CAROTENES FROM NATURAL SOURCES (ALGAL AND VEGETABLE)
First draft prepared by
Dr G.J.A. Speijers
National Institute of Public Health
and Environmental Protection
Laboratory for Toxicology, Bilthoven, The Netherlands
1. EXPLANATION
Carotenes from natural sources were reviewed at the eighteenth,
thirty-first and thirty-fifth meetings of the Committee (Annex 1,
references 35, 77 and 88). At its thirty-first meeting, the
Committee noted that, while there was a substantial toxicological
database relating to carotenes and an ADI had been established for
synthetic ß-carotene, the same ADI was not applicable to natural
carotenes as they did not comply with the specifications for
ß-carotene.
At the thirty-fifth meeting, the Committee concluded that there
was insufficient evidence to indicate that data relating to one
species of Dunaliella algae could be applied to others and that
the specifications of the test materials were so different from one
another that the results of the toxicity tests could not be
generalized. There were insufficient data to evaluate any of these
materials for the purpose of establishing an ADI. The Committee
concluded that carotene isolated from algal sources would be
acceptable for food additive use if it was of sufficient purity to
meet the specifications for synthetic ß-carotene. Acceptance of
algal biomass or crude extracts of carotene from algal sources for
use as food additives would be contingent on the provision of
evidence of the safety of such materials.
At its thirty-fifth meeting, the Committee considered limited
short-term toxicological studies on material stated to have been
prepared from three different algal species designated Dunaliella
bardawil, D. salina and D. kona (Annex 1, reference 88). At its
present meeting, the Committee was informed that Dunaliella
bardawil, D. kona and D. salina were identical, and that,
according to current nomenclature, the species used commercially was
Dunaliella salina. Some of the preparations produced from this
species were dehydrated powders prepared by lyophilization or spray-
drying and others were vegetable oil extracts.1
1 Although the Committee acccepts that this report does not cover
different Dunaliella species, the studies are summarized with
original names mentioned in the reports or publications
submitted.
With respect to the carotene preparations derived from
extraction of vegetables, mainly carrots, alfalfa or vegetable oil,
the Committee at the thirty-first meeting (Annex 1, reference 77)
felt that the need for toxicity tests may be obviated if detailed
analytical data were supplied to confirm that natural toxicants
occurring at low levels in food/feed stuffsare not concentrated in
the extract and that levels of use would not materially exceed the
levels of exposure that would result from normal use.
As no toxicological monograph has been prepared previously by
the Committee on carotenes from natural sources, data that have been
reviewed at previous meetings are incorporated in the present
monograph along with the new data that have become available.
Because this monograph covers the data on both algal and vegetable
carotene preparations, a modified form of the general monograph
format has been used, summarizing in order biological data on spray
dried concentrated, lyophilized or dehydrated preparations of
Dunaliella, then data on vegetable oil extracts of Dunaliella,
then data on carotene extracts from carrots, grass alfalfa and
vegetable oil.
ALGAL CAROTENE PREPARATIONS
SPRAY DRIED CONCENTRATED, LYOPHILIZED OR DEHYDRATED
PREPARATIONS OF DUNALIELLA SALINA
(syn DUNALIELLA BARDAWIL and DUNALIELLA KONA)
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion.
Male weanling CD rats were fed ad libitum a retinol-deficient
diet. After 60 days, the retinol content of the livers was 4-5 µg.
Depleted rats were allocated to 9 groups of 6 animals, housed
individually, and fed a retinol-deficient diet supplemented as
follows: Group 1, retinol at 7.5 mg/kg diet; Groups 2-4, all- trans
ß-carotene at 12, 29 or 48 mg/kg diet respectively; Groups 5-7,
lyophilized Dunaliella contributing 29, 58 or 112 mg ß-carotene/kg
diet respectively; Group 8, maize oil extract of Dunaliella
providing 16 mg ß-carotene/kg diet; and Group 9, no supplementation.
After seven days repletion, livers were taken for analysis for
retinol, retinol isomers and ß-carotene. The liver analyses
revealed a comparable content of retinol related to dose of
carotene, irrespective of source whether synthetic, algal biomass or
algal oil extract. Rats fed the algae or the algal extract-
supplemented diets accumulated 9- cis retinol in addition to the
all- trans isomer. Rats fed synthetic ß-carotene, lyophilized
algae or algal oil had a liver retinol:ß-carotene ratio of about
3:1. Rats fed algae or algal oil accumulated 9- cis ß-carotene and
all- trans ß-carotene in the liver in a ratio similar to that
present in the algae. It was concluded that dried Dunaliella
bardawil or an oil extract of the alga can serve as a dietary
natural ß-carotene source which can satisfy the total requirement of
retinol in rats (Ben-Amotz et al. 1988). [Note: this study is
again cited below in discussion of absorption of oil-extracted algal
carotene]
A 45-day-feeding study with 21-day old Sprague-Dawley rats was
performed to compare the bioavailability of four sources of
ß-carotene; Spray dried Dunaliella salina (0.61 % ß-carotene),
ß-carotene oil extract of Dunaliella salina (24.83 % ß-carotene),
oleoresin of carrots (11.5 % ß-carotene) and synthetic ß-carotene
(100 %). The diet with Dunaliella salina powder (59 g/kg diet)
was corrected for the amount of sucrose. The diets were made equal
in the percentage of ß-carotene, and the control diet contained
0.036 % ß-carotene.
The rats on the Dunaliella salina diets grew more rapidly.
Gross macroscopy at autopsy revealed no alterations in the rats.
The bioavailability of Dunaliella salina as a source of
ß-carotene and oil extract of ß-carotene from Dunaliella salina
was higher than that of ß-carotene from oleoresin of carrots or
synthetic ß-carotene. These results may be due to the presence of
the extra lipids in both the dried Dunaliella salina which
contains 8.6 % lipid and the oil extract of Dunaliella salina.
These results lead to the recommendation that dried Dunaliella
salina, when used as a source of ß-carotene, should be consumed in
oil to increase ß-carotene bioavailability. This is because
activity of carotene dioxygenase, the enzyme responsible for the
conversion of ß-carotene to vitamin A, is increased in the presence
of oil (Ghazi et al., 1992).
In one experiment, groups of 1-day-old white Leghorn chicks
received a retinol-deficient semi-purified diet or a similar diet
supplemented with 8.04 mg retinol/kg diet, or 30 mg synthetic
ß-carotene/kg diet or lyophilized D. bardawil at 1 g algae/kg
diet. The algal powder contained 30 g ß-carotene and 200 g NaCl/kg
and provided 30 mg ß-carotene/kg diet. In a second experiment, four
similar groups were used except that the lyophilized algal powder
was replaced with a similar concentration of drum-dried algae. The
drum-dried algal powder contained 34 g ß-carotene, 260 g NaCl, 180 g
glycerol, 5.5 g chlorophyll, 210 g protein, 170 g carbohydrate and
120 g lipid/kg (34 mg ß-carotene/kg diet). A third experiment
utilized three groups of 15 one-day-old chicks which received
retinol-deficient diet alone, or supplemented with lyophilized
D. bardawil at 0.58 g/kg diet, or with drum dried algae at 1 g/kg
diet. In each experiment, the chicks were assessed visually and
weighed daily for 5 weeks and at termination serum and liver were
analysed for retinol, ß-carotene and lutein.
After an initial lag, the chicks grew equally well on diets
containing retinol, ß-carotene or algae in all experiments. Serum
and liver concentrations of retinol were normal in all cases except
for the chicks receiving retinol-deficient diets without
supplements. The serum of chicks fed the algal-supplemented diets
contained lutein but no ß-carotene although the ratio of ß-carotene
to lutein in the algae exceeded 15:1.
In a separate experiment, two groups of 3 egg-laying hens
received a control diet containing 150 g maize meal/kg or the same
diet supplemented with 4 g lyophilized D. bardawil/kg. The algal
preparation contained 50 g ß-carotene and 300 g NaCl/kg (200 mg
ß-carotene/kg diet). Eggs from these hens showed an enhanced yolk
colour attributable to lutein; no ß-carotene was present in the egg
yolk (Ben-Amotz 1986).
2.1.2 Biotransformation
No information available.
2.1.3 Effects on enzymes and other biochemical parameters
No information available.
2.2 Toxicological studies
2.2.1 Acute toxicity studies
2.2.1.1 Mice
Acute toxicity of Dunaliella bardawil spray dried powder was
established in an LD50 test with mice. A single dose of 2.5, 5.0
or 10.0 g Dunaliella bardawil/kg diluted with CMC-Na solution was
administered by oral intubation. The observation period was 14
days, and mortality, general symptoms, body weights and gross
necropsy examination were recorded. The LD50 value was greater
than 10 g Dunaliella bardawil/kg for both male and female mice
(Aruga, 1987).
2.2.1.2 Rats
Male Sprague-Dawley rats were given dried Dunaliella by
gavage at a dose of 5 g/kg bw and observed for the subsequent 14
days. There were neither mortalities nor overt signs of toxicity
and all the rats gained weight during the observation period (Lock
1985).
2.2.2 Short-term toxicity studies
2.2.2.1 Mice
In the search for an antioxidative-anticarcinogenic substance,
the effects of repeated ingestion (no dose level given) of spray
dried Dunaliella bardawil on mammary growth and endocrine
parameters were examined in mice. In an additional group the mice
received also the vitamin A-deficient synthetic standard diet
supplemented with synthetic all-trans ß-carotene, whereas the
control animals received the normal synthetic diet adequate in
vitamin A. The concentration of ß-carotene in both test diets was
0.55 mg/kg. The ingestion of the Dunaliella bardawil-containing
diet between 20 and 120 days of age showed no deleterious side-
effects on mammary gland and uterine growth nor mammatrophic hormone
secretion, these results were similar to previously-observed results
in aged and mammary tumour-bearing mice. Puberty and body growth
were accelerated by Dunaliella bardawil compared to the synthetic
all-trans ß-carotene (Nagasawa et al., 1989).
2.2.2.2 Rats
Two groups of 10 male and 10 female weanling Sprague-Dawley
rats, caged individually, were given powdered diets containing 0 or
10% algal ß-carotene powder for 12 weeks. Body weight gain and food
intake were recorded at intervals up to and at termination when the
animals were autopsied. Weights of heart, lungs, liver, kidneys,
spleen, gonads and adrenals were determined at autopsy and blood was
collected for determination of serum glucose, ASAT, ALAT, alkaline
phosphatase, uric acid, BUN, triglycerides and cholesterol. One
male rat in the treated group died during the study from
"non-specific problems" not related to treatment.
Significant differences were observed in neither food intake
nor body weight gain between treated and untreated animals of either
sex and there were no treatment-related differences in organ
weights. Except for one treated male which displayed elevated ASAT
and ALAT levels, no significant differences were observed in any of
the clinical biochemical parameters between treated and untreated
animals of either sex. No histopathological examination was
performed (Majnarich 1988).
In a 28-day toxicity study spray dried Dunaliella bardawil
powder was orally administered to rats (5/group/sex) at dosages of
0.5 and 2.5 g Dunaliella bardawil/kg bw/dy. The control animals
(only) received 0.5 % aqueous solution of sodium CMC, which was used
as the vehicle for the preparation of the test article suspension.
Toxicological parameters recorded included food consumption, body
weight gain, urinalysis, ophthalmoscopy, haematology, serum
biochemistry, organ weights and histopathological examination.
In the males of the 2.5 g Dunaliella bardawil/kg bw/dy group
a significant increase in the relative weight of the kidneys was
noticed. Except slight changes in the thymus and the kidneys in a
few animals of the 2.5 g Dunaliella bardawil/kg bw/dy group, no
histopathological changes were reported. Although the effects were
observed mainly in the kidneys, it was suggested by the authors that
the dosage of 2.5 g Dunaliella bardawil/kg bw/dy was a NOEL
(Furahashi, 1989).
2.2.3 Long-term toxicity/carcinogenicity studies
No information available.
2.2.4 Reproduction studies
The safety of the alga Dunaliella bardawil for food use was
evaluated in a multigeneration study with rats. Four generations
were raised on diets containing 0, 50 and 100 g/kg of dehydrated
D. bardawil. The caloric value of the diets with the Dunaliella
bardawil preparations was adapted by lowering the amount of
starch. Each experimental group comprised 10 males and 20 females.
Starting with an F0-generation 3 other generations (F1 - F3)
were raised. The rats of the F0-generation were kept on the
different diets for 1 year and 5 male and 5 female rats were studied
for general toxicological effects. No significant differences were
observed between the rats consuming algae and the controls, of any
generation, in general appearance, behaviour, growth, reproductive
performance or gross pathology. The only effect of D. bardawil
powder observed was a significantly increased relative kidney
weights. The blood chemistry and haematology of the
first-generation animals, after 1 year on the diets, showed no
appreciable differences between the experimental and control
animals. The only differences in histopathology observed were a
decrease in some chronic inflammations, a slightly higher frequency
of metaplasia of the renal pelvis epithelium with ectopic
nephrocalcinosis in the renal papillae and an increased frequency of
focal bronchopneumonia in rats fed 10 g algae/kg feed when compared
with the controls. The latter effect may be attributed to the
powdery nature of the algal diet. Although at dose levels effect
were recorded on the kidneys and which were not explained, the
authors concluded that this multigeneration feeding study may be
indicative of the safety of D. bardawil for human consumption
(Mokady et al., 1989).
2.2.5 Special studies on genotoxicity
A mutagenicity study (Ames test) in Salmonella typhimurium TA
98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA both
with and without activation by a liver microsomal S-9 mix was
performed at dose levels of 312.5, 625, 1 250, 2 500 and 5 000 µg
Dunaliella bardawil paste/plate. Dunaliella bardawil was not
mutagenic in any strain (Aruga, 1988).
2.3 Observations in humans
Nine subjects were maintained on a low-carotene diet for two
weeks and serum carotene levels were then determined. For the next
ten days the volunteers took a daily dose of the powdered algal
preparation providing 75 000 IU ß-carotene (approx 135 mg) in
capsule form. Serum carotene was measured on days 7 and 10 of
treatment. There was considerable interindividual variation in
response to the same dose of carotenes, both in absolute values and
in the treatment-dependent increase in serum concentration of
carotene. In six of the subjects the serum level of carotene
continued to rise between the seventh and tenth day of the study
while in three others there was a slight fall in this period. One
subject with normal serum carotene levels at the outset showed
virtually no response to treatment. No adverse effects due to
ingestion of the algal preparation were reported (Cyanotech, 1988).
ALGAL CAROTENE PREPARATIONS
VEGETABLE OIL EXTRACT OF DUNALIELLA SALINA
(syn DUNALIELLA BARDAWIL and DUNALIELLA KONA)
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion.
In an experiment fully described in section 2.1.1 describing
results with spray dried concentrated, lyophilized or dehydrated
preparations of Dunaliella, male weanling CD rats were fed a
retinol-deficient diet ad libitum. After depletion, rats were
allocated to groups and fed a retinol-deficient diet supplemented
with one of: retinol; all- trans ß-carotene; lyophilized
Dunaliella; maize oil extract of Dunaliella; and no
supplementation. After seven days repletion, livers were taken for
analysis of retinol, retinol isomers and ß-carotene. The liver
analysis revealed a comparable content of retinol related to dose of
carotene, irrespective of source i.e. synthetic, algal biomass or
algal oil extract. It was concluded that dried Dunaliella bardawil
or an oil extract of the alga can serve as a dietary natural
ß-carotene source which can satisfy the total requirement of retinol
in rats (Ben-Amotz et al. 1988).
2.1.2 Biotransformation
No information available.
2.1.3 Effects on enzymes and other biochemical parameters
No information available.
2.2 Toxicological studies
2.2.1 Acute toxicity studies
No information available.
2.2.2 Short-term toxicity studies
No information available.
2.2.3 Long-term toxicity/carcinogenicity studies
No information available.
2.2.4 Reproduction studies
No information available.
2.2.5 Special studies on genotoxicity
The commercial, carotene-rich corn oil extract of Dunaliella
salina was inactive in an in vitro primary hepatocyte
unscheduled DNA synthesis assay (Cifone 1987).
The extract was negative in an assay of forward mutation at the
HGPRT locus in cultured Chinese hamster ovary cells, with or without
metabolic activation with rat liver S9 fraction. A dose-related
cytotoxicity was noted at concentrations above 2.0 µl/ml without S9
and above 10.0 µl/ml in the presence of S9 (Young 1987).
The material was not mutagenic in the Salmonella/microsome
assay (Ames test) with Salmonella typhimurium strains TA-1535,
TA-1537, TA-1538, TA-98 and TA-100 with or without metabolic
activation (Jagannath 1987).
In an in vivo mouse micronucleus assay using adult ICR mice,
the commercial carotene extract did not induce a significant
increase in micronuclei in bone marrow polychromatic erythrocytes
(Ivett 1987).
2.3 Observations in humans
After a depletion period of 10 days on a low-carotene diet, 12
male and 20 female healthy adults were randomly assigned to one of
five treatment groups. Two groups received capsules of carotene
obtained by vegetable oil extraction of Dunaliella salina
providing ß-carotene at levels of 8 or 24 mg and alpha-carotene at
levels of 1.1 or 3.2 mg respectively. Two further groups received
carrots (69.1 or 207.3 g respectively) that provided a similar
amount of ß-carotene to the Dunaliella salina extract groups; the
corresponding amounts of alpha-carotene were 6.3 and 18.9 mg
respectively. A fifth group received placebo capsules. The
subjects received the treatment for seven days and then underwent
another depletion phase of 7 days.
Treatment with carotene capsules or carrots led to an expected
increase in serum alpha- and ß-carotenes, with the higher dose
treatments being less efficient per mg carotene consumed. The
encapsulated algal carotenes were more efficient at raising serum
values per mg fed, consistent with other reports that carotenes are
better absorbed from oily solution than a vegetable matrix (Jensen
et al. 1985).
In a study on the bioavailability of cis- and trans-ß-
carotenes, 16 healthy adults, who had been on a low-carotene diet
for ten days, were fed either ß-carotene extracted from Dunaliella
salina alga, containing approximately equal amounts of all-trans-
ß-carotene and 9-mono-cis-ß-carotene, or ß-carotene in the form of
fresh carrots containing predominantly trans-ß-carotene, or avocado
oil-placebo capsules. Subjects were randomly divided into three
groups: they consumed daily in a single dose either 3 ß-carotene
capsules (24 mg ß-carotene), 207.3 g carrots (24 mg ß-carotene); or
3 ß-carotene free placebo capsules for seven days. HPLC
determinations of serum trans-cis ß-carotene ratios showed trans
ß-carotene to be the predominate serum isomer before and during all
treatments. Serum trans-ß-carotene concentrations were
significantly increased in the ß-carotene capsules and carrot
groups. Cis-ß-carotene concentrations were increased in the carrot
and placebo groups. However, the serum isomer increments for those
taking ß-carotene capsules and carrots strongly favoured trans-
ß-carotene over cis-ß-carotene. These data demonstrate a
predominant absorption of intact trans-ß-carotene over intact cis-
ß-carotene into human serum even when approximately equivalent
amounts of these isomers were ingested. This selective absorption
of intact ß-carotene isomers might be a factor in their biopotency
in humans (Jensen et al., 1987).
CAROTENE EXTRACTS FROM VEGETABLES
(Carrots, alfalfa and vegetable oil)
2. BIOLOGICAL DATA
2.1 Biochemical aspects
No information available.
2.2 Toxicological studies
No information available.
2.3 Observations in humans
The acute effects of consuming alpha- and ß-carotene from
carrots on serum alpha-carotene and ß-carotene levels were
investigated in 17 adult subjects (18-58 years of age). After a
10-day low-carotene diet, the subjects were randomized into three
groups based on day 6 ß-carotene levels. On day 11, fasting
baseline blood was drawn. Either 3 carrots, 1 carrot or 3 placebo
capsules were then consumed following a low-carotene breakfast.
Blood was drawn 1, 2, 3, 4, 5, 7 and 24 hours post-treatment and
alpha- and ß-carotene levels were determined by HPLC. Treatment of
3 carrots yielded significantly greater peak alpha- and ß-carotene
levels in serum at 5 hours post-treatment than did treatments with 1
carrot or 3 placebos. These results suggest the best condition for
drawing blood samples to assess the serum carotene status of adults
is at fasting state and that significant alterations in serum can
occur within 5 hours of a carotene rich meal (Jensen et al.,
1986).
3. COMMENTS
Few new toxicological data have become available since the
previous review by the Committee (Annex 1, reference 88). There
were no data from long-term toxicity or teratogenicity studies,
although a multigeneration study on dehydrated Dunaliella bardawil
(= salina) in rats did not reveal any adverse effects on
reproductive performance or gross fetal morphology. However, a NOEL
was not identified in this study, as animals of the F0-generation
maintained on diets containing 5% and 10% algal carotene for one
year showed renal pathological changes. In addition, there was
focal bronchopneumonia at the higher level. Although the renal
changes (metaplastic changes in the pelvic epithelium and
nephrocalcinosis) might have been due to nutritional imbalance, this
was not clearly established. The focal bronchopneumonia observed in
the lung at the higher dose level may have been associated with
inhalation of powdered diets, although both low-dose and control
rats also received powdered diets without showing similar effects.
The Committee considered that the available short-term toxicity
studies inadequate for establishing an ADI because of the small
numbers of animals tested, lack of or inadequate histopathological
examination, or inadequate reporting.
There were virtually no systematic toxicological studies
available on the oil-extract of alga. Available data on the dried
material could not be extrapolated to the oil extract since the
specifications are quite different and lipophilic materials may have
been concentrated during the oil extraction process.
4. EVALUATION
The Committee considered the data inadequate to establish an
ADI for the dehydrated algal carotene preparations or for the
vegetable oil extracts of Dunaliella salina. There is no history
of use of Dunaliella algae as food.
No relevant toxicological data on vegetable extracts were
available. However, the Committee concluded that there was no
objection to the use of vegetable extracts as colouring agents,
provided that the level of use did not exceed the level normally
present in vegetables. Implicit in this conclusion is that the
extracts should not be made toxic by virtue of the concentration of
toxic compounds (including toxicants naturally occurring in the
vegetables) nor by the generation of reaction products or residues
of a nature or in such amounts as to be toxicologically significant
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See Also:
Toxicological Abbreviations