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EC number: 208-901-2
CAS number: 546-46-3
Gene mutation (Bacterial reverse mutation assay / Ames test):
a) read across from citric acid and sodium dihydrogen citrate: negative
with and without activation in all strains tested (similar to OECD TG
b) read across from zinc oxide, zinc sulfate and zinc chloride: negative
with and without activation in all strains tested (similar to OECD TG
Cytogenicity in mammalian cells:
a) trizinc dicitrate positive for induction of micronuclei in cultured
human lymphocytes with activation (OECD TG 487)
b) read across from citric acid: positive for induction of micronuclei
in cultured human lymphocytes without activation (similar to OECD draft
Mammalian Chromosome Aberration test in rat (oral gavage administration)
(similar to OECD TG 475): read across from citric acid and zinc sulfate:
Rat dominant lethal Assay (oral gavage administration) (similar to EU
22): read across from citric acid and zinc sulfate: negative
Mammalian Erythrocyte Micronucleus Test in mouse (similar to OECD TG
474): read across from zinc sulfate: negative
Mammalian Erythrocyte Micronucleus Test in rat: read across from zinc
Single cell gel/comet assay in rodents for detection of DNA damage: read
across from zinc sulfate: positive
No information is available for trizinc dicitrate (CAS number 546-46-3),
except for an in vitro study a micronucleus assay (OECD 487). This study
lead to a positive result with metabolic activation and was negative
without metabolic activation. Other Information is available for the
closely related substances, sodium dihydrogen citrate and citric acid.
Information is available for citric acid (CAS number 77-92-9) and sodium
dihydrogen citrate (CAS number 18996-35-5) from reliable studies for
mutagenicity to bacteria. There is also information available from
studies of lower reliability that calcium citrate (CAS number 7693-13-2)
does not cause mutagenicity to bacteria, and that citric acid does not
cause chromosome aberrations in vitro. Information from reliable studies
indicate that citric acid does not cause chromosome damage in somatic or
in germ cells in vivo. A recent publication described positive results
in mammalian cells, including a chromosome aberration analysis and an in
vitro micronucleus assay. The micronucleus assay was chosen as key, as
the method was close to the draft guideline and effects were observed.
It is considered that this positive result does not affect the overall
genetic toxicity assessment of this substance as the effects seen in
vitro were not observed in vivo and are not considered biologically
relevant. In addition, data for zinc were included. Information is
available for zinc oxide (CAS number 00), zinc chloride (CAS number
7646-85-7), zinc monoglycerolate (CAS number 16754-68-0) and zinc
sulfate (CAS number 7733-02-0) from reliable studies on in vitro and in
vivo studies. Due to the huge amount of different studies only selected
studies were reported in detail in endpoint study records.
Data available in vitro and in vivo: overall conclusion negative.
Citric acid is not mutagenic in vitro with and without metabolic
activation. The active substance is not clastogenic in vitro without
metabolic activation. Citric acid did not induce mutagenic potential in
two in vivo studies (rat bone marrow and rat dominant lethal assay, see
endpoint study records).
The evaluation of citric acid as biocidal product (ECHA 2016) came to
the following conclusion: There is no evidence of genotoxic potential of
citric acid in in vitro or in vivo studies conducted in bacteria,
mammalian cells and rats. No genotoxic effects have been observed during
the long history of exposure via food, cosmetics and medicines.
A similar conclusion was given by the German MAK commission (1998).
Data available in vitro and in vivo: overall conclusion negative
The German MAK commission (2009) came to the following conclusion:
In vitro investigations on the genotoxicity of zinc compounds yielded no
mutagenic, but a clastogenic effect mainly in the high and in some cases
in the cytotoxic concentration range. As regards genotoxic effects in
soma cells in vitro, apart from a number of non-evaluable reports,
negative results in the micronucleus and chromosome aberration test are
available with one exception. Taking its shortcomings into account and
the presence of two negative studies performed in a comparable dose
range, this positive study is not sufficient to confirm genotoxicity in
vitro. It has to be taken into account that zinc was administered via
the intraperitoneal route, i.e. bypassing the mechanisms regulating
absorption. In mice and rats, the results of dominant lethal tests are
negative. As an essential trace element, zinc is subject to a strict
metal homoeostasis. If this is exceeded by increased zinc
concentrations, the metal is capable of binding to amino acids such as
cysteine, with enzyme inhibition as a result. In turn, this can induce
interactions with the energy metabolism, signal transmission and the
components of apoptosis, in addition to clastogenic or aneugenic effects
as well. One may assume that this mechanism of action is applicable at
least in vitro, which can thus explain the sporadically observed
positive findings for clastogenicity. However, no comparable zinc
concentrations are reached under workplace conditions.
The EU RAR (2008) came to a similar conclusion:
The available data indicate that the genotoxicity results vary widely.
Conflicting results have been found, even in the same test systems.
Overall, the results of the in vitro tests indicate that zinc has
genotoxic potential in vitro based on positive results in mammalian test
systems for gene mutations and chromosomal aberrations and on the
positive in vitro UDS test. In vivo, increases in chromosomal
aberrations were found in calcium-deficient mice exposed via the diet as
well as in mice with normal calcium status when dosed intraperitoneally.
In mice also negative results were obtained and even at higher
intraperitoneal dose levels. Rats tested negative for chromosomal
aberrations after oral dosing, either via gavage or via the diet. The
positive result for chromosomal aberrations in vitro is considered
overruled by negative in vivo tests for this endpoint. The positive
sperm head abnormality test is considered sufficiently counter-balanced
by two negative SLRL tests as well as two negative dominant lethal
tests. Moreover, this sperm test is not adequately reported and without
details on scoring criteria, interpretation of the observations is
rather subjective. In addition, sperm head abnormalities are indicative
rather than proof for genotoxicity. Based on the available data there is
insufficient ground to classify zinc as genotoxic. It should be noted
that the potential to induce gene mutations was not adequately tested in
vivo. However, there is no clear evidence from the available data that
zinc is genotoxic in vivo and without a clear indication for
carcinogenicity (see below) a guidance for further testing with respect
to target tissue is not available.
This section contains substantially new data.
Overall conclusion: negative
According to international accepted evaluation of genotoxicity data on
citric acid and zinc, the overall conclusion is negative, i.e. no
classification is required.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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