IPCS INCHEM Home

    DIMETHYLDICARBONATE (DMDC)

    First draft prepared by Dr M. Younes
    Max von Pettenkofer Institute of the Federal Health Office,
    Berlin, Germany.

    1.  EXPLANATION

         Dimethyldicarbonate (DMDC) has not been previously evaluated
    for acceptable daily intake by the Joint FAO/WHO Expert Committee on
    Food Additives.  DMDC is used as a cold sterilization agent for soft
    drinks and wines.  It has a broad antimicrobial range of action
    against yeasts, mould fungi, and bacteria. DMDC is unstable in
    aqueous solution and breaks down almost immediately after addition
    to beverages.  The principal breakdown products in wine and aqueous
    liquids are methanol and carbon dioxide.  Dimethylcarbonate (DMC)
    and methyl ethyl carbonate (MEC), as well as carbomethoxy adducts of
    amines, sugars, and fruit acids, are also formed in minor amounts. 
    In the presence of trace quantities of ammonia or ammonium ions
    (e.g. in wines), DMDC forms trace quantities of methylcarbamate
    (MC).  The data available on DMDC and DMDC-treated drinks, as well
    as the data on the breakdown products mentioned above, are
    summarized in the following monograph.

    DMDC AND DMDC-TREATED DRINKS

    2.  BIOLOGICAL DATA

    2.1  Biochemical effects

    2.1.1  Effects on enzymes and other biochemical parameters

         DMDC, like diethyldicarbonate, has a broad antimicrobial
    activity when added to drinks.  The inactivation of microorganisms
    proved to be strongly related to the inactivation of enzymes by
    protein modification, mainly through reaction with nucleophilic
    groups (imidazoles, amines, thiols) (Ough, 1983).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with DMDC is shown in
    Table 1.  

        Table 1:  Acute toxicity studies
                                                                                          
    Species     Sex       Route              LD50        LC50             Reference
                                             (mg/kg)     (mg/m3)          
                                                                                          
    Mouse       M         Oral               906.5                        Steinhoff, 1974

                F         Oral               752.7                        Steinhoff, 1974

                M         I.P.               44.9                         Steinhoff, 1974

                F         I.P.               44.5                         Steinhoff, 1974

                M         Inhal. (4h)                    850              Kimmerle, 1972

                F         Inhal. (4h)                    >1477            Kimmerle, 1972

    Rat         M         Oral               496.5                        Steinhoff, 1974

                F         Oral               334.6                        Steinhoff, 1974

                M         I.P.               186.0                        Steinhoff, 1974

                F         I.P.               186.0                        Steinhoff, 1974

                M         Inhal. (1h)                    approx. 2300     Kimmerle, 1972

                M         Inhal. (4h)                    520              Kimmerle, 1972
                                                                                          

    Table 1:  (contd)
                                                                                          
    Species     Sex       Route              LD50        LC50             Reference
                                             (mg/kg)     (mg/m3)          
                                                                                          
                M         Inhal (5x4h)                   >102             Kimmerle, 1972

                F         Inhal. (1h)                    >3017            Kimmerle, 1972

                F         Inhal. (4h)                    1350             Kimmerle, 1972

                F         Inhal. (5x4h)                  >102             Kimmerle, 1972

                                                                                          
    
    2.2.2  Short-term tests

    2.2.2.1  Rat

         The subchronic toxicity of DMDC-treated beverages was tested in
    Wistar (SPF) rats (28-32 days of age at the start of the
    experiment).  Groups of 15 male and 15 female rats received orange
    juice, black currant juice, beer (lager type), or wine (Riesling)
    without (control groups) or with 4000 mg/l DMDC instead of drinking
    water for a period of 3 months.  The animals were inspected daily. 
    Body weights and food consumption were recorded weekly, drink
    consumption daily.  Clinical chemical tests were performed on five
    rats of each sex for every group 1 and 3 months after the start of
    the experiment.  Haematological parameters investigated were
    haemoglobin concentration, haematocrit, erythrocyte and leukocyte
    counts, mean cell haemoglobin (MCH), mean cell volume (MCV), the
    reticulocyte count, differential blood count, and, at the end of the
    experiment, thromboblastin time.  Liver function was assessed by
    measurement of the activities of alkaline phosphatase (AP),
    glutamate-pyruvate-transaminase (GPT), glutamate-oxalacetate-
    transaminase (GOT), and glutamate dehydrogenase (GLDH), as well as
    the concentrations of bilirubin in the blood plasma.  Kidney
    function was assessed by measurement of plasma urea and creatinine
    concentrations as well as by urinalysis.  Blood glucose and
    cholesterol levels were also  determined.  Surviving animals were
    autopsied at the end of the experiment, animals that died earlier
    were autopsied immediately.  Samples from 22 different tissues were
    fixed for histopathological examinations.

         Animals receiving DMDC showed no differences in appearance,
    behaviour, or consumption of drink or food as compared to the
    corresponding controls.  Body weight gain was not significantly
    different in animals receiving DMDC in comparison with the
    respective controls.  Application of DMDC did not affect mortality. 

    No significant differences in haematological parameters or in
    parameters of liver and kidney function were observed between rats
    receiving DMDC and their controls.  Also, blood glucose and
    cholesterol concentrations remained within physiological ranges for
    all groups.

         On autopsy, no pathological changes attributable to the
    treatment were observed in any group.  Only slight and randomly
    distributed differences in organ weights were seen. 
    Histomorphological examination of fixed organs revealed no changes
    which were attributable to the administration of DMDC-treated
    drinks.  It was concluded that 4000 mg DMDC/l in fruit juice or
    alcoholic beverage was tolerated by rats without signs of toxicity
    (Löser, 1978). 

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Rat

         Fifty male and 50 female Wistar (SPF) rats (6-7 weeks old at
    the start of the experiment) received orange juice supplemented with
    4000 ppm DMDC as the only liquid over a period of 30 months.  Two
    control groups of the same size received either tap water (water
    controls) or untreated orange juice (juice controls).  Satellite
    groups consisting of 15 male and 15 female rats each were treated
    similarly and sacrificed after 6 months for interim examination. 
    The animals were inspected daily for signs of toxicity.  Body
    weights, as well as feed and consumption were recorded weekly. 
    Clinical laboratory tests were done 6, 12, 18, 24, and 30 months
    after the start of the experiment and covered haematological
    parameters, clinical chemistry parameters, and urinalysis.  At the
    end of the experiment, all surviving animals were killed and
    necropsied, as were the animals killed after 6 months and those that
    died or were killed in moribund state during the experiment.  For
    histopathological examinations, organs were fixed in buffered 10%
    formalin.  Additional liver specimens of rats killed after 6 months
    were fixed for fat determination.

         No differences in appearance or behaviour due to consumption of
    DMDC-treated juice was observed.  In both groups receiving orange
    juice (treated or untreated), feed consumption was lower and liquid
    intake higher than in the water control group.  No major differences
    in body weight gain were observed which indicated that animals
    drinking orange juice covered a part of their caloric intake via the
    juice.  No differences in the mortality rates were seen in any
    groups.  Few and randomly distributed changes in haematological
    parameters were observed in both groups consuming orange juice. 
    However, they were not considered to be of toxicological relevance. 
    Clinical chemical tests and urinalysis showed an increase in urinary
    protein content in the males of the DMDC/orange juice group after 30
    months.

         In the animals killed after 6 months, brains and adrenal glands
    of males consuming treated juice were heavier than those of males in
    the juice control group.  In both orange juice consuming groups,
    occasional elevation of pancreas weight was observed.  After 30
    months, significantly higher absolute weights of liver, kidney, and
    adrenal glands were seen in males of the juice control group. 
    However, no differences in the relative weights of these organs were
    observed.  In both groups which received orange juice, an absolute
    and relative pancreas weight was observed.  Gross macroscopic
    examinations revealed no toxic effects attributable to the intake of
    DMDC-treated orange juice.  Histopathological examinations revealed
    no treatment-related lesions and no carcinogenic effects of DMDC-
    treated juice.  It was concluded that under the described
    conditions, the administration of orange juice treated with 4000 ppm
    DMDC was tolerated with no indication of damage (Löser  et al.,
    1983).

         The chronic toxicity of wine treated with DMDC was investigated
    in Wistar (SPF) rats (6-7 weeks old at the start of the experiment). 
    Groups of 50 male and 50 female rats were given either tap water
    (water control group), untreated wine (wine control group), or wine
    treated with 4000 ppm DMDC (treatment group) as the only source of
    liquid for 30 months.  Additional groups of 15 rats of each sex were
    treated similarly, but were sacrificed after 12 months.  Animals
    were inspected daily.  Body weight was determined weekly for the
    first 6 months and biweekly thereafter.  Feed and liquid consumption
    were checked weekly.  Clinical laboratory investigations comprising
    haematological and clinical chemical investigations both in plasma
    and in urine were performed on 10 male and 10 female animals from
    each group 6, 12, 18, 24, and 30 months after the start of the
    experiment.  Autopsies were carried out on all animals that died
    during the experiment, as well as on all animals after 12 or 30
    months. The weights of thyroid, pancreas, heart, lungs, liver,
    spleen, brain, kidneys, adrenals, and testicles or ovaries were
    determined.  Samples of 29 organs and tissues, as well as any organs
    showing gross alterations, were fixed in buffered 10% formaldehyde. 
    Histological examination was performed on all material, including
    special staining techniques for tumour classification, as well as
    fat detection in frozen liver sections from 10 animals per group and
    sex.

         No differences in appearance, behaviour, vitality, or coat
    quality were observed between rats of the three groups.  Feed
    consumption was lower in both wine-treated groups, but no
    differences were observed between the treatment group and the wine
    control group.  Liquid consumption was higher in the test group as
    compared to both control groups.  No major differences in body
    weight gain were observed.  Among the haematological parameters
    measured, wine-consuming rats (both treated and untreated) had
    slightly lower leukocyte counts than the water control group.  The

    males of the treatment group displayed an elevation of the
    polymorphonuclear  neutrophil fraction and a reduced lymphocyte
    fraction after 18 months.  In isolated cases, polychromasia was
    observed in the treatment group, but this was also seen in the wine
    control group at the same time and in all animals at the end of the
    study.  These changes were regarded as incidental and of no
    toxicological importance.  No other haematologic effects were
    observed.

         Plasma enzyme activities and plasma substrate concentrations
    showed no toxicologically significant differences between the three
    groups.  All values lay within the range of biological variation. 
    Urinalysis also failed to show marked differences between groups. 
    Also, blood glucose and cholesterol levels were within the normal
    range of variation of these parameters.  No differences in
    histological findings including the nature, frequency, and time of
    occurrence of benign and malignant tumours discovered were observed
    between rats receiving DMDC-treated wine and either the wine control
    or the water control group.  It was concluded that under the
    described conditions, wine treated with 4000 ppm DMDC was tolerated
    by rats for 30 months without toxic effects (Eiben  et al., 1984).

    2.2.3.2  Dog

         The long term toxicity of DMDC-treated orange juice was
    examined in a one-year oral study in dogs.  Three groups of 6 male
    and 6 female beagle dogs, 12-15 weeks old, received as drinking
    fluid either orange juice treated with 4000 ppm DMDC (test group),
    untreated orange juice (juice control group), or tap water (water
    control group).  The animals were checked daily for health condition
    and behaviour.  Ophthalmoscopic examination was conducted on all
    dogs in week 0 (before the start of the experiment) as well as in
    weeks 17, 27, and 51.  A number of reflexes were tested at weeks 26
    and 52.  Body weight and feed and liquid intake were recorded
    weekly.  Haematologic and clinical chemical parameters were
    determined in blood obtained from the cephalic vein of all dogs in
    weeks 0, 6, 12, 26, and 51.  Urine was collected by cannulation of
    the bladder in weeks 0, 7, 11, 27, and 50 for urinalysis. At the end
    of the 52nd week of exposure, all animals were killed and
    necropsied.  Tissue samples were fixed for histopathological
    examination.

         No abnormalities in general health and behaviour or in
    neurological parameters attributable to the consumption of DMDC-
    treated juice were observed.  No statistically significant
    differences in body weight gain were seen between the DMDC-treated
    juice group and the juice control group.  Both groups showed
    slightly lower body weight gains as compared to the water control
    group.  The same was true for feed and liquid intake.

         No differences of toxicological significance in haematological
    findings were observed between the test group and the juice control
    group, except for a statistically significant increase in eosinophil
    number in females of the test group on Day 180.  A tendency towards
    higher values of haemoglobin concentration, red blood cell count,
    packed cell volume, and reticulocytes was evident in both juice
    drinking groups as compared to the water control group.  These and
    other incidental changes were considered to be of no toxicological
    relevance.  Among the clinical chemical parameters, higher values
    for alkaline phosphatase activity and cholesterol concentration, and
    lower urea concentrations were found in both juice-consuming groups. 
    Changes in plasma protein fractions after electrophoresis were
    observed, but these were not consistently present and were evident
    before the start of the experiment already.  No treatment related
    effects were seen in the urine.  On autopsy, no statistically
    significant differences in absolute or relative organ weights among
    the three groups were recorded.  Gross and microscopic examinations
    of all organs were not available (Lina & Till, 1983).

    2.2.4  Reproduction study

    2.2.4.1  Rat

         Groups of 10 male and female Wistar (SPF) rats (5-6 weeks old
    at the beginning of the experiment) received either tap water
    (control group), orange juice (juice control group) or orange juice
    treated with 4000 ppm DMDC (treatment group) as their sole source of
    liquid.  Weight development as well as feed and liquid intake were
    determined weekly.  F0 litters were treated for 70 days before
    pairing twice in succession.  While the F1a litters were killed
    after 4 weeks, males and females from the F1b litters were selected
    to form the F1 generation and were treated until the age of 100
    days before the first and second mating took place as in the case of
    the F0 rats.  The general condition of the F0 animals was
    unaffected by treatment.  Body weight gain was slightly lower in
    both groups receiving orange juice until week 6, after which only
    males from the juice control group continued to display a lower body
    weight gain.  No differences in liquid and feed consumption were
    seen between the treatment and the juice control groups.  No
    differences in reproduction parameters (fertility index, gestation
    index, viability index, lactation index, insemination index, litter
    size, sex ratio) were observed between the treatment group and the
    control groups. Also, no differences in the rate of mortality,
    behaviour,and appearance between rats in the different groups were
    evident.

         On autopsy of deceased or sacrificed parent and young rats, no
    evidence of any treatment related organ changes were observed, nor
    were any changes in organ weights attributable to treatment noted. 
    Histopathological examinations of organs of F1b-parent and F2b

    offspring, as well as those of deceased animals, did not reveal any
    damage due to consumption of DMDC-treated orange juice.  Thus, no
    adverse effects on reproduction resulted from the consumption of
    orange juice treated with 4000 ppm DMDC (Eiben  et al., 1983).

    2.2.5  Special study on embryotoxicity/teratogenicity

    2.2.5.1  Rat

         The potential of DMDC-treated orange juice to induce pre-
    implantation damage or to exert embryotoxic and/or teratogenic
    effects was investigated in FB 30 rat (Long Evans type).  Two groups
    of 25 female rats (2.5-3.5 months of age) were mated with 3-6 month
    old males by placing one male with two females in cages.  From day 0
    to day 20 of pregnancy the females were given orange juice only
    (control group) or orange juice treated with 4000 ppm DMDC (test
    group) instead of drinking water.  On day 20 of pregnancy, Caesarian
    sections were performed and foetuses were removed and examined.

         The treated female animals showed no adverse effects due to
    consumption of DMDC-treated orange juice.  Inspection of the litter
    showed no differences between the test and the control group with
    respect to implantation quota, litter size, reabsorption quota,
    average weight of foetuses, average weight of placenta, frequency of
    underdeveloped foetuses, frequency of foetuses with slight
    deviations in skeletal development, and deformation quota.  Thus,
    under these experimental conditions, DMDC-treated orange juice had
    no embryotoxic or teratogenic effect (Shlüter, 1980).

        2.2.5.2  Special studies on genotoxicity

    Table 2:  Results of genotoxicity assays on DMDC

                                                                                     
    Test system   Test Object         Concentration        Results       Reference
                                                                                     

    Ames Test     S. typhimurium      1.6-200 śg/plate     Negative      Herbold, 1978
    (1)           TA98, TA100,                                           
                  TA1535,                                                
                  TA1537                                                 


                                                                                     

    (1) Both with and without rat liver S-9 fraction.

    Table 3:  Results of genotoxicity assays on DMDC-treated drinks (1)
                                                                                            
    Test system        Test object       Concencentration      Results        Reference
                                                                                            

    Ames test (2)      S. typhimurium    25-500                Negative       Herbold, 1980
                       TA98, TA100,      µg/plate
                       TA1535, TA1537

    Ames test (2)      S. typhimurium    250-1000              Negative       Herbold, 1989a
                       TA98, TA100,      µg/plate
                       TA1535, TA1537

    Micronucleus       Mouse, bone       50 ml/kg              Negative       Herbold, 1989b
    test               marrow (in        p.o. 24, 
                       vivo)             48, 72 h

                                                                                            

    (1)  Orange juice treated with 4,000 ppm DMDC
    (2)  Both with and without rat liver S-9 fraction
    
    2.2.6  Special studies on skin irritation

    2.2.6.1  Rat

        Percutaneous application of 1000 µl/kg body weight of DMDC was
    tolerated by male and female Wistar II rats without any symptoms. 
    Cutaneous absorption was very low (Kimmerle, 1972).

    2.2.6.2  Rabbit

        Attachment of small pieces of wool holding 50 µl DMDC to the
    skin of rabbits caused swelling and reddening, which were still
    visible after 7 days.  Introduction of DMDC into the conjunctival
    sac of rabbits caused considerable irritation.  The cornea was still
    entirely cloudy after 7 days (Kimmerle, 1972).

        The skin-irritant effects of DMDC were investigated in 6 white
    New Zealand rabbits of both sexes with body weights of 3-4 kg. 
    Approximately 0.5 ml of the substance were applied to the shaved
    skin of every animal.  Exposure times were 30 min or 4 h.  At the
    end of the exposure period, skin areas were washed and dried.  Skin
    changes were recorded at 24, 48, and 72 h, as well as 7 days after
    the start of the exposure.  After an exposure for both 30 min and 4
    h, scale formation and necroses passing beyond the application area
    were observed.  The skin reactions were not reversible within the 7-
    day follow-up observation period.  Consequently, DMDC proved to be
    highly irritant and corrosive to rabbit skin (Pauluhn, 1982).

    2.3  Observations in humans

        No information available.

    METHANOL

    1.  EXPLANATION

        DMDC was added to wine and model solutions, and the methanol
    produced by hydrolysis of DMDC was measured.  The levels produced
    were linear with dose.  Also, the levels of ethyl methyl carbonate
    formed were found to be linear with substrate concentration and in
    the low mg/l range (Stafford & Ough, 1976).

        Hydrolysis of DMDC leads to the formation of 2 moles of methanol
    and 2 moles of CO2 per mole of DMDC.  On a weight basis, this
    corresponds to 47.8 mg of methanol for every 100 mg of DMDC.  At
    DMDC dosage of 250 mg/l, the methanol content would rise maximally
    by 119 mg/l.  Natural fruit juices contain up to 230 mg/l of
    methanol in their natural state, while wine may contain up to 350
    mg/l.  Assuming the consumption of a large amount of a drink treated
    with 250 mg/l DMDC (e.g., 21), the drink having an abnormally high
    natural methanol content (e.g. 230 mg/l), the total amount of
    ingested methanol would be approximately 700 mg/person
    (corresponding to 10 mg/kg on average).  The lowest toxic dose of
    methanol in primates, i.e., the dose showing evidence of metabolic
    acidosis, is 1000 mg/kg body weight.  Thus, the methanol content of
    the drink would be smaller than this value by a factor of 100.  The
    most alarming toxic effect of methanol reported in the working
    environment was impairment of vision at atmospheric concentrations
    of 1200 ml/m3 (=1560 mg/m3) air and over.  This corresponds to an
    intake of 171 mg/kg/day in man and is 17 times the estimated amount
    of methanol intake from DMDC-treated drinks.  Considering the fact
    that humans with normal eating habits metabolize 1000 to 2000 mg of
    methanol per day, it was concluded that there is a large margin of
    safety between the methanol intake and the amount which can be
    safely ingested (Bayer AG, 1987).

        The large body of data available on metabolism and toxicity of
    methanol cannot be specifically reviewed in this evaluation.

    DIMETHYL CARBONATE (DMC) AND
    METHYL ETHYL CARBONATE (MEC)

    1.  EXPLANATION

        During the course of purification of DMDC, dimethyl carbonate
    (DMC) may be formed through release of one mole of CO2 per mole of
    DMDC both under normal and reduced pressure.  Quality control
    specifications require that DMC be present at no higher a level than
    0.2%.  Thus, at a level of addition of 250 mg DMDC/l of a beverage,
    no more than 0.5 mg of DMC would be present.

        Methyl ethyl carbonate (MEC) is formed when DMDC is added to
    beverages with a minimum content of 1% (v/v) ethanol.  In addition,
    DMDC added to an ethanol containing beverage reacts with each
    percent by volume of ethanol present to yield MEC.  Thus, if 250 mg
    of DMDC are added to one liter of a beverage containing 11% (v/v) of
    ethanol, approximately 1.5 mg of MEC would be formed.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Biotransformation

        Incubation of MEC and DMC with liver or kidney homogenates of
    porcine or human origin resulted in hydrolysis of both compounds,
    MEC being more readily susceptible to hydrolytic decomposition
    (Rauenbusch, 1974).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

    Table 4:  Acute toxicity data on DMC

                                                                       
    Species     Sex      Route        LD50          Reference
                                   (mg/kg b.w.)     
                                                                       

    Mouse       F        oral      10 163           Steinhoff, 1973a

                F        i.p.       3 222           Steinhoff, 1973a

    Rat         F        oral      10 349           Steinhoff, 1973a

                F        i.p.       2 848           Steinhoff, 1973a
                                                                       

    Table 5:  Acute toxicity data on MEC


                                                            
    Species   Sex    Route       LD50       Reference
                              (mg/kg b.w.)
                                                            

    Mouse     F      oral       >15 000     Steinhoff, 1973b

              F      i.p.         3 637     Steinhoff, 1973b

    Rat       F      oral       >15 000     Steinhoff, 1973b

              F      i.p.         2 885     Steinhoff, 1973b

                                                            

    2.2.2  Short-term studies

    2.2.2.1  Rat (DMC)

        Groups of 20 male and 20 female Wistar (SPF) rats (4-5 weeks of
    age at the start of the experiment) received dimethylcarbonate (DMC)
    in their drinking water at doses of 0 (control group), 0.1, 0.3, or
    1.0% for 3 months.  Doses up to and including 1.0% DMC had no effect
    on behaviour or mortality of male or female rats.  Body weight gain
    was also not influenced by DMC.  Clinical laboratory investigations
    were carried out 1 and 3 months after the start of the experiment in
    5 male and 5 female rats of each group.  

        Haematological investigations revealed no adverse effects of DMC
    at any dose levels.  Clinical historical chemical determinations
    revealed no marked deviations from the values found in control
    groups and were all within the biological range.  Urinalyses carried
    out on urines from 5 male and 5 female rats of each group at 1 and 3
    months revealed no differences between control animals and dosed
    groups.

        Autopsies were performed on all animals which died during the
    study, as well as those which survived the 3 month treatment period. 
    The following organs were weighed: heart, lungs, thymus, liver,
    spleen, kidneys, adrenals, testes, and ovaries.  Treatment with DMC
    had no consistent effect on absolute or relative weights of these
    organs.  Samples of 29 tissues and organs were fixed in Bouin's
    solution for histological examination.  In addition, the left lobe
    of each liver was fixed in formol-calcium for fat detection. 
    Histopathological investigations revealed that neither the males nor
    the females treated with doses up to and including 1.0% DMC showed
    any increased incidence of organ changes.  Also, no substance-

    related increase in hepatic fat content was observed.  It was
    concluded that DMC was tolerated by rats for 3 months without damage
    up to and including a dosage of 1.0% in drinking water (Eiben  et
     al., 1982).

    2.2.2.2.  Rat (MEC)

        Groups consisting of 20 male and 20 female Wistar (SPF) rats
    (28-32 days of age) each were given methyl ethyl carbonate in their
    drinking water at concentrations of 0 (controls), 0.1, 0.3, or 1.0%
    over a period of 3 months.  The rats were inspected daily for
    clinical signs of toxicity and body weight, as well as food and
    drink consumption, which were recorded weekly.  MEC had no effect on
    body weight gain or mortality.  Haematological examination carried
    out on 5 male and 5 female rats of each group 1 and 3 months after
    the start of the experiment revealed no toxic effect of MEC. 
    Clinical chemical parameters and results of urinalyses gave no
    indication of liver or kidney toxicity.  Also, blood glucose and
    cholesterol concentrations were not influenced by MEC.  All animals
    were autopsied at the end of the experiment and organs were examined
    macroscopically.  No dose related effects on absolute or relative
    organ weights were observed.  Histopathological examinations of
    various organs did not reveal any morphological alteration  or
    variation from normal that was considered to be of toxicological
    significance.  It was concluded that methyl ethyl carbonate at
    concentrations up to and including 1.0% was tolerated by rats for
    three months without any adverse effect (Löser, 1973).

    2.2.3  Special study on embryotoxicity/teratogenicity

    2.2.3.1  Rat

        Groups of pregnant female FB 30 (Long Evans type) rats were
    given methyl ethyl carbonate in their drinking water at doses of 0%
    (controls), 0.01%, 0.1%, or 1.0% from day 6 to day 15, inclusive, of
    gestation.  On day 20 of gestation, all animals were killed for
    examination of their uterine contents.  None of the females showed
    any toxic response, but the liquid intake was lower in the highest
    dose group.  Also, body weight gain was slightly lower in dosed
    animals.  The numbers of implantation sites, resorptions, and viable
    young, as well as foetal and placental weights, were not influenced
    by the treatment.  Skeletal abnormalities were found in 4 fetuses
    and were randomly distributed among all groups.  No treatment-
    related malformations were observed.  The author concluded that MEC
    at doses of 1.0% and lower had no embryotoxic or teratogenic effects
    (Machemer, 1976).

    3.  Observations in humans

        No information available.

    CARBOXYMETHYLATION PRODUCTS

    1.  EXPLANATION

        DMDC added to beverages may form side products as a result of
    reaction with polyphenols, tannins, and amino acids.  Most of these
    adducts result from carboxymethylation of amino or hydroxy groups. 
    These adducts constitute, in general, no more than approximately 4
    mg/l of bound DMDC, when the latter is added at levels up to 250
    mg/l.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

        Upon oral administration of N-carbomethoxy alanine and N-
    carbomethoxy proline to rats, a high percentage of the dose was
    eliminated unchanged in the urine.  Enzymolysis of N-carbomethoxy
    alanine and liberation of the amino acid was observed with rat liver
    homogenates, while N-carbomethyoxy proline was more resistant
    towards enzymolysis (Schmidt, 1978).

    2.1.2  Biotransformation

        The hydrolytic decomposition of carbomethoxy compounds formed as
    reaction products of DMDC with amino acids, phenols, and lactate was
    investigated using homogenates of pig and human liver, as well as
    homogenates of pig and human kidney, as enzyme sources.  Most
    carbomethoxy compounds were easily hydrolyzed yielding the compounds
    from which they were formed.  Among the amino acid derivatives,
    carbomethoxy proline, di-carbomethoxy cystine, E-carbomethoxy
    lysine, and the aromatic amino acid derivatives were hydrolyzed very
    slowly and in part yielded different reaction products than the
    parent amino acids.  Carboxymethylated catechols were only partly
    hydrolyzed (Rauenbusch, 1974).

        2.2  Toxicological studies

    2.2.1  Acute toxicity studies

    Table 6
                                                                             
    Species      Sex      Route             LD50              Reference
                                         (mg/kg b.w.)
                                                                             

    N-carbomethoxy-tri-gallic acid:
    Mouse        F        oral                7 097           Steinhoff, 1973c

    N-carbomethoxy-glycine:
    Mouse        F        oral                6 275           Steinhoff, 1973c
    Rat          F        oral       approx.  6 000-7 000     Steinhoff, 1973c

    N-carbomethoxy-glutamic acid:
    Mouse        F        oral                6 390           Steinhoff, 1973c
                 F        oral                5 345*          Steinhoff, 1973c
    Rat          F        oral               >8 000           Steinhoff, 1973c
                 F        oral              >15 000*          Steinhoff, 1973c

    N-carbomethoxy-alanine:
    Mouse        F        oral                5 534           Steinhoff, 1973c
                 F        oral                3 707*          Steinhoff, 1973c
    Rat          F        oral        approx. 6 000-6 500     Steinhoff, 1973c
                 F        oral                7 102*          Steinhoff, 1973c

    N-carbomethoxyproline:
    Mouse        F        oral                9 115           Steinhoff, 1973c
    Rat          F        oral       approx. 12 000           Steinhoff, 1973c

    N-carbomethoxy-asparagine:
    Mouse        F        oral              >15 000           Steinhoff, 1973c
    Rat          F        oral       approx. 15 000           Steinhoff, 1973c

    N-carbomethoxy-proline:
    Mouse        F        oral                5 403           Steinhoff, 1973c
    Rat          F        oral               >6 000           Steinhoff, 1973c

    N-carbomethoxy-di-cysteine:
    Mouse        F        oral                6 397           Steinhoff, 1973c
    Rat          F        oral              >10 000           Steinhoff, 1973c

    N-carbomethoxy-phenylalanine:
    Mouse        F        oral                6 926           Steinhoff, 1973c

                                                                             

    Table 6 (contd)
                                                                             
    Species      Sex      Route             LD50              Reference
                                         (mg/kg b.w.)
                                                                             

    N-carbomethoxy-arginine:
    Mouse        F        oral              >15 000           Steinhoff, 1973c
    Rat          F        oral              >15 000           Steinhoff, 1973c

    N-carbomethoxy-leucine:
    Mouse        F        oral                4 633           Steinhoff, 1973c
    Rat          F        oral               >5 000           Steinhoff, 1973c

    N-carbomethoxy-monocysteine:
    Mouse        F        oral                4 733           Steinhoff, 1973c
    Rat          F        oral               >4 000           Steinhoff, 1973c
                                                                             

    *  Repeat study using a different batch of the same material.
    
    METHYL CARBAMATE (MC)

    1.  EXPLANATION

        Methyl carbamate (MC) is formed upon hydrolysis of DMDC in the
    presence of ammonium ions, which may be present in some wines and
    fruit juices.  In an experimental study, MC formed from DMDC added
    to model solutions and wines containing various amounts of ammonia
    at different pH-values was detected (recovery of MC by the
    analytical method employed was 51%).  MC-formation increased with
    increasing NH3-concentration and with increasing pH-value.  Under
    the most extreme conditions in normal commercial practices (pH <
    to 3.75; NH3-concentration < to 20 mg/l) less than 10 µg of MC
    per 1 would be formed following the addition of DMDC at 100 mg/l
    (Ough & Langbehn, 1976).

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

        Rats (100-200 g body weight) were dosed intraperitoneally with
    500 mg/kg of methylcarbamate.  Urine was collected for estimation of
    excretion rates.  7.25% of the dose was excreted within 24 h.  The
    authors concluded that MC was not concentrated in the kidneys as the
    proportion of the dose excreted in 24 h was of the same order as the
    ratio of urine excreted in 24 h to the total body water.  The
    distribution of methyl carbamate given i.p. to normal rats as well
    as rats with Walker carcinomata at doses of 500 or 1000 mg/kg body
    weight was studied.  Concentrations of methyl carbamate were
    determined in blood, lungs, and liver for 144 h.  MC was fairly
    quickly distributed in the body, but the concentrations in the
    tissues examined fell slowly.  An apparent elimination half-life of
    24 h was estimated (Boyland & Papadopoulos, 1952).

        In a comparative study designed to investigate the urinary
    excretion of carbamic acid esters and their N-hydroxy derivatives,
    methyl carbamate was injected  intraperitoneally (20% solution in
    water, w/v) into female rats in single doses of 0.3-1.0 g/kg.  Their
    urine was collected from 0-24 h and from 24-48 h following
    treatment.  In the first day, 3.3% of the dose was excreted
    unchanged, 0.008% as N-OH derivative, while in the second day 4.9%
    of the dose was excreted unchanged and 0.06% as the N-OH derivative. 
    I.P.-injection with 0.02-0.4 g/kg N-hydroxymethyl carbamate (5%
    solution in water, w/v) led to a urinary excretion of 29% N-
    hydroxymethyl carbamate and 4.1% MC in the first 24 h, and 3.9% N-
    hydroxylated and 5.7% MC in the second 24 h-collection period.  This
    indicates that N-hydroxylation takes place, but that dehydroxylation
    also occurs (Boyland & Nery, 1965).

        The renal elimination of methyl carbamate and its effect on
    activities of some xenobiotic-metabolizing enzymes was investigated
    in rats.  Groups of 5 male Wistar rats, as well as groups of 5 male
    Fischer 344 rats (13-15 weeks of age in both cases) were
    administered a single oral dose of 1000 mg/kg MC or received 7 daily
    doses of 800 mg/kg MC by gavage.  They were placed in metabolic
    cages and urine was collected in periods of 24 h.  Following a
    single oral administration, 16.2% of the MC dose was eliminated in
    the first 3 days in the urine of Wistar rats.  Excretion was only
    slightly lower in Fischer 344 rats (15.5% of dose).  Repeated dosing
    with MC over 7 days resulted in a gradual increase in the proportion
    of unchanged MC excreted renally reaching a value of 30% in Wistar
    rats and 32% in F 344 rats.  On all other days, Wistar rats excreted
    slightly higher amounts of MC with the urine.  These slight
    differences were interpreted as a cause of different hepatotoxic
    responses to MC in the two strains examined.

        The activities of the of the following enzymes was measured in
    the 10 000 g supernatants of liver homogenates obtained from control
    rats and from rats given 7x800 mg/kg MC:  7-ethoxycoumarin
    deethylase (EOD), aldrin epoxidase (ALD), biphenyl-4-hydroxylase
    (BH), epoxide hydrolase (EH), and GSH-transferase (GST).  Untreated
    Fischer rats displayed higher activities of BH and ALD and lower
    activities of EH and GST as compared to untreated Wistar rats. 
    Treatment with 7x800 mg/kg MC led to a slight decrease in hepatic
    ALD activity in Fischer rats and a slight increase in EOD activity
    in Wistar rats.  No other statistically significant differences were
    noted (Schmidt & Schmidt, 1987; Bomhard  et al., 1989).

        [Carbonyl-14C]-methyl carbamate was administered to male
    Fischer 344 rats and male B6C 3F1 mice orally at doses of 40, 400,
    and 1000 mg/kg, or i.v. at dose of 400 mg/kg (20 µCi/kg body weight
    in every case).  To study the metabolism to CO2, an i.v.-dose of
    0.4 mg/kg was applied.  Animals were housed in individual glass
    metabolism cages allowing for separate collection of urine, faeces,
    CO2 and other volatile compounds.  Although the initial
    distribution of MC was similar in both species, mice metabolized and
    cleared MC much more rapidly.  CO2 elimination accounted for 70% of
    the dose in 48 h in mice, but only for 18% of the dose in rats.  Of
    the material excreted in urine of both species, the parent compound
    accounted for 90%.  Less than 4% of the dose was excreted in the
    faeces of either species.  Repeated dosing with MC resulted in
    bioaccumulation of this compound in rats but not in mice, probably
    due to the lesser ability of the rat to metabolize MC.  Covalent
    binding of MC-derived radioactivity to DNA was detected in mouse
    liver, while binding to protein was found in muscle and liver tissue
    from both species (Ioannou  et al., 1988).

    2.1.2  Effects on enzymes and other biochemical parameters

        Male NMRI-mice (6-8 weeks of age) were injected with 375 mg/kg
    [3H]-methyl carbamate, 750 mg/kg [2-3H]-ethyl carbamate, or 375
    mg/kg [carboxy-14C]carbamate.  They were sacrificed 24 h later and
    liver RNA was isolated.  Radioactivity was incorporated into RNA. 
    Methyl carbamate led to a greater incorporation than ethyl
    carbamate.  Fractionation of RNA showed radioactive esters of
    cytosine-5-carboxylic acid to be present in the fractions where
    rapid RNA synthesis had occurred.  This, together with the finding
    that actinomycin D reduced the labelling of RNA, suggests that the
    cytosine-5-carboxylates were synthesized before incorporation into
    RNA chains.  Alternatively, a greater susceptibility of rapidly
    synthesized RNA to attack by chemically active metabolites of the
    carbamates might be postulated.  The methyl ester also caused a more
    rapid breakdown of RNA than ethyl carbamate.  Both esters of
    carbamic acid seemed to increase the RNA synthesis rate (Williams
     et al., 1971).

        The binding of [14C]-labelled methyl carbamate to the DNA of
    mouse liver, lung, and kidney as compared to ethyl, n-propyl, and n-
    butyl carbamates was studied.  Crackenbush mice were injected i.p.
    with 10 mg [Me-14C]-methyl carbamate (6 µCi) in sterile saline. 
    Animals were killed by cervical dislocation at different timepoints,
    the organs studied were excised and stored frozen.  DNA was
    extracted from the tissue and analyzed for bound radioactivity. 
    Radioactivity associated with DNA reached a maximum between 6 and 9
    h, and could still be detected after 24 h in liver and kidney.  Very
    little DNA-associated activity was seen in the lung.  However, the
    amount of binding was far below that seen with ethyl carbamate, and
    was considered to be of little relevance (Lawson & Pound, 1973).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

    Table 7
                                                                      
    Species    Sex      Route         LD50         Reference
                                  (mg/kg b.w.)
                                                                     

    Mouse      M        s.c.           4450        Pound, 1967

               ?        oral           6200        Srivalova, 1973

               M&F      oral          >2000        National Toxicology 
                                                   Program, 1987

    Rat        M&F      oral          >2000        National Toxicology 
                                                   Program, 1987

                                                                      

    2.3  Short-term studies

    2.3.1  Mouse

        Groups of 5 male and 5 female B6C 3F1 mice (7-8 weeks old) were
    administered 12 doses of 0, 250, 500, 1000, 2000, or 4000 mg/kg
    methyl carbamate p.o. in water over 16 days.  They were inspected
    twice daily and weighed on days 1, 8, and 15.  A necropsy was
    performed on all mice.  Histopathological examinations were carried
    out in the 1000 mg/kg dose groups.  Male mice that received 2000 or
    4000 mg/kg, female mice that received 4000 mg/kg, and 1/5 female
    mice that received 2000 mg/kg died.  No compound-related gross
    pathologic or histopathologic effects were seen in mice of either
    sex that received 1000 mg/kg methyl carbamate (National Toxicology
    Program, 1987).

        Groups of 10 male and 10 female B6C3F1 mice were treated orally
    on five days per week with methyl carbamate at doses of 0, 9.75,
    187.5, 375, 750, or 1500 mg/kg (males) or 0, 125, 250, 500, 1000, or
    2000 mg/kg (females) over a period of 13 weeks.  One of the female
    mice that received 2000 mg/kg died.  The dosed female mice had
    significantly greater liver weights than the vehicle controls. 
    Final mean body weights of all female mice were 5-10% lower than
    controls, that of the highest dose male mice 6% lower than controls. 
    Minimal to mild acute multifocal hepatocellular necroses and/or
    increased mitotic indices were observed in the livers of dosed male
    mice.  A hepatocellular adenoma was found in one high dose male
    mouse (National Toxicology Program, 1987; Quest  et al., 1987).

    2.3.2  Rat

        Methyl carbamate was administered orally in 5 ml/kg tap water at
    single doses of 0 (control group), 250, 500, and 1000 mg/kg to
    groups of 5 male Wistar (SPF) rats (initial body weight 199-207 g)
    daily for seven days.  The rats were inspected twice daily.   Body
    weight was determined at the beginning and at the end of the
    experiment, as were feed and water consumption.  One day after the
    final administration of the test compound, blood samples were
    collected for determination of plasma activities of alkaline
    phosphatase, GOT, and GPT and plasma concentrations of bilirubin,
    total protein, cholesterol, and triglycerides.  All animals were
    then sacrificed and autopsied.  Liver, testes, spleen, and sternum
    with bone marrow from all rats were fixed in 10% buffered
    formaldehyde, as were all organs with macroscopically visible
    changes.  Histopathological examinations were performed on the
    livers of all rats of the highest dose group.  In addition, frozen
    sections were stained for fat with Oil Red O.

        Rats treated with doses up to and including 500 mg/kg MC showed
    no changes in behaviour or physical appearance.  Rats of the highest
    dose group showed signs of poor condition from the fifth day
    onwards; in addition, lower water and feed (males only) intake was
    observed in this group.  Rats of the 1000 mg/kg dose group gained
    less weight than rats from other groups.  None of the rats died
    during the experiment.

        Apart from a decrease in alkaline phosphatase activity and
    triglyceride content in the plasma of the 1000 mg/kg dose group, no
    clinical chemical effects were observed.  Absolute and relative
    liver and spleen weights were significantly reduced in the 500 mg/kg
    and the 1000 mg/kg dose groups, while no changes were seen at a dose
    of 250 mg/kg MC and lower.  Gross pathological and histopathological
    examinations revealed no evidence of hepatotoxicity of MC up to and
    including a dose of 1000 mg/kg.  Roughness of the spleen surface was
    observed at doses of 500 mg/kg and above.  It was concluded that
    under these experimental conditions MC was tolerated at a dose of
    250 mg/kg and below without toxic effect (Bomhard & Kalina, 1984).

        Methyl carbamate was administered by gavage at daily doses of 0,
    250, 500, and 1000 mg/kg for seven days to groups of 5 male Fischer
    344 rats.  In addition, a group of 5 rats received N-methoxymethyl-
    O-methylurethane (MMU), the main impurity found in MC-preparations,
    at a daily dose of 100 mg/kg for seven days.  Rats were inspected
    daily.  Body weight, feed consumption and water intake were
    determined on days 0 and 7.  Blood samples were taken one day after
    the final dosing for clinical laboratory tests of plasma enzymes and
    substrates.  One day after the last administration of the test
    compound, rats were killed, dissected, and examined macroscopically. 
    Livers, spleens, and testes were weighed.  Liver, testes, spleen,

    and sternum (with bone marrow) of all rats, as well as all organs
    with visible changes were fixed in buffered formalin for
    histopathological examination.

        Animals of the 1000 mg/kg dose group displayed a poor general
    condition with impaired reflexes, uncoordinated movements, and
    weakness of rear extremities.  These signs were not seen in other
    dose groups.  Feed and water consumption, as well as body weight
    gain, were dose-dependently reduced at doses of 500 and 1000 mg/kg
    MC and slightly reduced in rats dosed with MMU.  None of the rats
    died during the experiment.  Plasma activities of GOT and GPT and
    concentrations of bilirubin and cholesterol were elevated, the
    activity of alkaline phosphatase and the concentrations of protein
    and triglycerides reduced in rats given 1000 mg/kg MC.  Changes of
    GOT, GPT, and cholesterol were also evident in the 500 mg/kg dose
    group, elevated GPT activities also in the 250 mg/kg dose group. 
    Treatment with MMU led to an elevation of plasma GPT activity and
    bilirubin and cholesterol concentrations.

        On autopsy, absolute and relative liver weights were reduced at
    a dose of 500 mg/kg and above, those of the spleen at a dose of 250
    mg/kg and above, and those of the testes of animals in the 1000
    mg/kg dose group.  Spleen weight (absolute and relative) was reduced
    in the MMU treatment group.  No substance-related gross
    morphological alterations were observed.  Histological examinations
    revealed treatment-related liver cell necroses (at 500 mg/kg and
    above), liver cell degeneration (at 500 mg/kg and above), increased
    fatty infiltration (1000 mg/kg), and iron-related pigmentation of
    Kupffer cells (1000 mg/kg).  In the livers of rats receiving 250
    mg/kg MC, only hyaline bodies were found sporadically in
    hepatocytes.  No treatment-related liver changes were seen in MMU-
    treated animals except for slight accumulation of iron-containing
    pigments in Kupffer cells. 

        Thus, clear MC-induced hepatotoxic effects were evident in
    Fischer 334 rats, which cannot be attributed to contamination with
    MMU, at all doses employed (Bomhard & Karbe, 1985a).

        Groups of 5 male and 5 female F344/N rats (6-8 weeks old) were
    administered 12 doses of 0, 250, 500, 1000, 2000, or 4000 mg/kg
    methylcarbamate in water by gavage over 16 days.  They were observed
    twice per day and were weighed on days 1, 8, and 15.  A necropsy was
    performed on male rats in the vehicle control, 1000, 2000, and 4000
    mg/kg dose groups, as well as on all female rats.  Histopathological
    examinations were carried out in the 500 mg/kg dose groups.  All
    rats dosed at 2000 or 4000 mg/kg and 3/5 male rats that received
    1000 mg/kg died during the study.  No compound-related gross
    pathologic or histopathologic effects were seen in rats of either
    sex that received 500 mg/kg (National Toxicology Program, 1987).

        Groups of 10 male and 10 female F 344/N rats (6-7 weeks old)
    received methyl carbamate in water by gavage at doses of 0, 50, 100,
    200, 400, or 800 mg/kg (males) or 0, 62.5, 125, 250, 500, or 1000
    mg/kg (females) on five days per week for 13 weeks.  Animals were
    checked twice daily, moribund animals were killed.  Survivors were
    killed after 13 weeks.  A necropsy was performed on all animals
    except those excessively autolyzed or cannibalized.  5/10 males that
    received 800 mg/kg and 4/10 females that received 1000 mg/kg died
    before the end of the experiment.  The final body weight of males
    was 14% or 31% lower than water controls for the 400 mg/kg or the
    800 mg/kg dose group, respectively.  Females of the 1000 mg/kg dose
    group had a final body weight that was 22% lower than that of
    controls.  Liver weight to body weight ratio was reduced in the two
    highest dose groups of males.  Compound-related lesions of the liver
    (toxic hepatitis), spleen (pigmented macrophages), bone marrow
    (atrophy), and testis (bilateral atrophy) were seen in the two
    highest dose groups of males and females (National Toxicology
    Program, 1987).

        Short-term toxicity of MC was assayed in F 344 rats.  MC was
    administered by gavage five times a week for 13 weeks to male (50,
    100, 200, 400, or 800 mg/kg) and female (62.5, 125, 250, 500, or
    1000 mg/kg) rats.  Each group consisted of 10 animals; control
    groups received distilled water (5 ml/kg).  Animals were observed
    twice daily for signs of morbidity or mortality and for clinical
    signs of toxicity.  All surviving animals were sacrificed at week
    13.  Complete histopathological examination was performed on all
    control and high dose group animals.  For those tissues where
    significant effects were seen at high dose levels, histopathological
    examinations were also conducted at progressively lower dose levels
    until a no-effect level was reached.  Mitotic index was determined
    on the liver of each rat (number of mitoses/100 hepatocytes); a
    minimum of 3000 hepatocytes were counted.   

        Deaths occurred at the highest doses in male (5/10) and female
    (4/10) rats.  Body weight gain was also slightly decreased in both
    groups.  Treatment resulted in dose-related lesions of the liver
    characterized by proliferative changes in hepatocytes consisting of
    foci of cellular alteration and frequent mitoses with atypical
    forms.  Toxic alterations consisted of focal hepatocellular
    necroses, pigmentation of Kupffer's cells, and the presence of
    basophilic inclusions resembling nuclei in hepatocyte cytoplasm. 
    Furthermore, testicular hyperplasia, bone marrow hyperplasia, and
    excessive pigmentation of the spleen were observed.  Liver changes
    were observed at doses of 200 mg/kg or higher in males, and at doses
    of 250 mg/kg or higher in females. The authors concluded that due to
    the proliferative nature of hepatic lesions observed, MC should be
    regarded as potentially carcinogenic (Quest  et al., 1987).

        Groups of 5 male and 5 female Wistar (SPF) rats (each 7-8 weeks
    old at the start of the experiment) received MC by stomach tube at 
    daily doses of 0, 200, 400, or 800 mg/kg for 13 weeks.  Two
    additional groups comprising 5 rats of each sex received 0 or 800
    mg/kg MC daily, but were killed after 4 weeks.  The rats were
    inspected daily.  Body weight, as well as feed and water intake,
    were measured weekly.  At weeks 4 and 13, blood samples were
    collected for determination of plasma activities of alkaline
    phosphatase (ALP), glutamate-oxalacetate transaminase (GOT), and
    glutamate-pyruvate transaminase (GPT) as well as of plasma
    concentrations of cholesterol, bilirubin, protein, and
    triglycerides.  Animals were killed at the end of the respective
    observation period (4 or 13 weeks), dissected, and examined
    macroscopically.  Liver, spleen, and testes were weighed.  The
    following organs of the animals killed after 4 weeks were fixed in
    10% buffered formaldehyde: liver, spleen, sternum (with bone
    marrow), and testes, as well as all organs with macroscopically
    visible changes.  On autopsy of the rats killed after 13 weeks,
    samples of 36 organs and tissues were fixed in buffered formalin. 
    Livers of all animals were examined histopathologically, including
    Oil Red O staining for fat.

        At 400 mg/kg and below, MC produced no changes in appearance,
    activity, coat condition, or behaviour.  Rats receiving 800  mg/kg
    displayed unspecific symptoms such as apathy, rough coat, poor
    general condition, and sunken flanks from week 2 onwards.  Feed and
    water consumption were dose-dependently reduced at 400 mg/kg and
    above, as was the body weight gain.  In week 4, one rat of the 800
    mg/kg dose group died.

        Clinical chemical investigations on weeks 4 and 13 revealed
    slight increases in serum ALP-, GOT-, and GPT-activities, as well as
    increases of cholesterol and triglyceride contents.  Gross
    pathological examinations revealed yellowish swellings or
    discoloration of the epididymis in two male rats of the 800 mg/kg
    dose group.  At week 13, no organ damage was found in the 200 mg/kg
    dose group.  At 800 mg/kg, testes, epididymis, and spleen were
    relatively small.  Also, dose- and time-related decreases in the
    absolute weights of liver, spleen, and testes were observed at 400
    mg/kg and above.  The same proved true, except for the liver, when
    relative organ weights were determined.  Relative spleen weight was
    also reduced in males of the 200 mg/kg dose group.  At week 4, no
    abnormalities attributable to treatment and no fat accumulation were
    observed in the livers of the dosed rats.  At week 13, liver of rats
    receiving 800 mg/kg MC had increased pigment contents, especially in
    Kupffer's cells.  The pigments contained iron.  Occasional focal
    round cell infiltrates, liver cell mitoses, and single cell
    necroses, as well as accumulation of fat droplets were observed in
    livers of all groups and were considered not to be treatment-
    related.

        An additional group of rats receiving MC with the drinking water
    to yield an average consumption of about 800 mg/kg MC daily, showed
    the same response as the group receiving 800 mg/kg MC by gavage.

        The authors concluded that at a dose of 200 mg/kg, MC was
    tolerated without adverse effects under the experimental conditions
    described.  The effects on the liver were found to be different than
    those observed in Fischer 344 rats (Bomhard & Karbe, 1985b).

    2.2.3.  Long-term/carcinogenicity studies

    2.2.3.1  Mouse

        A lifetime carcinogenicity study with methyl carbamate (>99.9%
    pure) was carried out in NMRI mice: the exposure was started  in
     utero to increase the sensitivity of the test.  Seventy-five male
    (35 g) and 75 female mice (30 g) were allocated to one of five
    groups which received 0 (control group), 0.5, 2.5, 12.5, or 62.5
    mg/kg/day MC with their drinking water.  They were then mated in a
    ratio of 1:1 under continuation of the treatment, the mating period
    allowed being 3 weeks.  The males were removed after mating, and the
    females continued to be treated until the end of the 4-week
    lactation period, after which they were also withdrawn from the
    experiments.  The offspring (F1-generation) were randomized at the
    age of 4 weeks and treated with the same concentrations of MC which
    had been received by the parents.  Only those litters that contained
    at least one male and one femals mouse were used, and only one mouse
    of each sex was chosen from every litter.  The number of animals in
    individual groups ranged therefore between 54 and 64 mice per sex.

        The treatment was continued throughout the lifespan of the F1-
    generation.  Mice were allowed to die spontaneously or were killed
    in moribund state.  All animals were necropsied, and all tumours, as
    well as all organs and tissues suspected of containing tumours, were
    fixed in every case: thyroid with parathyroids, heart, lung, liver
    with gall bladder, spleen, kidneys, adrenals, testes, ovaries,
    uterus, bladder, pituitary, stomach, oesophagus, pancreas, brain,
    and the entire intestine with mesenteric lymph nodes.  In addition,
    nasopharynx, skin from the mammary region, eyes, prostate, seminal
    vesicle, epididymis, mammary tissue, submandibular salivary glands
    with lymph nodes, spinal column with spinal cord, femur with muscle,
    larynx, and trachea of those animals which died or were killed from
    day 818 of the experiment onwards were fixed.  Histopathological
    examination of these organs was carried out in the case of mice from
    the two highest dose groups.

        The authors concluded that because the combined incidence of
    tumours was not changed in any group, and the variations in tumour
    incidence were not dose-related, methyl carbamate was not
    carcinogenic in this study.  The shift of tumour spectrum observed

    was attributed to the normal range of biological variation.  It
    should be noted that organ weights have not been indicated
    (Steinhoff, 1978).

        Three groups of Swiss mice (exact number not indicated) were
    treated as follows: two groups were with treated MC (800 mg/kg p.o.)
    or ethyl carbamate (EC) (590 mg/kg p.o.) on days 1, 10, and 25 after
    birth.  They were offspring of parent females which had been treated
    on days 13, 16, and 17 of gestation with the same doses of MC or EC. 
    A third group remained untreated and served as a control gorup,  The
    mice were allowed to live out their lifespan and were then examined
    for neoplasms.  MC did not increase tumour incidence above the level
    observed in control mice, whereas EC led to the development of
    multiple lung adenomas, lung carcinomas, and thymomas.  However, MC-
    treated mice displayed a higher mortality rate which might have
    covered carcinogenic effects.  In the opinion of the Committee, this
    is a major limitation of this study, and the relevance of its
    results is therefore questionable (Port & Ivankovich, 1979).

        A long-term toxicity study of MC was conducted by administering
    O, 500, or 1000 mg/kg methyl carbamate in water by gavage, 5 days
    per week for 103 weeks, to groups of 50 B6C3F1 mice of each sex. 
    Additional groups of 30 mice of each sex were administered 0 or 1000
    mg/kg MC on the same schedule.  Ten animals from each group were
    killed at 6, 12, or 18 months to follow the progressions of lesions. 
    Mice were checked daily for clinical signs of toxicity.  A complete
    necropsy was carried out on all animals that died or were killed in
    moribund state during the observation period.  All organs and
    tissues were examined for grossly visible lesions.  Histopathologic
    examinations were performed on all high dose and vehicle control
    mice, on all organs showing grossly visible lesions in all dose
    groups, and on potential target organs for chemically related
    effects.

        Compound-related neoplastic effects were not observed in mice in
    the 6-, 12-, or 18-month studies. In the 2-year study, the mean body
    weights of high dose (1000 mg/kg) male mice were about 8%-18% lower
    than those of the vehicle controls after week 24.  The mean body
    weights of high dose (1000 mg/kg) female mice were 16% lower than
    those of the vehicle controls after week 16 and 30% lower after week
    64.  Survival of dosed and vehicle control mice was similar (male:
    28/50, 35/50; 28/50; female: 38/50, 36/50; 32/50).

        In the 2-year studies, multinucleate giant cells in the liver
    were observed at increased incidence in dosed male mice (14/50;
    31/50; 31/49).  Adenomatous hyperplasia and histiocytosis of the
    lung were observed at increased incidence in high dose mice
    (adenomatous hyperplasia - male: 13/50; 19/50; 24/49; female: 9/49;
    10/50; 21/50).  There was no evidence of carcinogenic activity in
    male or female mice given MC at doses of 500 or 1000 mg/kg. 

    Adenomatous hyperplasia and histiocytosis of the lung, however, were
    observed in dosed mice of both sexes (National Toxicology Program,
    1987).

    2.2.3.2  Rat

        The carcinogenic activity of methyl carbamate was studied in
    Wistar W70 rats.  Seventy-five male and 75 female rats each were
    allocated to one of five dose groups receiving 0 (control group),
    0.5, 2.5, 12.5, or 62.5 mg/kg/day of MC in their drinking water. 
    They were then mated in a ratio of 1:1 under continuation of the
    treatment (a mating period of 3 weeks was allowed).  The females
    continued to be treated until the end of the 4-week lactation
    period.  The young (F1-generation) were randomized at four weeks of
    age and treated with the doses which had been received by the
    respective parent animals.  The control and test groups consisted of
    54-62 rats per sex.  The experiment was continued throughout the
    lifespan of the F1-generation.  The rats were allowed to die
    spontaneously or were killed in moribund state.

        The mating results did not differ between dosed groups and the
    control group, except for a slightly lower number of raised young in
    the highest dose group (in an additional experiment, treatment of
    rats with 312.5 mg MC/kg/day resulted in a marked reduction of
    litter size per mother animal and of the number of offspring
    raised).  No changes in appearance or behaviour were observed that
    were associated with treatment.  Also, no differences in survival
    time were evident between dosed groups and controls.  Body weight
    gain was slightly lower in the highest dose group as compared to the
    other treatment groups and to the control group. 

        All animals were autopsied at the end of the experiment.  All
    tumours and all organs and tissues suspected of containing tumours
    were fixed for histopathological examination. In addition, samples
    of 17 organs and tissues were fixed in every case.  Fixed samples
    were examined histologically in the case of all animals from either
    the control or the two highest dose groups.  There were no
    macroscopic lesions that were considered to be treatment-related. 
    Furthermore, there was no indication of a carcinogenic effect by MC. 
    The number of benign tumours was even lower in the highest dose
    group as compared to controls.  The authors concluded that lifetime
    treatment with MC up to a dose of 62.5 mg/kg/day had no carcinogenic
    effect in Wistar rats (Steinhoff  et al., 1977).

        Six groups of male and female Wistar rats (exact number not
    given) were treated as follows:  two groups were treated orally once
    on day 1 of life with 3100 mg/kg MC or 1300 mg/kg ethyl carbamate
    (EC).  Three other groups were formed from the offspring of parent
    females treated with 1300 or 1700 mg/kg MC or 1000 mg/kg EC on day
    19 of gestation .  A sixth group remained untreated and served as

    control.  The animals were allowed to live out their natural
    lifespan, and were subsequently examined for neoplasms.  There was
    no indication of a carcinogenic effect of MC.  Only rats treated
    postnatally with EC displayed a marginal carcinogenic effect.  In
    the opinion of the Committee, this study is poorly designed, and its
    results are therefore not conclusive (Port & Ivankovich, 1979).

        MC was administered by gavage, 5 days per week for 103 weeks, to
    groups of 50 F 344/N rats of each sex at doses of 100 or 200 mg/kg. 
    Control groups received distilled water only.  To follow the
    propagation of lesions, additional groups of 30 rats of each sex
    were administered 0 or 400 mg/kg MC on five days per week.  Ten
    animals of each sex from every group were killed at 6, 12, or 18
    months.  The rats were checked twice daily and clinical signs
    recorded once weekly.  All animals killed at the end of the
    respective observation period, as well as those that died or were
    killed in moribund state were necropsied.  All organs and tissues
    were examined for grossly visible lesions and were preserved in
    buffered formalin for histopathologic examination.  This was carried
    out on all high dose and vehicle control animals and on low dose
    rats dying through month 21 of the study.  In addition,
    histopathological examinations were performed on all grossly visible
    lesions in all dose groups, as well as on potential target organs
    and tissues in animals of the low dose group.

        In the 6-month studies, all vehicle control and dosed (400
    mg/kg) animals survived.  Cytologic alterations and atypical
    proliferative changes were observed in the livers of all dosed male
    and female rats, and neoplastic nodules of the liver were observed
    in 6/10 dosed male and 5/10 dosed female rats.  In the 12-month
    studies, all vehicle control male and female rats and dosed female
    rats survived.  One of 10 dosed male rats died.  Neoplastic nodules
    of the liver were observed in 7/10 dosed male and 9/10 dosed female
    rats, and hepatocellular carcinomas were observed in 8/10 dosed male
    and 6/10 dosed female rats.  In the 18-month studies, 1/10 dosed
    male and 8/10 dosed female and all vehicle control rats survived. 
    Hepatocellular carcinomas were observed in 9/10 dosed male and 8/10
    dosed female rats.

        In the 2-year studies, mean body weights of high dose (200
    mg/kg) male rats were generally 5%-9% lower than those of the
    vehicle controls after week 20.  Mean body weights of high dose
    female rats were 5%-8% lower than those of the vehicle controls
    after week 56. Survival of dosed and vehicle control rats was
    similar (male: vehicle control, 19/50; low dose, 26/50; high dose,
    29/50; female: 29/50; 36/50; 35/50).

        Chronic focal inflammation and cytologic alteration of the liver
    were observed at increased incidences in high dose rats of each sex. 
    Hyperplasia of hepatocytes was observed at increased incidence in
    dosed male and high dose female rats.  Neoplastic nodules or

    hepatocellular carcinomas (combined) in female rats occurred with a
    significant positive trend (0/50; 0/50; 6/49; P>0.01); the
    incidence of neoplastic nodules or hepatocellular carcinomas
    (combined) in high dose female rats was greater (P>0.03) than that
    in the vehicle controls.  Incidence of liver neoplasms in dosed male
    rats was not significantly increased (4/50; 0/50; 7/49). 
    Inflammation of the Harderian gland was observed at increased
    incidence in dosed rats (male: 4/50; 11/50; 16/50; female: 7/50;
    16/50; 30/50).  The lesions were considered to be chemically
    related.  In the 2-year studies in rats, significant decreases in
    tumour incidence included the following: leukaemia (both sexes),
    pituitary gland (male), adrenal gland (male), and mammary gland
    (female).

        Thus, there was clear evidence for carcinogenic activity for
    male and female F 344/N rats given MC, as indicated by increased
    incidence of hepatocellular neoplastic and/or proliferative changes. 
    Also, MC induced an inflammation of the Harderian gland (National
    Toxicology Program, 1987).

    Mathematical models (extrapolation to man)

        Using mathematical models, and assuming an average daily
    consumption of wine of 250 ml/person/day, and average human lifespan
    of 70 years, a daily consumption of wine for 50 years, a person's
    weight of 60 kg, and a methyl carbamate concentration of 0.01876
    mg/1 or less, a lifetime average daily dose (LADD) of 55.7x10-6
    mg/kg/day was calculated.  Under these conditions, the maximum
    lifetime risk (95% upper confidence limit) was estimated to be
    2.98x10-8.  If 1 liter of wine is consumed daily instead, the
    maximum lifetime risk would be 1.2 x 10-7 (Hahnemann & Schmitz,
    1986).

        Using a similar mathematical model, theoretical risk assessment
    of MC in soft drinks treated with DMDC was carried out assuming the
    least favourable case (direct genotoxic-carcinogenic potential, high
    exposure conditions).  Based on the assumption that DMDC-treated
    drinks are consumed in amounts of 1.0 or 1.5 l daily over a lifespan
    of 70 years, and that soft drinks are treated with 250 mg/l DMDC
    yielding an average of 6µg MC/l drink, the theoretical maximum
    lifetime risk was calculated to be smaller than 1x10-7 (Hahnemann,
    1987).

    2.2.4  Special studies on carcinogenicity

    2.2.4.1  Mouse

        The tumorigenic potential of MC and its covalent binding to DNA
    from various tissues was investigated in male mice and compared to
    the activities of several other carbamic acid esters.  For tumour

    induction experiments, groups of 30 mice (Hall strain, 7 weeks of
    age) were formed.  For each substance, one group was pretreated by a
    single application of 0.25ml of 0.075% (v/v) croton oil solution in
    acetone over the whole area of the skin of the back 18h before
    injection of the carbamates, another group was not pretreated.  MC
    and other carbamates were injected at dose of 27mEq/kg.  From 3
    weeks later onwards, 0.24 ml of croton oil solution was applied once
    per week for 18 weeks to skin and back, then the dosage of the
    croton oil was increased to 0.15% (v/v) and the applications
    continued until week 32. The number of skin tumours was determined
    at weeks 16, 22, 32, 36, 56, and 78.  One half of the animals were
    autopsied at week 56.  The remaining animals were autopsied at week
    78, as were all animals that died from week 36 onwards.  No
    differences in tumour incidence or in the number of tumours per
    mouse were observed between MC treated groups and the control groups
    with respect to the appearance of skin tumours, hepatomas, lung
    adenomas, and leukaemia.

        Binding to DNA was assessed following a single i.p. injection of
    6 mg of a carbamate (6µCi) into Crackenbush mice (7 weeks of age). 
    DNA was extracted at different time points and  bound radioactivity
    was measured.  MC bound covalently to DNA of the dermis and the
    epidermis, and maximal binding was observed between 6 and 12 h. 
    Painting of the skin with croton oil 16 h before MC treatment
    increased the covalent binding of MC to DNA (Pound & Lawson, 1976).

        The ability of a series of carbamates and aziridines to initiate
    lung tumours in strain A/He mice was investigated.  A group of 16
    mice (7-9 weeks old) received 12 intraperitoneal injections (three
    times per week) of MC in water yielding a total dose of 60 mg. 
    Twenty weeks after the last treatment, mice were killed and
    inspected for lung tumours.  MC-treated mice had a lung tumour
    incidence of 6% (water vehicle controls: 19%; no-treatment controls:
    7%), and the number of lung tumours per mouse averaged 0.1 (water
    vehicle controls: 0.2; no-treatment controls: 0.1).  In contrast to
    ethyl carbamate and several other derivatives of carbamic acid, MC
    proved non-carcinogenic in this study (Shimkin  et al., 1969).

        MC was injected intraperitoneally into groups of strain A mice
    of both sexes, a strain highly sensitive to lung carcinogens, at
    doses of 0.5 (46 mice), 1.0 (43 mice), and 2.0 g/kg body weight (49
    mice). The injections were carried out once weekly for 13 weeks. 
    Two to three weeks after the final injection, the mice were killed
    and examined for the incidence of lung tumours.  The lung tumour
    incidence was 16, 9, or 22% and the mean number of tumours 0.19,
    0.09, or 0.29 for the low, middle, or high dose, respectively. 
    These values were not different from control values observed in this
    study (tumour incidence: 17%; mean tumour number: 0.18
    tumours/mouse).  In contrast to ethyl carbamate, which proved
    carcinogenic in this study, MC showed no carcinogenic potential
    towards the lung (Larsen, 1974).

        In a study designed to investigate the skin tumour initiating
    properties of a variety of compounds, MC was applied topically as a
    25% solution to a group of 20 male "S"-mice (7-9 weeks old). 
    Fifteen weekly applications were made yielding a total dose of 1.12
    g of MC.  Three days following the start of MC-application, croton
    oil (0.3 ml of a 5% solution in acetone) was applied weekly for 18
    weeks. At the end of the croton oil treatment, 18 surviving mice
    were sacrificed.  One of the 18 mice developed a skin tumour as
    compared to 1 out of 20 control animals treated with croton oil
    only.  Thus, MC showed no skin tumour initiating properties in this
    model (Roe & Salsman, 1955).

        The ability of different carbamate derivatives (including methyl
    carbamate) to initiate skin tumours was investigated in male mice
    (strain "Hall").  Groups of 40 mice each were given an application
    of 0.25 ml of a 25% solution of acetic acid in acetone to the right
    side of the skin of the back.  Eighteen hours later, all groups,
    except for controls, were injected s.c. with a carbamate derivative. 
    MC was given at a dose of 40 mg/kg b.w.  In the first set of
    experiments, the mice received 24 weekly applications of 0.25 ml of
    a 0.075% solution of croton oil in acetone.  One week following the
    final application, the number of mice with tumours and the
    distribution of tumours were estimated.  In a second set of
    experiments, mice were treated similarly except that the repeated
    application of croton oil was omitted.  In contrast to several
    homologous and N-substituted derivatives of ethyl carbamate, MC
    showed no significant tumour initiating effect (Pound, 1967).

        The effect of co-administration of homologous carbamates and
    ethyl carbamate derivatives on skin tumour initiation by ethyl
    carbamate was studied in mice. Subcutaneous injection of MC at doses
    of 5, 10, or 20 mg/kg together with ethyl carbamate (25 mg/kg) did
    not enhance the tumour yield following treatment with ethyl
    carbamate only.  In all cases, the back skin was painted with 0.25
    ml of a 0.075% solution of croton oil in acetone once weekly for 28
    weeks.  The surviving animals were sacrificed at week 50 (Pound,
    1972).

        An unspecified number of random-bred male mice received 3 s.c.
    injections of 1 mg/kg b.w. MC at 2-day intervals and were observed
    for 3 months, another group received 3 s.c. injections of 0.1 mg/kg
    MC and were observed for 6 months.  Of those mice observed for 3
    months, 3/29 animals had lung adenomas compared with 3/22 controls
    and 23/27 mice given ethyl carbamate in addition to MC.  Of the
    animals observed for 6 months, 2/26 mice had lung adenomas compared
    with 0/26 controls and 6/28 mice given ethyl carbamate in addition
    to MC.  In contrast to ethyl carbamate, MC proved non-carcinogenic
    under the described experimental conditions (Yagubov & Suvalova,
    1973).


        2.2.5    Special studies on genotoxicity

    Table 8:  Results of genotoxicity assays on methyl carbamate (MC)
                                                                                          
    Test system        Test object         Concentration       Results        Reference
                                           of MC
                                                                                          

    Ames test          S. typhimurium      1 000 µg/plate      Negative       McCann et 
    (1)                TA98, TA100                                            al., 1975
                       TA1535, TA1537

    Ames test          S. typhimurium      1 000 µg/plate      Negative       Simmon, 
    (2)                TA98, TA100,                                           1979a
                       TA1535, TA1536,
                       TA1537, TA1538

    Ames test          S. typhimurium      100 & 250           Negative       Rosenkranz &
    (2)                TA1535, TA1538      µg/plate                           Poirier, 
                                                                              1979

    Ames test          S. typhimurium      100-10 000          Negative       National 
    (2)                TA97, TA98,         µg/plate                           Toxicology
                       TA100, TA1535                                          Program, 
                                                                              1987

    DNA damage         E. coli po1A-       500 µg              Negative       Rosenkranz &
                       (DNA-polymerase                                        Poirier, 
                       deficient)                                             1979

    DNA damage         E. coli             5 000 µg/plate      Negative       McCarrol et
    (2)                WP2uvrA, WP67,                                         al., 1981a
                       CM 611, WP100,
                       p3478 pol a-

                                                                                          

    Table 8 (contd)
                                                                                          
    Test system        Test object         Concentration       Results        Reference
                                           of MC
                                                                                          

    DNA damage         B. subtilis         5 000 µg/plate      Negative       McCarrol et
    (2)                H17rec- (repair                                        al., 1981b
                       deficient)

    Bacterial          B. subtilis         50 & 60 mg/ml       Negative       De Giovanni 
    back mutation      168i-(indole                                           et al., 1967
    assay              requiring)

    Bacterial          E. coli B/Sd-       40-80 mg/ml         Negative       Demerec et 
    back mutation      4/1,3,4,5 &                                            al., 1967
    assay              B/sd-4/3,4 
                       (Streptomycin-
                       dependent)

    Mitotic            S. cerevisiae       50 mg/ml            Negative       Simmon, 
    recombination      D3 (homozygous                                         1979b
    assay              ade2)

    Gene               Mouse lymphoma      2 821-21 208        Negative       Amacher &
    mutation (1)       frwd. mutation      µg/ml                              Turner, 1982
                       L5178Y/TK+/-

    Gene               Mouse lymphoma      1 049-5 000 µg/ml   Negative       National 
    mutation (2)       frwd. mutation                                         Toxicology
    L5178Y/TK+/-                                                              Program, 
                                                                              1987

    Unscheduled        Primary rat         1-1 000 µg/ml       Negative       National
    DNA symthesis      hepatocyte                                             Toxicology
                       cultures                                               Program, 
                                                                              1987

                                                                                          

    Table 8 (contd)
                                                                                          
    Test system        Test object         Concentration       Results        Reference
                                           of MC
                                                                                          

    Chromosone         A. nidulans-        0.4 mg/ml           Negative       Morpurgo et
    aberrations        P                                                      al., 1979

    Chromosome         Chinese             2000-5 000 µg/ml    Negative       National 
    aberrations        hamster ovary                                          Toxicology
    (2)                cells                                                  Program, 1987

    Sister             Chinese             160-5 000 µg/ml     Negative       National
    Chromatid          hamster ovary                                          Toxicology
    exchange (2)       cells                                                  Program, 
                                                                              1987

    Sister             Mouse alveolar      165 & 495           Negative       Cheng et 
    Chromatid          macrophages         mg/kg i.p.                         al., 1981a,b
    exchange           bone marrow &
                       liver (in vivo)

    Noeplastic         Syrian hamster      0.05 µg/ml          Negative       Dunkel et
    transformation     embryo cells                                           al., 1981
    assay

    Neoplastic         F344 rat            12.0 µg/ml          Positive       Dunkel et 
    transformation     embryo cells                                           al., 1981
                       infected with
                       Rauscher murine
                       leukaemia virus

    Sex-linked         Drosophila          25 000 ppm          Negative       National
    recessive          (in vivo)           (inj.); 35 000                     Toxicology, 
    lethal                                 & 50 000 ppm                       1987
    mutation                               (feeding)

                                                                                          

    Table 8 (contd)
                                                                                          
    Test system        Test object         Concentration       Results        Reference
                                           of MC
                                                                                          

    Dominant           Mouse (in           200 & 1 000 mg/kg   Negative       Epstein
    lethal             vivo)               i.p.                               et al., 1972
    mutations
                                                                                          

    (1)  With rat liver S-9 fraction
    (2)  Both with and without rat liver S-9 fraction
    (3)  Slight mutagenic effects in WP14 (but study design not convincing)
    

    2.2.6  Special study on immunotoxicity

    2.2.6.1  Mouse

        The immunotoxic potentials of methyl carbamate and ethyl
    carbamate were investigated in mice.  Female B6C3F1 (C57 BL)6N x
    C3H) hybrid mice (5-7 weeks of age) were given daily i.p. injections
    of ethyl carbamate at doses of 1, 2, or 4 mg/kg or MC at a dose of 4
    mg/kg in 0.2 ml of saline over a 14-day period.  Immune functions
    were assessed 3-5 days following the last treatment or after 6 weeks
    to determine long-term effects.  Parameters assessed included bone
    marrow cellularity and progenitor assays, humoral immunity, cellular
    immunity, macrophage function, and natural killer (NK) cell
    activity, as well as susceptibility to tumour cell challenge.  MC
    had no effect on any of the immunological parameters studied, while
    ethyl carbamate caused severe myelotoxicity associated with a marked
    depression of NK cell activity (Luster  et al., 1982).

    2.3.  Observations in humans

        No information available.

    3.  COMMENTS

        The Committee reviewed data from acute toxicity studies with
    DMDC in mice and rats, as well as short- and long-term toxicity
    studies in rats that received juices and alcoholic beverages, which
    had been treated with 4 g/l of DMDC in rats and a 1-year toxicity
    study in dogs.  Data from reproduction toxicity, embryotoxicity/
    teratogenicity, and genotoxicity studies with DMDC-treated beverages
    were also examined.  It was concluded that there was no evidence of
    toxic effects in mice and rats due to the consumption of DMDC-
    treated beverages.

        The Committee also reviewed data from acute toxicity studies
    with methylethylcarbonate, dimethylcarbonate, and several
    carboxymethylation products of amino and hydroxy acids, as well as
    short-term toxicity studies in rats with methylethylcarbonate and
    dimethylcarbonate, and an embryo-toxicity/teratogenicity study in
    rats with methylethylcarbonate.  No adverse effects due to the
    consumption of these decomposition products were observed.

        In the case of methylcarbamate, the Committee reviewed data from
    acute toxicity studies in mice and rats, short-term studies in mice
    and rats, long-term carcinogenicity studies in mice and rats, dermal
    carcinogenicity and DNA-binding studies in mice, a large number of
    studies on genotoxicity in bacterial and mammalian cells (including
     in vivo studies), and a special study on immunotoxicity in mice. 
    Methylcarbamate produced hepatocellular carcinomas in Fischer 344
    rats at high dose levels, but did not have such effects in Wistar
    rats or in mice.  Methylcarbamate was shown to be non-genotoxic.

        The no-observed-effect level for hepatic carcinogenesis in
    Fischer 344 rats was 100 mg per kg of body weight per day.  Since
    the estimated worst-case exposure of humans to methylcarbamate in
    beverages would be less than 20 µg/l at the concentrations of DMDC
    employed, a large margin of safety applies.  The Committee
    concluded, therefore, that the presence of methylcarbamate at the
    expected levels of use of DMDC (i.e., in accordance with Good
    Manufacturing Practice) would not be of risk to human health.

        The concentrations of methanol (up to 120 mg/l) resulting from
    the use of DMDC are similar to or less than those occurring
    naturally in many fruit juices and alcoholic beverages.  The
    Committee considered that the concentrations of methanol present
    after treatment of beverages with DMDC were of no toxicological
    concern.

    4.  EVALUATION

        DMDC was considered acceptable for use as a cold sterilization
    agent for beverages when used in accordance with Good Manufacturing
    Practice up to a maximum of 25 mg/l.

    5.  REFERENCES

    AMACHER, D.E. & TURNER, G.N. (1982).  Mutagenic evaluation of
    carcinogens and non-carcinogens in the L5178Y/TK assay utilizing
    postmitochondrial fractions (S9) from normal rat liver.   Mutat.
     Res., 97, 49-65.

    BAYER AG (1987).  Toxicological assessment of the small amounts of
    methanol anticipated in the sterilization of drinks with dimethyl
    dicarbonate.  Unpublished report.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    BOMHARD, E. & KALINA, G. (1984).  Methyl carbamate.  Exploratory
    subacute toxicity study on Wistar rats relating to the question of a
    hepatotoxic effect.  Seven-day gavage application experiment. 
    Unpublished report No. 13148 from Bayer AG, Institut fur
    Toxikologie, Wuppertal, FRG.  Submitted to WHO by Mobay Corporation,
    Pittsburgh, PA, USA.

    BOMHARD, E. & KARBE, E. (1985a).  Methyl carbamate; N-methoxymethyl-
    O-methylurethane.  Exploratory subacute toxicity on Fischer 344 rats
    relating to the question of a hepatotoxic effect.  Seven-day gavage
    application experiment.  Unpublished report No. 13275 from Bayer AG,
    Institut fur Toxikologie, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    BOMHARD, E. & KARBE, E. (1985b).  Methyl carbamate.  Subchronic
    toxicity study on Wistar rats (13-week experiment with
    administration of test compound by gavage or in drinking water). 
    Unpublished report No. 13542 from Bayer AG, Institut fur
    Toxikologie, Wuppertal, FRG.  Submitted to WHO by Mobay Corporation,
    Pittsburgh, PA, USA.

    BOMHARD, E., SCHMIDT, U., & KARBE, E. (1989).  Differences in liver
    sensitivity to methyl carbamate between Wistar and Fischer 344 rats. 
     Arch. Toxicol. Suppl., 13, 319-321.

    BOYLAND, E. & NERY, R. (1965).  The metabolism of urethane and
    related compounds.   Biochem. J., 94, 198-208.

    BOYLAND, E. & PAPADOPOULOS, D. (1952).  The metabolism of methyl
    carbamate.  Biochem. J., 52, 267-269.

    CHENG, M., CONNER, M.K., & ALARIE, Y. (1981a).  Potency of some
    carbamates as multiple tissue sister chromatid exchange inducers and
    comparison with known carcinogenic activities.   Cancer Res., 41,
    4489-4492.

    CHENG, M., CONNER, M.K., & ALARIE, Y. (1981b).  Multicellular SCE
    study of some carbamate esters.   Environ. Mutagen., 3, 385
    (Abstract).

    De GIOVANNI-DONNELLY, R., KOLBYE, S.M., & DIPAOLO, J.A., (1967). 
    The effect of carbamates on  Bacillus subtilis.  Mutat. Res., 4,
    543-551.

    DEMEREC, M., BERTANI, G., & FLINT, J. (1951).  A survey of chemicals
    for mutagenic action on E. coli.  American Naturalist, 85, 119-136.

    DUNKEL, V.C., PIENTA, R.J., SIVAK, A., & TRAUL, K.A. (1981). 
    Comparative neoplastic transformation responses of Balb/3T3 cells,
    Syrian hamster embryo cells, and Rauscher murine leukaemia virus-
    infected Fischer 344 rat embryo cell to chemical carcinogens.   J.
     Natl. Cancer Inst., 67, 1303-1315.

    EIBEN, R., LÖSER, E., & KALINA, G. (1982).  Dimethyl carbonate
    (DMC).  Subchronic toxicology study in rats.  3-month drinking
    experiment.  Unpublished report No. 11108 from Bayer AG, Institute
    of Toxicology, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    EIBEN, R., LÖSER, E., & JANDA, B. (1983).  Orange juice, sterilized
    with DMDC/Velcorin.  Generation test on rats (2-generations study). 
    Unpublished report No. 11969 from Bayer AG, Institute of Toxicology,
    Wuppertal, FRG. Submitted to WHO by Mobay Corporation, Pittsburgh,
    PA, USA.

    EIBEN, R., LÖSER E., LUCKHAUS, G., & JANDA, B. (1984).  Wine,
    sterilized with DMDC/Velcorin.  Chronic toxicity study in rats (30-
    month drinking study).  Unpublished report No. R 2989 from Bayer AG,
    Institute of Toxicology, Wuppertal, FRG. Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    EPSTEIN, S.S., ARNOLD, E., ANDREA, J., BASS, W., & BISHOP, Y.
    (1972).  Detection of chemical mutagens by the dominant lethal assay
    in the mouse.   Toxicol. Appl. Pharmacol., 23, 288-325.

    HAHNEMANN, S. (1987).  Multistage model risk assessment for methyl
    carbamate based on data from the NTP (U.S. National Toxicology
    Program)  Study on Fischer 344 rats after chronic oral
    administration.  Model:  soft drink.  Unpublished report from Bayer
    AG, Wuppertal, FRG.  Submitted to WHO by Mobay Corporation,
    Pittsburgh, PA, USA.

    HAHNEMANN, S. & SCHMITZ, H. (1986).  Theoretical risk assessment for
    methyl carbamate based on data from the U.S. National Toxicology
    Program bioassay for possible carcinogenicity.  Unpublished report
    from Bayer AG, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    HEMMERLY, J. & DEMEREC, M. (1955).  Tests of chemicals for
    mutagenicity.   Cancer Res., 15, 69-75.

    INSTITUT FUR TOXIKOLOGIE, WUPPERTAL, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    HERBOLD, B. (1978). DMDC (Dimethyldicarbonate).   Salmonella/
    microsome test for the investigation of point mutagenic effects. 
    Unpublished report No. 802 from Bayer AG, Institute of Toxicology,
    Wuppertal, FRG.  Submitted to WHO by Mobay Corporation, Pittsburgh,
    PA, USA.

    HERBOLD, B. (1980).  Velcorin-treated orange juice.  Salmonella/
    microsome test for the investigation of point mutagenic effects. 
    Unpublished report No. 9276 from Bayer AG, Institut fur Toxikologie,
    Wuppertal, FRG.  Submitted to WHO by Mobay Corporation, Pittsburgh,
    PA, USA.

    HERBOLD, B.A. (1989a).  Orange juice treated with 4000 ppm Velcorin. 
     salmonella/microsome test.  Unpublished report from Bayer AG,
    Fachbereich Toxicology, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    HERBOLD, B.A. (1989b).  Velcorin treated orange juice.  Micronucleus
    test on the mouse.  Unpublished report No. 18436 from Bayer AG,
    Fachbereich Toxicology, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    IARC (1974).  IARC (International Agency for Research on Cancer)
    Monographs on the evaluation of carcinogenic risk of chemicals to
    man:  Vol. 7, Some anti-thyroid and related substances, nitrofurans
    and industrial chemicals, Lyon, France, pp. 111-140.

    IARC (1976).  IARC (International Agency for Research on Cancer)
    Monographs on the evaluation of carcinogenic risk of chemicals to
    man.  Vol. 12, Some Carbamates, Thiocarbamates and carbazides 151-
    159, Lyon, France, pp 151-159.

    IOANNOU, Y.M., SANDERS, J.M., &  MATTHEWS, H.B. (1988).  Species-
    dependent variations in metabolism and clearance in rats and mice. 
     Drug Metab. Dispos., 16, 435-440.

    KIMMERLE, G. (1972).  Dimethyl dicarbonate acute toxicity study. 
    Unpublished report No. 3793 from Bayer AG, Institut fur Toxikologie,
    Wuppertal, FRG.  Submitted to WHO by Mobay Corporation, Pittsburgh,
    PA, USA.

    LARSEN, C.D. (1974).  Evaluation of the carcinogenicity of a series
    of esters of carbamic acid.   J. Natl. Cancer Inst., 8, 99-101.

    LAWSON, T.A. & POUND, A.W. (1973).  The interaction of carbon-14-
    labelled alkyl carbamates, labelled in the alkyl and carbonyl
    positions, with DNA  in vivo. Chem.-biol. Interact., 6,99-105.

    LINA, B.A.R. & TIL, H.P. (1983).  One-year oral toxicity study with
    DMDC-treated orange juice in dogs (Third interim report). 
    Unpublished report No. V 82.350/212101 from CIVO Institutes TNO,
    Zeist, Netherlands.  Submitted to WHO by Mobay Corporation,
    Pittsburgh, PA, USA.

    LÖSER, E. (1973)  Methyl ethyl carbonate (MEC).  Subchronic toxicity
    studies on rats (3-month experiment).  Unpublished report No. 4335
    from Bayer AG, Institut fur Toxikologie, Wuppertal, FRG.  Submitted
    to WHO by Mobay Corporation, Pittsburgh, PA, USA.

    LÖSER, E. (1978).  Dimethyl dicarbonte (DMDC) in fruit juices and
    alcohlic drinks.  Subchronic toxicity studies on rats (3-month
    drinking experiment).  Unpublished report No. 4808 from Bayer AG,
    Institut fur Toxikologie, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    LÖSER, E., EBIEN, R., SCHILDE, B., & JANDER, B. (1983).  Orange
    juice sterilized with DMDC/Velcorin.  Chronic toxicological tests on
    rats (30 months drinking test).  Unpublished report No. 11611 from
    Bayer AG, Toxicological Institute, Wuppertal, FRG.  Submitted to WHO
    by Mobay Corporation, Pittsburgh, PA, USA.

    LUSTER, M.I., DEAN, J.H., BOORMAN, G.A., DIETER, M.P., & HAYES, H.T. 
    (1982).  Immune functions in methyl and ethyl carbamate treated
    mice.   Clin. Exp. Immunol., 50, 223-230.

    MACHEMER, L. (1976).  Methyl ethyl carbonate.  Investigation for
    embryotoxic and teratogenic effects in rats after administration in
    drinking water.  Unpublished report N. 5887 from Bayer AG, Institute
    of Toxicology, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    McCANN, J., CHOI, E., YAMASAKI, E., & AMES, B.N. (1975).  Detection
    of carcinogens as mutagens in the  Salmonella/microsome test: Assay
    of 300 chemicals.   Proc. Natl. Acad. Sci. USA, 72, 5135-5139.

    McCARROL, N.E., PIPER, C.E., & KEECH, B.H. (1981a).  An  E. coli
    micro suspension assay for the detection of DNA damage induced by
    direct-acting agents and promutagens.   Environ. Mutagen., 3, 429-
    444.

    McCARROL, N.E., KEECH, B.H., & PIPER, C.E. (1981b).  A
    microsuspension adaptation of the  Bacillus subtilis "rec" assay. 
     Environ Mutagen., 3, 607-616.

    MORPURGO, G., BELLINCAMOI, D., GUALANDI, G., BALDINELLI, L., &
    CRESCENZI, O.S. (1979).  Analysis of mitotic nondisjunction with
     Aspergillus nidulans.  Environ. Hlth. Perspect., 31, 81-95.

    NATIONAL TOXICOLOGY PROGRAM (1987).  NTP technical report on the
    toxicology and carcinogenesis studies of methyl carbamate (CAS No.
    598-55-0) in F344/N rats and B6C3F1 mice.  NTP Technical Report
    Series No. 328, NIH Publication No. 88-2584.

    OUGH, C.S. (1983).  Dimethyl dicarbonate and diethyl dicarbonate. 
    In:  Branen, A.L. & Davidson, P.M. (eds.), Antimicrobials in Foods,
    Marcel  Dekker, Inc., New York, Basel, pp. 299-325.

    OUGH, C.S. & LANGBEHN L. (1976).  Measurement of methyl carbamate
    formed by the addition of dimethyl dicarbonate to model solutions
    and to wine.   J. Agric. Food Chem., 24, 428-430.

    PAULUHN, J. (1982). Velcorin.  Investigation into skin-irritant
    effects.  Unpublished report from Bayer AG, Wuppertal, FRG. 
    Submitted to WHO by Mobay Corporation, Pittsburgh, PA, USA.

    PORT, R.E. & IVANKOVICH, S. (1979).  Methyl carbamate:  No evidence
    for carcinogenicity following perinatal administration in Wistar
    rats and Swiss mice.  Unpublished report from the German Cancer
    Research Centre, Institutes of Biochemistry and Experimental
    Toxicology and Chemotherapy, Heidelberg, FRG.  Submitted to WHO by
    Mobay Corporation, Pittsburgh, PA, USA.

    POUND, A.W. (1967).  The initiation of skin tumours in mice by
    homologs and N-substituted derivatives of ethyl carbamate.   Aust.
     J. Exp. Biol. Med. Sci., 45, 507-516.

    POUND, A.W. (1972).  Tumour formation in mice by urethane
    administered with related carbamates.   Br. J. Cancer, 26, 215-225.

    POUND, A.W. & LAWSON, T.A. (1976).  Carcinogenesis by carbamic acid
    esters and their binding to DNA.   Cancer Res., 36, 1101-1107.

    QUEST, J.A., CAHN, P.C., CRAWFORD, D., KANAGALINAM, K.K., & HALL,
    W.C., (1987).  Thirteen-week oral toxicity study of methyl carbamate
    in rats and mice.   Fund. Appl. Toxiol., 8, 389-399.

    RAUENBUSCH, E. (1974).  Enzymatic hydrolysis of carbomethoxy
    compounds.  Unpublished report from Bayer AG, Wuppertal, FRG. 
    Submitted to WHO by Mobay Corporation, Pittsburgh, PA, USA.

    ROE, F.J.C. & SALAMAN, M.H. (1955).  Further studies on incomplete
    carcinogenesis:  Triethylene melamine (TEM), 1,2-benzanthracene and
    ß-propiolactone as initiators of skin tumour formation in the mouse. 
     Br. J. Cancer, 9, 177-203.

    ROSENKRANZ, H.S. & POIRIER, L.A. (1979).  Evaluation of the
    mutagenicity and DNA-modifying activity of carcinogens and
    noncarcinogens in microbial systems.   J. Natl. Cancer Inst., 62,
    873-891.

    SCHLÜTER, G. (1980).  Velcorin-treated orange juice.  Investigations
    of pre-implantation damage, embryotoxic and teratogenic effects
    following oral administration to rats.  Unpublished report No. 9328
    from Bayer AG, Institut fur Toxikologie, Wuppertal, FRG.  Submitted
    to WHO by Mobay Corporation, Pittsburgh, PA, USA.

    SCHMIDT, U. (1978).  Investigations of the enzymolysis of N-
    carbomethoxy proline.  Unpublished report No. 7224 from Bayer, AG,
    Institut fur Toxikologie, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    SCHMIDT, U. & SCHMIDT, W.M. (1987).  Methyl carbamate.  Renal
    elimination and liver enzyme activities after oral treatment of
    Wistar and Fischer rats.  Unpublished report No. 15783 from Bayer
    AG, Toxicology Section, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    SHIMKIN, M.B., WIEDER, R., McDONOUGH, M., FISHBEIN, L., & SWERN, D.
    (1969).  Lung tumour response in strain A mice as a quantitative
    bioassay of carcinogenic activity of some carbamates and aziridines. 
     Cancer Res., 29, 2184-2190.

    SIMMON, V.F. (1979a).   In vitro mutagenicity assays of chemical
    carcinogens and related compounds with  Salmonella typhimurium.  J.
     Natl. Cancer Inst., 62, 893-899.

    SIMMON, V.F. (1979b).   In vitro assays for recombinogenic activity
    of chemical carcinogens and related compounds with  Saccharomyces
     cerevisiae D3.   J. Natl. Cancer Inst., 62, 901-909.

    SRIVALOVA, T.I. (1973).  [Toxic and specific action of alkyl
    carbamates and their binary mixture] (article in Russian).
     Toksilol. Nov. Prom. Khim. Veshestu, 13, 86-91.  

    STAFFORD, P.A. & OUGH, C.S. (1976).  Formation of methanol and ethyl
    methyl carbonate by dimethyl dicarbonate and model solutions.   Am.
     J. Enol. Viticult., 27, 7-11.

    STEINHOFF, D. (1973a).  Akute toxikologische Untersuchungen. 
    Unpublished report from Bayer AG, Institut fur Toxikologie,
    Wuppertal, FRG.  Submitted to WHO by Mobay Corporation, Pittsburgh,
    PA, USA.

    STEINHOFF, D. (1973b).  Dimethyldicarbonat.  Akute toxikologische
    Untersuchungen mit Methylethylcarbonat.  Unpublished report from
    Bayer AG, Institut fur Toxikologie, Wuppertal, FRG.  Submitted to
    WHO by Mobay Corporation, Pittsburgh, PA, USA.

    STEINHOFF, D. (1973c).  Akute toxikologische Untersuchungen mit N-
    Carbomethoxy-Verbindungen.  Unpublished report from Bayer AG,
    Institut fur Toxikologie, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    STEINHOFF, D. (1974).  Akute toxikologische Untersuchungen mit
    Dimethyldicarbonat.  Unpublished report from Bayer AG, Institut fur
    Toxikologie, Wuppertal, FRG.  Submitted to WHO by Mobay Corporation,
    Pittsburgh, PA, USA.

    STEINHOFF, D. (1978).  Carcinogenicity study on mice orally dosed
    with methyl carbamate.  Unpublished report from Bayer AG, Institut
    fur Toxikologie, Wuppertal, FRG.  Submitted to WHO by Mobay
    Corporation, Pittsburgh, PA, USA.

    STEINHOFF, D., DYCKA, J., & ARTIK, N. (1977).  Carcinogenicity study
    on rats orally dosed with methyl carbamate.  Unpublished report from
    Bayer AG, Institut fur Toxikologie & Institut fur Dokumentation und
    Biometrie, Wuppertal, FRG.  Submitted to WHO by Mobay Corporation,
    Pittsburgh, PA, USA.

    WILLIAMS, K., KUNZ, W., PETERSEN, K., & SCHNIEDERS, B. (1971). 
    Changes in mouse liver RNA induced by ethyl carbamate (urethane) and
    methyl carbamate.   Z. Krebsforsch., 76, 69-82.

    YAGUBOV, A.S. & SUVALOVA, T.I. (1973).  Comparative evaluation of
    the blastomogenic action of a binary mixture of alkylcarbamates and
    its components (article in Russian).   Gig. Tr. Prof. Zabol., 8,
    19-22.


    See Also:
       Toxicological Abbreviations