Document ID: EPA-HQ-OW-2002-0043-0093
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-07-11T04:00Z

DRAFT
FOR
THE
DRINKING
WATER
CRITERIA
DOCUMENT
ON
GLYOXAL
AND
METHYLGLYOXAL
Prepared
Under
EPA
Contract
OW­
68­
C­
98­
141
CDM
Work
Assignment
No.
2­
13
for
Health
and
Ecological
Criteria
Division
Office
of
Science
and
Technology
Office
of
Water
U.
S.
Environmental
Protection
Agency
July
7,
2003
ii
DISCLAIMER
This
document
is
a
preliminary
draft.
It
has
not
been
released
by
the
Office
of
Water,
U.
S.
Environmental
Protection
Agency,
and
should
not
at
this
stage
be
construed
to
represent
Agency
Policy.
It
is
being
circulated
for
comments
on
its
technical
merit.
iii
CONTENTS
FOREWORD
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v
EXECUTIVE
SUMMARY
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vii
GLYOXAL
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1
I.
PHYSICAL
AND
CHEMICAL
PROPERTIES
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1
II.
TOXICOKINETICS
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2
A.
Absorption
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2
B.
Distribution
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2
C.
Metabolism
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2
D.
Excretion
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2
E.
Bioaccumulation
and
Retention
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2
III.
HUMAN
EXPOSURE
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3
A.
Occurrence
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3
B.
Fate
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5
C.
Exposure
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5
IV.
HEALTH
EFFECTS
IN
ANIMALS
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6
A.
Short­
Term
Exposure
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6
B.
Longer­
Term
Exposure
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6
C.
Reproductive/
Teratogenic
Effects
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7
D.
Mutagenicity
and
Genotoxicity
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8
E.
Carcinogenicity
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1
V.
HEALTH
EFFECTS
IN
HUMANS
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13
A.
Sensitive
Populations
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13
VI.
MECHANISM
OF
TOXICITY
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15
A.
Mechanism
for
DNA
Effects
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15
VII.
QUANTIFICATION
OF
TOXICOLOGICAL
EFFECTS
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16
A.
Noncarcinogenic
Effects
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16
B.
Carcinogenic
Effects
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21
METHYLGLYOXAL
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24
I.
PHYSICAL
AND
CHEMICAL
PROPERTIES
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24
II.
TOXICOKINETICS
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25
A.
Absorption
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25
B.
Distribution
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25
iv
CONTENTS
(
continued)

C.
Metabolism
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25
D.
Excretion
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27
E.
Bioaccumulation
and
Retention
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28
III.
HUMAN
EXPOSURE
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29
A.
Occurrence
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29
B.
Fate
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31
C.
Exposure
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31
IV.
HEALTH
EFFECTS
IN
ANIMALS
.
.
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32
A.
Short­
Term
Exposure
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32
B.
Longer­
Term
Exposure
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33
C.
Reproductive/
Developmental
Studies
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34
D.
Mutagenicity
and
Genotoxicity
.
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35
E.
Carcinogenicity
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39
V.
HEALTH
EFFECTS
IN
HUMANS
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43
A.
Sensitive
Populations
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44
VI.
MECHANISM
OF
TOXICITY
.
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46
A.
Mechanistic
Studies
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46
VII.
QUANTIFICATION
OF
TOXICOLOGICAL
EFFECTS
.
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49
A.
Noncarcinogenic
Effects
.
.
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.
49
B.
Carcinogenic
Effects
.
.
.
.
.
.
.
.
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53
REFERENCES
.
.
.
.
.
.
.
.
.
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56
v
FOREWORD
The
Safe
Drinking
Water
Act,
as
amended
in
1996,
requires
the
Administrator
of
the
U.
S.
Environmental
Protection
Agency
(
EPA)
to
publish
maximum
contaminant
level
goals
(
MCLGs)
and
promulgate
National
Primary
Drinking
Water
Regulations
for
each
contaminant
that,
in
the
judgment
of
the
Administrator,
may
have
an
adverse
effect
on
public
health
and
that
is
known
or
anticipated
to
occur
in
public
water
systems.
The
MCLG
is
nonenforceable
and
is
set
at
a
level
at
which
no
known
or
anticipated
adverse
health
effects
in
humans
occur
and
which
allows
for
an
adequate
margin
of
safety.
Factors
considered
in
setting
the
MCLG
include
health
effects
data
and
sources
of
exposure
other
than
drinking
water.

This
document
provides
the
health
effects
basis
to
be
considered
in
establishing
the
MCLG
for
glyoxal
and
methylglyoxal,
which
are
byproducts
of
water
ozonation.
To
achieve
this
objective,
data
on
pharmacokinetics,
human
exposure,
acute
and
chronic
toxicity
to
animals
and
humans,
epidemiology,
and
mechanisms
of
toxicity
were
evaluated.
Specific
emphasis
is
placed
on
data
providing
dose­
response
information.
Thus,
although
the
literature
search
and
evaluation
performed
in
support
of
this
document
were
comprehensive,
only
the
reports
considered
most
pertinent
in
the
derivation
of
the
MCLG
are
cited
in
this
document.
The
comprehensive
literature
search
in
support
of
this
document
includes
information
published
up
to
January
2003;
however,
more
recent
information
may
have
been
added
during
the
review
process.

When
adequate
health
effects
data
exist,
Health
Advisory
values
for
less
than
lifetime
exposure
(
1­
day,
10­
day,
and
longer
term,
approximately
10%
of
an
individual's
lifetime)
are
included
in
this
document.
These
values
are
not
used
in
setting
the
MCLG,
but
serve
as
informal
guidance
to
municipalities
and
other
organizations
when
emergency
spills
or
contamination
situations
occur.

The
Reference
Dose
(
RfD)
provides
information
on
long­
term
toxic
effects
other
than
carcinogenicity.
The
RfD
is
based
on
the
assumption
that
thresholds
exist
for
certain
toxic
effects
such
as
cellular
necrosis,
but
may
not
exist
for
other
toxic
effects
such
as
some
carcinogenic
responses.
It
is
expressed
in
terms
of
milligrams
per
kilogram
per
day
(
mg/
kg/
day).
In
general,
the
RfD
is
an
estimate
(
with
uncertainty
spanning
perhaps
an
order
of
magnitude)
of
a
daily
exposure
to
the
human
population
(
including
sensitive
subgroups)
that
is
likely
to
be
without
an
appreciable
risk
of
deleterious
effects
during
a
lifetime.
The
RfD
is
used
in
establishing
the
Lifetime
Health
Advisory
for
noncancer
effects.

The
carcinogenicity
assessment
provides
information
on
two
aspects
of
the
carcinogenic
risk
assessment
for
the
agent
in
question:
(
1)
the
EPA
classification
and
(
2)
quantitative
estimates
of
risk
from
oral
exposure.
The
classification
reflects
a
weight­
of­
evidence
judgment
of
the
likelihood
that
the
agent
is
a
human
carcinogen
and
the
conditions
under
which
the
carcinogenic
effects
may
be
expressed.
Quantitative
risk
estimates
are
presented
in
three
ways.
The
slope
factor
is
the
result
of
the
application
of
a
low­
dose
extrapolation
procedure
and
is
presented
as
the
risk
per
mg/
kg/
day.
The
unit
risk
is
the
quantitative
estimate
in
terms
of
risk
per
micrograms
per
liter
(

g/
L)
drinking
water.
The
third
form
in
which
risk
is
presented
is
a
drinking
water
concentration
providing
cancer
risks
of
1
in
10,000,
1
in
100,000,
or
1
in
1,000,000.
vi
Development
of
the
hazard
identification
and
dose­
response
assessments
for
glyoxal
and
methylglyoxal
has
followed
the
general
guidelines
for
risk
assessments
as
set
forth
by
the
National
Research
Council
(
1983)
and
The
Presidential/
Congressional
Commission
on
Risk
Assessment
and
Risk
Management
(
1997).
Other
guidelines
that
were
used
in
the
development
of
this
assessment
include
the
following:
Guidelines
for
Carcinogen
Risk
Assessment
(
U.
S.
EPA,
1986),
Proposed
Guidelines
for
Carcinogen
Risk
Assessment
(
U.
S.
EPA,
1996a,
1999),
Guidelines
for
Developmental
Toxicity
Risk
Assessment
(
U.
S.
EPA,
1991),
Guidelines
for
Reproductive
Toxicity
Risk
Assessment
(
U.
S.
EPA,
1996b),
Guidelines
for
Neurotoxicity
Risk
Assessment
(
U.
S.
EPA,
1998),
Recommendations
for
and
Documentation
of
Biological
Values
for
Use
in
Risk
Assessment
(
U.
S.
EPA,
1988),
and
Health
Effects
Testing
Guidelines
(
U.
S.
EPA,
1997).
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
vii
EXECUTIVE
SUMMARY
Glyoxal
and
methylglyoxal
are
small
geminal
(
neighboring)
dicarbonyl
compounds
that
are
produced
during
ozonation
of
potable
water.
Both
compounds
are
also
generated
endogenously
in
the
degradation
of
nutrients
or
xenobiotics.
They
are
metabolized
by
the
glyoxalase
enzyme
system.
When
present
in
excess,
they
can
modify
DNA,
RNA,
and
proteins.
Because
of
their
chemical
similarities,
glyoxal
and
methylglyoxal
are
considered
together
in
this
criteria
document.

Glyoxal
Administration
of
single
oral
doses
of
1000
mg/
kg
of
glyoxal
to
rats
resulted
in
decreased
protein
synthesis
in
liver
and
spleen.
Other
endpoints
of
toxicity
were
not
investigated.
Exposure
of
rats
to
glyoxal
in
drinking
water
for
30
to
180
days
resulted
in
decreased
food
and
water
intake
and
decreased
body
weight
gain
at
doses
of
239
mg/
kg/
day
and
above.
Body
weight
gain
was
slower
in
exposed
animals
than
in
pair­
fed
controls,
indicating
that
the
effect
on
body
weight
gain
was
not
entirely
due
to
taste
aversion.
Histological
examination
revealed
no
evidence
of
tissue
injury
except
for
a
slight
swelling
of
papillary
epithelial
cells
in
the
kidney
in
animals
exposed
to
298
to
315
mg/
kg/
day
for
180
days.
Analysis
of
blood
revealed
decreased
levels
of
some
serum
enzymes
and
total
protein
at
doses
of
107
mg/
kg/
day
and
above.

Glyoxal
administered
orally
to
rats
caused
cross­
linking
between
DNA
and
proteins
in
epithelial
cells
in
the
stomach.
In
vitro,
glyoxal
increased
the
incidence
of
sister
chromatid
exchange
(
SCE)
and
micronuclei
in
human
lymphocytes.
In
vitro
studies
of
mutagenicity
indicate
that
glyoxal
causes
both
point
and
frame
shift
mutations.
G:
C
base
pairs
appear
to
be
most
vulnerable
to
the
mutagenic
effects
of
glyoxal.

The
carcinogenic
potential
of
glyoxal
has
not
been
thoroughly
studied.
Exposure
of
rats
to
glyoxal
(
500
mg/
kg/
day)
in
drinking
water
for
32
weeks
did
not
result
in
tumors
or
hyperplastic
lesion
of
the
stomach,
but
too
few
animals
were
exposed
and
exposure
was
too
brief
to
draw
meaningful
conclusions.
Several
short­
term
studies
indicate
that
glyoxal
may
have
cancerinitiating
or
cancer­
promoting
potential.
Glyoxal
(
500
mg/
kg/
day
in
water
for
32
weeks)
caused
hyperplasia
and
increased
adenocarcinomas
in
the
stomach
of
rats
pretreated
with
N­
methyl­
Nnitro
N­
nitrosoguanidine
(
MNNG).

No
data
were
located
on
health
effects
in
humans
exposed
to
glyoxal
by
the
oral
route.
Several
studies
found
that
dermal
exposure
to
glyoxal
may
produce
skin
sensitization.
Diabetics
may
be
sensitive
to
glyoxal
because
of
compromised
glyoxylase
activity.
Glyoxal
exposure
may
increase
oxalate
excretion
in
individuals
with
hyperoxaluria.

The
mechanism
by
which
glyoxal
produces
adverse
effects
is
not
known,
but
is
presumably
related
to
the
reactivity
of
this
compound
and
its
ability
to
form
adducts
and
cross­
links
with
key
cellular
molecules
such
as
proteins
nucleic
acids.

The
available
data
on
glyoxal
are
inadequate
for
the
development
of
an
RfD
or
HA
values.
Neither
the
U.
S.
Environmental
Protection
Agency
(
EPA)
nor
the
International
Agency
for
the
Research
of
Cancer
(
IARC)
have
evaluated
the
cancer
weight
of
evidence
category
for
glyoxal,
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
viii
and
no
quantitative
estimates
of
cancer
potency
were
located.
Due
to
limitations
in
the
data,
the
carcinogenic
potential
for
glyoxal
cannot
be
determined
at
this
time.

Methylglyoxal
Oral
LD
50
values
for
methylglyoxal
in
rats
were
greater
than
(>)
1
mg/
kg;
the
LD
50
value
for
newborn
rats
was
less
than
half
that
for
adult
rats.
When
administered
intravenously
to
cats
and
rabbits,
methylglyoxal
seemed
to
cause
decreased
respiration
and
slowed
cardiac
function,
but
this
response
was
not
observed
when
administered
intraperitoneally.
Intraperitoneal
administration
of
methylglyoxal
to
rats
caused
mild
hepatotoxic
responses.
Pretreatment
with
10%
ethanol
or
1%
acetone
in
drinking
water
intensified
the
effects
of
methylglyoxal
in
the
liver.

Exposure
of
young
mice
to
about
5
g/
kg/
day
methylglyoxal
in
drinking
water
during
gestation,
lactation,
through
the
first
2
months
after
weaning
was
associated
with
depletion
of
erythrocyte
glutathione
and
decreased
activity
of
glutathione­
S­
transferase.
A
glucose
challenge
after
fasting
led
to
hyperglycemia
in
nearly
half
the
treated
mice.
Red
cells
from
treated
animals
had
decreased
resistance
to
oxidation
when
tested
in
vitro.

Exposure
of
rats
for
32
weeks
to
0.25%
methylglyoxal
in
drinking
water
(
a
dose
of
about
250
mg/
kg/
day)
did
not
result
in
any
obvious
effect
on
weight
gain
and
did
not
cause
any
obvious
signs
of
toxicity.
However,
histological
and
biochemical
studies
were
not
performed
so
these
data
do
not
identify
a
reliable
NOAEL.

Numerous
studies
indicate
that
methylglyoxal
is
mutagenic
in
bacterial
systems,
and
this
effect
is
increased
by
hydrogen
peroxide.
Methylglyoxal
was
weakly
clastogenic
in
rats,
causing
increased
micronuclei
in
liver
and
bone
marrow.
Methylglyoxal
also
has
produced
clastogenicity,
chromosomal
aberrations,
or
mutagenicity
in
yeast,
Chinese
hamster
ovary
(
CHO)
cells,
Chinese
hamster
lung
cells,
and
human
lymphocytes
exposed
in
vitro.

The
carcinogenic
potential
of
methylglyoxal
has
not
been
thoroughly
studied.
Exposure
of
rats
to
methylglyoxal
(
250
mg/
kg/
day)
for
32
weeks
did
not
result
in
tumors
or
hyperplastic
lesions
of
the
stomach,
but
too
few
animals
were
exposed
and
exposure
was
too
brief
to
draw
meaningful
conclusions.
Methylglyoxal
was
reported
to
produce
no
tumors
in
rats
exposed
to
20
mg/
kg/
day
for
2
years,
but
these
findings
have
been
presented
in
only
abstract
form.

Several
short­
term
studies
indicate
that
methylglyoxal
may
have
cancer­
initiating
or
promoting
potential.
Methylglyoxal
(
250
mg/
kg/
day
in
water
for
32
weeks)
induced
hyperplasia
in
the
stomach
of
rats
pretreated
with
N­
methyl­
N

­
nitro­
N­
nitrosoguanidine,
but
did
not
cause
tumors.
Exposure
of
rats
to
methylglyoxal
in
water
at
doses
of
5
to
20
mg/
kg/
day
for
6
weeks
resulted
in
an
increase
in
the
number
and
volume
of
 ­
glutamyl
transpeptidase
(
GGT)
foci
in
liver,
and
this
effect
was
increased
by
prior
exposure
to
2­
acetylaminofluorene.
Exposure
to
single
oral
doses
of
50
to
500
mg/
kg
resulted
in
increased
unscheduled
DNA
synthesis
and
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
ix
ornithine
decarboxylase
activity
in
stomach
mucosal
cells
of
rats.
Two
studies
indicate
that
subcutaneous
injection
of
methylglyoxal
may
result
in
injection
site
tumors.

No
data
were
located
on
health
effects
in
humans
exposed
to
methylglyoxal
by
the
oral
route.
Several
studies
indicate
that
the
levels
of
methylglyoxal
are
elevated
in
diabetic
subjects
and
are
associated
with
the
formation
of
advanced
glycylation
endproducts.
The
adducts
are
liberated
from
proteins
during
degradation
and
cleared
through
the
kidneys.
Although
the
adducts
form
in
healthy
subjects,
concentrations
are
increased
in
diabetes.
Thus,
diabetics
may
be
sensitive
to
direct
and
indirect
effects
of
methylglyoxal.
Advanced
glycylation
endproducts
are
believed
to
play
an
important
role
in
several
of
the
complications
of
long­
term
diabetes
including
atherosclerosis,
renal
problems,
and
changes
in
lens
proteins.
Individuals
with
kidney
problems
may
not
be
able
to
excrete
advanced
glycylation
endproducts
and
may
also
have
heightened
sensitivity
to
methylglyoxal.

The
mechanism
by
which
methylglyoxal
produces
adverse
effects
is
not
known,
but
is
presumably
related
to
the
reactivity
of
this
compound
and
its
ability
to
form
adducts
and
cross­
links
with
key
cellular
molecules.

The
available
data
on
methylglyoxal
are
inadequate
for
the
development
of
an
RfD
or
HA
values.
There
are
no
bioassay
data
on
methylglyoxal
that
can
be
used
to
classify
this
compound
for
its
potential
to
cause
cancer.
According
to
the
EPA
1999
guidelines,
the
carcinogenicity
of
methylglyoxal
cannot
be
determined
at
this
time.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
1
GLYOXAL
I.
PHYSICAL
AND
CHEMICAL
PROPERTIES
Glyoxal
is
a
white
crystalline
material
that
melts
near
room
temperature.
It
is
readily
soluble
in
water,
with
little
tendency
to
evaporate.
It
is
commercially
available
as
a
crystalline
dihydrate
and
in
30%
or
40%
aqueous
solutions
(
HSDB,
1999).
Other
physical­
chemical
properties
are
summarized
in
Table
1.

Glyoxal
is
prepared
by
oxidation
of
acetaldehyde
with
nitric
or
selenious
acid,
by
vaporphase
oxidation
of
ethylene
glycol,
or
by
hydrolysis
of
dichlorodioxane
(
Budavari
et
al.,
1996;
HSDB,
1999).
In
concentrated
solution
it
polymerizes
in
a
highly
exothermic
reaction.
In
diluted
aqueous
solution,
glyoxal
has
been
shown
to
exist
as
a
mixture
of
the
fully
hydrated
monomer,
dimer,
and
trimer,
with
the
monomer
and
dimer
being
the
predominant
species
(
Whipple,
1970,
as
cited
in
NTP,
1994).
Glyoxal
is
used
industrially
as
an
intermediate
in
the
synthesis
of
a
substituted
ethylene
urea
compound
used
in
postcuring
of
permanent­
press
fabrics,
as
a
crosslinking
and
insolubilizing
agent,
and
in
the
paper
industry
in
applications
such
as
sizing
and
washable
wallpaper.
Minor
uses
are
found
in
medicine,
bacteriology,
and
pest
control,
and
as
a
substitute
for
formaldehyde
in
embalming
fluids
(
HSDB,
1999).
There
is
no
indication
that
glyoxal
is
a
major
environmental
contaminant
as
a
result
of
these
industrial
uses
(
HSDB,
1999).

Table
1.
Physical
and
Chemical
Properties
of
Glyoxal
Parameter
Data
CASRN
107­
22­
2
Empirical
Formula
C2H2O2
(
OHCCHO)

Structure
CHO­
CHO
Molecular
Weight
58.04
Vapor
Pressure
255
mm
Hg
at
25

C
Water
Solubility
Very
Soluble
Conversion
Factors
(
air)
1
ppm
=
1
mg/
L
=
2.4
mg/
m3
at
25

C
422
ppm
at
25

C
Melting
Point
15

C
Boiling
Point
51

C
Density
1.14
g/
mL
Sources:
Budavari
et
al.,
1996;
HSDB,
1999.
Draft
 
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2003)
2
II.
TOXICOKINETICS
A.
Absorption
No
data
were
located
regarding
oral
absorption
of
glyoxal.
However,
based
on
its
chemical
structure,
it
is
expected
that
glyoxal
will
be
well
absorbed
from
the
gastrointestinal
tract.

B.
Distribution
No
data
were
located
regarding
tissue
distribution
of
glyoxal
following
oral
exposure.
However,
it
is
expected
that
glyoxal
would
be
widely
distributed
throughout
the
body.
The
aversge
concentration
of
glyoxal
in
human
blood
samples
is
about
200
pmol/
g
(
Thornalley,
1998).

C.
Metabolism
Glyoxal
is
a
substrate
for
the
glyoxalase
I
(
GLO
I)
and
glyoxalase
II
(
GLO
II)
system
(
Thornalley,
1998).
GLO
I
conjugates
glyoxal
to
glutathione
(
GSH)
forming
S­
glycolglutathione,
and
GLO
II
catalyzes
the
hydrolysis
of
S­
glycolglutathione
to
glycolate
(
hydroxyacetic
acid)
and
GSH
(
Thornalley,
1996).
Glycolate
can
be
oxidized
to
glyoxylate
and
then
converted
to
glycine
through
a
transamination
reaction
(
Montgomery
et
al.,
1990).

Arnold
et
al.
(
1983)
reported
that
glyoxal
reacts
in
vitro
with
the
enediol
portion
of
ascorbic
acid
to
yield
a
five­
membered
cyclic
acetal.
This
condensation
is
reversible
under
alkaline
conditions.
No
studies
were
performed
to
determine
whether
this
reaction
occurs
in
vivo.

Glyoxal
can
be
produced
endogenously
from
slow
oxidation
of
glucose,
degradation
of
glycosylated
proteins
and
lipid
peroxidation
(
Thornalley,
1998;
Thornalley
et
al.,
1999).
It
is
also
produced
during
the
metabolism
of
diethylene
glycol
and
alloxan
(
HSDB,
1999).

D.
Excretion
No
data
were
located
on
the
route
or
degree
of
glyoxal
excretion.
It
is
expected
that
glyoxal
or
its
metabolites
(
glycolate
glyoxylate,
oxylate)
will
be
excreted
mainly
in
urine.

E.
Bioaccumulation
and
Retention
No
data
were
located
on
the
bioaccumulation
or
retention
of
glyoxal.
Glyoxal
is
not
expected
to
accumulate
in
the
body.
Draft
 
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2003)
3
III.
HUMAN
EXPOSURE
A.
Occurrence
Air
There
was
little
information
in
the
available
literature
on
the
distribution
of
glyoxal
in
ambient
air.
It
is
apparently
generated
during
the
combustion
of
gasoline
and
has
been
reported
to
occur
in
the
exhaust
of
automobiles.
Glyoxal
was
identified
in
air
samples
collected
at
the
toll
plaza
for
the
San
Francisco
Bay
bridge
(
Destaillats
et
al.,
2002)
and
in
air
samples
collected
in
Los
Angeles
at
concentrations

1ppb
(
Kawamura
et
al.,
2000).
The
glyoxal
samples
from
downtown
Los
Angeles
were
higher
than
those
from
west
Los
Angeles.
Glyoxal
could
also
be
extracted
from
fine
particulate
matter
collected
from
the
Caldicott
Tunnel
near
Oakland,
CA
over
a
four
day
period
in
1997
(
Rao
et
al.,
2001)
Gasoline
combustion
appeared
to
produce
more
glyoxal
than
diesel
fuel.
Glyoxal
is
present
in
cigarette
smoke
(
NTP,
1994)
and
can
gain
access
to
the
atmosphere
from
this
source.

Food
Small
amounts
of
glyoxal
may
be
present
in
foods
as
metabolic
intermediates.
Nagao
et
al.
(
1986a)
reported
that
glyoxal
was
present
in
coffee
(
both
instant
and
brewed)
and
other
beverages
(
Table
2).
Glyoxal
has
also
been
detected
in
toast
and
soy
sauce
(
Nagao
et
al.,
1986a).
The
level
of
glyoxal
in
toast
was
0.5

g/
g.
In
soy
sauce,
there
was
4.9

g/
mL
glyoxal.
Small
amounts
of
glyoxal
can
be
produced
when
sugar­
containing
foods
are
cooked
at
high
temperatures
(
Nishi
et
al.,
1989);
it
is
found
in
alcoholic
beverages.

Table
2.
Concentrations
of
Glyoxal
in
Selected
Beveragesa
Beverage
Glyoxal
(

g/
mL)

Beer
b
0.07­
0.21
Bourbon
whiskey
a
0.39
Apple
brandy
a
0.33
Wine
a,
b
0.4­
1.5
Japanese
sake
a,
0.29
Instant
coffee
a,
c
0.34
Brewed
coffeea,
d
0.87
Black
teaa
trace
aNagao
et
al.,
1986a.
bBarros
et
al.,
1999
CPrepared
by
dissolving
1.5
g
powder
in
100
mL
water.
dPrepared
from
10
g
ground
coffee
beans
and
150
mL
water.
Draft
 
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2003)
4
Water
Glyoxal
is
formed
as
a
byproduct
from
ozonation
of
water
(
Glaze
et
al.,
1989;
Langlais
et
al.,
1991).
Ozonation
of
water
from
the
Los
Angeles
Aqueduct
Filtration
Plant
resulted
in
a
significant
increase
of
several
aldehydes
and
carboxylic
acids
compared
with
their
level
in
source
water.
For
example,
glyoxal
was
not
detectable
in
raw
water,
but
was
detected
at
concentrations
of
13

g/
L
following
ozonation
(
Langlais
et
al.,
1991).

No
trace
of
glyoxal
was
reported
by
Glaze
et
al.
(
1989)
in
surface
water
treatment
plant
influents.
However,
glyoxal
can
be
formed
during
the
ozonation
of
water
containing
humic
acids.
Following
ozonation,
the
concentration
of
glyoxal
in
potable
water
was
about
7

g/
L
after
10
minutes
and
about
2
to
8

g/
L
after
2
to
10
minutes
for
wastewater
(
Sayato
et
al.,
1989).
Lower
concentrations
are
expected
to
form
from
ground
water
sources
due
to
their
lower
organic
carbon
content.
Ueno
et
al.
(
1989)
studied
ozonated
water
containing
concentrated
humic
acids
isolated
from
three
different
sources
and
found
that
glyoxal
was
produced
in
all
three
cases.
Following
ozonation
of
surface
waters
to
which
odor­
causing
compounds
(
2­
methylesoborneal
and
geosmin)
had
been
added,
glyoxal
concentrations
ranged
from
0.3
to
0.8

g/
L.
Ozonation
was
carried
out
for
16
minutes
at
pH
7.6
or
7.7.
Odorant
concentrations
were
decreased
with
time
as
they
were
oxidized
to
simpler
compounds
(
Yamada
and
Somiya,
1989).

After
a
40­
minute
ozone
reaction
time,
the
concentration
of
glyoxal
was
increased
when
ozonation
was
carried
out
at
a
basic
pH
rather
than
an
acid
pH.
At
pH
10.5,
the
glyoxal
concentration
was
1.0

g/
L,
while
at
pH
6.6,
the
concentration
was
0.3

g/
L
(
Yamada
and
Somiya,
1989).

In
the
work
by
Yamada
and
Somiya
(
1989),
there
was
no
glyoxal
in
the
postfiltration
water
influent
to
the
ozone
contractors
at
a
demonstration
plant.
After
ozone
treatment,
the
concentration
of
glyoxal
was
0.3

g/
L.
Complete
removal
of
glyoxal
was
accomplished
using
a
granular
activated
filter
as
postozonation
treatment.
Glaze
et
al.
(
1989)
showed
that
glyoxal
is
also
removed
from
potable
water
by
reaction
with
chloramines
used
as
secondary
disinfectants.

As
part
of
the
Information
Collection
Rule
for
Disinfection
By­
Products,
quarterly
data
on
the
presence
of
glyoxal
were
collected
from
ozonation
water­
treatment
plants.
In
more
than
50%
of
the
samples,
no
glyoxal
was
detected.
The
maximum
concentration
detected
in
quarterly
samples
from
each
of
20
plants
was
22

g/
L
(
McLain
and
Blank,
2000).

Soil
There
was
no
information
in
the
available
literature
concerning
levels
of
glyoxal
in
soils.
There
is
no
reason
to
suspect
that
it
is
present
in
any
significant
concentrations
except
as
breakdown
products
of
organic
materials.
Draft
 
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2003)
5
B.
Fate
Glyoxal
undergoes
addition
and
condensation
reactions
with
amines,
amides,
aldehydes,
and
hydroxyl­
containing
materials
(
Ueno
et
al.,
1989).
Due
to
its
reactivity,
the
environmental
persistence
of
glyoxal
is
expected
to
be
minimal.

C.
Exposure
The
available
information
on
exposure
to
glyoxal
is
sparse.
Most
of
the
daily
exposure
is
likely
to
be
from
foods.
Exposure
from
drinking
water
is
expected
to
be
minimal.
Glyoxal
can
be
generated
endogenously
during
the
catabolism
of
energy
nutrients,
particularly
carbohydrates.
Exposure
from
endogenous
glyoxal
production
is
likely
to
exceed
that
from
exogenous
sources.
Draft
 
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2003)
6
IV.
HEALTH
EFFECTS
IN
ANIMALS
A.
Short­
Term
Exposure
Smyth
et
al.
(
1942)
reported
an
oral
acute
LD
50
of
2020
mg/
kg
in
albino
rats
and
760
mg/
kg
in
guinea
pigs
(
Ueno
et
al.,
1991c).
Oral
LD
50
values
of
400
to
800
mg/
kg
for
mice
and
1500
to
1800
mg/
kg
in
rats
are
cited
in
NTP
(
1994).
An
LD
50
of
200
to
400
mg/
kg
in
rats
is
also
cited
by
NTP
(
1994)
and
is
not
consistent
with
the
other
values
reported.

In
order
to
examine
the
effects
of
glyoxal
on
protein
synthesis,
Ueno
et
al.
(
1991c)
administered
single
oral
doses
of
0
or
1000
mg/
kg
of
glyoxal
to
groups
of
four
male
Sprague­
Dawley
rats
followed
by
an
intraperitoneal
injection
of
3H­
leucine
4
hours
later.
The
animals
were
sacrificed
2
hours
after
the
leucine
injection,
and
the
rate
of
protein
synthesis
in
liver,
kidney,
and
spleen
was
estimated
by
measuring
incorporation
of
3H­
leucine
into
trichloroacetic
acid­
insoluble
tissue
protein.
Exposure
to
1000
mg/
kg
glyoxal
resulted
in
a
significant
(
p<
0.01)
decrease
in
protein
synthesis
in
liver
and
spleen.
A
slight
decrease
was
noted
in
kidneys,
but
this
was
not
statistically
significant.
Comparable
effects
on
the
liver
were
noted
following
an
i.
v.
injection
of
150
mg/
kg.

B.
Longer­
Term
Exposure
Subchronic
Takahashi
et
al.
(
1989)
exposed
groups
of
10
male
Wistar
rats
to
0.5%
glyoxal
for
32
weeks.
Assuming
water
intake
of
0.10
L/
kg/
day,
this
corresponds
to
a
dose
of
about
500
mg/
kg/
day.
Animals
were
weighed
weekly.
Exposure
to
glyoxal
clearly
depressed
weight
gain.
There
was
very
little
change
in
average
weight
of
the
treated
animals
between
weeks
8
and
40,
the
period
of
glyoxal
administration.
In
fact,
there
was
a
decrease
in
average
body
weight
over
the
first
4
weeks
of
glyoxal
administration.
Gross
inspection
of
internal
organs
at
the
end
of
exposure
did
not
reveal
any
signs
of
toxicity.
Histological
evaluations
were
not
performed.

A
study
by
Ueno
et
al.
(
1991c)
examined
the
effects
of
glyoxal
exposure
via
drinking
water
on
groups
of
five
to
seven
male
Sprague­
Dawley
rats.
The
study
was
conducted
in
two
phases.
In
Phase
1,
test
animals
received
drinking
water
(
ad
libitum)
containing
glyoxal
at
0,
2000,
4000,
or
6000
mg/
L
for
30,
60,
or
90
days.
The
authors
indicated
that
average
associated
doses
were
188,
407,
and
451
mg/
kg/
day
at
the
30­
day
termination;
135,
239,
and
344
mg/
kg/
day
at
the
60­
day
termination;
and
107,
234,
and
315
mg/
kg/
day
at
the
90­
day
termination.

There
was
a
statistically
significant
(
p<
0.01)
dose­
related
decrease
in
average
daily
food
intake,
which
was
not
generally
significant
when
converted
to
g/
kg/
day.
There
was
a
statistically
significant
(
p<
0.01)
but
relatively
mild
decrease
in
mean
daily
water
consumption
per
rat
for
all
dose
groups
at
30­,
60­,
and
90­
day
terminations.
Terminal
body
weight
was
significantly
reduced
(
p<
0.01)
at
the
two
highest
doses
at
all
three
termination
times.
The
calculated
percent
change
in
terminal
body
weight
ranged
from
12%
to
14%
at
4000
mg/
L
and
from
26%
to
31%
at
6000
mg/
L.
Absolute
organ
weights
decreased
significantly
in
all
exposed
groups
at
all
three
termination
times
and
there
was
a
significant
increase
in
relative
kidney
weight
at
90
days
for
the
Draft
 
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2003)
7
high­
dose
group.
Analysis
of
blood
revealed
no
significant
effects
on
BUN,
creatine,
or
creatinine.
Serum
levels
of
aspartate
amino
transferase,
alanine
amino
transferase
and
lactate
dehydrogenase,
albumin,
and
total
protein
were
all
reduced
in
a
dose­
dependent
manner.
Several
effects
(
alanine
amino
transferase,
total
protein)
were
statistically
significant
at
all
doses.

At
the
30­
day
termination
but
not
for
the
other
durations,
there
was
an
increase
in
GLO
I
activity
in
erythrocytes
at
all
three
doses,
in
liver
at
the
two
highest
doses,
and
in
the
kidneys
only
at
the
highest
dose.
GLO
II
also
tended
to
increase
in
liver
and
erythrocytes,
but
the
effects
were
not
as
clear.
The
authors
identified
the
lowest
dose
(
107
mg/
kg/
day
over
the
90­
day
period)
as
a
LOAEL
based
on
its
effects
on
the
total
serum
protein.

In
Phase
2,
a
diet­
limited
control
group
was
run
concurrently
with
the
6000
mg/
L
exposure
group
and
a
control
group
that
was
not
diet­
limited.
The
diet­
limited
control
group
was
fed
the
same
amount
of
food
as
the
6000
mg/
L
exposure
group
in
order
to
distinguish
effects
of
reduced
food
consumption
from
glyoxal
toxicity.
This
study
was
carried
out
to
two
time
points,
90
days
and
180
days.
Based
on
measured
body
weights
and
water
intakes,
the
average
intake
was
315
mg/
kg/
day
at
90
days
and
298
mg/
kg/
day
at
180
days.
Reduced
food
consumption
could
account
for
only
about
half
of
the
body
weight
effect,
indicating
glyoxal
exposure
was
suppressing
growth.

Two
of
the
animals
exposed
to
glyoxal
died
within
30
days.
Necropsy
revealed
hemorrhage
of
the
glandular
stomach.
Food
intake
and
body
weight
gain
were
significantly
reduced
in
the
exposed
animals.
Inhibition
of
weight
gain
was
greater
in
the
exposed
animals
than
in
the
group
of
pair­
fed
controls,
suggesting
that
part
of
the
effect
on
body
weight
was
caused
by
systemic
toxicity.
Absolute
weights
of
organs
except
testes
and
brain
were
reduced,
and
an
increase
in
the
organ­
to­
body
weight
ratio
was
observed
in
liver,
kidney,
and
heart
at
both
90
and
180
days.
Gross
examination
revealed
stomach
lesions
(
hemorrhage,
polyps)
in
two
high­
dose
animals,
but
the
authors
did
not
consider
these
to
be
necessarily
treatment­
related.
Histological
examination
of
liver,
kidney,
spleen,
stomach,
thymus,
and
lymph
nodes
did
not
reveal
any
treatment­
related
effects
except
for
a
slight
swelling
of
papillary
epithelial
cells
in
the
kidneys.
Examination
of
blood
revealed
significant
decreases
in
serum
aspartate
amino
transferase
and
total
protein.
Based
on
body
weight,
serum
protein
levels,
and
histological
effects
in
kidney,
this
study
identifies
a
LOAEL
of
298
to
315
mg/
kg/
day.

C.
Reproductive/
Developmental
Effects
NTP
(
1991)
as
cited
in
NTP
(
1994)
conducted
a
pilot
developmental
study
using
groups
of
eight
Sprague­
Dawley
rats
and
doses
of
0,
200,
800,
1200,
1600,
or
2000
mg/
kg/
day
on
gestation
days
6­
15.
All
dams
in
the
two
highest
dose
groups
and
five
animals
in
the
1200
mg/
kg/
day
dose
group
died
or
had
to
be
euthanized
on
or
before
gestation
day
17.
Six
of
eight
litters
in
the
800
mg/
kg/
day
group
and
two
of
three
litters
in
the
1200
mg/
kg/
day
group
were
completely
resorbed.
The
NOAEL
in
the
pilot
study
was
200
mg/
kg/
day
and
the
LOAEL
was
800
mg/
kg/
day
based
on
complete
fetal
resorption
for
six
of
eight
litters.

NTP
(
1994)
conducted
a
developmental
toxicity
study
of
glyoxal
trimeric
dihydrate
in
Sprague­
Dawley
CD
®
rats.
Glyoxal
trimeric
dihydrate
was
chosen
because
it
is
more
stable
than
glyoxal
and
because
the
human
population
is
as
likely
to
be
exposed
to
the
trimeric
dihydrate
as
a
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
8
constituent
of
other
mixtures
as
to
glyoxal.
The
test
compound
was
administered
by
gavage
in
water
on
gestational
days
6
through
15
(
the
period
of
organogenesis)
to
time­
mated
groups
of
26
animals.
Doses
were
0,
50,
150,
or
300
mg/
kg/
day.
The
major
effect
noted
during
exposure
was
rooting
(
digging
in
the
bedding)
behavior
around
the
dosing
time
for
the
150
and
300
mg/
kg/
day
doses.

Animals
were
sacrificed
on
gestation
day
20
and
examined
for
maternal
body
and
organ
weights,
implant
status,
fetal
weight,
fetal
sex,
and
fetal
morphological
development.
Maternal
body
weight
of
the
exposed
animals
was
consistently
slightly
below
the
control
body
weight
but
the
difference
was
only
statistically
significant
for
the
highest
dose
on
gestation
day
12.
Maternal
body
weight
gain
of
animals
was
also
significantly
reduced
(
p<
0.05)
at
the
highest
dose
during
gestation
days
6
to
15,
which
corresponded
to
a
reduction
in
relative
food
consumption.
Maternal
relative
water
consumption
of
dosed
animals
was
generally
above
the
consumption
of
control
animals,
and
was
significantly
increased
at
all
dose
levels
on
gestation
days
15
to
20,
increasing
dramatically
after
cessation
of
exposure.
There
were
no
maternal
deaths
in
this
study,
and
maternal
toxicity
at
300
mg/
kg/
day
was
reflected
only
as
a
small
decrease
in
body
weight
and
food
consumption.
No
dose­
related
developmental
toxicity
was
observed
in
the
dose
range
tested.
For
the
dams,
the
NOAEL
was
150
mg/
kg/
day
and
the
LOAEL
300
mg/
kg/
day
for
minimal
maternal
toxicity
in
CD
rats.
The
highest
dose
(
300
mg/
kg/
day)
was
a
NOAEL
for
developmental
toxicity.

The
NOAEL/
LOAEL
data
from
the
NTP
(
1991,
1994)
studies
suggest
that
there
is
less
than
a
threefold
difference
between
the
dose
that
produces
no
effect
on
reproduction
(
300
mg/
kg/
day)
and
the
dose
that
produces
almost
complete
toxicity
(
800
mg/
kg/
day).

In
a
study
of
the
effects
of
ethylene
glycol
and
its
metabolites
on
development,
9.5
day­
old
embryo
were
harvested
from
pregnant
Wistar
rats
and
exposed
to
glyoxal
concentration
of
0,
3
or
6
mM
in
a
whole
embryo
culture
system
(
Klug
et
al.,
(
2000).
At
the
3
mM
concentration
the
number
of
dysmorphic
embryos
(
6%)
fell
within
the
range
for
historic
controls..
At
the
6
mM
concentration
78
%
of
the
embryos
were
dysmorphic.

D.
Mutagenicity
and
Genotoxicity
The
mutagenicity
and
genotoxicity
data
on
glyoxal
are
predominantly
positive.
Glyoxal
is
mutagenic
in
the
Ames
assay
with
many
strains
of
Salmonella
typhimurium
(
Kato
et
al.,
1989)
and
has
been
shown
to
cause
base­
pair
substitutions
and
some
frameshift
mutations
at
G:
C
base
pairs
(
Murata­
Kamiya
et
al.,
1997a).
It
has
also
been
found
to
damage
DNA
by
a
variety
of
tests,
including
causing
single­
strand
breaks
in
vivo
and
in
vitro
(
Ueno
et
al.,
1991b).

Gene
Mutation
Assays
Numerous
gene
mutation
assays
have
been
conducted
on
glyoxal,
most
using
S.
typhimurium,
not
only
to
determine
its
mutagenicity
but
also
to
study
its
mechanism
of
action
and
its
contribution
to
the
mutagenicity
of
complex
mixtures
to
which
the
human
population
is
routinely
exposed.
However,
no
Ames
assay
using
all
of
the
standard
recommended
strains
was
located.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
9
The
role
of
intercellular
enzymes
and
active
oxygen
species
associated
with
the
mutagenic
activity
of
glyoxal
was
evaluated
in
S.
typhimurium
strains
TA100
and
TA104
(
Ueno
et
al.,
1991a).
TA100
and
TA104
were
exposed
to
glyoxal
in
the
presence
of
various
active
oxygen
scavengers,
or
with
suppression
of
intercellular
levels
of
superoxide
dismutase
(
SOD),
catalase
(
CAT),
or
GSH.
The
mutagenic
activity
of
glyoxal
was
reduced
by
addition
of
GSH
in
both
strains
and
was
further
reduced
by
the
addition
of
GLO
I
and/
or
GLO
II
with
GSH
(
Ueno
et
al.,
1991a).
The
mutagenic
activity
in
TA100
was
not
reduced
by
addition
of
SOD,
CAT,
SOD+
CAT,
or
D­
mannitol;
however,
addition
of
CAT
resulted
in
a
slight
inhibition
of
mutagenic
activity
in
TA104.
Mutagenic
activity
of
glyoxal
did
not
vary
with
the
suppression
of
SOD
activity
in
the
cells
but
was
enhanced
by
the
GSH
depleters,
with
about
a
40%
decrease
of
GSH
level
in
TA100.

Dorado
et
al.
(
1992)
examined
the
mutagenicity
of
glyoxal
(
and
eight
other
dicarbonyl
compounds)
against
S.
typhimurium
strain
TA100
using
the
standard
plate
incorporation
Ames
assay
without
preincubation
and
without
metabolic
activation.
Glyoxal
was
found
to
be
mutagenic,
whereas
sodium
glyoxylate
(
CAS
2706­
75­
4)
was
found
not
to
be
mutagenic
to
strain
TA100.
The
authors
suggested
that
the
difference
in
mutagenicity
between
glyoxal
and
sodium
glyoxylate
was
that
glyoxal
forms
adducts
with
the
guanine
residues
of
nucleic
acids,
whereas
sodium
glyoxylate
cannot
form
stable
adducts
with
guanine.
Because
strain
TA100
reverts
to
His+
from
base­
pair
mutations,
this
study
demonstrated
that
the
mutagenic
activity
of
glyoxal
resulted
from
base­
pair
transversions,
as
opposed
to
frame­
shift
mutations.

Mellado
and
Montoya
(
1994)
examined
equilibrium
constants
of
the
adduct
formation
reactions
with
guanine
and
guanosine
for
several
dicarbonyl
compounds
in
order
to
determine
a
mutagenic
mechanism.
Glyoxal,
methylglyoxal,
and
phenylglyoxal
were
evaluated.
The
values
of
K
F
and
 
H
F
related
to
the
stability
of
the
adduct
formation
and
the
chemical
reactivity.
The
higher
the
K
F,
the
greater
the
stability,
and
the
more
negative
the
 
H
F
the
greater
the
reactivity
of
the
compound.
The
order
of
compounds
with
the
highest
K
F
and
lowest
 
H
F
was
found
to
be:
glyoxal
>
methylglyoxal
>
phenylglyoxal.

The
correlation
coefficients
between
K
F
and
mutagenicity
and
 
H
F
and
mutagenicity
in
S.
typhimurium
strain
TA100
were
determined.
A
strong
correlation
was
found
between
adduct
formation
with
the
purinic
bases
guanine
and
guanosine,
and
mutagenicity
(
average
r
=
0.94).
The
authors
concluded
that
the
mutagenic
activity
for
glyoxal
was
directly
related
to
reactions
with
the
guanine.

Murata­
Kamiya
et
al.
(
1997b)
used
S.
typhimurium
strains
TA7001,
TA7002,
and
TA7003,
which
detect
base­
pair
substitutions
at
A:
T
pairs;
strains
TA7004,
TA7005,
and
TA7006,
which
detect
base­
pair
substitutions
at
G:
C
pairs;
and
strain
TA98,
which
detects
frameshift
mutations
at
G:
C
pairs.
The
plate
incorporation
method
was
used,
with
preincubation
used
to
increase
sensitivity.
Strain­
specific
positive
controls
were
used.
Glyoxal
was
tested
at
20­
100

g/
plate,
with
no
mention
of
metabolic
activation,
and
was
mutagenic
in
strains
TA7004,
TA7005,
TA7006,
and
TA98,
the
strains
that
detect
base­
pair
substitutions
at
G:
C
pairs
plus
frameshift
mutations
at
G:
C
pairs.
This
reference
supported
results
of
others
that
glyoxal
reacts
with
guanine,
and
provided
insight
into
the
mechanism
of
genotoxicity.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
10
Murata­
Kamiya
et
al.
(
1997b)
attempted
to
gain
a
better
understanding
of
glyoxal's
mutagenic
mechanisms
by
analyzing
the
mutations
induced
in
the
chromosomal
lacI
gene
of
wildtype
E.
coli.
This
study
strongly
suggested
that
glyoxal
is
mutagenic
to
E.
coli.
Cell
death
and
mutation
frequency
increased
with
dose,
and
glyoxal
induced
base­
pair
reversions
predominately
at
G:
C
base­
pairs.

Chromosomal
Aberration
Assays
Nishi
et
al.
(
1989)
described
the
clastogenic
activity
of
thermally
decomposed
products
of
carbohydrates
in
cultured
Chinese
hamster
V79
cells.
Products
included
glyoxal
and
methylglyoxal.
The
mitotic
index
was
calculated
as
the
percentage
of
metaphases
among
more
than
2000
interphase
nuclei.
Glyoxal
and
methylglyoxal
induced
a
significant
number
of
chromosome
aberrations
and
lowered
the
mitotic
index
significantly.

Glyoxal
was
tested
at
doses
of
100,
200,
300,
and
400

g/
mL.
The
highest
dose
showed
evidence
of
cytotoxicity.
The
percentage
of
aberrant
cells
was
6%
in
the
solvent
control,
and
17%,
41%,
48%,
and
32%
for
the
four
increasing
doses.
Chromatid
gaps
and
breaks
and
exchanges
predominated.
Glyoxal
had
only
approximately
one­
tenth
the
potency
of
methylglyoxal.

Other
Assays
A
series
of
chlorinated
chemicals
and
aldehydes,
representing
chlorination
and
ozonation
byproducts,
was
tested
by
Matsui
et
al.
(
1989)
using
the
liquid
Bacillus
subtilis/
microsome
recassay
to
detect
DNA
damage.
Glyoxal
was
classified
as
strongly
DNA
damaging
both
with
and
without
metabolic
activation.
Only
acrolein
and
formaldehyde
produced
stronger
responses
without
activation,
and
only
formaldehyde
produced
a
stronger
response
with
activation.

Hellmer
and
Bolcsfoldi
(
1992)
employed
the
differential
DNA
repair
test
by
using
DNA
repair
deficient
and
proficient
strains
of
E.
coli
K12/
343/
113
in
an
in
vitro
test.
Results
were
considered
positive
if
the
DNA
repair
deficient
strain
viability
was
significantly
reduced
at
a
lower
concentration
than
the
DNA
repair
proficient
strain.
However,
if
both
strains
were
reduced
at
the
same
concentration,
then
a
genotoxic
mechanism
was
not
likely.
The
glyoxal
result
was
positive
with
and
without
metabolic
activation.
Results
for
49
out
of
the
61
compounds
were
in
agreement
with
the
Ames
test,
indicating
an
80%
concordance
between
the
two
tests.
Thus,
the
study
found
these
bacterial
strains,
and
differential
DNA
repair
as
an
endpoint,
to
be
sufficiently
accurate
as
an
indicator
of
genotoxicity.

Ueno
et
al.
(
1991b)
applied
the
alkaline
elution
technique
to
evaluate
the
damage
to
rat
hepatic
DNA
following
in
vitro
and
in
vivo
exposure
to
glyoxal.
For
in
vitro
exposures,
primarycultured
hepatocytes
were
exposed
to
0.1
to
0.6
mg/
mL
glyoxal
for
60
minutes.
For
in
vivo
exposures,
male
Sprague­
Dawley
rats
were
administered
200,
500,
or
1000
mg/
kg
body
weight
of
glyoxal
via
gastric
intubation.
After
1
to
24
hours
of
exposure,
animals
were
sacrificed
and
hepatocytes
prepared
for
analysis.
After
60
minutes
of
exposure,
the
in
vitro
study
found
dosedependent
DNA
single­
strand
breaks
while
no
DNA
cross­
link
was
observed.
Within
2
hours,
DNA
lesions
were
detected
in
the
liver
following
in
vivo
exposure
to
glyoxal
at
200
to
1000
Draft
 
Do
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or
Quote
(
July,
2003)
11
mg/
kg
body
weight.
The
DNA
lesions
reached
a
maximum
around
9
hours
after
exposure
and
returned
to
control
levels
24
hours
after
exposure
to
all
doses.
This
study
strongly
suggested
that
glyoxal
is
genotoxic
to
rat
hepatocytes,
and
able
to
cause
DNA
lesions
by
single­
strand
breaks.
However,
it
also
suggested
that
the
DNA
damage
was
readily
repaired.

Furihata
and
Matsushima
(
1989)
and
Furihata
et
al.
(
1989)
applied
the
alkaline
elution
technique
to
evaluate
the
DNA
damage
of
rat
pyloric
mucosa
of
the
stomach
following
in
vivo
exposure
to
glyoxal.
Male
F344
were
administered
glyoxal
via
gastric
intubation
at
doses
of
5,
50,
500,
and
550
mg/
kg
body
weight.
The
results
of
this
study
showed
a
dose­
dependent
increase
in
the
elution
rate
constant
after
administration
of
glyoxal,
indicating
glyoxal
induced
DNA
damage
to
the
pyloric
mucosa
of
rat
stomach
after
in
vivo
administration.
Pyloric
mucosal
DNA
damage
was
observed
with
a
dose
of
50
mg/
kg
body
weight
but
no
effects
were
seen
with
the
5
mg/
kg
dose.

Glyoxal
and
kethoxal
were
tested
in
human
peripheral
lymphocytes
to
determine
whether
the
responses
were
typical
of
mammalian
cells
or
were
unique
to
AUXB1
cells
(
Tucker
et
al.,
1989).
Although
glyoxal
produced
small
but
significant
SCE
increases
at
every
dose,
it
lacked
a
dose
response.
On
the
other
hand,
kethoxal
produced
a
linear
SCE
response.
Neither
of
these
agents
induced
ERCs.

E.
Carcinogenicity
Takahashi
et
al.
(
1989)
exposed
10
male
Wistar
rats
to
glyoxal
[
5000
mg/
L;
617
mg/
kg/
day
using
EPA
(
1988
conventions)]
in
drinking
water
for
32
weeks.
Assuming
water
intake
of
0.10
L/
kg/
day,
this
corresponds
to
a
dose
of
about
500
mg/
kg/
day.
At
termination,
all
internal
organs
were
examined
macroscopically,
and
the
stomachs
were
fixed
and
examined
histologically.
No
gastric
carcinomas
or
hyperplastic
lesions
were
detected
in
animals
exposed
to
glyoxal.
This
negative
finding
is
limited
by
the
small
number
of
animals
tested,
the
relatively
short
exposure
period,
and
the
absence
of
a
follow­
up
observation
period.

Takahashi
et
al.
(
1989)
tested
the
cancer­
promoting
activity
of
glyoxal
in
rats.
Groups
of
30
male
Wistar
rats
were
exposed
for
8
weeks
to
N­
methyl­
N'­
nitro­
N­
nitrosoguanidine
(
MNNG)
in
drinking
water
(
100
mg/
L).
Following
this,
animals
were
exposed
to
glyoxal
(
5000
mg/
L)
in
drinking
water
for
32
weeks.
Assuming
consumption
of
0.10
L/
kg/
day,
this
corresponds
to
a
dose
of
about
500
mg/
kg/
day.
Control
animals
were
exposed
to
MNNG
for
8
weeks
and
then
given
untreated
water
for
32
weeks.
At
termination,
animals
were
sacrificed,
internal
organs
were
examined
macroscopically,
and
the
stomach
was
fixed
and
examined
histologically.
Exposure
to
glyoxal
increased
the
incidence
of
hyperplasia
and
adenocarcinoma
of
the
stomach,
especially
in
the
pylorus.
No
toxic
or
neoplastic
changes
were
noted
in
any
other
organs.
These
findings
were
taken
to
indicate
that
glyoxal
exerts
a
cancer­
promoting
effect
in
the
stomach.

Furihata
et
al.
(
1985b)
studied
the
initiating
and
promoting
effects
of
glyoxal
in
rat
glandular
stomach.
Groups
of
five
male
F344
rats
were
given
single
oral
doses
of
300
to
1500
mg/
kg
of
glyoxal
by
stomach
tube,
and
the
rates
of
unscheduled
DNA
synthesis
(
UDS)
and
ornithine
decarboxylase
(
ODC)
activity
were
measured
in
gastric
mucosa
over
the
next
45
to
68
hours.
Glyoxal
increased
both
UDS
and
ODC,
with
a
maximal
effect
about
16
hours
after
exposure.
The
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
12
authors
took
the
increases
in
UDS
and
ODC
to
suggest
that
glyoxal
had
potential
tumor­
initiating
and
­
promoting
activities
in
the
glandular
stomach.

Miyakawa
et
al.
(
1991)
tested
pyrolysates
of
carbohydrates,
including
glyoxal
and
methylglyoxal,
in
a
two­
stage
mouse
skin
carcinogenesis
model.
Initiation
involved
treatment
with
0.1
mL
aliquots
of
test
chemical
or
positive
control
on
the
shaved
backs
of
female
CD­
1
mice
twice
weekly
for
5
weeks.
One
week
after
the
last
initiation
treatment,
12­
Otetradecanoylphorbol
13­
acetate
(
TPA)
in
0.1
mL
acetone
or
acetone
alone
was
applied
twice
weekly
for
47
weeks,
as
a
promoter.
Glyoxal
produced
an
average
of
0.2
skin
tumors
per
mouse,
which
was
not
statistically
significant.

Several
intermediate­
term
rat
liver
bioassays
using
similar
protocols
have
been
reported
for
glyoxal.
Hasegawa
and
Ito
(
1992)
made
use
of
the
two­
step
theory
of
hepatocarcinogenesis,
giving
F344
male
rats
a
single
intraperitoneal
initiating
dose
of
diethylnitrosamine,
followed
2
weeks
later
by
6
weeks
of
exposure
to
the
test
chemical.
At
week
3,
rats
had
a
two­
thirds
partial
hepatectomy
to
stimulate
growth
of
new
liver
tissue.
Results
were
considered
to
be
positive
if
there
was
a
significant
(
p<
0.05)
increase
in
either
the
number
or
area
of
GST­
P
foci.
The
results
for
glyoxal
were
negative.
In
fact,
glyoxal
(
5000
mg/
L
in
drinking
water)
caused
a
statistically
significant
decrease
(
p<
0.05)
in
both
the
number
and
area
of
glutathione
S­
transferase
placental
form
(
GST­
P)
positive
foci
when
compared
with
the
diethylnitrosamine
treated
control.
The
results
reported
indicated
that
glyoxal
exposure
after
diethylnitrosamine
treatment
resulted
in
6.06
foci
per
square
centimeter,
with
an
area
of
0.40
square
millimeter
per
square
centimeter
of
liver
tissue.
The
corresponding
results
reported
for
dimethylnitrosamine
alone
were
7.83
foci
per
square
centimeter
and
an
area
of
0.55
square
millimeter
per
square
centimeter
of
liver
tissue.
The
significance
of
the
role
glyoxal
may
have
played
in
inhibiting
the
formation
of
GST­
P
foci
from
diethylnitrosamine
is
unknown.

Hasegawa
et
al.
(
1995)
evaluated
the
hepatocarcinogenicity
potential
of
coffee
and
its
constituent
compounds
(
methyl
xanthines
and
glyoxal)
using
the
same
procedure
employed
in
their
1992
study
above.
The
test
group
was
initially
exposed
to
diethylnitrosamine
(
DEN)
intraperitoneally
to
initiate
hepatocarcinogenesis
followed
by
dietary
exposure
to
the
test
compound.
Glyoxal
was
administered
at
a
concentration
of
0.2%
(
w/
v)
in
drinking
water
and
again
in
this
experiment
exerted
an
inhibitory
effect
on
both
the
number
and
areas
of
GST­
P
positive
foci.
The
results
reported
indicated
that
glyoxal
exposure
after
diethylnitrosamine
treatment
resulted
in
6.06
foci
per
square
centimeter,
with
an
area
of
0.40
square
millimeter
per
square
centimeter
of
liver
tissue.
The
corresponding
results
reported
for
dimethylnitrosamine
alone
were
7.83
foci
per
square
centimeter
and
an
area
of
0.55
square
millimeter
per
square
centimeter
of
liver
tissue.

The
results
reported
in
the
two
Hasegawa
papers
were
identical,
although
the
concentrations
of
glyoxal
tested
were
reported
to
be
5000
mg/
L
in
the
first
study
and
0.2%
(
2000
mg/
L)
in
the
second
study.
It
is
difficult
to
determine
if
the
results
reported
are
from
one
experiment
with
an
error
in
the
glyoxal
concentration
in
one
case
or
two
different
experiments
with
identical
results.
Draft
 
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or
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(
July,
2003)
13
V.
HEALTH
EFFECTS
IN
HUMANS
No
data
were
located
on
health
effects
in
humans
exposed
to
glyoxal
by
the
oral
route.

Hindson
and
Lawlor
(
1982)
described
a
case
of
a
woman
who
developed
eczema
on
her
hands
while
working
in
an
occupation
that
involved
wrapping
pipe
with
fiberglass
and
coating
it
with
a
polyvinyl
resin.
The
resin
contained
vinyl
acetate,
glyoxal,
formaldehyde,
and
ammonium
persulfate.
Patch
testing
of
the
exposed
woman
revealed
a
positive
response
to
1%
glyoxal,
whereas
10
other
people
did
not
respond.
This
finding
suggests
that
repeated
dermal
contact
with
glyoxal
may
lead
to
skin
sensitization,
but
the
presence
of
the
other
chemicals
in
the
resin
limits
the
confidence
in
this
conclusion.

Schnuch
et
al.
(
1998)
reviewed
the
results
of
31,849
patch
tests
for
allergic
sensitization
collected
over
a
three
year
period
by
the
German
Information
Network
of
Departments
of
Dermatology.
The
results
for
different
dermal
sensitizing
agents
were
compared
for
health
care
workers
and
the
rest
of
the
population.
The
percentage
of
health
care
workers
responding
to
glyoxal
was
4.2%
compared
to
1.4%
for
individuals
from
other
occupations.
Glyoxal
is
an
ingredient
in
some
disinfectants
applied
to
the
surfaces
of
medical
equipment
and
instruments.
Dermal
sensitization
is
caused
by
exposure
of
a
sensitive
individual
to
a
sensitizing
dose.
Thus,
the
increased
incidence
for
positive
patch
test
results
among
health
care
workers
is
more
likely
a
consequence
of
increased
exposure
than
biological
sensitivity.
However,
once
a
person
has
been
sensitized,
that
person
is
more
likely
to
have
a
dermatological
response
at
a
lower
dose
than
the
remainder
of
the
population.

In
a
similar
study,
2689
patients
were
tested
for
their
reaction
to
1%
glyoxal
in
petrolatum
at
33
centers
of
the
German­
Austrian
Information
Network
of
Departments
of
Dermatology
(
Uter
et
al.,
2001)
Allergic
reaction
were
observed
in
1.6%
of
the
patients
and
irritant
reaction
in
0.3%.
Readings
were
equivocal
in
0.6
%
of
the
patients.
Many
of
the
patients
that
were
sensitive
to
glyoxal
worked
in
the
health
care
professions.

Uter
et
al.
(
2001)
also
reported
on
the
testing
of
189
patients
at
a
dermatitis
clinic
in
Osnabruk,
Germany.
Eleven
(
5.8%)
had
positive
reactions
to
1%
glyoxal
in
water
and/
or
petrolatum;
81%
of
those
tested
were
women.
Three
people
had
what
were
classified
as
irritant
reactions
and
two
had
equivocal
reactions.

A.
Sensitive
Populations
Possible
Childhood
Susceptibility
No
references
were
located
related
to
childhood
susceptibility
to
glyoxal.

Possible
Gender
Differences
No
references
were
located
related
to
gender
differences
in
susceptibility
to
glyoxal.

Other
Draft
 
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or
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(
July,
2003)
14
Diabetics
may
be
sensitive
to
glyoxal
because
of
their
compromised
glyoxalase
activity.
A
study
by
Shinohara
et
al.
(
1998)
focused
on
chronic
hyperglycemia
and
its
role
in
diabetic
microvascular
disease.
The
autoxidation
of
glucose
results
in
glyoxal,
which
can
further
react
to
produce
advanced
glycation
end
products
by
condensation
with
protein
(
Thornalley
et
al.,
1999).
Formation
of
advanced
glycation
end
products
has
been
associated
with
low
catalytic
efficiency
of
aldehyde
reductase
in
the
diabetic
rat
kidney.
This
study
not
only
points
out
the
importance
of
glyoxal
in
normal
metabolism
but
also
may
identify
diabetics
as
a
sensitive
subpopulation
for
glyoxal
exposure.
However,
there
was
no
increase
in
cellular
glyoxal
levels
in
vitro
in
cultures
of
human
fetal
endothelial
cells
exposed
to
30
mM
glucose.

Carboxymethyllysine
(
CML)
is
the
reaction
product
of
glyoxal
with
lysine
and
is
characterized
as
an
advanced
glycation
endproduct.
Dedenhardt
et
al.
(
1998)
found
that
CML
in
human
lens
proteins
increased
linearly
with
age
and
Dyer
et
al.
(
1993)
observed
a
similar
phenomenon
in
skin
collagen.
The
amount
of
CML
in
the
skin
collagen
of
diabetic
rats
was
about
twice
that
for
controls
when
the
animals
(
12­
13
female
Sprague­
Dawley
rats)
were
evaluated
about
28
weeks
after
induction
of
diabetes
(
Dedenhardt
et
al.,
1998).
In
humans,
the
amounts
of
CML
are
increased
in
diabetics
as
compared
to
the
non­
diabetic
populations
(
Dyer
et
al.,
1993)
and
the
concentrations
of
CML
are
significantly
correlated
to
the
duration
of
diabetes,
and
the
incidence
of
retinopathy
and
nephropathy
(
McCance
et
al.,
1993).
However
the
CML
is
believed
to
result
from
the
slow
Maillard
modification
of
fructoslysine,
the
initial
product
from
the
reaction
of
glucose
with
protein
(
McCance
et
al.,
1993),
rather
than
from
direct
reaction
of
glyoxal
with
protein.

CML
dimerizes
to
form
bis(
lysyl)
imidazolium
crosslink
(
glyoxal
lysyl
dimer:
GOLD),
an
advanved
glycation
endproduct.
Endothelial
cells,
monocytes,
and
macrophages
carry
receptor
sites
for
advanced
glycation
endproducts
through
which
they
can
take
up
the
modified
proteins
for
degradation
(
Thornalley
et
al.,
1999).
Odani
et
al.
(
1998
found
that
the
te
levels
of
GOLD
in
the
serum
of
uremic
patients
(
n=
15)
was
increased
four­
to
five­
fold
over
that
for
controls
(
n­
15).

Individuals
with
the
rare
genetic
disorder
(
hyperoxaluria
Type
1)
would
be
susceptible
to
elevated
exposures
to
glyoxal.
In
this
condition,
the
inability
to
convert
glyoxalate
to
glycine
leads
to
the
formation
and
excretion
of
oxalate
produced
by
oxidation
of
glyoxalate
(
Montgomery
et
al.,
1990).

Glyoxal,
previously
identified
as
a
radiosensitizer
at
the
cellular
level,
was
studied
in
solution
by
UV
spectroscopy
with
and
without
DNA
and
with
and
without
irradiation
(
Peinado
et
al.,
1989).
The
presence
of
DNA
did
not
alter
the
radiosensitivity
of
glyoxal.
Draft
 
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or
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(
July,
2003)
15
VI.
MECHANISM
OF
TOXICITY
The
mechanism
of
action
of
glyoxal
is
not
known.
However,
aldehydes
and
 ­
ketoaldehydes
are
relatively
reactive
compounds,
and
form
adducts
with
DNA,
RNA,
proteins,
or
thiols
(
Conroy,
1979;
Krymkiewicz,
1973).
It
seems
likely
that
the
toxic
and
mutagenic
activities
of
glyoxAL
is
ultimately
attributable
to
ITS
reaction
with
nucleophilic
groups
(
amines,
thiols,
alcohols)
present
in
key
cellular
molecules.
Glyoxal
can
also
participate
in
aldol­
condensation
reactions
with
other
carbonyl
compounds
A.
Mechanism
for
DNA
Effects
It
has
been
hypothesized
that
glyoxal
can
be
produced
endogenously
within
the
cell
nucleus
as
a
result
of
free
radical
reaction
damage
to
DNA.
A
study
by
Murata­
Kamiya
et
al.
(
1995)
found
that
glyoxal
was
produced
during
DNA
exposure
to
an
oxygen
radical
forming
system.
Calf
thymus
DNA
was
exposed
to
5
mM
FeSO
4­
EDTA,
a
metal
containing
oxygen
radical
forming
system,
at
37

C
for
60
minutes.
Glyoxal
was
identified
as
a
reaction
product
and
the
amount
produced
was
17
times
higher
than
8­
hydroxy­
deoxyguanosine,
a
common
biomarker
for
free
radical­
induced
DNA
damage.
Glyoxal
produced
from
DNA
will
readily
react
with
neighboring
DNA
bases,
especially
those
with
amino
functional
groups
(
adenine,
guanine,
and
cytosine)
causing
mutations.

Thornalley
et
al.
(
1999)
incubated
50
mM
glucose
in
phosphate
buffer
(
100
mM)
at
pH
7.4
and
37
C
and
measured
the
production
of
glyoxal
over
20
days.
Glyoxal
was
formed
at
a
rate
of
1.72
+/­
0.05
mmol/
day.
Methylglyoxal
and
3­
deoxyglucosone
were
other
products
of
the
reaction
but
were
produced
at
slower
rates
and
at
lower
concentrations.
In
a
second
part
of
this
same
study,
50
mM
glucose
and
NtBOC
lysine
were
incubated
under
the
same
pH
and
temperature
conditions.
Fructosameine,
glyoxal,
methylglyoxal
and
3­
deoxyglucosone
were
formed.
Among
the
alpha
oxoaldehydes
produced,
glyoxal
was
present
at
the
highest
concentration.
The
rates
of
formation
for
the
alpha
oxoaldehydes
were
increased
in
the
presence
of
the
N­
t­
BOC
lysine;
there
was
a
five­
fold
enhancement
of
glyoxal
production.
This
study
provides
support
for
the
hypothesis
that
the
formation
of
glyoxal
is
involved
in
the
process
of
the
glycation
of
lysine.
However
there
were
no
actual
measurements
for
carboxymethyllysine
or
GOLD
Draft
 
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July,
2003)
16
VII.
QUANTIFICATION
OF
TOXICOLOGICAL
EFFECTS
Quantification
of
toxicological
effects
of
a
chemical
consists
of
separate
assessments
of
noncarcinogenic
and
carcinogenic
health
effects.
Chemicals
that
do
not
produce
carcinogenic
effects
are
believed
to
have
a
threshold
dose
below
which
no
adverse,
noncarcinogenic
health
effects
occur.
Carcinogens
are
assumed
to
act
without
a
threshold
unless
there
are
data
elucidating
a
nonmutagenic
mode
of
action
and
demonstrating
a
threshold
for
the
precursor
events
that
commit
a
cell
to
an
abnormal
tumorigenic
response.

A.
Noncarcinogenic
Effects
1.
Reference
Dose
In
the
quantification
of
noncarcinogenic
effects,
a
Reference
Dose
(
RfD)
(
formerly
called
the
Acceptable
Daily
Intake
[
ADI])
is
calculated.
The
RfD
is
"
an
estimate
(
with
uncertainty
spanning
perhaps
an
order
of
magnitude)
of
a
daily
exposure
to
the
human
population
(
including
sensitive
subgroups)
that
is
likely
to
be
without
appreciable
risk
of
deleterious
effects
during
a
lifetime"
(
U.
S.
EPA,
1993).
The
RfD
is
derived
from
a
no­
observed­
adverse­
effect
level
(
NOAEL),
lowest­
observed­
adverse­
effect
level
(
LOAEL),
or
a
NOAEL
surrogate
such
as
a
benchmark
dose
identified
from
a
subchronic
or
chronic
study,
and
divided
by
a
composite
uncertainty
factor(
s).
The
RfD
is
calculated
as
follows:

RfD
=
NOAEL
(
LOAEL)
UF
.
MF
where:

NOAEL
=
No­
observed­
adverse­
effect
level:
A
highest
exposure
level
at
which
there
are
no
statistically
or
biologically
significant
increases
in
the
frequency
or
severity
of
adverse
effects
between
the
exposed
population
and
its
appropriate
control.

LOAEL
=
Lowest­
observed­
adverse­
effect
level:
The
lowest
exposure
level
at
which
there
are
statistically
or
biologically
significant
increases
in
frequency
or
severity
of
adverse
effects
between
the
exposed
population
and
its
appropriate
control
group.

UF
=
Uncertainty
factor
chosen
according
to
EPA/
NAS
guidelines
MF
=
Modifying
factor
Selection
of
the
uncertainty
factor
to
be
employed
in
the
calculation
of
the
RfD
is
based
on
professional
judgment,
while
considering
the
entire
database
of
toxicological
effects
for
the
chemical.
To
ensure
that
uncertainty
factors
are
selected
and
applied
in
a
consistent
manner,
the
Office
of
Water
(
OW)
employs
a
modification
to
the
guidelines
proposed
by
the
National
Academy
of
Sciences
(
NAS,
1977,
1980).
According
to
the
EPA
approach
(
U.
S.
EPA,
1993),
Draft
 
Do
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or
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(
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2003)
17
uncertainty
is
broken
down
into
its
components
and
each
dimension
of
uncertainty
is
given
a
quantitative
rating.
The
total
uncertainty
factor
is
the
product
of
the
component
uncertainties.
The
individual
components
of
the
uncertainty
are
as
follows:

UF
H
A
1,
3,
or
10­
fold
factor
used
when
extrapolating
from
valid
data
in
studies
using
long­
term
exposure
to
average
healthy
humans.
This
factor
is
intended
to
account
for
the
variation
in
sensitivity
(
intraspecies
variation)
among
the
members
of
the
human
population.

UF
A
An
additional
factor
of
1,
3,
or
10
used
when
extrapolating
from
valid
results
of
long­
term
studies
on
experimental
animals
when
results
of
studies
of
human
exposure
are
not
available
or
are
inadequate.
This
factor
is
intended
to
account
for
the
uncertainty
involved
in
extrapolating
from
animal
data
to
humans
(
interspecies
variation).

UF
S
An
additional
factor
of
1,
3,
or
10
used
when
extrapolating
from
less­
than­
chronic
results
on
experimental
animals
when
there
are
no
useful
long­
term
human
data.
This
factor
is
intended
to
account
for
the
uncertainty
involved
in
extrapolating
from
less­
than­
chronic
NOAELs
to
chronic
NOAELs.

UF
L
An
additional
factor
of
1,
3,
or
10
used
when
deriving
an
RfD
from
a
LOAEL,
instead
of
a
NOAEL.
This
factor
is
intended
to
account
for
the
uncertainty
involved
in
extrapolating
from
LOAELs
to
NOAELs.

UF
D
An
additional
1­,
3­
or
10­
fold
factor
used
when
deriving
an
RfD
from
an
"
incomplete"
database.
This
factor
is
meant
to
account
for
the
inability
of
any
single
type
of
study
to
consider
all
toxic
endpoints.
The
intermediate
factor
of
3
(
approximately
½
log
10
unit,
i.
e.,
the
square
root
of
10)
is
often
used
when
there
is
a
single
data
gap
exclusive
of
chronic
data.
It
is
often
designated
as
UF
D.

On
occasion,
EPA
also
uses
a
modifying
factor
in
the
determination
of
the
RfD.
A
modifying
factor
is
an
additional
uncertainty
factor
greater
than
zero
and
less
than
or
equal
to
10.
The
magnitude
of
the
MF
reflects
the
scientific
uncertainties
of
the
study
and
database
not
explicitly
treated
with
standard
uncertainty
factors
(
e.
g.,
the
number
of
species
tested).
The
default
value
for
the
MF
is
1.

In
establishing
the
UF
or
MF,
it
is
recognized
that
professional
scientific
judgment
must
be
used.
The
total
product
of
the
uncertainty
factors
and
modifying
factor
should
not
exceed
3000.
If
the
assignment
of
uncertainty
results
in
a
UF/
MF
product
that
exceeds
3000,
then
the
database
does
not
support
development
of
an
RfD.

2.
Drinking
Water
Equivalent
Level
The
drinking
water
equivalent
(
DWEL)
is
calculated
from
the
RfD.
The
DWEL
represents
a
drinking­
water­
specific
lifetime
exposure
at
which
adverse,
noncarcinogenic
health
effects
are
not
anticipated
to
occur.
The
DWEL
assumes
100%
exposure
from
drinking
water.
The
DWEL
Draft
 
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2003)
18
provides
the
noncarcinogenic
health
effects
basis
for
establishing
a
drinking
water
standard.
For
ingestion
data,
the
DWEL
is
derived
as
follows:

DWEL
=
(
RfD)
×
BW
WI
where:

BW
=
70
kg
adult
body
weight
WI
=
Drinking
water
intake
(
2
L/
day)

3.
Health
Advisory
Values
In
addition
to
the
RfD
and
the
DWEL,
EPA
calculates
health
advisory
(
HA)
values
for
noncancer
effects.
HAs
are
determined
for
lifetime
exposures
as
well
as
for
exposures
of
shorter
duration
(
1­
day,
10­
day,
and
longer
term).
The
shorter
duration
HA
values
are
used
as
informal
guidance
to
municipalities
and
other
organizations
when
emergency
spills
or
contamination
situations
occur.

The
shorter
term
HAs
are
calculated
using
an
equation
similar
to
the
RfD
and
DWEL;
however,
the
NOAELs
or
LOAELs
are
derived
from
acute
or
subchronic
studies.
The
HAs
are
derived
as
follows:

HA
=
NOAEL
or
LOAEL
×
BW
UF
×
WI
where:

NOAEL
or
LOAEL
=
No­
or
lowest­
observed­
adverse­
effect
level
in
mg/
kg
bw/
day
BW
=
Assumed
body
weight
of
a
child
(
10
kg)
or
an
adult
(
70
kg)

UF
=
Uncertainty
factor
in
accordance
with
EPA
or
NAS/
OW
guidelines
WI
=
Assumed
daily
drinking
water
intake
of
a
child
(
1
L/
day)
or
an
adult
(
2
L/
day)

Using
the
above
equation,
the
following
drinking
water
HAs
are
developed
for
noncarcinogenic
effects:

°
1­
day
HA
for
a
10
kg
child
ingesting
1
L
water
per
day
°
10­
day
HA
for
a
10
kg
child
ingesting
1
L
water
per
day
°
Longer­
term
HA
for
a
10
kg
child
ingesting
1
L
water
per
day
°
Longer­
term
HA
for
a
70
kg
adult
ingesting
2
L
water
per
day
Draft
 
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(
July,
2003)
19
Each
of
these
shorter­
term
HA
values
assumes
that
the
total
exposure
to
the
contaminant
comes
from
drinking
water.

The
lifetime
HA
is
calculated
from
the
DWEL
and
takes
into
account
exposure
from
sources
other
than
drinking
water.
It
is
calculated
using
the
following
equation:

Lifetime
HA
=
DWEL
×
RSC
where:

DWEL
=
Drinking
water
equivalent
level
RSC
=
Relative
source
contribution.
The
fraction
of
the
total
exposure
allocated
to
drinking
water.
The
default
value
is
20%
of
the
total
exposure.

The
lifetime
HA
becomes
the
MCLG
for
a
chemical
that
is
not
a
carcinogen.

The
following
paragraphs
evaluate
the
data
available
to
support
development
of
HA
values
for
glyoxal.

1­
Day
Health
Advisory
The
1­
day
HA
calculated
for
a
10
kg
child
assumes
a
single
acute
exposure
to
the
chemical
and
is
generally
derived
from
a
study
of
fewer
than
7
days'
duration.

Ueno
et
al.
(
1991c)
reported
that
a
single
oral
dose
of
glyoxal
caused
inhibition
of
tissue
protein
synthesis
in
rats,
but
a
NOAEL
was
not
identified.
In
the
absence
of
suitable
data
on
the
acute
NOAEL
for
glyoxal,
no
1­
day
HA
value
can
be
derived
at
present.
LD
50
data
for
the
oral
route
indicate
that
glyoxal
has
low
to
moderate
acute
toxicity.

10­
Day
Health
Advisory
The
10­
day
HA
assumes
a
limited
exposure
period
of
1
to
2
weeks
and
is
generally
derived
from
a
study
of
fewer
than
30
days'
duration.
Because
no
data
were
located
on
the
short­
term
oral
toxicity
of
glyoxal,
no
10­
day
HA
values
can
be
derived
at
present.

Longer­
Term
Health
Advisory
A
longer­
term
HA
is
derived
for
both
a
10
kg
child
and
a
70
kg
adult
and
assumes
an
exposure
period
of
approximately
7
years
(
or
10%
of
an
individual's
lifetime).
A
longer­
term
HA
is
generally
derived
from
a
study
of
subchronic
duration
(
exposure
for
10%
of
animal's
lifetime).

Takahashi
et
al.
(
1989)
reported
that
exposure
of
rats
to
500
mg/
kg/
day
of
glyoxal
for
32
weeks
resulted
in
suppression
of
normal
weight
gain.
Ueno
et
al.
(
1991c)
reported
that
exposure
of
rats
to
glyoxal
at
doses
of
298
to
315
mg/
kg/
day
for
90
to
180
days
resulted
in
decreased
weight
gain
and
mild
histological
changes
in
kidney.
Decreased
levels
of
serum
aspartate
amino
Draft
 
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July,
2003)
20
transferase
and
total
serum
protein
were
detected
at
a
LOAEL
of
107
mg/
kg/
day
averaged
over
90
days.
None
of
these
studies
identifies
a
NOAEL
for
glyoxal.
The
effects
detected
at
107
mg/
kg/
day
appear
to
be
mild,
suggesting
that
the
NOAEL
is
not
far
below
this
dose.
However,
given
the
limitation
of
the
data
(
one
sex;
5
to
7
animals/
dose
group,
lack
of
a
NOAEL),
the
data
are
inadequate
for
risk
assessment
and
thus
were
not
used
to
derive
a
longer­
term
HA.
A
more
complete
description
of
the
database
limitations
is
provided
below.

Reference
Dose,
Drinking
Water
Equivalent
Level,
and
Lifetime
Health
Advisory
Table
3
summarizes
studies
that
provide
quantitative
data
on
the
noncancer
adverse
effects
associated
with
oral
exposure
to
glyoxal.
The
suitability
of
these
studies
for
derivation
of
an
RfD
value
and
lifetime
HA
is
evaluated
below.

No
lifetime
data
were
located
on
the
oral
toxicity
of
glyoxal,
and
there
are
data
from
only
two
subchronic
toxicity
studies:
Takahashi
et
al.
(
1989)
and
Ueno
et
al.
(
1991c).
Both
studies
are
limited
by
the
small
number
of
animals
evaluated
(
5
to
10)
and
the
use
of
male
rats
only.

The
Takahashi
et
al.
(
1989)
study
did
not
evaluate
either
tissue
histopathology
or
biomarkers
of
adverse
organ
effects.
The
toxicological
evaluation
conducted
by
Ueno
et
al.
(
1991c)
was
more
complete
(
organ
weights,
histopathology,
serum
markers
for
biochemical
abnormalities)
and
identified
decrements
in
hepatic
protein
synthesis
as
the
critical
effect
at
a
LOAEL
of
107
mg/
kg/
day
averaged
over
the
90­
day
intake
period.
Higher
doses
had
adverse
effects
on
weight
gain
and
were
associated
with
marginal
pathological
changes
in
the
kidney
epithelium.

Data
from
a
developmental
study
using
doses
of
0,
5,
150,
or
300
mg/
kg/
day
in
groups
of
26
Sprague­
Dawley
rats
showed
a
deficit
in
weight
gain
and
food
intake
at
the
highest
dose
over
the
9­
day
administration
period
(
NTP,
1994).
The
data
from
this
study
support
the
observations
of
Takahashi
et
al.
(
1989)
and
Ueno
et
al.
(
1991c).

Each
of
these
studies
identified
decrements
in
weight
gain
following
exposures
to
300
to
500
mg/
kg/
day
glyoxal
for
periods
of
9
days
to
32
weeks.
Other
effects
observed
included
Table
3.
Summary
of
Quantitative
Studies
on
the
Oral
Toxicity
of
Glyoxal
Reference
Duration
Species
Route
LOAEL
(
mg/
kg/
day)
Effect
Ueno
et
al.
(
1991c)
Single
exposure
Rat
Gavage
1000
Decreased
protein
synthesis
in
liver
and
spleen
Ueno
et
al.
(
1991c)
30
 
90
days
Rat
Water
107
 
188
Decreased
serum
proteins
Decreased
weight
gain
Ueno
et
al.
(
1991c)
90
 
180
days
Rat
Water
298
 
315
Decreased
body
weight,
mild
swelling
in
kidneys,
decreased
serum
protein
Takahashi
et
al.
(
1989)
32
weeks
Rat
Water
500
Decreased
weight
gain
NTP
(
1994)
9
days
Rat
Gavage
300
mg/
kg/
day
Decreased
weight
gain
(
maternal)
Draft
 
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July,
2003)
21
apparent
deficits
in
protein
synthesis
at
a
LOAEL
of
107
mg/
kg/
day
and
mild
effects
on
the
kidney.
However,
the
number
of
animals
evaluated
was
small,
only
males
were
tested,
and
there
was
no
monitoring
of
hematological
parameters.
There
are
no
chronic
data
and
two
subchronic
studies,
neither
of
which
monitored
all
standard
subchronic
endpoints.
Accordingly,
the
database
is
not
considered
adequate
to
support
development
of
an
RfD
for
lifetime
glyoxal
exposures.

B.
Carcinogenic
Effects
In
1986,
EPA
established
a
five­
category,
alphanumeric
system
for
carcinogen
with
the
publication
of
Guidelines
for
Carcinogen
Risk
Assessment
(
U.
S.
EPA,
1986).
The
five
categories
were
as
follows:

Group
A:
Human
Carcinogen.
Group
B:
Probable
Human
Carcinogen.
Group
B1:
Limited
evidence
in
humans.
Group
B2:
inadequate
evidence
in
humans;
sufficient
evidence
in
animals.
Group
C:
Possible
Human
Carcinogen.
Group
D:
Not
classified
as
to
Human
Carcinogenicity.
Group
E:
Evidence
of
Noncarcinogenicity
for
Humans.

In
1996
the
Agency
issued
proposed
revisions
to
Guidelines
for
Carcinogen
Risk
Assessment
for
public
comment.
The
1996
proposal
was
later
refined
and
released
as
a
revised
draft
in
1999.
Although
the
1999
version
of
the
Guidelines
for
Carcinogen
Risk
Assessment
has
not
yet
been
formally
adopted
by
the
agency,
use
of
the
1986
guidelines
ceased
in
2000
with
the
publication
of
a
directive
from
the
administrator
(
Federal
Register,
2001
)
specifying
that
the
1999
guidelines
are
to
be
used
on
an
interim
basis.

Under
the
U.
S.
EPA
(
1999)
Guidelines
for
Carcinogen
Risk
Assessment:
Review
Draft,
the
U.
S.
EPA
presents
the
carcinogenic
potential
of
a
chemical
compound
in
a
narrative
fashion,
and
uses
one
of
the
following
five
standard
descriptors
to
express
the
conclusion
regarding
the
weight
of
evidence
for
carcinogenic
hazard
potential:

°
Carcinogenic
to
humans
°
Likely
to
be
carcinogenic
to
humans
°
Suggestive
evidence
of
carcinogenic
potential,
but
not
sufficient
to
assess
human
carcinogenic
potential
°
Inadequate
information
to
assess
human
carcinogenic
potential
°
Not
likely
to
be
carcinogenic
to
humans
Each
standard
descriptor
is
presented
only
in
the
context
of
a
chemical­
specific,
weight­
ofevidence
narrative.
Additionally,
more
than
one
conclusion
may
be
reached
for
an
agent
(
e.
g.,
an
agent
is
"
likely
to
carcinogenic"
by
inhalation
exposure
and
"
not
likely
to
be
carcinogenic"
by
oral
exposure.

In
cases
where
the
toxicological
evidence
leads
to
the
classification
of
the
contaminant
as
a
carcinogen
or
likely
to
be
a
carcinogen,
mathematical
models
are
used
to
calculate
the
estimated
1
A"
point
of
departure"
(
POD)
marks
the
beginning
of
extrapolation
to
lower
doses.
The
POD
is
an
estimated
dose
(
expressed
in
human­
equivalent
terms)
near
the
lower
end
of
the
observed
range,
without
significant
extrapolation
to
lower
doses
(
U.
S.
EPA,
2003).

2LED10
=
Lower
bound
on
the
dose
associated
with
an
increased
tumor
incidence
of
10%

Draft
 
Do
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July,
2003)
22
excess
cancer
risk
associated
with
ingestion
of
the
contaminant
in
drinking
water.
The
data
input
to
the
models
usually
come
from
lifetime­
exposure
studies
in
animals.
In
order
to
predict
the
risk
for
humans,
animal
doses
must
be
converted
to
equivalent
human
doses.
The
conversion
can
include
corrections
for
noncontinuous
exposure,
less­
than­
lifetime
studies,
and
allometric
scaling
of
the
animal
body
weight.
The
dose­
response
assessment
is
performed
in
two
stages.
A
mathematical
assessment
of
experimental
dose
data
is
used
to
derive
a
point
of
departure
(
POD)
1.
Extrapolation
from
the
POD
may
assume
either
linearity
or
nonlinearity
of
the
dose­
response
relationship,
or
both.
The
linear
approach
(
slope
factor)
is
used
for
mutagenic
carcinogens,
i.
e.
those
with
linear
mode
of
action,
or
where
the
mode
of
action
cannot
be
determined.
For
carcinogens
with
a
well­
substantiated
nonlinear
mode
of
action,
an
RfD
approach
is
used.
In
both
cases
a
range
of
models
are
available.

With
the
linear
approach
the
slope
of
the
line
from
the
point
of
departure
(
LED
10)
2
to
the
origin
is
calculated.
The
slope
factor
(
q
1*)
is
a
reflection
of
the
cancer
potency.
It
is
used
to
calculate
the
concentration
in
drinking
water
that
is
equivalent
to
a
specific
risk
to
the
population.
Risk
estimates
are
generally
presented
for
the
one­
in­
ten
thousand
risk
(
1/
10.000;
E­
4),
the
onein
one
hundred
thousand
(
1/
100,000;
E­
5)
risk
and
the
one­
in
a
million
(
1/
1,000,000;
E­
6)
risk
using
the
following
equation:

Concentration
in
Drinking
Water
(
mg/
L)
=
Risk
x
Body
Weight
(
Kg)
Slope
Factor
(
risk/
mg/
kg/
day)
x
2
L/
day
It
is
assumed
that
the
average
adult
human­
body
weight
is
70
kg
and
that
the
average
water
consumption
of
an
adult
human
is
two
liters
of
water
per
day.
Drinking
water
regulations
target
the
E­
4
to
E­
6
risk
range
as
determined
from
the
lower
confidence
bound
on
the
POD.

The
data
base
used
to
calculate
and
support
the
setting
of
cancer
risk
rates
has
an
inherent
uncertainty
due
to
systematic
and
random
errors
in
scientific
measurement.
Thus,
there
is
uncertainty
when
the
risk
is
extrapolated
from
epidemiological
or
animal
data
to
the
entire
humans
population.
When
developing
cancer­
risk
rates,
some
of
the
uncertainties
that
exist,
include
incomplete
knowledge
concerning
the
health
effects
of
contaminants
in
drinking
water,
the
impact
of
the
experimental
animal's
age,
sex
and
species,
the
nature
of
the
target­
organ
system(
s)
examined,
and
the
actual
rate
of
exposure
of
the
internal
targets
in
experimental
animals
or
humans.
Dose
response
data
usually
are
available
only
for
high
levels
of
exposure,
not
for
the
lower
levels
at
which
a
standard
may
be
set.
When
there
is
exposure
to
more
than
one
contaminant,
additional
uncertainty
results
from
a
lack
of
information
about
possible
synergistic
or
antagonistic
effects.
The
true
risk
to
humans,
while
not
identifiable,
is
not
likely
to
exceed
the
upper
limit
estimate
and,
in
fact,
may
be
lower
or
even
zero.
Draft
 
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2003)
23
Categorization
of
Carcinogenic
Effects
Numerous
studies
indicate
that
glyoxal
is
genotoxic
(
see
Section
V­
D),
but
there
are
only
minimal
data
on
its
carcinogenicity.
Several
groups
of
researchers
have
reported
that
glyoxal
has
initiating
and/
or
promoting
activity
in
various
short­
term
tests
(
Takahashi
et
al.,
1989;
Martelli
et
al.,
1988;
Furihata
et
al.,
1985a,
b).

The
carcinogenic
potential
of
glyoxal
cannot
be
determined
using
the
proposed
Guidelines
for
Carcinogen
Risk
Assessment
(
U.
S.
EPA,
1999)
because
no
adequate
epidemiological
studies
exist
and
because
no
properly
conducted
long­
term
animal
bioassay
has
been
conducted.
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24
METHYLGLYOXAL
I.
PHYSICAL
AND
CHEMICAL
PROPERTIES
Methylglyoxal
is
a
yellow
liquid.
It
is
readily
soluble
in
water,
with
little
tendency
to
evaporate.
Aqueous
solutions
are
a
mixture
of
anhydrous,
monohydrated
and
dihydrated
forms
in
a
1:
71:
28
ratio
(
Thornalley,
1996).
Other
physical­
chemical
properties
are
summarized
in
Table
4.

Methylglyoxal
is
obtained
by
warming
isonitrosoacetone
with
dilute
sulfuric
acid
(
Budavari
et
al.,
1996).
It
does
not
have
widespread
industrial
use.
It
is
used
as
a
reagent
in
organic
synthesis
and
is
an
intermediate
in
the
metabolism
of
acetone
and
acetone
derivatives
in
various
biological
systems
(
Inoue
and
Kimura,
1995).
It
is
found
naturally
in
several
beverages
such
as
coffee
and
tea,
in
foods
(
IARC,
1991),
and
is
also
listed,
under
its
synonym
pyruvaldehyde,
as
a
direct
food
additive
in
21
CFR
172.515
"
Synthetic
flavoring
substances
and
adjuvants"
(
FDA,
1997).

Table
4.
Physical
and
Chemical
Properties
of
Methylglyoxal
Parameter
Data
CASRN
78­
98­
8
Empirical
Formula
C3H4O2
Structure
CH3COCHO
Molecular
Weight
72.06
Water
Solubility
Very
soluble
Boiling
Point
72

C
Density
1.05
g/
mL
Sources:
Budavari
et
al.,
1996;
Patnaik,
1992.
Draft
 
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2003)
25
II.
TOXICOKINETICS
A.
Absorption
No
data
were
located
regarding
oral
absorption
of
methylglyoxal.
However,
based
on
its
chemical
structure
and
aqueous
solubility,
it
is
expected
that
methylglyoxal
will
be
well
absorbed
from
the
gastrointestinal
tract.

B.
Distribution
Few
data
were
identified
regarding
tissue
distribution
of
methylglyoxal
following
oral
exposure.
Disposition
studies
by
Phillips
et
al.
(
1993),
as
cited
by
Thornalley
(
1996),
identified
methylglyoxal
in
the
liver,
kidney,
lungs,
lens,
blood,
and
sciatic
nerve
of
rats.
The
highest
concentrations
were
found
in
the
lens
and
sciatic
nerve
(
2.1
and
2.4
nmol/
g,
respectively).
Concentrations
in
the
liver
and
kidney
were
about
1
nmol/
g.
The
level
of
methylglyoxal
in
the
blood
was
0.16

mol/
L.

Diabetics
form
higher
levels
of
endogenous
methylglyoxal
than
nondiabetic
subjects.
McLellan
et
al.
(
1994)
found
that
the
median
serum
methylglyoxal
concentration
was
80
pmol/
g
blood
(
range
25
to
892
pmol/
g)
in
21
normal
subjects.
The
difference
between
the
median
and
the
high
end
of
the
range
demonstrates
wide
variability
among
individuals.
In
a
group
of
42
Type
I
(
juvenile)
diabetics,
the
median
value
was
471
pmol/
g
with
a
range
of
86
to
1040
pmol/
g.
In
a
group
of
105
Type
II
(
adult
onset)
diabetics,
the
median
value
was
287
pmol/
g
with
a
range
of
55
to
2370
pmol/
g.
These
distributions
are
obviously
skewed
with
some
individuals
having
rather
high
levels
of
serum
methylglyoxal.

C.
Metabolism
Methylglyoxal
is
produced
from
the
triose
phosphates:
glyceraldehyde­
3­
phosphate
and
dihydroxyacetone
phospate,
by
a
spontaneous
reaction
during
glycolysis
(
Figure
1;
Phillips
and
Thornalley,
1993).
Methylglyoxal
originates
during
the
triose
phosphate
isomerase
reaction
by
phosphate
elimination
from
the
3­
phospho­
2,3­
ene­
diol
intermediate.

In
bacteria,
methylglyoxal
is
formed
from
dihydroxyacetone
phosphate
through
the
action
of
methylglyoxal
synthase
(
Inoue
and
Kimura,
1995).
Thus,
intestinal
bacteria
can
contribute
to
the
exposure
of
humans
to
methylglyoxal.
Methylglyoxal
synthase
has
not
been
identified
in
mammalian
tissues.

Methylglyoxal
is
also
produced
endogenously
in
the
metabolism
of
acetone
and
the
amino
acid
threonine
(
Figure
1)
(
Peinado
et
al.,
1986;
Thornalley,
1996).
Production
from
threonine
is
a
two­
step
reaction
requiring
threonine
dehydrogenase
and
amine
oxidase.
Acetone
is
oxidized
to
methylglyoxal
through
the
action
of
acetone
monooxidase
in
reactions
utilizing
molecular
oxygen
and
NADPH.
Draft
 
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26
Figure
1.
Methylglyoxal
metabolism.
Draft
 
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2003)
27
The
catabolism
of
methylglyoxal
occurs
primarily
through
the
action
of
glyoxalase
I
(
GLO
I)
and
glyoxalase
II
(
GLO
II),
leading
to
the
production
of
D
lactate,
which
can
be
converted
to
pyruvate
(
Argiles,
1986;
Thornalley,
1990,
1996).
A
portion
of
the
D­
lactate
enters
circulation
and
is
excreted
by
the
kidneys.
Pyruvate
is
metabolized
to
carbon
dioxide
and
water
by
way
of
the
citric
acid
cycle
and
electron
transport
chain.

GLO
I
is
a
cytosolic
enzyme
(
Armeni
et
al.,
1998)
that
conjugates
methylglyoxal
to
reduced
glutathione
(
GSH),
forming
S­
D­
lactoyl
glutathione
(
Thornalley,
1990,
1996).
GLO
II
is
present
in
the
cytosol
and
mitochondria;
it
removes
the
conjugated
glutathione
from
S­
D­
lactoyl
glutathione
by
hydrolysis,
regenerating
GSH
and
forming
D­
lactate
(
Thornalley,
1996).
It
has
been
hypothesized
that
S­
D­
lactoyl
glutathione
may
function
to
transport
GSH
into
the
mitochondria
(
Armeni
et
al.,
1998).

The
glyoxalase
pathway
for
methylglyoxal
disposition
is
least
active
in
the
kidney,
where
an
alternate
pathway
seems
to
predominate
(
Thornalley,
1996).
The
alternate
pathway
utilizes
aldose
reductase
and
NADPH
to
form
hydroxyacetone
(
acetol)
and
D­
lactaldehyde
in
a
95%:
5%
ratio
(
Vander
Jagt
et
al.,
1992).
Both
products
are
further
reduced
to
propanediol.
In
humans,
Aldose
reductase
is
most
active
in
the
kidney.
It
is
also
active
in
the
heart
muscle,
lens,
and
peripheral
neurons,
and
its
generation
of
alpha­
ketoaldehydes
from
sugars,
along
with
generation
of
hydroxyacetone
from
methylglyoxal
may
play
a
role
in
development
of
the
long­
term
effects
of
diabetes
(
Vander
Jagt
et
al.,
1992).
Reaction
of
albumin
with
glucose,
methylglyoxal
and
hydroxyacetone
in
vitro
results
in
modified
proteins
similar
to
advanced
glycation
endproducts
(
Vander
Jagt
et
al.,
1992).

There
are
differences
in
the
activities
of
erythrocyte
GLOI
and
GLO
II
in
diabetic
and
normal
subjects
(
McLellan
et
al.
1994).
The
significance
of
the
effect
on
GLO
I
activity
was
greater
than
that
for
GLO
II
in
noninsulin­
dependant
diabetics
(
P<
0.001
versus
P<
0.05).
In
the
case
of
insulin­
dependant
diabetics
the
increase
in
enzyme
activity
was
only
significant
for
GLO
I
(
P<
0.001).
There
were
no
significant
differences
in
the
blood
levels
of
glutathione
but
concentrations
of
methylglyoxal
metabolites
were
significantly
elevated
in
the
diabetic
subjects.

In
situations
where
methylglyoxal
concentrations
exceed
the
cellular
capacity
to
metabolize
it,
methylglyoxal
can
form
conjugates
with
DNA,
RNA,
and/
or
protein
(
Thornalley,
1996).
Guanine
is
the
DNA/
RNA
base
most
vulnerable
to
modification.
Adenine
and
cytosine
are
much
less
reactive
(
Krymkiewicz,
1973,
as
cited
by
Thornalley,
1996).
In
each
case
the
methylglyoxal
reacts
with
the
amino
functional
group
of
the
nucleotide
base.
The
product
of
the
reaction
with
guanine,
[
3­(
2'­
deoxyribosyl)­
6,7­
dihydro­
6­
methylimidazole
[
2,3­
b]
purine­
9
(
8)
one],
has
been
detected
in
extracts
of
DNA
from
human
peripheral
leucocytes
incubated
with
methylglyoxal
(
Vaca
et
al.,
1994,
as
cited
in
Thornalley,
1996).

In
proteins,
the
side
chains
of
arginine,
lysine,
and
cysteine
are
vulnerable
to
conjugation
with
methylglyoxal
(
Thornalley,
1996).
The
product
of
the
initial
conjugation
of
methylglyoxal
with
the
guanidino
functional
group
in
arginine
proceeds
to
form
a
terminal
imidazole
ring,
elongating
and
changing
the
acid/
base
properties
of
the
side
chain.
The
reaction
of
the
lysine
terminal
amino
functional
group
can
lead
to
cross­
linking
with
other
lysines
within
the
polypeptide
chain
or
between
polypeptide
chains
(
Thornalley,
1996).
In
the
case
of
cysteine,
the
Draft
 
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28
methylglyoxal
adds
to
the
sulfhydryl
functional
group,
forming
a
hemithio
acetate
(
Thornalley,
1996).
Each
of
these
reactions
modifies
the
conformation
of
the
affected
polypeptide,
altering
function
and
contributing
to
cytotoxicity.

Arnold
et
al.
(
1983)
reported
that
methylglyoxal
reacts
in
vitro
with
the
enediol
portion
of
ascorbic
acid
to
yield
a
5­
membered
cyclic
acetal.
This
condensation
is
reversible
under
alkaline
conditions.
No
studies
were
performed
to
determine
whether
this
reaction
occurs
in
vivo.

D.
Excretion
Few
data
were
located
on
the
route
or
degree
of
methylglyoxal
excretion.
A
small
amount
of
methyl
glyoxal
(
4

mol/
day)
and
about
40

mol
of
its
metabolite
D­
lactate
are
excreted
in
urine.
Other
potential
metabolic
endproducts
such
as
lactate
or
propanediol
may
form
when
concentrations
of
methylglyoxal
are
elevated
but
are
water­
soluble,
and
should
be
excreted
mainly
in
urine.
Some
methylglyoxal
will
be
metabolized
to
carbon
dioxide
in
the
citric
acid
cycle
via
pyruvate.

E.
Bioaccumulation
and
Retention
No
data
were
located
on
the
bioaccumulation
or
retention
of
methylglyoxal.
This
compound
is
unlikely
to
accumulate
in
the
body
because
of
its
aqueous
solubility
and
minimal
hydrophobicity.
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29
III.
HUMAN
EXPOSURE
A.
Occurrence
Air
There
was
little
information
in
the
available
literature
on
the
distribution
of
methylglyoxal
in
air.
It
is
apparently
generated
during
the
combustion
of
gasoline
and
has
been
reported
to
occur
in
the
exhaust
of
automobiles.
Methylglyoxal
was
identified
in
three
air
samples
collected
at
the
toll
plaza
for
the
San
Francisco
Bay
bridge
at
concentrations
ranging
from
0.039
to
0.101

g/
m3
(
Destaillats
et
al.,
2002).
It
was
also
detected
in
air
samples
collected
in
Los
Angeles
at
concentrations

0.5
ppb
(
Kawamura
et
al.,
2000).
The
methylglyoxal
concentrations
in
samples
from
downtown
Los
Angeles
were
higher
than
those
from
west
Los
Angeles
and
methylglyoxal
concentrations
were
lower
than
those
for
glyoxal.
It
occurs
in
cigarette
smoke
(
50
to
100

g/
cigarette)
and
may
be
present
in
ambient
air
from
this
source
(
IARC,
1991;
Kalapos,
1999).

Food
Small
amounts
of
methylglyoxal
is
present
in
foods
as
a
metabolic
intermediate
(
Table
5:
Barros
et
al.,
1999;
Hayashi
and
Shibamoto,
1985;
Nagao
et
al.,
1986a)
.
It
is
an
FDA­
approved
synthetic
flavoring
agent
(
21
CFR,
172.515).
Methylglyoxal
was
present
in
coffee
(
both
instant
and
brewed)
and
other
beverages(
Barros
et
al.,
1999;
Hayashi
and
Shibamoto,
1985;
Nagao
et
al.,
1986a).
The
methods
of
preparing
the
coffee
and
tea
samples
varied
as
did
the
analytical
methods
employed;
several
wine
and
beer
products
were
assayed.
Methylglyoxal
was
also
detected
in
toast
and
soy
sauce
(
Nagao
et
al.,
1986b).
The
level
of
glyoxal
in
toast
was
0.5

g/
g.
In
soy
sauce,
there
was
8.7

g/
mL
methylglyoxal.
Because
methylglyoxal
forms
from
sugars,
when
they
are
heated,
it
is
present
in
cane
sugar,
maple
syrup,
and
products
containing
these
ingredients
(
Hayashi
and
Shibamoto,
1985;
IARC,
1991).

Water
Methylglyoxal
is
formed
as
a
byproduct
from
ozonation
of
water
(
Glaze
et
al.,
1989;
Langlais
et
al.,
1991).
Ozonation
of
water
from
the
Los
Angeles
Aqueduct
Filtration
Plant
resulted
in
a
significant
increase
of
several
aldehydes
and
carboxylic
acids
compared
to
their
levels
in
source
water.
For
example,
methylglyoxal
was
not
detectable
in
raw
water,
but
was
detected
at
concentrations
of
28

g/
L
following
ozonation
(
Langlais
et
al.,
1991).
Draft
 
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30
Table
5.
Concentrations
of
Methylglyoxal
in
Selected
Beverages
Beverage
Methylglyoxal
(

g/
mL)

Apple
brandy
c
0.32
Apple
juice
b
0­
2.6
Beer
a
0.08­
0.24
Black
tea
c
trace­
2.4
Bourbon
whiskey
c
1.5
Brewed
coffee
c
7.0­
25
Cocoa
b
1.2
Cola
b
0.23
Dry
milk,
nonfat
b
1.4
Instant
coffee
a,
c
1.6­
23
Japanese
sake
c
0.26
Maple
syrup
b
2.5
Orange
juice
b
0.04
Root
beer
b
0.76
Soy
sauce
b
3.0­
7.7
Tomato
Juice
b
0.06
Wine
a,
c
0.11­
2.88
a
Barros
et
al.,
1999.
b
Hayashi
and
Shibamoto,
1985.
c
Nagao
et
al.,
1986a.

Following
ozonation
of
surface
waters
to
which
odor­
causing
compounds
(
2­
methylesoborneal
and
geosmin)
had
been
added,
methylglyoxal
concentrations
ranged
from
0.1
to
0.2

g/
L
(
Yamada
and
Somiya,
1989).
Ozonation
was
carried
out
for
16
minutes
at
pH
7.6
or
7.7.
Odorant
concentrations
decreased
with
time
as
they
were
oxidized
to
simpler
compounds.

After
a
40­
minute
ozone
reaction
time,
the
concentration
of
methylglyoxal
was
increased
when
ozonation
was
carried
out
at
basic
pHs
as
apposed
to
acid
pHs.
At
pH
10.5,
the
methylglyoxal
conentration
was
1.5

g/
L,
whereas
at
pH
6.6,
no
methylglyoxal
was
detected
(
Yamada
and
Somiya,
1989).

In
the
work
by
Yamada
and
Somiya
(
1989),
there
was
no
methylglyoxal
in
the
postfiltration
water
influent
to
the
ozone
contactors
at
a
demonstration
plant.
After
ozone
treatment,
there
was
0.3

g/
L
glyoxal.
Complete
removal
of
methylglyoxal
was
accomplished
using
a
granularactivated
filter
as
postozonation
treatment.
It
has
been
shown
that
methylglyoxal
is
also
removed
Draft
 
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2003)
31
from
potable
water
by
reaction
with
chloramines
used
as
secondary
disinfectants
(
Glaze
et
al.,
1989).

Concentrations
of
methylglyoxal
in
the
treated
water
from
ozonation
plants
were
monitored
under
the
Disinfection
Byproduct
Information
Collection
Rule
over
an
18­
month
period.
Twenty
ozonation
plants
participated
in
the
project.
One
was
a
ground
water
plant,
the
remainder
used
a
surface
water
source.
After
1
year
more
than
50%
of
the
sites
did
not
detect
any
methylglyoxal
in
the
finished
water.
The
maximum
concentration
reported
was
17

g/
L.

Soil
No
information
was
available
in
the
literature
concerning
levels
of
methylglyoxal
in
soils.
There
is
no
reason
to
suspect
that
it
is
present
in
any
significant
concentrations
except
as
breakdown
products
of
organic
materials.

B.
Fate
Methylglyoxal
undergoes
addition
and
condensation
reactions
with
amines,
amides,
aldehydes,
and
hydroxyl­
containing
materials
(
Ueno
et
al.,
1989).
Because
of
its
reactivity,
the
environmental
persistence
of
methylglyoxal
is
expected
to
be
minimal.

C.
Exposure
The
available
information
on
exposure
to
methylglyoxal
is
sparse.
Most
of
the
daily
exogenous
exposure
is
likely
to
be
from
foods.
Exposure
from
drinking
water
is
expected
to
be
minimal.
Methylglyoxal
can
form
endogenously
in
humans
during
the
catabolism
of
energy
nutrients
(
proteins,
carbohydrates,
triglycerides).
About
120

mol/
kg/
day
is
generated
in
normal
adult
humans;
the
amounts
generated
daily
in
diabetic
subjects
is
elevated
2­
to
3­
fold
(
Thornalley,)
Draft
 
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32
IV.
HEALTH
EFFECTS
IN
ANIMALS
A.
Short­
Term
Exposure
The
oral
LD
50
for
adult
male
rats
was
1990
mg/
kg
and
in
female
rats
it
ranged
from
1165
to
1623
mg/
kg
depending
on
the
age
or
state
of
pregnancy
(
IARC,
1991).
Neonates
appeared
to
be
more
sensitive
to
the
acute
effects
of
methylglyoxal.
The
acute
LD
50
of
531
mg/
kg
for
neonates
is
less
than
50%
of
the
adult
value
(
IARC,
1991).
Conroy
(
1979)
reported
that
the
intravenous
LD
50
for
methylglyoxal
was
108
mg/
kg/
day
when
given
daily
for
10
days.
Doses
of
144
mg/
kg/
day
were
lethal
if
given
for
5
days
or
more.
Clinical
signs
included
disorientation
and
generalized
central
nervous
system
depression,
but
no
detailed
study
of
the
cause
of
death
was
performed.

Jerzykowski
et
al.
(
1975)
investigated
changes
in
cardiovascular,
respiratory,
and
biochemical
indices
resulting
from
methylglyoxal
injection.
Anesthetized
cats
and
rabbits
were
given
single
intravenous
doses
of
0,
10,
25,
100,
or
250
mg/
kg.
Doses
in
excess
of
25
mg/
kg
resulted
in
diminished
respiratory
function,
blood
pressure
instability,
and
dose­
related
slowing
of
cardiac
action
in
both
rabbits
and
cats.
In
contrast,
doses
of
up
to
1000
mg/
kg
administered
intraperitoneally
had
no
effect
on
the
circulatory
or
respiratory
parameters
measured,
although
biochemical
parameters
such
as
lactate
dehydrogenase,
aspartate
aminotransferase
(
AST),
and
creatine
kinase
levels
were
elevated
in
rabbits
at
200
mg/
kg
and
in
rats
at
500
mg/
kg.

High
doses
of
methylglyoxal
can
be
hepatotoxic.
A
single
intraperitoneal
injection
of
400
mg/
kg
led
to
a
significantly
increased
relative
liver
weight,
increased
serum
AST
activity,
and
decreased
levels
of
hepatic
GSH
in
groups
of
six
male
CFLP
mice
within
24
hours
of
compound
administration
(
Kalapos,
1999).
In
a
similar
study
by
Chaudhary
et
al.
(
1997),
doses
of
0,
50,
100,
200,
or
400
mg/
kg
in
groups
of
six
male
Swiss
albino
mice
were
associated
with
significant
effects
on
serum
AST
activity
(
p<
0.05)
after
24
hours
for
all
dose
groups.
Hepatic
GSH
was
decreased
for
all
doses
except
the
lowest.
More
pronounced
GSH
depletion
was
seen
in
all
dose
groups
at
6
and
12
hours.
Lipid
peroxidation
was
observed
in
the
liver
at
6,
12,
and
24
hours:
all
dose
groups
were
affected
at
12
and
24
hours
and
all
but
the
lowest
dose
group
at
6
hours.
When
Kalapos
(
1999)
examined
the
liver
tissues
microscopically,
he
observed
increased
vacuolization
of
cells
in
the
centrilobular
area.

The
hepatic
toxicity
of
methylglyoxal
was
increased
in
rats
pretreated
for
1
week
prior
to
methylglyoxal
administration
with
10%
ethanol
or
1%
acetone
in
their
drinking
water
(
Kalapos,
1999).
The
effects
of
pretreatment
were
more
severe
with
acetone
pretreatment
than
with
ethanol.
Changes
in
biochemical
biomarkers
of
toxicity
(
serum
AST,
GSH)
were
more
pronounced
in
the
pretreated
animals,
as
was
the
vacuolization
of
the
centrilobular
liver
cells.
Pretreatment
with
dimethylsulfoxide
had
little
effect
on
methylglyoxal
hepatic
toxicity.

Ethanol,
acetone,
and
dimethylsulfoxide
are
inducers
of
CYP
2E1.
Since
cytochrome
P­
450
enzymes
are
capable
of
converting
acetone
to
methylglyoxal,
the
severity
of
the
effects
of
acetone
pretreatment
likely
reflect
an
increased
total
methylglyoxal
internal
dose.
Draft
 
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33
Kalapos
(
1999)
also
measured
the
effects
of
methylglyoxal
and
a
combination
of
methylglyoxal
and
CYP­
2E1
inducers
on
hepatic
carbohydrate
metabolism.
Neither
the
methylglyoxal
injection
alone
or
in
combination
with
the
CYP
2E1­
inducer
pretreatment
led
to
significant
changes
in
blood
glucose
levels.
However,
pretreatment
appeared
to
decrease
liver
glycogen.
Tissue
glycogen
was
determined
by
staining
tissue
samples
with
periodic
acid
Schiff
reagent.

B.
Longer­
Term
Exposure
Although
methylglyoxal
does
not
appear
to
be
severely
toxic,
it
can
cause
metabolic
perturbations
of
concern
and
may
disproportionally
affect
selected
sensitive
populations.
Ankrah
and
Appiah­
Opong
(
1999)
conducted
a
study
in
juvenile
mice
exposed
during
gestation,
lactation,
and
weaning.
This
study
did
not
evaluate
standard
reproductive
or
teratology
parameters
but
did
evaluate
a
series
of
biochemical
parameters
(
GSH
levels,
GST
activity,
GGT
activity,
fasting
blood
glucose,
glucose
tolerance,
bile
production,
and
hemoglobin
oxidation)
in
a
group
of
37
juvenile
DPY
mice
as
compared
with
a
group
of
37
control
mice.
It
is
accordingly
not
classified
as
a
reproductive
or
teratology
study.

The
experimental
animals
were
exposed
to
10
g/
L
(
1%)
concentration
of
methylglyoxal
in
drinking
water
5
days/
week.
Prenatal
and
lactation
exposures
occurred
through
the
dams.
After
weaning,
the
mice
were
given
drinking
water
with
the
same
10
g/
L
concentration
for
a
2­
month
period.

The
authors
reported
an
exposure
of
1.7

mol/
mouse
(
5
mg/
kg/
day
based
on
a
25
g
body
weight
for
the
dams).
However,
this
value
is
not
consistent
with
the
10
g/
L
(
1%)
methylglyoxal
concentration
in
water
because
the
1.7

mol
dose
would
be
equivalent
to
a
water
intake
of
only
0.01
mL/
day,
an
unreasonably
low
intake.
Accordingly,
it
appears
as
if
the
intake
of
1.7

mol/
mouse
should
have
been
1.7
mmol/
mouse
and
that
the
dose
should
be
5
g/
kg/
day.
To
achieve
this
dose,
the
water
intake
would
have
been
10
mL/
day.
This
value
is
reasonably
close
to
the
7­
8
mL
water
intake
estimate
proposed
by
EPA
(
1988)
for
B6C3F1
mice
in
studies
with
a
subchronic
duration.
Accordingly,
the
dose
that
will
be
used
in
all
discussion
of
this
study
will
be
1.7
mmol
or
5
g/
kg/
day
rather
than
the
dose
presented
in
the
published
paper.

The
methylglyoxal
exposure
was
associated
with
a
significant
decrease
in
erythrocyte
GSH
and
in
the
activity
of
GST
transferase,
an
enzyme
family
responsible
for
conjugation
of
the
endproducts
of
cytochrome
P­
450
oxidation
with
GSH
for
excretion
(
Stipanuk,
2000).
However,
there
was
no
change
in
the
activity
of
 ­
glutamyl
transferase,
an
enzyme
involved
in
processing
glutathione
conjugates.

Parallel
with
a
decrease
in
erythrocyte
GSH
there
was
a
loss
in
hemoglobin's
ability
to
resist
oxidation.
When
aliquots
of
red
blood
cells
from
exposed
mice
were
first
exposed
to
oxidizing
conditions
in
vitro
there
were
no
differences
in
hemoglobin
oxidation
compared
with
controls.
However,
with
continued
exposure
of
the
red
cells
to
oxidation
for
a
3­
hour
period,
the
red
cells
of
the
methylglyoxal­
treated
mice
had
three
times
more
methemoglobin
than
the
control
cells.
The
authors
suggested
that
individuals
that
carry
the
G6PD
trait
(
homozygots
and
heterozygots)
Draft
 
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2003)
34
may
have
heightened
sensitivity
to
methylglyoxal.
Additional
studies
will
be
necessary
to
confirm
this
hypothesis.

It
has
been
proposed
that
diabetics
and
individuals
with
impaired
glucose
tolerance
may
be
sensitive
to
methylglyoxal.
In
order
to
test
this
hypothesis,
Ankrah
and
Appiah­
Opong
(
1999)
drew
blood
samples
from
fasting
juvenile
mice
exposed
to
0
or
5
g/
kg/
day
methylglyoxal.
The
animals
were
then
given
an
oral
bolus
dose
of
1.7
g
glucose/
kg,
and
blood
samples
were
collected
after
2
hours.
Fasting
blood
glucose
levels
for
the
exposed
mice
did
not
differ
from
those
for
the
controls.
However,
blood
glucose
levels
increased
to
levels
outside
the
normal
range
after
the
glucose
challenge
for
17/
37
of
the
mice
given
methylglyoxal
compared
with
only
3/
37
of
the
control
mice.
Thus,
glucose
tolerance
was
impaired
in
the
exposed
mice
and
they
were
more
likely
to
become
hyperglycemic
than
the
controls.

The
study
by
Ankrah
and
Appiah­
Opong
(
1999)
is
not
suitable
for
dose­
response
assessment
because
only
a
single
dose
was
tested.
However,
the
fact
that
a
drinking
water
route
of
administration
was
used
and
that
effects
were
seen
with
doses
of
about
5
g/
kg/
day
makes
it
an
important
study
in
hazard
identification.
The
effects
observed
are
biologically
plausible
and
consistent
with
the
limited
toxicology
data
for
methylglyoxal.
Thus,
this
study
suggests
the
need
for
a
validating
study
in
laboratory
animals,
expanding
the
investigation
of
Ankrah
and
Appiah­
Opong
(
1999)
and
using
a
series
of
doses
to
both
confirm
the
effects
observed
and
delineate
the
dose­
response.

Takahashi
et
al.
(
1989)
exposed
groups
of
10
male
Wistar
rats
to
0.25%
methylglyoxal
for
32
weeks.
Assuming
water
intake
of
0.10
L/
kg/
day,
this
corresponds
to
doses
of
about
250
mg/
kg/
day.
Animals
were
weighed
weekly.
The
methylglyoxal­
treated
animals
gained
weight
during
the
exposure
period,
but
because
there
was
no
comparable
control
group,
it
was
not
possible
to
determine
if
degree
of
weight
gain
was
normal.
These
data
are
not
adequate
to
identify
a
NOAEL
or
a
LOAEL
for
methylglyoxal.

Fujita
et
al.
(
1986)
exposed
groups
of
40
male
F344
rats
to
0%
or
0.5%
methylglyoxal
in
drinking
water
for
2
years.
The
average
daily
dose
in
the
exposed
animals
was
reported
to
be
7.7
mg/
rat
(
about
20
mg/
kg/
day).
[
NOTE:
This
corresponds
to
a
water
intake
of
only
1.5
mL/
day,
suggesting
that
the
authors
reported
either
the
dose
or
the
concentration
incorrectly.]
Average
body
weight
was
about
15%
less
in
the
exposed
groups
than
in
the
controls.
These
findings
were
reported
in
abstract
form
only,
so
the
reliability
of
the
data
is
uncertain.

C.
Reproductive/
Developmental
Studies
No
information
on
reproductive/
developmental
effects
of
methylglyoxal
was
located
from
any
primary
source.
A
review
paper
on
teratogenicity
results
for
a
number
of
chemicals
by
Schardein
(
1993)
lists
a
negative
result
for
methylglyoxal
(
pyruvaldehyde)
in
rats
from
a
study
by
Peters
et
al.
(
1977).

Clerici
et
al.
(
1990)
found
that
methylglyoxal,
at
concentrations
from
10­
5
to
10­
3
mol/
L
(
0.56
to
56
mg/
L),
interfered
with
blastocyst
formation
in
vitro
in
embryos
recovered
at
the
2­
cell
stage
from
superovulated
female
CD­
1
mice.
Methylglyoxal
appeared
to
exert
its
greatest
effect
Draft
 
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2003)
35
when
morulae
became
blastocysts
and
was
classified
by
the
authors
as
embryocidal.
Unfortunately,
the
abstract
did
not
provide
many
experimental
details.

Amicarelli
et
al.
(
1988)
examined
the
effect
of
exogenous
methylglyoxal
on
Bufo
bufo
(
common
toad)
embryo
development.
Embryonic
jelly
was
removed
from
adult
Bufo
bufo
treated
with
150,
300,
or
600

M
(
11,
22,
or
43
mg/
L)
solutions
for
morphological
and
histological
analysis.
The
results
showed
that
300
and
600

M
methylglyoxal
causes
a
marked
inhibition
of
cell
division
in
the
early
development
stages
that
yields
morulae
and
blastulae
with
cells
swollen
and
reduced
in
number
with
respect
to
the
controls.
Stages
19,
20,
22,
and
25
showed
various
malformations
such
as
oedema
and
severe
caudal
and
dorsal
abnormalities.
At
42
hours
after
fertilization,
the
treated
embryos
showed
a
significant
delay
in
development
and
strong
abnormalities.
At
12
days
following
fertilization,
the
treated
embryos
displayed
the
complete
absence
of
differentiated
intestine,
and
failure
to
incorporate
yolk.
Tail
abnormalities
determined
by
fractures
of
notochord
were
also
observed.
The
percentage
of
viability
in
the
embryos
developed
in
the
presence
of
methylglyoxal
was
markedly
reduced
compared
with
controls.
All
effects
were
evident
at
150

M
methylglyoxal
and
were
dose­
dependent.

D.
Mutagenicity
and
Genotoxicity
Gene
Mutation
Assays
Methylglyoxal
has
been
tested
extensively
for
its
mutagenic
and
genotoxic
properties.
The
studies
described
below
have
been
grouped
by
the
type
of
assay
and
test
organism.
Methylglyoxal
is
chemically
reactive
in
Schiff­
base­
type
reactions
with
amine
functional
groups
or
conjugation
with
thiol
functional
groups.
Accordingly,
it
can
form
adducts
with
several
DNA
bases
and
can
also
react
with
RNA
and
nuclear
proteins.
It
can
impact
DNA
replication
and
transcription
through
both
genetic
and
epigenetic
mechanisms.

Salmonella
typhimurium
Gene
mutation
assays
using
various
strains
of
S.
typhimurium
(
Ames
assay)
have
been
conducted
by
many
investigators.
However,
these
studies
were
conducted
to
determine
selective
aspects
of
methylglyoxal
mutagenicity,
and
no
study
using
the
full
range
of
standard
Ames
tester
strains
was
located.
Most
Ames
assays
have
used
strains
98,
100,
102,
and
104.
TA
98
detects
frame
shift
mutations,
and
TA
100
detects
base­
pair
mutations
as
does
TA104.
TA102
detects
changes
caused
by
a
variety
of
oxidants
and
cross­
linking
agents
(
Mitchell,
1993).
The
data
indicate
that
methylglyoxal
causes
point
mutations
and/
or
adducts
in
the
presence
and
absence
of
microsomal
activation
(
TA100,
TA104,
TA102).
The
response
is
intensified
in
the
presence
of
hydrogen
peroxide
and
appears
to
be
weakened
by
microsomal
metabolism
(
Aeschbacher
et
al.,
1989;
Bronzetti
et
al.,
1987;
Friederich
et
al.,
1985).
Frame
shift
changes
are
also
possible
in
the
absence
of
microsomal
activation
(
TA98)
but
are
not
seen
after
microsomal
metabolism
of
methylglyoxal
(
Fujita
et
al.,
1985a,
b;
Furukawa
et
al.,
1986;
Kato
et
al.,
1989;
Migliore
et
al.,
1990;
Nagao
et
al.,
1986a;
Shane
et
al.,
1988).

Methylglyoxal
was
used
as
the
positive
control
in
an
experiment
investigating
the
mutagenic
mechanisms
of
four
nitro­
group
 
containing
aromatic
amines
(
Chen
et
al.,
1997).
The
Draft
 
Do
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or
Quote
(
July,
2003)
36
standard
plate
incorporation
assay
was
used,
without
metabolic
activation.
When
tested
at
10

g/
plate,
methylglyoxal
was
mutagenic
in
strains
TA100
and
TA104,
but
not
in
strains
TA4001
and
TA4006,
which
detect
TA

CG
and
CG

GC
transitions,
respectively.
Inhibition
by
the
histidine
analogue
DL­
1,2,4­
triazole­
3­
alanine
was
used
to
distinguish
histidine­
locus
from
suppressor
gene
mutations.
In
strain
TA100,
methylglyoxal
produced
100%
histidine­
locus
mutations
(
CG

AT
and
CG

TA)
rather
than
suppressor
gene
mutations
(
TA

CG).
In
strain
TA104,
methylglyoxal
produced
17%
histidine­
locus
mutations
(
TA

GC
and
TA

AT)
and
83%
suppressor
gene
mutations
(
TA

AT,
CG

TA,
and
CG

AT).

Escherichia
coli
Various
E.
coli
strains
are
useful
in
evaluating
the
mutagenic
activity
of
chemicals
toward
causing
base
pair
substitutions.
Tests
of
methylglyoxal
in
E.
coli
in
general
support
the
Ames
assay
results,
although
Nunoshiba
et
al.
(
1989,
1991)
found
that
hydrogen
peroxide
weakened
rather
than
potentiated
the
response.
The
Nunoshiba
et
al.
study
was
the
only
one
identified
with
E.
coli
that
used
hydrogen
peroxide.

Kranendonk
et
al.
(
1996)
described
the
development
and
characterization
of
strain
MX100,
a
derivative
of
E.
coli
K12
laboratory
strain
AB1157.
This
strain
has
sensitivity
toward
the
detection
of
base­
substitution
mutagens.
A
comparison
of
mutagenicity
results
obtained
in
MX100
versus
routinely
used
tester
strains,
namely
E.
coli.
WP2
and
Salmonella
strains
revealed
that
the
response
of
MX100
matched
that
found
in
the
other
strains.
Methylglyoxal
was
tested
over
a
dose
range
of
0
to
0.349

M/
plate
and
showed
a
dose­
dependent
increase
in
the
number
of
revertants
in
MX100.
MX100
shows
sensitivity
toward
the
detection
of
oxidative
and
carbonyl
mutagens.

Murata­
Kamura
et
al.
(
2000)
exposed
KS40
E.
coli
vector
plasmid
PMY189
to
methyl
glyoxal
(
0
to
70
ug)
and
transvected
the
treated
plasmid
into
Simian
kidney
COS7
cell
cultures.
After
24
hours
the
amplified
plasmids
were
recovered
from
the
treated
COS7
cells
and
introduced
into
E.
colu
strain
HB101.
The
E.
coli
were
plated
on
culture
media
and
held
overnight
at
37

C.
The
number
of
colonies
decreased
with
increasing
methyl
glyoxal
concentration
indicating
a
partial
blockage
of
DNA
replication.

The
amplified
plasmid
was
also
introduced
into
Ks40
PKY241
E.
coli
and
plated
on
a
medium
that
would
select
cells
with
a
mutated
SUP1
gene.
After
overnight
incubation
at
37

C,
the
colonies
were
counted
and
the
mutation
frequencies
calculated.
Mutation
frequency
increased
with
methylglyoxal
dose.
Fifty
percent
of
the
mutations
were
the
result
of
deletions
(
8
to
320
base
pairs);
35%
of
the
mutations
were
base
pair
substitutions.
Among
the
base
pair
substitutions,
89
%
involved
G:
C
pairs
with
transition
from
G:
C
to
C:
G
or
C:
C
to
T:
A.
The
may
reflect
the
vulnerability
of
guanine
to
modification
by
methylglyoxal.

Other
Mutagenicity
Assays
Bronzetti
et
al.
(
1987)
studied
the
genotoxic
potential
of
methylglyoxal
in
Saccharomyces
cerevisiae
D7
through
the
study
of
mitotic
gene
conversion
and
reverse
point
mutations.
Yeast
cells
were
incubated
in
concentrations
of
10,
15,
20,
and
40
mM
methylglyoxal,
with
or
without
Draft
 
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or
Quote
(
July,
2003)
37
metabolic
activation.
The
40
mM
dose
was
cytotoxic
in
the
absence
of
the
S9
fraction
with
no
surviving
colonies,
and
only
25%
of
colonies
survived
at
the
20
mM
dose.
Methylglyoxal
induced
both
mitotic
gene
conversion
and
reverse
point
mutation
in
S.
cerevisiae
D7.

Cajelli
et
al.
(
1987)
reported
that
exposure
of
V79
Chinese
hamster
lung
cells
to
methylglyoxal,
at
concentrations
ranging
from
0.16
to
1.5
mM,
caused
a
dose­
dependent
increase
in
the
frequency
of
mutations
in
the
hypoxanthine­
guanosine
phosphoribosyl
transferase
(
HGPRT)
gene
locus.
This
effect
was
reduced
when
cells
were
co­
cultured
with
freshly
prepared
rat
hepatocytes.
The
authors
speculated
that
this
was
caused
by
metabolism
of
methylglyoxal
to
lactate
or
pyruvate.

Chromosomal
Aberration
Assays
Chromosomal
aberration
assays
including
monitoring
for
strand
breaks,
sister­
chromatid
exchange
(
SCE)
micronuclei,
and
cross­
linking
have
been
used
to
study
the
genetic
damage
produced
by
methylglyoxal,
as
well
as
by
other
dicarbonyl
compounds
and
by
complex
mixtures
containing
these
compounds.
Both
in
vitro
and
in
vivo
assays
tend
to
be
positive
although
the
in
vivo
response
is
weaker
than
the
in
vitro
response.

Tucker
et
al.
(
1989)
studied
the
effect
of
methylglyoxal
on
the
induction
of
SCEs
and
endoreduplicated
cells
(
ERCs)
in
CHO
AUXB1
cells
and
human
peripheral
lymphocytes.
SCEs
were
significantly
elevated
at
methylglyoxal
concentrations
of
200
to
500

M.
Endoreduplicated
cells
were
significantly
increased
at
methylglyoxal
concentrations
ranging
from
100
to
500

M.
The
relative
percent
cell
survival
decreased
from
100%
in
the
control
cells
to
31%
in
cells
exposed
to
methylglyoxal
concentrations
of
500

M.

Nishi
et
al.
(
1989)
described
the
clastogenic
activity
of
thermally
decomposed
products
of
carbohydrates
in
cultured
Chinese
hamster
V79
cells.
Products
included
glyoxal
and
methylglyoxal.
The
mitotic
index
was
calculated
as
the
percentage
of
metaphases
among
more
than
2000
interphase
nuclei.
Glyoxal
and
methylglyoxal
both
induced
a
significant
number
of
chromosome
aberrations
and
also
lowered
the
mitotic
index.
The
doses
of
methylglyoxal
that
induced
comparable
numbers
of
aberrant
cells
were
about
1/
10
of
the
glyoxal
doses.
This
assay
was
conducted
only
without
metabolic
activation,
and
positive
controls
were
not
included
in
the
study
design.

Migliore
et
al.
(
1990)
studied
the
effect
of
methylglyoxal
on
chromosomal
aberrations,
SCEs,
and
micronuclei
in
human
lymphocytes,
in
both
the
presence
and
the
absence
of
metabolic
activation.
In
the
absence
of
metabolic
activation,
micronuclei
were
increased
at
methylglyoxal
concentrations
of
3.0
and
4.5
mM,
structural
chromosomal
aberrations
(
excluding
gaps)
were
significantly
increased
at
concentrations
of
1.5
mM
or
greater,
and
SCEs
increased
significantly
at
concentrations
greater
than
or
equal
to
0.5
mM.
A
significant
correlation
was
observed
between
the
concentration
of
methylglyoxal
and
the
number
of
chromosome
aberrations.
Metabolic
activation
resulted
in
reduction
in
the
chromosome
aberrations
and
SCE
induction,
although
the
differences
were
not
statistically
significant.
Addition
of
metabolic
activation
largely
prevented
the
increase
in
micronuclei
observed
in
the
absence
of
activation.
Draft
 
Do
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or
Quote
(
July,
2003)
38
The
micronucleus
assay
was
conducted
in
both
liver
and
bone­
marrow
cells
of
male
Sprague­
Dawley
albino
rats
using
two
dosage
protocols
(
Martelli
et
al.,
1989).
In
the
first,
800
mg/
kg
of
methylglyoxal
was
administered
by
gavage
to
animals
that
had
been
partially
hepatectomized
20
hours
before
treatment.
In
the
second,
rats
were
given
methylglyoxal
in
drinking
water
at
a
dose
of
400
mg/
kg/
day
for
5
successive
days,
a
partial
hepatectomy
was
done
48
hours
after
the
last
dose,
and
liver
and
bone­
marrow
cells
were
obtained
48
hours
later.
Methylglyoxal
was
weakly
positive
in
terms
of
frequency
of
micronucleated
hepatocytes
at
the
single
high
dose,
but
was
negative
in
cells
of
animals
given
successive
lower
doses.
It
was
negative
in
bone
marrow
in
both
protocols.
The
authors
hypothesized
that
methylglyoxal
was
positive
only
at
doses
where
the
glyoxalase
system
 
which
metabolizes
it
to
lactate
 
and
the
alpha­
ketoaldehyde
dehydrogenase
system
 
which
metabolizes
it
to
pyruvate
 
became
saturated.

Migliore
et
al.
(
1990)
assessed
the
potential
genotoxic
activity
of
methylglyoxal
using
in
vivo
cytogenetic
tests
on
mouse
intestinal
cells.
Swiss
CD1
mice
were
dosed
with
400
or
600
mg/
kg
methylglyoxal
by
oral
gavage.
Only
a
weak
positive
response
was
obtained
for
the
induction
of
SCEs
at
the
highest
dose
of
600
mg/
kg
body
weight
in
duodenal
cells.
Methylglyoxal
appeared
to
be
nongenotoxic
in
intestinal
cells.

Other
Assays
for
Genetic
Damage
DNA
damage
caused
by
methylglyoxal
has
been
investigated
in
a
number
of
studies.
Much
of
the
focus
has
been
on
strand
breaks
and
adduct
formation,
although
other
endpoints
have
also
been
used.

Brambilla
et
al.
(
1985)
studied
the
capacity
of
methylglyoxal
to
induce
DNA
damage
and
repair
in
CHO
cells.
DNA
damage
was
evaluated
using
an
alkaline
elution
technique
to
measure
the
formation
of
DNA­
protein
cross­
links.
Increasing
concentrations
of
methylglyoxal
(
1.7
to
7.8
mM)
resulted
in
a
dose­
dependent
decrease
in
cell
survival.
Cross­
linking
was
observed
at
concentrations
of
1.5
and
4.5
mM
methylglyoxal.
CHO
cells
exposed
to
methylglyoxal
in
the
presence
of
a
rat
liver
metabolic
system
showed
significantly
reduced
incidence
of
cross­
linking.

Rahman
et
al.
(
1990)
investigated
the
ability
of
methylglyoxal
to
produce
strand
breaks
in
double­
stranded
DNA
by
incubating
calf
thymus
DNA
with
methylglyoxal
in
DNA
base
pair
to
methylglyoxal
molar
ratios
of
1:
1
to
1:
16
for
2
hours,
followed
by
incubation
for
3
hours
with
S
1
nuclease
to
determine
the
extent
of
hydrolysis.
The
percentage
of
DNA
hydrolyzed,
suggesting
destabilization
possibly
through
strand
breaks,
was
22%
in
DNA
not
exposed
to
methylglyoxal
and
25%,
25%,
36%,
and
43%
at
DNA
base
pair:
methylglyoxal
ratios
of
1:
1,
1:
2,
1:
8,
and
1:
16,
respectively.
To
determine
whether
the
damage
was
really
caused
by
strand
breaks,
the
alkaline
unwinding
assay
was
used.
The
number
of
breaks
per
unit
DNA
increased
from
0.3
at
a
1:
1
DNA
base
pair
to
methylglyoxal
molar
ratio
to
3.6
at
a
1:
16
ratio,
strongly
suggesting
the
formation
of
single­
strand
breaks
causing
increased
susceptibility
to
hydrolysis
by
S
1
nuclease.

Thermal
denaturation
of
DNA,
as
determined
by
thermal
melting
profiles,
was
also
used
to
study
the
effects
of
methylglyoxal
(
Rahman
et
al.,
1990).
The
midrange
melting
temperature
of
unexposed
double­
stranded
DNA
was
75oC,
but
was
reduced
to
68oC
at
the
1:
8
molar
ratio
of
DNA:
methylglyoxal
suggesting
destabilization
and
was
increased
to
80oC
at
the
1:
16
ratio
Draft
 
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or
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(
July,
2003)
39
suggesting
stabilization,
perhaps
by
formation
of
cross­
links.
Cross­
links
were
studied
by
thermally
degrading
DNA
that
had
been
exposed
to
methylglyoxal
(
1:
16),
and
then
determining
its
ability
to
reanneal,
suggesting
that
the
DNA
strands
were
held
together
by
covalent
cross­
links.
Some
evidence
of
reannealing
was
found.

The
acetylation
of
guanine
residues
in
DNA
by
methylglyoxal
was
studied
by
Tada
et
al.
(
1996)
using
HPLC
in
combination
with
32P­
postlabeling,
using
calf­
thymus
DNA
exposed
to
methylglyoxal.
The
adduct
was
detected
at
2
per
106
nucleotides
in
double­
stranded
DNA
and
at
1
per
105
nucleotides
in
single­
stranded
DNA.
This
work
supported
earlier
work
on
adduct
formation.

Methylglyoxal
can
be
formed
in
mammals
by
enteric
bacteria
in
the
intestinal
system.
Isolated
villus
and
crypt
cells
from
rat
small
intestine
and
isolated
colonocytes
from
rat
colon
were
studied
(
Baskaran
and
Balasubramanian,
1990)
in
cell
suspensions
exposed
to
methylglyoxal
in
the
range
of
0.1
to
3.0
mM.
Incorporation
of
radiolabeled
leucine,
thymidine,
and
uridine
was
used
to
study
the
effects
on
protein
synthesis,
DNA
synthesis,
and
RNA
synthesis,
respectively.
Methylglyoxal
inhibited
synthesis
of
all
three
types
of
macromolecules
in
a
dose­
dependent
manner.
The
thiol
compounds
cysteine
and
glutathione
were
not
protective
of
this
inhibition.

E.
Carcinogenicity
Fujita
et
al.
(
1986)
exposed
groups
of
40
male
F344
rats
to
0%
or
0.5%
methylglyoxal
in
drinking
water
for
2
years.
The
average
daily
dose
in
the
exposed
animals
was
reported
to
be
7.7
mg/
rat
(
about
20
mg/
kg/
day).
[
NOTE:
This
corresponds
to
a
water
intake
of
only
1.5
mL/
day,
suggesting
that
the
authors
reported
either
the
dose
or
the
concentration
incorrectly.]
Average
body
weight
was
about
15%
less
in
the
exposed
groups
than
in
the
controls.
No
increase
in
tumor
incidence
was
detected
by
either
gross
or
histological
examinations.
The
authors
speculated
that
methylglyoxal
might
react
with
amines
or
thiols
in
the
stomach,
thereby
reducing
its
mutagenic
and
carcinogenic
potential.
These
findings
were
reported
in
abstract
form
only,
so
the
reliability
of
these
data
is
uncertain.

Takahashi
et
al.
(
1989)
exposed
10
male
Wistar
rats
to
methylglyoxal
(
2500
mg/
L)
in
drinking
water
for
32
weeks.
Assuming
water
intake
of
0.10
L/
kg/
day,
this
corresponds
to
doses
of
about
250
mg/
kg/
day.
At
termination,
all
internal
organs
were
examined
macroscopically,
and
the
stomachs
were
fixed
and
examined
histologically.
No
gastric
carcinomas
or
hyperplastic
lesions
were
detected
in
animals
exposed
to
methylglyoxal.
This
negative
finding
is
limited
by
the
small
number
of
animals
tested,
the
relatively
short
exposure
period,
and
the
absence
of
a
followup
observation
period.

Takahashi
et
al.
(
1989)
tested
the
cancer­
promoting
activity
of
methylglyoxal
in
rats.
Groups
of
30
male
Wistar
rats
were
exposed
for
8
weeks
to
N­
methyl­
N'­
nitro­
Nnitrosoguanidine
(
MNNG)
in
drinking
water
(
100
mg/
L).
Following
this,
animals
were
exposed
to
methylglyoxal
(
2500
mg/
L)
in
drinking
water
for
32
weeks.
Assuming
consumption
of
0.10
L/
kg/
day,
this
corresponds
to
doses
of
about
250
mg/
kg/
day.
Control
animals
were
exposed
to
MNNG
for
8
weeks,
and
than
given
untreated
water
for
32
weeks.
At
termination,
animals
were
sacrificed,
internal
organs
were
examined
macroscopically,
and
the
stomach
was
fixed
and
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2003)
40
examined
histologically.
Methylglyoxal
increased
the
incidence
of
hyperplasia
in
the
pylorus,
but
did
not
increase
the
incidence
of
gastric
carcinomas.
No
toxic
or
neoplastic
changes
were
noted
in
any
other
organs.
These
findings
were
taken
to
indicate
that
methylglyoxal
may
exert
a
cancerpromoting
effect
in
the
stomach.

Furihata
et
al.
(
1985a)
studied
the
initiating
and
promoting
activity
of
methylglyoxal
in
gastric
mucosal
cells
following
oral
exposure
of
rats.
Groups
of
five
male
F344
rats
were
given
single
oral
doses
of
50
to
100
mg/
kg
by
gastric
tube,
and
the
level
of
ODC
and
UDS
in
gastric
mucosa
were
measured
over
the
next
48
to
72
hours.
Methylglyoxal
caused
a
clear
dosedependent
increase
in
both
UDS
and
ODC
activity,
peaking
about
16
hours
after
exposure.
The
increased
rate
of
UDS
was
taken
to
be
an
indicator
of
possible
initiating
activity,
and
the
increased
level
of
ODC
was
taken
to
be
an
index
of
possible
promoting
activity.

Takayama
et
al.
(
1984)
exposed
10
male
and
10
female
F344
rats
to
1.3
mg
of
methylglyoxal
(
65.5%
pure)
by
subcutaneous
injection
twice
weekly
for
10
weeks.
Assuming
a
body
weight
of
0.25
kg,
this
corresponds
to
an
average
daily
dose
of
about
0.24
mg/
kg/
day.
Controls
(
10
males
plus
10
females)
received
injections
of
saline
solution.
After
70
weeks,
subcutaneous
tumors
were
found
in
two
experimental
animals.
No
additional
experimental
details
were
provided.

Nagao
et
al.
(
1986a)
exposed
8
male
and
10
female
F344
rats
to
methylglyoxal
by
subcutaneous
injection.
Doses
of
2
mg
were
administered
twice
a
week
for
10
weeks.
Assuming
a
body
weight
of
0.25
kg,
this
corresponds
to
an
average
daily
dose
of
about
2.3
mg/
kg/
day.
A
control
group
of
21
males
and
19
females
received
only
saline.
After
20
months,
3
males
and
1
female
from
the
treated
rats
had
fibrosarcomas
at
the
injection
site.
No
tumors
were
observed
in
the
control
group.

Several
intermediate­
term
carcinogenesis
studies
have
been
done
on
methylglyoxal.
Hasegawa
and
Ito
(
1992)
made
use
of
the
two­
step
theory
of
hepatocarcinogenesis,
dosing
male
F344
rats
with
a
single
intraperitoneal
dose
of
diethylnitrosamine,
followed
by
a
2­
week
recovery
period
and
then
by
6
weeks
of
exposure
to
the
test
chemical.
At
week
3,
rats
had
a
two­
thirds
partial
hepatectomy
to
stimulate
growth
of
new
liver
tissue.
Two
control
groups
were
used.
One
control
group
received
diethylnitrosamine
and
partial
hepatectomy,
but
no
chemical,
at
the
same
time
points
as
the
chemically
exposed
group.
The
second
control
group
received
saline
instead
of
diethylnitrosamine,
partial
hepatectomy,
and
exposure
to
the
test
chemical.
Both
the
numbers
and
areas
of
induced
glutathione­
S­
transferase
placental
(
GST­
P)
form
positive
foci
in
the
livers
were
then
determined.
Methylglyoxal,
when
dosed
at
2000
ppm
in
drinking
water,
caused
a
statistically
significant
increase
(
p<
0.05)
in
the
number
of
GST­
P
positive
foci,
and
an
increase
in
foci
area
that
was
not
statistically
significant
(
p>
0.05),
giving
overall
weak
results.
No
effects
other
than
GST­
P
positive
foci
were
monitored
or
reported.
This
in
vivo
test
did
correlate
with
short­
term
in
vitro
tests
such
as
the
Ames
test
for
methylglyoxal.

Hasegawa
et
al.
(
1995)
examined
the
potential
carcinogenicity
of
coffee
and
its
related
compounds,
at
doses
broadly
comparable
with
those
usually
consumed
by
humans,
to
cause
hepatocarcinogenicity.
An
8­
week
medium­
term
liver
bioassay
(
as
described
above),
based
on
the
induction
of
GST­
P
form
positive
foci
in
F344
male
rats
was
used
to
determine
potential
Draft
 
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2003)
41
carcinogenicity.
Coffee,
theophylline,
and
caffeine
exerted
no
effect
on
GST­
P
positive
foci
development.
Hence,
these
coffee­
related
compounds
examined
demonstrated
no
obvious
enhancing
potential
and
were
not
considered
carcinogenic
for
rat
liver.
The
increase
in
number
of
foci
for
methylglyoxal
was
statistically
significant
at
p<
0.05,
but
the
value
was
within
the
range
of
historical
control
values.
A
positive
result
was
not
found
for
the
other
parameter,
area
of
foci.
Also,
no
foci
larger
than
0.2
mm
in
diameter
were
induced
without
prior
diethylnitrosamine
initiation.
Therefore,
the
modifying
effects
of
methylglyoxal
observed
were
considered
negligible.
Because
Hasegawa
and
Ito
(
1992)
and
Hasegawa
et
al.
(
1995)
both
report
the
number
of
foci
per
square
centimeter
as
7.97
±
1.91
and
the
area
as
0.49
±
0.18
square
millimeters
per
square
centimeter
and
likely
describe
the
same
data,
there
has
been
some
reinterpretation
of
their
significance.
Body
weight
and
relative
and
absolute
liver
weight
effects
were
also
reported
in
Hasegawa
et
al.
(
1995).
Body
weight
and
absolute
but
not
relative
liver
weight
in
the
methylglyoxal
experiment
were
significantly
different
from
the
saline
with
methylglyoxal
controls
but
were
not
significantly
different
from
controls
that
received
diethylnitrosamine
without
methylglyoxal
treatment.
No
effects
other
than
body
weight,
liver
weight,
and
foci
were
reported.

Martelli
et
al.
(
1988)
studied
the
direct­
acting,
initiating,
and
promoting
ability
of
0.05%
and
0.2%
methylglyoxal
in
drinking
water
in
a
series
of
experiments
using
male
F344
rats
given
partial
hepatectomies
during
the
exposure
period,
and
using
the
induction
of
gammaglutamyltranspeptidase
(
GGT)­
positive
foci
for
measurement
as
a
marker
of
carcinogenic
potential.
2­
Acetylaminofluorene
(
2­
AAF)
was
used
as
initiator
in
some
experiments
and
as
promoter
in
others.
N­
Nitrosodiethylamine
was
used
as
an
initiation­
positive
control
and
sodium
phenobarbital
as
a
promotion­
positive
control.
The
number
of
positive
foci
was
reported
in
terms
of
square
centimeters
and
cubic
centimeters,
and
total
foci
volume
was
also
reported.
There
were
two
9­
week
study
protocols.

To
test
methylglyoxal
as
an
initiator,
it
was
given
for
6
weeks
in
drinking
water
with
partial
hepatectomy
at
day
7,
followed
by
a
1­
week
recovery
and
2
weeks
of
exposure
to
0.02%
2­
acetylaminofluorene
in
the
diet
with
1
mL/
kg
carbon
tetrachloride
given
by
gavage
on
day
6.
To
test
methylglyoxal
as
a
promoter,
the
2­
week
exposure
to
0.02%
2­
acetylaminofluorene
with
carbon
tetrachloride
at
day
7
was
given
first,
followed
by
the
1­
week
recovery
period,
followed
by
6
weeks
of
exposure
to
methylglyoxal
with
partial
hepatectomy
at
day
7
into
this
latter
exposure
(
day
28
of
the
experiment).
Methylglyoxal
was
also
tested
without
initiation
or
promotion.

Statistically
significant
positive
results
were
obtained
with
methylglyoxal
as
initiator
and
2­
AAF
as
promoter,
with
methylglyoxal
as
the
promoter
and
2­
AAF
as
the
initiator,
and
with
methylglyoxal
alone
when
compared
with
partially
hepatectomized
controls.
Statistical
significance
was
achieved
only
at
the
high
dose
(
0.2%
in
drinking
water)
when
compared
with
controls
given
2­
acetylaminofluorene/
carbon
tetrachloride
as
initiator
or
promoter
and
partial
hepatectomy.
The
authors
concluded
that
although
there
was
substantial
interanimal
variability
in
results,
methylglyoxal
could
be
a
complete
carcinogen
in
the
rat
liver.
A
chronic
study
would
be
required
to
confirm
this.
No
effects
other
than
number
and
volume
of
foci
were
reported.

A
two­
stage
stomach
carcinogenesis
model
using
male
outbred
Wistar
rats
was
also
used
to
test
glyoxal
and
methylglyoxal
for
tumor­
promoting
potential
(
Takahashi
et
al.,
1989).
Groups
of
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42
30
animals
were
dosed
with
N­
methyl­
N

­
nitro­
N­
nitrosoguanidine
at
100
mg/
L,
plus
10%
NaCl
as
a
supplement,
in
drinking
water
for
8
weeks.
They
were
then
dosed
with
drinking
water
containing
0.25%
methylglyoxal
for
32
weeks
and
sacrificed.
Groups
of
10
animals
exposed
to
no
chemical
or
the
test
chemical,
but
not
the
nitrosamine,
served
as
controls.
Methylglyoxal
significantly
increased
the
incidence
of
hyperplasia,
but
not
carcinoma,
in
the
pylorus
of
the
glandular
stomach,
indicating
some
tumor­
promoting
potential.
No
effects
were
seen
in
the
forestomach
or
in
other
thoracic
or
abdominal
organs.

These
intermediate­
term
bioassays
provide
relevant
information
for
assessing
the
carcinogenic
risk
of
methylglyoxal.
However,
the
limited
number
of
dose
levels,
the
use
of
only
male
rats,
and
the
measurement
and
observation
of
only
a
few
endpoints
diminishes
the
use
of
these
studies
for
quantitative
and
noncancer
risk
assessment.
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43
V.
HEALTH
EFFECTS
IN
HUMANS
No
epidemiology
or
case
study
data
were
located
for
methylglyoxal.
However,
several
studies
with
human
tissues
or
cell
lines
provide
some
insight
as
to
the
impact
of
methylglyoxal
on
cells.

Leoncini
et
al.
(
1989)
studied
the
effect
of
methylglyoxal
on
glucose
metabolism
by
human
red
blood
cells
in
vitro.
Concentrations
of
0.5
to
5
mM
methylglyoxal
(
36
to
360
mg/
L)
caused
a
dose­
dependent
decrease
in
glucose
utilization,
primarily
by
inhibiting
glycolysis.
This
resulted
in
a
decrease
in
lactate
and
pyruvate,
an
increase
in
several
intermediates
of
the
glycolytic
pathway,
and
a
decrease
in
ATP
levels.
No
studies
were
performed
to
determine
whether
these
effects
occurred
in
vivo,
but
it
seems
unlikely
that
such
high
serum
concentrations
of
methylglyoxal
could
occur
in
humans
exposed
to
methylglyoxal
via
drinking
water.

The
activities
of
GLO
I
and
GLO
II,
as
well
as
glyceraldehyde­
3­
phosphate­
dehydrogenase,
in
Carl­
1
human
melanotic
melanoma
cells,
exposed
to
0,
200,
or
400

M
concentrations
of
methylglyoxal,
were
studied
by
Amicarelli
et
al.
(
1998)
in
order
to
investigate
cellular
mechanisms
which
that
control
the
methylglyoxal
level.
Activities
of
all
three
enzymes
increased
in
a
dosedependent
manner.
Production
of
L­
lactate
decreased
in
a
dose­
dependent
manner
up
to
400

M,
and
methylglyoxal
had
an
enhancing
effect
on
the
apoptotic
response,
which
was
maximized
and
stabilized
at
100

M
and
above.
Methylglyoxal
was
an
inhibitor
of
cell
proliferation
in
these
cells
and
there
appeared
to
be
a
feedback
mechanism
whereby
increased
glycolysis
was
used
to
defend
against
the
antiproliferative
effect
of
methylglyoxal.
Glyoxalase
inhibitors
had
been
proposed
for
use
as
antiproliferative
agents.

The
production
of
methylglyoxal
increased
during
the
intermediary
process
of
the
glycation
reaction,
especially
under
diabetic
conditions
(
Okado
et
al.,
1996).
Although
methylglyoxal
has
a
reactive
group
whereby
it
can
accelerate
the
glycation
process,
it
can
also
modify
essential
cellular
components.
The
mechanism
by
which
methylglyoxal
exerts
its
cytotoxicity
is
unknown
at
the
present.
The
authors
investigated
the
cytotoxic
effects
of
methylglyoxal
and
found
that
in
certain
cells
it
triggered
apoptotic
cell
death.
They
investigated
the
cytotoxic
effects
of
methylglyoxal
at
physiological
concentrations
on
monocytic
leukemia
U937
cells,
RAW264.7,
K562,
HL60,
and
KATO
III
cells.
Methylglyoxal
induced
the
ladder
formation
of
DNA
as
well
as
nuclear
fragmentation
in
U937
cells
and
in
the
RAW264.7
cells
at
100

M,
but
not
in
the
other
cell
lines.
Levels
of
intracellular
oxidants
increased
in
the
cells
in
which
apoptotic
cell
death
was
induced
by
methylglyoxal
treatment,
as
judged
by
measuring
DCFH
fluorescence
intensity,
but
not
in
the
other
cells.
Apoptosis
and
intracellular
oxidant
levels
were
enhanced
by
buthionine
sulfoximine,
an
inhibitor
of
glutathione
biosynthesis,
and
partially
blocked
by
N­
acetylcysteine,
an
antioxidant.
However,
methylglyoxal
had
minimal
effect
on
the
levels
of
intracellular
glutathione
in
these
cell
lines.
Therefore,
it
did
not
interfere
with
the
normal
role
of
glutathione
in
acting
as
an
antioxidant
to
protect
cells
against
various
forms
of
oxidative
stress.
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44
A.
Sensitive
Populations
Possible
Childhood
Susceptibility
No
studies
were
located
that
evaluated
childhood
susceptibility
to
methylglyoxal;
however,
Ankrah
and
Appiah­
Opong
(
1999)
suggested
that
individuals
who
carry
the
G6PD
trait
(
homozygots
and
heterozygots)
might
be
more
sensitive
to
oxidation
of
hemoglobin
because
of
the
ability
of
methylglyoxal
to
deplete
cellular
glutathione.
Infants
that
carry
the
G6PD
trait
are
especially
vulnerable
to
hemoglobin
oxidation
(
methemoglobinemia)
because
of
the
normal
developmental
delay
in
the
activity
of
hemoglobin
reductase,
the
enzyme
that
controls
blood
levels
of
methemoglobin.
Carriers
of
the
G6PD
trait
have
a
diminished
capacity
to
maintain
glutathione
in
its
reduced
form
in
red
blood
cells.
Methylglyoxal­
induced
depletion
of
GSH
could
aggravate
this
situation.

LD50
data
reported
by
IARC
(
1991)
suggest
that
methylglyoxal
is
more
acutely
toxic
to
newborn
rats
than
to
adults
rats.
The
LD50
value
for
the
newborn
rats
was
531
mg/
kg
and
for
the
adults
rats
ranged
from
1165
to
1990
mg/
kg.

Possible
Gender
Differences
No
references
were
located
related
to
gender
differences
in
susceptibility
to
methylglyoxal.

Other
There
are
data
that
suggest
that
diabetics
may
have
an
elevated
risk
for
adverse
effects
following
exposure
to
methylglyoxal
(
Ankrah
et
al.,
1990;
Dedenhardt
et
al.,
1998;
Schalkwijk
et
al.,
1998).
This
appears
to
be
a
consequence
of
problems
in
methylglyoxal
clearance
and
the
presence
of
elevated
levels
of
serum
methylglyoxal
in
diabetic
subjects.
As
reported
by
McClellan
et
al.
(
199h),
median
serum
concentrations
of
methylglyoxal
in
Type
I
and
Type
II
diabetics
are
three
to
six
times
higher
in
diabetic
subjects
than
in
controls.
Diabetic
patients
with
retinopathy,
neuropathy,
and/
or
nephropathy
had
significantly
higher
glyoxylase
activities
and
modified
hemaglobin
than
patients
without
diabetic
complications.

Protein
cross­
linking
is
one
factor
that
contributes
to
some
of
the
long­
term
complications
of
diabetes,
particularly
changes
in
vasculature,
retinal
changes,
and
problems
with
kidney
basement
membranes.
Methylglyoxal
reacts
with
lysine
residues
in
proteins
to
form
N (
carboxyethyl)
lysine
(
CEL)
and
imidazolium
cross­
link
methylglyoxal­
lysine
dimer
(
MOLD).
CEL
and
MOLD
are
dicarbonyl­
derived
advanced
glycation
endproducts
(
AGEs),
which
accumulate
in
the
tissue
proteins
with
age
and
chronic
diseases
such
as
diabetes
and
atherosclerosis.
Dedenhardt
et
al.
(
1998)
described
work
with
CEL
and
MOLD.
They
found
that
CEL
and
MOLD
are
present
at
lower
concentrations
in
skin
collagen
than
in
lens
proteins
and
increase
by
two­
to
fourfold
in
uremia.
The
levels
of
CEL
in
skin
collagen
increase
by
two­
to
threefold
in
diabetic
rat
skin
collagen.
MOLD
has
not
been
measured
in
collagen
of
human
diabetic
subjects.

Hyperlipidemia
and
atherosclerosis
are
another
common
complication
of
diabetes.
Schalkwijk
et
al.
(
1998)
studied
the
effect
of
methylglyoxal
on
the
physiochemical
and
biological
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45
properties
of
low­
density
lipoproteins
(
LDL),
the
lipoprotein
complex
that
carries
endogenous
lipids
and
cholesterol
from
the
liver
to
peripheral
tissues.
In
patients
with
both
insulin­
dependent
and
non­
insulin
dependent
diabetes
mellitus,
the
concentration
of
methylglyoxal
was
found
to
increase
two­
to
sixfold.
Isolated
human
LDL
was
then
used
to
determine
if
methylglyoxal
could
modify
the
properties
of
LDL.
LDL
was
incubated
in
vitro
for
18
hours
with
increasing
levels
of
methylglyoxal
(
0.1,
1.0,
and
10
mM/
L).
Methylglyoxal
modified
LDL
in
a
time­
dependent
manner
leading
to
changes
in
both
physiochemical
and
biological
properties.

The
study
by
Schalkwijk
et
al.
(
1998)
found
that
modified
methylglyoxal­
LDL
did
not
bind
well
to
the
LDL
receptors
of
fibroblasts
and
found
no
indication
of
activation
of
endothelial
cells
by
modified
methylglyoxal­
LDL.
This
study
may
indicate
that
the
elevated
levels
of
methylglyoxal
seen
in
persons
with
non­
insulin
dependent
and
insulin­
dependent
diabetes
mellitus
could
interfere
with
cellular
uptake
of
LDL,
thereby
increasing
circulating
LDL
levels
and
increasing
the
risk
for
atherosclerosis
Ankrah
and
Appiah­
Opong
(
1999)
found
that
glucose
tolerance
was
impaired
in
nondiabetic
mice
exposed
to
about
5
g/
kg/
day
methylglyoxal
during
development
and
for
a
2­
month
period
after
weaning.
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46
VI.
MECHANISM
OF
TOXICITY
The
mechanism
of
action
of
methylglyoxal
has
not
been
definitively
identified.
However,
aldehydes
and
 ­
ketoaldehydes
are
relatively
reactive
compounds
and
form
adducts
with
DNA,
RNA,
proteins,
or
thiols
(
Conroy,
1979,
Dedenhardt
et
al.,
1998,
Krymkiewicz,
1973;
Thornalley,
1996).
It
seems
likely
that
the
toxic
and
mutagenic
activities
of
methylglyoxal
are
ultimately
attributable
to
its
reaction
with
nucleophilic
groups
(
amines,
thiols,
alcohols)
present
in
key
cellular
molecules.

A.
Mechanistic
Studies
Several
references
were
located
describing
various
mechanisms
of
action
for
methylglyoxal,
both
as
a
normal
metabolite
and
as
a
toxic
chemical.

Mutagenicity
Fazal
et
al.
(
1994)
investigated
the
formation
of
superoxide
anions
and
hydroxyl
radicals
by
methylglyoxal
as
a
possible
mechanism
for
the
increased
mutagenicity
of
hydrogen
peroxide
in
the
presence
of
methylglyoxal.
Superoxide
anion
generation
was
followed
in
a
0.18
mM
methylglyoxal
solution
containing
nitroblue
tetrazolium
dye.
Production
of
superoxide
ion
was
indicated
by
a
steady
increase
in
absorbance
over
a
90­
minute
period
because
of
the
reduction
of
nitroblue
tetrazolium
dye.
Addition
of
superoxide
dismutase
completely
quenched
the
formation
of
superoxide
anion
and
the
effect
on
nitroblue
tetrazolium.
Formation
of
superoxide
anion
increased
approximately
linearly
until
just
below
0.2
mM,
and
then
began
to
level
off
at
higher
concentrations
of
methylglyoxal.

Hydroxyl
radicals
were
measured
using
salicylate
as
a
receptor
molecule
in
a
buffer
solution
containing
Fe(
III)
(
Fazal
et
al.,
1994).
The
hydroxyl
radical
concentration
increased
with
methylglyoxal
concentration
from
11.94
nM
at
0.07
mM
methylglyoxal
to
30.60
nM
at
1.40
mM
methylglyoxal.
Since
the
formation
of
hydroxyl
radicals
was
almost
completely
inhibited
by
catalase
(
94.1%)
and
by
superoxide
dismutase
(
92.3%),
the
authors
concluded
that
superoxide
and
hydrogen
peroxide
were
intermediates
in
the
hydroxyl
radical
formation.
Inhibition,
to
lesser
extent,
by
mannitol,
albumin,
and
sodium
formate
proved
that
it
was
the
hydroxyl
radical
that
was
being
measured.
Hydrogen
peroxide
was
shown
to
change
supercoiled
plasmid
pBR322
DNA
to
relaxed,
open
circles.
This
process
was
accelerated
by
methylglyoxal,
and
also
resulted
in
formation
of
some
linear
molecules.

Cytotoxicity
Elevated
concentrations
of
methylglyoxal
appear
to
cause
apoptosis
in
insulin­
secreting
cells
based
on
in
vitro
data
(
Sheader
et
al.,
2001.)
Cultured
RINm5F
cells
,
derived
from
rat
insulolinoma
and
similar
in
many
respects
to
pancreatic
beta
cells,
were
exposed
to
0,
0.1,
1
and
10
mM
methylglyoxal
and
processed
sto
stain
the
cells
for
visualization
of
apototic
cells.
There
was
a
dose­
related
increase
in
cellular
apoptosis
which
achieved
significance
for
the
1
mM
and
10
mM
concentrations.
Over
a
24
hour
period
the
percent
of
apoptotic
cells
increased
to
the
point
where
about
70%
were
affected
by
the
1
mM
methylglyoxal
concentration.
The
rate
of
apoptosis
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2003)
47
was
greatest
during
the
first
six
hours
after
which
the
rate
of
increase
leveled
off.
The
authors
hypothesized
that
cytotoxicity
from
methylglyoxal
might
play
a
role
in
damage
to
insulinproducing
cells
as
a
result
of
chronic
hyperglycemia.

Unregulated
accumulation
of
methylglyoxal
in
tissues
can
be
cytotoxic
because
it
inhibits
protein
synthesis
and
glycolysis
and
damages
DNA.
The
glyoxalase
system
prevents
accumulation
of
methylglyoxal
in
the
tissues.
Ankrah
et
al.
(
1990)
investigated
whether
dietary
levels
of
aflatoxin
B
1
could
interfere
with
the
metabolic
role
of
GLO
I
in
methylglyoxal
disposal.
The
GLO
I
activity
was
examined
in
colon
and
liver
of
ddy
mice.
Mice
were
chosen
as
the
experimental
animal
because
they
metabolized
aflatoxin
B
1
in
a
manner
similar
to
humans.

Male
and
female
mice
were
fed
a
diet
containing
0.0166
ng
aflatoxin
B
1
per
10
g
food
from
8
to
10
weeks
of
age.
They
were
mated
and
allowed
to
deliver
offspring.
The
male
offspring
were
kept
on
the
aflatoxin­
dosed
food
after
weaning
and
were
sacrificed
at
3
or
6
months
of
age.

Levels
of
S­
D
lactoylglutathione
in
liver
and
colon
were
measured
by
UV
spectroscopy
as
an
indicator
of
GLO
I
activity.
In
the
male
offspring
of
mice
fed
the
aflatoxin
B
1
supplemented
food
there
was
a
decrease
in
colon
GLO
I
activity
by
39%
and
50%
at
3
and
6
months
of
age,
respectively.
In
the
liver,
the
decrease
was
fairly
small
at
3
months,
but
was
approximately
40%
at
6
months.
There
were
significant
decreases
in
serum
protein
and
glucose
levels
in
aflatoxin
B
1
fed
mice
that
received
three
intraperitoneal
doses
of
methylglyoxal
at
6
months
of
age.
The
test
mice
exhibited
high
mortality
rate,
and
the
postmortem
observations
were
consistent
with
a
decreased
capacity
to
dispose
of
methylglyoxal.
The
study
thus
concluded
that
the
aflatoxin
B
1
mediated
fall
in
GLO
I
activity
could
lead
to
decreased
disposal
of
endogenous
methylglyoxal.
This
study
provided
evidence
that
methylglyoxal
regulation
could
be
disrupted
by
exposure
to
environmental
chemicals.

Advanced
Glycation
Endproducts
Thronalley
et
al.
(
1999)
incubated
50
mM
glucose
in
phosphate
buffer
(
100
mM)
at
pH
7.4
and
37
C
and
measured
the
production
of
glyoxal
over
20
days.
Methyllyoxal
was
formed
at
a
rate
of
0.098
+/­
0.0003
mmol/
day.
This
was
far
less
than
the
rate
of
formation
for
glyoxal
and
3­
deoxygluconone..
In
a
second
part
of
this
same
study,
50
mM
glucose
and
NtBOC
lysine
were
incubated
under
the
same
pH
and
temperature
conditions.
Fructosameine,
glyoxal,
methyl
glyoxal
and
3­
deoxyglucosone
were
formed.
Among
the
alpha
oxoaldehydes
produced,
glyoxal
was
present
at
the
highest
concentration
and
methylglyoxal
at
the
lowest
concentration.
The
rates
of
formation
for
the
alpha
oxoaldehydes
were
increased
in
the
presence
of
the
N­
t­
BOC
lysine;
there
was
a
32­
fold
enhancement
of
methylglyoxal
production.
This
study
provides
support
for
the
hypothesis
that
the
formation
of
methylglyoxal
is
involved
in
the
process
of
the
glycation
of
lysine.
However
there
were
no
actual
measurements
for
carboxyethyllysine
or
MOLD
Other
Effects
Kawase
et
al.
(
1996)
examined
the
activity
of
the
methylglyoxal
pathway
in
rats
during
the
progression
of
hepatocarcinogenesis.
One
group
of
25
male
Donryu/
Crj
(
SPF/
VAF)
test
rats
was
fed
0.064%
3'­
methyl­
4­
dimethylaminoazobenzene
(
MDAB)
in
their
diet
for
up
to
21
weeks,
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2003)
48
while
a
control
group
received
food
without
the
test
chemical.
Changes
in
the
concentrations
of
D­
lactate,
pyruvate,
methylglyoxal,
and
glutathione,
and
in
the
activities
of
GLO
I
and
II,
were
measured
in
plasma
or
liver.
During
the
initial
phase
of
hepatic
carcinogenesis,
there
was
an
increase
in
hepatic
concentrations
of
methylglyoxal
and
D­
lactate
by
several
fold
over
those
of
controls.
However,
after
21
weeks,
the
D­
lactate
level
declined
but
remained
slightly
elevated
over
that
of
controls
whereas
that
of
methylglyoxal
remained
at
the
same
level.
The
hepatic
GLO
I
activity
reached
its
peak
at
4
weeks
in
the
treated
group
and
remained
elevated,
whereas
that
of
GLO
II
showed
an
initial
increase
followed
by
a
decline
to
55%
of
the
control
at
21
weeks.
Glutathione
also
attained
a
maximum
level
at
4
weeks
and
thereafter
decreased.
The
authors
concluded
that
an
increase
in
the
activity
of
GLO
I
and
II
during
the
early
stages
of
carcinogenesis
led
to
increased
production
of
methylglyoxal
and
D­
lactate.
In
the
later
stage,
glutathione
and
GLO
II
activity
had
decreased
while
GLO
I
activity
remained
elevated.
This
led
to
an
increase
in
hepatic
methylglyoxal
content
and
a
subsequent
decrease
in
D­
lactate
content
in
the
liver.
Draft
 
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49
VII.
QUANTIFICATION
OF
TOXICOLOGICAL
EFFECTS
Quantification
of
toxicological
effects
of
a
chemical
consists
of
separate
assessments
of
noncarcinogenic
and
carcinogenic
health
effects.
Chemicals
that
do
not
produce
carcinogenic
effects
are
believed
to
have
a
threshold
dose
below
which
no
adverse,
noncarcinogenic
health
effects
occur.
Carcinogens
are
assumed
to
act
without
a
threshold
unless
there
are
data
elucidating
a
nonmutagenic
mode
of
action
and
demonstrating
a
threshold
for
the
precursor
events
that
commit
a
cell
to
an
abnormal
tumorigenic
response.

A.
Noncarcinogenic
Effects
1.
Reference
Dose
In
quantification
of
noncarcinogenic
effects,
a
Reference
Dose
(
RfD)
(
formerly
called
the
Acceptable
Daily
Intake
(
ADI))
is
calculated.
The
RfD
is
"
an
estimate
(
with
uncertainty
spanning
approximately
an
order
of
magnitude)
of
a
daily
exposure
to
the
human
population
(
including
sensitive
subgroups)
that
is
likely
to
be
without
appreciable
risk
of
deleterious
effects
over
a
lifetime"
(
U.
S.
EPA,
1993).
The
RfD
is
derived
from
a
no
observed
adverse
effect
level
(
NOAEL),
lowest
observed
adverse
effect
level
(
LOAEL),
or
a
NOAEL
surrogate
such
as
a
benchmark
dose
identified
from
a
subchronic
or
chronic
study,
and
divided
by
a
composite
uncertainty
factor(
s).
The
RfD
is
calculated
as
follows:

RfD
=
NOAEL
(
LOAEL)
UF
.
MF
where:

NOAEL
=
No­
observed­
adverse­
effect
level:
A
highest
exposure
level
at
which
there
are
no
statistically
or
biologically
significant
increases
in
the
frequency
or
severity
of
adverse
effects
between
the
exposed
population
and
its
appropriate
control.

LOAEL
=
Lowest­
observed­
adverse­
effect
level:
The
lowest
exposure
level
at
which
there
are
statistically
or
biologically
significant
increases
in
frequency
or
severity
of
adverse
effects
between
the
exposed
population
and
its
appropriate
control
group.

UF
=
Uncertainty
factor
chosen
according
to
EPA/
NAS
guidelines
MF
=
Modifying
factor
Selection
of
the
uncertainty
factor
to
be
employed
in
calculation
of
the
RfD
is
based
on
professional
judgment,
while
considering
the
entire
database
of
toxicological
effects
for
the
chemical.
To
ensure
that
uncertainty
factors
are
selected
and
applied
in
a
consistent
manner,
the
Office
of
Water
(
OW)
employs
a
modification
to
the
guidelines
proposed
by
the
National
Academy
of
Sciences
(
NAS,
1977,
1980).
According
to
the
EPA
approach
(
U.
S.
EPA,
1993),
Draft
 
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2003)
50
uncertainty
is
broken
down
into
its
components,
and
each
dimension
of
uncertainty
is
given
a
quantitative
rating.
The
total
uncertainty
factor
is
the
product
of
the
component
uncertainties.
The
individual
components
of
the
uncertainty
are
as
follows:

UF
H
A
1,
3,
or
10­
fold
factor
used
when
extrapolating
from
valid
data
in
studies
using
long­
term
exposure
to
average
healthy
humans.
This
factor
is
intended
to
account
for
the
variation
in
sensitivity
(
intraspecies
variation)
among
the
members
of
the
human
population.

UF
A
An
additional
factor
of
1,
3,
or
10
used
when
extrapolating
from
valid
results
of
long­
term
studies
on
experimental
animals
when
results
of
studies
of
human
exposure
are
not
available
or
are
inadequate.
This
factor
is
intended
to
account
for
the
uncertainty
involved
in
extrapolating
from
animal
data
to
humans
(
interspecies
variation).

UF
S
An
additional
factor
of
1,
3,
or
10
used
when
extrapolating
from
less­
than­
chronic
results
on
experimental
animals
when
there
are
no
useful
long­
term
human
data.
This
factor
is
intended
to
account
for
the
uncertainty
involved
in
extrapolating
from
less­
than­
chronic
NOAELs
to
chronic
NOAELs.

UF
L
An
additional
factor
of
1,
3,
or
10
used
when
deriving
an
RfD
from
a
LOAEL,
instead
of
a
NOAEL.
This
factor
is
intended
to
account
for
the
uncertainty
involved
in
extrapolating
from
LOAELs
to
NOAELs.

UF
D
An
additional
1,3­
or
10­
fold
factor
used
when
deriving
an
RfD
from
an
"
incomplete"
database.
This
factor
is
meant
to
account
for
the
inability
of
any
single
type
of
study
to
consider
all
toxic
endpoints.
The
intermediate
factor
of
3
(
approximately
½
log
10
unit,
i.
e.,
the
square
root
of
10)
is
often
used
when
there
is
a
single
data
gap
exclusive
of
chronic
data.
It
is
often
designated
as
UF
D.

On
occasion,
EPA
also
uses
a
modifying
factor
in
the
determination
of
the
RfD.
A
modifying
factor
is
an
additional
uncertainty
factor
that
is
greater
than
zero
and
less
than
or
equal
to
10.
The
magnitude
of
the
MF
reflects
the
scientific
uncertainties
of
the
study
and
database
not
explicitly
treated
with
standard
uncertainty
factors
(
e.
g.,
the
number
of
species
tested).
The
default
value
for
the
MF
is
1.

In
establishing
the
UF
or
MF,
it
is
recognized
that
professional
scientific
judgment
must
be
used.
The
total
product
of
the
uncertainty
factors
and
modifying
factor
should
not
exceed
3000.
If
the
assignment
of
uncertainty
results
in
a
UF/
MF
product
that
exceeds
3000,
then
the
database
does
not
support
development
of
an
RfD.

2.
Drinking
Water
Equivalent
Level
The
drinking
water
equivalent
(
DWEL)
is
calculated
from
the
RfD.
The
DWEL
represents
a
drinking­
water­
specific
lifetime
exposure
at
which
adverse,
noncarcinogenic
health
effects
are
not
anticipated
to
occur.
The
DWEL
assumes
100%
exposure
from
drinking
water.
The
DWEL
Draft
 
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51
provides
the
noncarcinogenic
health
effects
basis
for
establishing
a
drinking
water
standard.
For
ingestion
data,
the
DWEL
is
derived
as
follows:

DWEL
=
(
RfD)
×
BW
WI
where:

BW
=
70
kg
adult
body
weight
WI
=
Drinking
water
intake
(
2
L/
day)

3.
Health
Advisory
Values
In
addition
to
the
RfD
and
the
DWEL,
EPA
calculates
Health
Advisory
(
HA)
values
for
noncancer
effects.
HAs
are
determined
for
lifetime
exposures
as
well
as
for
exposures
of
shorter
duration
(
1­
day,
10­
day,
and
longer­
term).
The
shorter
duration
HA
values
are
used
as
informal
guidance
to
municipalities
and
other
organizations
when
emergency
spills
or
contamination
situations
occur.

The
shorter­
term
HAs
are
calculated
using
a
similar
equation
to
the
RfD
and
DWEL;
however,
the
NOAELs
or
LOAELs
are
derived
from
acute
or
subchronic
studies
and
identify
a
sensitive
noncarcinogenic
endpoint
of
toxicity.
The
HAs
are
derived
as
follows:

HA
=
NOAEL
or
LOAEL
×
BW
UF
×
WI
where:

NOAEL
or
LOAEL
=
No­
or
lowest­
observed­
adverse­
effect­
level
in
mg/
kg
bw/
day
BW
=
Assumed
body
weight
of
a
child
(
10
kg)
or
an
adult
(
70
kg)

UF
=
Uncertainty
factor,
in
accordance
with
EPA
or
NAS/
OW
guidelines
WI
=
Assumed
daily
drinking
water
intake
of
a
child
(
1
L/
day)
or
an
adult
(
2
L/
day)

Using
the
above
equation,
the
following
drinking
water
HAs
are
developed
for
noncarcinogenic
effects:

°
1­
day
HA
for
a
10
kg
child
ingesting
1
L
water
per
day.
°
10­
day
HA
for
a
10
kg
child
ingesting
1
L
water
per
day.
°
Longer­
term
HA
for
a
10
kg
child
ingesting
1
L
water
per
day.
°
Longer­
term
HA
for
a
70
kg
adult
ingesting
2
L
water
per
day.
Draft
 
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2003)
52
Each
of
these
shorter­
term
HA
values
assumes
that
the
total
exposure
to
the
contaminant
comes
from
drinking
water.

The
lifetime
HA
is
calculated
from
the
DWEL
and
takes
into
account
exposure
from
sources
other
than
drinking
water.
It
is
calculated
using
the
following
equation:

Lifetime
HA
=
DWEL
×
RSC
where:

DWEL
=
Drinking
water
equivalent
level
RSC
=
Relative
source
contribution.
The
fraction
of
the
total
exposure
allocated
to
drinking
water.
The
default
value
is
20%
of
the
total
exposure.

The
lifetime
HA
is
the
MCLG
for
a
chemical
that
is
not
a
carcinogen.

The
following
paragraphs
evaluate
the
data
available
to
support
the
development
of
HA
values
for
methylglyoxal.

1­
Day
Health
Advisory
The
1­
day
HA
calculated
for
a
10
kg
child
assumes
a
single
acute
exposure
to
the
chemical
and
is
generally
derived
from
a
study
of
less
than
7
days'
duration.
The
only
short­
term
data
for
oral
exposure
to
methylglyoxal
are
LD50
data
and
are
not
appropriate
for
the
derivation
of
a
1­
day
HA.

10­
Day
Health
Advisory
The
10­
day
HA
assumes
a
limited
exposure
period
of
1
to
2
weeks
and
is
generally
derived
from
a
study
of
less
than
30­
day
duration.
No
data
were
identified
that
are
appropriate
for
the
derivation
of
a
10­
day
HA.

Longer­
Term
Health
Advisory
A
longer­
term
HA
value
is
derived
for
both
the
10
kg
child
and
the
70
kg
adult
and
assumes
an
exposure
period
of
approximately
7
years
(
or
10%
of
an
individual's
lifetime).
A
longer­
term
HA
is
generally
derived
from
a
study
of
subchronic
duration
(
exposure
for
10%
of
animal's
lifetime).

In
the
study
by
Ankrah
and
Appiah­
Opong
(
1999),
groups
of
37
mice
were
exposed
to
0
or
5
g/
kg/
day
methylglyoxal
in
their
drinking
water
during
gestation,
lactation,
and
weaning.
This
study
did
not
evaluate
standard
reproductive
or
teratology
parameters
but
did
evaluate
a
series
of
biochemical
parameters
(
GSH
levels,
GST
activity,
 ­
glutamyl
transferase
activity,
fasting
blood
glucose,
glucose
tolerance,
bile
production,
and
hemoglobin
oxidation).
Draft
 
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2003)
53
Methylglyoxal
exposure
was
associated
with
a
significant
decrease
in
erythrocyte
GSH
and
in
the
activity
of
GST
transferase,
an
enzyme
family
responsible
for
conjugation
of
the
end
products
of
cytochrome
P­
450
oxidation
with
GSH
for
excretion
(
Stipanuk,
2000).
However,
there
was
no
change
in
the
activity
of
GGT,
an
enzyme
involved
in
the
processing
of
glutathione
conjugates.

Parallel
with
a
decrease
in
serum
GSH
there
was
a
loss
in
hemoglobin's
ability
to
resist
oxidation.
When
aliquots
of
red
blood
cells
from
exposed
mice
were
first
exposed
to
oxidizing
conditions
in
vitro
there
were
no
differences
in
hemoglobin
oxidation
compared
with
controls.
However,
with
continued
exposure
of
the
red
cells
to
oxidation
for
a
3­
hour
period,
the
red
cells
of
the
methylglyoxal
treated
mice
had
three
times
more
methemoglobin
than
the
control
cells.
Ankrah
and
Appiah­
Opong
(
1999)
suggest
that
methylglyoxal
may
be
more
toxic
to
individuals
who
carry
the
G6PD
trait
(
homozygots
and
heterozygots).
Additional
studies
will
be
necessary
to
confirm
this
hypothesis.

The
effects
of
methylglyoxal
on
glucose
tolerance
also
were
evaluated
by
Ankrah
and
Appiah­
Opong
(
1999).
Blood
samples
were
drawn
from
fasting
animals.
The
animals
were
then
given
an
oral
bolus
dose
of
1.7
g
glucose/
kg
body
weight,
and
blood
samples
were
collected
after
2
hours.
Fasting
blood
glucose
levels
for
the
exposed
mice
did
not
differ
from
those
for
the
controls.
However,
blood
glucose
levels
increased
to
levels
outside
the
normal
range
after
the
glucose
challenge
for
17/
37
of
the
mice
given
methylglyoxal
compared
with
only
3/
37
of
the
control
mice.
Thus,
glucose
tolerance
was
impaired
in
the
exposed
mice
and
they
were
more
likely
to
become
hyperglycemic
than
the
controls.

The
study
by
Ankrah
and
Appiah­
Opong
(
1999)
is
not
suited
to
dose­
response
assessment
because
only
a
single
dose
was
tested.
There
are
also
questions
about
the
exact
dose
that
was
tested
(
see
section
IV
B).
Although
the
effects
observed
are
early­
stage
markers
of
toxicity,
lack
of
corroborating
and
supporting
data
from
a
multidose
standard
subchronic
study
limits
its
use
for
risk
assessment.
Accordingly,
a
longer­
term
HA
has
not
been
derived.

Reference
Dose,
Drinking
Water
Equivalent
Level,
and
Lifetime
Health
Advisory
No
standard
subchronic
or
chronic
studies
were
located
on
the
noncancer
effects
of
methylglyoxal.
Accordingly,
no
lifetime
HA
is
derived
for
this
chemical.
The
study
by
Ankrah
and
Appiah­
Opong
(
1999)
discussed
above
is
not
suitable
for
derivation
of
an
RfD
or
lifetime
HA
because
of
its
less­
than­
lifetime
duration
and
the
lack
of
data
for
many
standard
markers
of
systemic
toxicity.

B.
Carcinogenic
Effects
In
1986,
EPA
established
a
five­
category,
alphanumeric
system
for
carcinogen
with
the
publication
of
Guidelines
for
Carcinogen
Risk
Assessment
(
U.
S.
EPA,
1986).
The
five
categories
were
as
follows:

Group
A:
Human
Carcinogen.
Group
B:
Probable
Human
Carcinogen.
3
A"
point
of
departure"
(
POD)
marks
the
beginning
of
extrapolation
to
lower
doses.
The
POD
is
an
estimated
dose
(
expressed
in
human­
equivalent
terms)
near
the
lower
end
of
the
observed
range,
without
significant
extrapolation
to
lower
doses
(
U.
S.
EPA,
2003).

Draft
 
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2003)
54
Group
B1:
Limited
evidence
in
humans.
Group
B2:
inadequate
evidence
in
humans;
sufficient
evidence
in
animals.
Group
C:
Possible
Human
Carcinogen.
Group
D:
Not
classified
as
to
Human
Carcinogenicity.
Group
E:
Evidence
of
Noncarcinogenicity
for
Humans.

In
1996
the
Agency
issued
proposed
revisions
to
Guidelines
for
Carcinogen
Risk
Assessment
for
public
comment.
The
1996
proposal
was
later
refined
and
released
as
a
revised
draft
in
1999.
Although
the
1999
version
of
the
Guidelines
for
Carcinogen
Risk
Assessment
has
not
yet
been
formally
adopted
by
the
agency,
use
of
the
1986
guidelines
ceased
in
2000
with
the
publication
of
a
directive
from
the
Administrator
(
Federal
Register,
2001
)
specifying
that
the
1999
guidelines
are
to
be
used
on
an
interim
basis.

Under
the
U.
S.
EPA
(
1999)
Guidelines
for
Carcinogen
Risk
Assessment:
Review
Draft,
the
U.
S.
EPA
presents
the
carcinogenic
potential
of
a
chemical
compound
in
a
narrative
fashion,
and
uses
one
of
the
following
five
standard
descriptors
to
express
the
conclusion
regarding
the
weight
of
evidence
for
carcinogenic
hazard
potential:

°
Carcinogenic
to
humans
°
Likely
to
be
carcinogenic
to
humans
°
Suggestive
evidence
of
carcinogenic
potential,
but
not
sufficient
to
assess
human
carcinogenic
potential
°
Inadequate
information
to
assess
human
carcinogenic
potential
°
Not
likely
to
be
carcinogenic
to
humans
Each
standard
descriptor
is
presented
only
in
the
context
of
a
chemical­
specific,
weight­
ofevidence
narrative.
Additionally,
more
than
one
conclusion
may
be
reached
for
an
agent
(
e.
g.,
an
agent
is
"
likely
to
carcinogenic"
by
inhalation
exposure
and
"
not
likely
to
be
carcinogenic"
by
oral
exposure.

In
cases
where
the
toxicological
evidence
leads
to
the
classification
of
the
contaminant
as
a
carcinogen
or
likely
to
be
a
carcinogen,
mathematical
models
are
used
to
calculate
the
estimated
excess
cancer
risk
associated
with
ingestion
of
the
contaminant
in
drinking
water.
The
data
input
to
the
models
usually
come
from
lifetime­
exposure
studies
in
animals.
In
order
to
predict
the
risk
for
humans,
animal
doses
must
be
converted
to
equivalent
human
doses.
The
conversion
can
include
corrections
for
noncontinuous
exposure,
less­
than­
lifetime
studies,
and
allometric
scaling
of
the
animal
body
weight.
The
dose­
response
assessment
is
performed
in
two
stages.
A
mathematical
assessment
of
experimental
dose
data
is
used
to
derive
a
point
of
departure
(
POD)
3.
Extrapolation
from
the
POD
may
assume
either
linearity
or
nonlinearity
of
the
dose­
response
relationship,
or
both.
The
linear
approach
(
slope
factor)
is
used
for
mutagenic
carcinogens,
i.
e.
those
with
linear
mode
of
action,
or
where
the
mode
of
action
cannot
be
determined.
For
4LED10
=
Lower
bound
on
the
dose
associated
with
an
increased
tumor
incidence
of
10%

Draft
 
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2003)
55
carcinogens
with
a
well­
substantiated
nonlinear
mode
of
action,
an
RfD
approach
is
used.
In
both
cases
a
range
of
models
are
available.

With
the
linear
approach
the
slope
of
the
line
from
the
point
of
departure
(
LED
10)
4
to
the
origin
is
calculated.
The
slope
factor
(
q
1*)
is
a
reflection
of
the
cancer
potency.
It
is
used
to
calculate
the
concentration
in
drinking
water
that
is
equivalent
to
a
specific
risk
to
the
population.
Risk
estimates
are
generally
presented
for
the
one­
in­
ten
thousand
risk
(
1/
10.000;
E­
4),
the
onein
one
hundred
thousand
(
1/
100,000;
E­
5)
risk
and
the
one­
in
a
million
(
1/
1,000,000;
E­
6)
risk
using
the
following
equation:

Concentration
in
Drinking
Water
(
mg/
L)
=
Risk
x
Body
Weight
(
Kg)
Slope
Factor
(
risk/
mg/
kg/
day)
x
2
L/
day
It
is
assumed
that
the
average
adult
human­
body
weight
is
70
kg
and
that
the
average
water
consumption
of
an
adult
human
is
two
liters
of
water
per
day.
Drinking
water
regulations
target
the
E­
4
to
E­
6
risk
range
as
determined
from
the
lower
confidence
bound
on
the
POD.

The
data
base
used
to
calculate
and
support
the
setting
of
cancer
risk
rates
has
an
inherent
uncertainty
due
to
systematic
and
random
errors
in
scientific
measurement.
Thus,
there
is
uncertainty
when
the
risk
is
extrapolated
from
epidemiological
or
animal
data
to
the
entire
humans
population.
When
developing
cancer­
risk
rates,
some
of
the
uncertainties
that
exist,
include
incomplete
knowledge
concerning
the
health
effects
of
contaminants
in
drinking
water,
the
impact
of
the
experimental
animal's
age,
sex
and
species,
the
nature
of
the
target­
organ
system(
s)
examined,
and
the
actual
rate
of
exposure
of
the
internal
targets
in
experimental
animals
or
humans.
Dose
response
data
usually
are
available
only
for
high
levels
of
exposure,
not
for
the
lower
levels
at
which
a
standard
may
be
set.
When
there
is
exposure
to
more
than
one
contaminant,
additional
uncertainty
results
from
a
lack
of
information
about
possible
synergistic
or
antagonistic
effects.
The
true
risk
to
humans,
while
not
identifiable,
is
not
likely
to
exceed
the
upper
limit
estimate
and,
in
fact,
may
be
lower
or
even
zero..

Characterization
of
Carcinogenic
Effects
Methylglyoxal
is
mutagenic
in
several
strains
of
S.
typhimurium
with
and
without
metabolic
activation
(
Kato
et
al.,
1989;
Aeschbacher
et
al.,
1989;
Shane
et
al.,
1988;
Chen
et
al.,
1997),
indicating
it
can
be
a
direct­
acting
mutagen.
In
in
vitro
assays
methylglyoxal
is
capable
of
producing
chromosomal
aberrations
(
Martelli
et
al.,
1989;
Migliore
et
al.,
1990)
and
SCEs
(
Tucker
et
al.,
1989),
but
in
vivo
assays
were
negative
except
for
weakly
positive
results
at
high
doses
(
Martelli
et
al.,
1989;
Migliore
et
al.,
1990).
Methylglyoxal
caused
a
number
of
other
positive
genotoxic
responses
including
induction
of
the
SOS
response
indicative
of
repair
of
DNA
damage;
the
ability
to
cause
single­
strand
breaks
and
cross­
links
in
DNA;
and
the
ability
to
form
adducts
and
reaction
products
with
DNA.
The
overall
weight
of
evidence
for
genotoxicity
is
strong,
although
whether
this
genotoxicity
is
expressed
in
vivo
is
not
yet
clear.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
56
There
are
weak
mechanistic
data
for
carcinogenicity
in
several
animal
studies
using
male
rats.
In
an
intermediate­
term
liver
carcinogenesis
bioassay
(
Hasegawa
and
Ito,
1992;
Hasegawa
et
al.,
1995)
methylglyoxal
at
a
dose
of
0.2%
in
drinking
water
significantly
increased
the
number
but
not
the
area
of
GST­
P­
positive
liver
foci.
In
another
intermediate­
term
liver
carcinogenesis
bioassay
(
Martelli
et
al.,
1988),
methylglyoxal
at
doses
of
0.05%
and
0.2%
in
drinking
water
significantly
increased
the
number
of
foci
and
total
foci
volume
when
compared
with
partially
hepatectomized
controls,
but
only
showed
significant
effects
at
the
high
dose
when
compared
with
controls
exposed
to
0.02%
2­
acetylaminofluorene
as
initiator
or
promoter
without
methylglyoxal
exposure.
In
a
medium­
term
gastric
carcinogenesis
bioassay
(
Takahashi
et
al.,
1989),
methylglyoxal
at
a
dose
of
0.25%
in
drinking
water
significantly
increased
hyperplasia
but
not
carcinoma
in
the
pylorus
of
the
rat
glandular
stomach.

Methylglyoxal
exposure
caused
no
increase
in
tumors
in
rats
exposed
to
via
their
drinking
water
for
2
years
at
an
average
dose
of
about
20
mg/
kg/
day
(
Fujita
et
al.,
1986).
However,
these
data
were
reported
in
abstract
form
only,
so
the
reliability
of
the
study
is
uncertain.
Takahashi
et
al.
(
1989)
found
no
increase
in
gastric
tumors
or
hyperplastic
lesions
in
rats
exposed
to
either
glyoxal
(
700
mg/
kg/
day)
or
methylglyoxal
(
350
mg/
kg/
day)
for
32
weeks.
However,
the
short
duration
of
this
exposure
limits
the
significance
of
the
study.

In
contrast
to
the
negative
tumorigenicity
studies
above,
several
groups
of
researchers
have
reported
that
methylglyoxal
has
initiating
and/
or
promoting
activity
in
various
short­
term
tests
where
methylglyoxal
was
administered
following
an
initial
exposure
to
a
known
carcinogen
(
Takahashi
et
al.,
1989;
Martelli
et
al.,
1988;
Furihata
et
al.,
1985a,
b).
Methylglyoxal
has
also
been
found
to
produce
injection
site
tumors
in
two
subcutaneous
exposure
studies
in
rats
(
Takayama
et
al.,
1984;
Nagao
et
al.,
1986a,
b).

There
are
no
human
exposure
data
to
assist
in
this
evaluation.
Because
methylglyoxal
is
a
normal
metabolite
and
is
found
in
many
foods
and
beverages,
the
body
would
be
expected
to
handle
small
amounts
through
normal
metabolic
pathways
or
repair
mechanisms.
Additional
in
vivo
genotoxicity
tests
may
help
to
clarify
the
situation,
although
they
cannot
fully
substitute
for
a
standard
2­
year
carcinogen
bioassay.

An
IARC
monograph
(
1991)
on
methylglyoxal
summarized
the
status
of
toxicity
testing
to
date
and
concluded
that
methylglyoxal
was
not
classifiable
as
to
its
carcinogenicity
to
humans.
Under
the
proposed
Guidelines
for
Carcinogen
Risk
Assessment
(
U.
S.
EPA,
1996,
1999),
the
carcinogenic
potential
of
methylglyoxal
cannot
be
determined,
because
no
adequate
human
epidemiological
studies
exist
and
because
no
properly
conducted
long­
term
animal
bioassay
has
been
done
to
form
the
basis
for
such
a
determination.

REFERENCES
Aeschbacher
HU,
Wolleb
U,
Loliger
J,
Spadone
JC,
Liardon
R.
1989.
Contribution
of
coffee
aroma
constituents
to
the
mutagenicity
of
coffee.
Food
Chem
Toxicol
27(
4):
227­
32.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
57
Amicarelli
F,
Bucciarelli
T,
Poma
A,
Aimola
P,
Di
Ilio
C,
Ragnelli
AM,
Miranda
M.
1998.
Adaptive
response
of
human
melanoma
cells
to
methylglyoxal
injury.
Carcinogenesis19(
3):
519­
523.

Ankrah
N,
Appiah­
Opong
R.
1999.
Toxicity
of
low
levels
of
methylglyoxal:
Depletion
of
blood
glutathione
and
adverse
effect
on
glucose
tolerance
in
mice.
Toxicol
Lett
109:
61­
17.

Ankrah
N­
A,
Ekuban
FA,
Asamoah
EB.
1990.
Depressant
effect
of
very
low
levels
of
aflatoxin
B
1
on
mouse
glyoxylase­
I
activity
and
methylglyoxal
disposal.
Biochem
Pharmacol
39(
7):
1261­
1263.

Argiles
JM.
1986.
Has
acetone
a
role
in
the
conversion
of
fat
to
carbohydrate
in
mammals?
Trends
Biochem
Sci
11:
61­
63.

Armeni
T,
Pieri
C,
Marra
M,
Saccucci
F,
Principato
G.
1998.
Studies
on
the
life
prolonging
effect
of
food
restriction:
glutathione
levels
and
glyoxylase
enzymes
in
rat
liver.
Mech
Ageing
Devel
101:
101­
110.

Arnold
R,
Fodor
G,
Mathelier
H,
Mohacsi
T,
Szent­
Gyorgyi
A,
Veltri
RW.
1983.
Recent
aspects
of
the
chemistry
of
vitamin
C.
NRCF
Cancer
Res
Assoc
Symp
2:
197­
219.

Barros,
A,
Rodrigues,
JA,
Almeida,
PJ,
Oliva­
Teles,
MT.
1999.
Determination
of
glyoxal,
methylglyoxal,
and
diacetyl
in
selected
beer
and
wine,
by
HPLC
with
UV
spectophotometric
detection
after
deriivatization
with
o­
phenylenediamine.
J
Liq
Chromm
&
Rel
Technol
22(
13):
2061­
2069.

Baskaran
S,
Balasubramanian
KA.
1990.
Toxicity
of
methylglyoxal
towards
rat
enterocytes
and
colonocytes.
Biochem
Internat
21(
1):
165­
174.

Brambilla
G,
Sciaba
L,
Faggin
P,
Finollo
R,
Bassi
AM,
Ferro
M,
Marinari
UM.
1985.
Methylglyoxal­
induced
DNA­
protein
cross­
links
and
cytotoxicity
in
CHO
cells.
Carcinogenesis
6:
683­
686.

Bronzetti
G,
Corsi
C,
Del
Chiaro
D,
Boccardo
P,
Vellosi
R,
Rossi
F,
Paolini
M,
Cantelli
FG.
1987.
Methylglyoxal:
Genotoxicity
studies
and
its
effect
in
vivo
on
the
hepatic
microsomal
monoxygenase
system
of
the
mouse.
Mutagenesis
2:
275­
277.

Budavari
S,
O'Neil
MJ,
Smith
A,
Heckelman
PE,
Kinneary
JF,
eds.
1996.
The
Merck
index:
An
encyclopedia
of
chemicals,
drugs,
and
biologicals.
Whitehouse
Station,
NJ:
Merck.

Cajelli
E,
Canonero
R,
Martelli
A,
Brambilla
G.
1987.
Methylglyoxal­
induced
mutation
to
6­
thioguanine
resistance
in
V79
cells.
Mutat
Res
190:
47­
50.

Chaudhary
D,
Chandra
D,
Kale
R.
1997.
Influence
of
methylglyoxal
on
antioxidant
enzymes
and
oxidative
damage.
Toxicol
Lett
93:
141­
152.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
58
Chen
S­
C,
Wong
TY,
Chung
K­
T.
1997.
Base­
pair
mutation
caused
by
four
nitro­
group
containing
aromatic
amines
in
S.
typhimurium
TA100,
TA104,
TA4001,
and
TA4006.
Mutat
Res
395:
223­
227.

Clerici
L,
Mascheroni
G,
Sacco
MG,
Lucchini
F.
1990.
The
embryotoxicity
of
methyglyoxal
on
mouse
fertilized
eggs
cultured
in
vitro.
Mutat
Res
234:
375.

Conroy
PJ.
1979.
Carcinostatic
activity:
Methylglyoxal
and
related
substances
in
tumour­
bearing
mice.
CIBA
Found
Sympos
67:
271­
298.

Dedenhardt
TP,
Thorpe
SR,
Baynes
JW.
1998.
Chemical
modifications
of
proteins
by
methylglyoxal.
Cell
Mol
Biol
44(
7):
1139­
1145.

Destaillats,
H,
Spaulding,
R,
Charles,
MJ.
202.
Ambient
air
measurement
of
acrilein
and
other
carbonyls
at
the
Oakland­
San
Francisco
Bay
Bridge
Toll
Plasa.
Environ
Sci
Technol
36:
2227­
2235.

Dorado
L,
Montoya
MR,
Mellado
JMR.
1992.
A
contribution
to
the
study
of
the
structuremutagenicity
relationship
for
 ­
dicarbonyl
compounds
using
the
Ames
test.
Mutat
Res
269:
301­
306.

Dyer,
DG,
Dunn,
JA,
Thorpe,
SR,
Ballie,
KE,
Lyons,
TJ,
McCance,
DR,
Baynes,
JW.
1993.
Accumulation
of
Maillard
reaction
products
in
skin
collegen
in
diabetes
and
aging.
J
Clin
Invest
91:
2463­
2469.

Fazal
F,
Ahmed
MA,
Rahman
A,
Hadi
SM.
1994.
Generation
of
superoxide
anion
and
hydroxyl
radical
by
methylglyoxal.
Med
Sci
Res
22:
21­
22.

Food
and
Drug
Administration
(
FDA).
1997.
Code
of
Federal
Regulations,
Vol.
21.
Food
and
Drugs.
Section
172.515.

Friederich
U,
Hann
D,
Albertini
S,
Schlatter
C,
Wurgler
FE.
1985.
Mutagenicity
studies
on
coffee.
The
influence
of
different
factors
on
the
mutagenic
activity
in
the
Salmonella/
mammalian
microsome
assay.
Mutat
Res
156:
39­
52.

Fujita
Y,
Wakabayashi
K,
Nagao
M,
Sugimura
T.
1985a.
Characteristics
of
major
mutagenicity
of
instant
coffee.
Mutat
Res
142:
145­
148.

Fujita
Y,
Wakabayashi
K,
Nagao
M,
Sugimura
T.
1985b.
Implication
of
hydrogen
peroxide
in
the
mutagenicity
of
coffee.
Mutat
Res
144:
227­
230.

Fujita
Y,
Wakabayashi
K,
Ohgaki
H,
Nagao
M,
Sugimura
T.
1986.
Absence
of
carcinogenicity
of
methylglyoxal
in
F344
rats
by
oral
administration
(
meeting
abstract).
Proc
Ann
Meet
Jpn
Cancer
Assoc
45:
64.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
59
Furihata
C,
Hatta
A,
Sato
Y,
Matsushima
T.
1989.
Alkaline
elution
of
DNA
from
stomach
pyloric
mucosa
of
rats
treated
with
glyoxal.
Mutat
Res
213:
227­
231.

Furihata
C,
Matsushima
T.
1989.
Prediction
of
possible
carcinogens,
tumor­
promoters
and
antitumor
promoters
in
the
glandular
stomach
(
Abstract).
Environ
Mol
Mutagen
14(
Suppl
15):
63.

Furihata
C,
Sato
Y,
Matsushima
T,
Tatematsu
M.
1985a.
Induction
of
ornithine
decarboxylase
and
DNA
synthesis
in
rat
stomach
mucosa
by
methylglyoxal.
Carcinogenesis
6:
91­
94.

Furihata
C,
Yoshida
S,
Matsushima
T.
1985b.
Potential
initiating
and
promoting
activities
of
diacetyl
and
glyoxal
in
rat
stomach
mucosa.
Jpn
J
Cancer
Res
76:
804­
814.

Furukawa
H,
Kawai
K,
Kawai
N,
Nagano
H.
1986.
Modulation
of
mutagenic
activities
by
interaction
between
amines
and
carbonyl
compounds
such
as
methylglyoxal,
aflatoxin
B1
and
aflatoxin
G1.
Mutat
Res
164:
265­
266.

Glaze
WH,
Koga
M,
Cancilla
D,
Wang
K,
McGuire
MJ,
Liang
S,
Davis
MK,
Tate
CH,
Aieta
EM.
1989.
Evaluation
of
ozonation
by­
products
from
two
California
surface
waters.
J
Am
Water
Works
Assoc
8:
66­
73.

Hasegawa
R,
Ito
N.
1992.
Liver
medium­
term
bioassay
in
rats
for
screening
of
carcinogens
and
modifying
factors
in
hepatocarcinogenesis.
Food
Chem
Toxicol
30(
11):
979­
992.

Hasegawa
R,
Ogiso
T,
Imaida
KT,
Shirai
T,
Ito
N.
1995.
Analysis
of
the
potential
carcinogenicity
of
coffee
and
its
related
compounds
in
a
medium­
term
liver
bioassay
of
rats.
Food
Chem
Toxicol
33(
1):
15­
20.

Hayashi,
T,
Shibamoto,
T.
1985.
Analysis
of
methyl
glyoxal
in
foods
and
beverages.
J
Agric
Food
chem
33:
1090­
1093.

Hazardous
Substances
Data
Bank
(
HSDB)
[
database
online].
1999.
National
Library
of
Medicine,
National
Toxicology
Information
Program.
Bethesda,
MD.
http://
toxnet.
nlm.
nih.
gov/.

Hellmér
L,
Bolcsfoldi
G.
1992.
An
evaluation
of
the
E.
coli
K­
12
uvrB/
recA
DNA
repair
hostmediated
assay.
I.
In
vitro
sensitivity
of
the
bacteria
to
61
compounds.
Mutat
Res
272:
145­
160.

Hindson
C,
Lawlor
F.
1982.
Allergy
to
glyoxal
in
a
polyvinyl
resin
emulsion.
Contact
Dermatitis
8:
213.

Inoue
Y,
Kimura
A.
1995.
Methylglyoxal
and
regulation
of
its
metabolism
in
microorganisms.
In:
Poole
RK,
ed.
Advances
in
microbial
physiology,
Vol.
37.
London:
Academic
Press.
pp.
178­
227.

International
Agency
for
the
Research
of
Cancer
(
IARC).
1991.
Methylglyoxal.
IARC
Monographs
on
the
Evaluation
of
Carcinogenic
Risk
to
Humans
51:
443­
458.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
60
Jerzykowski
T,
Matuszewski
LO,
Tarnawski
R,
Luinter
R,
Herman
ZS,
Sokola
A.
1975.
Changes
of
certain
pharmacological
and
biochemical
indices
in
acute
methylglyoxal
poisoning.
Arch
Immunol
Ther
Exper
23:
549.

Kalapos
MP.
1999.
Influence
of
acetone
on
the
hepatotoxicity
of
methylglyoxal.
Med
Sci
Res
27:
617­
620.

Kato
F,
Araki
A,
Nozaki
K,
Matsushima
T.
1989.
Mutagenicity
of
aldehydes
and
diketones
(
Abstract).
Mutat
Res
216:
366­
367.

Kawamura,
K,
Steinberg,
S,
Kaplan,
IR.
2000.
Homologous
series
of
C1­
C10
monocarboxyls
acids
and
C1­
C6
carbonyls
in
Los
Angeles
air
and
motor
vehicle
exhausts.
Atmos
Environ
34:
4175­
4191
Kawase
M,
Tada
M,
Akagi
S,
Ohmori
S.
1996.
Changes
in
concentrations
of
methylglyoxal,
Dlactate
and
glyoxalase
activities
in
liver
and
plasma
of
rats
fed
a
3'­
methyl­
4­
dimethylaminoazobenzene
rich
diet.
Res
Exp
Med
196:
251­
259.

Klug,
S,
Merker,
HJ,
Jackh,
R.
2001.
Effects
of
ethylene
glycol
and
metabolites
on
in
vitro
development
of
rat
embryos
during
organogenesis.
Toxicol
in
Vitro
15:
635­
642.

Kranendonk
M,
Pintado
F,
Mesquita
P,
Laires
A,
Vermeulen
NP,
Rueff
J.
1996.
MX100,
a
new
Escherichia
coli
tester
strain
for
use
in
genotoxicity
studies.
Mutagenesis
11(
4):
327­
333.

Krymkiewicz
N.
1973.
Reactions
of
methylglyoxal
with
nucleic
acids.
FEBS
Lett
29:
51­
54.

Langlais
B,
Reckhow
DA,
Brink
DR,
eds.
1991.
Ozone
in
water
treatment:
Application
and
engineering.
Chelsea,
MI:
Lewis
Publishers.
pp.
238­
245.

Leoncini
G,
Maresca
M,
Buzzi
E.
1989.
Inhibition
of
the
glycolytic
pathway
by
methylglyoxal
in
human
platelets.
Cell
Biochem
Func
7:
65­
70.

Martelli
A,
Allavena
A,
Ottonello
M,
Marinari
UM.
1989.
Evaluation
of
methylglyoxal
genotoxicity
in
the
rat
using
the
micronucleus
assay
in
liver
and
bone­
marrow
cells.
Biosciences
76:
159­
163.

Martelli
A,
Ghia
M,
Mereto
E,
Marinari
UM,
Brambilla
G.
1988.
Induction
and
promotion
of
gamma­
glutamyltrans­
peptidase­
positive
foci
in
the
rat
liver
by
methylglyoxal.
Jpn
J
Cancer
Res
79:
666­
669.

Matsui
S,
Yamamoto
R,
Yamada
H.
1989.
The
Bacillus
Subtilis/
microsome
rec­
assay
for
the
detection
of
DNA
damaging
substances
which
may
occur
in
chlorinated
and
ozonated
waters.
Water
Sci
Technol
21:
875­
887.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
61
McCance,
DR,
Dyer,
DG,
Dunn,
JA,
Baillie,
KE,
Thorpe,
SR,
Baynes,
JW,
Lyons,
TJ.
1993.
Maillard
reaction
products
and
their
relation
to
complications
in
insulin­
dependant
diabetes
mellitus.
J
Clin
Invest
91:
2470­
2478.

McLain
J,
Blank
V.
2000.
ICR
DBP
Occurrence
Data.
Office
of
Ground
Water
and
Drinking
Water.
U.
S.
Environmental
Protection
Agency.
Personal
Communication,
Feb
2,
2000.

McLellan
CC,
Thornalley
PJ,
Benn
J,
Sonksen
PH
1994.
The
glyoxylase
system
in
clinical
diabetes
mellitus
and
correlation
with
diabetic
complications.
Clin
Sci
87:
21­
29
(
as
cited
in
Thornalley
1996).

Mellado
JMR,
Montoya
MR.
1994.
Correlations
between
chemical
reactivity
and
mutagenic
activity
against
S.
Typhimurium
TA100
for
 ­
dicarbonyl
compounds
as
a
proof
of
the
mutagenic
mechanism.
Mutat
Res
304:
261­
264.

Migliore
L,
Barale
R,
Bosco
E,
Giogelli
F,
Minunni
M,
Scarpato
R,
Loprieno
N.
1990.
Genotoxicity
of
methylglyoxal:
Cytogenetic
damage
in
human
lymphocytes
in
vitro
and
in
intestinal
cells
of
mice.
Carcinogenesis
11(
9):
1503­
1507.

Mitchell
AD.
1993.
Genetic
toxicology.
In:
Stacey
NS,
ed.
Occupational
toxicology.
Bristol,
PA:
Taylor
and
Francis.
pp.
123­
147.

Miyakawa
Y,
Nishi
Y,
Kato
K,
Sato
H,
Takahashi
M,
Hayashi
Y.
1991.
Initiating
activity
of
eight
pyrolysates
of
carbohydrates
in
a
two­
stage
mouse
skin
tumorigenesis
model.
Carcinogenesis
12(
7):
1169­
1173.

Montgomery
R,
Conway
TW,
Spector
AA.
1990.
Biochemistry:
A
case­
oriented
approach.
St.
Louis,
MO:
CV
Mosby.

Murata­
Kamiya
N,
Kamiya
H,
Kaji
H,
Kasai
H.
2000.
Methylglyoxal
induces
G:
C
to
C:
G
and
G:
C
to
T:
A
transversions
in
the
supF
gene
on
a
shuttle
vector
plasmid
replicated
in
mammalian
cells.
Mutat
Res
468:
173­
182.

Murata­
Kamiya
N,
Kaji
H,
Kasai
H.
1997a.
Types
of
mutations
induced
by
glyoxal,
a
major
oxidative
DNA­
damage
product,
in
Salmonella
typhimurium.
Mutat
Res
377:
13­
16.

Murata­
Kamiya
N,
Kamiya
H,
Kaji
H,
Kasai
H.
1997b.
Mutational
specificity
of
glyoxal,
a
product
of
DNA
oxidation,
in
the
lacI
gene
of
wild­
type
Escherichia
coli
W3110.
Mutat
Res
377:
255­
262.

Murata­
Kamiya
N,
Kamiya
H,
Iwamoto
N,
Kasai
H.
1995.
Formation
of
a
mutagen,
glyoxal,
from
DNA
treated
with
oxygen
free
radicals.
Carcinogenesis
16(
9):
2251­
2253.

Nagao
M,
Fujita
Y,
Wakabayashi
K,
Nukaya
H,
Kosuge
T,
Sugimura
T.
1986a.
Mutagens
in
coffee
and
other
beverages.
Environ
Health
Perspect
67:
89­
91.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
62
Nagao
M,
Fujita
Y,
Sugimura
T,
Kosuge
T.
1986b.
Methylglyoxal
in
beverage
and
foods:
Its
mutagenicity
and
carcinogenicity.
IARC
Sci
Publ
70:
283­
291.

National
Academy
of
Sciences
(
NAS).
1977.
Drinking
water
and
health.
Washington,
DC.

NAS.
1980.
Drinking
water
and
health,
Vol.
3.
Washington,
DC:
National
Academy
Press.

National
Research
Council
(
NRC),
National
Academy
of
Sciences.
1983.
Risk
assessment
in
the
Federal
Government:
Managing
the
process.
Washington,
DC:
National
Academy
Press.

National
Toxicology
Program
(
NTP).
1991.
Range
finding
studies:
developmental
toxicity,
glyoxal
dihydrate
when
administered
via
gavage
in
CD
Sprague­
Dawley
rats
(
as
cited
in
NTP,
1994).
Study
No.
NTP­
90­
RF/
DT­
014;
Contract
Number
NO1­
ES­
94259.

NTP.
1994.
Final
report
on
the
developmental
toxicity
of
glyoxal
trimeric
dihydrate
(
CAS
No.
4405­
13­
4)
in
Sprague­
Dawley
CD
®
rats
on
gestational
days
6
through
15.
National
Toxicology
Program,
Research
Triangle
Park,
NC.
Publication
No.
PB94­
151974.

Nishi
Y,
Miyakawa
Y,
Kato
K.
1989.
Chromosome
aberrations
induced
by
pyrolysates
of
carbohydrates
in
Chinese
hamster
V79
cells.
Mutat
Res
227:
117­
123.

Nishimura,
C,
Furue,
M,
Ito,
T,
Omori,
Y,
Tanimoto,
T.
1993.
Quantitative
determination
of
human
aldose
reductase
by
enzyme­
linked
immunosorbent
assay.
Biochem
Pharmacol
46(
1):
21­
28.

Nunoshiba
T,
Hashimoto
M,
Nishioka
H.
1989.
Cross
adaptive
response
between
hydrogen
peroxide
and
aldehydes
in
E.
coli
(
Abstract).
Environ
Mol
Mutagen
14(
Suppl
15):
144.

Nunoshiba
T,
Hashimoto
M,
Nishioka
H.
1991.
Cross­
adaptive
response
in
Escherichia
coli
caused
by
pretreatment
with
H
2
O
2
against
formaldehyde
and
other
aldehyde
compounds.
Mutat
Res
255:
265­
271.

Odani,
H,
Shinzato,
T,
Usami,
J,
Matsumoto,
Y,
Frye,
EB,
Baynes,
JW,
Maeda,
K.,
1998.
Inidazolium
crosslinks
derived
from
reaction
of
lysine
with
glyoxal
and
methylglyoxal
are
increased
in
serum
proteins
of
uremic
patients:
evidence
for
increased
oxidative
stress
in
uremia.
FEBS
Letters
427:
381­
385.

Okado
A,
Kawasaki
Y,
Hasuike
Y,
Takahashi
M,
Teshima
T,
Fujii
J,
Taniguchi
N.
1996.
Induction
of
apoptotic
cell
death
by
methylglyoxal
and
3­
deoxyglucosone
in
macrophage­
derived
cell
lines.
Biochem
Biophys
Res
Commun
225:
219­
224.

Patnaik
P.
1992.
A
comprehensive
guide
to
the
hazardous
properties
of
chemical
substances.
New
York:
Van
Nostrand
Reinhold.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
63
Peinado
J,
Lopez­
Soriano
FJ,
Argiles
JM.
1986.
The
metabolism
of
acetone
in
the
pregnant
rat.
Biosci
Rep
6:
983­
989.

Peinado
LS,
Ramirez
PC,
Lopez
C.
1989.
The
in
vitro
interaction
with
DNA
of
several
hypoxic
radiosensitizers.
Chem
Biol
Interact
71:
245­
253.

Peters
MA,
Jurgelski
W,
Hudson
PM.
1977.
Toxicity
of
methylglyoxal
to
pregnant
and
nonpregnant
rats.
Congress
of
Toxicology
Abstracts.
p.
528.

Phillips
SA,
Thornalley
PJ.
1993.
The
formation
of
methylglyoxal
from
triose
phosphates.
Eu
J
Biochem
2(
2):
101­
105.

Phillips
SA,
Mirrlees
D,
Thornalley
PJ.
1993.
Modification
of
the
glyoxylase
system
in
streptozotocin­
induced
diabetic
rats.
Effect
of
the
aldose
reductase
inhibitor
Statil,
Biochem
Pharmacol
46:
805­
811.

Presidential/
Congressional
Commission
on
Risk
Assessment
and
Risk
Management.
1997.
Framework
for
environmental
health
risk
management:
risk
assessment
and
risk
management
in
regulatory
decision
making.
Gail
Charnley,
Ed.
277
pp.

Rahman
A,
Shahabuddin,
Hadi
SM.
1990.
Formation
of
strand
breaks
and
interstrand
cross­
links
in
DNA
by
methylglyoxal.
J
Biochem
Toxicol
5:
161­
166.

Rao,
X,
Kobayashi,
R,
White­
Morris,
R,
Spaulding,
R,
Frazey,
P,
Charles,
MJ.
2001.
GC/
ITMS
measurements
of
carbonyls
and
multifunctional
carbonyls
in
PM2.5
particles
emitted
from
motor
vehicles.
J
AOA
Int.
84:
669­
705.

Sayato
Y,
Nakamuro
K,
Ueno
H.
1989.
Mutagenicity
on
chlorination
of
products
formed
by
ozonation
of
naphthoresorcinol
in
water.
Mutat
Res
226:
151­
155.

Schalkwijk
CG,
Vermeer
MA,
Stenhouwer
CDA,
te
Koppele
J,
Pricen
HMG,
Vanttinsbergh
VWM.
1998.
Effect
of
methylglyoxal
on
the
physio­
chemical
and
biological
properties
of
lowdensity
lipoprotein.
Biochim
Biophys
Acta
1394:
187­
198.

Schardein
JL.
1993.
Miscellaneous
chemicals.
In:
Chemically
induced
birth
defects,
2nd
ed.
New
York:
Marcel
Dekker.
pp.
842­
869.

Schnuch
A,
Uter
W,
Geier
J,
et
al.
1998.
Contact
allergies
in
health
care
workers:
Results
from
the
IVDK.
Acta
Derm
Venereol
78:
358­
363.

Shane
BS,
Troxclair
AM,
McMillin
DJ,
Henry
CB.
1988.
Comparative
mutagenicity
of
nine
brands
of
coffee
to
S.
typhimurium
TA100,
TA102,
and
TA104.
Environ
Mol
Mutagen
11:
195­
206.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
64
Shinohara
M,
Thornalley
PJ,
Giardino
I,
Beisswenger
P,
Thorpe
SR,
Onorato
J,
Brownlee
M.
1998.
Overexpression
of
glyoxalase­
I
in
bovine
endothelial
cells
inhibits
intracellular
advanced
glycation
endproduct
formation
and
prevents
hyperglycemia­
induced
increases
in
macromolecular
endocytosis.
J
Clin
Invest
101(
5):
1142­
1147.

Smyth
HF,
Seaton
J,
Fischer
L.
1942.
The
single
dose
toxicity
of
some
glucols
and
derivatives.
J
Ind
Hyg
Toxicol
23:
259­
238.

Stipanuk
MH.
2000.
Biochemical
and
physiological
aspects
of
human
nutrition.
Philadelphia,
PA:
WB
Saunders.

Tada
A,
Wakabayashi
K,
Totsuka
Y,
Sugimura
T,
Tsuji
K,
Nukaya
H.
1996.
P­
postlabeling
analysis
of
a
DNA
adduct,
an
N2­
acetyl
derivative
of
guanine,
formed
in
vitro
by
methylglyoxal
and
hydrogen
peroxide
in
combination.
Mutat
Res
351:
173­
180.

Takahashi
M,
Okamiya
H,
Furukawa
F,
Toyoda
K,
Sato
H,
Imaida
K,
Hayashi
Y.
1989.
Effects
of
glyoxal
and
methylglyoxal
administration
on
gastric
carcinogenesis
in
Wistar
rats
after
initiation
with
N­
methyl­
N'­
nitro­
N­
nitrosoguanidine.
Carcinogenesis
10(
10):
1925­
1927.

Takayama
S,
Nagao
M,
Suwa
Y,
Sugimura
T.
1984.
Long­
term
carcinogenicity
studies
on
caffeine
instant
coffee
and
methylglyoxal
in
rats.
In:
MacMahon
B,
Sugimura
T,
eds.
Banbury
report.
Coffee
and
Health,
Symposium,
Oct.
1983.
Cold
Spring
Harbor,
NY:
Cold
Spring
Harbor
Laboratory
17:
99­
104.

Thornalley
PJ.
1990.
The
glyoxylase
system:
New
developments
towards
functional
characterization
of
a
metabolic
pathway
fundamental
to
biological
life.
Biochem
J
269:
1­
11.

Thornalley
PJ.
1996.
Pharmacology
of
methylglyoxal:
Formation,
modification
of
proteins
and
nucleic
acids
and
enzymatic
detoxification
 
a
role
in
pathogenesis
and
antiproliferative
chemotherapy.
Gen
Pharmacol
27(
4):
565­
573.

Thornalley,
PJ.
1998.
Glutathione­
dependent
detoxification
of
 ­
oxoaldehydes
by
the
glyoxalase
system:
involvement
in
disease
mechanisms
and
antiproliferative
activity
of
glyoxalase
I
inhibitors.
Chemico­
Biol
Interact
111­
112:
137­
151.

Thornalley,
PJ,
Langborg,
A,
Minhas,
HS.
1999.
Formation
of
glyoxal,
methylglyoxal,
and
3­
deoxyglucosone
in
the
glycation
of
proteins
by
glucose.
Biochem
J
344:
109­
115.

Tucker
JD,
Taylor
RT,
Christensen
ML,
Strout
CL,
Hanna
ML,
Carrano
AV.
1989.
Cytogenetic
response
to
1,2­
dicarbonyls
and
hydrogen
peroxide
in
CHO
AUXB1
cells
and
human
peripheral
lymphocytes.
Mutat
Res
224:
269­
279.

Ueno
H,
Segawa
T,
Nakamuro
K,
Sayato
Y,
Okada
S.
1989.
Mutagenicity
and
identification
of
products
formed
by
aqueous
ozonation
of
humic
acids
of
different
origins.
Chemosphere
19:
1843­
1852.
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
65
Ueno
H,
Nakamuro
K,
Sayato
Y,
Okada
S.
1991a.
Characteristics
of
mutagenesis
by
glyoxal
in
Salmonella
typhimurium:
Contribution
of
singlet
oxygen.
Mutat
Res
251:
99­
107.

Ueno
H,
Nakamuro
K,
Sayato
Y,
Okada
S.
1991b.
DNA
lesion
in
rat
hepatocytes
induced
by
in
vitro
and
in
vivo
exposure
to
glyoxal.
Mutat
Res
260:
115­
119.

Ueno
H,
Segawa
T,
Hasegawa
T,
Nakamuro
K,
Maeda
H,
Hiramatsu
Y,
Okada
S,
Sayato
Y.
1991c.
Subchronic
oral
toxicity
of
glyoxal
via
drinking
water
in
rats.
Fund
App
Toxicol
16:
763­
772.

U.
S.
Environmental
Protection
Agency
(
U.
S.
EPA).
1986.
Guidelines
for
carcinogen
risk
assessment.
Fed
Reg
51(
85):
33992­
34003.

U.
S.
EPA.
1988.
Recommendation
for
and
documentation
of
biological
values
for
use
in
risk
assessment.
Environmental
Criteria
and
Assessment
Office,
Office
of
Health
and
Environmental
Assessment,
Office
of
Research
and
Development.
EPA
600/
6­
87/
008,
NTIS
PB88­
179874/
AS.

U.
S.
EPA.
1991.
Guidelines
for
developmental
toxicity
risk
assessment.
Fed
Reg
56(
234):
63798­
63826.

U.
S.
EPA.
1993.
Reference
dose
(
RfD):
Description
and
use
in
health
risk
assessments.
Integrated
Risk
Information
System
(
IRIS).
Online.
Intra­
Agency
Reference
Dose
(
RfD.
Work
Group,
Office
of
Health
and
Environmental
Assessment,
Environmental
Criteria
and
Assessment
Office.
Cincinnati,
OH.

U.
S.
EPA.
1996a.
Proposed
guidelines
for
carcinogen
risk
assessment.
Fed
Reg
61(
79):
17960­
18011.

U.
S.
EPA.
1996b.
Guidelines
for
reproductive
toxicity
risk
assessment.
Fed
Reg
61(
212):
56273­
56322.

U.
S.
EPA.
1997.
Health
effects
testing
guidelines.
40
CFR
Part
798.
OPPTS
series
870.

U.
S.
EPA.
1998.
Guidelines
for
neurotoxicity
risk
assessment.
Fed
Reg
63(
93):
26926­
26958.

U.
S.
EPA.
1999.
Guidelines
for
carcinogen
risk
assessment.
Review
draft.
Office
of
Research
and
Development.
Washington,
DC.
NCEA­
F­
0644.

Uter,
W,
Schwanitz,
HJ,
Lessmann,
H,
Schnuch,
A.
2001.
Glyoxal
is
an
important
allergen
for
(
medical
care)
cleaning
ataff.
Int
J
Hyg
Environ
Health
204:
251­
253.

Vaca
CE,
Fang
JL,
Conradi
M,
Hou
S­
M.
1994.
Development
of
a
32P­
postlabelling
method
for
the
analysis
of
2'­
deoxyguanosine­
3'­
monophosphate
and
DNA
adducts
of
methylglyoxal.
Carcinogenesis15(
9):
1887­
1894.

Vander
Jagt,
DL,
Robinson,
B,
Tayleo,
KK,
Hunsaker,
LA.
1992.
Reduction
of
trioses
by
NADP­
Draft
 
Do
Not
Cite
or
Quote
(
July,
2003)
66
dependent
aldo­
keto
reductases.
J
Biol
Chem.
267(
7):
4364­
4369.

Whipple
EB.
1970.
The
structure
of
glyoxal
in
water.
J
Am
Chem
Soc
97:
7183­
7186.

Yamada
H,
Somiya
I.
1989.
The
determination
of
carbonyl
compounds
in
ozonated
water
by
the
PFBOA
method.
Oxone
Sci
Eng
11:
127­
141.