363

Mutation Research, 259 (1991) 3 6 3 - 3 8 5 © 1991 Elsevier Science Publishers B.V. 0 1 6 5 - 1 2 1 8 / 9 1 / $ 0 3 . 5 0 ADONIS 0 1 6 5 1 2 1 8 9 1 0 0 0 6 9 0

M U T G E N 00042

Aldehydes: occurrence, carcinogenic potential, m e c h a n i s m of action and risk assessment V.J. Feron, H.P. Til, Flora de Vrijer, R.A. Woutersen, F.R. Cassee and P.J. van Bladeren TNO-CIVO Toxicology and Nutrition Institute, Zeist (The Netherlands) ( R e c e i v e d 14 M a y 1990) ( A c c e p t e d 27 A u g u s t 1990)

Keywords: A l d e h y d e s ; Occurrence; Carcinogenicity; M e c h a n i s m of action; R i s k a s s e s s m e n t

Contents S u ~

...................................................................................

Introduction ................................................................................ C h e m i c a l p r o p e r t i e s a n d b i o t r a n s f o r m a t i o n of a l d e h y d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O c c u r r e n c e in food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formaldehyde ............................................................................... Occurrence ............................................................................... Genotoxicity .............................................................................. Carcinogenicity ............................................................................ Risk assessment ............................................................................ Acetaldehyde ................................................................................ Occurrence ............................................................................... Genotoxicity .............................................................................. Carcinogenicity ............................................................................ Risk assessment ............................................................................ Malondialdehyde ............................................................................. Occurrence ............................................................................... G e n o t o x i c i t y , c a r c i n o g e n i c i t y a n d risk a s s e s s m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acrolein ................................................................................... Occurrence ............................................................................... Genotoxicity and carcinogenicity ............................................................... Risk assessment ............................................................................ Crotonaldehyde .............................................................................. Occurrence ............................................................................... G e n o t o x i c i t y , c a r c i n o g e n i c i t y a n d risk a s s e s s m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cinnamaldehyde ............................................................................. Occurrence ............................................................................... G e n o t o x i c i t y , c a r c i n o g e n i c i t y a n d risk a s s e s s m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benzaldehyde ................................................................................ Occurrence ............................................................................... G e n o t o x i c i t y , c a r c i n o g e n i c i t y a n d risk a s s e s s m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C o r r e s p o n d e n c e : Dr. V.L Feron, T N O - C I V O T o x i c o l o g y a n d N u t r i t i o n Institute, Z e i s t (The Netherlands).

364

364 365 366 367 367

368

: .........

...................

368 369 371 371 371 3"/1 372 372 372 373 373 373 373 374 374 374 374 375 375 375 375 375 375

364

Furfural

...................................................................................

Occurrence

376

...............................................................................

376

Genotoxicity .............................................................................. Carcinogenicity ............................................................................

376 376

Risk assessment ............................................................................ Glycidaldehyde .............................................................................. Occurrence ...............................................................................

377 377 377

Genotoxicity, carcinogenicity and risk,assessment .................... Citral ..................................................................................... Occurrence

, ................................

...............................................................................

377 378 378

G e n o t o x i c i t y , c a r c i n o g e n i c i t y a n d risk a s s e s s m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anisaldehyde ................................................................................ Occurrence ............... ~ ...............................................................

378 378 378

G e n o t o x i c i t y , c a r c i n o g e n i c i t y a n d risk a s s e s s m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vanillin .....................................................................................

378 378

Occurrence ............................................................................... G e n o t o x i c i t y , c a r c i n o g e n i c i t y a n d risk a s s e s s m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

378 378

Other aldehydes ..............................................................................

378

Concluding remarks

379

...........................................................................

Summary Aldehydes constitute a group of relatively reactive organic compounds. They occur as natural (flavoring) constituents in a wide variety of foods and food components, often in relatively small, but occasionally in very large concentrations, and are also widely used as food additives. Evidence of carcinogenic potential in experimental animals is convincing for formaldehyde and acetaldehyde, limited for crotonaldehyde, furfural and glycidaldehyde, doubtful for malondialdehyde, very weak for acrolein and absent for vanillin. Formaldehyde carcinogenesis is a high-dose phenomenon in which the cytotoxicity plays a crucial role. Cytotoxicity may also be of major importance in acetaldehyde carcinogenesis but further studies are needed to prove or disprove this assumption. For a large number of aldehydes (relevant) data on neither carcinogenicity nor genotoxicity are available. From epidemiological studies there is no convincing evidence of aldehyde exposure being related to cancer in humans. Overall assessment of the cancer risk of aldehydes in the diet leads to the conclusion that formaldehyde, acrolein, citral and vanillin are no dietary risk factors, and that the opposite may be true for acetaldehyde, crotonaldehyde and furfural. Malondialdehyde, glycidaldehyde, benzaldehyde, cinnamaldehyde and anisaldehyde cannot b e evaluated on the basis of the available data. A series of aldehydes should be subjected to at least mutagenicity, cytogenicity and cytotoxicity tests. Priority setting for testing should be based on expected mechanism of action and degree of human exposure.

Introduction The chemical as well as the biological properties of aldehydes are governed by the presence of a ketone moiety. From a mechanistic point of view, in addition to saturated or simple aldehydes (e.g., formaldehyde, acetaldehyde, propionaldehyde), a, fl-unsaturated aldehydes (e.g., acrolein and crotonaldehyde), and aldehydes containing a second functional group (e.g., furfural, cinnamaldehyde, glycidaldehyde) can be distinguished. Some

aldehydes such as acetaldehyde occur in a large number of foods, whereas others occur only in a few food products but may constitute the major component of a product, e.g., cinnamaldehyde which is the chief ingredient (up to 90%) of cinnamon oil (Mantovani et al., 1989). Carcinogenic potential in experimental animals has been demonstrated for several aldehydes: formaldehyde vapor has been reported to induce nasal carcinomas in rats, and possibly in mice (Kerns et al., 1983); prolonged inhalation ex-

365 posure to acetaldehyde vapor has been found to induce nasal carcinomas in rats (Woutersen et al., 1986) and laryngeal carcinomas in hamsters (Feron et al., 1982); 2-year oral gavage studies with furrural produced clear evidence of carcinogenic activity in mice and some evidence of carcinogenicity in rats (Anonymous, 1989). No aldehyde is found among the agents for which there is conclusive evidence of carcinogenic activity in humans (IARC, 1987b; Tomatis, 1989). According to IARC (1982, 1987b) formaldehyde is probably carcinogenic to humans. Marcus and Feinman (1988) reviewed the cancer epidemiology of formaldehyde and concluded that some studies produced evidence of an association of formaldehyde with cancer of the nasopharynx and nasal sinuses. However, for no malignancy in man is there convincing evidence of a relationship with formaldehyde exposure, and, if a relationship does exist, the excess cancer risk must be small (Anonymous, 1988) and within the confidence intervals (Ulm, 1989). The present paper deals with some general remarks on chemical properties and biotransformation of aldehydes, their presence in food, and the carcinogenicity, mode of action and risk assessment of major representatives occurring in foods. Moreover, major gaps in our knowledge are identified and recommendations are made for further research.

Chemical properties and biotransformation of aldehydes Aldehydes constitute a group of relatively reactive organic compounds characterized by the presence of a polarized carbon-oxygen double bond. The oxygen atom is highly electronegative compared to the carbon atom and therefore aldehydes have substantial dipole moments. The carbonyl carbon is an electrophilic site and reacts easily with nucleophiles (McMurry, 1984). When carbons 2 and 3 (also known as a and fl) possess a double bond (alkenals, e.g., acrolein) the molecule becomes even more reactive. Because of its conjugation with the carbonyl group, the fl-carbon becomes positively polarized, and will be the site of nucleophilic attack. The partially positive charge can be influenced by substituents. For example, a

4-hydroxy group has an electron-withdrawing effect and as a consequence the fl-carbon becomes even more electrophilic. Comparative studies on the influence of halogen atoms have been performed by Rosen et al. (1980). These facts allow some predictions with respect to the behavior of aldehydes in biological systems. Aldehydes can be subdivided into 3 classes, with regard to the reactions they can undergo. (1) Saturated or simple aldehydes: a range of aldehyde dehydrogenases are available to oxidize the functional group to carboxylic acid. Conjugation with molecules containing thiol or amino groups has been reported as well as cross-linking properties, but oxidation seems to be favored. Oxidation occurs enzymatically by various aldehyde dehydrogenases, some of which have a high substrate specificity, while others are able to accommodate a variety of substrates (Schauenstein et al., 1977; Brabec, 1981; Siew, 1976; Weiner, 1980; Casanova-Schmitz et al., 1984; Sladek et al., 1989). The oxidation products are the corresponding carbon acids. The oxidation reaction is dependent on NAD(P) ÷ and in the case of the (specific) formaldehyde dehydrogenase also on reduced glutathione. The general reaction scheme of the oxidation of simple aldehydes is: R-CHO + N A D + +

H20

--, R-COOH + N A D H + H + Reduction is less important in the: biotransformation of aldehydes, because t h e K = values for aldehyde reductase- and alcohol dehydrogenasecatalyzed reactions are in general 1 - 3 magnitudes higher than for the aldehyde dehydrogenases (Sladek et al., 1989). The reduction to an alcohol is also easily reversible, whereas oxidation of the aldehydes to carboxylic acids is essentially irreversible (Sladek et al., 1989). Nucleophilic attack on the carbonyl moiety by thiols and amines resuits in hemiacetals and Schiff's bases, respectively. The hemiacetal of formaldehyde and glutathione is the actual substrate for the dehydrogenase reaction (Koivusalo et al., 1989). From the initial adduct with cysteine or giutathione, a thiazolidine can be formed. Attack of a second thiol

366 H

I

R--C--N--R' H

I

H

R--C~O

H

I

HS--R" H ~ ~ +

/R' HN H

/S--R

R/C~N__R

R--C--O--H

,, ,

I

H

I

S--R" Fig. 1. Generalreactionschemeof thiol and amineinteractionswithaldehydemoieties. or amine on the initial adducts can result in protein-protein, DNA-protein or DNA-DNA crosslinking (see Fig. 1). Several authors have reported the ability of aldehydes to form covalent crosslinks between DNA and proteins (Feldman, 1977; Chaw et al., 1980; Swenberg et al., 1983; Ma and Harris, 1988). The main targets in these molecules are amino groups, for example in guanosine. (2) a,fl-Unsaturated aldehydes: these will generally be conjugated with glutathione or other thiol-containing molecules. The fl-carbon of the unsaturated aldehydes is a prime target for soft electrophiles like glutathione or cysteine. Several studies on the interactions of this type of aldehyde with glutathione or cysteine have been published (Schauenstein et al., 1977; Esterbauer et al., 1976; Patel et al., 1980; Ohno et al., 1985; ,Alin et al., 1985; Rikans, 1987; Witz, 1989; Mitchell and Petersen, 1989). In principle the reaction is reversible, and the alkylating aldehydes might be released at some other site (Monks et al., 1990). On reaction with cysteine, cyclic thiazolidines will be formed (Schauenstein et al., 1977). From 4-hydroxy-2-alkenals very stable hemiacetals are obtained (Schauenstein et al., 1977; Witz, 1989). Oxidation, when it occurs, seems to take place only after conjugation (Mitchell and Petersen, 1988). Interactions with DNA and proteins have been reported, mainly as the result of reactions with amino groups, especially those from guanosine (Chung et al., 1984; Brambilla et al., 1986; Witz, 1989). (3) Halogenated or otherwise substituted aldehydes: the way in which these aldehydes will be metabolized depends on the character of the other functional groups. Some are oxidized like furfural

(Flek and Sedivec, 1978), benzaldehyde (Laham et al., 1988), malondialdehyde (Sugimoto et al., 1976; Hjelle and Petersen, 1983), while others are mainly conjugated with for instance glutathione or cysteine, as is the case for cinnamaldehyde (Delbressine et al., 1981) and chloroacetaldehyde (Van Duuren, 1975), or with serine, as found for malondialdehyde (Hadley and Draper, 1988). Potent DNA- and protein-binding aldehydes are glutaraldehyde (Schauenstein et al., 1977), malondialdehyde (Brooks and Klamerth, 1968; Moschel and Leonard, 1976) and chloroacetaldehyde (Van Duuren, 1975; Keller and Heck, 1988). Metabolites of chloral (trichloroacetaldehyde) have been found to bind to D N A in vitro (Miller and Guengerich, 1983), but DNA-protein cross-links could not be demonstrated in vivo (Keller and Heck, 1988), which has been suggested to be caused by the strong hydration of chloral. It must be emphasized that aldehydes can be metabolized via the different aforementioned pathways concurrently, while particular pathways may be preferred depending on the structure of the aldehydes. Occurrence in food

More than 300 aldehydes have been identified in over 300 different foods or food components. In general, only qualitative data are available, but for some foods quantitative data have been published (Volatile Compounds in Food, 1989). Table 1 contains a selection of aldehydes identified in foods. An aldehyde is included in the table when: (a) it has been identified in 30 or more foods or food components; (b) it occurs in a very high concentration in one food or a few

367 TABLE 1 A SELECTION O F A L D E H Y D E S I D E N T I F I E D IN F O O D S * Compound

N u m b e r of foods in which the compound has been identified

Maximum concentration measured (ppm)

Food in which m a x i m u m concentration was measured

Remarks * *

Formaldehyde Acetaldehyde Malondialdehyde * * * Butanal Acrolein Crotonaldehyde Cirmamaldehyde Furfural Glycidaldehyde * * * Citral Anisaldehyde Vanillin Propanal 2-Methylpropanal 2-Methylbutanal 3-Methylbutanal Pentanal 2-Pentenal Hexanal 2-Hexenal Heptanal 2-Heptenal 2,4-Heptadienal Octanal 2-Octenal Nonanal 2-Nonenal 2,4-Nonadienal Decanal 2-Decenal 2,4-Decadienal Undecanal Citronellal Benzaldehyde Phenylacetaldehyde

40 150 . 60 35 35 30 150 . 15 10 30 100 80 40 80 100 30 100 80 80 40 50 80 30 80 50 30 30 30 80 30 25 150 80

98 1,060 . 51 3.8 0.7 815,000 255 . 130,000 25,000 23,200 31 14 2 73 14 6 300 76 100 20 15 8,100 100 24,000 410 1.5 22,000 200 500 310 2,000 3,000 14

Shellfish Vinegar

d a

Wheaten bread Red wine Red wine Cassia (bark oil) Coffee

b e a c

Lime, peel oil Anise Vanilla Wheaten bread Whiskey Potato chips Cocoa Red wine Heated butter Orange, peel oil Banana Orange, peel oil Orange, peel oil Heated butter Grape, peel oil Orange, peel oil Orange, peel oil Kumquat, peel oil Heated butter Orange, peel oil Orange, peel oil Tangerine, peel oil Lime, peel oil Orange, peel oil Cinnamon Tea, fermented

a b a b b b b c b b b c c b c b c c a b c b b a b

.

.

.

.

* Data in this table are mainly from Volatile C o m p o u n d s in Foods (1989); for selection criteria see text. * * From Food Chemical News Guide (1990). * * * F o u n d in rancid foods. Generally recognized as safe under par. 182.60 (Synthetic flavoring substances and adjuvants). b Cleared under par. 172.515 (Synthetic flavoring substances and adjuvants). c Deemed to be generally recognized as safe by the Flavor and Extract Manufacturers' Association. d Cleared under par. 176.180 (Components of paper and paperboard in contact with dry, aqueous and fatty foods). e Cleared under par. 172.892 (Food starch-modified).

foods; or (c) it is known to occur in substantial concentrations in rancid foods. Formaldehyde Occurrence

Formaldehyde is a widely produced industrial

chemical (3.5 × 1 0 9 kg/year) and is used, e.g., in medical applications, as a preservative in food, in cosmetics and household cleaning agents. It is present in the environment as a result of natural processes and from man-made sources. In the troposphere it is formed in large quantities by oxidation of hydrocarbons (4 × 1011 kg/year). An

368 important source of indoor formaldehyde intake is cigarette smoke (WHO, 1987). It occurs as a natural constituent in a variety of raw fruits (pears, apples, tomatoes and white radish in amounts varying from 3.7 to 60 ppm) and raw vegetables (cabbage, carrot, green onion, spinach in amounts varying from 3.3 to 26.3 ppm). Formaldehyde has been found in meat (5.7-20 ppm), in milk and milk products (1-3.3 ppm), in fish (8.8-20 ppm) and in shellfish (1-98 ppm). The quantity of formaldehyde ingested via the food is difficult to estimate but may range from 1.5 to 14 rag/person/day. Drinking water contains about 0.1 rag/l, resulting in a daily intake of about 0.2 rag/person (WHO, 1987). In a recent paper Owen et al. (1990) concluded that using a 'worst-case' scenario formaldehyde intake in drinking water would be a factor of about 55 less than the normal dietary intake, and a factor of 70 less than the level of endogenous formaldehyde circulating in the blood of humans (Malorny et al., 1965).

Genotoxicity Formaldehyde has been found to be mutagenic in Salmonella typhimurium (Connor et al., 1983; Haworth et al., 1983; Pool et al., 1984; Schmid et al., 1986) and in Escherichia coil (Wilkins and McLeod, 1976; Takahashi et al., 1985), whereas Gocke et al. (1981), Brusick (1983) and Hughes et al. (1984) reported formaldehyde to be inactive in the Salmonella test. The effects of metabolic activation systems on the mutagenicity of formaldehyde are also inconsistent. Sasaki et al. (1978) and Temcharoen and Thilly (1983) reported reduction, Haworth et al. (1983) and Schmid et al. (1986) enhancement of the mutagenic response. It was shown that by increasing the dose the mutagenic activity was reduced due to cytotoxicity of formaldehyde; moreover, the mutagenic activity appeared to be higher in the presence of an activation system because then formaldehyde is less toxic (Temcharoen and Thilly, 1983; Pool et al., 1984; Schmid et al., 1986). Several mutagenicity studies in animals appeared to be negative (IARC, 1987a), although at lethal doses positive findings have also been reported (Litton, 1982; Brusick, 1983). It is positive in Drosophila (reviewed by Auerbach et al., 1977),

induces SCE in Chinese hamster ovary cells (Obe and Beek, 1979; Brusick, 1983; Natarajan et al., 1983) and chromosome aberrations (Natarajan et al., 1983) and genotoxic effects in a variety of human cell lines (Martin et al., 1978; Obe and Beek, 1979; Grafstrom et al., 1983, 1984, 1985; Schmid et al., 1986). Two studies on occupational exposure to formaldehyde showed negative results (Fleig et al., 1982; Thomson et al., 1984); Bauchinger and Schmid (1985) found a slight increase in dicentrics and Yager et al. (1986) showed increases in SCE. In conclusion, although the genotoxicity data on formaldehyde are conflicting, the compound is considered to possess weak mutagenic activity and is capable of inducing DNA-protein cross-links both in vitro and in vivo, the genotoxic potential becoming manifest principally, if not exclusively, at cytotoxic doses.

Carcinogenicity Following inhalation exposure at levels causing cell damage and hyperproliferative changes in the epithelium of the nasal cavity, formaldehyde has been found to cause nasal cavity tumors (mainly squamous cell carcinomas) in rats (Kerns et al., 1983; Tobe et al., 1989; Sellakumar et al., 1985; Feron et al., 1989) and probably in mice (Kerns et al., 1983) but not in hamsters (Dalbey, 1982). Hexamethylenetetramine (HMT), which gradually decomposes to formaldehyde and ammonia, has been tested for carcinogenicity in several long-term oral studies with rats and mice using dose levels that varied from 0.1 to 5% either in the drinking water or in the diet (Della Porta et al., 1968; JECFA, 1974). These studies produced no or no convincing evidence of carcinogenicity. Takahashi et al. (1986) reported that rats given 0.5% formalin in their drinking water for 32 weeks developed papillomas in the forestomach, and non-neoplastic changes in the glandular stomach. Groups of 70 male and 70 female Wistar rats received formaldehyde in their drinking water at dose levels of 0, 1.2, 15 or 81 (males) and 0, 1.8, 21 or 109 (females) mg/kg b.w./day, for 2 years (corresponding to 20, 60, or 1900 mg/1 for the low-, mid- and high-dose groups, respectively). Papillary epithelial hyperplasia frequently accompanied by hyperkeratosis and focal ulceration in

369 the forestomach was observed in high-dose rats as were chronic atrophic gastritis, ulceration, and hyperplasia in the glandular stomach. The 'no-observed-adverse-effect level' of formaldehyde was 15 mg/kg b.w./day for male rats and 21 mg/kg b.w./day for female rats. No compound-related gastric tumors or tumors at other sites were found (Til et al., 1989). In another study, in which groups of 20 male and 20 female Wistar rats received formaldehyde in the drinking water at doses of 0, 0.02, 0.10 or 0.5% for 2 years, erosions and/or ulcers, hyperplasia and hyperkeratosis were found in the forestomach of high-dose rats. Hyperkeratosis in the forestomach was also found in 2 mid-dose rats. Erosions, ulcers, and submucosal cell infiltration occurred in the glandular stomach of the high-dose rats. No signs of intoxication Were observed at the low dose of 0.02%. There was no significant increase in tumor incidence in any test group compared with controls (Tobe et al., 1989). Soffritti et al. (1989) administered formaldehyde in the drinking water to groups of 50 male and 50 female, 7-week-old, Spragne-Dawley rats at levels varying from 10 to 1500 ppm for a period of 104 weeks. A group of 100 males and 100 females served as controls, and one group of 50 males and 50 females received 15 mg/1 methanol in their drinking water. Moreover, male and female breeders (25 weeks old) of the same strain and their offspring were given drinking water containing 0 or 2500 ppm formaldehyde for 104 weeks, starting the formaldehyde administration at day 12 of pregnancy. An increased incidence of hemolymphoreticular neoplasms (lymphoblastic leukemia, lymphoblastic lymphosarcoma, immunoblastic lymphosarcoma and other types of leukemias and hemolymphoreticular sarcomas) was reported to occur at 500, 1000 and 1500 ppm in comparison with methanol-treated rats, at 50 ppm and higher in comparison with the controls, and in the breeders exposed to 2500 ppm formaldehyde. Moreover, gastrointestinal tumors (adenomas, adenocarcinomas, squamous cell carcinomas, papillomas, acanthomas, leiomyomas and leiomyosarcomas) were found in breeders and their offspring exposed to 2500 ppm formaldehyde, and in the groups receiving 10, 50, 1000 or 1500 ppm, but not in controls, methanol-treated rats or rats given 100 or 500 ppm formaldehyde. In addition to the

striking distribution of the gastrointestinal tumors over the various groups, the strong heterogeneity of both the gastrointestinal and the hemolymphoreticular neoplasms was remarkable. It is also striking that, apart from gastric papillomas, none of the tumor types reported has been observed in any of the other long-term oral or inhalation studies with formaldehyde. In summary, there is clear evidence that formaldehyde is carcinogenic in several animal species, producing carcinomas of the nasal respiratory epithelium in rats and probably also in mice after inhalation exposure to high, cytotoxic concentrations. In 3 long-term oral studies in rats administration of formaldehyde caused severe damage to the gastric mucosa including papillary epithelial hyperplasia, but did not result in gastric or other tumors, apart from a few (benign) forestomach papillomas reported in only one of the 3 studies. In a recent paper on a long-term oral study in Sprague-Dawley rats, an increased incidence of hemolymphoreticular tumors and tumors of the gastrointestinal tract was reported in formaldehyde-treated rats. Since, however, crucial information on procedures and histopathology of non-neoplastic changes is lacking, the adequacy of this study and the relevance of the data can hardly be judged, if at all.

R i s k assessment

Formaldehyde is a highly irritating, directacting genotoxic carcinogen capable of inducing DNA-protein cross-links and squarnous cell carcinomas of the nasal respiratory epithelium in rats (Ma and Harris, 1988). The squamous cell carcinomas were observed only at exposure levels that also caused severe damage to the nasal respiratory mucosa (Swenberg et al., 1983; Woutersen et al., 1989). Moreover, from analysis of the dose-response relationship and the relationship between non-neoplastic and neoplastic lesions in the respiratory epithelium it may be concluded that the dose-response curve for nasal tumors, which appears to be very steep and non-linear, was nearly identical to those for cell turn-over, DNA-protein cross-links and hyperproliferation, indicating an association between the cytotoxic and carcinogenic effects. It has been shown that in

370

rats recurrent tissue damage and repair must be accompanied by exposure to high, cytotoxic concentrations of formaldehyde in order to induce nasal carcinomas (Feron and Woutersen, 1989; Woutersen et al., 1989). Correspondingly, if the nasal tissue is not sufficiently injured, exposure to relatively low, non-cytotoxic levels of formaldehyde can be assumed to represent a negligible cancer risk. Therefore, human exposure to formaldehyde vapor should be minimized for its potency to damage nasal tissue (Feron and Woutersen, 1989; Feron et al., 1989). In addition to severe damage to the gastric mucosa including hyperkeratosis and papillary hyperplasia in the forestomach, oral administration of a high dose of formaldehyde to rats has been shown to induce DNA-protein cross-links in the forestomach epithelium (Minini, 1985). Therefore, analogous to what happens in the nose, prolonged oral administration of high, cytotoxic doses of formaldehyde to rats may result in gastric tumors. Indeed, 2 of the chronic oral studies with formaldehyde in rats revealed gastric tumors (Takahashi et al., 1986; Soffritti et al., 1989). However, in 2 other long-term oral studies with formaldehyde in rats, no gastric tumors were found (Til et al., 1989; Tobe et al., 1989). Moreover, oral administration of hexamethylenetetramine did not result in gastric tumors (Della Porta et al., 1968; Natvig et al., cited by JECFA, 1974). These conflicting results cannot be ascribed to differences in dose levels because these were very similar in the various studies. Different criteria for the classification of a gastric lesion as papilloma or papillary hyperplasia may explain the difference between the papillomas reported by Takahashi et al. (1986) and the papillary hyperplasia described by Til et al. (1989) and Tobe et al. (1989). Clearly, this difference in classification is not valid for the adenocarcinomas, leiomyomas and leiomyosarcomas reported by Soffritti et al. (1989), but the relationship of these tumors to formaldehyde ingestion is at least questionable. Soffritti et al. (1989) used Sprague-Dawley rats, whereas in the other studies Wistar rats were used. Maybe Sprague-Dawley rats are more sensitive to the carcinogenic properties of formaldehyde than Wistar rats. Soffritti et al. (1989) also reported an increased number of hemolymphoreticular tumors

in formaldehyde-treated rats. This is the only formaldehyde study in which compound-related tumors at sites remote from the port of entry have been observed. Moreover, in nasal carcinogenesis the cytotoxicity of formaldehyde is thought to play a highly significant role. Since Soffritti et al. (1989) did not report any toxic effects on the hemolymphoreticular tissue, for the time being it seems justifiable to consider the hemolymphoreticular tumors unrelated to formaldehyde and to ignore them in risk assessment. Although there is no convincing evidence to suggest that formaldehyde is a gastric carcinogen, drawing a parallel between the nasal and gastric toxicity of formaldehyde unavoidably leads to the conclusion that prolonged oral intake of huge cytotoxic doses of formaldehyde may constitute a gastric cancer risk factor. In this light it seems legitimate to assess the possible gastric cancer risk of oral formaldehyde intake for humans. Formaldehyde occurs in a large number of foods and drinks and its maximum oral intake is estimated to be 14.2 m g / p e r s o n / d a y . The 'no-toxiceffect level' in rats was found to be 15 m g / k g b.w./day, which is equivalent to 1050 m g / d a y for a person of 70 kg, indicating a safety margin of at least a factor 74 (1050 : 14.2). However, the effects of formaldehyde on the stomach may be more dependent on the concentration of formaldehyde in foods than on the total dose ingested. Formaldehyde concentrations of 5-10 m g / k g product are often found, but concentrations of up to 50 or 60 m g / k g food or higher are very exceptional and have been found only in special products such as shellfish. Ignoring the inveracious results of one of the chronic oral studies (Soffritti et al., 1989), the toxicity data demonstrate the absence of any gastric effect at a drinking water concentration of 260 mg/1, and only minor hyperkeratosis in the forestomach of a few rats at a level of about 800 mg/1. Thus, also with respect to the concentration of formaldehyde in foods there is a safety factor of at least 5 for some special products and of 25-50 for the more common formaldehyde-containing foods. The overall conclusion is that the occurrence of formaldehyde in foods, beverages and drinking water is low enough to avoid cytotoxic effects on the gastric mucosa, and, therefore, does not constitute a cancer risk factor in humans.

371

Acetaldehyde Occurrence Acetaldehyde has been identified as a natural constituent of many fruits (e.g., apples, apricots, bananas, cherries, citrus fruits, wild berries, currants, grapes, melons, papayas, peaches, pears, pineapples, raspberries, blackberries and strawberries in amounts varying from 0.0005 to 230 ppm), vegetables (such as cabbage, carrot, celery, cucumber, lettuce, onion, peas, potatoes, rutabaga, tomatoes in amounts varying from 0.2 to 400 ppm), alcoholic beverages (0.6-104 ppm), bread (0.1-96 ppm), cheese, eggs, fish, meat, hops, aniseed, ginger, mint, vinegar, parsley, cocoa, coffee, tea (0.2-0.6 ppm), nuts and other foods (Volatile Compounds in Food, 1989). Acetaldehyde is also used as an important component of many flavors added to food products, such as milk products (fruit yoghurt), baked foods, fruit juices, candies, desserts, soft drinks and margarine. The acetaldehyde concentration in 18 European beers was reported to be in the range of 2.6-13.5 ppm. It has been detected in commercial wine samples in Japan at levels of 0.2-1.2 ppm. In vinegar a concentration of 1060 ppm has been detected (Volatile Compounds in Food, 1989). Acetaldehyde is 'generally recognized as safe' by the FDA (1988b) for its intended use as a synthetic flavoring substance and adjuvant. Acetaldehyde has been detected in mother's milk in the U.S.A. (Pellizzari et al., 1987). It is formed during intracellular oxidation of ethanol. Genotoxicity Acetaldehyde has been shown to induce chromosomal aberrations, micronuclei a n d / o r SCE in cultured mammalian cells including human lymphocytes (Obe and Beck, 1979; Bird et al., 1982; BiShlke et al., 1983; Jansson, 1982; de Raat et al., 1983; He and Lambert, 1985; Norppa et al., 1985). Available evidence suggests that acetaldehyde produces similar cytogenetic effects in vivo (Dellarco, 1988). The production of cytogenetic effects may be related to the ability of acetaldehyde to form DNA-DNA a n d / o r DNA-protein cross-links (Riskow and Obe, 1978; Lambert et al., 1985; Minini, 1985; Lam et al., 1986; Dellarco, 1988). Acetaldehyde has been found to produce gene

mutations (sex-linked recessive lethals) in Drosophila (Woodruff et al., 1985), but most tests in bacteria have been negative and no information is available on the ability of acetaldehyde to induce gene mutations in cultured mammalian cells (Dellarco, 1988). It can be concluded that acetaldehyde is a genotoxic cross-linking agent with limited or no ability to induce gene mutations in bacteria and mammalian cells.

Carcinogenicity Inhalation studies in hamsters have shown that acetaldehyde at concentrations of 2750 m g / m 3 and higher is capable of inducing laryngeal and nasal carcinomas accompanied by inflammatory changes and severe hyper- and meta-plasia of the epithelium in the upper segments of the respiratory tract (Kruysse et al., 1975; Feron, 1979; Feron et at., 1982). Also in rats the nose and larynx appeared to be the main target sites of acetaldehyde vapor (Appelman et al., 1982). From a 28month carcinogenicity study in rats, using exposure levels of 0, 1375, 2750 and 5500/1830 m g / m 3, it was clear that acetaldehyde vapor produced nasal carcinomas at each exposure level tested (Woutersen et al., 1984, 1986). It was emphasized that at each exposure concentration, in addition to nasal tumors, degenerative hyperplastic and metaplastic changes of the nasal mucosa were also observed. The results of a recovery study suggested that non-neoplastic, hyperproliferative changes of the nasal epithelium seen at the end of the exposure period (52 weeks) may progress to malignant tumors despite discontinuation of treatment (Woutersen and Feron, 1987). In conclusion, there is sufficient evidence that acetaldehyde is carcinogenic to hamsters and rats, producing tumors of the laryngeal and nasal epithelium after inhalation exposure. There are only limited data on the oral toxicity of acetaldehyde. In a 4-week drinking-water study in rats, using dose levels of 0, 25, 125 and 625 mg acetaldehyde/kg b.w./day, hyperkeratosis of the forestomach epithelium was found in the highestdose group, pointing to an irritating effect of acetaldehyde on the forestomach (Til et al., 1988). In this study the 'no-observed-adverse-effect level' appeared to be 125 mg/kg b.w./day. No data are

372 available on the chronic oral toxicity of acetaldehyde. R i s k assessment

The distribution of nasal lesions induced by acetaldehyde correlated with regional acetaldehyde dehydrogenase deficiencies, suggesting that regional susceptibility to the toxic effects of acetaldehyde may be due, at least in part, to a lack of aldehyde dehydrogenase in the susceptible regions (Bogdanffy et al., 1986). To understand the mechanism of nasal carcinogenesis by acetaldehyde, Lam et al. (1986) carried out in vitro and in vivo studies to determine whether acetaldehyde can react with D N A in target tissues of the rat nasal mucosa. The results clearly indicate that acetaldehyde can form DNA-protein cross-links in the olfactory mucosa both in vitro and in vivo. Moreover, acetaldehyde appeared positive in a series of 'genotoxicity tests' (Dellarco, 1988). Therefore, it is reasonable to assume that acetaldehyde possesses initiating activity, which played a crucial role in the induction of nasal and laryngeal tumors in rodents. In fact, acetaldehyde meets the widely accepted criteria for a complete carcinogen. Since, in analogy to formaldehyde (Woutersen et al., 1986), acetaldehyde also may react preferentially with single-stranded D N A and the incidence of dividing cells in the normal intact nasal epithelium is low, it seems conceivable that the initiating potential of acetaldehyde in concentrations not leading to cell damage is very low, whereas concentrations of acetaldehyde causing recurrent tissue damage may be very effective with respect to initiation. Thus, indeed the cytotoxic effects of acetaldehyde may be very important for the induction of nasal carcinomas in experimental animals. However, in contrast to formaldehyde, acetaldehyde has not been tested for carcinogenicity at a cytotoxic exposure concentration not leading to (nasal) tumors (the lowest concentration tested, 1375 m g / m 3, is cytotoxic and was found to induce carcinomas of the nasal olfactory epithelium), and more importantly, has not been tested for carcinogenicity at a non-cytotoxic exposure concentration. As a consequence it cannot be excluded that acetaldehyde at non-cytotoxic concentrations is capable of inducing nasal cancer in rats.

In summary, acetaldehyde is an irritating genotoxic carcinogen, of which the cytotoxicity probably but not certainly plays a crucial role in its carcinogenicity. As long as the role of the cytotoxicity in the carcinogenicity of acetaldehyde has not been demonstrated more clearly, the genotoxicity rather than the cytotoxicity should be considered the determining factor in cancer risk assessment. Information on the chronic oral toxicity of acetaldehyde including carcinogenicity is badly needed, because (a) acetaldehyde occurs in a large number of foods and food products (Volatile Compounds in Food, 1989), (b) has been shown to induce hyperkeratosis in the forestomach of rats (Til et al., 1988) and DNA-protein cross-links in the rat stomach (Minini, 1985), and (c) data on its carcinogenic potential after oral administration are lacking. W i t h o u t f u r t h e r i n f o r m a t i o n acetaldehyde should be considered a potential dietary cancer risk factor for humans. Consequently, for the time being oral exposure of humans to acetaldehyde should be diminished as far as possible. Clearly, this conclusion is at variance with the 'generally recognized as safe' status (FDA, 1988b) and may have far-reaching consequences for the use of acetaldehyde as a food additive.

Malondialdehyde Occurrence

Malondialdehyde (propanedial) occurs in many foodstuffs and is often present in high concentrations in rancid foods. It has been detected in fish meat, fish oil, rancid salmon oil, rancid nuts, rancid flour, orange juice essence, vegetable oils, fats, fresh frozen green beans, milk, milk fat, rye bread, and in raw, cured and cooked meats (Apaja, 1980; Newburg and Concon, 1980). Concentrations of free malondialdehyde in commercial samples of refined groundnut oils ranged from 0.04 to 0.14 ppm, and the level in sunflower oil was 0.08 ppm. The total malondialdehyde concentrations were 0.53 ppm in groundnut oils and 0.98 ppm in sunflower oil (Arya and Nirmala, 1971). Increased levels of malondialdehyde have been found in hamburger, chicken and beef as a result of cooking under a variety of conditions (e.g., microwave, frying, baking and boiling) (Newburg and Con-

373 con, 1980). Among several meats, beef had the highest content of malondialdehyde (up to 13.7 ppm in sirloin steak). Turkey and cooked chicken also had high levels, but most cheeses, pork and fish contained only small amounts, and many vegetables, fruits and canned foods had either minute quantities or no malondialdehyde at all (Shamberger et al., 1977).

either conflicting or are based on inadequate studies, and may be compromised due to the use of impure test substance. Since malondialdehyde occurs in so many foodstuffs and is often present in relatively high concentrations in rancid foods, relevant genotoxicity tests and long-term oral studies in rats and mice, using highly purified test material, are clearly indicated.

Genotoxicity, carcinogenicity and risk assessment Highly purified malondialdehyde is weakly mutagenic to Salmonella typhimurium TA102, TA104, TA2638 and his D3052 (Levin et al., 1982; Basu and Marnett, 1983; Marnett et al., 1985), and also to Escherichia coli (Yonei and Furui, 1981). Negative results were reported in Salmonella typhimurium TA1535, TA1537 and TA1538 (Mukai and Goldstein, 1976; Marnett and Tuttle, 1980). The activity of malondialdehyde in strain his D3052 as well as other Salmonella typhimurium strains (Mukai and Goldstein, 1976; Shamberger et al., 1979) may be attributable to mutagenic impurities (Marnett and Tuttle, 1980). Malondialdehyde induced somatic mutations, but no sexlinked recessive lethal mutations, in Drosophila melanogaster (Szabad et al., 1983). In cultured rat skin fibroblasts, the compound induced micronuclei, chromosomal aberrations and aneuploidies (Bird and Draper, 1980; Bird et al., 1982). Sodium malondialdehyde and malondialdehyde bis(dimethylacetal) were given to mice in drinking water for 12 months or for life. However, these studies were inadequate for evaluation due to either the short duration of the study or the high mortality and lack of untreated controls (IARC, 1985). After topical application, no increase in the incidence of skin tumors occurred in a study by Fischer et al. (1983). In a 2-stage mouse skin assay, a high dose of malondialdehyde (possibly containing impurities) showed initiating activity (Shamberger et al., 1974, 1975). In 2 other 2-stage assays using lower doses of malondialdehyde, no initiating or promoting activity was observed (Fischer et al., 1983). Based on these data, IARC (1985) concluded that there is inadequate evidence for the carcinogenicity of malondialdehyde in experimental animals. Genotoxicity and carcinogenicity data are

Acrolein

Occurrence Acrolein (2-propenal) occurs in a wide variety of fruits (apples, grapes, raspberries, strawberries, brambles in amounts varying from < 0.01 to 0.05 ppm), vegetables (cabbage, carrots, potatoes, tomatoes in amounts up to 0.59 ppm) and beverages (beer, wine, coffee, tea, rum, whiskey, brandy, cognac, in red wine levels up to 3.8 ppm have been found), and also in cheese, caviar, lamb, and hops (Volatile Compounds in Food, 1989). Greenhoff and Wheeler (1981) detected acrolein in fresh lager beer at levels of 0.0011-0.002 ppm. It has also been detected in souring salted pork (Cantoni et al., 1969), the fish odor of cooked horse mackerel (Shimomura et al., 1971), the aroma volatiles of white bread (Mulders and Dhont, 1972) and the aroma volatiles of raw chicken breast muscle (Grey and Shrimpton, 1967). Furthermore, acrolein has been found among the products from heating animal fats or vegetable oils (Bauer et al., 1977; Izard and Libermann, 1978). It was also detected among the decomposition products of cellophane used in sealing meat packages (Robles, 1968). Genotoxicity and carcinogenicity Acrolein was muta'genic to Salmonella typhimurium TA104 without an exogenous metabolic system (Marnett et al., 1985). Both positive and negative results have been reported in strains TA98 and TA100 under various test conditions (Florin et al., 1980; Lijinsky and Andrews, 1980; Loquet et al., 1981; Haworth et al., 1983). A weak mutagenic response was reported in strain TA1535 in the presence of a hepatic microsomal fraction from phenobarbital-induced rats (Hales, 1982). A slight mutagenic effect was reported with Escherichia coli WP2 uvrA (Hemminki et al., 1980). Acrolein did not cause DNA cross-links or

374

D N A breaks in Saccharomyces cerevisiae (Fleer and Brendel, 1982). It was reported to induce SCE in Chinese hamster ovary cells (Au et al., 1980), and D N A single-strand breaks and DNA-protein cross-links in human bronchial epithelial cells (Grafstrom et al., 1986, 1988). Acrolein appeared to be negative in the dominant lethal test in male mice (Epstein et al., 1972). It was not mutagenic to normal human fibroblasts, but strongly mutagenic to cells from xeroderma pigmentosum patients (Curren et al., 1988). Syrian hamsters exposed to 4.0 ppm acrolein vapor for 52 weeks (7 h / d a y , 5 days/week) showed inflammatory changes and metaplasia of the olfactory epithelium of the nose (Feron and Kruysse, 1977). After a withdrawal period of 6 months the affected mucosa had partially recovered in most animals. Treatment-related nasal tumors or tumors at other sites were not encountered. Very similar nasal effects have also been observed in rats and rabbits exposed to acrolein vapor for 13 weeks (Feron et al., 1978; Kutzman et al., 1981). In a long-term (100 weeks) study in which F344 rats were given drinking water containing 0-625 ppm acrolein, 5 out of 20 female animals given 625 ppm acrolein developed adrenal cortical adenomas and 2 out of 20 had neoplastic nodules in the adrenal cortex as compared to none in female and 1 in male controls (Lijinsky, 1984). In summary, there is no convincing evidence that acrolein possesses genotoxic or carcinogenic activity. Risk assessment Although the design and conduct of the 78-week inhalation study in hamsters (Feron and Kruysse, 1977) and the 100-week drinking-water study in rats (Lijinsky, 1984) do not meet present-day standards for long-term carcinogenicity studies in rodents, these studies allow the conclusion that acrolein is not a potent carcinogen. Since, in addition, the genotoxicity tests produced equivocal results, it seems reasonable to conclude that acrolein in foods and drinks is not a major cancer risk factor. On the other hand, human exposure to this very irritating and strongly cytotoxic agent should be low enough to avoid tissue damage. In this way any possible cancer risk would also be minimized.

Crotonaidehyde Occurrence Crotonaldehyde (2-butenal) is an a, fl-unsaturated carbonyl compound and a homologue of acrolein. It is used in the manufacturing of nbutanol and sorbic acid, in the production of flavors and in numerous other applications. Crotonaldehyde naturally occurs in many fruits (e.g., apples, guavas, grapes, strawberries, tomatoes in amounts < 0.01 ppm), vegetables (e.g., cabbage, carrots, celery leaves, cauliflower, Brussels sprouts in amounts varying from 0.02 to 0.1 ppm), bread, cheese, milk, meat, fish, beer (0-0.04 ppm), wine (0-0.7 ppm) and chips (Volatile Compounds in Food, 1989). Crotonaldehyde occurs as a contaminant in drinking water (Simmon et al., 1977) and has also been found in cigarette smoke at relatively high concentrations (Wynder et al., 1965). Genotoxicity, carcinogenicity and risk assessment Crotonaldehyde has been shown to modify deoxyguanosine under physiological conditions without metabolic activation (Chung and Hecht, 1983; Chung et al., 1984). In agreement with its alkylating activity it is mutagenic without metabolic activation (Lutz et al., 1982; Marnett et al., 1985). Crotonaldehyde is irritating to the respiratory system and causes damage to the lung (Skog, 1950). Oral administration of crotonaldehyde by gavage at doses of 0, 2.5, 5, 10, 20 and 40 m g / k g to F344 rats and B6C3F 1 mice for 13 weeks resulted in hyperplasia of the forestomach epithelium (at 20 and 40 m g / k g in rats) and in forestomach inflammation at 40 m g / k g in r~fice and at 10 m g / k g and above in rats. Forestomach hyperkeratosis, ulcers and necrosis were also seen in rats at 40 mg/kg. Acute inflammation in the nasal cavity was observed in rats at 5 m g / k g and higher (Wolfe et al., 1987). Crotonaldehyde administered in the drinking water at levels of 42 or 420 rag/1 to groups of 23-27 male F344 rats for 113 weeks caused liver tumors (Chung et al., 1986). At 42 mg/1 hepatocellular neoplasms (hepatocellular carcinomas in 2 rats and neoplastic nodules in 9 rats) and altered liver cell foci were observed. At 420 mg/1 hepato-

375 cellular neoplasms were observed in only I animal; however, moderate to severe liver damage (fatty metamorphosis, focal necrosis, fibrosis, cholestasis and mononuclear cell infiltration) but no preneoplastic or neoplastic lesions were seen in 10 out of 23 high-dose rats. The remaining 13 rats in this group showed altered liver cell loci, and 1 of these animals also had a preneoplastic liver nodule. Furthermore, in the 42-mg/1 group one papilloma of the forestomach and in the 420-mg/1 group one adenocarcinoma of the glandular stomach and 2 bladder tumors were observed, whereas such tumors were not seen in controls. In summary, there is evidence that crotonaldehyde possesses genotoxic activity; it is irritant to the respiratory tract of rats and to the stomach of rats and mice and hepatotoxic and carcinogenic to rats, producing in this species a variety of nonneoplastic and neoplastic liver lesions including hepatocellular carcinomas, and possibly also tumors in other organs. Since crotonaldehyde is a very common natural food compound and is used in many widely varying applications, long-term oral (and inhalation) carcinogenicity studies are recommended to verify the results of the study by Chung et al. (1986). In view of its genotoxic potential and hepatocarcinogenic effects in rats, pending further information on the carcinogenicity of crotonaldehyde, it should be regarded as a potential oral carcinogen and thus a dietary cancer risk factor for humans. For the time being the available data do not allow a quantitative risk assessment.

Genotoxicity, carcinogenicity and risk assessment Cinnamaldehyde has been found to be mutagenic to Salmonella typhimurium strain TA100 when tested in the presence or absence of a metabolic activation system. It also induced chromosomal aberrations in Chinese hamster fibroblasts (Ishidate et al., 1984). Hyperkeratosis of the forestomach has been observed in rats fed a diet containing 1% cinnamaldehyde for a period of 16 weeks (Hagan et al., 1967). No data were found on the carcinogenic potential of cinnamaldehyde. However, its structural similarity to crotonaldehyde and acrolein, resulting in very similar metabolic pathways (Delbressine et al., 1981), renders it likely that the adverse effects will also be similar. This assumption in combination with the wide use of cinnamon oil in foods, often in high concentrations, indicates the necessity of further genotoxicity tests, and in particular adequate long-term oral studies in rodents.

Cinnamaldehyde

Genotoxicity, carcinogenicity and risk assessment Benzaldehyde was negative in Salmonella typhimurium both with and without metabolic activation (Sasaki and Endo, 1978; Florin et al., 1980; Kasamaki et al., 1982; Haworth et al., 1983; Nohmi et al., 1985). Benzaldehyde caused increases in SCE in mammalian cells in culture with and without activation (Galloway et al., 1987) and in human lymphocytes (Jansson et al., 1988). Chromosomal damage was observed in Chinese hamster cells by Kasamaki et al. (1982) and Sofuni et al. (1985) but not by Galloway et al. (1987). Benzaldehyde given in doses of 400 or 800 m g / k g b.w. by stomach tube on 5 days/week for 13 weeks caused mild epithelial hyperplasia and

Occurrence Cinnamaldehyde is the main ingredient (up to 90%) of cinnamon oil (Mantovani et al., 1989). It is a powerful flavoring agent with wide use in beverages, ice-creams, confectionery, baked foods, chewing gums, condiments and meat preparations at concentrations ranging from 8 ppm (ice-creams) to 700 ppm (sweets) (Mantovani et al., 1989). Cinnamaldehyde has also been identified in the essential oils of several plants (e.g., myrrh, hyacinth)-that are used in perfumes for the manufacture of household products, detergents, soaps, medicinal products and liquors.

Benzaldehyde Occurrence Benzaldehyde has been identified in more than 150 of 300 food products examined. It naturally occurs in alcoholic beverages (0.01-0.8 ppm), dairy products, meat (0.03-0.13 ppm), poultry, fruits (in amounts varying from 0.0003 to 8.9 ppm), vegetables at levels up to 1.2 ppm, coffee, tea, cocoa at levels varying from 0.7 to 7.4 ppm and spices. A concentration as high as 3000 ppm has been found in cinnamon (Volatile Compounds in Food, 1989).

376 keratosis in the forestomach of rats, and also damage to the brain and kidneys at 800 m g / k g b.w. No forestomach lesions were observed at doses of 200 m g / k g b.w. or lower. In mice, benzaldehyde at doses of 600 or 1200 m g / k g b.w. (5 days/week for 13 weeks) induced renal tubular necrosis, but not at doses of 300 m g / k g or lower (Kluwe et al., 1983). There were no treatment-related changes in gross and microscopic pathology of the major tissues in groups of 5 male and 5 female rats fed approximately 50 or 500 m g / k g b . w . / d a y for 26-28 weeks, or 16 weeks, respectively (Hagan et al., 1967). Carcinogenicity studies with benzaldehyde are presently in progress. Doses of 200 or 400 m g / k g b.w. are given by stomach tube to rats and male mice, and doses of 300 or 600 m g / k g b.w. to female mice (NTP, 1988). Preliminary results indicate the absence of treatment-related tumors of the forestomach in mice (Federal Register, 1988). In summary, benzaldehyde is negative in the Ames test but may damage chromosomes of mammalian cells in vitro. In rat, it is irritant to the forestomach and, in high doses, also neuro- and nephro-toxic after oral administration by gavage. Although the preliminary data of long-term gavage studies in rats and mice do not indicate carcinogenicity, it is prudent to await the final results before any judgment on the carcinogenic potential and the cancer risk is made. Fudural

Occurrence Furfural (a heterocyclic aldehyde) has a pungent, aromatic odor reminiscent of almonds when freshly distilled. It is a colorless oily liquid which is used in large quantities as an industrial solvent; it has a wide variety of other uses such as chemical intermediate, ingredient of phenolic resins, weed killer, fungicide and flavoring agent (Kirk-Othmer, 1984). Furfural as a natural volatile compound has been identified in many foods such as fruits (apples, apricots, cherries, citrus fruits, berries, grapes, etc. at levels up to 0.34 ppm), vegetables (carrots, cabbage, onions, potatoes at levels up to 0.01 ppm), alcoholic beverages (beer, brandies, rum, whiskey, wine at levels up to 67 ppm), coffee at levels up to 255 ppm and bread and bread prod-

ucts (at levels up to 14 ppm) (Volatile Compounds in Food, 1989).

Genotoxicity No mutagenic activity of furfural appeared from the bacterial repair test using Escherichia coli, the prophage induction test and the chloroplast bleaching test using Euglena gracilis (Soska et al., 1981). Moreover, furfural did not produce reciprocal translocations in Drosophila (Woodruff, 1985) and unscheduled D N A synthesis in nasal epithelium (Wilmer et al., 1987). Furfural administered by intraperitoneal injection was negative in tests for induction of SCE and chromosomal aberrations in mouse bone marrow cells (Anonymous, 1989). However, mutagenicity of furfural was measured in the Salmonella test but only with tester strain TA100 and at a dose close to the toxic dose (Zdzienicka et al., 1978; Loquet et al., 1981; Mortelmans et al., 1986). In addition, furfural was mutagenic in the rec-assay with Bacillus subtilis (Shinohara, 1986), caused sex-linked recessive lethal mutants in Drosophila (Woodruff, 1985), induced chromosome aberrations in cultured CHO cells (Stich et al., 1981), was positive in the mouse lymphoma assay (McGregor et al., 1988), caused spindle fiber damage in cultured human blood cells (Gomez-Arroyo and Souza, 1985), and produced breaks in calf thymus duplex D N A (Hadi et al., 1989). Reynolds et al. (1987) found activated ras and raf oncogenes in mouse liver tumors obtained from animals exposed to furfural. In conclusion, although furfural exhibits a somewhat inconsistent pattern of genotoxic activity it is considered a genotoxic compound capable of inducing gene mutations in certain bacteria and in Drosophila, chromosome damage in cultured mammalian cells but not in mouse bone marrow cells, breaks in double-stranded DNA, and presumably oncogene activation in mouse liver. Carcinogenicity Subchronic and chronic inhalation exposure of Syrian golden hamsters to furfural vapor at concentrations ranging from 448 to 2165 m g / m 3 resuited in damage to the nasal olfactory mucosa; the changes were characterized by atrophy of the epithelium, degeneration of Bowman's glands, downward growth of sensory cells and cyst-like

377 structures in the lamina propria (Feron and Kruysse, 1978; Feron et al., 1979). No compound-related nasal tumors or tumors at other sites were detected. Also a 78-week study in hamsters receiving 36 weekly intratracheal instillations of 3 mg furfural did not produce any evidence of furfural possessing carcinogenic activity (Feron, 1972). As regards other species, rabbits exposed to comparable concentrations of furfural vapor (500 or 1000 m g / m 3) for periods of at most 4 weeks (4 h/day, 5 days/week) showed pathological changes in liver and kidneys, viz., hepatic degeneration, necrosis and fibrosis, and tubular nephropathy (Castellino et al., 1963). Male rats fed diets containing 20-40 ml furfural/kg diet for 90-120 days developed cirrhotic livers (Shimizu and Kanisawa, 1986); 180 days after discontinuation of the furfural feeding cirrhotic changes were still visible, but no (pre)cancerous changes were observed. In 13-week studies with F344/N rats and B6C3F1 mice furfural was administered in corn oil by gavage at doses ranging from 11 to 180 mg/kg b.w. for rats and from 75 to 1200 mg/kg for mice (Anonymous, 1989). Liver and kidney lesions were found at all dose levels. In a subsequent 2-year study using doses of 0, 30 and 60 mg/kg for rats, and 0, 50, 100 and 175 mg/kg for mice, mild liver toxicity occurred at increased incidences in all furfural-treated groups of mice and rats. Bile duct dysplasia was observed in 2 out of 50 male highest-dose rats and cholangiocarcinoma in 2 other males of this group, whereas such lesions were not found in controls or low-dose rats and the historical incidence of bile duct neoplasms in oil vehicle control male rats is 3 out of 2145 (0.1%). The incidences of hepatocellular adenomas and carcinomas in male mice and hepatocellular adenomas in female mice were significantly increased in the highest-dose group compared to those in the vehicle controls. In addition, there was some evidence of a higher incidence of renal cortical adenomas/carcinomas in furfural-exposed male mice, and of forestomach papillomas in highest-dose female mice (Anonymous, 1989). In conclusion, inhalation and intratracheal instillation studies in hamsters produced no evidence of furfural possessing carcinogenic activity. Two-year gavage studies in rats and mice pro-

duced clear evidence of carcinogenicity of furfural in male mice and some evidence of carcinogenicity in male rats and female mice. Risk assessment In both the short-term and 2-year oral studies with furfural in rats and mice, the liver appeared to be the target organ and indeed exhibited the greatest susceptibility to the test compound (Shimizu and Kanisawa, 1986; Anonymous, 1989). At the doses used in the 2-year studies, mild furfural-related liver toxicity was observed (centrilobular necrosis in male rats, and slight multifocal chronic inflammation and pigmentation in dosed male and female mice). These results and the tumor responses in mice and rats indicate the adequacy of the doses used in the 2-year studies for evaluating the carcinogenic potential of furfural without producing significant toxic injury in the primary target organ (Anonymous, 1989). Although the genotoxic activity of furfural is somewhat inconsistent and the overall evidence of carcinogenic activity after oral administration is limited, it seems justifiable to consider furfural an oral genotoxic carcinogen of low potency, the use of which in foods should be discouraged and reduced where possible.

Glycidaldehyde Occurrence Glycidaldehyde (2,3-epoxypropanal) occurs in sunflower oil, and its concentration increases with increasing periods of storage. Glycidaldehyde has also been detected in rancid samples of commercial lard (Woller and Nagy, 1968). It has not been identified in normal foods and food compounds. Genotoxicity, carcinogenicity and risk assessment Exposure of bacteriophage T4 to a 300-fold dilution of glycidaldehyde induced mutations by producing base-pair transitions, frameshifts and some deletions (Corbett et al., 1970). Glycidaldehyde has also produced base-pair mutations in 2 strains of Salmonella typhimurium (McCann et al., 1975). A 1% solution in ethanol induced reverse base-pair mutations in Saccharomyces cerevisiae strain $211; a 2-4% solution induced cytoplasmic

378 mutations in strain N123 of the same organism (Izard, 1973). IARC (1987b) has classified glycidaldehyde as an animal carcinogen since it appeared to be carcinogenic in mice by skin application (Van Duuren et al., 1965, 1967; Shamberger et al., 1974) and by subcutaneous injection (Van Duuren et al., 1966) and in rats by subcutaneous injection (Van Duuren et al., 1966, 1967). To the best of our knowledge no data have been reported on the carcinogenic potential of glycidaldehyde after oral administration. In summary, glycidaldehyde is mutagenic in bacteria and yeast, and carcinogenic to mice and rats after subcutaneous injection and to mice also after skin application. Since oral carcinogenicity data are lacking it is difficult to estimate whether the consumption of oils and rancid foods containing glycidaldehyde brings on any cancer risk. Since glycidaldehyde has not been found in normal foods information on the oral carcinogenicity would be needed only when prevention of its formation in stored foods or food compounds is impracticable.

Anisaldehyde Occurrence Anisaldehyde (4-methoxybenzaldehyde) has been identified in several food products such as fruits (apricots, cranberries, black currants, cloudberries, tomatoes at levels up to 0.1 ppm), anise, alcoholic beverages (beer, grape brandy, sherry, anise brandy), tea, basil and vanilla. In anise a concentration up to 25,000 ppm has been found (Volatile Compounds in Food, 1989). Genotoxicity, carcinogenicity and risk assessment In an oral toxicity study rats were fed diets containing 10,000 ppm for 15 weeks or 1000 ppm for 27-28 weeks. No deleterious effects were found (Hagan et al., 1967). Since no data were found on the genotoxic or carcinogenic potential of anisaldehyde, some relevant genotoxicity testing is deemed desirable, especially because some anisebased specialities may contain high concentrations of this aldehyde.

Vanillin Citral Occurrence Citral (3,7-dimethyl-2,7-octadienal), neral (ycitral) a n d / o r geranial (fl-citral) have been identified in over 30 food products such as fruits, especially citrus fruits, spices (at levels up to 13,500 ppm in ginger), and tea. The highest concentration (130,000 ppm) has been found in lemon peel oil (Volatile Compounds in Food, 1989). Citral is listed as an adjuvant and is employed as a natural or artificial flavor in brewing at levels that are kept confidential, and is deemed to be generally recognized as safe for this purpose (Food Chemicals News Guide, 1990). Genotoxicity, carcinogenicity and risk assessment In both an Ames and a chromosome aberration test citral showed no mutagenic activity (Ishidate et al., 1984). In a 13-week oral toxicity study with rats 1000, 2500 or 10,000 ppm citral showed no deleterious effect (Hagan et al., 1967). No data were found on the carcinogenic potential of citral. The limited toxicity data available do not point to citral as a health risk factor in foods.

Occurrence Vanillin (4-hydroxy-3-methoxybenzaldehyde) has been identified in over 40 food products; in fruits (berries, currants up to 0.02 ppm), in asparagus (1.2 ppm), in spices (mint, nutmeg), crispbread, alcoholic beverages (at levels varying from 0 to 9.6 ppm), coffee, tea and vanilla. The highest concentration, up to 23,000 ppm, has been found in vanilla (Volatile Compounds in Food, 1989). Genotoxicity, carcinogenicity and risk assessment In both an Ames and a chromosome aberration test vanillin showed no mutagenic activity (Ishidate et al., 1984). Rats fed 50,000 ppm vanillin for 1 year or 10,000 ppm for 2 years showed no deleterious effect (Hagan et al., 1967), pointing to vanillin as a rather harmless food component.

Other aldehydes In addition to the aldehydes so far discussed in some detail, a large number of aldehydes are used in foods, mainly as flavoring ingredients. Major

379 representatives are: 1-hexanal, hexen-2-al, heptanal, heptanal dimethylacetal, 1-octanal, 1-nonanal, decanal (capraldehyde), tetradecanal (myristic aldehyde), undecanal, undecenal, dodecanal (lauryl aldehyde), 2-methyl undecenal, hexyl cinnamaldehyde, methyl cinnamaldehyde, hydrocirmamaldehyde, amylcinnamaldehyde, veratraldehyde (3,4-dimethoxybenzaldehyde), dimethyl heptenal, cyclamen aldehyde (2-methyl-3-(p-isopropylphenol)-propionaldehyde) and cuminaldehyde (4-isopropylbenzaldehyde). All of these aldehydes were granted a 'generally recognized as safe' status by the Flavoring Extract Manufacturers' Association (1965), were approved by the U.S. Food and Drug Administration for food use (FDA, 1988a), and were either included in the list of artificial food flavoring ingredients or given an 'acceptable daily intake' value by the Council of Europe (1974). Available toxicological data on these compounds are generally restricted to acute oral and dermal toxicity data, and to data on skin irritation and sensitization; occasionally (e.g., hexen-2-al) subchronic oral toxicity data have been reported (Gaunt et al., 1971). These aldehydes invariably have low oral and dermal toxicity, and do not produce sensitization reactions, but are often slightly or moderately irritating to the rabbit skin (Opdyke, 1979). 3-(N-Nitrosomethylamino)propionaldehyde, found to induce DNA single-strand breaks in human buccal cells, may be of importance in the induction of cancer in betel-quid chewers (Sundqvist et al., 1989). Many more aldehydes have been identified in a limited number of foods or food products (Volatile Compounds in Food, 1989). However, to the best of our knowledge there are data neither on their use nor on their toxicity.

Concluding remarks Most aldehydes discussed in the present paper are irritants producing tissue damage at the site of application, and possess genotoxic activity that may be based on the formation of DNA-protein or DNA-DNA cross-links. The combination of both properties has been shown to be crucial for the induction of nasal carcinomas in rats by formaldehyde vapor, indicating that exposure to

non-cytotoxic concentrations of formaldehyde is virtually safe. This may also be true for acetaldehyde but further studies of the mechanism of its carcinogenicity are needed to substantiate this assumption and to allow quantitative risk assessment. On the other hand, tissue-specific factors may also play a role in the induction of cancer by aldehydes as is demonstrated by the presence of formaldehyde-induced nasal carcinomas in rats, and the absence of forestomach carcinomas in rats with severe, formaldehyde-induced damage to the gastric mucosa. Dissolved in water some aldehydes seem to be less irritating than as a vapor; for instance acrolein vapor is extremely irritating to the nasal and tracheal mucosa of rodents but administration in the drinking water to rats has not been reported to cause gastric damage. Ifi addition, furfural vapor may induce severe damage to the nasal olfactory mucosa in hamsters but no cytotoxic damage to the gastric mucosa has been reported after prolonged dietary administration of this compound at doses that appeared to cause non-neoplastic and neoplastic liver changes. On the other hand, crotonaldehyde shows a different behavior; being a respiratory tract irritant as a vapor, it induces gastritis and hyperplasia of the forestomach in rats after oral administration by gavage and at comparable levels causes liver damage in rats and mice and liver cancer in rats after long-term administration in the drinking water. For both furfural and crotonaldehyde further oral carcinogenicity studies are needed to verify the available data and to permit quantitative risk assessment. For reasons already discussed genotoxicity and carcinogenicity studies are clearly indicated for malondialdehyde and cinnamaldehyde, and may be needed for giycidaldehyde if its formation in foods during storage cannot be prevented. Although the toxicological data base for aldehydes such as citral, anisaldehyde and vanillin is limited, there is no reason to suspect carcinogenicity, and, therefore, further testing does not seem to be indicated. Toxicological data on a large number of aldehydes occurring in foods and drinks are completely lacking or very deficient. This is unacceptable from a toxicological point of view, and, therefore, at least information on the cytotoxic and

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