TO Dr. C. I. Ayres
Dr. D.G. Felton
Dr. R.E. Thornton
Dr. R.R. Baker
REF
[)PR/BTI-.1/114/466
FROM D.P. Robinson
DATE
13 th April, 1983
HIGH LEVELS OF IRRITANTS IN SIDESTREAM PLUMES
A paper by Ayer and Yeager W, - recently
referenced in STAB (No.
137/48), concluded that measured concentrations of
the irritants
formaldyhyde and acrolein in sidestream are up to
three orders of magnitude
above occupational limits. Typically,
concentrations up to 110 ppm for
To-rmaidehyde and up to 70 ppm for acrolein were
reported. In the
discussion of these results the authors point out
that formaldehyde
causes irritation at levels far below the
occupational limit and cauces
cancer in the nasal cavity of rats. Furthermore,
based on their experi-
mental data, they associate the irritants with the
particulate p-hase of
the sidestream and as a consequence imply that
formaldehyde could produce
bronchogenic carcinomas in man, rather than nasal
cancer, since smoke
particles are carried into the respiratory tract.
However, it is apparent that the "measured"
concenVations, which
lead to some rather bold statements, are based on
calculated properties
of the sidestream plume which are at variance with
data I have recently
published in RD 1890 (2). In particular Ayer and
Yeager measured the
velocity of the sidestream plume by two
independent techniques and
reported an average value of 0.2 m/s. Together
with a plume diameter of
2 mm this gave a calculated plume flowrate of 6 x
10-7 m3/s. Thi s
calculated flowrate was then subsequently used to
obtain the "measured"
concentration. With reference to RD 1890 it is
apparent that a single
valued velocity for the plume is a gross
ovor-simplifi-cation of the real
situation. The plume is part of a three
dimensional hydrodynamic field
which accelerates as it moves away from the
cigarette. Furthermore, the
diameter of the plume increases monotonically with
distance from the
cigarette. With both these factors taken into
account a typical plume
mass flowrate 3cm above the cigarette (Figure 16
RD 1890) would be
around 60 mg/s. This corresponds to 600 x 1P m3/s
for a plume
density of 1 kg/m3. This is 100 times greater than
that measured by
Ayer and Yeager and would give rise to a
corresponding reduction in the
plume concentrations. It can be argued that the
air entrained into the
plurne results in a substantial dilution of any
containinant being
transported by the plume.
Cont'd/...
00
LN
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2
& . Ve
It would appear that we are in a strong posftion
to refute the
observations made by Ayer and Yeager if the need
should arise. Perhaps
publication in the open literature of the data
reported in RD 1890 might
be opportune so that researchers are aware of the
features we have
observed.
Finally, as part of our sidestream investigation,
we have developed
a 3D mathematical model of the sidestream thermal
-hydraulics which appears
to be in excellent agreement with our experimental
data. The next stage
of model development will be the inclusion of
species concentrations
which will enable us to predict the contaminant
concentrations directly.
r alm_~ -
D.P. ROBINSON
ir
(1) Ayer, H.E. and Yeager, D.W.
American Journal Public Health (1982), Vol. 72,
No. 11, P. 1283-1285
Irritants in Cigarette Smoke Plumes
(2) Robinson, D.P.
RD 1890 Restricted (24.11.82)
Hydrodynamic Characteristics of the Cigarette
Sidestream Plume.
CO
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E U G E N 0 L
Author: Dr. P. M. M. Godden
Date: April 1983
4 W
DISTRIBUTION:
Dr. R. E. Thornton,
Dr. G. Smith,
Dr. E. D. Massey,
Mr. J. L. Beven.
PMMG/LAP/46D
13 April, 1983
Its"
and
Q)
LN
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EUGENOL
OCH3 Synonyms
4 allyl-2-methoxyphenol
4 allylguaiacol
2 methoxy-4-prop-2-enyl phenol
1-hydroxy-2-methoxy-4-allylbenzene
XH,-CH=
H2-CH=CH2
Physical properties: colourless liquid
strong odour of clove, pungent taste
Melting point -9.10C
Boiling point 1270C
Occurrence: Main constituent of several important
essential oils such as oil of clove,
clove stem and leaf, pimenta berry and
leaf, bay and cinnamon leaf, cinnamon
bark, oananga, calamus and ylang ylang
Use: Flavours, fragrance, disinfectant,
local anodyne in dentistry.
ir
ACUTE TOXICITY
The acute oral dose required to kill 50% of
treated animals
2.68 g/kg rat
3.00 g/kg mouse
2.13 g/kg guinea pig
(Hagan et al, 1965).
Oral doses of 150 mg eugenol to rats and guinea
pigs produced
histological damage consisting of desquamatation
of the
epithelium and punctate haemorrhages in the pylo
ric and glandular
regions of the stomach (Hartiala et al, 1966).
Degenerative
and reparative changes were observed in the*
gastric mucous
barrier after -application of a 5% eugenol
emulsion to the mucosa
of Heidenhain's pouches in dogs (Hollander and
Goldfischer,
1949; discussed by Opdyke, 1975). When the dose of
eugenol
given orally to rats was gradually increased to
4.00 g/kg
c0
____j
LA
(JI
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-2-
there was considerable mortality. Histological
examination of
the forestomach revealed moderately severe
hyperplasia and
hyperkeratosis of the stratified squamous
epithelium with
focal ulceration (Hagan et al, 1965; 1967; see
Opdyke, 1975).
Part of the effects in the stomach may have been
due to the
irritant action of eugenol or impurities found in
commercial
preparations (Webb and Bussell, 1981).
Rats treated with 4.00 g eugenol/kg (Hagan et al,
1967; see
Opdyke, 1975) exhibited a small degree of
osteoporosis, there
was also enlargement of the liver, liver cell .
size and the
adrenal glands. Four daily doses of 900 mg
eugenol/kg to rats
produced liver damage consisting of discoloration,
mottling and
blunting of the lobe edges (Taylor et al, 1964;
see Opdyke,
1975). Eugenol thus has some hepatotoxic activity.
Eugenol had no adverse effects on growth rate,
organ weight,
histology of any of the major tissues or
haemtology in rats fed
1% eUgenol in the diet (Hagan et al, 1967; see
Opdyke, 1975).
PHARMACOLOGICAL EFFECTS
Cardiovascular System.
Eugenol affects the peripheral aspects of the
cardiovascular
system. The heart is not the principal site of
action since
eugenol has little effect on the electrical
activity and only
slightly reduces the contractile force unless a
near fatal dose
is used (Sticht and Smith, 1971). The intravenous
injection of
eugenol (up to 0.5 ml) to mongrel dogs caused a
drop in arterial
blood pressure. Increased blood flow observed
after intra-
arterial and intravenous injections suggests that
the blood
CO
vessels are the.main site of action within the
cardiovascular
system. Large doses of eugenol (0.033-0.06 ml/kg)
may damage
CN
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-3-
capillary membranes as suggested by the release of
a bloody
exudate from the respiratory tract. stiffening of
the limbs
has been observed after large intra-arterial doses
of eugenol
to dogs (Sticht and Smith, 1971).
Nervous System
All concentrations between 0.1% and 100% eugenol
blocks the
transmission of evoked impulses in frog sciatic
nerve tissue
(Kozam, 1977).
Eugenol affects the central nervous system. It is
an
anaesthetic in mice and dogs at doses in excess of
300 mg/kg
(Sticht and Smith, 1971; Dallmeier and Calini,
1981). Larger
doses lengthen the sleeping time (Dallmeier and
Carlini, 1981).
Barbiturate sleeping time is increased by eugenol
(see Sell and
Carlini, 1976). Vomitting as long as two hours a
4er the oral
administration of eugenol to dogs (Lauber et al,
1950, see
Sticht and Smith, 1971) may have been the result
of local
irritation in the stomach or an effect on the
vomitting centre
in the brain.
Activity and Behaviour
Spontaneous motor activity was not affected in
mice at a
dose of 100 mg/eugenol/kg (de Mello et al, 1973
Eugenol has
a myorelaxant activity in mice at doses exceeding
50 mg/kg
(Dallmeier and Carlini, 1981). Rope climbing time
was depressed
by doses in excess of 160 mg/kg in rats. Some of
these animals
also showed paralysis of hindquarters which also
impaired
climbing performance (de Mello et al, 1973). Motor
co-ordination
is lost in dogs treated with 0.033-0.06 ml/kg
(Sticht and Smith,
1971). QJ-4
C)O
In mice eugenol (200 mg/kg) protects against
convulsion and
has acatatonic effect although the latter response
may have been
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-4-
non-specific because the animals were very
depressed (de Mello
et al, 1973; Dallmeier and Carlini, 1981).
Other aspects
Eugenol reduces body temperature in rats at doses
exceeding
50 mg/kg (Dallmeier and Carlini, 1981). An
increase in salivary
flow was observed in dogs injected wth 0.033-0.06
ml.eugenol/kg
(Sticht and Smith, 1971).
From quantum chemical calculations, the ability of
halluc-
inogenic amines to donate electrons has been
related to their
activity. No such relationship has been seen with
eugenol or
its analogues (discussed by de Mello et al, 1973).
THE MUTAGENICITY AND CARCINOGENICITY OF EUGEMOL
The potential mutagenicity and carcinogenicity of
both
4 K
eugenol and its metabolites are of particular
interest and
importance to the tobacco industry following
studies on a
chemically similar compound called safrole.
Safrole is not
mutagenic in the Ames Test using a wide range of
strains of
S. typhimurium in the absence of metabolic
activation (Dorange
et al, 1977; Swanson et al, 1979). Some mutagenic
activity
was observed in the presence of metabolic
activation (Green
and Savage, 1978). There is good evidence that
safrole is a
weak hepatocarcinogen (Miller et al, 1979). In
contrast eugenol
is non-mutagenic in several strains of S.
typhimurium with or
without the presence of metabolic activation
(Dorange et al
1977; Swanson et al, 1979). To date there has been
no evidence
to demonstrate a significant carcinogenic effect
of eugenol in
any species.
Eugenol may have some activity to promote skin
tumours in mice
c0
pretreated with the initiator
7,12-dimethylbenz(a)anthracene
U-4
cc
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-5-
(Van Duuren et al 1966; see Opdyke 1975). The
co-administration
of eugenol and benzo(a)pyrene to the skin of- mice
partially
inhibited benzo(a)pyrene carcinogenicity (Van
Duuren and
Goldschmidt, 1976). This may be due to the
-metabolism of
eugenol by, and thus reducing the availability of
enzymes
required for, the activation of benzo(a)pyrene to
the active
carcinogen. In a limited study in mice eugenol did
not potentiate
the tumorogenic Qffects of a known carcinogen
3-methylchol-
anthrene (Hitchco"ck, 1952; see Opdyke, 1975).
THE METABOLISM OF EUGENOL
Eugenol and other related allyl benzenes can be
metabolised
in the methoxy group or the allyl side chain The
methylene
dioxy group of safrole can be cleaved with loss of
the carbon
atom. The major metabolite is ally1catechol or its
isomer
propeny1catechol. Eugenol is a secondary
metabolite (Ioannides
et al, 1981). Demethylation of eugenol can also
occur in rats
(Weinberg et al, 1972) probably also resulting in
the formation
of ally1catechol. Little is known about the
mutagenic or
carcinogenic potential of these compounds.
There are some similarities between the metabolism
of the
allyl side chains of eugenol and safrole. Both
compounds - can
be converted to the respective 2',3'-epoxide and
dihydrodiol
(see Figure 1) by rat liver cells and both rat and
mouse liver
microsomes and are found in the urine and liver
homogenates of
rats pretreated with eugenol or safrole (Delaforge
et al,
1980). The epoxides of both compounds are
mutagenic in the
Ames Test (Swanson et al, 1979). The
2',3'-epoxides react
non-enzymically with guanosine at neutral pH
(Swanson et al, co
_1 J
L-J
\10
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-6-
1981). Guanosine is a riboside found in D.NA, the
genetic
material of the cell, and RNA. The 2',31-epoxide
of safrole
but not eugenol had DNA repair inducing activity.
DNA repair
inducing activity is greatest following the
addition to the In
vitro assay system of a direct acting carcinogen.
However, a
negative result does not mean that the compound is
not carcin-
ogenic or mutagenic (Francis et al, 1981).
Formation of 2',3'-epoxy eugenol is greater in
male than
female rats. Female mice exhibited a higher rate
of formation
of epoxide than male mice. The rate of epoxidation
of eugenol
in both species was much slower than that of
estragole or
safrole (Swanson et al, 1981). The metabolism of
estragole and
safrole to the 2',3'-epoxides and I'hydroxides
depends on
cytochrome P450 and the presence of NADPH (Swanson
et al, 1981).
ir
This has not been determined for eugenol.
The 2',3'-epoxides of eugenol, estragole and
safrole were
all very susceptible to the action of epoxide
hydrase in rat
and mouse liver microsomes resulting in the
formation of
dihydrodiols. The reactions could be inhibited by
the presence
of a cytochrome P450 inhibitor trichlo'ropropylene
oxide (Swanson
et al, 1981).
Safrole and estragole can be metabolized to a
l'-hydroxy
metabolite. l'-Hydroxysafrole forms covalently
bound adducts
with hepatic DNA, rRNA and protein in vivo in the
rat and mouse.
The I'hydroxy metabolites are weakly mutagenic,
carcinogenic,
can be metabolized to the I I -hydroxy, 2',
3'-epox ides which are
also weakly mutagenic and carcinogenic (Miller et
al, 1979).
The I'-hydroxy,2' 3'-epoxides are more resistant
to the action
of epoxide hydrase than the simple 2',3'-epoxides.
The hydroxyl
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-7-
vicinal to the epoxide ring confers resistance of
the epoxide
to hydrolysis by microsomal epoxide hydrase,
reduces the activity
as direct acting mutagens, but retains or enhances
the activity
of the compounds to initiate papilloma formation
in mouse skin
(Swanson et al, 1981). Esterification of
I'hydroxy-safrole and
estragole by rat and mouse liver cytosol yields
electrophilic
esters which can form hepatic DNA adducts (Swanson
et al, 1981)
and could be carcinogenic. Comparable metabolism
of eugenol
has not been established but could possibly occur.
Any
metabolites so formed might reflect the mutagenic
and carcin-
ogenic activities of the saf role and estragole
analogues. On the
other hand, an absence of metabolism of eugenol at
the l'
position of the allyl side chain might be
associated with its
lack of carcinogenicity in rats and mice. if
After a single oral administration of 200 mg of
eugenol to
rats, a large amount of ether-type glucuronide was
found in the
urine up to 24 hours later. In contrast there was
little change
in urinary content of the ester type glucuronide.
Eugenol
increased the specific activity of UDP
glucuronyltransf erase
(the enzyme which catalyses the conjugation of a
hydroxyl
metabolite of a drug with glucuronic acid). The
time course of
induction was similar to that of cytochrome P450
or any other
drug inducible metabolizing enzymes in liver
microsomes (Yuasa,
1974). Thus after treatment with eugenol the body
increases its
ability to metabolize the compound to a polar
conjugate which can
be more readily excreted.
co
THE EFFECT OF EUGENOL ON METABOLISM
Eugenol inhibited mouse liver microsomal
hydroxylation
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-8-
of dimethylaminopyrene and hexobarbital (Jaffe et
al, 1968).
This may have been due to the preferential use of
available
enzymes to metabolize eugenol.
Respiration of isolated mitochondria prepared from
rat
livers is inhibited by low concentrations of
eugenol. High
concentrations of eugenol uncoupled oxidation and
phosphory-
lation (Cotmore et al, 1979). These actions may
contribute to
the hepatotoxicity of eugenol. Noradrenaline
induced oxidative-
metabolism in hamsters isolated brown fat cells is
also inhibited
by eugenol (Petterson et al, 1980).
There have been no studies investigating the
metabolism of
eugenol administered by inhalation. Similarly
there are no
studies on the' effects of inhaled eugenol on
metabolism.
ir
00
r\J
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FIGURE 1
-9-
THE MgrABOLISM OF EUGENOL AND SAFROLE
OH
O-OH
CH2--CH=CH2
OH
O-OCH
3
CH2-M=CH2
eugenol
['2
I
0
3
CH2 2
-V
0
2',3'-epoxyeugenol
CH2-CH=CH2
Safrole
I
/CH2
0
0
--CH=CH2
1'hiroxysafroie
..* CH2
0
0
-CH=CH3
0- ester
dihydroxy-eugenol
3
CH21-'CH2
I
OH OH
CH2
?- I
CH2-'CH-<:H2
2 3 'frole
.,CH2
0 k
0
0
T-CH--CH2
OH
1 hydroxy~2 3 -epoxy
safrole
C)o
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_10-
REFERENCES
COTMORE, J.M., Burke, A., Lee, N.H. and Shapiro,
I.M. (1979)
Respiratory inhibition of isolated rat liver
mitochondria
by eugenol.
Arch. Oral. Biol., 24, 565-568.
DALLMEIER, K. and Carlini, E.A. (1981)
Anaesthetic, hypothermic, myorelaxant and
anticonvulsant
effects of synthetic derivatives and natural
analogues.
Pharmacology, 22, 113-127.
DELAFORGE, M., Janiaud, P. , Levi, P. and Morizot,
J.P. (1980)
Biotransformation of allylbenzene analogues in
vivo and
in vitro through the epoxide-diol pathway.
Xenobiotica, 10, 737-744.
DE MELLO, A.C., Carlini, E.A., Dressler, K.,
Green, J.P.,
Kang, S. and Margolis, S. (1973)
Behavioural observations on compounds found in
nutmeg.
Psychopharmacologia, 31, 349-366.
DORANGE, J.L., Delaforge, M. , Janiaud, P. and
Padieu, P.
(1977) 1
Pouvoir mutagene de me"tabolites de la voie
'epoxyde-diol du
dafrol et ' d 'analogues. Etude sur Salmonella
typhimurium.
C.R. Seanc. Soc. Biol., 171, C 1041-1048.
FRANCIS, A.A., Snyder, R.D., Dunn, W.C. and Regan,
J.D. (1981)
Classification of chemical agents as to their
4ability to
induce long- or short-patch DNA repair in human
cells.
Mut. Res. 83, 159-169.
GREEN, N.R. and Savage, J.R. (1978)
Screening of safrole, eugenol, their ninhydrin
positive
metabolites and selected secondary amines for
potential
mutagenicity.
Mut. Res. 57, 115-121.
HAGAN, E.C., Jenner, P.M. Jones, W.I., Fitzhugh,
O.G.,
Long, E.L., Brouwer, J.G. and Webbf W.K. (1965)
Toxic properties of compounds related to safrole.
Toxicol. Appl. Pharmacol., 7, 18-24.
HARTIALA, K.J.W., Pulkkinen, M. and Ball, P*.
(1966)
Inhibition of B-D-glucosiduronic acid conjugation
by
eugenol.
Nature, 210, 739-740.
IOANNIDES, C., Delaforge, M. and Parke, D.V.
(1981)
Safrole: Its metabolism, carcinogenicity and
interactions
with cytochrome P-450.
Fd. Cosmet. Toxicol. 19, 657-666.
JAFFE, H., Futii, K., Sengupta, M, Guerin, H. and
Epstein, S.S. (1968)
In vivo inhibition of mouse liver microsomal
hydroxylation
systems by methylenedioxyphenyl insecticidal
synergists and
related compounds. CDo
Life Sci. 7, 1051-1062.
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KOZAM, G. (1977)
Eugenol plays on the
Fd. Cosmet. Toxicol.,
nerves.
17, 175-176.
MILLER, J.A. Swanson, A.B. and Miller, E.C. (1979)
The metabolic activation of safrole and related
naturally
occurring alkenylbenzenes in relation to
carcinogenesis by
these agents.
In: Naturally occurring carcinogens-mutagens and
modulators
of carcinogenesis. Eds. E.C. Miller et al. Univ.
Park
Press, Baltimore, pp 111-125.
OPDYKE, D.J.L. (1975)
Monographs on fragrance raw materials, Eugenol.
Fd. Cosmet. Toxicol., 13, 545-554.
PETTERSSON, B., Curvall, M. and Enzell, C.R.
(1980)
Effects of tobacco smoke compounds on the
noradrenaline
induced oxidative metabolism in isolated brown fat
cells.
Toxicology, 18, 1-5.
SELL, A.B. and Carlini, E.A. (1976)
Anaesthetic action of methyleugenol and other
eugenol
derivatives.
Pharmacology, 14, 367-377.
STICHT, F.D. and Smith, R.M. (1971)
Eugenol: Some pharmacologic observations.
J. Dent. Res., 50, 1531-1535.
SWANSON, A.B., Chambliss, D.D. Blomquist, J..C.
Miller, E.C.
and Miller, J.A. (1979)
The mutagenicities of safrole, estragole, eugenol,
trans-
anethole, and some of their known or possible
metabolites
for Salmonella typhimurium mutants.
Mut. Res., 60, 143-153.
SWANSON, A.B., Miller, E.C. and Miller, J.A.
(1981)
The side chain epoxidation and hydroxylation of
the
hepatocarcinogens safrole and estragole and some
related
compounds by rats and mouse liver microsomes.
Biochim. Biophys. Acta, 673, 504-516.
VAN DUUREN, B.L. and Goldschmidt, B.M. (1976)
Cocarcinogenic and tumour-promoting agents in
tobacco
carcinogenesis.
J. Natl. Cancer Inst., 56, 1237-1242.
WEBB, J.A. and Bussell, N.E. (1981)
A comparison of the inflammatory response produced
by
commercial eugenol and purified eugenol.
J. Dent. Res.. 60, 1724-1728.
WEINBERG, J.E., Rabinowitz, J.L., Zanger, M. and
Gennaro,
A.R. (1972)
14 C-Eugenol: I. Synthesis, polymerization and
use.
J. Dent. Res., 51, 1055-1061.
CN
co
-
-Nj
_rIZI.
Qn
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-12-
YUASA, A. (1974)
Experimental studies on glucuronidation. III
UDP-glucuronyl-
transferase activity and glucuronide excretion
enhanced by
oral administration of eugenol.
Jap. J. Vet. Sci., 36, 427-432.
CO
01 \
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