SMOKE FLAVOURINGS
EXPLANATION
Smoke condensates and liquid smoke were considered at the
nineteenth meeting of the Joint FAO/WHO Committee on Food Additives
(Annex 1, reference 38). Inadequate information was available at that
time for an evaluation.
The present Committee reviewed both the specifications and safety
data for this group of products. It noted that smoke flavourings were
complex mixtures of varying composition, primarily prepared by the
condensation of smoke generated by pyrolysis of certain hardwoods in
the absence of or in the presence of a limited amount of air. Woods
commonly used for the preparation of smoke flavourings include oak
(Quercus spp.), hickory (Carya spp.), beech (Fagus spp.), alder
(Alnus spp.), and maple (Acer spp.). No toxicity data are
available on pyroligneous acid preparations which are condensates
derived from the pyrolysis of wood in the absence of air. The smoke
flavourings reviewed in this monograph are derived from the smoke
condensate of wood burned in a limited amount of air.
The initial smoke condensate separates into an aqueous phase and
a tarry phase. The smoke condensate may be separated into fractions by
physical separation techniques or solvent extraction. These fractions
may be further purified, if necessary, to remove hazardous
constituents known to be present in smoke. Smoke flavourings include
smoke condensates, fractions thereof, and mixtures of such fractions.
SMOKE FLAVOURINGS (AQUEOUS PHASE)
BIOLOGICAL DATA
Biochemical aspects
No information available.
Toxicological studies
Special study on carcinogenicity
An aqueous wood smoke flavouring, which previously had been shown
to be inactive in a Salmonella typhimurium mutation assay but
positive in an assay utilizing TK6 human lymphoblasts, was tested in
the mouse lung adenoma assay. In this study, newborn Swiss Webster
mice were injected with the smoke flavouring on days 1, 8, and 15, and
then maintained until 26 weeks of age. The total dose of test
substance administered ranged from 17.5 µl to 31.5 µl. The test
substance did not induce lung tumours or rumours at other sites.
Toxicity was observed at 15 weeks in some of the test animals, which
included hyperplastic kidneys and abnormalities in the colon and
rectum (Braun et al, 1986).
Special studies on mutagenicity
Smoke flavouring preparations were tested using Salmonella
typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 and in
E. coli WP2 (UVRA), both with and without metabolic activation, at
doses ranging from 3 to 10,000 µg/plate. Toxicity was observed at the
highest dose tested without activation. The preparations were not
mutagenic in these tests (Mortelmans & Eckford, 1980; Rattech, 1981).
An aqueous wood smoke flavouring which was not mutagenic in the
Salmonella typhimurium forward mutation assay (using strain TM677),
at concentrations up to 18 µg/ml (of dissolved solids), induced a
significant increase in mutation frequency of TK6 human lymphoblasts
(Braun et al., 1986).
Acute toxicity
No information available.
Short-term studies
Rats
Four groups, each containing 10 male and 10 female rats, were
maintained for 90 days on diets containing 0, 0.3, 2.5, or 20% of a
liquid smoke preparation (maple or hickory). All diets were adjusted
to contain 20% added water. Slight growth reduction was observed at
the high-dose level, but this was associated with decreased feed
intake. Haematologic and urine analyses at the end of the study showed
no abnormalities in any of the test groups. At autopsy, organ-weight
analyses of the principal organs and tissues showed no significant
changes in absolute organ weights. Gross and microscopic examinations
of these tissues showed no compound-related effects (WARF, 1961).
Three groups of 25 male and 25 female rats were maintained for 90
days on diets containing 0, 0.25, or 2.0% of one liquid smoke
flavouring or 2.0% of another smoke flavouring preparation. Growth and
food intake were similar among test and control groups. Haematologic
values at week 6 and at termination of the study were within normal
limits in all groups. At autopsy, organ-weight analyses and gross
pathology and histopathology of the principal organs were carried out.
No compound-related effects were observed, with the exception of minor
degenerative changes in the liver and kidneys in one of the 2.0% dose
groups and slight bone marrow hypoplasia in both 2.0% dose groups
(WRC, 1963; FCT, 1965).
Analytical studies on the preparation fed at 0.25 and 2.0% of the
diet showed the absence of 3,4-benzo(a)pyrene, but the presence of 5
non-carcinogenic polycyclic aromatic hydrocarbons, namely benzo(a)
anthracene, carbazole, chrysene, pyrene, and fluouranthene at levels
of 0.012, 0.20, 0.011, 0.013, and 0.032 ppm of the liquid smoke
preparation, respectively. The other preparation contained formic,
propionic, vanillic, and siringic acids, dimethylphenol, methyl
glyoxal, furfural, acetaldehyde, acetone, ethanol, and benzopyrenes
(< 1 ppb benzo(a)pyrene, 6 ppb pyrene, 3 ppb flouranthene, and 18 ppb
phenanthrene (Lijinsky & Shubik, 1965).
A series of studies was carried out with two liquid smoke
flavourings. The concentrations of total acids, phenols, and carbonyl
compounds in these preparations are outlined in Table 1.
Table 1. Constituents of liquid smoke 1 (LS 1) and liquid smoke
2 (LS 2)
Constituent LS 1 LS 2
Total acids (acetic acid) 1.8 g/l 2.0-2.8 g/l
Total carboxylic compounds 2.0 g/l 3.0-3.6 g/l
Phenols 0.018 g/l 0.9-1.3 g/l
PAHs (3,4-benzo(a)pyrene) -- 0.2 ppb
Five groups, each containing 6 male and 6 female Colworth Wistar
rats, were fed diets containing 1.25, 2.5, 5, 10, or 20% LS 2 or 20%
LS 1 for 28 days. Another group of 12 male and 12 female rats was fed
control diets. Body-weight gains of both male and female rats fed 20%
of either preparation, or 5 or 10% LS 2, were less than those of
controls. Plasma analysis at termination of the study showed sporadic
variation in a number of constituents, but these variations were not
dose-related. However, decreased plasma alkaline phosphatase was
observed at all dose levels in male rats and female rats in the
high-dose groups showed elevated thyroxine, (T4) and
triiodothyronine (T3) levels. Haematologic changes at the high-dose
level included decreased platelet counts in male rats administered LS
1 and in haemoglobin and red cell counts in male rats fed LS 2. At
autopsy, organ-weight analysis showed increased relative liver and
kidney weights in high-dose male rats fed LS 2 and increased thyroid
weight in high-dose female rats fed LS 2. Histopathological
examination of tissues and organs showed no compound-related effects,
with the exception of severe corticomedullary nephrocalcinosis in
females fed 20% LS 2 (Parish, 1986a).
In a paired feeding study, groups of 6 male and 6 female Colworth
Wistar rats were fed diets containing 2 or 20% LS 1 or LS 2. A control
group of 12 male and 12 female rats was fed control diet
ad libitum. Minor differences in weight gain were observed between
test animals and paired controls. At autopsy, serum chemistry values
of test animals and paired controls were similar. Organ-weight
analysis showed significantly, increased liver and kidney weights in
rats fed liquid smoke when compared to paired controls. Macroscopic
evaluation of organs and microscopic examination of the livers and
kidneys showed no compound-related effects (Parish, 1986b).
Three groups of 20 male and 20 female Carworth Wistar rats were
maintained on diets containing LS 2 for 13 weeks. The levels of liquid
smoke in the diet were adjusted biweekly, so that the intake in mg/kg
b.w. was constant. The levels of liquid smoke in the diet were
0.5-1.0, 1.5-3.0, and 3.0-6.0%, which were equivalent to daily intakes
of 0.6, 1.9, and 3.8 g/kg b.w./day for males and 0.7, 2.2, and
4.4 g/kg b.w./day for females, respectively. Additional groups at the
high-dose level and the control group were maintained for another 13
weeks on control diets. No animals died during the course of the
study. No significant differences in body weights were observed at
weeks 13 or 26, although there were significant reductions in
body-weight gain during weeks 4-8 in both male and female rats in the
high-dose group. At termination of the study, serum analysis showed a
number of changes in both sexes in the high-dose group. Haematologic
changes were also observed in this group, which included increased
haemoglobin and packed RBC volume in male rats and decreased WBC
counts and lymphocytes in females. Organ-weight analyses showed
significant increases in relative liver and kidney weights of male and
female rats in the high-dose group. Relative liver weights of female
rats were also increased in the 1% group. Histopathology of the
tissues and organs at week 13 of the study showed no treatment-related
changes. All the changes observed in rats at 13 weeks were no longer
apparent after the 13-week recovery period (Parish, 1986c).
In a study designed to investigate possible effects of smoke
flavouring on the thyroid, groups of 3 male and 3 female rats received
diets containing from 0.625 to 20% LS 1 for 28 days. Another group was
administered 0.025% 2-thiouracil in the diet, and another group of 6
male and 6 female rats was fed control diet. Body-weight gains of
female rats fed 20% liquid smoke were significantly lower than those
of control animals. Food intake of rats in the 2-thiouracil group was
significantly depressed. At termination, plasma studies, including
determination of thyroid hormones, were carried out. There were some
minor changes in plasma constituents in the high-dose group; however,
thyroid hormones were not affected, except in the 2-thiouracil group.
No significant histological changes were observed in any of the organs
of the rats fed LS 1 (Parish, 1986d).
In a study comparing the toxicity of two types of smoke
flavourings, 5 groups of 20 male and 20 female rats were maintained
for 90 days on diets containing 0, 0.3, or 3.0% of a basic aqueous
condensate or 0.025 or 0.25% of an oil soluble smoke flavouring
derived from the tar-like precipitate of a freshly prepared
condensate. Ten animals died during the course of the study, but death
was not related to the administration of test compound. Weight gain
was similar in test and control animals. No haematological changes
were observed at week 5 or at termination of the study. At termination
of the study, organ-weight analyses and gross pathological and
histopathological studies of the major organs showed no
compound-related effects (Eschenberger, 1963).
Long-term studies
No information available.
Observations in man
No information available.
LIQUID SMOKE FLAVOURINGS (TARRY EXTRACT)
BIOLOGICAL DATA
Biochemical aspects
No information available.
Toxicological studies
Many of the studies summarized here were carried out using a
liquid smoke flavouring preparation derived from the tarry fraction of
smoke condensate prepared from alder wood (Alnus incana). Diethyl
ether was used for the extraction process. Seventy-seven substances
have been identified in the smoke flavouring. The major components
were phenols and their alkylated derivatives (alkylated phenols,
guaicols, and siringols). PAHs were present below 10 ppb, and no
nitrosamines were detected (limit of detection, 5 ppb) (Miler, 1978).
Special studies on mutagenicity
A smoke flavouring derived from hickory was negative in the Ames
Salmonella microsome plate test, with or without activation, at
levels of 0.005 µl to 10 µl per plate (Jagannath & Brusick, 1979).
The smoke flavouring preparation from alder wood was tested using
Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, with
or without metabolic activation, at dose levels ranging from 28.3 to
2292 µg per plate. No mutagenic effects were observed (Jensen, 1986).
Acute toxicity1
Species Sex Route LD50 Reference
(g/kg b.w.)
Mouse (Swiss albino) M oral 2.8 Fitko, 1979a
F 2.3
Rat (Wistar) M 4.0 Fitko, 1979b
F 3.5
Pig M & F 3.6 Fitko, 1979c
1 The tested substance was the smoke flavouring preparation from
alder wood.
Short-term studies
Mice
Swiss albino mice were maintained for 90 days on diets containing
0, 0.1, 0.5, 1.0, or 2.0% of the smoke flavouring derived from alder
wood. The number of mice was 35 females and 25 males in each of the
control and high-dose groups and 12-14 of each sex in each of the
intermediate groups. Eleven animals died during the course of the
study. Deaths occurred in all groups and were not compound-related.
Body-weight gains in all but the lowest-dose animals were increased
over controls, and were associated with increased food intake.
Haematologic studies, urinalysis, and clinical chemistry studies at
weeks 6 and 13 showed no significant differences between the control
and high-dose animals. At autopsy, organ-weight analysis showed
significant increases in weights of the kidneys and thyroid.
Histological examination showed compound-related effects in the liver,
kidneys, gastrointestinal tract, lungs, and spleen in the test groups,
with the exception of the 0.1% group. The effects that were noted
included parenchymatous degeneration of the renal tubules of the
kidneys, oedema and hyperaemia of the spleen, parenchymatous
degeneration of the liver, and oedema and hyperaemia in the mucosa of
the stomach and the submucosa of the small intestine (Fitko, 1979d).
NOTE: The pathology terms used in this study and all the other
studies authored by Fitko that are summarized in this
monograph are those of the author. No criteria were
available for the terms used in the pathology reports, hence
complete evaluations of these studies are difficult because
of the lack of definitions of the terms used.
Rats
Four groups, each containing 15 male and 15 female Wistar rats,
were fed diets containing 0, 0.1, 0.3, or 1.01 of the tarry fraction
from a smoke flavouring preparation for 13 weeks. Weight gain was
significantly decreased in the high-dose group. At autopsy,
organ-weight analysis showed a significant increase in thyroid weight
of both male and female rats in the high-dose group. The thyroid
glands of animals in the high- and medium-dose groups were noticably
enlarged, compared to the controls. Histological examination indicated
dose-related changes in the thyroid, characterized by areas of atrophy
and large acini, a condition similar to colloidal goitre in man
(Hercules, 1977).
Five groups of equal numbers of male and female Wistar rats were
maintained for 90 days on diets containing 0, 0.1, 0.5, 1.0, or 2.0%
of the smoke flavouring derived from alder wood. The number of rats
was 35 of each sex in each of the control and high-dose groups and 15
of each sex in each of the intermediate groups. Haematologic, serum,
clinical chemistry, and urine analyses carried out at 0, 6, and 13
weeks showed no significant differences between the control and
high-dose animals. Body-weight gains were similar in test and control
animals, except for the high-dose group in which the females showed a
decrease, and males an increase, in body-weight gain compared to
controls. Food consumption was higher in the high-dose group than in
the controls. At autopsy, organ-weight analysis showed significant
increases in liver, kidney, and heart weights, except in animals in
the low-dose group. Macroscopic examination of the organs showed the
following compound-related changes; catharrhal inflamation of the
mucosa and small intestine and hyperaemia of the mucosa of the stomach
in dosed groups, with the exception of the low-dose group.
Histopathological examination revealed, in addition to changes in the
gastrointestinal tract, changes in the liver (parenchymal degeneration
and hyperaemia), kidneys (parenchymal degeneration of the renal
glomerulus), and lungs (hyperaemia). No significant effects were
observed in the low-dose group (Fitko, 1979e).
Pigs
Five groups, each containing 3 male and 3 female "large white"
pigs, were administered in the diet the smoke flavouring derived from
alder wood at dose levels equivalent to 0, 200, 600, 1000, or
1400 mg/kg b.w./day for 90 days. Each dose was given to the animals in
2 portions. Body-weight gains showed significant decreases with
increasing dietary concentration of the test compound. However, this
was associated with decreased food intake. Haematologic, serum,
clinical chemistry, and urine analyses of the control and high-dose
animals at weeks 0, 6, and 13 showed no consistent compound-related
effects, with the exception of a dose-related increase in serum
bilirubin. A liver function test (BSF retention) of high-dose and
control animals at week 13 showed no significant differences. At
autopsy, organ weight analysis showed no compound-related effects
except for the liver and the uterus. In the case of the liver, there
was a significant decrease in weight in the lower-dose groups, and an
increase in weight in the high-dose groups. The uterus showed a
dose-related increase in weight. Macroscopic examination of the organs
showed inflammation of the stomach and intestines, as well as changes
in the kidneys and lungs. Histological examination of the tissues
showed dose-related changes in the gastrointestinal tract, ranging
from inflammation to prominent proliferation of lymphoid tissue into
the submucosa of the stomach and small intestines and oedema of the
mucosa in these organs. In addition, parenchymal degeneration of the
liver and renal glomeruli and hyperaemia in the liver and lungs were
observed. At the lowest dose fed, the only significant effects were
inflammation of the stomach mucosa and lymphoid cell infiltration in
the submucosa (Fitko, 1979f).
In another study, four groups of 3 male and 3 female pigs were
fed diets containing the smoke flavouring derived from alder wood for
52 weeks at doses equivalent to 0, 200, 600, or 1000 mg/kg b.w./day.
The daily dose was given to the test animals in two portions. There
were no compound-related deaths during the course of the study. There
was a dose-related decrease in mean body weights, and this was
associated with decreased feed efficiency. Haematologic studies, serum
clinical chemistry studies, and urinalyses were performed at 0, 1, 3,
6, and 12 months on animals in the control and high-dose groups. Serum
cholesterol and direct bilirubin showed dose-related increases in the
test animals. There were sporadic variations in haematologic
parameters and certain serum enzymes (aspartate aminotransferase and
alkaline phosphatase). Liver function tests (BSP retention) at 0, 6,
and 12 months were similar at all test periods. At termination of the
study, organ-weight analysis showed significantly increased relative
weights of the liver and brain and decreased relative weights of the
thymus in the high-dose group, and increased relative kidney weights
in the intermediate group. Macroscopic examination of the organs
showed inflammation of the gastrointestinal tract of all treated
animals, and liver necrosis in the high-dose group. Histopathology
studies showed changes ranging from inflammation of the
gastrointestinal tract in all animals to liver necrosis in the
high-dose group. Effects on the kidneys, which included parenchymal
degeneration, hyperaemia, and necrosis, appeared to be most severe in
the high-dose group (Fitko, 1979g).
Long-term studies
Mice
Groups of equal numbers of male and female Swiss albino mice were
maintained for 16 months on diets containing smoke flavouring derived
from alder wood at dose levels equivalent to 0, 150, 920, or
1650 mg/kg b.w./day for females, and 0, 150, 760, or 1720 mg/kg b.w./
day for males. The control group and highest-dose group consisted of
60 mice of each sex, and the intermediate doses consisted of 25 mice
of each sex. One hundred thirty-three of the 340 animals died during
the course of the study. There were no significant differences between
survival in control and test groups. Gross pathology of 68/133 mice
autopsied showed inflammation of the gastrointestinal tract in 34
animals and liver degeneration in 33 animals. These and other
pathological changes were not dose related. No rumours were found in
animals dying before sacrifice. Weight gain was slightly reduced in
the high-dose group compared to the control and appeared to be
associated with a decrease in feed efficiency. Haematology, serum,
clinical chemistry studies, and urinalysis were carried out at 1, 3,
6, 12, and 16 months. There were no significant compound-related
changes. At termination of the study, organ-weight analysis showed an
increase in liver weights and a decrease in thyroid weights. Changes
in weights of other organs appeared to be sporadic and not dose
related. Histological studies showed a dose-related increase in the
frequency of fatty degeneration of the liver, with liver necrosis in
the highest-dose group. Inflammation of the gastrointestinal tract was
observed in all groups. Other effects observed in the high-dose group
included lymphocytic infiltration in the adrenals and lungs and
hypertrophy of mucosa in the uterus. The number of test animals with
rumours did not differ significantly from the number in the control
group (the total number of animals examined in each group ranged from
19 to 24) (Fitko, 1979h).
Rats
Groups of rats were maintained for 24 months on test diets
containing smoke flavouring derived from alder wood at dose levels
equivalent to 0, 48, 260, or 630 mg/kg b.w./day for males and 59, 360,
or 850 mg/kg b.w./day for females. The high-dose group consisted of
118 male and 117 female rats, the intermediate groups of 45 male and
45 female rats, and the control group of 117 males and 118 females.
During the study 201 animals died; mortality was equally distributed
among all groups. Lung disease followed by inflammation of the
gastrointestinal tract and degeneration of the liver were listed as
causes of death. No rumours were reported. Weight gain was lower in
all test groups compared to controls and was associated with decreased
feed efficiency. Haematology, serum clinical chemistry analyses, and
urinalyses were carried out at 1, 3, 12, 18, and 24 months. There were
sporadic changes in various parameters, but these were not dose
related, and were within normal ranges. At termination of the study
organ-weight analysis showed weight increases of the uterus, thyroid,
lungs, and kidneys in females in the high-dose group, and in the
hypophysis and thymus of males in the high-dose group. Histological
studies of the tissues showed inflammation of the gastrointestinal
tract, and necrosis of the liver and kidney in the two high-dose
groups. There were no significant differences in the frequency
distribution of any type of observed neoplasms between test and
control animals. The data were evaluated using the methods described
by Peto (1974) (Fitko, 1979i).
Observations in man
No information available.
COMMENTS
Toxicity data are available for liquid smoke flavourings derived
from the aqueous and tar fractions.
The fractions were not mutagenic in bacterial systems
(Salmonella typhimurium and E. coli strains), with or without
activation. However, one liquid smoke preparation (aqueous fraction)
which was inactive in the Salmonella test caused a significant
increase in mutation frequency in the TK6 lymphoblast system. This
compound was not positive in a lung rumour biossay system utilizing
newborn mice, although toxic effects were observed.
Short-term studies were available for several types of liquid
smoke flavourings (aqueous extract). The major adverse effects noted
were significant increases in liver and kidney weights of the test
animals. In one study, these changes were shown to be reversible. Only
minor histological changes were reported in these animals. Minor
changes in serum chemistry have also been reported. In these studies
the no-effect level ranged from 0.25% to 3% of the liquid smoke
preparation in the diet. It was also noted that in some studies there
were significant increases in thyroid weights in the high-dose groups.
However, changes in serum levels of T3 and T4 were reported in
only one study.
Almost all the studies with smoke flavourings (tarry extract)
relate to a single product. This product was tested in a number of
species (mice, rats, and pigs). The tests include long-term studies in
mice and rats. The major effects observed in the high-dose groups, in
all species tested, were decreased body-weight gain and increased
liver and kidney weights. Histopathological changes were primarily
related to the gastrointestinal tract, liver and kidneys that included
inflammation of the gastrointestinal tract and necrosis of the liver
and kidney. These effects were observed in the groups fed the highest
levels of the test material, and were minimal or absent in the
low-dose groups. In lifetime feeding studies in mice and rats, there
were no compound-related increases in rumour incidence or type in the
treated animals. However, the lack of any neoplastic foci of the liver
in rats or mice is unusual in studies of this duration. A complete
evaluation of these studies is difficult because of the lack of
definition of the terms used in the pathology report. High levels of
smoke flavourings (tarry extracts) in the diets of rats also resulted
in a significant increase in thyroid weights, and in one case
histological changes were observed.
EVALUATION
The Committee viewed the use of smoke flavourings generically,
keeping in mind that smoke flavourings are a replacement for
traditional smoking practices, and as such they represent a definite
improvement in that a large number of potentially toxic compounds are
eliminated in their production. A similar view has been expressed by
the Council of Europe (Resolution AP185/2).
The Committee concluded that so complex a group of products might
not be amenable to the allocation of an ADI, and that smoke
flavourings of suitable specifications could be used provisionally to
flavour foods traditionally treated by smoking. However, as the safety
data for these products were limited, new or novel uses of smoke
flavourings should be approached with caution.
The Committee concluded that detailed information on the
production and composition of smoke flavourings is required, and that
it would be desirable to have further safety studies on a well-defined
spectrum of smoke flavourings.
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