722. Dimethyldicarbonate (DMDC) (WHO Food Additives Series 28)

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 F_1a litters were killed
after 4 weeks, males and females from the F_1b 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 F_1b-parent and F_2b

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 œg/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.

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    See Also:
       Toxicological Abbreviations