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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:

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 471)

b) read across from zinc oxide, zinc sulfate and zinc chloride: negative with and without activation in all strains tested (similar to OECD TG 471)

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 TG 487)

Genetic toxicity in vivo

Description of key information

In vivo

Mammalian Chromosome Aberration test in rat (oral gavage administration) (similar to OECD TG 475): read across from citric acid and zinc sulfate: negative

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 monoglycerolate: negative

Single cell gel/comet assay in rodents for detection of DNA damage: read across from zinc sulfate: positive

Additional information

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.

Citric acid:

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).

Zinc

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

Justification for classification or non-classification

According to international accepted evaluation of genotoxicity data on citric acid and zinc, the overall conclusion is negative, i.e. no classification is required.