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Magnesium stearate, a widely-Used food additive, exhibits a lack of in vitro and in vivo genotoxic potential

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Magnesium stearate is widely used in the production of dietary supplement and pharmaceutical tablets, capsules and powders as well as many food products, including a variety of confectionery, spices and baking ingredients. Although considered to have a safe toxicity profile, there is no available information regarding its potential to induce genetic toxicity. To aid safety assessment efforts, magnesium sulfate was evaluated in a battery of tests including a bacterial reverse mutation assay, an in vitro chromosome aberration assay, and an in vivo erythrocyte micronucleus assay. Magnesium stearate did not produce a positive response in any of the five bacterial strains tested, in the absence or presence of metabolic activation. Similarly, exposure to magnesium stearate did not lead to chromosomal aberrations in CHL/IU Chinese hamster lung fibroblasts, with or without metabolic activation, or induce micronuclei in the bone marrow of male CD-1 mice. These studies have been used by the Japanese government and the Joint FAO/WHO Expert Committee on Food Additives in their respective safety assessments of magnesium stearate. These data indicate a lack of genotoxic risk posed by magnesium stearate consumed at current estimated dietary exposures. However, health effects of cumulative exposure to magnesium via multiple sources present in food additives may be of concern and warrant further evaluation.
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Toxicology Reports
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Magnesium stearate, a widely-used food additive, exhibits a lack of in vitro
and in vivo genotoxic potential
Cheryl A. Hobbs
a,
, Kazuhiko Saigo
b
, Mihoko Koyanagi
c
, Shim-mo Hayashi
c
a
Toxicology Program, Integrated Laboratory Systems, Inc., PO Box 13501, Research Triangle Park, NC 27709, USA
b
Drug Safety Research Laboratories, Shin Nippon Biomedical Laboratories, Ltd., 2438 Miyanoura-cho, Kagoshima-City, Kagoshima 891-1394, Japan
c
Global Scientic and Regulatory Aairs, San-Ei Gen F.F.I., Inc., 1-1-11 Sanwa-cho, Toyonaka, Osaka 561-8588, Japan
ARTICLE INFO
Keywords:
Genotoxicity
Food additive
Magnesium stearate
DNA damage
Dietary supplement
Joint FAO/WHO Expert Committee on Food
Additives (JECFA)
ABSTRACT
Magnesium stearate is widely used in the production of dietary supplement and pharmaceutical tablets, capsules
and powders as well as many food products, including a variety of confectionery, spices and baking ingredients.
Although considered to have a safe toxicity prole, there is no available information regarding its potential to
induce genetic toxicity. To aid safety assessment eorts, magnesium sulfate was evaluated in a battery of tests
including a bacterial reverse mutation assay, an in vitro chromosome aberration assay, and an in vivo erythrocyte
micronucleus assay. Magnesium stearate did not produce a positive response in any of the ve bacterial strains
tested, in the absence or presence of metabolic activation. Similarly, exposure to magnesium stearate did not
lead to chromosomal aberrations in CHL/IU Chinese hamster lung broblasts, with or without metabolic acti-
vation, or induce micronuclei in the bone marrow of male CD-1 mice. These studies have been used by the
Japanese government and the Joint FAO/WHO Expert Committee on Food Additives in their respective safety
assessments of magnesium stearate. These data indicate a lack of genotoxic risk posed by magnesium stearate
consumed at current estimated dietary exposures. However, health eects of cumulative exposure to magnesium
via multiple sources present in food additives may be of concern and warrant further evaluation.
1. Introduction
Magnesium stearate is the magnesium salt of the fatty acid, stearic
acid (Fig. 1). It has been widely used for many decades in the food
industry as an emulsier, binder and thickener, as well as an antic-
aking, lubricant, release, and antifoaming agent. It is present in many
food supplements, confectionery, chewing gum, herbs and spices, and
baking ingredients. Magnesium stearate is also commonly used as an
inactive ingredient in the production of pharmaceutical tablets, cap-
sules and powders.
For food applications, magnesium stearate is typically manufactured
by one of two processes. The direct or fusion process involves direct
reaction of fatty acids with a source of magnesium, such as magnesium
oxide, to form magnesium salts of the fatty acids. In the indirect or
precipitation process, a sodium soap is produced by reacting fatty acids
with sodium hydroxide in water and precipitating the product through
addition of magnesium salts to the soap. The fatty acids used as raw
material are derived from edible fats and oils and consist mainly of
stearic and palmitic acid. The nal product contains 4.0-5.0% magne-
sium, on a dried basis, and the fatty acid fraction is composed of 90%
stearic and palmitic acids, at least 40% of which are stearic acid. It is a
very ne powder that is greasy to the touch and practically insoluble in
water.
Upon ingestion, magnesium stearate is dissolved into magnesium
ion and stearic and palmitic acids. Magnesium is absorbed primarily in
the small intestine, and to a lesser extent, in the colon. Magnesium is an
essential mineral, serving as a cofactor for hundreds of enzymatic re-
actions and is essential for the synthesis of carbohydrates, lipids, nu-
cleic acids and proteins, as well as neuromuscular and cardiovascular
function [1,2]. The majority of magnesium content in the body is stored
in bone and muscle [1,3]. A small amount (1%) is present in serum
and interstitial body uid, mostly existing as a free cation while the
remainder is bound to protein or exists as anion complexes [3]. The
kidney is largely responsible for magnesium homeostasis and
http://dx.doi.org/10.1016/j.toxrep.2017.10.003
Received 17 July 2017; Received in revised form 28 September 2017; Accepted 13 October 2017
Corresponding author.
E-mail addresses: chobbs@ils-inc.com,cahobbsriter@bellsouth.net (C.A. Hobbs).
Abbreviations: 2AA, 2-aminoanthracene; 9AA, 9-aminoacridine hydrochloride monohydrate; ADI, acceptable daily intake; AF-2, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; DMSO,
dimethyl sulfoxide; EFSA, European Food Safety Authority; FAO, Food and Agriculture Organization of the United Nations; ENNG, N-ethyl-N'-nitro-N-nitrosoguanidine; FDA, U.S. Food
and Drug Administration; GLP, Good Laboratory Practice; JECFA, Joint FAO/WHO Expert Committee on Food Additives; MMC, mitomycin C; MN, micronucleus or micronuclei; MN-PCE,
micronucleated polychromatic erythrocyte(s); OECD, Organization for Economic Cooperation and Development; PCE, polychromatic erythrocyte(s); WHO, World Health Organization
Toxicology Reports 4 (2017) 554–559
Available online 16 October 2017
2214-7500/ © 2017 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
MARK
maintenance of serum concentration [1,3]. Excretion occurs primarily
via the urine, but also occurs in sweat and breast milk. Stearic and
palmitic acids are products of the metabolism of edible oils and fats for
which the metabolic fate has been well established. These fatty acids
undergo ß-oxidation to yield 2-carbon units which enter the tri-
carboxylic acid cycle and the metabolic products are utilized and ex-
creted [4].
Magnesium stearate is permitted for use in the European Union and
other countries including China, Japan, Australia and New Zealand, and
was granted generally recognized as safe (GRAS) status in the United
States [5]. However, there are no published data available related to the
genotoxic potential of magnesium stearate. For the safety assessment of
food ingredients, the U.S. Food and Drug Administration (FDA) re-
commends a bacterial reverse mutation test [68],anin vitro test for
chromosomal damage or gene mutation in mammalian cells, as well as
an in vivo test for chromosomal damage using mammalian hemato-
poietic cells [9], such as the rodent erythrocyte micronucleus assay
[10,11] which has proven utility for predicting carcinogens [12]. The
European Food Safety Authority (EFSA) guidances [13,14] recommend
a similar, albeit tiered, approach for assessing genotoxic potential. To
provide requested genotoxicity data to support initial assessment of the
safety of magnesium stearate conducted by the Japanese government,
and subsequently, reassessment by the Joint FAO/WHO Expert Com-
mittee on Food Additives [15,16], magnesium stearate was evaluated in
a bacterial gene mutation assay using Salmonella and E. coli tester
strains, an in vitro mammalian chromosome aberration assay using
Chinese Hamster Lung cells and a micronucleus assay in the bone
marrow of male CD-1 mice. This combination of tests is commonly used
to evaluate the genotoxicity of food additives [17,18]. The studies re-
ported here were performed as Good Laboratory Practice (GLP) ex-
periments in accordance with Japanese Ministry of Health, Labour and
Welfare and OECD testing guidelines current at the time the studies
were conducted [1922].
2. Material and methods
2.1. Chemicals
All genotoxicity assays were GLP-compliant; however, analysis of
dose formulations for concentration was not mandated by the Japanese
regulatory agency requesting these studies and was not performed.
Magnesium stearate (99% relative content of stearic and palmitic acid;
CAS No. 557-04-0; San-Ei Gen F.F.I., Inc., Osaka, Japan) was stored at
room temperature. Formulations were prepared just prior to use by
adding vehicle to the weighed test substance and solubilizing with ul-
trasound; lower concentrations were prepared by serial dilution.
Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich Japan
K.K. (Shinagawa-ku, Japan). 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide
(AF-2), 2-aminoanthracene (2AA), sodium carboxymethyl cellulose,
and mitomycin C (MMC) were purchased from Wako Pure Chemical
Industries, Ltd., Osaka, Japan. 9-Aminoacridine hydrochloride mono-
hydrate (9AA) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) were
purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Japanese
Pharmacopeia saline was purchased from the Otsuka Pharmaceutical
Factory, Inc. (Tokushima, Japan).
2.2. Bacterial reverse mutation assay
A bacterial mutagenicity assay of magnesium stearate, with and
without metabolic activation, was conducted using the preincubation
method using Salmonella typhimurium strains TA100 and TA1535 and
Escherichia coli strain WP2uvr A as detection systems for base-pair
substitution mutations, and S. typhimurium strains TA98 and TA1537
for detection of frame-shift mutations [68]. All strains (National In-
stitute of Health Sciences, Japan) were checked for maintenance of
genetic markers prior to the study. Based on results of a range-nding
assay using all tester strains at 2 plates per concentration (Supplemental
Data Table S1), a top concentration of 5 mg/plate, with and without
metabolic activation, was chosen as recommended by expert group [7]
as well as OECD [21] and Japanese [22] guidelines for non-cytotoxic
compounds. The doses tested were 5000, 2500, 1250, 625, 313, and
156 μg/plate. Strain specic positive controls tested without metabolic
activation were AF-2 (TA98 and TA100), ENNG (TA1535 and E. coli
WP2), and 9AA (TA1537). 2-AA was used as the positive control for all
strains tested with metabolic activation. Metabolic activation was
provided by a 10% phenobarbital/5,6-benzoavone-induced rat liver
S9 preparation (Kikkoman Corp., Co., Noda, Japan) with added cofac-
tors (glucose-6-phosphate dehydrogenase, NADPH, and NADH, Oriental
Yeast Co., Ltd, Tokyo, Japan). Test solutions were prepared in DMSO.
The assay tubes were pre-incubated at 37 °C for 20 min with shaking
before addition of top agar and plating onto minimal agar. Two test
plates per concentration were inverted and cultured at 37 °C for 48 h
and then revertant colonies counted using an automatic colony counter
(CA-7A, Toyo Sokki Co., Ltd., Japan). The use of two plates for all the
tester strains with no evidence of a positive response in the rangender
assay justies the use of only two plates per concentration in the de-
nitive assay, in accordance with the OECD test guideline that states
that duplicate plating is acceptable when scientically justied; this
study design is also acceptable to the Japanese regulatory authority.
Appropriate control plates were included to verify sterility of the ve-
hicle, test chemical solutions, and S9 mix. Criteria for a positive re-
sponse were a 2-fold increase in the average plate count compared to
the vehicle control for at least one concentration level, a dose response
over the range of tested concentrations in at least one strain with or
without metabolic activation, and reproducibility between the range
nding and denitive mutagenicity studies.
2.3. In vitro chromosome aberration assay
The mycoplasma-free CHL/IU Chinese hamster lung broblast cell
line was obtained from the Division of Laboratory Products, Dainippon
Pharmaceutical Co., Ltd. This cell line has an approximate cell doubling
time of 15 h. Cells were cultured in Eagle MEM medium containing
10% heat inactivated fetal bovine serum at 37 °C with 5% CO
2
and high
humidity. S9 liver homogenate, prepared from male rats treated with
phenobarbital and 5,6-benzoavone (Kikkoman Corporation, Noda,
Japan), was added at a nal concentration of 30% to a ltered coen-
zyme solution containing 1.7 mg/mL glucose-6-phosphate dehy-
drogenase, 3.35 mg/mL NADPH, 4 mM HEPES, 5 mM MgCl
2
·6H
2
O, and
33 mM KCl. The assay was performed for short-term (6-h) and con-
tinuous treatments as described previously [23,24] and in accordance
with JMHLW guidelines current at the time. Based on the results of a
range nding study of magnesium stearate (Supplemental Data Table
S2), the 50% growth-inhibitory concentrations were estimated to be
49 μg/mL for the short-term treatment without metabolic activation,
784 μg/mL for the short-term treatment with metabolic activation,
9μg/mL for the continuous 24-h treatment, and 4 μg/mL for the con-
tinuous 48-h treatment. The top concentrations of magnesium stearate
selected for the chromosomal aberration test were 1000 and 50 μg/mL
for the short-term treatment with and without metabolic activation,
Fig. 1. Chemical structure of magnesium stearate. Magnesium stearate, also known as
octadecanoic acid, exists as a salt containing two stearate anions and a magnesium cation.
C.A. Hobbs et al. Toxicology Reports 4 (2017) 554–559
555
respectively, and 10 and 5 μg/mL for the continuous 24- and 48-h
treatments, respectively; 0.5% sodium carboxymethyl cellulose was
used as the vehicle.
Freshly thawed cells were cultured for 72 h, then diluted to 1 × 10
4
cells/mL; 5 mL of the suspension were transferred to each of two 6-cm
plastic Petri dishes per treatment group, and cultured for 72 h. Then,
2.5 mL of the culture medium were removed from each petri dish and
0.5 mL S9 mix (nal concentration of 5%) or culture medium was
added for tests with and without metabolic activation, respectively. The
nal volume of vehicle, magnesium stearate, or MMC (20 μg/mL nal
concentration) formulations added to culture medium was 10%; B[a]P
was added at 0.5% (0.15 μg/mL nal concentration). After culturing for
6 h, the cells were rinsed once with physiological saline, 5 mL of fresh
medium added, and cells cultured for an additional 18 h. For con-
tinuous exposures, 72 h after the start of the culture, 0.5 mL of the
magnesium stearate formulation, vehicle, or MMC solution (nal con-
centration of 0.05 μg/mL) was added and the cells were cultured for 24
or 48 h. Colcemid was added to each petri dish at a nal concentration
of 0.1 μg/mL 2 h before the end of the culture period.
Following treatment, cells were trypsinized and viability de-
termined using trypan blue exclusion. The cell suspension was cen-
trifuged at 1000 rpm for 5 min, and hypotonic treatment was per-
formed by adding 5 mL of 0.075 M KCl at 37 °C to the cell pellet and
incubating at 37 °C for 30 min. Next, 0.5 mL of chilled Carnoy's xative
(3:1 ratio of methanol and glacial acetic acid) was added the cells and
the mixture was centrifuged (1000 rpm for 5 min). Approximately 4 mL
of xative was added to the cell pellet and the mixture was centrifuged;
this cycle was repeated 3 times. Two drops of the xed cell suspension
were applied to each clean slide. These slides were air dried, stained
with 3.0% Giemsa pH 6.8 (Merck, Kenilworth, NJ) for 15 min, rinsed in
water, and allowed to dry. The slides were coded in a double-blind
manner and one hundred metaphase spreads were observed per slide at
magnications up to 600×. Structural and numerical aberrations ob-
served in cells with 25 ± 2 chromosomes were recorded. Structural
aberrations were classied as a chromatid break, chromatid exchange,
chromosome break, chromosome exchange, and others in accordance
with the Atlas on Chromosomal Aberrations[25]. Interchromosomal
exchanges (triradial or quadriradial) and intrachromosomal exchanges
(ring chromatid) were recorded as chromatid exchanges, and dicentric
chromosomes and ring chromosomes were recorded as chromosome
exchanges. Many gaps (chromatid or chromosome) and breaks were
considered as fragmentation, and 10 or more breaks and exchanges
were considered as multiple aberrations, both of which were classied
as otheraberrations. If two or more aberrations were observed in a
single cell, each of the aberrations was counted as a single aberration.
Gaps were not included in the tabulation of structural aberrations. A
gap was dened as an achromatic region that was narrower than the
chromatid width, and a break was dened as an achromatic region that
was wider than the chromatid width. For evaluation of numerical
aberrations, metaphase spreads with 38 chromosomes were con-
sidered as polyploids and distinguished from endoreduplication. Eagle
MEM, fetal bovine serum and colcemid were purchased from Gibco BRL
(Grand Island, NY).
2.4. Animal husbandry
Male Crj: CD-1 (ICR) mice (Charles River Laboratories Japan, Inc.)
were 7 weeks of age at the time of treatment. Animals were housed in
aluminum cages with absorbent bedding (White Flakes, Charles River
Laboratories Japan, Yokohama, Japan) in a specic pathogen free fa-
cility with a 12-h light/12-h dark cycle. Mice were provided cobalt-60
irradiated solid feed (CE-2, CLEA Japan, Inc., Tokyo, Japan) and water
ad libitum.
2.5. In vivo erythrocyte micronucleus (MN) assay
In a dose range nding study (Supplemental Data Table S3), no
death was observed at doses up to 2000 mg/kg magnesium stearate.
The frequency of micronucleated polychromatic erythrocytes (MN-PCE)
was normal in the bone marrow at 24, 48, and 72 h in all groups. The
percentage of polychromatic erythrocytes (PCE) decreased in a dose-
dependent manner 24 h, and to a lesser extent, 48 h, following ad-
ministration of magnesium stearate without excessive cytotoxicity.
Therefore, for the denitive study, male CD-1 mice (6 animals/dose
group) were administered magnesium stearate at 2000, 1000, or
500 mg/kg or vehicle (0.5% sodium carboxymethyl cellulose) once by
gastric tube, or the positive control compound, MMC in Japanese
Pharmacopeia saline at 2 mg/kg, once by intraperitoneal injection.
Twenty-four hours after administration, mice were sacriced by cer-
vical dislocation. Both femurs were removed, ends cut, and bone
marrow cells ushed out with fetal bovine serum (FBS; GIBCO BRL,
Grand Island, NY). The cells were centrifuged at 1000 rpm for 5 min
and most of the supernatant was discarded. A few drops of the con-
centrated cell suspension were applied to a degreased slide, smeared
using a cover glass, and allowed to dry at room temperature. The bone
marrow slides were xed in methanol for 5 min, stained for 30 min with
a Giemsa stain that had been diluted to 3% with 6.7 mM sodium-po-
tassium phosphate buer (pH 6.8), and gently rinsed with sodium-po-
tassium phosphate buer. The slides were then treated with 0.004%
citric acid solution for approximately 3 s, rinsed with distilled water,
and allowed to dry.
The frequency of MN-PCE was determined by counting the number
of micronuclei (MN) in 2000 PCE per animal using coded specimens
and an oil immersion lens (nal magnication: 1000×). Five hundred
erythrocytes [PCE + normochromatic erythrocytes (NCE)] from each
animal were scored to determine the percentage of PCE in total ery-
throcytes as an index of chemical-induced growth suppression of bone
marrow cells.
2.6. Statistical analyses
A chi-square test (one-sided, p< 0.05) was used to compare the
frequency of cells with chromosomal aberrations in each of the test
substance groups with that in the vehicle control group; the test was
considered positive if the frequency of cells with chromosomal aber-
rations was signicantly increased and a dose dependency or re-
producibility was observed. For the in vivo MN assay, signicant dif-
ferences in the frequency of MN-PCE between the vehicle control group,
each of the magnesium stearate groups, and the positive control group
were analyzed using the Kastenbaum and Bowman method [26].Ifthe
frequency of MN-PCE in the test substance group was signicantly
higher than in the vehicle control group at a signicance level of 5%,
the test substance was considered to induce MN in mouse bone marrow
cells. A Student's t-test was used to determine if the % PCE, an index of
growth inhibition of bone marrow cells, was signicantly dierent
between a magnesium stearate-exposed group and the vehicle control
group.
3. Results
3.1. Bacterial reverse mutation assay
A mutagenicity assay was conducted to assess the potential of
magnesium stearate to induce gene mutations in bacteria up to the
recommended maximum concentration for non-cytotoxic chemicals
(5000 μg/plate). Growth inhibition of the test strains was not observed
at any concentration; precipitation of magnesium stearate was observed
at concentrations 313 μg/plate. Average plate counts for each set of
replicate plates are provided in Table 1. Consistent with the results of
the range nding assay (Supplemental Data Table S1), a positive
C.A. Hobbs et al. Toxicology Reports 4 (2017) 554–559
556
mutagenic response to magnesium stearate was not produced in any of
the ve Salmonella or E. coli strains tested either with or without me-
tabolic activation. Average revertant values for positive control che-
micals, both with and without metabolic activation, were at least 2-fold
above concurrent solvent controls. The numbers of revertant colonies in
the vehicle and positive control groups were within the range of la-
boratory historical data. The lack of induction of an increase in re-
vertant colonies or any apparent concentration-dependent response
indicates that, under the assay conditions tested, magnesium stearate is
not mutagenic in the bacterial reverse mutation assay.
3.2. In vitro chromosome aberration assay
Precipitation was observed at the start and end of the treatment for
magnesium stearate concentrations of 6.25 μg/mL and higher for short
exposures and at doses of 5 μg/mL for continuous exposures. Excessive
cytotoxicity precluded evaluation of chromosomal aberrations in cells
exposed to 10 μg/mL for 24 h and 5 μg/mL for 48 h. Exposure to
magnesium stearate did not induce increased frequencies of structural
or numerical aberrations under any of the test conditions (Table 2). The
positive control chemicals, MMC and B[a]P, induced statistically posi-
tive increases in the frequency of cells with structural, but not numer-
ical, aberrations. Numerical aberrations would not be expected in re-
sponse to MMC, a clastogen. B[a]P was observed to produce
aneuploidy, but not polyploidy, at 2.510 μg/mL in V79-MZ Chinese
hamster lung cells [27]; since only cells with 25 ± 2 chromosomes
were scored in this study, aneuploidy would not have been detected
even if induced at the much lower B[a]P concentration used in this
study.
3.3. In vivo MN assay
In a preliminary dose setting study, indication of chemical exposure
without evidence of excessive cytotoxicity or MN induction was ob-
served in the bone marrow of mice 24, 48, or 72 h following a single
administration of magnesium stearate up to 2000 mg/kg (Supplemental
Data Table S3). Based on these results, a MN assay was conducted in
which male CD-1 mice were administered magnesium stearate orally
once at 500, 1000, and 2000 mg/kg and bone marrow evaluated 24 h
following chemical administration. The selection of a 24-h timepoint
was in accordance with a published recommendation that if the fre-
quency of MN-PCE did not increase signicantly at any dose levels or
sampling times tested up to 72 h in a dose setting study, the sampling
time for the denitive study should be set at 24 or 30 h [28]. This study
design was acceptable to the Japanese regulatory authority at the time
the study was conducted [29]. All animals survived to termination.
Results of analysis of MN-PCE and PCE frequencies are summarized in
Table 3. Under the conditions used in the MN study, no increase in the
frequency of MN-PCE was observed in mice administered magnesium
stearate. Decreases in the % PCE relative to the vehicle control animals
were measured in mice in all magnesium stearate dose groups, in-
dicating some bone marrow cytotoxicity reective of chemical exposure
at the tested doses. There was a statistically signicant increase in MN-
PCE and suppression of PCE in the bone marrow of animals adminis-
tered the concurrent positive control, MMC. The %MN-PCE and %PCE
values for the vehicle and positive control groups fell within the la-
boratory historical control range.
4. Discussion
These studies were conducted to produce genotoxicity information
to aid safety assessment of magnesium stearate used as a food additive.
In a bacterial reverse mutation assay, magnesium stearate did not
produce a positive response in any of the ve test strains, either with or
without metabolic activation, up to the OECD-recommended limit dose.
These results are consistent with results provided in a study report
submitted to the FDA [30]. Likewise, exposure to magnesium stearate
did not induce chromosomal aberrations in hamster lung broblasts or
micronuclei in the bone marrow of CD-1 mice.
Upon ingestion, magnesium stearate dissolves into its component
ions, magnesium and stearic and palmitic acids. Therefore, the safety
assessment should be based on its constituent cations and anions. Fatty
acids are normal constituents of coconut oil, butter and other edible oils
and have not been considered to pose a toxicological risk [4,31].As
such, it was concluded that stearic and palmitic acids used as avouring
agents do not present a safety concern [32]. This position has been
supported by results of recent studies demonstrating a lack of geno-
toxicity and toxicity of some fatty acids containing stearic and palmitic
acids [33,34].
The results of the genetic toxicity tests reported here were used by
the Japanese government in its assessment of the safety of magnesium
stearate, leading to its approved use as a food additive for certain ap-
plications in Japan in 2006. The safety of magnesium stearate was most
recently reviewed internationally at the 80th meeting of the Joint FAO/
WHO Expert Committee on Food Additives in 2015 [15,16]. The results
of the studies reported here were provided to the Committee and served
Table 1
Results of bacterial reverse mutation assay of magnesium stearate.
Dose (μg/plate) Mean revertants/plate ( ± SD) without rat liver S9 Mean revertants/plate ( ± SD) with rat liver S9
TA100 TA98 TA1535 TA1537 WP2 uvrA TA100 TA98 TA1535 TA1537 WP2 uvrA
0 127 ± 4 27 ± 1 12 ± 0 8 ± 1 23 ± 3 138 ± 1 36 ± 3 13 ± 1 10 ± 1 22 ± 1
156 137 ± 1 34 ± 4 15 ± 4 5 ± 0 20 ± 1 148 ± 2 46 ± 1 17 ± 5 8 ± 5 18 ± 4
313 132 ± 10 29 ± 0 12 ± 1 5 ± 3 20 ± 4 147 ± 3 39 ± 5 12 ± 0 8 ± 4 21 ± 4
625 129 ± 8 33 ± 6 13 ± 0 6 ± 1 22 ± 4 132 ± 5 34 ± 6 16 ± 7 11 ± 1 19 ± 6
1250 132 ± 4 27 ± 8 11 ± 4 9 ± 1 18 ± 1 144 ± 1 47 ± 6 12 ± 2 10 ± 3 16 ± 1
2500 138 ± 11 33 ± 2 13 ± 3 11 ± 1 18 ± 5 143 ± 1 40 ± 2 14 ± 1 9 ± 2 22 ± 4
5000 130 ± 1 24 ± 1 15 ± 6 6 ± 1 21 ± 1 139 ± 0 44 ± 6 16 ± 5 10 ± 0 22 ± 3
Positive control 382 ± 13
a
337 ± 6
b
260 ± 14
c
692 ± 62
d
976 ± 49
e
1238 ± 21
f
647 ± 39
g
241 ± 42
h
222 ± 1
h
232 ± 46
i
a
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide administered at 0.01 μg/plate.
b
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide administered at 0.1 μg/plate.
c
N-Ethyl-N'-nitro-N-nitrosoguanidine administered at 5 μg/plate.
d
9-Aminoacridine hydrochloride administered at 80 μg/plate.
e
N-Ethyl-N'-nitro-N-nitrosoguanidine administered at 2 μg/plate.
f
2-Aminoanthracene administered at 1 μg/plate.
g
2-Aminoanthracene administered at 0.5 μg/plate.
h
2-Aminoanthracene administered at 2 μg/plate.
i
2-Aminoanthracene administered at 10 μg/plate.
C.A. Hobbs et al. Toxicology Reports 4 (2017) 554–559
557
as the primary basis for its opinion that magnesium stearate is not
genotoxic. The Committee also evaluated a range of other toxicological
studies and assessed dietary exposure. It concluded that the toxicity of
magnesium stearate should not be evaluated dierently than other
magnesium salts and conrmed the previously recommended [31] ac-
ceptable daily intake (ADI) of not speciedfor magnesium salts of
stearic and palmitic acids. Subsequently, the Japanese Ministry of
Health, Labour and Welfare conducted a re-evaluation of magnesium
stearate, including the data from this genetic toxicity test battery, and
expanded the existing use standards beyond foods for specied health
uses and with nutrient function claims, to include foods not in con-
ventional food form such as tablet confectioneries and capsules or ta-
blets with functional claims (http://members.wto.org/crnattachments/
2017/SPS/JPN/17_2276_00_e.pdf).
Toxicology data from animal studies relevant to evaluation of
magnesium stearate are lacking (e.g., doses that wont lead to a dietary
imbalance, known composition of material tested, appropriate admin-
istration route, etc.) [15]. There are also no human data related to
magnesium stearate toxicity. It has been noted that infants are parti-
cularly sensitive to the sedative eects of magnesium salts and that
individuals with chronic renal impairment retained 1530% of ad-
ministered magnesium, which may cause toxicity [31]. Moreover,
diarrhea and other gastrointestinal eects have been observed with
excessive magnesium intake resulting from use of various magnesium
salts for pharmacological/medicinal purposes. Many magnesium-con-
taining food additives have been evaluated individually, but not
Table 2
Results of chromosome aberration assay in CHL cells exposed to magnesium stearate.
Dose (μg/
mL)
Viability (%) Structural Chromosomal Aberrations Numerical
Chromosome
Aberrations
Endoreduplication Total
Mean Chromatid
Break
Chromatid
Exchange
Chromosome
Break
Chromosome
Exchange
Others Gaps
a
Total
b
Polyploid
6 h Exposure without S9
0 100.0 1 0 0 0 0 1 1 1 0 1
1.56 101.3 0 1 1 0 0 0 2 1 0 1
3.12 90.7 0 0 0 0 0 1 0 1 0 1
6.25
c
78.8 2 0 1 0 0 0 3 2 0 2
12.5
c
69.5 1 0 0 0 0 2 1 3 0 3
25
c
53.6 0 1 1 0 0 1 2 3 0 3
50
c
44.4 2 1 0 0 0 1 3 3 0 3
MMC
(0.15)
62.3 11 40 0 0 0 0 43 0 0 0
6 h Exposure with S9
0 100.0 1 1 0 0 0 0 2 2 0 2
31.3
c
96.5 0 0 0 0 0 0 0 2 0 2
62.5
c
90.0 0 1 1 0 0 1 2 3 0 3
125
c
77.1 1 1 0 0 0 1 2 4 0 4
250
c
62.9 0 0 0 0 0 1 0 4 0 4
500
c
51.2 2 0 0 0 0 0 2 4 0 4
1000
c
44.7 0 1 1 0 0 2 2 5 0 5
B(a)P (20) 45.9 7 46 0 0 0 0 50 0 0 0
24 h Exposure without S9
0 100.0 0 1 0 0 0 1 1 1 0 1
0.313 94.0 1 0 1 0 0 0 2 1 0 1
0.625 83.3 2 0 0 0 0 1 2 2 0 2
1.25 64.3 0 1 0 0 0 1 1 3 0 3
2.5 53.6 1 0 1 0 0 1 2 2 0 2
5
c
39.9 1 1 0 0 0 1 2 3 0 3
10 30.4 NA NA NA NA NA NA NA NA NA NA
MMC
(0.05)
69.0 15 44 0 0 0 0 49 0 0 0
48 h Exposure without S9
0 100.0 1 0 0 0 0 1 1 2 0 2
0.156 94.9 1 1 0 0 0 0 2 2 0 2
0.313 87.0 1 0 0 0 0 1 1 3 0 3
0.625 71.8 1 0 1 0 0 1 2 3 0 3
1.25 57.9 0 1 0 0 0 2 1 3 0 3
2.5 45.4 0 1 0 0 0 1 1 3 0 3
5
c
29.6 NA NA NA NA NA NA NA NA NA NA
MMC
(0.05)
56.0 11 49 0 0 0 0 54 0 0 0
NA = Not analyzed due to excessive cytotoxicity.
MMC = Mitomycin C; B(a)P = Benzo[a]pyrene.
a
Gaps include both chromatid-type aberration and chromosome-type aberration.
b
Gaps not included in total of structural aberrations.
c
Precipitate observed.
Table 3
Results of micronucleus assay in mice administered magnesium stearate.
Dose (mg/kg) % PCE
a
%MN-PCE
a
0 51.6 ± 2.1 0.10 ± 0.05
500 49.7 ± 2.2 0.11 ± 0.04
1000 48.7 ± 1.9
b
0.09 ± 0.04
2000 43.6 ± 2.0
b
0.11 ± 0.04
MMC 37.9 ± 2.7
b
3.32 ± 0.30
c
MMC = mitomycin C administered at 2 mg/kg.
a
Group mean ± standard deviation.
b
Signicant at p< 0.05 (Student's t-test).
c
Signicant at p< 0.05 (Kastenbaum and Bowman's method).
C.A. Hobbs et al. Toxicology Reports 4 (2017) 554–559
558
collectively, for laxative eects. Based on the recent dietary exposure
assessment to magnesium stearate and concern that use of magnesium
salts in many food additives may result in cumulative exposure that
could lead to a laxative eect, JECFA reiterated its earlier re-
commendation [35] that total dietary exposure to magnesium from
food additives and other sources in the diet be assessed [15]. Although
eects of cumulative exposure to magnesium via food additives should
be evaluated, the studies reported here indicate a lack of genotoxic risk
posed specically by magnesium stearate consumed at current esti-
mated dietary exposures.
5. Funding
This research did not receive any specic grant from funding
agencies in the public, commercial, or not-for-prot sectors.
Acknowledgements
This work was conducted at Shin Nippon Biomedical Laboratories,
Ltd. and funded by San-Ei Gen, F.F.I., Inc., a manufacturer/supplier of
magnesium stearate. Shin Nippon Biomedical Laboratories, Ltd was
responsible for the study design, the collection, analysis, and inter-
pretation of data, and the writing of the nal study reports. ILS, Inc.
reviewed the study reports and wrote the manuscript at the request of
San-Ei Gen, F.F.I., Inc.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.toxrep.2017.10.003.
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