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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN 
    FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES: 42





    Prepared by the Fifty-first meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)





    World Health Organization, Geneva, 1999
    IPCS - International Programme on Chemical Safety

    FURFURAL

    First draft prepared by
    R. Kroes
    Research Institute of Toxicology, Utrecht University, 
    Utrecht, Netherlands 

         Explanation
         Biological data
              Biochemical aspects
                   Absorption, distribution, biotransformation, and 
                        excretion
                   Aldehyde reactivity 
              Toxicological studies 
                   Acute toxicity
                   Short-term studies of toxicity 
                   Long-term studies of toxicity and carcinogenicity
                        Special studies on carcinogenicity 
                   Genotoxicity 
                   Reproductive toxicity
         Human intake 
         Comments 
         Evaluation 
         References

    1.  EXPLANATION

         Furfural was evaluated previously at the thirty-ninth meeting of
    the Committee (Annex I, reference 101), but an ADI was not established
    because of evidence of its genotoxicity and carcinogenicity. At that
    time, the Committee considered that its direct addition as a flavour
    was not appropriate, that its use as a solvent should be restricted to
    situations in which alternatives are not available, and that its
    transfer into food should be reduced to the lowest extent technically
    feasible. At the present Meeting, the Committee considered furfural as
    a flavouring agent.

         Furfural is used as a flavouring agent in a variety of food
    products and alcoholic and non-alcoholic beverages. Furfural and many
    of its derivatives occur widely as natural constituents of the food
    supply.

         Since the last evaluation, additional data, including those from
    studies of the metabolism of furfural, its potential genotoxicity, and
    effects in the liver have become available.

         This consolidated monograph includes relevant information from
    the previous monograph and the results of studies reviewed for the
    first time at the present Meeting.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, biotransformation, and excretion

     Laboratory animals

         Furfural is well absorbed after administration by any route. In
    rats, 85% of 14C-furfural administered by gavage in corn oil was
    recovered in urine within 72 h (National Toxicology Program, 1987).
    Similar results were found for rats and mice (Nomeir et al., 1992;
    Parkash & Caldwell, 1994). After oral administration, furfural is
    rapidly absorbed from the gastrointestinal tract and distributed to
    the tissues, principally the liver and kidney. The tissue
    concentrations are generally proportional to the dose.

         Furfural is metabolized primarily by oxidation of the aldehyde
    function in rats (Paul et al., 1949; Rice, 1972; Nomeir et al., 1992;
    Parkash & Caldwell, 1994) and mice (Parkash & Caldwell, 1994).
    Oxidation yields furoic acid, which, as the coenzyme A (CoA)
    thioester, is either conjugated with glycine and excreted or condensed
    with acetyl CoA to form the chain-lengthened metabolite
    2-furanacryloyl CoA (Figure 1). 2-Furanacryloyl CoA conjugates with
    glycine and is excreted primarily in the urine. In rats and mice,
    furoic acid appears to decarboxylate to a very minor extent (~1%)
     via oxidation of the furan ring to yield carbon dioxide; the
    mechanism is unknown. The absorption (83-90%), tissue distribution
    (primarily liver and kidney), relative amounts of metabolites (urine,
    76-105%; faeces, 1-7%; breath, 4-7%; tissues, 1%), and pattern of
    excretion in mice and rats were linear over the range of doses
    investigated (0.1-200 mg/kg bw of furfural) (Nomeir et al., 1992;
    Parkash & Caldwell, 1994).

    FIGURE 1

         In animals, the condensation reaction of 2-furoyl CoA with acetyl
    CoA to yield furanacryloyl CoA is reversible, favouring formation of
    2-furoyl CoA (Parkash & Caldwell, 1994). The observation that furoic
    acid is excreted in the urine of dogs given furanacrylic acid
    (Friedmann, 1911) supports this conclusion. Analogous reversible
    reactions between the CoA thioesters of benzoic acid and cinnamic acid
     in vivo favour benzoic acid (Nutley et al., 1994). Excretion of
    unconjugated furoic acid and furanacrylic acid at higher doses
    suggests that glycine conjugation in laboratory animals is limited,
    probably by the supply of endogenous glycine (Gregus et al., 1993). As
    mentioned earlier, the principal metabolite in rodents, furoic acid,
    may also be metabolized to a very minor extent  via oxidation of the
    heteroaromatic ring. Since heteroaromatic and aromatic carboxylic
    acids do not normally undergo decarboxylation   in vivo (Caldwell,
    1982), it can be assumed that oxidation of the furan ring system of
    furoic acid precedes the loss of carbon dioxide. On the basis of this
    assumption, epoxidation (Ramsdell & Eaton, 1990) or hydroxylation
    (Ravindranath & Boyd, 1985; Koenig & Andreesen, 1990) of the furan
    ring may yield reactive intermediates (e.g. furfural-2,3-epoxide,
    acetylacrolein, and alpha-ketoglutaric acid) which readily undergo
    decarboxylation. Biochemical changes in the lungs and livers of
    animals exposed to furfural indicate that ring oxidation may be
    catalysed by a cytochrome P450b isoenzyme, yielding an intermediate
    which subsequently conjugates with glutathione (Gupta et al., 1991;
    Mishra et al., 1991).

     Humans

         As in laboratory animals, the predominant pathway for
    detoxification of furfural in humans is oxidation of the aldehyde to
    yield furoic acid, which either conjugates with amino acids or
    condenses with acetyl CoA to produce furanacrylic acid.

         After inhalation or dermal absorption, furfural is efficiently
    and rapidly absorbed ithrough the lungs and skin, with 20-30% of the
    amount absorbed by the lungs (Flek & Sedivec, 1978). When six
    volunteers were exposed to 30 mg/m3 for 8 h, the mean pulmonary
    retention was 78% (range, 75-82%). Retention was independent of the
    concentration of furfural vapour and the length of exposure. An
    average 8-h exposure maximum was equivalent to a dose of 1.9 mg/kg bw.
    The biological half-life of absorbed furfural in humans is only about
    2 h. Essentially all of the absorbed furfural could be accounted for:
    97% (range, 93-100%) was oxidized to 2-furoic acid and excreted as the
    glycine conjugate, 0.5-5% was excreted as furanacrylic acid, and less
    than 1% was exhaled unchanged. 

    2.1.2  Aldehyde reactivity

         Furfural contains a heteroaromatic furan ring with a reactive
    aldehyde functional group at the 2 position. The reactivity of the
    aldehyde function of aldehydes of low molecular mass, like furfural,
    suggests that such compounds are not absorbed intact at doses that do
    not saturate the oxidation or condensation reactions associated with

    the aldehyde function in digestive fluids. Likewise, when furfural
    reaches the body fluids, it is likely to react before entering the
    cell. The reactivity of the aldehyde group has been demonstrated, for
    example, for acetaldehyde and formaldehyde, both normal metabolic
    intermediates, which may have toxic effects including the induction of
    tumours when administered under non-physiological conditions at high
    doses (Til et al., 1989).

         Aldehydes of low molecular mass have been reported to bind with
    soluble proteins, protein components of cell membranes, and
    thiol-containing molecules to form unstable and stable adducts.
    Formation of adducts with albumin (Nagasawa et al., 1980; Donahue et
    al., 1983; Tuma et al., 1984), haemoglobin (Peterson & Nguyen, 1985),
    and erythrocyte membranes (Gaines et al., 1977) has been demonstrated
     in vitro and  in vivo. In membranes, binding is usually with the
    protein component (Nomura & Lieber, 1981). Aldehydes also bind
    glutathione (Videlia et al., 1982). Aldehyde dehydrogenase-catalysed
    oxidation of low-molecular-mass aldehydes requires glutathione
    (Eckfeldt & Yonetani, 1982), suggesting that free aldehyde is rapidly
    conjugated with glutathione  in vivo to form a thiohemiacetal which
    is subsequently oxidized to the corresponding acid (Brabec, 1981).
    Thus, the reactivity of the aldehyde function will significantly
    curtail the intracellular concentration of free aldehyde.

         When an aldehyde is present in a cell, it undergoes rapid
    oxidation, with conjugation of the resulting carboxylic acid (Gregus
    et al., 1993). The various metabolic processes, i.e. oxidation,
    conjugation, and condensation (see section 2.1.1) effectively
    eliminate the reactive aldehyde functional group when not saturated by
    high, non-physiological doses.

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         On the basis of the oral LD50 values in various species
    (Table 1), furfural is acutely toxic at lower doses in rats than in
    mice. 

         Wistar/Slc rats given a single dose of 50 mg/kg bw furfural by
    gavage had sporadic eosinophilic degeneration of hepatic cells with
    nuclear pyknosis and eosinophilic necrosis and increased hepatocyte
    mitosis. No massive or zonal necrosis was observed. Since the control
    rats were killed at the beginning of the experiment, no appropriate
    controls were available (Shimizu & Kanisawa, 1986). Decreased
    activities of succinic dehydrogenase and ATPase and increased
    activities of acid phosphatase and acid DNase II were reported in male
    Wistar rats injected intraperitoneally with 20 or 50 mg/kg bw (Jonek
    et al., 1975).

    Table 1. Acute toxicity of furfural

                                                                          

    Species  Route            LD50 (mg/kg bw)        Reference
                              or LC50 (mg/m3)
                                                                             

    Rat      Oral             127                    Jenner et al. (1964)
    Rat      Intraperitoneal  120                    Tiunov et al. (1970)
    Mouse    Oral             333                    Boyland (1940)
    Mouse    Subcutaneous     200                    Tiunov et al. (1970)
    Mouse    Subcutaneous     223 (1-day survival)   Castellino et al.(1963)
                              119 (10-day survival)
    Rabbit   Dermal           > 310                  Moreno (1976)
    Hamster  Inhalation       2500                   Kruysse (1972)
    Bird     Oral             > 98                   Schafer et al. (1983)
                                                                             


    2.2.2  Short-term studies of toxicity

     Rats

         In rats receiving 0.05, 2.5, or 25 mg/kg bw per day furfural for
    35 days, no hepatic impairment was seen (Kuznetsov, 1966). When male
    rats were injected intraperitoneally with furfural in increasing doses
    from 29 to 58 mg/kg bw for 30 days, damage and increased intracellular
    catabolic processes were seen in liver and kidney cells (Kaminska,
    1977).

         In a 16-day study, groups of five male and five female Fischer
    344/N rats were given furfural at doses of 0, 15, 30, 60, 120, or
    240 mg/kg bw per day in corn oil by gavage, five days per week for a
    total of 12 doses. At the end of the study, the surviving animals were
    killed, necropsied, and examined histopathologically. No
    treatment-related abnormalities were found. Eight of ten rats at the
    highest dose had died by the third day due to gavaging accidents.
    Animals at 120 mg/kg bw appeared to be less active. The body weights
    of rats given doses < 120 mg/kg bw per day did not differ
    significantly from those of controls.

         In a 16-day study, groups of five male and five female B6C3F1
    mice were given furfural at doses of 0, 25, 50, 100, 200, or 400 mg/kg
    bw per day in corn oil by gavage on five days per week for a total of
    12 doses. At the end of the study, the surviving animals were killed,
    necropsied, and examined histopathologically. The mean body weights of
    the treated mice did not differ significantly from those of controls.
    One male at the highest dose had died by day five, and two female mice
    died from gavaging accidents. No treatment-related abnormalities were
    found.

         In a 13-week study, groups of 10 male and 10 female B6C3F1 mice
    were given furfural at doses of 0, 75, 150, 300, 600, or 1200 mg/kg bw
    per day in corn oil by gavage on five days per week. Necropsies were
    performed on all survivors, and histopathological examinations were
    performed on those at the two highest doses and the controls. All the
    mice at the two highest doses died before the end of the study, except
    for one male and one female at 600 mg/kg bw per day. The body weights
    of the males treated with doses > 150 mg/kg bw per day were
    slightly lower than those of controls. Female mice at 75-300 mg/kg bw
    per day and male mice at 300 mg/kg bw per day showed significantly
    increased relative liver weights as compared with controls.
    Centrilobular coagulative necrosis of hepatocytes was observed in most
    male mice at the two highest doses and in one male per group treated
    with 150 or 300 mg/kg bw per day. Among the females, only two at the
    highest dose showed similar lesions. Animals with coagulative necrosis
    also had inflammation of the liver, with minimal to mild mononuclear
    inflammatory cell infiltrate (National Toxicology Program, 1990).

         A group of 48 male, six-week-old Wistar/Slc rats was fed furfural
    at a concentration of 20 ml/kg diet (equivalent to approximately 1200
    mg/kg bw per day) for one week, 30 ml/kg diet for one week, and then
    40 ml/kg diet on days 15-90; on days 90-120, the rats were given the
    diet containing 40 ml/kg for only five days per week to prevent
    reduced weight gain. The concentration of furfural in the feed was not
    analysed after preparation of the diet. A control group of 40 male
    rats of the same age was fed the basal diet. 

         Treated rats sacrificed after 90 days had marked
    cholangiofibrosis, with areas of increased density containing red
    nodules. The nodules showed fibrous widening of Glisson's sheath,
    bile-duct proliferation, and destruction of the limiting plates. The
    parenchymal damage consisted of bridging necrosis and hydropic
    degeneration of hepatocytes. The parenchyma often showed no cirrhotic
    changes in areas other than those with fibrosis, suggesting a
    regenerative process in the liver. Increased numbers of cells
    undergoing mitosis were seen. In rats killed on day 120 of the study,
    similar but more marked findings were reported. The incidence of
    hepatic fibrosis was increased in the furfural-treated rats. No
    atypical hepatocyte growth was noted. Furfural did not cause
    hepatocellular hyperplastic changes (Shimizu & Kanisawa, 1986). 

         In a study designed to examine the mechanism of furfural-induced
    hepatocytic injury, six groups of six male Wistar/Slc rats were
    maintained on diets containing furfural at doses calculated to result
    in average daily intakes of 20 ml/kg diet (approximately 1200 mg/kg bw
    per day) for the first 30 days and 30 ml/kg diet (approximately
    1700 mg/kg bw per day) thereafter. Each group also included four
    control animals. The furfural-containing diets were replaced after 15,
    30, 60, 90, 120, or 150 days, and animals were killed 14 days after
    termination of furfural administration. Increased duration of exposure
    to furfural was accompanied by increased numbers and size of foci
    positive for the placental form of glutathione- S-transferase in
    hepatocytes (Shimizu et al., 1989). Such foci have been reported to be

    a marker for early detection of preneoplastic or neoplastic cells
    (Pickett & Lu, 1988). 

         In a 13-week study, groups of 10 male and 10 female Fischer 344/N
    rats were given furfural at doses of 0, 11, 22, 45, 90, or 180 mg/kg
    bw per day in corn oil by gavage on five days per week. All survivors
    were necropsied, and histopathological examinations were performed on
    those at the two highest doses and the controls. Only one male rat at
    the highest dose survived to the end of the study; four of the deaths
    were considered to be related to gavage, as were two deaths among
    controls and four among rats at the second highest dose. The mean body
    weights of treated rats did not differ from those of controls. The
    relative and absolute weights of the livers and kidneys of males given
    90 and 180 mg/kg bw per day were significantly higher than those of
    controls. An increased incidence of cytoplasmic vacuolization in
    hepatocytes was observed in all groups of treated males: control,
    4/10; 11 mg/kg bw per day, 10/10; 22 mg/kg bw per day, 10/10; 45 mg/kg
    bw per day, 10/10; 90 mg/kg bw per day, 9/10. No lesions were reported
    in the kidneys of these rats (National Toxicology Program, 1990).

     Hamsters

         Groups of 10 Syrian golden hamsters of each sex were exposed to
    furfural vapour at 0, 77, 448, or 2165 mg/m3 for 6 h per day, five
    days per week over 13 weeks. The main findings were mild growth
    retardation, irritation of the eyes and nose, and hyperplastic atrophy
    of the nasal epithelium, all at the highest dose. The NOEL was 77
    mg/m3, since mild nasal epithelial degeneration was observed at 448
    mg/m3 (Feron & Kruysse, 1978).

    2.2.3  Long-term studies of toxicity and carcinogenicity

     Mice

         In a two-year study, 50 male and 50 female B6C3F1 mice were
    given furfural at doses of 0, 50, 100, or 175 mg/kg bw per day in corn
    oil by gavage on five days per week. The animals were weighed weekly
    for the first 13 weeks and monthly thereafter until the conclusion of
    the study, when surviving animals were killed. All animals were
    necropsied and examined histologically. Furfural had no significant
    effect on body weight or survival. Microscopic examination revealed
    lesions in the liver, kidneys, and forestomach. In the liver,
    pigmentation, necrosis, and chronic inflammation were seen in males at
    the intermediate and high doses and in females at the high dose (Table
    2). Hepatocellular adenomas and carcinomas were seen in all groups,
    including controls, and the increased incidence was statistically
    significant only in males at the high dose. Carcinomas occurred at the
    same incidence in females at the high dose as in controls.


        Table 2. Incidences of liver lesions and hepatocellular adenomas and carcinomas in male and 
    female B6C3F1 mice given furfural by gavage

                                                                                                       

    Lesion                             Dose (mg/kg bw)
                                                                                                       
                                       0               50              100             175
                                                                                                        
                                       M       F       M       F       M       F       M       F
                                                                                                       

    Pigmentation                        0/50   0/50     0/50   0/50     8/49   0/50    18/50   11/50
    Necrosis, focal and multifocal      3/50   2/50     2/50   1/50     7/49   3/50    10/50    2/50
    Chronic Inflammation                0/50   0/50     0/50   0/50     8/49   1/50    18/50    8/50
    Hepatocellular adenoma              9/50   1/50    13/50   3/50    11/49   5/50    19/50    8/50
    Hepatocellular carcinoma            7/50   4/50    12/50   0/50     6/49   2/50    21/50    4/50
    Combined rates                     16/50   5/50    22/50   3/50    17/49   7/50    32/50   12/50
                                                                                                       

    From National Toxicology Program (1990)
    M, male; F, female
    

         Hyperplasia of the renal tubules was found in one male at the low
    dose. One male at the intermediate dose, one at the high dose, and one
    female at the low dose had a cortical adenoma, and one male at the low
    dose had a cortical carcinoma. Owing to the low incidence and lack of
    dose-response relationship, these tumours were not considered as
    evidence for carcino-genicity. Marginal increases in the incidences of
    hyperplasia and papillomas in the forestomach of female mice were
    considered to be of no biological significance and were probably
    related to the irritating effects of chronic administration of the
    compound by gavage or the method of gavage itself. None of the animals
    showed evidence of malignant forestomach lesions (National Toxicology
    Program, 1990).

     Rats

         In a two-year study, 50 male and 50 female Fischer 344/N rats
    were given furfural at doses of 0, 30, or 60 mg/kg bw per day in corn
    oil by gavage on five days per week. The animals were weighed at
    specific intervals, and the survivors were killed at the end of the
    study, necropsied, and examined histologically. Overall survival was
    slightly reduced for female rats at the highest dose (78% at week 97),
    due to accidental gavage-related deaths. Furfural had no effect on
    body weight or survival. Congestion and foreign bodies were observed
    in a dose-related manner in the lungs of female rats. A small increase
    in the incidence of squamous-cell carcinomas and papillomas  in the
    forestomachs of treated rats was observed but was considered unrelated
    to treatment. Mild centrilobular hepatocellular necrosis was observed
    in all groups (vehicle control: males, 3/50; females, 10/50; low dose:
    males, 9/50; females, 9/50; high dose: males, 12/50; females, 4/50).
    Bile-duct hyperplasia occurred in 45/50 control males, 36/50 control
    females; 41/50 males and 28/50 females at the low dose; and 44/50
    males and 24/50 females at the high dose. Focal bile-duct dysplasia
    was noted in one male at the intermediate dose, and bile-duct
    hyperplasia accompanied by fibrosis (cholangiofibrosis) was seen in
    two males at the high dose. One control male had a hepatocellular
    adenoma, and two males at the high dose had a cholangiocarcinoma. The
    incidence of the latter lesion was not statistically significant, but
    some evidence of carcinogenicity was consluded since
    cholangiocarcinomas were rare in historical controls (3/2145)
    (National Toxicology Program, 1990).

    2.2.3.1  Special studies of carcinogenicity

         The aldehydes citral and heptaldehyde inhibit the growth of
    spontaneous mammary tumours in mice; however, furfural and its
    furfuracrylic acid metabolite at a daily dose of 2.5 mg had no effect
    on the growth of spontaneous mammary tumours in mice, although they
    were weakly active against sarcomata (Boyland, 1940).

         Studies have been designed to evaluate the effect of furfural on
    the carcinogenic potential of known carcinogens. In the first study,
    three groups of 35 male and 35 female Syrian golden hamsters were
    provided with 0.2 ml of 1.5% furfural in physiological saline (about

    25 mg/kg bw), 0.5% benzo [a]pyrene (8.3 mg/kg bw), or 1.5% furfural
    plus 0.5% benzo [a]pyrene intratracheally once weekly for 36 weeks.
    Furfural in combination with benzo [a]pyrene led to earlier
    development of hyper- and metaplastic changes in the tracheobronchial
    epithelium, a shorter latency for tracheobronchial tumours, a few more
    bronchial and peripheral squamous-cell carcinomas, and a substantially
    reduced number of tracheal squamous-cell carcinomas. The incidence of
    peritracheal sarcomas was increased, which may indicate a
    co-carcinogenic effect of furfural. Irritation but no tumorigenic
    effect was observed after administration of furfural alone (Feron,
    1972).

         In a subsequent study, groups of 126 Syrian golden hamsters of
    each sex were exposed to atmospheres containing 400 ppm (1600 mg/m3)
    furfural for 7 h per day, five days per week for nine weeks, then 330
    ppm (1300 mg/m3) for 11 weeks, and 250 ppm (970 mg/m3) for an
    additional 32 weeks. The effects on the respiratory tract included
    atrophy and downward growth of the olfactory epithelium, degenerative
    changes in Bowman's glands, and the appearance of cyst-like structures
    in the lamina propria beneath the olfactory epithelium. Although
    furfural was irritating to the olfactory epithelium, treatment did not
    result in hepatic toxicity or carcinogenicity. Furfural did not
    potentiate the carcinogenic effect of benzo [a]pyrene or
     N-nitrosodiethylamine (Feron & Kruysse, 1978). 

         Localized irritation of the nasal cavity and respiratory tract,
    with no hepatic effects, were also observed in B6C3F1 mice and
    Fischer 344/N rats exposed to atmospheres containing 0, 2, 4, 8, 16,
    or 32 ppm of the metabolic precursor furfuryl alcohol for 13 weeks
    (Mellick et al., 1991).

         SPF Wistar rats were given furfural at doses of 20 ml
    (approximately equal to 1200 mg/kg bw per day) for one week, 30 ml for
    the second week, and 40 ml for the following 10 weeks to produce
    hepatic cirrhosis. Half of the remaining rats in this study (38
    treated and 32 controls) were fed a diet containing 0.03%
     N-2-fluorenylacetamide for three weeks, followed by one week of
    normal diet; the other half received only the normal diet. All
    surviving rats were killed 12 weeks later, and their livers were
    examined. Rats that had received furfural only had no hyperplastic
    changes in the liver but did have cirrhosis. Rats that had been
    treated with  N-2-fluorenylacetamide developed multiple hyperplastic
    nodules which stained for alpha-fetoprotein, and this response was
    markedly potentiated in the rats previously treated with furfural. The
    authors concluded that furfural-induced hepatic cirrhosis increases
    the susceptibility of rats to potent hepatocarcinogens (Shimizu,
    1986).

         Samples of mouse liver tumours from the National Toxicology
    Program carcinogenesis bioassay programme were assessed for
    transforming gene activity by Southern blot analysis for H-, K-, and
    N- ras oncogens. The pattern of mutations in the oncogenes from liver
    tumours that occurred spontaneously differed from that which occurred

    in some furfural-treated animals: The activated  ras genes were
    H- ras in 15 of 17 spontaneous tumours, with one  raf and one
    unknown oncogene; activating point mutations occurred at codon 61 in
    six tumours, codon 13 in two tumours, and codon 117 in one tumour. The
    authors concluded that the spectrum of activating mutations in the
    H- ras gene and the pattern of  ras gene activation in liver tumours
    derived from furfural-treated mice differed from those in liver
    tumours derived from untreated animals, and indicates a direct
    genotoxic effect of furfural. An alternative mechanism-induction of
    mutations by an indirect, secondary genotoxic pathway as a result of a
    cytotoxic event -- was mentioned but excluded by the authors on the
    basis of the absence of cytotoxic lesions in the livers of similar
    mice after 90 days of treatment with the same dose of furfural. The
    authors do not mention, however, that necrosis, pigmentation, and
    chronic inflammation were seen regularly in mice at the intermediate
    and high doses in the carcinogenicity experiment (see Table 2). It can
    be concluded that the effect was either due to a direct genotoxic
    event or occurred  via an indirect, secondary genotoxic pathway
    (Reynolds et al., 1987).

    2.2.4  Genotoxicity

         Studies of the genotoxicity of furfural are summarized in
    Table 3. Testing of furfural for mutagenicity  in vitro at
    concentrations up to those causing cytotoxicity provided no evidence
    that it is mutagenic to  Salmonella typhimurium strains TA98, TA102,
    TA1535, or TA1537 or ito two strains of  Escherichia coli, with or
    without metabolic activation (Sasaki & Endo, 1978; Zdzienicka et al.,
    1978; McMahon et al., 1979; Loquet et al., 1981; Marnett et al., 1985;
    Mortelmans et al., 1986; Shinohara et al., 1986; Nakamura et al.,
    1987; Shane et al., 1988; Aeschbacher et al., 1989; Kato et al., 1989;
    National Toxicology Program, 1990; Dillon et al., 1992). The majority
    of these studies also showed negative results for  S. typhimurium  
    TA104, although a single positive result was reported (Shane et al.,
    1988). Furfural was not mutagenic when incubated with
     S. typhimurium TA100 in most reports (Sasaki & Endo, 1978; McMahon
    et al., 1979; Mortelmans et al., 1986; Shinohara et al., 1986; Kim et
    al., 1987; Shane et al., 1988; Aeschbacher et al., 1989; Kato et al.,
    1989; National Toxicology Program, 1990; Eder et al., 1991; Dillon et
    al., 1992) but was weakly mutagenic in other studies (Zdzienicka et
    al., 1978; Loquet et al., 1981; Shane et al., 1988). When fufural was
    co-incubated with benzo [a]pyrene and  S. typhimurium TA100, it did
    not alter the mutagenic activity of benzo [a]pyrene in this strain
    (Zdzienicka et al., 1978). Furfural showed no mutagenic potential in
    two  rec assays with  Bacillus subtilis by a direct streaking method
    (Osawa & Namiki, 1982; Matsui et al., 1989) but also gave positive
    results in this test system (Shinohara et al., 1986).


        Table 3. Results of assays for the genotoxicity of furfural

                                                                                                                                              

    End-point             Test object                         Concentration             Result                     Reference
                                                                                                                                              

    In vitro
    Reverse mutation      S. typhimurium TA100                NR                        Negative                   Eder et al. (1991)
    Reverse mutation      S. typhimurium TA100, TA102, and    NR                        Negativea                  Dillon et al. (1992)
                          TA104
    Reverse mutation      S. typhimurium TA100, TA98, and     < 36 mol/plate           Weakly positive (TA100)b   Loquet et al. (1981)
                          TA1535                              60 mol/plate             Negativec
    Reverse mutation      S. typhimurium TA100, TA98, and     < 1.2 mmol/plate          Negative                   Aeschbacher et al. (1989)
                          TA102                                                                                    
    Reverse mutation      S. typhimurium TA100 and TA98       0.165-0.660 mol/plate    Negativea                  Shinohara et al. (1986)
    Reverse mutation      S. typhimurium TA100, TA102, and    5-500 g/plate            Positive (TA104)           Shane et al. (1988)
                          TA104
    Reverse mutation      S. typhimurium TA100, TA98, and     NR                        Negativea                  Kato et al. (1989)
                          TA104; E. coli WP2uvrA/PKM101
    Reverse mutation      S. typhimurium TA104 and TA102      1 mol/plate              Negative                   Marnett et al. (1985)
    Reverse mutation      S. typhimurium TA98, TA100,         < 6667 g/plate           Negativea                  Mortelmans et al. (1986) 
                          TA1535, and TA1537
    Reverse mutation      S. typhimurium TA100 and TA98       7 l/plate                Positivea (TA100)          Zdzienicka et al. (1978) 
    Reverse mutation      S. typhimurium TA100, TA1535,       < 20 l/plate             Negativea                  McMahon et al. (1979)
                          TA1537, TA1538, TA98, G46; 
                          E. coli WP2uvrA
    Reverse mutation      S. typhimurium TA100 and TA98       NR                        Negativea                  Sasaki & Endo (1978)
    Reverse mutation      S. typhimurium TA100                4.44 mol/plate           Negativea                  Kim et al. (1987)
    umu gene expression   S. typhimurium TA1535/pSK/002       1932 g/ml                Negativea                  Nakamura et al. (1987)
    Reverse mutation      S. typhimurium TA100                < 1 mg                    Negativea                  Osawa & Namiki (1982)
    Reverse mutation      S.typhimurium TA98, TA100,          33-6666 g/plate          Negativea                  National Toxicology Program 
                          TA1535, and TA1537                                                                       (1990a)
    Reverse mutation      S.typhimurium TA98, TA100, and      33-6666 g/plate          Negativea                  National Toxicology Program
                          TA1535                                                        (TA100 equivocal)          (1990b)
    rec Gene mutation     B. subtilis H17 and M45             < 1 mg                    Negativea                  Osawa & Namiki (1982)
    rec Gene mutation     B. subtilis H17 and M45             1.7-17 mg/disk            Positivea                  Shinohara et al. (1986)
    rec Gene mutation     B. subtilis H17 and M45             0.6 ml                    Negativea                  Matsui et al. (1989)

    Table 3. (continued)

                                                                                                                                              

    End-point             Test object                         Concentration             Result                     Reference
                                                                                                                                              

    Chromosomal           Chinese hamster ovary cells         < 40 mmol/L               Positivea                  Stich (1981a)
    aberration
    Chromosomal           Chinese hamster ovary cells         3 mg/ml                   Positive                   Stich (1981b)
    aberration
    Forward mutation      L5178Y tk+/- mouse lymphoma cells   25-400 g/ml              Positiveb                  McGregor et al. (1988) 
    Sister chromatid      Chinese hamster ovary cells         < 1170 g/ml              Positivea                  National Toxicology Prohgram 
    exchange/Chromosomal                                                                                           (1990)
    aberration
    Sister chromatid      Human lymphocytes                   < 0.14 mmol/L             Positive                   Gomez-Arroyo & Souza (1985)
    exchange
    Unscheduled DNA       Human liver slices                  0-25 mmol/L               Negative                   Lake et al. (1998)
    synthesis

    In vivo
    Sex-linked recessive  D. melanogaster                     1000 ppm in diet          Negative                   Woodruff et al. (1985) 
    lethal mutation
    Sex-linked recessive  D. melanogaster                     100 ppm by injection      Positive                   Woodruff et al. (1985)
    lethal mutation
    Sex-chromosome loss   D. melanogaster males mated with    Fed or injected with      Negative                   Rodriguez-Arnaiz et al. (1992)
                          repair-proficient females           < 33% lethal dose
    Sex-chromosome loss   D. melanogaster males mated with    Fed or injected with      Positive only after        Rodriguez-Arnaiz et al. (1992)
                          repair-deficient females            < 33% lethal dose         injection
    Sex-chromosome loss   D. melanogaster                     < 6500 ppm by             Negative                   Rodriguez-Arnaiz et al. (1989)
                                                              injection 
    Sex-linked recessive  D. melanogaster                     < 6500 ppm by             Negative                   Rodriguez-Arnaiz et al. (1989)
     lethal mutation                                          injection 
    Reciprocal            D. melanogaster                     100 ppm by injection      Negative                   Woodruff et al. (1985) 
     translocation
    Sister chromatid      B6C3F1 mouse bone-marrow cells      50-200 mg/kg by           Negative                   National Toxicology Program 
    exchange/Chromosomal                                      injection                                            (1990)
     aberration

    Table 3. (continued)

                                                                                                                                              

    End-point             Test object                         Concentration             Result                     Reference
                                                                                                                                              

    Somatic chromosomal   Swiss albino mouse bone-marrow      < 4000 ppm for 5 days     Negatived                  Subramanyam et al. (1989)
     mutation             cells
    Sperm-head            Swiss albino mouse                  < 4000 ppm for 5          Negatived                  Subramanyam et al. (1989)
     abnormalities                                            weeks
    Unscheduled DNA       Fischer 344 rat hepatocytes         5.0, 16.7, or 50 mg/kg    Negative                   Phillips et al. (1997)
     synthesis                                                bw orally
                                                                                                                                              

    NR, not reported
    a   With and without metabolic activation
    b   Without metabolic activation
    c   With metabolic activation
    d   Abstract only: reported to have positive effects at 4000 ppm, but no details available
    

         Furfural produced a dose-dependent increase in the frequency of
    chromatid breaks and exchanges in Chinese hamster ovary cells with and
    without metabolic activation (Stich et al., 1981a,b; National
    Toxicology Program, 1990) and induced chromosomal aberration in these
    cells at a cytotoxic concentration (National Toxicology Program,
    1990). An increased frequency of sister chromatid exchange was
    reported in human lymphocytes incubated with furfural at a
    concentration of 0.07 or 0.14 mmol/L, but not at 0.035 mmol/L
    (Gomez-Arroyo & Souza, 1985). In this study, furfuryl alcohol  did not
    increase the frequency of sister chromatid exchange at a concentration
    of 3.3, 6.6, or 9.9 mmol/L.

         Furfural increased trifluorothymidine resistance when incubated
    with L5178Y  tk+/- mouse lymphoma cells at a concentration of 200 or
    400 mg/ml without metabolic activation, but showed no mutagenic
    activity at 25, 50, or 100 mg/ml (McGregor et al., 1988).
    Concentrations of 400 and 800 mg/ml were cytotoxic in separate trials.

          In vivo, furfural did not induce sex-linked recessive lethal
    mutations or reciprocal translocations in male  Drosophila 
     melanogaster when incorporated into their diet at 1000 ppm.
    Mutations were induced when furfural was administered by injection at
    a concentration of 100 ppm, but no reciprocal translocations occurred.
    A similar pattern of responses was reported for other simple aldehydes
    (e.g. acetaldehyde and  trans-cinnamaldehyde) when given via the
    intraperitoneal (positive) and oral (negative) routes of
    administration (Woodruff et al., 1985). No sex-chromosome loss was
    observed when male  D. melanogaster were fed or injected with
    furfural at concentrations equivalent to 25-33% of the lethal dose of
    furfural and then mated with repair-proficient females
    (Rodrigues-Arnaiz et al., 1992), but sex-chromosome loss was reported
    by these authors when treated males were mated with repair-deficient
    females. No sex-linked recessive lethal mutation or sex-chromosome
    loss was reported when male  D. melanogaster were injected with
    furfuryl alcohol (Rodrigues-Arnaiz et al., 1989). 

         Tests of mammals treated  in vivo with furfural have produced
    mainly negative results. Furfural did not induce sister chromatid
    exchange or chromosomal aberrations in B6C3F1 mouse bone-marrow cells
    after intraperitoneal injections of furfural at doses of 50-200 mg/kg
    bw (National Toxicology Program, 1990). No genotoxic effects or
    spermhead abnormalities were reported in mice given up to 4000 ppm
    furfural daily for five weeks, and only the highest dose tested was
    associated with an increased frequency of somatic (liver)-cell
    chromosomal mutations when given for 24 or 48 h but not after 72 h
    (Subramanyam et al., 1989). Furfural administered by gavage to male
    Fischer 344/N rats in corn oil at doses of 5, 16.7, or 50 mg/kg bw did
    not induce unscheduled DNA synthesis in hepatocytes (Phillips et al.,
    1997). 

         Chromosomal aberrations were not observed in lymphocytes taken
    from workers occupationally exposed to furfural or furfuryl alcohol
    (Gomez-Arroyo & Souza, 1985). 

         Liver slices prepared from samples from four human donors were
    cultured in medium containing 3H-thymidine and 0-25 mmol/L furfural
    for 24 h. In a preliminary experiment, furfural was markedly toxic at
    concentrations > 10 mmol/L. As positive controls, liver slices were
    also cultured with three known genotoxic agents,
    2-acetylaminofluorene, aflatoxin B1, and
    2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). Unscheduled
    DNA synthesis was quantified as the changes in mean nuclear and
    cytoplasmic grain counts relative to controls, net grain count (i.e.
    mean nuclear - mean cytoplasmic grain counts), and the percentage of
    centrilobular hepatocyte nuclei with > 5 and > 10 net grains. In
    comparison with control liver slice cultures (in dimethylsulfoxide),
    those treated with 0.02 or 0.05 mmol/L 2-acetylamino-fluorene, 0.002
    or 0.02 mmol/L aflatoxin B1, and 0.003 or 0.05 mmol/L PhIP had
    significantly higher net grain counts in centrilobular hepatocytes,
    due primarily to large increases in mean nuclear grain counts. The
    three genotoxic agents also significantly increased the number of
    centrilobular hepatocyte nuclei with > 5 and > 10 net grains. 

         Treatment with 0.005-0.5 mol/L furfural had no significant effect
    on net grain count or mean nuclear or cytoplasmic grain counts. A
    small but statistically significant increase in net grain counts
    observed at 2, 5, and 10 mmol/L furfural was the result of a
    significant concentration-dependent decrease in mean cytoplasmic grain
    counts accompanied by a smaller increase in nuclear grain counts.
    Furfural had no significant effect on the percentage of hepatocyte
    nuclei with > 5 or > 10 net grain counts, except at a concentration
    of 2 mmol/L and only in liver cultures in which marked toxicity was
    noted at 10 mmol/L. The decreases in mean cytoplasmic and nuclear
    grain counts and the correlation between cytotoxicity and changes in
    net grains count and nuclei with > 5 net grains support the
    conclusion that furfural at 2, 5, or 10 mmol/L induces a cytotoxic
    response in cultured human liver slices. In comparison with the three
    genotoxins tested, furfural did not induce unscheduled DNA synthesis
    in cultured human liver slices (Lake et al., 1998). 

         Furfural was reported to increase the frequency of single-strand
    DNA breaks in isolated calf-thymus duplex DNA (Hadi et al., 1989).
    When incubated with plasmid DNA, furfural decreased the transformation
    capacity of  Escherichia coli host cells; however, the damaged
    plasmids were repaired after propagation in the host (Khan & Hadi,
    1993).

         Thus, negative results were obtained with furfural in most tests
    for genotoxicity  in vitro. Positive results were reported at
    relatively high doses in three of 16 assays for reverse mutation in
     S. typhimurium and in one of three assays for gene mutation in
     B. subtilis. Weakly positive results were obtained in tests for
    chromosomal aberrations at relatively high doses. Positive results

    were found in two assays for sister chromatid exchange and one for
    forward mutation in mouse lymphoma cells. The results of all tests for
    genotoxicity  in vivo were negative, except in  D. melanogaster 
    injected with furfural.

    2.2.5  Reproductive toxicity

     Rats

         A summary report of an investigation to define the doses of
    furfural for a study of developmental toxicity was submitted to the
    Toxic Substances Control Administration of the US Environmental
    Protection Agency. Five groups of eight Crl:CD(SD)BR rats received
    furfural in water by gavage at doses of 10, 50, 100, 500, or 1000
    mg/kg bw per day during gestation days 6-15. Because of excessive
    deaths at 500 and 1000 mg/kg bw per day, the breeding phase of the
    study was re-initiated to characterize the potential maternal and
    developmental toxicity at 150, 250, and 350 mg/kg bw per day. Two
    concurrent control groups of eight rats received only the vehicle
    (reverse osmosis-treated water) by gastric intubation. Clinical
    observations, body weights, and food consumption were recorded. On
    gestation day 20, laparohysterectomy was performed on all surviving
    animals; the uteri and ovaries were examined, and the numbers of
    fetuses, early and late resorptions, total implantation, and corpora
    lutea were recorded. The uteri and fetuses were weighed and examined
    for external malformations, variations, and sex. 

         Because of excessive mortality on day 6 after dosing, all females
    at 250, 350, 500, and 1000 mg/kg bw per day were killed. The clinical
    signs in these animals included lethargy, effects on respiration, and
    whole-body tremor. The maternal animals at other doses survived,
    except for one female at 150 mg/kg bw per day group, which died within
    I h of dosing. The surviving females at 150 mg/kg bw per day had
    reduced body-weight gain and reduced food consumption; however, the
    mean body weight, net body weight, net body-weight gain, and gravid
    uterine weight were unaffected. Body weight and food consumption of
    animals at 10, 50, and 100 mg/kg bw per day were unaffected.
    Intrauterine growth and survival were not affected at 10, 50, 100, or
    150 mg/kg bw per day. The only external malformation noted
    (omphalocele) occurred in offspring of rats at 10 mg/kg bw per day. No
    developmental variations were noted (US Environmental Protection
    Agency, 1997).

    3.  HUMAN INTAKE

         On the basis of the most recently reported annual volumes, 3600
    kg in Europe (International Organization of the Flavor Industry, 1995)
    and 590 kg in the United States (National Academy of Sciences, 1987),
    the estimated daily  per capita intake of 'eaters only' of furfural
    from use as a flavouring substance is approximately 9 g/kg bw per day
    in Europe and 2 g/kg bw per day in the United States. Furfural and
    the corresponding furfuryl alcohol are virtually ubiquitous in nature
    (Table 4). They are formed from the acid hydrolysis or heating of

        Table 4. Natural occurrence of furfural

                                                                                        

    Food item                                                          Concentration of
                                                                        furfural (ppm)
                                                                                        

    Apple (raw), apple juice, apricot (Prunus armeniaca L.), sweet       0.02-0.05
      cherry (Prunus avium L.), sour cherry (Prunus cerasus L.)
    Orange juice (Citrus sinensis L. Osbeck)                             Trace
    Orange peel oil, grapefruit juice (Citrus paradisi)                  0.34
    Bilberry (Vaccinium myrtillus L.)                                    0.02
    American cranberry (Vaccinium macrocarpon Ait.)                      0.1-0.3
    Lingonberry (Vaccinium vitis idaea L.)                               0.02
    Black currents, berries, guava (Psidium guajava L.)                  0.0014-0.19
    Grape (dried, sultana), peach (Prunus persica L.), pineapple         0.01
      (Ananas comosus), raspberry (Rubus idaeus L.), strawberry 
      (Fragaria species), asparagus (raw), asparagus (cooked)
    Carrot (Daucus carota L.), celery leaves (raw), onion (roasted),     0.005
       leek (heated), potato (raw), potato (cooked), bell pepper 
      (Capsicum annuum)
    Wheaten bread, sauerkraut, tomato (Lycopersicon                      0.8-26
      esculentum Mill.), cinnamon (Cinnamomum zeylanicum Blume), 
      cloves (Eugenia caryophyllata Thunberg), Mentha species 
    Crispbread, bread, other types, blue cheeses, parmesan,              0.02
      butter, yogurt, milk, chicken and turkey (raw), beef (boiled/
      cooked), beef (grilled/roasted)
    Lamb and mutton, pork (heated), hop oil, beer                        0-0.3
    Cognac                                                               0.6-33
    Armagnac                                                             2
    Weinbrand                                                            0.2-4.3
    Grape brandy, other types, rum (all categories), rum (category I:    22
      total volatiles > 3600 ppm)
    Rum (category II: total volatiles 1100-3600 ppm)                     Trace-25
    Rum (category III: total volatiles 240-1100 ppm)                     Trace
    Bourbon whiskey                                                      2-11.6
    Irish whiskey                                                        0.8-13.6
    Malt whisky                                                          10-37
    Scotch blended whisky                                                1.1-30
    Canadian whiskey                                                     0.3-0.8
    Japanese whiskey                                                     0.5-4.5
    Cider, sherry, white wine                                            Trace-10.3
    Red wine                                                             0.005-0.05
    Rose wine, port wine                                                 2-34
    Special wine, botrytized wine                                        0.13
    Cocoa, coffee                                                        55-255
    Black tea                                                            2-7
    Green tea                                                            0.1
    Microbial fermented tea, tea (brewed)                                0.3-0.8
                                                                                        

    Table 4. (Continued)

                                                                                        

    Food item                                                          Concentration of
                                                                        furfural (ppm)
                                                                                        

    Barley (roasted), filbert (roasted, Corylus avellano), peanut        0.08-0.2
      (roasted, Arachis hypogea), pecan (roasted), popcorn, potato 
      chips (American)
    Oat flakes (toasted), honey, soybean, arctic bramble (Rubus          Trace
      arcticus L., Rubus stell.), cloudberry (Rubus chamaemorus L.)
    Passion fruit juice (yellow), passion fruit (yellow), plum (raw),    2.58
      plum (salted and pickled)
    Beans (Phaseolus vulgaris L.), mushroom (raw)                        0.05
    Trassi (cooked), plum brandy, almond (roasted, Prunus                9
      amygdalus)
    Macadamia nut (roasted, Macadamia integrifolia), sesame              0-0.1
      seed (roasted), mango (raw)
    Mango (canned), cauliflower (cooked), tamarind (Tamarindus           7
      indica), pear brandy
    Apple brandy, beetroot (cooked),artichoke (cooked, Cynarus           < 0.01
      scolymus L.), gin, rice bran, traditional rice (cooked), quince 
      (Cydonia oblonga), radish (fermented), shoyu (fermented soya 
      hydrolysate), bacuri (Platonia insignis), cupuacu (Theobroma 
      grandiflora), muruci (Brysonima crassifolia)
    Potato (sweet, heated), sukiyaki, licorice (Glycyrrhiza glabra L.),  < 0.01
      matsutake (Tricholoma matsutake), strawberry wine, pumpkin 
      (Cucurbita pepo L.), sake, oat groats, maize, cashew apple 
      (Anacardium occidentale), basil (Ocimum basilicum), malt, 
      peated malt, wort, bonito (dried, katsuobishi), elderberry 
      (Sambucus nigra L.), mangosteen (Garcinia mangostana),
       cherimoya (Annona cherimola), bilberry wine, buchu oil, 
      vanilla, mountain papaya (Carica pubescens)
    Wild rice (Zizania aquatica), chicory (Cichorium intrybus L.),       0-0.2
      endive (Cichorium endivia L.), ouzo
    Sapodilla fruit (Achras sapota L.)                                   Trace
    Aubergine (Solanum melongena L.), pistachio nut (roasted,            17.2
      Pistachia vera), arrack
    Nectarine                                                            < 0.01
                                                                                        

    From Maarse & Visscher (1994)
    Intake (g/kg bw per day) calculated as follows: 
    {[(annual volume, kg)  (1  109 mg/kg)  (1/60 kg bw)]/[population  0.6  
    365 days]}, where population (10%, 'eaters only') = 32  106 for Europe and 
    24  106 for the United States; 0.6 represents the assumption that only 60% 
    of the flavour volume was reported in the survey (National Academy of Sciences, 
    1987; International Organization of the Flavor Industry, 1995). Slight 
    variations may occur from rounding off.
    
    polysaccharides which contain pentose and hexose fragments. Furfural
    has been detected in a broad range of fruits and fruit juices, wines,
    whiskeys, coffee, and tea (Maarse et al., 1994). The highest
    concentrations of furfural in foods have been reported in cocoa and
    coffee (55-255 ppm), alcoholic beverages (1-33 ppm), and whole-grain
    bread (26 ppm). Furfuryl alcohol, which is readily converted to
    furfural  in vivo (Rice, 1972; Nomeir et al., 1992), has been found
    in the highest concentrations in heated skim milk (230 ppm) and coffee
    (90-881 ppm). The total potential daily  per capita intake of
    furfural and precursors of furfural (i.e. furfuryl alcohol and
    furfuryl esters) from consumption of foods in which they occur
    naturally (Stofberg & Grundschober, 1987) is approximately 0.3 mg/kg
    bw per day (i.e. about 300 g/kg bw per day) in the United States.
    Thus, the intake of furfural and furfuryl derivatives from use as
    flavouring substances represent 1-3% of the total intake.

    4.  COMMENTS

         In both humans and rodents, furfural is efficiently metabolized
    by oxidation of the aldehyde function to furoic acid, most of  which
    is  conjugated with glycine and excreted. A minor proportion (< 5%)
    of furoic acid is condensed with acetyl coenzyme A to form
    furanacryloyl coenzyme A; the resulting furanacryloic acid is
    conjugated with glycine and excreted in the urine.  About 5% of
    [carboxyl-14C]-labelled furfural is eliminated by rats and mice as
    14C-carbon dioxide. The metabolic pathway, which could involve direct
    decarboxylation or epoxidation and ring opening, has not been defined.

         In short-term studies, furfural was clearly hepatotoxic at doses
    > 90 mg/kg bw per day in male rats and > 150 mg/kg per day in
    mice. Minor changes reported in the livers of male rats given furfural
    at lower doses by gavage in corn oil for 13 weeks were also present to
    a lesser extent in vehicle controls. In a study of developmental
    toxicity, furfural was not toxic to rats at 150 mg/kg bw per day, the
    highest dose tolerated.

         Furfural, like other aldehydes such as endogenous acetaldehyde,
    is a reactive aldehyde; it is reported to bind to soluble proteins and
    protein components of cell membranes. Various metabolic processes
    (i.e. oxidation, conjugation, and condensation) effectively eliminate
    the reactive aldehyde functional group when these metabolic pathways
    are not saturated by high, unphysiological doses.

         Furfural was not genotoxic in most tests  in vitro. Positive
    results were reported at relatively high concentrations in only three
    of 16 assays for reverse mutation in  Salmonella typhimurium and in
    one of three  rec assays in  B. subtilis. A few chromosomal
    aberrations were seen in Chinese hamster ovary cells exposed to
    relatively high concentrations. Sister chromatid exchanges and forward
    mutations were induced in mouse lymphoma cells. This weak activity of
    furfural  in vitro in some tests for genotoxicity might be explained
    by its aldehyde reactivity. Recent studies of unscheduled DNA
    synthesis in hepatocytes of rats treated  in vivo and in human liver

    slices gave negative results. Negative results were found in tests for
    genotoxicity  in vivo, except in  Drosophila injected with furfural. 

         In a two-year study of carcinogenicity in rats given furfural in
    corn oil by gavage at doses of 30 or 60 mg/kg bw per day, bile-duct
    hyperplasia and cholangiofibrosis were seen in essentially all rats,
    the controls having the highest incidence. Mild hepatocellular
    necrosis was seen in all groups, however, at higher rates in males in
    all treated groups. Two cholangio-carcinomas were observed in males at
    the high dose, but the incidence was not statistically significant.

         In a study of carcinogenicity in mice, the combined incidence of
    adenomas and carcinomas of the liver (64%) was significantly
     (p < 0.01) increased in males at the high dose (175 mg/kg bw per
    day) but not in females. Hepatocellular adenomas and carcinomas also
    occurred in the controls and in animals at the low (50 mg/kg bw per
    day) and intermediate (100 mg/kg bw per day) doses, at incidences of
    34-44% in males and 6-14% in females. The incidences in historical
    controls were 38% (14-50%) in males and 6.2% (0-30%) in females.
    Hepatotoxicity, manifested by features such as focal and multifocal
    necrosis and chronic inflammation, was seen in all groups, including
    the controls, but was considerably more frequent in mice at the high
    dose.

         Studies of oncogene activation in samples of liver tumours from
    treated mice in the study of carcinogenicity revealed some differences
    in the pattern of mutations from those in liver tumours of controls.
    As it was not possible to identify the animals from which the tumours
    originated and since hepatoxicity was seen at the intermediate and
    high doses, it was not possible to determine whether a direct
    genotoxic event or a secondary genotoxic pathway is involved.

    5.  EVALUATION

         Because of concern about the tumours observed in male mice given
    furfural and the fact that no NOEL was identified for hepatotoxicity
    in rats, the Committee was unable to allocate an ADI. Before reviewing
    the substance again, the Committee would wish to review the results of
    studies of DNA binding or adduct formation  in vivo to clarify
    whether furfural interacts with DNA in mice and of a 90-day study of
    toxicity in rats to identify a NOEL for hepatotoxicity.

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    Boyland, E. (1940) Experiments on the chemotherapy of cancer. 4.
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    Dillon, D.M., McGregor, D.B., Combes, R.D. & Zeiger, E. (1992)
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    See Also:
       Toxicological Abbreviations
       Furfural (ICSC)
       Furfural (WHO Food Additives Series 30)
       Furfural (WHO Food Additives Series 46)
       FURFURAL (JECFA Evaluation)
       Furfural (IARC Summary & Evaluation, Volume 63, 1995)