Ethyl carbamate: analytical methodology, occurrence, formation, biological activity and risk assessment

Mutation Research, 259 (1991) 325-350 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100067W

325

M U T G E N 00040

Ethyl carbamate: analytical methodology, occurrence, formation, biological activity and risk assessment B. Zimmerli and J. Schlatter * Swiss Federal Office of Public Health, Division of Food Science, Berne (Switzerland) (Received 14 February 1990) (Accepted 31 July 1990)

Keywords: Ethyl carbamate; Analytical methodology; Occurrence; H u m a n exposure; Formation; Possible reaction pathways; Biological activity; Carcinogenicity; Risk assessment; Review

Contents Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction and historical overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analytical methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemistry and general . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gas-chromatographic detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occurrence and human exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alcoholic beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Foodstuffs and tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daily intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies on formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General and overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stone-fruit brandies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Whisky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible reaction pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidation of hydrogen cyanide . . . . . . . . . ........................................................ Carbamyl phosphate . . . . . . . . . . . . . . .......................................................... Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions for other foodstuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutagenieity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Low-dose extrapolation and risk assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Correspondence: Dr. B. Zimmerli, Swiss Federal Office of Public Health, Division of Food Science, Haslerstrasse 16, P.O. Box, CH-3000 Berne 14 (Switzerland).

326 326 327 327 329 330 330 330 332 332 332 332 334 334 336 337 338 338 339 340 341 341 342 342 343 344

* Address: Institute of Toxicology, Federal Institute of Technology and University of Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach (Switzerland).

326 Summary Ethyl carbamate (EC) is a genotoxic compound in vitro and in vivo, it binds covalently to D N A and is an animal carcinogen. Today, EC is mainly found as a natural trace constituent in different alcoholic beverages and in fermented food items. Data on analytical methodology and the levels of EC in different food items are summarized and the daily burden of humans is estimated. Under normal dietary habits excluding alcoholic beverages, the unavoidable daily intake is 10-20 n g / k g b.w. On the basis of the evaluation of all toxicity data and its mode of action a conventional risk assessment of EC indicates that this level represents a negligible lifetime cancer risk ( < 0.0001%). Individual habits may greatly enhance the risk. Regular drinking of table wine (500 m l / d a y ) would increase the risk up to 5 times, regular drinking of stone-fruit distillates (20-40 m l / d a y ) would raise the calculated hypothetical tumor risk to near 0.01%. Human exposure to carcinogenic compounds should be as low as reasonably achievable. In order to take reliable measures to reduce EC levels in beverages and foods, it is crucial to know the mode of its formation. For its natural formation the presence of ethanol is absolutely required. In stone-fruit distillates hydrogen cyanide together with photochemically active substances are crucial to form EC. The main part of EC is formed after the distillation involving photochemical reactions. In wine (and probably bread) significant EC formation seems to depend on heat treatment. While in distillates hydrogen cyanide is the most important single precursor, in wine different carbamyl compounds, mainly urea, seem to be involved in EC formation. Despite this apparent difference a common EC formation pathway is discussed for all alcoholic beverages by assuming cyanic-/isocyanic acid as an important ultimate reactant with ethanol. Some ideas are presented as to the possible course of future work.

Introduction and historical overview

Ethyl carbamate (NH2COOCH2CH 3, EC, CAS No. 51-79-6), also known as urethane, is the ethyl ester of carbamic acid (NH2COOH). In the 1940s, EC was used as a hypnotic in man at doses of 1 g / d a y and as an anesthetic for laboratory animals. This latter use led in 1943 to the discovery of its carcinogenicity in animals. Three years later, the activity of EC against leukemia in man was described, and since 1948 it has been known that EC is mutagenic in Drosophila melanogaster. Today, EC is still used as an anesthetic agent for laboratory animals (Field and Lang, 1988) but it is no longer used as a therapeutic agent in man. In 1971, the occurrence of small amounts of EC was detected in fruit juices which had been treated with the antimicrobial agent diethyl pyrocarbonate ((CH3CH2OCO)20 , DEPC). DEPC reacts with ammonia to form EC (Anonymous, 1972; Solymosy et al., 1978; Ough, 1976b, 1978). One year later the regulation permitting the use of DEPC in the U.S.A. was rescinded (Canas et al.,

1989). In several other countries (e.g., Switzerland) this treatment of beverages had never been authorized. That EC may also occur naturally in fermented foods like bread or wine in the lower n g / g range was first demonstrated in 1976 (Ough, 1976a). At that time the national health authorities considered the low levels of EC found in foods and wine as insignificant with respect to human health. However, even then a number of commercial sake samples were found to contain 100-600 n g / m l (Ough, 1978). At that time it was assumed that these amounts resulted from the use of DEPC to sterilize the mash prior to fermentation (Ough, 1976a, 1978). In 1985 the Liquor Control Board of Ontario (Canada) detected relatively high levels of EC in some wines and distilled spirits (Anonymous, 1985). These high levels of EC were probably first attributed to the legal addition of urea as a nutrient for the yeast (nitrogen source) because it has been known since 1840 (WiShler) that EC is formed by reacting urea with ethanol (Adams and Baron,

327 1965). As a consequence, the Canadian health authorities prohibited the use of urea in alcoholic fermentation and established guideline levels for EC * (Anonymous, 1985). However, it was also stated that the use of urea was only one of the factors involved in the high levels of EC in Canadian alcoholic beverages (Conacher et al., 1986). In Switzerland, where only ammonium phosphate or sulfate are legal yeast nutrients, early studies showed clearly that the extremely high EC levels of stone-fruit brandies must involve other factors than urea (Baumann and Zimmerli, 1986b; Zimmerli et al., 1986; Tanner, 1985, 1986b).

Analytical methodology Chemistry and general EC (MW 89.1) has a melting point of 48-50 ° C and a boiling point of 1 8 2 - 1 8 4 ° C (760 mm Hg). At 25 o C its solubility in water is about 2 g/ml, in organic solvents generally less (IARC, 1974). This together with its relatively high vapor pressure, in the order of 1 mm Hg at 30-40 ° C (Weast and Astle, 1980), may give rise to some special problems in analysing EC at trace levels (e.g., extraction efficiency, losses during extract concentration steps). The esters of carbamic acid exhibit some characteristic properties of carboxylic esters and amides. Some of their reactions are those of esters, amides, enols and apparently of cyanic acid ** When EC is treated with aqueous alkali, a mixture of alkali cyanate and, subsequently, alkali carbonate results. In non-aqueous medium, the alkaline hydrolysis was restricted to the cyanate state. By treating EC with ammonia at 1 5 0 ° C in a sealed system, urea was obtained. In general, carbamates are more readily acetylated than ordinary acid amides. When EC is treated with thionyl chloride, ethyl allophanate is formed in exceptionally high yields. Under similar conditions amides are usu-

* Table wines 30 ng/g, fortified wines (ports and sherries) 100 ng/g, distilled spirits 150 ng/g, sake 200 ng/g, fruit brandies and liqueurs 400 ng/g. ** In the following the name cyanic acid (IN-=C-_Q-H) will be used although it is known that isocyanic acid (HN=C=~_) represents the more stable form (Maier et al., 1988).

ally dehydrated to nitriles (Adams and Baron, 1965). !

Overview of methods Table 1 gives an overview of the methods for the detection of EC in distilled spirits published since 1986. These methods differ from earlier ones (Walker et al., 1974; Ough, 1976a; Joe et al., 1977) mainly in the use of capillary columns in gas chromatography (GC) instead of packed columns (e.g., polar liquid phases, polyethylene glycols) and the use of mass spectrometry (MS) as G C detector. However, a method based on G C with matrix isolation of EC and Fourier transform infrared spectrometry detection has also been published (Mossoba et al., 1988). Methods which incorporate a minimum of sample clean-up and capillary GC with MS, Hall or TEA detection are in general also appropriate for the analysis of wines and alcoholic beverages other than distillates (Gaetano and Matta, 1987; Cairns et al., 1987; Conacher et al., 1987; Clegg and Frank, 1988; Canas et al., 1 9 8 8 ) a s well as for foods (Canas et al., 1989; Dennis et al., 1989).

Extraction Adjustment of the pH in wine to the alkaline range (Joe et al., 1977) before extraction with dichloromethane, the most commonly used solvent, or the use of solid phase extraction, e.g., Extrelut, results in reduced difficulties with emulsions. In addition the solid phase extraction can also be combined with a clean-up step, e.g., elution with n-pentane eliminates apolar coextractives (Baumann and Zimmerli, 1986a). Chlorine-containing solvents should be absent in the final extract when using a Hall or a thermionic detector (NPD) (Dennis et al., 1986).

Recovery, detection limit, internal standards The recovery of EC in general depends on the concentration level o f EC, the type of alcoholic beverage as well as on the method used. The achieved recoveries of EC without any corrections (e.g., internal standard) are always >~ 60%, mostly > 80%. The published detection limits for alcoholic beverages are in the range of - 1 - - 100 n g / m l depending on the type of beverage and the method. For further research on the occurrence of

328 TABLE 1 SUMMARY OF THE METHODOLOGY OF ETHYL CARBAMATE ANALYSIS IN DISTILLED SPIRITS Principle of sample preparation

Detector (References) FID

Direct Liquid-liquid

Solid-liquid

without concentration, addition of salts CH2CI 2 (petroleum ether, ethyl acetate) addition of salts (NaCI, K2CO3] or Na2SO4) [ extraction with CH2C12, CHC13 or diethyl ether [ (Florisil clean-up (10)) ] Extrelut (or Chem-Elut) clean-up with n-pentane or petroleum ether extraction with CH2C12 or n-hexane-ethyl acetate mixture (Florisil-Sep-Pak(15- 17))

1

NPD

Hall

TEA

7, 12 1

12,22,27

2, 25

20, 28

29

MS

19

3, 4, 7, 8

5, 12 18

10, 14

15-17

3,6,9,10, 13, 21, 25, 26

11, 15-17

12, 15-18, 23, 24

FID, flame ionization detector; NPD, thermionic detector with special sensitivity for N- and P-containing substances; TEA, thermal energy analyzer; MS, mass spectrometer, includes EI-MS and CI-MS, MS-MS as well as ion trap (ITD); Hall, electrolytic conductivity detector. References: 1, Adam and Postel, 1987, 1990; 2, Andrey, 1987; 3, Aylott eta., 1987; 4, Bailey et al., 1986; 5, Baumann and Zimmerli, 1986; 6, Bebiolka and Dunkel, 1987; 7, Bertrand and Barros, 1988; 8, Bertrand and Triquet-Pissard, 1986; 9, Brumley et al., 1988; 10, Cairns et al., 1987; 11, Canas et al., 1988; 12, Christoph et al., 1986; 13, Clegg and Frank, 1988; 14, Conacher et al., 1987; 15, Dennis et al., 1986; 16, Dennis et al., 1988; 17, Dennis et al., 1989; 18, Drerder and Schmid, 1989; 19, Gaetano and Matta, 1987; 20, Kobayashi et al., 1987; 21, Lau et al., 1987; 22, MacNamara et al., 1989; 23, Mildau et al., 1987; 24, Ough et al., 1988; 25, Pierce et al., 1988; 26, Riffldn, et al., 1989b; 27, Schulz and Renner, 1986; 28, van Ingen et al., 1987; 29, Wasserfallen and Georges, 1987.

EC, the d e t e c t i o n limits should b e in the o r d e r of 0.5 n g / g for foodstuffs, 2 n g / m l for table wines, a n d a b o u t 50 n g / m l for distilled spirits. C a r b a m a t e s such as m e t h y l c a r b a m a t e , n - p r o p y I c a r b a m a t e or n - b u t y l c a r b a m a t e as well as tert.-butyl c a r b a m a t e have m o s t l y b e e n u s e d as i n t e r n a l s t a n d a r d s for G C / N D P and GC/MS. Under s t r o n g l y alkaline e x t r a c t i o n c o n d i t i o n s the possib i l i t y exists t h a t E C m a y be f o r m e d f r o m such c a r b a m a t e s ( A d a m s a n d Postel, 1987). A s dim e t h y l p y r o c a r b o n a t e ( ( C H 3 O C O ) 2 0 ) has rec e n t l y b e c o m e a n a c c e p t e d a d d i t i v e for wines in the U.S.A. ( A n o n y m o u s , 1988a), it is also p o s s i b l e that they a l r e a d y c o n t a i n small a m o u n t s of m e t h y l carbamate.

Collaborative studies O n l y two o f the p u b l i s h e d m e t h o d s have b e e n tested in c o l l a b o r a t i v e studies, one o f t h e m for stone-fruit b r a n d i e s b a s e d on G C / F I D w i t h o u t

c l e a n - u p ( A n d r e y , 1987) a n d the o t h e r for different a l c o h o l i c beverages ( C o n a c h e r a n d Page, 1986; C o n a c h e r et al., 1987). I n a c o o p e r a t i v e s t u d y (7 l a b o r a t o r i e s , 6 m e t h o d s ) 4 of the 7 l a b o r a t o r i e s m e t the chosen a c c e p t a b i l i t y criteria for E C in w h i s k y (level 4 0 - 1 7 0 n g / m l ) , while for the b e e r trial (level < 1 - 2 0 n g / m l ) all the l a b o r a t o r i e s were sufficiently close to these s t a n d a r d s ( D e n n i s et al., 1990). T h e r e a l i z a t i o n o f i n t e r - l a b o r a t o r y c o l l a b o r a t i v e studies s h o u l d be encouraged. H o w ever, it m u s t b e k e p t in m i n d t h a t to check the r e l i a b i l i t y o f a n a l y t i c a l results, at least in the trace c o n c e n t r a t i o n range, in general a f o r m a l q u a l i t y a s s u r a n c e p r o g r a m m a y be of greater i m p o r t a n c e t h a n s t a n d a r d m e t h o d s o f analysis.

Chemical derivatives So far o n l y 3 c h e m i c a l derivatives of E C have b e e n d e s c r i b e d for a n a l y t i c a l p u r p o s e s : N,N-dim e t h y l ethyl c a r b a m a t e (Bailey et al., 1986), N-tri-

329 fluoracetyl ethyl carbamate (Walker et al., 1974), and N,N-dimethylaminomethylene ethyl carbamate (Kobayashi et al., 1987). According to the structural differences, the NP detector response for the mentioned derivatives should be lowest for the first and highest for the last. After some minor modifications of the reaction conditions and after introducing an additional clean-up, good results have been obtained with the methylation procedure. The method allows analysis or confirmation of EC in wine in the concentration range of 1-5 n g / m l with G C / N P D (Zoller et al., 1990). When our laboratory tried to prepare the trifluoroacetic anhydride derivate of EC (or urea) at trace levels (standard solutions) we always found trifluoroacetic acid amide, identified by G C / M S , as a main product (Zoller et al., 1990)!

Stability A special problem in analyzing EC is its stability in certain samples during storage and processing. Heating of wine samples, e.g., those containing traces of urea, raises the EC concentration (see below). As a consequence analysis of EC in table wines (116 samples), involving steam distillation as a part of the sample clean-up (Funch and Lisbjerg, 1988), resulted in distinctly higher EC concentrations compared to other studies. In stone-fruit brandies, especially recently distilled ones, EC concentration can drastically increase upon exposure to light while heat alone does not seem to raise its concentrations significantly (see below). For the measurement of the actual concentration of EC in stone-fruit brandies at the time of sampling and to obtain comparable results in different laboratories it is, therefore, essential that the samples are protected from light (Andrey, 1986; Tuor, 1986; Zimmerli et al., 1986; Mildau et al., 1987). However, with respect to possible future food regulations it may be also important to know the potential amount of EC which can be formed in stone-fruit brandies and possibly in wines (Baumann and Zimmerli, 1988). EC may also be partially destroyed if the EC extraction is performed under strongly alkaline conditions. At least two such methods have been described (Adam and Postel, 1987; Bebiolka and Dunkel, 1987). EC may be partially hydrolysed to ethanol within 0.5 h if for analysis of distilled

spirits steam distillation at pH 2 is used to remove ethanol (Wasserfallen and Georges, 1987).

Gas-chromatographic detectors A variety of GC detectors are used in EC analysis, mainly selective ones (thermal energy analyzer (TEA), electrolytic conductivity detector (Hall), alkali-flame ionization or thermionic detector (NPD)), but the most widespread seems to be the mass spectrometer (selected ion monitoring (SIM)). The use of capillary columns in connection with selective or specific detectors often allows a reduced or simplified clean-up step. However, for certain extracts insufficient clean-up may lead to interference. Flame ionization detector (FID) In 5 publications the application of the FID has been described for the analysis of EC in distilled spirits, especially for stone-fruit brandies (Andrey, 1987) as well as for other distillates'(see Table 1). However, it has been demonstrated that an additional clean-up (alumina) was often necessary (Pierce et al., 1988): potential interference in wine extracts eluted just before EC on certain polar columns. The substance has been identified as diethyl succinate (Bertrand and Triquet-Pissard, 1986; Clegg and Frank, 1988; Lau et al., 1987). For the G C / F I D analysis of EC in sake (including a Florisil clean-up), a derivatization of EC to N, N-dimethylaminomethylene ethyl carbamate was necessary, because it had been shown by G C / M S that the peak eluting at the retention time of EC consists of at least 2 substances (Kobayashi et al., 1987). Therefore the G C / F I D method seems to be inadequate for EC assays, especially in non-distilled samples. In practice, it is not possible to predict a priori whether a particular matrix will be amenable to G C / F I D analysis. However, a method based on a columnswitching technique by which EC is transferred from an apolar column to a polar one before FID detection (heart cutting) seems to offer a promising and reliable alternative t o conventional G C / F I D (Van Ingen et al., 1987). Thermionic detector (NPD) NPDs offer a real and low-cost alternative to the expensive and difficult to handle instrumenta-

330 tions such as Hall, MS or TEA. In 10 publications the successful use of G C / N P D has been described (see Table 1). However, in our opinion no reliable results, except for distilled spirits, can be expected with procedures without an appropriate extract clean-up (e.g., on Florisil or silica gel) and the confirmation of positive samples (Zoller et al., 1990).

Mass spectrometer (MS) Low-resolution quadrupole mass spectrometers in the selected ion monitoring mode (SIM) have been used in many of the published studies, either as a G C detector on its own (see Table 1) or for confirmation purposes (Joe et al., 1977; Conacher et al., 1987; Kobayashi et al., 1987; Pierce et al., 1988; Canas et al., 1988; Dennis et al., 1989). Additional techniques including ion trap detector (Clegg and Frank, 1988), high-resolution SIM (Lau et al., 1987), and methane or isobutane chemical ionization (Schmeltz et al., 1978; Joe et al., 1977; Bebiolka and Dunkel, 1987; Cairns et al, 1987; Brumley et al., 1988; Lau et al., 1987), as well as M S / M S have also been been applied for the analysis of EC in different matrices. When operated in the electron impact ionization mode (El) the spectrum of EC is dominated by fragment ions at m / z 44 [NH2CO'] +, 45 [C/HsO] + and 62 [M-CzH3] +. The first 2 ions and the molecular ion ( m / z 89) are either not sufficiently specific or not intense enough to be useful in trace analysis. In low-resolution MS interferences at m / z 44 and especially at m / z 74 (contribution from C3H602 from diethyl succinate, Lau et al., 1987) are relatively often observed, e.g., when wine extracts are analyzed without an efficient clean-up (Zoller et al., 1990). However, m / z 62 provides a high selectivity and sensitivity, m / z 74 [M-CH3] + and 89 [M] + are of low relative abundance but also characteristic of EC (Dennis et al., 1986; Brumley et al., 1988; Lau et al., 1987). The ion at m / z 62 is also characteristic of most of the non-N-substituted carbamates except for those that do not contain a r-hydrogen in the alkoxyl chain such as methyl carbamate or 2,2-dimethyl carbamate (Lau et al., 1987). In the ion trap detector (ITD) the molecular ion of EC may be protonated and appears at m / z 90 (Clegg and Frank, 1988). Whether enhanced (M + 1) +

peaks with ITD are the result of self-chemical ionization or of other phenomena is still a matter of discussion (Eichelberger et al., 1987; McLucky et al., 1988; Brodbelt et al., 1987). When the MS is operated in the chemical ionization mode (CI), isobutane produces almost exclusively the [M + H] + ion, whereas methane yields additionally m / z 62 of almost equal intensity. Methane CI is recommended over isobutane CI since it provides both structurally significant fragments and molecular weight information (MacNamara et al., 1989). However, the presence of only 2 ions at the correct retention time cannot always be considered as providing the ideal criterion for unambiguous proof of identity. With the ability to select the protonated molecular ions for collision-activated dissociation, daughter ions can be produced ( M S / M S ) that are structurally related to the parent ion, thereby providing a more sophisticated level of confirmation and quantitation (Cairns et al., 1987; Brumley et al., 1988). The decomposition of [M + H] + to m / z 62 appears to be absolutely specific for EC at least in beverages (Brumley et al., 1988) using the isolation procedure described by Conacher et al. (1987). Occurrence and human exposure

Alcoholic beverages The occurrence of EC in alcoholic beverages varies over a wide range. Table 2 gives an overview, summarizing the available worldwide data mainly from surveys in Canada, U.S.A., Germany, U.K. and Switzerland (for details see Battaglia et al., 1990). The highest EC concentrations were clearly found in fruit brandies derived from plums, apricots or cherries ('stone' fruits), 1-2 orders of magnitude higher than in other distilled spirits. The enormously wide range of EC concentrations in stone-fruit brandies reflects its light-induced and time-dependent formation after distillation (see below). The lowest EC levels in distilled spirits are found in the highly rectified ones, e.g., vodka. Apple wine, and with some exceptions, also beer usually seem to contain low EC levels.

Foodstuffs and tobacco Compared to the data on EC concentrations in alcoholic beverages, information on other food

331 TABLE 2 BEST ESTIMATES O F E T H Y L C A R B A M A T E LEVELS IN A L C O H O L I C BEVERAGES (A S U M M A R Y O F D A T A ) a Beverage Stone-fruit brandies (cherry, plum, apricot, etc.) Sake, rice wine Bourbon whiskies Other whiskies Other distilled spirits (e.g., apple, grape) a n d liqueurs Gin, vodka Table wines

Range (ng/g)

Median (ng/g)

References

2000

Baumann, Zimmerli, 1986; Battaglia et al., 1990; Tanner, 1985 Battaglia et al., 1990 Battaglia et al., 1990 Battaglia et al., 1990

100-20000 10-900 < 30-350 < 10-170

130 90 40

< 10-200

20 < 30 <1 10-15

< 1-10 < 1-110

Sweet wines (malaga, port, sherry) Beer

10-250 0.3-18

Apple wines

50 1b

< 1-3

1b

Battaglia et al., 1990 Battaglia et al., 1990 Battaglia et al., 1990; Zoller et al., 1990; Christmann, 1988; Clegg et al., 1988 Battaglia et al., 1990 Battaglia et al., 1990; Dennis et al., 1989; Canas et al., 1989 Canas et al., 1989

a The actual concentrations m a y be less as a result of changes in the production processes. b Mean.

items or tobacco is only scarce. Based on studies from U.K., U.S.A. and Switzerland, Table 3 summarizes the EC content of different food items. The available data confirm, in general, the low levels first reported by Ough (1976a). The highest levels are found in soy sauces. However, in similar non-fermented protein-based condiments EC was

not detectable (Hartman and Rosen, 1989). The main sourde of EC exposure of humans by foods seems to be bread. Toasting of bread (American type) raises the concentration of EC on average about 3-fold (Canas et al., 1989). Whether the higher concentration in bread (Table 3) found in a preliminary Swiss investigation is a real difference

TABLE 3 BEST ESTIMATES O F E T H Y L C A R B A M A T E LEVELS IN N O N - A L C O H O L I C F O O D S a Food item

Mean (ng/g)

Soy sauce Bread Bread Yogurt

18 ' 2 7 <1

< 1-95 < 1-8 3 - 15 0.3-3

Cheese Black tea (1 b a g / 2 5 0 ml water) Apple cider Beer, alcohol-free

<1 <1 <1 0.5

< 1-6 < 0.2-0.6

a Data from U.K., U.S.A. and Switzerland.

Range (ng/g)

References Canas et al., 1989; H a r t m a n and Rosen, 1989 Canas et al., 1989; Dennis et al., 1989 Zimmerli et al., 1986 Canas et al., 1989; Dennis et al., 1989; Zimmerli et aL, 1986 Dennis et ai., 1989; Canas et al., 1989 Canas et al., 1989 Canas et al., 1989 Battaglia et al., 1990; Canas et al., 1989

332 from the results of English or American surveys or not (e.g., different types of bread) remains to be elucidated. EC was found as a natural constituent in tobacco in a concentration range of about 310-375 ng/g. In mainstream smoke 20-48 ng/cigarette was found (Schrneltz et al., 1978).

Daily intake Taking into account all available data on EC concentrations in different food items, an exogenous mean daily EC intake for adults in the order of 10-20 n g / k g b.w. can be estimated assuming bread to be the main source. Today, this daily burden is unavoidable. Smoking 20 cigarettes per day can double the daily intake of EC. But only the drinking of alcoholic beverages can increase this daily burden substantially: the daily consumption of 200-300 ml table wine with an average EC concentration of 10 n g / m l may triple the baseline daily intake, while the daily drinking of 30 ml stone-fruit brandy (half the ethanol dose of 300 ml wine) increases the daily burden about 60-fold. Whether EC can also be formed endogenously, e.g., as a result of ethanol consumption, is at present not known. In this context it is interesting to note that Wikman-Coffelt and Berg (1976) found evidence that goat and human sera may have antibody activity against EC. Studies on formation

General and overview Although some very valuable results on the formation of EC have been accumulated since 1985, the mechanisms of its formation in alcoholic beverages and in fermented foods are still obscure. Two main groups of beverages and foods appear to be distinguishable. The first includes stone-fruit brandies and the initial distillate in the production of Scotch whisky, in which the major part of EC is formed after the distillation already in the cold. The second group comprises wine, sake and probably bread, in which significant EC formation often seems to depend on heat treatment. Potential EC precursors, which react with ethanol in the cold, are carbamyl phosphate and cyanic acid. Those which tend to need heat in order to form considerable amounts of EC in

model aqueous ethanol solutions are urea and other N-carbamyl group-containing substances, such as citrullin, allantoin, fl-ureidopropionic acid as well as N-carbamyl compounds of other ct- or t - a m i n o acids (Zimmerli et al., 1986; Baumann and Zimmerli, 1986b; Ough et al., 1988a; Hara et al., 1988). On the other hand, orotic acid, hydroorotic acid, and dihydrouracil (Ough et al., 1988a) as well as biuret (Hara et al., 1988) did not react.

Stone-fruit brandies Although most of the EC in stone-fruit brandies is formed after distillation under the influence of light, small amounts are also formed in the mash (Battaglia et al., 1988). The EC concentration increases markedly when the mash is heated (Christoph et al., 1986). Freshly distilled stonefruit brandies normally show EC levels only in the order of 100 n g / m l . It has been demonstrated that hydrogen cyanide, a natural constituent of stone and other fruits, is the main precursor of EC in fruit brandies, and that a photochemical process is involved in its time-dependent formation (Zimmerli et al., 1986; Baumann and Zimmerli, 1986b; Christoph et al., 1987b), in which light with wavelengths of 350-450 nm is the most efficient (Mildau et al., 1987; Andrey, 1986, 1987). Depending on the type of samples and the ethanol concentration, the rate of daylight-induced EC formation may decrease with time and the EC concentrations may reach plateau values after days, weeks or months (Mildau et al., 1987; Baumann and Zimmerli, 1987). When the light source is removed, the reaction continues to some degree in the dark (Baumann and Zimmerli, 1988; Christoph et al., 1987b). From the data on the temperature dependence of light-induced EC formation in a sample of a stone-fruit brandy (mercury arc) an overall energy of activation of about 15 kcal/mole can be estimated (Baumann and Zimmerli, 1988). When the EC concentration has reached a plateau value, the concentration of hydrogen cyanide (total) is not necessarily zero (Andrey, 1987; Zimmerli et al., 1990). It has been speculated that besides cyanide other unidentified, unstable substances, such as benzoylimido ethyl ester, could also be responsible for light-induced EC formation in the distillates (Crhistoph et al., 1987a). However, when distillates are free of cyanide (total

333 TABLE 4 E T H Y L C A R B A M A T E F O R M A T I O N F R O M H Y D R O G E N C Y A N I D E IN M O D E L S O L U T I O N S A N D IN D I S T I L L A T E S Addition

Effect of temperature

Ethyl carbamate formation dark

Model solution a none copper(II) ions methyl glyoxal biacetyl biacetyl, copper(II) ions biacetyl, iron(III) ions diacetyl peroxide benzaldehyde

?

-

+ + .9 .9 ? ? ?

+ + + + _ + + + + ?

pentanedione-2,3

?

Stone-fruit distillates none none methyl glyoxal ascorbic acid

? + + +

hydrogen peroxide iron(II) or (III) ions n-butanol, 10 and 20% ( v / v )

++ + + + + + + + + _ b ? + + +++

--

+ +

_

+ + + e

+++ +++

? ?

+

+++

?

? .9

?

_b

_

References

daylight

+ + d

B a u m a n n and Zimmerli, 1986b, Zimmerli et al., 1990 Baumarm and Zimmerli, 1986b, Baumarm and Zimmerli, 1986b, Zimmerli et al., 1990 Zimmerli et al., 1990 B a u m a n n and Zimmerli, 1987 B a u m a n n and Zimmerli, 1987; Christoph et al., 1987b B a u m a u n and Zimmerfi, 1986b,

1987 1987 1987

1987

Baumarm and Zimmerli, 1988 B a u m a n n and Zimmerli, 1988 B a u m a n n and Zimmerli, 1988 Tanner, 1986c; B a n m a n n and Zimmerli, 1988 Tanner, 1986e; B a u m a n n a n d Zimmerli, 1988 A d a m and Postel, 1987 Zimmerli et al., 1990

Hydrogen cyanide (20 p g / m l ) in ethanol-water (40% v / v ) in borosilicate glass vessels. b Cyanide will probably be complexed. c High-pressure mercury arc (Heraeus T Q 150). d Formation of n-butyl carbamate (BC), on a molar basis the s u m of EC and BC remains practically unchanged. +/-, mainly qualitative effects, also depending on the m o u n t s of reagents added; ?, not studied.

amount < 0.1 #g/ml) no EC is formed in appreciable amounts under light exposure (Baumann and Zimmerli, 1988; Christoph et al., 1987b, 1988; Schmidt, 1988). On the other hand several findings indicate that besides hydrogen cyanide one or several further species are additionally needed to form EC in stone-fruit distillates exposed to light (Baumann and Zimmerli, 1986b, 1987, 1988; Christoph et al., 1987b; Schmidt, 1988). Although copper ions, which are always present in traditional stone-fruit brandies (up to 5 pg/ml, typically 0.1-0.2 #g/ml), may increase the yield of light-induced EC considerably, they seem not to be indispensable for its formation (Zimmerli et al., 1990). Based on model experiments, vicinal dicarbonyl compounds, such as biacetyl and pentanedione-2,3 (Zimmerli et al., 1986; Baumann and Zimmerli, 1986b, 1987, 1988), benzaldehyde (Christoph et al., 1987b) or polyun-

saturated fatty acids, their esters or peroxides (Tourli6re, 1989), have been proposed to be essential in EC formation together with hydrogen cyanide *. However, in model solutions biacetyl seemed to be more effective in light-induced EC formation than benzaldehyde (Baumann and Zimmerli, 1987). Table 4 summarizes the results of model experiments on the EC formation from hydrogen cyanide (for a discussion see below). The relation between the consumed amount of hydrogen cyanide (measured as total cyanide) and the amount of EC formed in different samples of relatively fresh distillates from 3 producers (40% v / v ethanol) after their exposure to artificial daylight (Philips LP-7, 64 days, at about 30 ° C) is

* All these c o m p o u n d s are trace constituents (in the order of 0.1-20 p g / m l ) of stone-fruit distillates.

334 presented in Fig. 1 (data from Andrey, 1987). The slope of the regression line ( A H N C = a + bAEC), passing through the origin (a = 0.015 + 0.026), is 4.3 + 0.2 M / M ( + standard error). This seems to indicate that in stone-fruit distillates the maximum yield of light-induced EC from cyanide amounts to about 25% of the theoretical maximum. However, with another set of distillates, probably including older ones, and other illumination conditions (7-10 days) variable yields in the range of about 2-30% were obtained (Zimmerli et al., 1990). The addition of n-butanol to a cherry distillate (10 and 20% v / v ) and exposure to artificial daylight resulted in a reduction of the amount of EC formed and in formation of an about equimolar amount of n-butyl carbamate (Zimmerli et al., 1990). It seems reasonable to postulate that the most simple and probable initial step in the formation of EC from hydrogen cyanide involves its oxidation to cyanic acid (Baumann and Zimmerli, 1987; Christoph et al., 1987b; Battaglia et al., 1990). After exposure to light the existence of an oxidizing environment capable of oxidizing iodide to free iodine has been demonstrated in otherwise stable model solutions containing ethanol, hydrogen cyanide and biacetyl or pentanedione-2,3 (Baumann and Zimmerli, 1987). Acetoin, the reduced form of biacetyl, as well as ammonia (probably from hydrolysis of cyanic acid) have been identified in such a solution (Battaglia et al., 1990). In a study on the production of a plum brandy

1.D

0.5

0

,

0.i0

,

0.20 AEC (raM)

,o 0 .5

Fig. 1. Light-induced ethyl carbamate formation in different samples of stone-fruit distillates (64 days): o cherry, • plum. AEC: amount of EC formed (mM), AHCN: amount of cyanide consumed (mM).

(Battaglia et al., 1988), it was found that in the distillates, with one exception, only the initial biacetyl concentrations (benzaldehyde not measured) were strongly positively correlated with the light-induced EC formation (Battaglia et al., 1990). An analogous correlation for different samples of distillates has also been found by Andrey (1987). He also demonstrated that biacetyl disappears during light-induced EC formation. Table 5 summarizes the proposed procedures for the reduction of EC levels in commercial stone-fruit brandies. Procedures I, II, IV and VI seem to be the most promising ones. Wh/sky

Riffldn et al. (1989a,b) have conducted studies on reducing EC formation during the traditional double-distillation procedure used in the manufacture of Scotch whiskies. In the first distillates (low wines), it was shown that EC formation depends on the pH (optimum between p H 4 and 6), the temperature, the time, and the crucial presence of copper ions. Exposure to light seemed to have no effect on EC formation (Riffkin et al., 1989b). Riffkin et al. (1989a) also discussed a Cu z+peptide/protein complex, formed during foaming in the upper parts of the traditional pot still, as a precursor for the EC formation in low wines: refluxing ethanol (or n-propanol) solutions of essential amino acids in the presence of copper did not result in a significant formation of EC (or n-propyl carbamate), whereas a similar reflux with bovine serum albumin or wheat gluten did. It might be that the precursor of EC in the low wines is hydrogen cyanide, which will be first oxidized to cyanic acid (Table 4). Although cyanide itself can be expected to be present only in trace amounts (1-10 n g / g ) in the raw materials used for whisky production (Lehmann et al., 1979), its content may increase considerably during germination (Erb et al., 1981). On the other hand trace amounts of hydrogen cyanide can also be formed during the distillation of a mixture of carbohydrates and amino acids in the presence of oxygen (Lehmann and Zinsmeister, 1979; M~511er, 1984). Wine

Most of the EC content of wines is formed after fermentation, by a reaction between ethanol

335 TABLE 5 PROPOSED PROCEDURES FOR THE REDUCTION OF THE ETHYL CARBAMATE LEVELS IN STONE-FRUIT BRANDIES Stage Mash

Procedure I

Measure undertaken

Results in

References

addition of copper (I or II) salts a

reduced cyanide levels in the distillate, probably by complex formation in the mash reduced cyanide levels in the distillate, probably by complex formation in the mash reduced cyanide levels in the distillate reduced cyanide levels in the distillate delay or inhibition of light-induced EC formation reduced cyanide levels in the distillate formation of EC, being separated during the second distillation precipitation of free cyanide

1, 2, 3

copper(I) chloride Distillation

II III

Distillate (freshly distilled)

IV V VI VII

distillation columns filled with copper rings distillate passes an anionexchange resin storage in brown glass or stoneware bottles addition of copper powder and redistillation after some weeks addition of ascorbic acid and redistillation after some weeks addition of silver salts and filtration

2, 4 5 6, 10 7 3 8 9

a Cu(CN)~- being much more stable than Cu(CN) 2-. References: 1, Tanner, 1986a; 2, Christoph et al., 1988; 3, Schmidt, 1988; 4, Christoph, 1989; 5, Ritzmann and Flih, 1987, 1988; 6, Tuor, 1986; 7, Mildau et al., 1987; 8, Tanner, 1986c; Tanner et al., 1987; 9, Christoph et al., 1987b; 10, Bertrand et al., 1990.

and E C precursors. Hence, the concentrations of ethanol and EC precursors as well as the temperature and the time of storage are important parameters for E C formation. D a y h g h t does not seem to have any significant influence (Tegmo-Larsson and Spittler, 1990). T h e easy reaction of carbamyl phosphate (CP), p r o d u c e d by yeast, with ethanol to form EC was p r o p o s e d b y O u g h (1976a) as an explanation for the occurrence of trace amounts of E C in fermented foods, such as bread, yogurt and wine. However, C P already reacts with ethanol in the cold, and its concentration p r o d u c e d b y the yeast is expected to be very low. Most y o u n g wines do not contain E C ( < 10 n g / m l ) , unless heat treatment is applied, e.g., 48 h at 70 ° C (potential EC). Therefore, C P is considered not to be a significant source of E C in wines (Ough et al., 1988a, b). H y d r o g e n cyanide also seems not to be an imp o r t a n t precursor of EC, because only very low concentrations (tOtal a m o u n t 2 - 1 0 n g / m l ) were measured in wines of different origins (Wiirdig

and Miiller, 1988), and in a commercial grape-juice sample (17 n g / m l ) (MSller, 1984). In wine (including rice wine (sake), H a r a et al., 1988), urea is considered to be a main precursor of EC. It can result f r o m the b r e a k d o w n of arginine to f o r m ornithine and urea by the yeast (arginases). A l o n g with proline, arginine is one of the major amino acids in grapes. At the end of the growth phase and at steady state of the yeast, the urea which has not been c o n s u m e d could be excreted from the cells and subsequently generate E C in the presence of ethanol (Ough et al., 1988b; Monteiro et al., 1989; Totsuka et al., 1988). There is evidence that the formation of urea m a y also depend on the yeast strain, the a - a m i n o nitrogen content of the juices (Ough et al., 1990), the addition of d i a m m o n i u m phosphate as well as on the presence of oxygen during fermentation (Ough et al., 1988b; Henschke, 1989). It has also been suggested that heavily fertilized vineyards m a y be the m a j o r cause of high urea contents in wine, because of high nitrogen nutrients in the juice (Almy and

336 Ough, 1989). A positive correlation was found between EC in wines from a fertilization trial stored for 10 years ( 1 2 - 1 4 ° C ) and the arginine levels originally present in the juice. The EC levels in these wines were also correlated with the remaining urea content after the storage period (Ough et al., 1989). To remove urea from wine (and sake, Kobashi et al., 1988) the use of an acid-tolerant urease has been proposed. However, it seems that certain types of wine show some inhibitory effect on the enzyme system (Ough and Trioli, 1988). Recently, the construction of an arginase-deficient yeast has been described which produces much less urea from arginine (Suizu et al., 1990). Although it is very probable that urea as such is a major EC precursor in wine, it should be noted that it seems not to be the only one (Henschke, 1989; Tegmo-Larsson, 1989; Ingledew et al., 1987). A comparison of the EC content of fermented and non-fermented grape juice from grapes with low amino acid concentrations showed that the non-fermented juice samples produced as much EC as fermented samples (11-24 n g / m l ) when adjusted to the same ethanol concentration (12% v / v ) and heated (48 h, 70°C). These findings suggest that EC precursors already exist in the fresh grape juice (HCN?) and that no further precursors are produced by the yeast during fermentation. Neither the juices nor the wines contained any detectable amount ( < 5 #g/m1) of urea. In a similar experiment with non-fermented grape juices no correlation was found between the potential amount of EC and the concentration of citrulline and arginine. Since these studies were conducted with grapes with low amino acid concentrations, the observed effect might not be applicable to grapes with higher concentrations of available nitrogen (Tegmo-Larsson and HenickKling, 1990a,b). Results of experiments with grape juice concentrate also indicate that EC is not formed during fermentation ( < 20 n g / m i ) even if urea or ammonium phosphate or amino acid-containing yeast nutrients have been used in excess. However, heating of the supematant at the end of the fermentation led to EC formation only if urea was used. Since at the end of the fermentation no free urea was measurable, the authors concluded that

it must have been bound as a precursor of EC (Ingledew et al., 1987). It has also been suggested that EC might be formed in wine during malolactic fermentation (Bertrand and Triquet-Pissard, 1986). However, a systematic study on Chardonnay wine with different strains of lactic bacteria demonstrated that malolactic fermentation has no effect on EC (Tegmo-Larsson et al., 1989). A series of commercial wines with low amino acid contents were stored for extended periods on yeast lees after completion of the alcoholic fermentation. N o increase in EC concentration was found. Apparently, no additional EC precursors were released from the yeast during extended lees contact. However, this effect might not be applicable to grapes with higher concentrations of available nitrogen (Tegmo-Larsson and Henick-Kling, 1990a). Compared to stone-fruit brandies, EC formation in wine seems to be more complex.

Possible reaction pathways In order to take reliable measures to reduce EC levels in beverages and foods, it is crucial to know the mode of its formation. From the preceding chapter on the different experimental results on EC formation it appears that a variety of precursors are involved in EC formation. Theoretically many different reaction pathways may finally lead to EC, e.g., reactions of organic cyanates * or of compounds formed from cyanogen (CN)2 * *. However, cyanic acid as a common ultimate reactant with ethanol is postulated. Be-

* Ethyl cyanate (C2HsOCN) reacts with water to form EC and with ethanol to form diethyl ether and cyanic acid which in turn forms EC and ethyl aliophanate. Ethyl allophanate results from the reaction of EC with cyanic acid (Jensen et al., 1966). Acetyl cyanate has been proposed as a possible intermediate in the light-induced EC formation from biacetyl and hydrogen cyanide (Baumann and Zimmerli, 1987). ** The reaction of cyanogenwith pure ethanol in the presence of anhydrous hydrogen chloride results in diethyl oxaldiimidate, diethyl oxalate and EC. Under acid conditions it may be hydrolyzed to oxamide (NH2CO)2. Cyanogen can also dismutate into cyanic acid and hydrogen cyanide, especially in alkaline solutions but also in an acid environment (Brotherton and Lynn, 1959).

337 low the different precursors for cyanic acid and its reaction mechanism are outlined and discussed: Hydrogen cyanide Carbamyl phosphate Urea, other N-, S- or Ocarbamyl compounds

Cu(CN) 3-, which could favor cyanogen formation. On the other hand copper(II) ions could also

OMdation

Dissociation

Dissociation

)

C N O - + H 3 0 + # HNCO + H20

f

Oxidation of hydrogen cyanide It seems that mainly the free, unbound hydrogen cyanide is essential for light-induced EC formation in stone-fruit brandies (Adam and Postel, 1987; Christoph et al., 1987b; Schmidt, 1988). Whereas in fresh distillates most of the hydrogen cyanide is free in its undissociated form, it will later be reversibly bound as cyanohydrins. Cyanohydrins of aliphatic aldehydes are the most stable ones. Highly enolized ketones, such as oxaloacetic ester, do not form cyanohydrins (Friederich and Wallenfels, 1970). Considering some dissociation constants of cyanohydrins * that are important in this context, it might be expected that in distillates relatively rich in benzaldehyde and poor in acetaldehyde (and/or formaldehyde) more free hydrogen cyanide should be available and vice versa. Formally the simplest way for the postulated oxidation of hydrogen cyanide to cyanic acid seems to be its exothermic reaction with hydrogen peroxide (Schwarzer, 1975), showing a reaction enthalpy of about - 7 0 kcal/mole (Cicerone and Zellner, 1983). However, the reaction of oxidants such as hydrogen peroxide with hydrogen cyanide in acid solutions produces cyanogen. Copper(II) ions are also capable of forming cyanogen from hydrogen cyanide (Cu 2+ + C N - ~ Cu + + 1/2(CN)2) (Brotherton and Lynn, 1959). As cyanogen will probably be the result of a combination of 2 cyanide radicals its formation in very diluted solutions seems rather improbable. However, it can be assumed that copper ions exist, at least partially, as cyanide complexes, e.g., as Cu(CN) 2- or

react with suitable organic matter by forming copper(I) ions (Jardim et al., 1986) which in their turn are able to reduce dissolved oxygen * * to hydrogen peroxide (2Cu++ 02 + 2 H + ~ 2Cu2++ H202) (Ito et al., 1988). Ascorbic acid (AH2) also reduces oxygen to hydrogen peroxide, where minute amounts of copper(II) or iron(III) ions strongly catalyze the reaction to dehydroascorbic acid (A) (AH 2 + 02 ~ A + H202) as well as the decomposition of the hydrogen peroxide formed. During the reaction short-lived intermediates, such as superoxide ion (O~') and radicals (e.g., OH') (Martell, 1982; Scarpa et al., 1983; Tadera et al., 1988), seem to be formed which in their turn could also oxidize cyanide to cyanate. Methylglyoxal as a strong reducing agent could formally react in a similar way as ascorbic acid. It is also known that aldehydes, and especially benzaldehyde, easily add oxygen. This auto-oxidation resulting in peroxides is catalyzed by light and metal ions. The light-induced EC formation has to be discussed in view of the formation of electronically excited states of molecules (e.g., triplet states). In this context the most interesting substances are vicinal carbonyl compounds and benzaldehyde, which all also absorb light above 350 nm. The most probable reactions of their triplet states in the distillates may be those with dissolved oxygen in its ground state (302) forming initially a diradical by addition or the superoxide ion radical (O~') by electron transfer. Using appropriate rate constants and the concentrations of the potential reactants it can be supposed that the typical hydrogen abstraction reaction from ethanol by their

* In water at 25oC: mandelonitrile5.1 x 10 - 3 M, lactonitrile 5.7×10 -s M, glyconitrile2.2x10-6 M (Schlesinger and Miller, 1973).

** Its concentration in ethanol-water mixtures can be assumed to be in the order of 10-3-10 -4 M at 25°C.

338

triplet states will be less important: a maximum of about 10% for benzaldehyde *. The reactions mentioned may lead to oxidative species capable of initiating chain reactions find/or of oxidizing hydrogen cyanide. Because the energies of the triplet states of vicinal dicarbonyl compounds are about 14 kcal/mole lower than those of benzaldehyde the possibility exists that benzaldehyde in the triplet state may efficiently transfer ( k - 1 0 9 - 1 0 m / M / s ) its energy to these compounds (Turro, 1978). However, it should be kept in mind that the cyanide complexes of iron and copper are also photochemically active. Ultraviolet irradiation of an aqueous solution of potassium hexacyanoferrate(III) yielded cyanogen (Brotherton and Lynn, 1959).

Carbamyl phosphate Carbamyl phosphate (H2NCO2PO3H2, CP) can be formed biologically or chemically, by the latter from phosphate and cyanate (Jones and Lipmann, 1960). The aqueous hydrolysis of CP has been studied by different authors (e.g., Halmann et al., 1962; Allen and Jones, 1964; Vogels et al., 1970). For the release of phosphate from monoanion and dianion, which occurs predominantly at p H 2 - 4 and 6 - 8 respectively, the first-order rate constants are very similar (half-life at 37 ° C about 45 rain). In general, it is believed that below pH 4 (monoanion) PO2 - , CO 2 and N H 3 are released in a reaction sequence involving carbamic acid (or cyanic acid?) as an unstable intermediate. At higher p H values the decomposition seems to proceed by a monomolecular elimination of cyanic acid, which is relatively stable in alkaline solutions (see below). Urea The conversion of ammonium cyanate to urea and its dissociation, followed by hydrolysis, has been extensively studied with kinetic methods. Due to the equilibration between possible pairs of reactants, the ionic and molecular mechanisms are

kinetically indistinguishable. However, by analogy to other reactions of the isocyanic group, the non-ionic mechanism is accepted at present as the more probable one (Frost and Pearson, 1964; Smyth, 1967; Stark, 1965a). (NH2)2CO (

k+l

) NH~- + N C O -

k 1 ~ NH 3 + HNCO

A rise of temperature from 0 ° C to 1 0 0 ° C increases the equilibrium constant K = k+l/k_ ~ by a factor of about 100 and consequently the relative amount of cyanate by a factor of about 10. Furthermore, the time to reach the state of equilibrium is reduced by a factor of 105 ** For aqueous urea solutions in the concentration range of 5-100 # g / m l , which could occur in mashes or wines, the degree of dissociation of urea can be calculated to be in the range of 50-90% at 100 o C. The cyanic acid formed may hydrolyze or react with suitable nucleophiles other than water, e.g., ethanol, and, depending on the pH, especially with amino a n d / o r sulfhydryl groups of proteins or amino acids (Stark et al., 1960; Stark, 1964, 1965a, b; Smyth, 1967; Cejka et al., 1968). The hydrolysis of urea in pure aqueous solution is not catalyzed by acid and its energy of activation (Ea) is 32.7 k c a l / m o l e (Frost and Pearson, 1964). From the data given by Monteiro et al. (1989), a rough estimate of E a for the hydrolysis of urea in a water-ethanol mixture (20% v / v , p H = 3.2) is about 43 kcal/mole and for the formation of EC from urea about 22 kcal/mole. Thus, at higher temperatures hydrolysis of urea proceeds at a faster rate than the formation of EC, while at low temperatures the reverse is the case. Estimates of E, for EC formation from the results of other model experiments (pH range 3.1-4.3) are: from urea 27 kcal/mole (Ough et al., 1988a) or 36 kcal/mole (Hara et al., 1988), and from citrullin 29 k c a l / m o l e (Ough et al., 1988a). K=k+l/k_l= ([NH~ ][NCO- ]/[(NH)2CO] = [(NH2)2CO]o-a2/(1 - a) (a

* In pure water the equilibrium constant * Biacetyl in its triplet state may abstract hydrogen from ethanol, but it is unlikely that it does so from water. In aqueous solutions of biacetyl a reaction with oxygen and a photolytic hydrolysis resulting in acetaldehyde and acetic acid seem to be important (Stevens and Dubois, 1962).

= degree of dissociation), increases from 8 × 10 -6 M [5.9 × 1 0 - 1 2 / s / 7 . 7 × 1 0 - 7 / M / s ] at 0 ° C to 8 × 1 0 -4 M [4× 1 0 - 5 / s / 5 × 1 0 - 2 / M / s ] at 100°C; at 1 8 ° C K = 2 . 5 × 1 0 -5 M (Hagel et al., 1971).

339

It can be supposed that other N-carbamyl compounds, which have been demonstrated to form EC in aqueous ethanol solutions, may react by a similar pathway as urea by directly forming either cyanic acid or urea. It has been shown that allantoate (diureidoacetate) was converted to ureidoglycolate and urea at pH values below 7, whereas at pH above 7 cyanate, ammonia, glyoxylate and urea were formed. The reaction rate constant of this latter conversion was similar to those measured in urea and N-carbamylglycine (hydantoate) hydrolyses (Vogels and Van der Drift, 1969).

Cyanic acid Werner and Gray (1947) demonstrated that the sole product of the reaction of cyanate (2 M) with ethanol is EC, if an excess of hydrochloric acid was avoided. In absolute ethanol the yield of EC was 60%, in a 50% ethanol-water mixture only 2.3% (and 1.8% ethyl allophanate). There is very little or no carbamylation of ethanol by potassium cyanate in aqueous ethanol at pH 7 (Stark, 1965b). However, when the reaction of cyanate with alcohols occurs in inert solvents (benzene, dichloromethane) in the presence of trifluoroacetic acid, the carbamates are formed with 60-90% yields (Loev and Kormendy, 1963). On the assumption of a molecular mechanism *, the following competitive reactions consisting in nucleophilic attacks upon the carbon atom of isocyanic acid would be essential in EC formation: kl

HNCO + C2HsOH HNCO

, EC k2a(k2b)

+ H30+(H20

)

>

(1) products

(e.g., C 0 2 , HCO 3 , NH 3, NH~-) HNCO + X

k3

> carbamyl compounds

concentration of ethanol. At pH values higher than 4 in pure water, each increase of a pH unit decreases theportion of undissociated cyanic acid by a factor of about 10 * *. Hence above pH 7, where mainly cyanate exists, which hydrolyses very slowly (Vogels et al., 1970; Hagel et al., 1971), the rate of EC formation is expected to be very small. On the other hand, at pH values less than 5, where the undissociated acid predominates, its rate of hydrolysis (which is catalyzed by hydrogen ions at pH < 2) will be faster (Vogels et al., 1970; Hagel et al., 1971; Jensen, 1958) * * *. According to such a mechanism the rate of EC formation as a function of pH should have an optimum pH value. This is exactly what has been observed (optimum between pH 4 and 6) in the first distillates of Scotch whisky (Riffkin et al., 1989b) as well as in the light-induced EC formation in a cherry distillate (Zimmerli et al., 1990), thus indicating that the same reactive species may be involved in both cases. However, the observed pH dependence could also include pH-dependent preceding reactions, e.g., the oxidation of hydrogen cyanide to cyanic acid. The light-induced EC formation in stone-fruit distillates resulted in one set of samples with a constant yield of EC (AEC/AHCN) of about 25% (Fig. 1), whereas other sets yielded values from about 2% to 30% (see above). Likewise, the relatively high yield of about 30% seems to contrast with the results of model experiments on EC formation from carbamyl phosphate (Ough, 1976a; Ough et al., 1988a), cyanate (Hara et al., 1988) and urea (Ough et al., 1988a; Monteiro et al., 1989) which resulted in apparent yields of less than 5%, in spite of the fact that cyanic acid can

(2) (3)

X: e.g., NH3, RNH2, RSH, ROH, RCOOH, RCONH2, urea, carbamates, phenols. When neglecting reaction (3) it is obvious that the rate of EC formation at a given temperature must depend on the pH of the solution and the * The carbon of cyanate ion will be much less electrophilic than that of cyanic acid.

** p K a = 3.70 at 2 0 - 3 0 ° C in water (Boughton and Keller, 1966). In water-ethanol mixtures the p K a of cyanic acid will probably be higher than in water, because of the lower dielectric constant of the solvent, resulting probably in a more rapid hydrolysis of cyanic acid. The latter effect has been demonstrated experimentally by Stark (1965b). *** In diluted aqueous solution (data of Jensen, 1958, expressed as bimolecular rate constants with u n l t s / M / s ) : H N C O + H 3 0 + ~ ; k2a = 6.0 × 10- 2 / M / s at 18 o C, E a = 15.1 kcal/mole. H N C O + H 2 0 ~ ; k2b = 1.4× 1 0 - 5 / M / s at 18°C, E~ =19.9 kcal/mole. At pH 3-7 only reaction 2b is essential, at least in aqueous solutions.

340 always be assumed as an intermediate. However, the ratio of the apparent rate constants k a / k 3 which can be calculated from the results of the experiments with n-butanol (Table 4) yielded about 1.2 (ca. 30 o C). This result seems to be in good agreement with the quite comparable nucleophilic reactivity of ethanol and n-butanol toward an electrophilic species, such as cyanic acid. As discussed above the pH value of the distillate and its possible change during light exposure should influence the reaction rates of cyanic acid. Hence, different pHs of the samples could offer a possible explanation for the different yields observed in different distillates *. However, the yields of EC (based on the amount of hydrogen cyanide that has disappeared) could also be influenced by constituents of the distillates present in variable amounts, which catalyze either the rate of hydrolysis of cyanic acid, such as succinate and maleate (Vogels et al., 1970), or the rate of its reaction with ethanol to EC. For a detailed discussion of the yields of light-induced EC in stone-fruit distillates further quantitative studies are needed. If ammonia, amino or sulfhydryl groups are available reaction (3) may become important depending on their concentrations, their p K a values, and the pH of the solution. At p H values less than 7, typical for mashes, and wine, the most im, portant reactions of cyanic acid would be those with sulfhydryl, carboxyl or carbamyl groups (Stark, 1964, 1965a,b). These types of principally reversible reactions could offer an explanation for the results obtained in fermentation experiments where urea as a yeast nutrient has been added excessively (Ingledew et al., 1987), and which demonstrated the existence of a type of ' b o u n d urea' as an EC precursor. Monteiro et al. (1989) also noted that in wine there was a greater conversion of urea to some compounds other than EC than in the buffer solutions.

Conclusions for other foodstuffs Apart from the reaction of diethylpyrocarbonate with ammonia, the presence of ethanol

* In general stone-fruit distillates show pH values in the range of 3.3-4.8, with a tendency to increase during storage.

is a prerequisite for the formation of EC in all kinds of food. Most probably, undissociated cyanic acid is the ultimate reactant with ethanol. Cyanic acid may originate from different sources such as carbamyl phosphate, the oxidation of hydrogen cyanide, N-carbamyl compounds such as urea and citrullin, or from hitherto unknown substances with labile carbamyl groups. Because of the possible formation of EC from carbamyl compounds and ethanol there is no reason to believe that EC may not be present in any food, drug or natural flavoring extract. Many products other than alcoholic beverages or fermented foods, in which ethanol and heat have been used during processing, may potentially contain EC. After the addition of ethanol to non-fermented grape juices and subsequent heating, up to about 50 n g / m l of EC has been found (TegmoLarsson, 1989; Tegmo-Larsson and Henick-Kling, 1990a,b). Hence, little fermented grape juice, which has been pasteurized to stop fermentation (called 'Sauser' in Switzerland), may potentially contain EC. Ough (1976a) has demonstrated that a h e a t acid hydrolysis of bread prior to the extraction of EC results in a marked increase of its concentration, although the natural ethanol concentrations in bread are very low ( < 0.02-0.3% m / m ) , typically about 0.1% (Anonymous, 1989). Although a preliminary estimation of the mean daily intake of urea compounds in Switzerland resulted in only 3 m g / p e r s o n (Shephard et al., 1987) the question may be raised what amount of EC could be formed in the stomach after the ingestion of a meal consisting of bread and wine. The proposed use of ethanol for mold inhibition in bread, doughs and prebaked bread (Briimmer and Morgenstern, 1984; Pafumi and Durham, 1987) demands further studies, but it seems that its use is already accepted in certain countries, e.g., in Italy for sliced toast bread (Anonymous, 1988b). Possible alterations of amino acids have been studied after extraction of foods with supercritical carbon dioxide (Weder, 1984; Weder and Hegarty, 1989). At present, the possibility of formation of trace amounts of urea compounds or carbamic acid derivates as precursors for EC cannot be excluded with certainty. On the other hand heterocyclic derivates of Ncarbamyl glycine (Nofre et al., 1989) or amides (Nofre and Tinti, 1989) have recently been pro-

341 posed as sweeteners. Another example of potential EC formation is the proposed use of urea complexation of fatty acids in the preparation of omega-3 polyunsaturated fatty acid concentrates from fish oil, in the course of which mixtures of urea and fatty acids are refluxed with ethanol (Ratnayake et al., 1988). The use of azodicarbonamide (NH2CO-N--N-CONH2) also merits attention. It may be used legally in certain countries, either as a flour improver in reducing thiol groups (Anonymous, 1967; Ewart, 1988) or as blowing agent for plastics. It appears that the latter use in manufacturing beer bottle caps may contribute to the EC levels in beer (Anonymous, 1990). Cyanide or cyanogenic glycosides like amygdalin can be found in practically all plant materials. Unprocessed cassava (Manihot esculenta Crantz) is known to contain toxic levels, but bitter almonds, linseed and the seeds of stone fruits also show relatively high concentrations. In certain regions of Africa cassava is also used as a starchy material for traditional beer brewing. Hence, these beers should also be analyzed for EC. Cyanide (total amounts) is also contained in cereals (< 111 ng/g), European beers (28-164 ng/ml), fruit juices (< 20-2120 ng/ml), as well as in fruit.containing baby foods (20-370 ng/g) (Lehmann and Mt~ller, 1981; Lehmann et al., 1979; M/511er, 1984), It has also been mentioned that fermentation of cyanide-containing plant material is not ab. solutely necessary to get EC, it seems sufficient that such material comes into contact with ethanol (Wucherpfennig et al., 1987; Tourli&e, 1989), Hence, ethanol-containing jam which is commercially available as a speciality should also be analyzed for EC. However, trace amounts of hydrogen cyanide can also be formed during the MaiUard reaction, if oxygen is present (Lehmann and Zinsmeister, 1979; M~ller, 1984). Also, the possibility may exist that by the use of oxidants as flour improvers cyanide is oxidized to cyanic acid, which could react with flour constituents to form additional precursors resulting in EC formation during bread making. Thus, much more research is needed about the occurrence of EC in the human diet beside alcoholic beverages as well as on its potential endogenous formation.

Biological activity Recently, the data on metabolism, mutagenicity and carcinogenicity have been extensively reviewed and a risk assessment of EC for humans at dietary levels has been performed (Schlatter and Lutz, 1990). With respect to a possible health hazard at low exposure levels, it is important to characterize the toxic potential of EC, including its mechanism of action. In this context, the acute toxicity of EC can be neglected as it is relatively low, in the order of 2500 mg/kg b.w. (LDs0 rat, oral application; NIOSH, 1985).

Metabolism EC is rapidly distributed throughout the body regardless of the route of administration. The major part of the compound (up to 90%) is completely degraded to CO2, H20 and NH 3 within 24 h. Minor pathways also exist, leading to the formation of different urinary metabolites (up to 6% of the compound is excreted as unchanged ethyl carbamate, N-hydroxy ethyl carbamate, N-acetylN-hydroxy ethyl carbamate, ethyl mercapturi¢ acid or N-acetyl-S-ethoxy carbonylcysteine; for reviews see Mirvish, 1968; IARC, 1974). The metabolism of EC is saturable (O'Flaherty and Sichak, 1983). The dose required to saturate metabolism is species-dependent and is in the order of 5 mg/kg b.w.i.v, for male Fischer 344 rats and in the order of 50 mg/kg b.w. for male B6C3F1 mice (Nomeir et al., 1989). EC crosses the placental barrier, and in newborn mice the elimination of EC is even slower than in the adult mouse (IARC, 1974). In male A/Jax mice Hurst et al. (1990) found a half-life of disappearance from blood of 30-40 rain after an oral EC dose of 11.1 mg/kg b.w. (the data presented were insufficient to calculate a terminal elimination half-life). Thus metabolism of EC is rather fast. Yamamoto et al. (1988) showed that radiolabeled EC disappeared from the blood almost completely within 4 h. However, a single high dose of ethanol (5 g / k g b.w., initial blood ethanol level of about 0.5%) leads to a high, constant blood level of EC which persists for 8 h after oral administration and only declines thereafter (Waddell et al., 1987; Yamamoto et al., 1988). A detailed analysis of these data led to the conclusion that acute administra-

342

tion of high doses of ethanol may postpone the metabolism of EC, possibly by blocking metabolizing enzymes, including the group of cytochromes P-450. The toxic effects might, therefore, merely be postponed (Schlatter and Lutz, 1990). It should also be kept in mind that chronic administration of ethanol, as opposed to the acute situation, may lead to induction of metabolizing enzyme systems such as P-450 (Lieber et al., 1987a,b) and consequently have a modulatory effect on the carcinogenicity of EC (see below). Gupta and Dani (1989) demonstrated the occurrence of oxidation reactions at the ethyl group upon incubation of EC in vitro with microsomes from rat lung (leading to the formation of N-hydroxy ethyl carbamate, N-hydroxy vinyl carbamate, epoxy ethyl carbamate). The generation of vinyl carbamate and its epoxy derivative may represent the proximate and ultimate electrophilic metabolites responsible for genotoxicity and carcinogenicity.

Mutagenicity Vogt (1948) already described EC as a chromosome-breaking agent able to induce the same type of mutations in Drosophila which are also induced by different types of high-energy irradiation or mustard gas. Many studies have been published concerning the mutagenicity of EC in a wide range of organisms, including plants (Bateman, 1976; Kada and Ishidate, 1980; de Serres and Ashby, 1981; Field and Lang, 1988). In bacterial test systems the results were mainly negative. An explanation for this may be that in the standard Salmonella/microsome assay, using rat liver $9, the first step in the metabolic activation, the oxidation of EC to vinyl carbamate, is insufficient to give positive results. The fact that vinyl carbamate gives positive results in the Ames test (Dahl et al., 1980) strongly supports this hypothesis. In tests with eukaryotic cells, positive and negative findings are about equal in frequency. It seems that positive results were obtained only under conditions of appropriate metabolic activation. EC was genotoxic in the somatic mutation and recombination test in Drosophila melanogaster (number and shape of wing hairs after treatment of larvae), in a standard strain and in a strain in

which genetic control of cytochrome P-450-dependent enzyme systems have been altered (constitutively increased P-450 enzyme activities) (FrtShlich and Wiirgler, 1988; Frt~hlich, 1989). The effects were dose-dependent and the modified strain was more sensitive to EC by about one order of magnitude than the standard strain. This further suggests that the P-450 enzyme system is involved in the activation of EC. More than 10 publications are available with quantitative data on DNA-adduct formation by EC. All tissues investigated showed DNA-adduct formation upon EC exposure, the liver almost always had the highest values a few hours after single-dose administration (Lutz, 1979; Fossa et al., 1985; Scherer et al., 1986; Svensson, 1988). Several authors proposed a metabolic pathway which leads to the formation of vinyl carbamate and, after epoxidation, to DNA and RNA adducts (Ribovich et al., 1982; Miller and Miller, 1983; Leithauser et al., 1990). Recently, this hypothesis has been supported by the already mentioned study of Gupta and Dani (1989), who identified N-hydroxyvinyl carbamate and an epoxy derivative of EC as metabolites. Furthermore, 7-(2oxoethyl)-guanine was identified as a DNA adduct in mouse and rat liver after injection of labeled EC (Miller and Miller, 1983; Scherer et al., 1986; Leithauser et al., 1990). In addition, vinyl carbamate was shown to lead to the same DNA adducts as EC, although the potency of the former was much higher (Scherer et al., 1986). Since 7-(2oxoethyl)-guanine is also formed by vinyl chloride (Laib et al., 1981), Svensson (1988) postulated a common ultimate carcinogen of the 2 compounds and compared their potency to form DNA adducts with their respective carcinogenic potencies. Schlatter and Lutz (1990) also compared the level of DNA-adduct formation in the liver per dose administered: EC was amongst the moderately potent genotoxic (hepato-) carcinogens, a factor 100-1000 below the carcinogenic potency of aflatoxin B1 and a factor 10 below the potency of vinyl chloride.

Carcinogenicity The data discussed above indicate that EC is genotoxic in vitro and in vivo. It is therefore not surprising that this compound was also reported

343 to be carcinogenic. There is a vast literature on EC carcinogenicity (Mirvish, 1968; IARC, 1974; National Cancer Institute, 1978-1980). Doses of 100-2000 mg EC/kg b.w., typically one single dose of 1000 mg/kg b.w., have been shown to induce tumors in rats, mice and hamsters following administration by inhalation or by oral, dermal, subcutaneous or intraperitoneal routes. A tumor incidence of 40-100% is typical after administration of 100-1000 mg EC/kg b.w. via drinking water, depending on the duration of treatment. A high tumor incidence (80-100% lung tumors) was also found in newborn mice after a single 'standard' dose of 1000 mg EC/kg b.w. given by oral gavage. If pregnant mice received a single intraperitoneal injection of this standard dose 1 day before delivery, 100% of the offspring developed lung tumors within 6 months (9-10 tumors per mouse). Tumor induction in offspring was also demonstrated when EC was administered to lactating mice. The main target organ in mice seems to be the lung, and in rats the mammary gland (Schlatter and Lutz, 1990), but various other organs were also susceptible to EC tumor induction: lymphomas, vascular tumors, skin tumors, and hepatomas have been found. A comparison of tumor susceptibility among various organs of fetal, young and adult mice showed a high tumor susceptibility in rapidly proliferating and undifferentiated cells (Nomura, 1976). The data published so far deafly indicate that ethyl carbamate is a pluripotent carcinogen with respect to tumor induction in different species, organs, and stages of development of the animals. Low-dose extrapolation and risk assessment Human exposure to carcinogenic compounds should in general be kept as low as reasonably achievable. For human risk assessment, animal carcinogenicity data from long-term low-dose exposure are often used in the absence of human carcinogenicity data. At present, only 2 experimental carcinogenicity studies in rats and mice with doses in the lower mg/kg b.w. range are available in the literature (Dahl et al., 1980; Port et al., 1976; Schm~ihl et al., 1977). Only in the latter study was EC administered for the entire lifetime but the quantitative data were reported as tumor incidences pooled for all organs only. Re-

cently, sex-specific and target organ-specific data have been compiled from original data (Schlatter and Lutz, 1990). Based on these data and by using a linear-at-low-dose, no-threshold model, Schlatter and Lutz (1990) calculated a 'virtually safe dose' for a lifetime risk level of 1/106 for 2 organs and 2 animal species. The resulting daily dose for this risk level ranged between 20 and 80 ng/kg b.w./ day. As outlined earlier, the data on human exposure levels to EC are far from complete today. Nevertheless, an estimate of the dally burden can be made taking into account confounding factors (e.g., sample handling, variability in analytical methods). Considering all the data together, human exposure under normal dietary habits, excluding alcoholic beverages (10-20 ng/kg b.w./ day) represents a negligible hypothetical risk. However, individual habits may greatly enhance the risk above the 'widely accepted' level: if 500 ml table wine is consumed dally, the risk is increased by a factor of up to 5, relative to the above-mentioned risk under normal dietary habits. Regularly drinking 20-40 ml stone-fruit brandy per day would raise the calculated hypothetical tumor risk to near 1/104 . In this context it is important to note that this amount of alcohol is generally not yet considered to be a significant health risk per se. The question raised by the data published by Yamamoto et al. (1988) on EC metabolism, namely the possibility of a modulatory effect of the simultaneous administration of EC and ethanol on the tumorigenicity of EC, has been addressed recently by Kristiansen et al. (1990). Combinations of ethanol (0, 5, 10, 20% v/v) and EC (0, 200, 500, 1000 gg/g) were added to the drinking water of female A / P h mice for 12 weeks (daily EC intake about 0, 35, 90, 175 mg/kg b.w.; daily ethanol intake 0, 9, 18, 35 g/kg b.w.). After 12 weeks of treatment all animals developed primary lung adenomas (40-67% incidence in the control groups without EC). The number of tumors per mouse induced by EC was significantly reduced with 10 and 20% ethanol as the only source of fluid for the animals (numbers of tumors per mouse for the 90 (175) mg/kg b.w. EC dose are 45.4 (70.9), 46.0 (61.3), 22.1 (39.3) and 9.6 (21.6) for the 0, 5, 10 and 20% ethanol groups). The authors concluded that 'a clearcut inhibitory action of concomitant

344 ethanol intake upon the development of lung adenomas in mice given high urethane doses indicates that co-administration of ethanol may inhibit carcinogenic action of urethane.' However, it must be kept in mind that the blood levels of ethanol in the 10% and 20% ethanol groups must be constantly high (effective blood levels were not measured), leading to increased relative liver weights within 12 weeks in the higher EC treatment groups. However, with a dosing regimen more closely related to the human exposure situation in non-alcoholics, namely by bolus application allowing the ethanol blood level to decrease between doses, different results were obtained. In a study just completed at the Bundesgesundheitsamt, Berlin (Germany), female N M R I mice were treated daily with EC (0, 90, 180 m g / k g b.w.) by gastric intubation either in water or in 20% ( v / v ) ethanol (daily ethanol intake 0, 2.3 g / k g b.w.) for 8 weeks (Altmann et al., 1990). After another 8 weeks without any treatment all EC-treated animals developed lung adenomas, but here the dose-dependent increase in the number of lung adenomas per mouse was not influenced by the co-administration of ethanol (0 vs. 20% ethanol group: 32 vs. 40 and 81 vs. 78 lung tumors per mouse for the 90 and 180 m g / k g b.w. EC dose). In 2 studies done in the Soviet Union and reported by the IARC (1988), an increase in the number of tumors per mouse was even found after co-administration of ethanol (40%, 4 g / k g b.w., EC 100 m g / k g b.w., 2-weekly bolus doses). From these studies, together with the metabolism data described earlier, it could be speculated that a constantly high ethanol blood level (at or above 0.15%; Yamamoto et al., 1988) reduces the carcinogenicity of EC by altering the rate of formation of reactive intermediates of EC, while a dosing regimen of ethanol allowing ethanol blood levels to decrease below 0.15% does not influence the carcinogenicity of EC. The above risk assessment is not in line with the Canadian guidelines mentioned earlier (see Introduction). However, at the time these guidelines were set 'the assessment of the magnitude of this risk [carcinogenicity] was extremely difficult in view of deficiencies in the available toxicity data base' (Anonymous, 1986). Despite this fact,

the Canadian authorities assumed a N O E L (no observable effect level) for rodents to be 1500 /~g/kg b.w. and applied a safety factor of 5000 to this N O E L to estimate a tolerable daily intake for humans of 300 n g / k g b.w. (Anonymous, 1986). The guideline levels for the different types of alcoholic beverages were thereafter calculated based on daily consumption figures (table wines 650 g / d a y , fortified wines 200 g / d a y , distilled spirits 125 g / d a y and fruit brandies 50 g/day). Today a more convenient risk assessment is possible based on an evaluation of all toxicity data, including mechanistic studies on the mode of action of EC (see Schlatter and Lutz, 1990). There is presently no need to alter the risk assessment presented by Schlatter and Lutz (1990) because of the available data on E C / e t h a n o l interaction. However, further studies on the nature (mechanism, kinetics) of E C / e t h a n o l interaction are needed.

Acknowledgements We gratefully acknowledge the help of Prof. Dr. W. Grunow, Federal Health Office, D-1000 Berlin 33 (Germany) who kindly provided us with the data of their study on e t h a n o l / E C interaction, as well as Dr. M. Tegma-Larsson of the New York State Agricultural Experiment Station, Geneva, N Y (U.S.A.) who kindly provided us with the manuscripts of her publications. Thanks are also expressed to Dr. D. Andrey, Laboratorium der Urkantone, CH-6440 Brunnen (Switzerland), Dr. H. Tanner (now retired), Eidg. Forschungsanstalt fiir Obst- und Weinbau, CH-8820 W~idenswil (Switzerland), and Dr. A. Tuor, Kantonales Laboratorium, CH-6002 Luzem (Switzerland), for their personal communications and helpful discussions.

Appendix After preparation of the manuscript 4 excellent and informative publications on the EC formation in grain-based spirits were sent to us by Dr. G. Cochrane of the United Distillers International Research Center, Menstrie, Clackmannanshire (U.K.). Because these reports give important additional information on EC precursors and confirm some of our assumptions on the precursors of

345

EC in whisky as well as on the possible involvement of cyanic acid in EC formation the data are summarized below. Aylott et al. (1990) have demonstrated that in freshly distilled grain whisky the post-distillation EC formation may be accelerated by light, and that EC is also formed in the dark during maturation in oak casks, probably by components extracted from the wood (e.g., methyl glyoxal). Initial EC precursors include anions such as cyanate, cyanide and copper-cyanide complexes. Other species potentially involved in EC formation include thiocyanate and cyanogen (Aylott et al., 1990). Lactonitrile was determined to be the major cyanohydrin in freshly distilled grain spirit, but is not considered to be an important EC precursor. The conversion of 'measurable cyanide' (hydrogen cyanide, copper-cyanide complexes and cyanohydrins) into EC during maturation of grain-based spirits has been determined to be in the order of 30%. However, studies on EC formation from added H14CN (1 /tg/ml) to a sample of freshly distilled grain spirit of 94.2% ethanol (v/v) (4 days, dark, 25°C, 10 # g / m l Cu 2+) resulted in an EC yield of less than 1% (McGill and Morley, 1990). It was assumed that the incorporation of cyanide into EC can be greatly influenced by other spirit congeners. Analytical studies on the characterization of the corrosion deposits in the distillation apparatus (Coffey still) demonstrated the existence of a range of copper salts, incorporating cyanide, cyanate and thiocyanate as well as thiosulfate (MacKenzie et al., 1990). It can be assumed that the copper complexes, being nonvolatile, descend the rectifier column from areas at and above the spirit take-off point. The hydrogen cyanide in the distillates as well as in Coffey still deposits originates from the thermal decomposition of the cyanohydrin of isobutyraldehyde which is present in fermented mash. This compound arises during alcoholic fermentation by the hydrolytic action of yeast fl-glucosidase on a naturally occurring cyanogenic glycoside contained in malted barley (absent in unmalted barley), and identified as epiheterodendrin (Cook et al., 1990). References Adam, L., and W. Postel (1987) Gaschromatographische Bestimmung von Ethylcarbamat (Urethan) in Spirituosen, Brarmtweinwirtschaft, 127, 66-68.

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