INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
CONCISE INTERNATIONAL CHEMICAL ASSESSMENT DOCUMENT NO. 26
BENZOIC ACID AND SODIUM BENZOATE
This report contains the collective views of an international group of
experts and does not necessarily represent the decisions or the stated
policy of the United Nations Environment Programme, the International
Labour Organization, or the World Health Organization.
First draft prepared by Dr A. Wibbertmann, Dr J. Kielhorn, Dr G.
Koennecker, Dr I. Mangelsdorf, and Dr C. Melber, Fraunhofer Institute
for Toxicology and Aerosol Research, Hanover, Germany
Published under the joint sponsorship of the United Nations
Environment Programme, the International Labour Organization, and the
World Health Organization, and produced within the framework of the
Inter-Organization Programme for the Sound Management of Chemicals.
World Health Organization
Geneva, 2000
The International Programme on Chemical Safety (IPCS),
established in 1980, is a joint venture of the United Nations
Environment Programme (UNEP), the International Labour Organization
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WHO Library Cataloguing-in-Publication Data
Benzoic acid and sodium benzoate.
(Concise international chemical assessment document ; 26)
1.Benzoic acid - toxicity 2.Sodium benzoate - toxicity
3.Risk assessment 4.Environmental exposure
I.International Programme on Chemical Safety II.Series
ISBN 92 4 153026 X (NLM Classification: QD 341.A2)
ISSN 1020-6167
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TABLE OF CONTENTS
FOREWORD
1. EXECUTIVE SUMMARY
2. IDENTITY AND PHYSICAL/CHEMICAL PROPERTIES
3. ANALYTICAL METHODS
4. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
4.1. Natural sources of benzoic acid
4.2. Anthropogenic sources
4.2.1. Benzoic acid
4.2.2. Sodium benzoate
4.3. Uses
4.3.1. Benzoic acid
4.3.2. Sodium benzoate
4.4. Estimated global release
5. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, TRANSFORMATION, AND ACCUMULATION
5.1. Transport and distribution between media
5.1.1. Benzoic acid
5.1.2. Sodium benzoate
5.2. Transformation
5.2.1. Benzoic acid
5.2.2. Sodium benzoate
5.3. Accumulation
5.3.1. Benzoic acid
5.3.2. Sodium benzoate
6. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
6.1. Environmental levels
6.2. Human exposure
7. COMPARATIVE KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS
7.1. Precursors of benzoic acid
8. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS
8.1. Single exposure
8.2. Irritation and sensitization
8.2.1. Benzoic acid
8.2.2. Sodium benzoate
8.3. Short-term exposure
8.3.1. Oral exposure
8.3.2. Inhalation exposure
8.3.3. Dermal exposure
8.4. Long-term exposure
8.4.1. Subchronic exposure
8.4.2. Chronic exposure and carcinogenicity
8.4.3. Carcinogenicity of benzyl acetate, benzyl alcohol, and benzaldehyde
8.5. Genotoxicity and related end-points
8.5.1. Benzoic acid
8.5.2. Sodium benzoate
8.6. Reproductive and developmental toxicity
8.6.1. Fertility
8.6.2. Developmental toxicity
8.6.3. Reproductive toxicity of benzyl acetate, benzyl alcohol, and benzaldehyde
9. EFFECTS ON HUMANS
10. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD
10.1. Aquatic environment
10.2. Terrestrial environment
11. EFFECTS EVALUATION
11.1. Evaluation of health effects
11.1.1. Hazard identification and dose-response assessment
11.1.2. Criteria for setting tolerable intakes or guidance values for benzoic acid and sodium benzoate
11.1.3. Sample risk characterization
11.2. Evaluation of environmental effects
12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES
REFERENCES
APPENDIX 1 -- SOURCE DOCUMENTS
APPENDIX 2 -- CICAD PEER REVIEW
APPENDIX 3 -- CICAD FINAL REVIEW BOARD
APPENDIX 4 -- INTERNATIONAL CHEMICAL SAFETY CARD
RÉSUMÉ D'ORIENTATION
RESUMEN DE ORIENTACI²N
FOREWORD
Concise International Chemical Assessment Documents (CICADs) are
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all available data on a particular chemical; rather, they include only
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upon which the CICAD has been based.
Risks to human health and the environment will vary considerably
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of locally measured or predicted exposure scenarios. To assist the
reader, examples of exposure estimation and risk characterization are
provided in CICADs, whenever possible. These examples cannot be
considered as representing all possible exposure situations, but are
provided as guidance only. The reader is referred to EHC 1701 for
advice on the derivation of health-based tolerable intakes and
guidance values.
1 International Programme on Chemical Safety (1994)
Assessing human health risks of chemicals: deriviation of
guidance values for health-based exposure limits. Geneva, World
Health Organization (Environmental Health Criteria 170).
While every effort is made to ensure that CICADs represent the
current status of knowledge, new information is being developed
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In the event that a reader becomes aware of new information that would
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Procedures
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These procedures are designed to take advantage of the expertise that
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The first draft is based on an existing national, regional, or
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A standard outline has been developed to encourage consistency in
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1. EXECUTIVE SUMMARY
This CICAD on benzoic acid and sodium benzoate was prepared by
the Fraunhofer Institute for Toxicology and Aerosol Research, Hanover,
Germany. The two compounds are being considered together because it is
undissociated benzoic acid that is responsible for its antimicrobial
activity. As benzoic acid itself is only slightly soluble in water,
sodium benzoate -- which, under acid conditions, converts to
undissociated benzoic acid -- is often used instead.
This CICAD was based on reviews compiled by the German Advisory
Committee on Existing Chemicals of Environmental Relevance (BUA,
1995), the US Food and Drug Administration (US FDA, 1972a), and the
Joint FAO/WHO Expert Committee on Food Additives (JECFA) (WHO, 1996)
to assess potential effects of benzoic acid and sodium benzoate on the
environment and on humans. A comprehensive literature search of
relevant databases was conducted in September 1999 to identify any
relevant references published subsequent to those incorporated in
these reports. Information on the preparation and peer review of the
source documents is presented in Appendix 1. Information on the peer
review of this CICAD is presented in Appendix 2. This CICAD was
approved as an international assessment at a meeting of the Final
Review Board, held in Sydney, Australia, on 21-24 November 1999.
Participants at the Final Review Board meeting are listed in Appendix
3. The International Chemical Safety Card (ICSC 0103) for benzoic
acid, produced by the International Programme on Chemical Safety
(IPCS, 1993), has also been reproduced in this document (Appendix 4).
Benzyl acetate, its hydrolysis product, benzyl alcohol, and the
oxidation product of this alcohol, benzaldehyde, are extensively
metabolized to benzoic acid in experimental animals and humans.
Therefore, toxicological data on these precursors were also utilized
in the assessment of the potential health effects of benzoic acid.
Benzoic acid (CAS No. 65-85-0) is a white solid that is slightly
soluble in water. Sodium benzoate (CAS No. 532-32-1) is about 200
times more soluble in water. Benzoic acid is used as an intermediate
in the synthesis of different compounds, primarily phenol (>50% of
the amount produced worldwide) and caprolactam. Other end products
include sodium and other benzoates, benzoyl chloride, and diethylene
and dipropylene glycol dibenzoate plasticizers. Sodium benzoate is
primarily used as a preservative and corrosion inhibitor (e.g., in
technical systems as an additive to automotive engine antifreeze
coolants). Benzoic acid and sodium benzoate are used as food
preservatives and are most suitable for foods, fruit juices, and soft
drinks that are naturally in an acidic pH range. Their use as
preservatives in food, beverages, toothpastes, mouthwashes,
dentifrices, cosmetics, and pharmaceuticals is regulated. The
estimated global production capacity for benzoic acid is about
600 000 tonnes per year. Worldwide sodium benzoate production in 1997
can be estimated at about 55 000-60 000 tonnes. Benzoic acid occurs
naturally in many plants and in animals. It is therefore a natural
constituent of many foods, including milk products. Anthropogenic
releases of benzoic acid and sodium benzoate into the environment are
primarily emissions into water and soil from their uses as
preservatives. Concentrations of naturally occurring benzoic acid in
several foods did not exceed average values of 40 mg/kg of food.
Maximum concentrations reported for benzoic acid or sodium benzoate
added to food for preservation purposes were in the range of 2000
mg/kg of food.
After oral uptake, benzoic acid and sodium benzoate are rapidly
absorbed from the gastrointestinal tract and metabolized in the liver
by conjugation with glycine, resulting in the formation of hippuric
acid, which is rapidly excreted via the urine. To a lesser extent,
benzoates applied dermally can penetrate through the skin. Owing to
rapid metabolism and excretion, an accumulation of the benzoates or
their metabolites is not to be expected.
In rodents, the acute oral toxicity of benzoic acid and sodium
benzoate is low (oral LD50 values of >1940 mg/kg body weight). In
cats, which seem to be more sensitive than rodents, toxic effects and
mortality were reported at much lower doses (about 450 mg/kg body
weight).
Benzoic acid is slightly irritating to the skin and irritating to
the eye, while sodium benzoate is not irritating to the skin and is
only a slight eye irritant. For benzoic acid, the available studies
gave no indication of a sensitizing effect; for sodium benzoate, no
data were identified in the literature.
In short-term studies with rats, disorders of the central nervous
system (benzoic acid/sodium benzoate) as well as histopathological
changes in the brain (benzoic acid) were seen after feeding high doses
(>1800 mg/kg body weight) over 5-10 days. Other effects included
reduced weight gain, changes in organ weights, changes in serum
parameters, or histopathological changes in the liver. The information
concerning long-term oral exposure of experimental animals to benzoic
acid is very limited, and there is no study available dealing
specifically with possible carcinogenic effects. From a limited
four-generation study, only a preliminary no-observed-(adverse-)effect
level (NO(A)EL) of about 500 mg/kg body weight per day can be derived.
With sodium benzoate, two long-term studies with rats and mice gave no
indication of a carcinogenic effect. However, the documentation of
effects is inadequate in most of these studies; therefore, no reliable
NO(A)EL values can be derived. Data on its precursors support the
notion that benzoic acid is unlikely to be carcinogenic.
Benzoic acid tested negative in several bacterial assays and in
tests with mammalian cells, while in vivo studies were not
identified. Sodium benzoate was also inactive in Ames tests, whereas
tests with mammalian cells gave consistently positive results. In one
in vivo study (dominant lethal assay with rats), a positive result
was obtained. At present, a genotoxic activity of sodium benzoate
cannot be ruled out entirely.
For benzoic acid, two limited studies gave no indication of
adverse reproductive or developmental effects. With sodium benzoate,
several studies on different species have been performed, and
embryotoxic and fetotoxic effects as well as malformations were seen
only at doses that induced severe maternal toxicity. In a dietary
study in rats, a NO(A)EL of about 1310 mg/kg body weight was
established. Data on its precursors support the notion that benzoic
acid is unlikely to have adverse reproductive effects at dose levels
not toxic to the mother.
In humans, the acute toxicity of benzoic acid and sodium benzoate
is low. However, both substances are known to cause non-immunological
contact reactions (pseudoallergy). This effect is scarce in healthy
subjects; in patients with frequent urticaria or asthma, symptoms or
exacerbation of symptoms was observed. A provisional tolerable intake
of 5 mg/kg body weight per day can be derived, although benzoates at
lower doses can cause non-immunological contact reactions
(pseudoallergy) in sensitive persons. As there are no adequate studies
available on inhalation exposure, a tolerable concentration for
exposure by inhalation cannot be calculated.
From their physical/chemical properties, benzoic acid and sodium
benzoate emitted to water and soil are not expected to volatilize to
the atmosphere or to adsorb to sediment or soil particles. From the
results of numerous removal experiments, the main elimination pathway
for both chemicals should be biotic mineralization. Data from
laboratory tests showed ready biodegradability for both substances
under aerobic conditions. Several isolated microorganisms (bacteria,
fungi) have been shown to utilize benzoic acid under aerobic or
anaerobic conditions. From the experimental data on bioconcentration,
a low to moderate potential for bioaccumulation is to be expected.
From valid test results available on the toxicity of benzoic acid
and sodium benzoate to various aquatic organisms, these compounds
appear to exhibit low to moderate toxicity in the aquatic compartment.
The lowest EC50 value of 9 mg/litre (cell multiplication inhibition)
reported in a chronic study was observed in the cyanobacterium
Anabaena inaequalis. EC50/LC50 values for the other aquatic
species tested were in the range of 60-1291 mg/litre. Immobilization
of Daphnia magna has been demonstrated to be pH dependent, with a
lower 24-h EC50 (102 mg/litre) at acidic pH. For the freshwater fish
golden ide (Leuciscus idus), a 48-h LC50 of 460 mg/litre has been
determined. Developmental effects have been found in frog (Xenopus)
embryos at a concentration of 433 mg/litre (96-h EC50 for
malformation). For sodium benzoate, exposure of juvenile stages of
aquatic organisms in a multispecies test (including Daphnia magna,
Gammarus fasciatus, Asellus intermedius, Dugesia tigrina,
Helisoma trivolvis, and Lumbriculus variegatus) resulted in 96-h
LC50 values of greater than100 mg/litre. A 96-h LC50 of 484
mg/litre has been determined in the freshwater fish fathead minnow
(Pimephales promelas). Owing to the limited available data on
exposure levels in water, a quantitative risk characterization with
respect to aquatic organisms in surface waters could not be performed.
Taking into account the rapid biodegradability, the low to moderate
bioaccumulation potential, the low toxicity to most aquatic species,
and the rapid metabolism of these substances, benzoic acid and sodium
benzoate will -- with the exception of accidental spills -- pose only
a minimal risk to aquatic organisms.
The few available data indicate that benzoic acid and sodium
benzoate have only a low toxicity potential in the terrestrial
environment. Except for the antimicrobial action of benzoic acid,
characterized by minimum microbiocidal concentrations ranging from 20
to 1200 mg/litre, no data on toxic effects of benzoic acid on
terrestrial organisms were available. For sodium benzoate, bacterial
and fungal growth were inhibited in a pH-dependent manner by
concentrations ranging from 100 to 60 000 mg/litre. Owing to the lack
of measured exposure levels, a sample risk characterization with
respect to terrestrial organisms could not be performed.
2. IDENTITY AND PHYSICAL/CHEMICAL PROPERTIES
Benzoic acid (CAS No. 65-85-0; C7H6O2; C6H5COOH;
benzenecarboxylic acid, phenyl carboxylic acid [E 210
(EU No. Regulation on Labelling of Foodstuffs)]; molecular weight
122.13) is a white solid that starts to sublime at 100°C, with a
melting point of 122°C and a boiling point of 249°C. Its solubility
in water is low (2.9 g/litre at 20°C), and its solution in water
is weakly acid (dissociation constant at 25°C = 6.335 × 10-5;
Maki & Suzuki, 1985; p Ka 4.19). It is soluble in ethanol
and very slightly soluble in benzene and acetone. It has an
octanol/water partition coefficient (log Kow) of 1.9. Its
vapour pressure at 20°C ranges from 0.11 to 0.53 Pa. Its calculated
Henry's law constant at 20°C was given as 0.0046-0.022 Pa.m3/mol
(BUA, 1995). Additional physical and chemical properties are
presented in the International Chemical Safety Card reproduced in
this document (Appendix 4).
Sodium benzoate (CAS No. 532-32-1; C7H5O2Na; benzoic acid,
sodium salt [E 211 (EU No. Regulation on Labelling of Foodstuffs)];
molecular weight 144.11) has a melting point above 300°C. It is very
soluble in water (550-630 g/litre at 20°C) and is hygroscopic at a
relative humidity above 50%. Its pH is about 7.5 at a concentration of
10 g/litre water. It is soluble in ethanol, methanol, and ethylene
glycol. Dry sodium benzoate is electrically charged by friction and
forms an explosive mixture when its dust is dispersed in air (Maki &
Suzuki, 1985).
3. ANALYTICAL METHODS
Analytical methods for the determination of benzoic acid include
spectrophotometric methods, which need extensive extraction procedures
and are not very specific; gas chromatographic (GC) methods, which are
more sensitive and specific but need lengthy sample preparation and
derivatization prior to determination; and high-performance liquid
chromatography (HPLC), which has a high specificity and minimum sample
preparation and does not require derivatization.
A direct determination of benzoic acid in air by flash desorption
at 240°C with helium into capillary-GC gave a detection limit of
0.1 ppm (0.5 mg/m3) in a 20-litre sample (=10 µg benzoic acid).
This method has been developed and used for monitoring occupational
exposure (Halvorson, 1984).
A method for the determination of benzoic acid in solid food at
0.5-2 g/kg levels involves extraction with ether into aqueous sodium
hydroxide and methylene chloride, conversion to trimethylsilyl esters,
and detection by GC and flame ionization (Larsson, 1983; AOAC, 1990).
For margarine, a method using HPLC and ultraviolet (UV) detection has
been described with prior extraction with ammonium acetate/acetic
acid/methanol (Arens & Gertz, 1990).
When benzoic acid is used as a preservative in soft drinks and
fruit drinks, other additives, colouring agents, and other acids
(e.g., sorbate) may interfere with its analysis. Liquid
chromatographic methods were developed to overcome this (e.g., Bennett
& Petrus, 1977; Puttemans et al., 1984; Tyler, 1984). For the
sensitive determination of benzoic acid in fruit-derived products, a
clean-up pretreatment with solid-phase extraction followed by liquid
chromatography with UV absorbance detection is described (Mandrou et
al., 1998). The detection limit is 0.6 mg/kg, with a range of
quantification of 2-5 mg/kg. For soft drinks, a simultaneous
second-order derivative spectrophotometric determination has been
developed (detection limit 1 mg/litre) (Castro et al., 1992). Sodium
benzoate was measured in soya sauce, fruit juice, and soft drinks
using HPLC with a UV spectrophotometric detector. Before injection,
all samples were filtered (Villanueva et al., 1994).
GC determination of low concentrations (down to 10 ng/ml) of
benzoic acid in plasma and urine was preceded by diethyl ether
extraction and derivatization with pentafluorobenzyl bromide (Sioufi &
Pommier, 1980). Detection was by 63Ni electron capture. HPLC methods
have been developed for the simultaneous determination of benzoic acid
and hippuric acid -- the metabolite of sodium benzoate that is
eliminated in the urine -- that require no extraction step (detection
limit for both, 1 µg/ml; Kubota et al., 1988). Hippuric acid and
creatinine levels have been determined simultaneously by HPLC, and
measured hippuric acid levels corrected for urinary creatinine
excretion (Villanueva et al., 1994).
4. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
4.1 Natural sources of benzoic acid
Benzoic acid is produced by many plants as an intermediate in the
formation of other compounds (Goodwin, 1976). High concentrations are
found in certain berries (see section 6.1). Benzoic acid has also been
detected in animals (see section 6.1). Benzoic acid therefore occurs
naturally in many foods, including milk products (Sieber et al., 1989,
1990).
4.2 Anthropogenic sources
4.2.1 Benzoic acid
Benzoic acid is produced exclusively by the liquid-phase
oxidation of toluene (Srour, 1998).
According to Srour (1998), the estimated global production
capacity of benzoic acid is 638 000 tonnes per year, although over
half of this is converted directly to phenol. The major producers of
benzoic acid are the Netherlands (220 000 tonnes per year) and Japan
(140 000 tonnes per year), followed by the USA (125 000 tonnes per
year). Another reference gives the total European capacity as less
than 153 000 tonnes (SRI, 1998).
Benzoic acid is detected in car exhaust gases, presumably as an
oxidation product of toluene (Kawamura et al., 1985), and in Japanese
cigarettes (12 and 28 µg per cigarette in mainstream and sidestream
smoke, respectively; Sakuma et al., 1983). It can also be produced
through the photochemical degradation of benzoic acid esters used as
fragrance ingredients (Shibamoto & Umano, 1985; Shibamoto, 1986).
Benzoic acid has been detected in wastewater from the wood production
industry in Norway and Sweden (Carlberg et al., 1986; Lindström &
Österberg, 1986) and in foundry waste leachates (Ham et al., 1989), as
well as in extracts of fly ash from municipal incinerators
(Tong et al., 1984).
4.2.2 Sodium benzoate
Sodium benzoate is produced by the neutralization of benzoic acid
with sodium hydroxide. Worldwide sodium benzoate production in 1997
can be estimated at about 55 000-60 000 tonnes (Srour, 1998). The
largest producers are the Netherlands, Estonia, the USA, and China.
4.3 Uses
4.3.1 Benzoic acid
In 1988, of the benzoic acid produced in Europe, about 60% was
further processed to phenol and 30% to caprolactam (for nylon fibres).
Five per cent was used for the production of sodium and other
benzoates, 3% for benzoyl chloride, and the rest for alkyd resins,
benzoate esters, such as methyl benzoate, and various other products
(Srour, 1989). These percentages are still approximately correct today
(Srour, 1998). Caprolactam seems to be produced only by European
companies (Srour, 1998).
Benzoic acid is increasingly used in the production of diethylene
and dipropylene glycol dibenzoate plasticizers in adhesive
formulations (about 40 000 tonnes in 1997). It is also used to improve
the properties of alkyd resins for paints and coatings and as a "down
hole" drilling mud additive in secondary oil production. Its use as a
rubber polymerization retarder is diminishing (Srour, 1998).
Benzoic acid and sodium benzoate (see section 4.3.2) are used as
preservatives in beverages, fruit products, chemically leavened baked
goods, and condiments, preferably in a pH range below 4.5.
A disadvantage is the off-flavour they may impart to foods (Chipley,
1983). Owing to their inhibitory effect on yeast, they cannot be used
in yeast-leavened products (Friedman & Greenwald, 1994). Examples of
upper concentrations allowed in food are up to 0.1% benzoic acid (USA)
and between 0.15% and 0.25% (other countries) (Chipley, 1983). The
European Commission limits for benzoic acid and sodium benzoate are
0.015-0.5% (EC, 1995).
Benzoic acid and its salts and esters are found in 11 of 48 (23%)
toothpastes (Sainio & Kanerva, 1995) to a maximum of 0.5% (Ishida,
1996) and in mouthwashes and dentifrices. Benzoic acid is also used in
cosmetics (in creams and lotions with pH values under 4, up to 0.5%)
(Wallhäusser, 1984). Sixteen out of 71 deodorants tested contained
benzoic acid (Rastogi et al., 1998).
Benzoic acid is a breakdown product of benzoyl peroxide, which is
used as an additive at levels of between 0.015% and 0.075% to bleach
flour (Friedman & Greenwald, 1994) and in dermatological antifungal
preparations (BMA, 1998). Benzoic acid is reported to leach from
denture-base acrylic resins, where benzoyl peroxide is added as a
polymerization initiator (Koda et al., 1989, 1990).
Benzoic acid can be used in combination with salicylic acid
(Whitfield's ointment) as a fungicidal treatment for ringworm (BMA,
1998).
4.3.2 Sodium benzoate
Although undissociated benzoic acid is the more effective
antimicrobial agent for preservation purposes, sodium benzoate is used
preferably, as it is about 200 times more soluble than benzoic acid.
About 0.1% is usually sufficient to preserve a product that has been
properly prepared and adjusted to pH 4.5 or below (Chipley, 1983).
A major market for sodium benzoate is as a preservative in the
soft drink industry, as a result of the demand for high-fructose corn
syrup in carbonated beverages. Sodium benzoate is also widely used as
a preservative in pickles, sauces, and fruit juices (Srour, 1998).
Benzoic acid and sodium benzoate are used as antimicrobial agents in
edible coatings (Baldwin et al., 1995).
Sodium benzoate is also used in pharmaceuticals for preservation
purposes (up to 1.0% in liquid medicines) and for therapeutic regimens
in the treatment of patients with urea cycle enzymopathies
(see section 9).
Possibly the largest use of sodium benzoate, accounting for
30-35% of the total demand (about 15 000 tonnes of benzoic acid), is
as an anticorrosive, particularly as an additive to automotive engine
antifreeze coolants and in other waterborne systems (Scholz &
Kortmann, 1991; Srour, 1998). A new use is the formulation of sodium
benzoate into plastics such as polypropylene, to improve strength and
clarity (BFGoodrich Kalama Inc., 1999). Sodium benzoate is used as a
stabilizer in photographic baths/processing (BUA, 1995).
4.4 Estimated global release
From data provided by the German producers, emissions of benzoic
acid from industrial processes were less than 525 kg per year into the
atmosphere, less than 3 tonnes per year into the River Rhine, and
8 tonnes per year into sewage or water purification plants (BUA,
1995). No data were available from other countries.
Other anthropogenic releases of benzoic acid and sodium benzoate
into the environment are emissions into water and soil from their uses
as preservatives in food, toothpastes, mouthwashes, dentifrices, and
cosmetics. There were no data available on the emission of benzoic
acid from the disposal of antifreeze mixtures and waterborne cooling
systems and other miscellaneous industrial uses.
The amount of benzoic acid emitted to air from car exhaust gases
as an oxidation product is not quantifiable from the available data.
5. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, TRANSFORMATION,
AND ACCUMULATION
5.1 Transport and distribution between media
5.1.1 Benzoic acid
From its use pattern (see section 4), it can be expected that
benzoic acid is released to surface waters and (from dumping sites) to
leaching water (and groundwater). Minor amounts are expected to be
emitted to the atmosphere. From its physicochemical properties (vapour
pressure, Henry's law constant; see section 2), a significant
volatilization of benzoic acid from water or soil is not expected.
Owing to its solubility in water (see section 2), wet deposition from
air may occur. Experimental data on wet and dry deposition from air
are not available.
5.1.2 Sodium benzoate
No information on the environmental transport and distribution of
sodium benzoate could be identified. Owing to its use pattern, which
is similar to that of benzoic acid, most of the amounts released to
the environment are also expected to be emitted to aquatic
compartments (e.g., surface waters).
5.2 Transformation
5.2.1 Benzoic acid
The experimental determination of the photodegradation of benzoic
acid in aqueous solution (25°C; lambda = 240-300 nm) in terms of
quantum yield (average number of photons absorbed) resulted in very
low values -- in the order of 6 × 10-2 mol/einstein1 (Oussi et
al., 1998). However, benzoic acid adsorbed on silica gel (SiO2) and
irradiated with UV light (lambda > 290 nm) for 17 h showed 10.2%
photodegradation (Freitag et al., 1985). This may be due to a
photocatalytic effect, which was also observed with other oxides,
notably zinc oxide (ZnO) and titanium dioxide (TiO2). When benzoic
acid was irradiated with sunlight in aqueous suspensions of zinc or
titanium dioxide, 67% (after 2-3 h) or 90% (after 24 h) of the applied
amount was mineralized (Kinney & Ivanuski, 1969; Matthews, 1990).
1 An einstein is a unit of light energy used in photochemistry,
equal to Avogadro's number times the energy of one photon of light
of the frequency in question.
Indirect photolysis by reaction with hydroxyl radicals is
expected to be low. Hydroxyl radical rate constants (kOH) for
benzoic acid and its anion have been estimated to be approximately
0.5 × 10-12 and 2 × 10-12 cm3/s, respectively (Palm et al., 1998).
Standardized tests on ready (MITI, 1992) or inherent (Zahn &
Wellens, 1980) biodegradation showed benzoic acid to be readily
biodegraded. The degrees of aerobic degradation were as follows:
MITI I 85% (100 mg/litre; (MITI, 1992)
test 2 weeks; OECD
No. 301C)
Zahn-Wellens >90% (508 mg/litre; (Zahn & Wellens,
test 2 days) 1980)
Easy degradation of benzoic acid to methane and carbon dioxide
was also observed in different non-standardized experiments using
sewage sludge as inoculum (BUA, 1995). Benzoic acid was found to be
degraded by adapted anaerobic sewage sludge at 86-93% after 14 days
(Nottingham & Hungate, 1969), by aerobic activated sludge (adapted) at
>95% after 5-20 days (Pitter, 1976; Lund & Rodriguez, 1984), and by
unadapted aerobic activated sludge at 61-69% after 2-3 days with a
preceding lag time of 2-20 h (Urano & Kato, 1986). The use of a
synthetic sewage inoculated with laboratory bacterial cultures led to
complete degradation of benzoic acid after 14 days under anaerobic
conditions (Kameya et al., 1995).
A greater variability in degradation (0-100%) was seen in tests
using environmental matrices (e.g., rain, lake water, seawater, soil,
etc.). It depended mainly on substance concentration and time for
acclimation (see Table 1). Test durations exceeding 2 days resulted in
removal of >40% when initial concentrations were below 20 mg/litre.
A rapid mineralization occurred in groundwater and subsurface soil
samples. In groundwater, a half-life of 41 h has been found for
benzoic acid (initial concentration 1-100 µg/litre; metabolized to
14CO2) under aerobic conditions (Ventullo & Larson, 1985).
Half-lives of 7.3 h and 18.2 h, respectively, have been observed for
aerobic and anaerobic degradation of benzoic acid (initial
concentration 1 mg/kg dry weight; metabolized to 14CO2) in
subsurface soils of septic tank tile fields (Ward, 1985). Anaerobic
degradation of benzoic acid (initial concentration 250 mg
carbon/litre) in a methanogenic microcosm (consisting of aquifer
solids and groundwater) required 4 weeks of adaptation, followed by
nearly complete depletion after 8 weeks of incubation (Suflita &
Concannon, 1995).
Several isolated microorganisms have been shown to utilize (and
therefore probably degrade) benzoic acid under aerobic or anaerobic
conditions. They include, among others, fungal species such as
Rhodotorula glutinis and other yeast-like fungi (Kocwa-Haluch &
Lemek, 1995), the mould Penicillium frequentans (Hofrichter &
Fritsche, 1996), and bacteria, such as Alcaligenes denitrificans
(Miguez et al., 1995), Rhodopseudomonas palustris, several strains
of denitrifying pseudomonads (Fuchs et al., 1993; Elder & Kelly, 1994;
Harwood & Gibson, 1997), and Desulfomicrobium escambiense
(Sharak Genthner et al., 1997).
Although benzoic acid is primarily metabolized to hippuric acid
in rats (see section 7), some other species do excrete other
metabolites, such as dibenzoylornithine (hen), benzoylglutamic acid
(Indian fruit bat), benzoylarginine (tick, insects), or benzoyltaurine
(southern flounder, Paralichthys lethostigma) (Parke, 1968; Goodwin,
1976; James & Pritchard, 1987).
5.2.2 Sodium benzoate
Experimental data on photodegradation of sodium benzoate are not
available. As with benzoic acid, photolysis in aqueous solution is
assumed to be unlikely with regard to its known UV spectra
(Palm et al., 1998). Indirect photolysis by reaction with hydroxyl
radicals plays only a minor role, with estimated and measured hydroxyl
rate constants of about 0.33 × 10-11 cm3/s
(Palm et al., 1998).
Sodium benzoate was readily biodegradable under aerobic
conditions in several standard test systems:
Modified 84% (100 mg/litre; (King & Painter,
MITI test 10 days) 1983)
Modified 80-90% (50 mg/litre; (Salanitro et al.,
Sturm test 7 days) 1988)
Closed bottle 75-111% (5 mg/litre; (Richterich &
test 30 days) Steber, 1989)
Degradation assays using seawater as test medium ("natural
water") or as inoculum (marine filter material given into a synthetic
marine medium) according to an adapted Organisation for Economic
Co-operation and Development (OECD) guideline (301B) resulted in a
degradation of 85% and 97%, respectively (10 mg/litre; carbon dioxide
measurement; 28 days) (Courtes et al., 1995).
Anaerobic mineralization of sodium benzoate (50-90 mg/litre) by
domestic sewage sludge varied from 50% to 96.5% (measurement of carbon
dioxide and methane; 28-61 days) (Birch et al., 1989). In another
study using anaerobic sludge from sewage works receiving a mixture of
domestic and industrial wastewaters, 93% mineralization was observed
after 1 week of incubation (measurement of carbon dioxide and methane;
initial concentration 50 mg carbon/litre) (Battersby & Wilson, 1989).
Table 1: Removal of benzoic acid in freshwater, marine, and soil matrices.
Matrix Initial Conditions Duration Removal Measured Reference
concentration (days) (%) parameter
(mg/litre
or mg/kg)
Rainwater 0.001 22°C; shaking 2 0 benzoic acid Kawamura &
once per day; dark 7 40 Kaplan (1990)
45 100
Lake water 0.059 29°C; 7 98.7 14C (in CO2, Rubin et al.
(eutrophic/ no shaking; dark biomass) (1982)
mesotrophic)
Seawater 20°C; dark; 14C (in Shimp &
(estuary) rotary shaking CO2, biomass) Young (1987)
USA 20 30 <10
0.005 8 70-80
Canada 20 16 60
0.005 10 >70
Seawater 2 5 75 BODa Takemoto
et al. (1981)
Soil 20 2 mg benzoic acid 70 63 14CO2 Haider et al.
(grey soil, in 0.1 ml acetone (1974)
alkaline) + 100 g soil
+ 10 ml H2O
Soil 0.05 24°C; 20-25% 15 40 14CO2 Federle (1988)
(sand; moisture content
18.9 m depth)
a BOD = biological oxygen demand.
Benzoate-acclimated sludges were reported to be capable of completely
degrading benzoate concentrations of 3000 mg/litre within 5-7 days
(Kobayashi et al., 1989).
5.3 Accumulation
5.3.1 Benzoic acid
The n-octanol/water partition coefficient (log Kow) of 1.9
(see section 2) indicates a low potential for bioaccumulation.
Consistently, measured bioconcentration factors (BCFs) found in
aquatic biota were low. BCFs of <10 (based on wet weight) have been
determined for fish (golden ide, Leuciscus idus melanotus) and green
algae (Chlorella fusca) after 3 and 1 days, respectively
(Freitag et al., 1985). A 6-day BCF of 7.6 has been reported for
another green alga (Selenastrum capricornutum) (Mailhot, 1987),
and a 5-day BCF of 1300 (based on dry weight) in activated sludge
(Freitag et al., 1985). The following 24-h bioaccumulation factors
(focusing on uptake via medium plus feed within food chain members)
have been obtained in aquatic model ecosystems operated with
0.01-0.1 mg of radiolabelled benzoic acid per litre: 21 (mosquitofish,
Gambusia affinis), 102 (green alga, Oedogonium cardiacum),
138 (mosquito larvae, Culex quinquifasciatus), 1772 (water flea,
Daphnia magna), and 2786 (snail, Physa sp.). Except for
Daphnia and snail, the values were low (Lu & Metcalf, 1975).
However, the very low exposure concentrations could likely have
resulted in the calculation of the high BCF values, even with moderate
uptake. Moreover, because this was a radiolabel study, it remains
unclear if the label was still the parent compound.
Geoaccumulation of benzoic acid has also been found to be low.
Depending on soil depth, sorption coefficients (Kd) of 0.62 (18.9
m) to 1.92 (0.4 m) have been measured (Federle, 1988). Mobility
determinations of 14C-labelled benzoic acid in different soils by
means of thin-layer chromatography showed benzoic acid to be
moderately mobile. Its mobility was positively correlated with soil pH
and negatively correlated with aluminium and iron contents and
effective anion exchange capacity (Stolpe et al., 1993).
5.3.2 Sodium benzoate
No experimental data on bioaccumulation or geoaccumulation of
sodium benzoate have been identified. From the information on benzoic
acid, a significant potential for accumulation is not to be expected.
6. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
6.1 Environmental levels
Generally, benzoic acid can occur in almost all environmental
compartments. Whether it exists in the undissociated or dissociated
form depends on the specific physicochemical conditions. Above pH 6,
the benzoate anion prevails (Chipley, 1983).
There is a series of reports on positive qualitative analyses of
benzoic acid in various environmental media, such as air (Belgium:
Cautreels & van Cauwenberghe, 1978; Germany: Helmig et al., 1989),
rain or snow (Norway: Lunde et al., 1977; Germany: Winkeler et al.,
1988), surface waters (Norway, river: Schou & Krane, 1981), and soils
(United Kingdom, heathland soil: Jalal & Read, 1983; Germany, river
terrace soil: Cordt & Kußmaul, 1990), but these do not provide
quantitative measurements.
Semiquantitative measurements of concentrations of benzoic acid
in urban air in Pasadena, California (USA) were in the range of
0.09-0.38 µg/m3 (Schuetzle et al., 1975). This was comparable to
quantitative measurements performed in 1984 in Los Angeles, California
(USA), which resulted in atmospheric concentrations of 0.005-0.13
µg/m3 (n = 8) (Kawamura et al., 1985). Most of the quantitative
data compiled in Table 2 with respect to water samples refer to
concentrations of benzoic acid in groundwater, with a maximum of 27.5
mg/litre measured in the vicinity of a point source.
Benzoic acid occurs naturally in free and bound form in many
plant and animal species. It is a common metabolite in plants and
organisms (Hegnauer, 1992). Appreciable amounts have been found in gum
benzoin (around 20%) and most berries (around 0.05%) (Budavari et al.,
1996). For example, ripe fruits of several Vaccinium species (e.g.,
cranberry, V. vitis idaea; bilberry, V. macrocarpon) contain as
much as 300-1300 mg free benzoic acid per kg fruit (Hegnauer, 1966).
Benzoic acid is also formed in apples after infection with the fungus
Nectria galligena (Harborne, 1983) or in Pinus thunbergii callus
inoculated with a pathogenic pine wood nematode (Bursaphelenchus
xylophilus) (Zhang et al., 1997). Among animals, benzoic acid has
been identified primarily in omnivorous or phytophageous species,
e.g., in viscera and muscles of the ptarmigan (Lagopus mutus)
(Hegnauer, 1989) as well as in gland secretions of male muskoxen
(Ovibos moschatus) (Flood et al., 1989) or Asian bull elephants
(Elephas maximus) (Rasmussen et al., 1990).
Owing to its occurrence in many organisms, benzoic acid is
naturally present in foods (review in Sieber et al., 1989, 1990). Some
typical examples specifying reported ranges of means in selected foods
have been compiled from Sieber et al. (1989) as follows:
Milk traces - 6 mg/kg
Yoghurt 12-40 mg/kg
Cheese traces - 40 mg/kg
Fruits (excluding traces - 14 mg/kg
Vaccinium species)
Potatoes, beans, cereals traces - 0.2 mg/kg
Soya flour, nuts 1.2-11 mg/kg
Honeys from different floral sources (n = 7) were found to
contain free benzoic acid at concentrations of <10 mg/kg (n = 5)
and of <100 mg/kg (n = 2) (Steeg & Montag, 1987).
Because benzoic acid and its compounds are used as food
preservatives (see section 4), some processed foods contain
artificially elevated concentrations of these substances (see section
6.2).
6.2 Human exposure
The main route of exposure of the general population to benzoic
acid or sodium benzoate is likely via foodstuffs that contain the
substances naturally or added as antimicrobial agents. There are a few
analyses of processed foodstuffs available. They refer to different
types of food items (juice, soft drinks, soya sauce varieties) from
the Philippines (a total of 44 samples) and from Japan (a total of 31
samples) and to orange drinks sampled in England. The concentrations
of sodium benzoate in the Philippine dietary samples ranged from 20 to
>2000 mg/litre. The range in the Japanese products was 50-200
mg/litre, thus reflecting the lower maximum level of sodium benzoate
allowed to be added to food in Japan as compared with the Philippines
(Villanueva et al., 1994). Orange drinks from England contained sodium
benzoate at concentrations ranging from 54 to 100 mg/litre (mean 76.7
mg/litre; n = 6) (Freedman, 1977).
Generally, the actual uptake depends on the individual's choice
of food to be consumed and the different limit values in different
countries. Several intake estimations have been published. Three
Japanese studies reported average daily intakes of benzoic acid from
processed foodstuffs to be 10.9 mg per person (Toyoda et al., 1983a)
and 1.4 mg per person (Toyoda et al., 1983b; Yomota et al., 1988),
corresponding to 0.02-0.2 mg/kg body weight (for persons with a body
weight of 50-70 kg). Both of the latter studies used the market basket
method for intake calculations, whereas the first-mentioned study
calculated intakes using the results of a national nutrition survey.
The concentrations of benzoic acid in 3319 food samples analysed for
this study (Toyoda et al., 1983a) ranged from not detected to 2100
mg/kg food. The maximum was found in salted fish (n = 7; mean 754
mg/kg). Another survey refers to the United Kingdom, where analyses of
benzoic acid in foods and drinks in which it is permitted as well as
intake estimates have been performed (UK MAFF, 1995). Sixty-five out
of 122 samples tested contained detectable benzoic acid. The highest
Table 2: Concentrations of benzoic acid in rain, snow, groundwater, and leachate samples.
Medium Location; sampling date Concentration Reference
(µg/litre)
Los Angeles area, California, Sum concentrationsa Kawamura & Kaplan
USA; 1982-1983 (1986)
Rain: urban 0.06-10.2 (n = 6)
Rain: semirural 0.02 (n = 1)
Snow: rural 0.04-0.1 (n = 3)
Groundwater Wyoming, USA (near underground 16-860 (n = 3) Stuermer et al.
coal gasification site; 15 months (1982)
after the end of gasification)
Groundwater Florida, USA (near wood treatment 10-27 500 (n = 3) Goerlitz et al.
facility); 1984 (1985)
Groundwater Ontario, Canada (near traces (n = 2) Barker et al.
landfillb); 1983 (1988)
Groundwater Barcelona area, Spain up to 0.21 (n = 3) Guardiola et al.
(near landfillb) (1989)
Leachate Ontario, Canada; 1981 <0.1->1000 (n = 5) Reinhard & Goodman
(from landfillb) (1984)
Leachate Ontario, Canada; 1983 traces (n = 2) Barker et al.
(from landfillb) (1988)
Leachate USA; 1986-1988 200-400c (n = 3) Ham et al.
(from foundry (1989)
wastes)
a Including benzoic acid, 3-methyl benzoic acid, and 4-methyl benzoic acid.
b Receiving rural, municipal (domestic), and industrial wastes.
c Concentrations estimated from gas chromatography/mass spectrometry data.
concentrations were found in sauces (mean 388 mg/kg; n = 20; range
71-948 mg/kg), reduced sugar jam (mean 216 mg/kg; n = 4; range
<20-333 mg/kg), non-alcoholic drinks (mean 162 mg/kg; n = 20; range
55-251 mg/kg), and semipreserved fish product (653 mg/kg; n = 1).
The survey found that the concentrations of benzoic acid detected
would lead to a dietary intake below 5 mg/kg body weight per day, even
for adults with an above-average consumption.
A frequent contributor to dietary exposure is soft drinks. A
rough estimation based on the average daily consumption in Germany of
such drinks (372 ml non-alcoholic beverages, excluding bottled water;
BAGS, 1995) by 19- to 24-year-old men, assuming the concentration of
benzoic acid present corresponds to a maximum allowable level of
150 mg/litre (EC, 1995), would result in a mean daily intake of
55.8 mg benzoic acid per person (or 0.80 mg/kg body weight, assuming
a 70-kg body weight). For comparison, a similar calculation with
sugar-free marmalade, jam, and similar spreads, which are allowed to
contain higher levels of benzoic acid (500 mg/kg; EC, 1995), would
result in a possible intake of 4.1 mg per person per day, or
0.06 mg/kg body weight per day (assumes a daily consumption of 8.2 g,
according to BAGS, 1995). This was more than a possible intake via
fruits containing natural benzoic acid. For example, a daily
consumption of 40.4 g of fruits (BAGS, 1995) would lead to a possible
intake of 0.57 mg benzoic acid per person per day (or 0.008 mg/kg body
weight for a 70-kg person), if the reported maximum of 14 mg benzoic
acid/kg (see section 6.1) were present.
The Joint FAO/WHO Expert Committee on Food Additives (JECFA)
assessed the intake of benzoates from information provided by nine
countries (Australia, China, Finland, France, Japan, New Zealand,
Spain, United Kingdom, and USA) (WHO, 1999). Because diets differ
among countries, the foods that contribute to benzoate intake would be
expected to vary. The food category that contributed most to benzoate
intake was soft drinks (carbonated, water-based, flavoured drinks) for
Australia/New Zealand, France, the United Kingdom, and the USA. In
Finland, 40% was in soft drinks. Soya sauce was the main source of
benzoate in China and the second most important in Japan. The best
estimates of national mean intakes of benzoates by consumers ranged
from 0.18 mg/kg body weight per day in Japan to 2.3 mg/kg body weight
per day in the USA. These estimates were based on analyses involving
either model diets or individual dietary records and maximum limits
specified by national governments or the European Union. The estimated
intake by high consumers of benzoates, based on food additive levels
in national standards, was 7.3 mg/kg body weight per day in the USA
and 14 mg/kg body weight per day in China.
Benzoates have been detected in groundwater, but not in
drinking-water.
Quantitative information on (oral or dermal/mucosal) exposure via
cosmetic, hygienic, or medical products is rare, but the data
available indicate a remarkable contribution to exposure. There are
reports on leaching of benzoic acid from denture-base acrylic resins.
After 10 days of immersion in artificial saliva, concentrations of up
to about 3 mg/litre have been observed for benzoic acid, which is
formed as a degradation product of the benzoyl peroxide that is added
as a polymerization initiator (Koda et al., 1989, 1990). In Japan,
commercial toothpastes have been found to contain benzoic acid at
concentrations ranging from 800 to 4450 mg/kg (n = 18). Use of the
toothpaste with the highest concentration (by 40 20-year-old female
students) would result in a calculated daily intake of about 2.23 mg
per person. This was about the same amount as their estimated intake
from diet (Ishida, 1996). Benzoic acid is also used in dermatology as
a fungicidal topical treatment for ringworm (Tinea spp.). The
emulsifying ointment preparation contains benzoic acid at 6% and is
applied twice daily (Goodman et al., 1990; BMA, 1998).
Recent quantitative monitoring data on concentrations of benzoic
acid or salts in ambient or indoor air are not available. Considering
the few (low) levels of benzoic acid measured in urban air in the
past, with a maximum of 0.38 µg/m3 (see section 6.1), inhalation may
contribute only marginally to exposure of the general population.
Using this maximum, a daily inhalative dose of 8.74 µg per person (or
0.12 µg/kg body weight) is obtained (assuming a daily inhalation
volume of 23 m3 for a 70-kg adult male; WHO, 1994).
Few quantitative data on occupational exposure have been
identified. Nevertheless, there is a potential for inhalation or
dermal contact in the chemical and allied product industries as well
as in workplaces where these products are used. Air samples (n = 50)
collected in an industrial environment (no further details given) over
a year's time showed benzoic acid concentrations ranging from not
detected to 1.5 mg/m3 (Halvorson, 1984). On the basis of the latter
value, an inhalative dose of 14.4 mg per person per 8-h working time
(or 0.2 mg/kg body weight) would result (assuming an inhalation volume
of 9.6 m3 for an 8-h exposure with light activity; WHO, 1994).
However, because of the lack of information on specific working
operations and conditions involved (e.g., duration of exposure, use of
protective clothes, etc.), it is impossible to derive a realistic
estimate of occupational exposure.
7. COMPARATIVE KINETICS AND METABOLISM IN LABORATORY ANIMALS
AND HUMANS
After oral ingestion of benzoic acid and sodium benzoate, there
is a rapid absorption (of undissociated benzoic acid) from the
gastrointestinal tract in experimental animals or humans (US FDA,
1972a, 1973). From the figures on excretion given below, 100%
absorption can be assumed. In humans, the peak plasma concentration is
reached within 1-2 h (Kubota et al., 1988; Kubota & Ishizaki, 1991).
Benzoic acid is not completely absorbed by the dermal route. In a
study with six human subjects, Feldmann & Maibach (1970) found an
uptake of 36% of the applied dose (14C-labelled benzoic acid
dissolved in acetone; 4 µg/cm2; circular area of 13 cm2; ventral
surface of the forearm; non-occlusive) within 12 h. The total uptake
within 5 days was 43%. In a second study with 6-7 subjects (comparable
method; application of 3, 400 or 2000 µg/cm2), the percent
absorption decreased from 35% to 14% within 24 h. However, the total
uptake per cm2 increased from 1 to 288 µg (Wester & Maibach, 1976).
For sodium benzoate, no data concerning dermal uptake were identified
in the literature.
In vivo dermal studies with benzoic acid in experimental
animals (e.g., guinea-pigs, mice, rats, pigs, dogs, rhesus monkeys)
confirm the results with humans (Hunziker et al., 1978;
Andersen et al., 1980; Wester & Noonan, 1980; Bronaugh et al., 1982a;
Reifenrath et al., 1984; Carver & Riviere, 1989; Maibach & Wester,
1989; Bucks et al., 1990). Absorption ranged from 25% in pigs
(Reifenrath et al., 1984; Carver & Riviere, 1989) to 89% in rhesus
monkeys (Wester & Noonan, 1980; Maibach & Wester, 1989; Bucks et al.,
1990). Due to the good database on humans and animals in vivo,
in vitro studies performed with animal or human skin are not
considered further (Franz, 1975; Bronaugh et al., 1982b;
Hotchkiss et al., 1992; MacPherson et al., 1996).
No information is available on absorption via inhalation.
After oral and dermal uptake, benzoate is metabolized in the
liver by conjugation with glycine, resulting in the formation of
hippuric acid (Feldmann & Maibach, 1970; US FDA, 1972a; WHO, 1996;
Feillet & Leonard, 1998). The rate of biotransformation in humans is
high: after oral doses of 40, 80 or 160 mg sodium benzoate/kg body
weight, the transformation to hippuric acid was independent of the
dose -- about 17-29 mg/kg body weight per hour, corresponding to about
500 mg/kg body weight per day (Kubota & Ishizaki, 1991). Other authors
obtained higher values of 0.8-2 g/kg body weight per day (US FDA,
1972a, 1973; WHO, 1996). Hippuric acid is rapidly excreted in urine.
In humans, after oral doses of up to 160 mg/kg body weight, 75-100% of
the applied dose is excreted as hippuric acid within 6 h after
administration, and the rest within 2-3 days (Kubota et al., 1988;
Fujii et al., 1991; Kubota & Ishizaki, 1991).
The limiting factor in the biosynthesis of hippuric acid is the
availability of glycine. The utilization of glycine in the
detoxification of benzoate results in a reduction in the glycine level
of the body. Therefore, the ingestion of benzoic acid or its salts
affects any body function or metabolic process in which glycine is
involved; for example, it leads to a reduction in creatinine,
glutamine, urea, and uric acid levels (US FDA, 1972a, 1973; Kubota &
Ishizaki, 1991; WHO, 1996).
Another metabolite of benzoate is the benzoyl glucuronide. For
example, the dog excretes considerable amounts of this metabolite in
the urine (20% after a single dose of 50 mg/kg body weight; Bridges et
al., 1970). In other species, this metabolite appears only after
higher doses of about 500 mg/kg body weight (see above) of benzoic
acid or sodium benzoate, resulting in a depletion of the glycine pool
(Bridges et al., 1970; US FDA, 1972a; Kubota et al., 1988). In cats,
glucuronidation is generally very low (Williams, 1967).
In some species, including humans, minor amounts of benzoic acid
itself are also excreted in the urine (Bridges et al., 1970; Kubota &
Ishizaki, 1991).
Experiments on the distribution and elimination of 14C-benzoate
in the rat have shown no accumulation of sodium benzoate or benzoic
acid in the body (US FDA, 1972a, 1973).
In the acid conditions of the stomach, the equilibrium moves to
the undissociated benzoic acid molecule, which should be absorbed
rapidly. Benzoate from sodium benzoate would change from the ionized
form to the undissociated benzoic acid molecule. As a result, the
metabolism and systemic effects of benzoic acid and sodium benzoate
can be evaluated together.
7.1 Precursors of benzoic acid
Benzyl acetate, its hydrolysis product, benzyl alcohol, and the
oxidation product of this alcohol, benzaldehyde, are precursors of
benzoic acid in experimental animals and humans. Benzyl acetate is
metabolized to benzoic acid and further to hippuric acid and benzoyl
glucuronide to an extent of >90% both in mice and in rats of
different strains. Benzyl alcohol was metabolized to benzoic acid and
its conjugates in preterm infants. Benzaldehyde is metabolized to
benzoic acid and its conjugates in rabbits to an extent of
approximately 90% (WHO, 1996).
8. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS
8.1 Single exposure
With oral LD50 values (administration by gavage) of 3040 mg
benzoic acid/kg body weight in rats (Bio-Fax, 1973) and 1940-2263 mg
benzoic acid/kg body weight in mice (McCormick, 1974; Abe et al.,
1984), the acute toxicity of benzoic acid is low. Clinical signs of
intoxication (reported for rats only) included diarrhoea, muscular
weakness, tremors, hypoactivity, and emaciation (Bio-Fax, 1973). With
oral LD50 values of 2100-4070 mg sodium benzoate/kg body weight in
rats, the acute toxicity of sodium benzoate is similar to that of
benzoic acid, as are the symptoms (Smyth & Carpenter, 1948;
Deuel et al., 1954; Bayer AG, 1977).
In four cats given diets containing 0 or 1% benzoic acid
(approximately 0 or 450-890 mg/kg body weight), aggression,
hyperaesthesia, and collapse starting 14-16 h after feed uptake were
seen at a dose level equal to 630 mg/kg body weight. The duration of
the syndrome was about 18-176 h, and the mortality rate was 50%. The
histopathological examination of the two cats that died revealed
degenerative changes in liver, kidneys, and lung, but no pathological
findings in brain or spinal cord (Bedford & Clarke, 1972). The authors
attributed the higher toxicity of benzoic acid in cats compared with
other species to the low capacity of cats for glucuronidation
(see section 7).
In rats, exposure by inhalation to 26 mg/m3 over 1 h caused no
mortality, but generalized inactivity and lacrimation were noted. The
gross autopsy gave no significant findings (no further information
available; Bio-Fax, 1973).
In a limit test with rabbits, no mortality or signs of
intoxication were seen after dermal application of 10 000 mg/kg body
weight. The gross autopsy gave no significant findings (no further
information available; Bio-Fax, 1973).
8.2 Irritation and sensitization
8.2.1 Benzoic acid
Although there is a wide range of results from mostly
non-standardized tests using various scoring systems, it can be
concluded that benzoic acid is slightly irritating to the skin and
irritating to the eyes.
In different experiments with rabbits, which have not been
performed according to current guidelines, benzoic acid applied as dry
powder or in the form of a paste was not irritating to slightly
irritating to the skin (score 1.66/8: Bio-Fax, 1973; no score given:
Bayer AG, 1978; primary skin irritation index 0.5 [no further
information available]: RCC Notox, 1988a).
In an acute eye irritation/corrosion study with rabbits conducted
according to OECD Guideline 405, some eye irritation was reported
after application of benzoic acid in the form of a paste. Within 72 h,
the scores for chemosis, reddening of the conjunctivae, iritis, and
keratitis always remained at <2 (Bayer AG, 1986).
In different non-standardized experiments with the solid
substance, moderately irritating to severely irritating effects on the
eye were noted (score 65/110: Bio-Fax, 1973; no score given: Bayer AG,
1978; score up to 108/110 [eyes rinsed after instillation] or up to
50/100 [eyes not rinsed]: Monsanto Co., 1983; score 35 according to
the scheme of Kay & Calandra, 1962: RCC Notox, 1988b).
In a maximization test, none of 15 guinea-pigs reacted positively
after induction and challenge with a 10-20% solution of benzoic acid
in water (Gad et al., 1986). In addition, the substance also tested
negative in a Buehler test with guinea-pigs and in an ear swelling
test and local lymph node assay with mice (Gad et al., 1986; Gerberick
et al., 1992). The concentrations used for induction and challenge
were 10-20% in acetone or water.
However, a dose-dependent positive result was obtained in an ear
swelling test with five guinea-pigs (induction with 0.2, 1, 5, or 20%
in absolute ethyl alcohol; no challenge) used as a model for detecting
agents causing non-immunological contact urticaria in humans. At
several other regions (back, abdomen, flank site), a concentration of
20% failed to produce any reactions (Lahti & Maibach, 1984).
8.2.2 Sodium benzoate
An acute dermal irritation/corrosion study with rabbits conducted
according to OECD Guideline 404 (no data about physical state; score
0: RCC Notox, n.d., a) as well as a non-standardized experiment with
the solid substance (score not given: Bayer AG, 1977) gave no
indication for skin irritating effects.
In a study performed according to OECD Guideline 405 (no data
about physical state; RCC Notox, n.d., b), sodium benzoate was only
slightly irritating to the eye (score 9.3, according to the scheme of
Kay & Calandra, 1962). The application of the solid substance in a
non-standardized experiment caused no irritation (score not given:
Bayer AG, 1977).
For sodium benzoate, no data on sensitizing effects were
identified in the available literature.
8.3 Short-term exposure
8.3.1 Oral exposure
In general, the database for benzoic acid and sodium benzoate is
limited, and there are no studies available performed according to
current guidelines. In addition, the documentation of these studies in
most cases is insufficient. Detailed information is given in Table 3.
From the available studies, it can be assumed that the toxicity
of benzoic acid after short-term oral exposure is low. In high-dosed
rats given approximately 2250 mg/kg body weight per day via diet over
5 days, excitation, ataxia, convulsions, and histopathological changes
in the brain were seen. The mortality was about 50%; in some cases,
bleeding into the gut was noted (Kreis et al., 1967). In two other
studies with rats dosed with approximately 825 mg/kg body weight per
day over 7-35 days (Kreis et al., 1967) or with 65-647 mg/kg body
weight per day over 28 days (Bio-Fax, 1973), no clear
treatment-related effects occurred. The reduced weight gain at 2250
and 825 mg/kg body weight per day may be attributed to reduced food
intake in the study by Kreis et al. (1967). The relevance of the
reduced relative kidney weight at 324 mg/kg body weight per day, which
was not dose-related and not accompanied by changes in
histopathological examinations, is unclear (Bio-Fax, 1973). As given
in Table 3, both studies have several limitations (i.e., missing
haematological and clinical chemical investigations, incomplete
histopathological examinations); therefore, both of these studies were
inadequate for derivation of a NO(A)EL.
More information on dose-response can be gained from the study of
Fujitani (1993), in which rats received sodium benzoate for 10 days in
feed. At the lowest tested concentration of 1358 mg/kg body weight per
day, changes in serum cholesterol levels occurred in females. At doses
of 1568 mg/kg body weight per day and above, changes in further serum
parameters and an increased relative liver weight were described.
Histopathological changes of the liver, increased relative kidney
weights, and disorders of the central nervous system (convulsions)
were seen after dosing via diet with approximately 1800 mg/kg body
weight per day. In several other studies listed in Table 3, adverse
effects were seen only at higher doses after feeding sodium benzoate
over periods from 10 to 42 days, so that a
lowest-observed-(adverse-)effect level (LO(A)EL) of 1358 mg sodium
benzoate/kg body weight per day for short-term exposure can be
derived.
With cats (Bedford & Clarke, 1972), also described in Table 3,
the effect levels with benzoic acid were lower. However, due to the
differences in the metabolism of benzoic acid in cats compared with
other experimental animals and humans, this study was not taken into
further consideration (see section 7).
Table 3: Toxicity of benzoic acid and sodium benzoate after short-term oral exposure.
Species; strain; Treatment Duration Organs examined in Resultsa Reference
number of animals (days) histopathology, clinical
per dosea chemistry, haematology
Benzoic acid
cat; 4 m 0 or 0.5% 3-4 liver, kidney, heart, mild hyperaesthesia, Bedford & Clarke
in diet stomach, lung, brain, apprehension, and (1972)
(approx. 0 spinal cord (only depression starting 48-92
or 300-420 mg/kg animals that died were h after uptake; duration of
body weight) examined); blood samples the syndrome: about 20-48 h;
were taken from surviving mortality rate: 50%;
cats degenerative changes in
liver, kidneys, and lung,
but no pathological findings
in brain or spinal cord;
surviving cats: urea and
serum alanine
aminotransferase (S-ALAT) *,
indicating liver and kidney
damage
cat; 4 m a) 100 or 200 a) 15 only blood samples no adverse effects Bedford & Clarke
mg/kg body weight were taken were reported (1972)
via diet
b) 0 or 0.25% in b) 23
diet (approx. 0
or 130-160 mg/kg
body weight)
Table 3 (cont'd)
Species; strain; Treatment Duration Organs examined in Resultsa Reference
number of animals (days) histopathology, clinical
per dosea chemistry, haematology
rat; Wistar; 0 or 3% in diet 1-5 heart, liver, spleen, body weight gain **; Kreis et al.
5-15 m (approx. 0 or kidney, brain in rats dosed over 5 days, (1967)
2250 mg/kg body disorders of the central
weight) nervous system (excitation,
ataxia, tonoclonic
convulsions); mortality
rate approx. 50%; in some
cases, bleeding into the
gut; brain damage (necrosis
of parenchymal cells of the
stratum granulosum of the
fascia dentata and the
cortex of the lobus
piriformis) in most animals
dosed over 3-5 days (still
present after 35 days)
rat; Wistar; 0 or 1.1% in 7-35 heart, liver, spleen, body weight gain **; Kreis et al.
5-10 m diet kidney, brain no clinical signs of (1967)
(approx. 0 or intoxication
825 mg/kg body
weight)
rat; albino; 10 m 0, 760, 3800, or 28 liver, kidney, no deaths or signs of Bio-Fax (1973)
7600 ppm via diet adrenals, testes intoxication
(approx. 0, 65, 324 mg/kg body
324, or 647 mg/kg weight: relative kidney
body weight) weights **; no further
information available
Table 3 (cont'd)
Species; strain; Treatment Duration Organs examined in Resultsa Reference
number of animals (days) histopathology, clinical
per dosea chemistry, haematology
Sodium benzoate
rat; F344/Ducrj; 0, 1.81, 2.09, or 10 liver, kidney; >1358 mg/kg body weight: Fujitani (1993)
6 m/f 2.4% in diet standard clinical changes in serum levels
(approx. 0, 1358, chemistry (cholesterol ** (f))
1568, or 1800 >1568 mg/kg body weight:
mg/kg body relative liver weight * (m);
weight) changes in serum levels
(albumin * (m), total
protein * (m))
1800 mg/kg body weight:
1/6 males died
(hypersensitivity,
convulsions); body
weight ** (m/f); relative
liver weight * (f); relative
kidney weights * (m/f);
absolute weights of spleen
and thymus ** (m);
absolute/relative weights
of thymus ** (f); changes
in serum levels
(gamma-glutamyltranspeptidase
(GGT) * (m), albumin * (f),
cholinesterase ** (f));
eosinophilic foci around
periportal vein and
enlargement of hepatocytes
with glassy cytoplasm in
the periportal area of the
liver (m); no changes in
the kidney (m)
Table 3 (cont'd)
Species; strain; Treatment Duration Organs examined in Resultsa Reference
number of animals (days) histopathology, clinical
per dosea chemistry, haematology
rat; Sherman; 0, 2, or 5% in 28 no data available 2200 mg/kg body weight: Fanelli & Halliday (1963)
6 m/f diet (approx. 0, slight depression of
2200, or 6700 body weight gain (m)
mg/kg body 6700 mg/kg body weight:
weight) mortality 100% within
11 days; signs of
intoxication included
hyperexcitability, urinary
incontinence, and
convulsions
no further information
available
rat; 28 (no 0 or 5% in diet 28 no data available mortality about 100% within Kieckebusch & Lang
further data) (approx. 0 or 3 weeks; decreased feed (1960)
3750 mg/kg body intake, diarrhoea,
weight) intestinal haemorrhage and
crusted blood in the nose;
no further information
available
rat; 5 (no 0 or 5% in diet >28 no data available mortality 80% within Kieckebusch & Lang
further data) (approx. 0 or 4-5 weeks; decreased (1960)
3750 mg/kg body weight; no further
body weight) information available
Table 3 (cont'd)
Species; strain; Treatment Duration Organs examined in Resultsa Reference
number of animals (days) histopathology, clinical
per dosea chemistry, haematology
rat; F344; 0, 0.5, 1, 2, 4, 42 histopathology performed, >375 mg/kg body weight: Sodemoto & Enomoto
10-11 m/f or 8% in diet but not further specified hypersensitivity after (1980)
(approx. 0, 375, dosing
750, 1500, 3000, >3000 mg/kg body weight:
or 6000 mg/kg mortality about 100% within
body weight) 4 weeks; apart from atrophy
of the spleen and lymph
nodes, no other
morphological changes were
noted
rat; Sherman; 0 or 16-1090 30 adrenals, upper intestine, no adverse effects were Smyth & Carpenter
5 m/f mg/kg body kidney, liver, spleen reported; no further (1948)
weight via diet information available
mouse; B6C3F1; 0, 2.08, 2.5, or 10 liver, kidney; standard „3750 mg/kg body weight: Fujitani (1993)
4-5 m/f 3% in diet clinical chemistry changes in serum levels
(approx. 0, 3000, (cholinesterase * (m))
3750, or 4500 4500 mg/kg body weight:
mg/kg body weight) hypersensitivity in all
animals; convulsions
1/5 males and 2/5 females
(both females died);
absolute/relative liver
weight * (m/f); relative
kidney weight * (f);
changes in serum levels
(cholesterol * (m),
phospholipids * (m));
Table 3 (cont'd)
Species; strain; Treatment Duration Organs examined in Resultsa Reference
number of animals (days) histopathology, clinical
per dosea chemistry, haematology
enlarged hepatocytes,
single cell necrosis
and vacuolation of
hepatocytes in all
livers (m); no changes
in the kidney (m/f)
mouse; albino 0, 0.5, 1, 2, 4, 35 survival, chemical 3000 mg/kg body weight: Toth (1984)
Swiss; 4 m/f or 8% via consumption, histological "suitable for lifelong
drinking-water changes (not further treatment" based on
(approx. specified) (prestudy four parameters: survival,
0-12 000 mg/kg for carcinogenicity study) body weight, chemical
body weight) consumption, and histology
6000 mg/kg body weight:
mortality 75% in m/f;
body weight of surviving
mice ** (m/f)
12 000 mg/kg body weight:
mortality 100% within
3 weeks
a m = male; f = female.
8.3.2 Inhalation exposure
Ten CD rats per sex per group were exposed to 0, 25, 250, or
1200 mg benzoic acid dust aerosol/m3 (analytical concentration; mass
aerodynamic diameter [MAD]/sigma g (standard deviation): 0, 4.6/3.1,
4.4/2.1, 5.2/2.1; mass median aerodynamic diameter [MMAD]: 4.7 µm) for
6 h per day and 5 days per week over 4 weeks. After this time, various
serum biochemical, haematological, organ weight, and histopathological
examinations were conducted. At >25 mg/m3, an increased incidence
of interstitial inflammatory cell infiltrate and interstitial fibrosis
in the trachea and lungs in treated animals compared with controls was
seen. Although the number of these microscopic lesions was higher in
treated animals than in controls, there was no clear dose dependency
for this effect. A concentration of >250 mg/m3 resulted in upper
respiratory tract irritation, as indicated by inflammatory exudate
around the nares, and significantly decreased absolute kidney weights
in females. In the highest dose group, one rat per sex died, and the
body weight gain was significantly decreased in males and females
compared with controls. In addition, a significant decrease in
platelets (males/females), absolute/relative liver weights (males),
and trachea/lung weights (females) was noted (Velsicol Chemical
Corp., 1981).
Studies concerning repeated exposure by inhalation to sodium
benzoate were not identified in the available literature.
8.3.3 Dermal exposure
Studies concerning repeated dermal exposure to benzoic acid or
sodium benzoate were not identified in the available literature.
8.4 Long-term exposure
In general, the database for benzoic acid and sodium benzoate is
limited, and there are no studies available performed according to
current guidelines. In addition, the documentation in most cases is
limited. Detailed information is given in Table 4.
8.4.1 Subchronic exposure
In a 90-day study with rats dosed with 0, 1, 2, 4, or 8% sodium
benzoate via diet, the mortality in the highest dose group (approx.
6290 mg/kg body weight per day) was about 50%. Other effects in this
group included a reduced weight gain, increased relative weights of
liver and kidneys, and pathological changes (not further specified) in
these organs (Deuel et al., 1954).
Table 4: Results of studies concerning long-term oral exposure to benzoic acid and sodium benzoate.
Species; strain; Treatment Duration Examinations; Resultsa Reference
number of animals organs in
per dosea histopathology,
clinical chemistry,
haematology
Benzoic acid
rat; Wistar; 0 or 1.5% in diet 18 months no data available reduced weight gain with Marquardt (1960)
dose group: (approx. 0 or decreased feed intake;
30 m/20 f; 750 mg/kg body increased mortality rate
controls: weight) (15/50 vs. 3/25 in
13 m/12 f controls); no further
information available
(only provisional results
are given)
rat; Wistar or 0 or 1.5% in diet 18 months no data available reduced weight gain with Marquardt (1960)
Osborne-Mendel; (approx. 0 or decreased feed intake;
dose group: 750 mg/kg body no further information
20 m; controls: weight) available (only
10 m provisional results are
given)
rat; not given; 0, 0.5, or 1% in generation 1 histopathology in no effects on growth and Kieckebusch & Lang
20 m/f diet and 2: animals of organ weights; feeding of (1960)
(approx. 0, 250, lifelong generation 3 0.5% led to prolongation
or 500 mg/kg body generation 3: (not further specified) of survival compared with
weight) 16 weeks controls; no further
generation 4: information available
until breeding
Table 4 (cont'd)
Species; strain; Treatment Duration Examinations; Resultsa Reference
number of animals organs in
per dosea histopathology,
clinical chemistry,
haematology
Sodium benzoate
rat; Sherman; 0, 1, 2, 4, or 8% 90 days histopathology 6290 mg/kg body weight: Deuel et al.
5 m/f in diet (approx. 0, performed, but mortality about 50%; (1954)
640, 1320, 2620, not further specified weight gain **;
or 6290 mg/kg body relative weights of
weight) liver and kidneys *;
pathological lesions
(not further specified)
in liver and kidneys
rat; F344; 0, 1, or 2% in 18-24 months histopathology average mortality rate of Sodemoto & Enomoto
dose group: diet performed, but not all animals during the (1980)
50 m/52 f; (m: approx. 0, 700, further specified first 16 months: 14.5%
controls: or 1400 mg/kg (all dead rats showed
body weight; f: pneumonia with abscess);
25 m/43 f about 100 rats including
approx. 0, 290, controls died after
or 580 mg/kg 16 months due to
body weight) haemorrhagic pneumonia
(infection); no adverse
clinical signs and no
differences in average
body weight and mortality
in dosed animals compared
with controls;
non-carcinogenic effects
not reported
Table 4 (cont'd)
Species; strain; Treatment Duration Examinations; Resultsa Reference
number of animals organs in
per dosea histopathology,
clinical chemistry,
haematology
mouse; 0 or 2% via lifelong liver, spleen, kidney, no difference in survival Toth (1984)
albino Swiss; drinking-water bladder, thyroid, rates in treated animals
dose group: (approx. 0 or heart, pancreas, compared with controls;
50 m/f; 5960-6200 mg/kg testes, ovaries, brain, no pathological or
controls: 99 m/f body weight) nasal turbinates, lung statistical evidence of
tumour induction
a m = male; f = female.
8.4.2 Chronic exposure and carcinogenicity
In two studies with rats given 1.5% benzoic acid via diet
(approximately 750 mg/kg body weight per day), the animals showed a
reduced weight gain with decreased feed intake after dosing over
18 months. In one of these studies, mortality was increased
(15/50 rats of both sexes versus 3/25 in controls) (Marquardt, 1960).
No further information on these studies is available, as only
provisional results were published. In a four-generation study with
rats, no effects on life span, growth rate, or organ weights were
reported after dosing with up to 1% in the diet (approximately
500 mg/kg body weight per day) (Kieckebusch & Lang, 1960). Only
animals of the third generation were autopsied after 16 weeks, but
it is not clear if a complete histopathological investigation was
performed.
With sodium benzoate, two long-term studies with rats
(administration of up to 1400 mg/kg body weight per day via diet over
18-24 months; Sodemoto & Enomoto, 1980) or mice (lifelong application
of up to 6200 mg/kg body weight per day via drinking-water; Toth,
1984) are available. The results gave no indication of a carcinogenic
effect in the tested animals. Although the study with mice was not
performed according to current guidelines, the results seem to be
reliable, due to a sufficient number of animals and detailed
histopathological examinations. However, the results from the study
with rats are uncertain, due to a very high mortality in animals of
all dose groups, including controls (from an "infection" after
16 months), no detailed information about dosing regimen (only mean
values given), and the considerable differences in the body weight of
male and female rats (the body weight of females was about twice that
of males).
8.4.3 Carcinogenicity of benzyl acetate, benzyl alcohol, and
benzaldehyde
As benzyl acetate, benzyl alcohol, and benzaldehyde are
practically quantitatively metabolized via benzoic acid (see section
7.1), data on their carcinogenicity from 2-year studies may be used as
supportive evidence in the assessment of the hazards associated with
benzoic acid.
Benzyl acetate was administered in corn oil via gavage to F344/N
rats (0, 250, or 500 mg/kg body weight per day) or B6C3F1 mice
(0, 500, or 1000 mg/kg body weight per day). In high-dose male rats,
the incidence of acinar cell adenomas of the exocrine pancreas was
increased, whereas there was no evidence of carcinogenicity in female
rats. In high-dose male and female mice, benzyl acetate caused
increased incidences of hepatocellular adenomas and squamous cell
neoplasms of the forestomach (US NTP, 1986). In contrast to these
findings, no such tumours were observed in another study with the same
strain of rats and mice when benzyl acetate was administered via diet
(rats: <575 mg/kg body weight per day; mice: <375 mg/kg body
weight per day) (US NTP, 1993).
With benzyl alcohol, no treatment-related increase in tumours was
observed in F344/N rats or B6C3F1 mice after administration of
<400 mg/kg body weight per day in rats or <200 mg/kg body weight
per day in mice by gavage in corn oil (US NTP, 1989).
In B6C3F1 mice dosed with benzaldehyde in corn oil by gavage
(males: 0, 200, or 400 mg/kg body weight per day; females: 0, 300, or
600 mg/kg body weight per day), the incidences of squamous cell
papillomas of the forestomach were significantly greater in both
exposure groups than in controls. A dose-related increase in the
incidence of forestomach hyperplasia was also observed. In F344/N rats
dosed with <400 mg/kg body weight per day, there was no evidence of
carcinogenic activity (US NTP, 1990).
8.5 Genotoxicity and related end-points
8.5.1 Benzoic acid
Benzoic acid tested negative in several Ames tests and in one DNA
damage assay with different Salmonella typhimurium strains in the
presence or absence of metabolic activation (McCann et al., 1975;
Ishidate et al., 1984; Nakamura et al., 1987; Zeiger et al., 1988).
Only in one recombination assay with Bacillus subtilis H17 and M45
was a positive result obtained (Nonaka, 1989). However, due to missing
experimental details (only results given), the validity of this study
cannot be judged. There was no indication of genotoxic activity
(chromosome aberrations, sister chromatid exchange) in tests with
mammalian cells (Chinese hamster CHL and CHO cells, human
lymphoblastoid cells, human lymphocytes) without metabolic activation
(Oikawa et al., 1980; Tohda et al., 1980; Ishidate et al., 1984;
Jansson et al., 1988).
In vivo studies with benzoic acid were not identified in the
literature.
8.5.2 Sodium benzoate
Sodium benzoate also gave negative results in some Ames tests and
in Escherichia coli in the presence or absence of metabolic
activation (Ishidate et al., 1984; Prival et al., 1991). As with
benzoic acid in recombination assays with Bacillus subtilis H17 and
M45, positive results were obtained (Ishizaki & Ueno, 1989; Nonaka,
1989). Although sodium benzoate tested negative in a cytogenetic assay
with WI-38 cells in the absence of metabolic activation (US FDA,
1974), consistently positive results (in contrast to the negative
results of benzoic acid) were obtained in tests on sister chromatid
exchange and chromosome aberrations with CHL/CHO and DON cells or
human lymphocytes without metabolic activation (Abe & Sasaki, 1977;
Ishidate & Odashima, 1977; Ishidate et al., 1984, 1988; Xing & Zhang,
1990). However, from the limited information given in the publications
(i.e., only results given), it cannot be judged if these positive
results may have been attributable to cytotoxic effects.
In a valid in vivo study performed by the US FDA (1974), sodium
benzoate tested negative in a cytogenetic assay (bone marrow) in rats
after single or multiple oral application of doses up to 5000 mg/kg
body weight. In a study with mice (comparable dosing scheme), there
was also no indication of mutagenic activity in a host-mediated assay
(US FDA, 1974).
However, in a dominant lethal assay with rats (comparable dosing
scheme; males were mated with untreated females following 7 or 8 weeks
of dosing), some statistically significant and dose-related findings
were reported in week 7: decreased fertility index for both treatment
regimens and an increased number of preimplantation losses after
single dosing (US FDA, 1974).
In summary, the in vitro studies with benzoic acid gave no
indications for genotoxic effects, whereas in vivo studies were not
identified. Sodium benzoate was also inactive in bacterial test
systems, whereas tests with mammalian cells gave consistently positive
results. In addition, in an in vivo study with sodium benzoate
(dominant lethal assay in rats), a positive result was obtained. As a
result, a genotoxic activity of sodium benzoate cannot be ruled out
entirely at present.
Detailed information concerning the genotoxicity of benzoic acid
and sodium benzoate in vitro is given in Table 5.
8.6 Reproductive and developmental toxicity
8.6.1 Fertility
There are no studies available dealing specifically with the
effects of benzoic acid or sodium benzoate on fertility that have been
conducted according to current protocols.
In a four-generation study with male and female rats, no adverse
effects on fertility or lactation (only investigated parameters) were
seen after dosing with benzoic acid at up to 1% in the diet
(approximately 500 mg/kg body weight per day) (see also section 8.4.2;
Kieckebusch & Lang, 1960).
In studies with repeated oral application, no effects on the
testes were observed in rats after dosing with benzoic acid at up to
647 mg/kg body weight per day in the diet for 4 weeks (see also Table
3; Bio-Fax, 1973) or in mice after lifelong application of 6200 mg
sodium benzoate/kg body weight per day via drinking-water (see also
Table 4; Toth, 1984).
In summary, no clear statement can be given as to the possible
effects of benzoic acid or sodium benzoate on fertility.
8.6.2 Developmental toxicity
In a study with pregnant rats given only one oral dose of benzoic
acid (510 mg/kg body weight on gestation day 9), there was no
indication of an increase in resorption rates or malformations
(Kimmel et al., 1971).
For sodium benzoate, several teratogenicity studies are available
that have been performed with different species. As given in Table 6,
no effects were seen in dams or offspring of rats, mice, rabbits, or
hamsters given oral doses of up to 300 mg/kg body weight per day
(highest dose tested) during gestation (US FDA, 1972b). In a study
with rats by Onodera et al. (1978), doses of 4% or 8% via diet (uptake
of 1875 or 965 mg/kg body weight per day) induced severe maternal
toxicity (no weight gain/loss in body weight, increased mortality) and
were associated with embryotoxic and fetotoxic effects as well as
malformations. However, the authors suggested that the effects on the
dams and fetuses at >4% dietary levels were caused by reduced
maternal feed intake, leading to malnutrition. The intake of sodium
benzoate in the highest dose group (8%) was lower than that at 2%,
where no adverse effects were seen. From this study, a NO(A)EL of
about 1310 mg/kg body weight per day can be derived. In a study with
rats by Minor & Becker (1971), however, fetotoxic and teratogenic
effects occurred at 1000 mg/kg body weight per day. In this study,
sodium benzoate was applied by intraperitoneal injection. Therefore,
differences in pharmacokinetics between oral and intraperitoneal
administration may be the reason for the higher sensitivity.
Studies performed with eggs of leghorn hens (single injection of
<5 mg per egg), chick embryo neural retina cells
(lowest-observed-effect concentration [LOEC] of 34.7 mmol/litre), and
a chick embryotoxicity screening test (single injection of <0.1 mg
per embryo) gave no indication of embryotoxic or teratogenic effects
(Verrett et al., 1980; Jelinek et al., 1985; Daston et al., 1995).
8.6.3 Reproductive toxicity of benzyl acetate, benzyl alcohol,
and benzaldehyde
As benzyl acetate and benzyl alcohol are practically
quantitatively metabolized via benzoic acid (see section 7.1), data on
their reproductive toxicity may be used a