K. H., and Eisenbrand, G. (1988)Nitrosation of bromhexine and aminophenazone in gastric juice after high nitrate loading in the diet. An ex-vivohn-vitro study in humans. Arzneim. Forsch. 38, 1365-1368. Loeppky, R. N., Outram, J. R., Tomasik, W., and Faulconer, J . M. (1983)Rapid nitrosamine formation from a tertiary amine: the nitrosation of 2-(N,N-dimethylaminomethyl)pyrrole. Tetrahedron Lett. 24,4271-4274. Poocharoen, B., Barbour, J. F., Libbey, L. M., and Scanlan, R. A. (1992)Precursors of N-nitrosodimethylamine in malted barley. 1. Determination ofhordenine and gramine. J. Agric. Food Chem. 40,2216-2221. Mirvish, S. S.(1975)Formation of N-nitroso compounds: Chemistry, kinetics and in vivo occurrence. Toxicol. Appl. Pharmacol. 31, 325-351. Druckrey, H., Preussmann, R., Ivankovic, S., and Schmaehl, D. (1967)Organotropic carcinogenic effects of 65 different N-nitroso compounds on BD-rats. 2.Krebsforsch. 69, 103-201. Preussmann, R., and Stewart, B. W. (1984)N-Nitroso carcinogens. In Chemical Carcinogens, 2nd ed., Vol. 2,ACS Monograph 182, pp 643-828,American Chemical Society, Washington, DC. Sander, J., Schweinsberg, F., and Menz, H. P. (1968)Formation of carcinogenic nitrosamines in the stomach. Hoppe Seyler’s 2. Physiol. Chem. 349, 1691-1697. Tannenbaum, S. R., Sinskey, A. J., Weisman, M., and Bishop, W. (1974)Nitrite in human saliva. Its possible relation to nitrosamine formation. J . Natl. Cancer Znst. 53, 79-84. Spiegelhalder, B., Eisenbrand, G., and Preussmann, R. (1976) Influence of dietary nitrate on nitrite content in human saliva: possible relevance to in vivo formation of N-nitroso compounds. Food Cosmet. Toxicol. 14,545-548. Calmels, S.,Ohshima, H., and Bartsch, H. (1988)Nitrosamine formation by denitrifymg and non-denitrifymg bacteria: implication of nitrite reductase and nitrate reductase in nitrosation catalysis. J . Gen. Microbiol. 134, 221-226. Loeppky, R. N., Bao, Y. T., Bae, J. Y., Yu, L., and Shevlin, G. (1994)Blocking nitrosamine formation: Understanding the chemistry of rapid nitrosation. In Nitrosamines and Related N-Nitroso Compounds: Chemistry and Biochemistry (Loeppky, R. N., and Michejda, C. J., Eds.) pp 52-65, American Chemical Society, Washington, DC.