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Genetic toxicity in vitro

Description of key information

No genetic toxicity study with zirconium, acetate lactate oxo ammonium complexes is available, thus the genetic toxicity will be addressed with existing data on the assessment entities zirconium, acetate, lactate and ammonium.

The substance zirconium, acetate lactate oxo ammonium complexes is not expected to be genotoxic, since the individual moieties zirconium, acetate, lactate and ammonium have not shown gene mutation potential in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No genetic toxicity study with zirconium, acetate lactate oxo ammonium complexes is available, thus the genetic toxicity will be addressed with existing data on the individual moieties zirconium, acetate, lactate and ammonium

Zirconium

Genetic toxicity in vitro

Bacterial Reverse Mutation Test:

Laus (2008) and the Chemicals Inspection and Testing Institute, Japan (1997) performed a bacterial reverse mutation study according to OECD guideline 471 and EU method B13/14. Both studies were used in a weight-of-evidence approach. Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were exposed to 50 to 4998 ug/plate with and without metabolic activation in 2 independent experiments in the Laus (2008) study and S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvr A were exposed to 0.156 to 5000 ug/plate with and without metabolic activation in 2 independent experiments in the study performed by Chemicals Inspection and Testing Institute (1997, Japan). Vehicle and positive controls included in both studies were valid. Zirconium dioxide did not induce mutation in both studies with and without metabolic activation and no cytotoxicity was observed.

In vitro cytogenicity assay:

NOTOX B.V. (2010) performed a chromosome aberration test according to OECD guideline 473. Cultured peripheral human lymphocytes were exposed for 3 hours to 10, 33 and 100 µg zirconium dioxide/mL culture medium with and without S9-mix (dose range finding test/first cytogenetic assay); at 24 and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100, 333 and 1000 µg zirconium dioxide/mL culture medium without S9-mix. A second cytogenicity test was performed as follows: without S9-mix: 10, 33 and 100 µg/mL culture medium (24 and 48h exposure time, 24h and 48h fixation time); with S9-mix: 10, 33 and 100 µg/mL culture medium (3h exposure time, 48h fixation time). Vehicle and positive control substances were tested simultaneously and considered valid. Zirconium dioxide tested negative with and without metabolic activation. No cytotoxicity was observed.

In vitro gene mutation assay:

NOTOX B.V. (2010) performed a mouse lymphoma test according to OECD guideline 476. Mouse lymphoma L5178Y cells were exposed to 0.03, 0.1, 1, 3, 10, 33 and 100 µg/mL zirconium dioxide with and without metabolic activation. In a first experiment, cell cultures were exposed for 3 hours to zirconium dioxide in exposure medium in the absence and presence of S9-mix. In a second experiment, cell cultures were exposed to Zirconium dioxide in exposure medium for 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Zirconium dioxide tested negative in both experiments with and without metabolic activation. No cytotoxicity was observed, and positive and vehicle controls were considered valid.

In vitro comet assay:

Braz (2008) performed a Comet assay according to following guideline: Single Cell Gel/Comet Assay: Guidelines for in vitro and in vivo genetic toxicology testing. Human lymphocytes were exposed to 1, 100 and 1000 ug/L zirconium dioxide without metabolic activation. Vehicle and positive control were tested simultaneously and considered valid. Zirconium dioxide tested negative in human lymphocytes without metabolic activation. No cytotoxicity was observed.

Zirconium dioxide did not induce any mutation or chromosome aberration in 5 different in vitro studies and therefore, the substance should not be classified for genetic toxicity.

 

Genetic toxicity in vivo

No reliable data were available on the genetic toxicity in vivo endpoint. Zirconium dioxide tested negative in in vitro toxicity tests (Ames test, mammalian chromosome aberration test, mouse lymphoma assay and comet assay) and therefore no in vivo mutagenicity tests should be performed with this substance.

 

Acetate

Genetic toxicity in vitro

A registration dossier shall contain information on the human health hazard assessment (regulation 1907/2006, Art.10). However, it is considered that the information requirements for acetate as laid down in annex VII to IX can be fulfilled by adaptation of the standard testing regime according to Annex XI, points 1.2. and 1.3. as presented in the following:

Acetic acid, calcium acetate, and sodium diacetate have a well-established history of use in food where they are considered safe at any concentration level, consistent with their intended physical, nutritional or other technical effect. They are also widely used in human and veterinary medicine, cosmetics, as plant protection agents and in a variety of household products as buffering agents or because of their anti-microbial properties. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded “Acetic acid, sodium diacetate, and calcium acetate are permitted food additives that may be added directly to food intended for human consumption without any limitation. This authorisation followed the assessment of safety by JECFA (1974, 1998) and the EU Scientific Committee on Food (SCF, 1990). JECFA considered acetic acid, calcium acetate, and sodium diacetate separately although data on acetic acid were primarily considered in each evaluation as no specific studies on sodium diacetate and calcium acetate were identified at that time. JECFA allocated an ADI of “not limited” (i.e., “not specified”) to acetic acid and its calcium salt in 1974 and this conclusion was retained when JECFA evaluated a group of saturated linear primary alcohols, aldehydes, and acids that included acetic acid in 1998.”(EFSA 2012)  The assessment of safety by JECFA (1974, 1998) also reported results of various genotoxicity tests with acetic acid. It is noteworthy that culture conditions of low pH and high osmolality, which may occur upon incubation with acidic substances, have been shown to produce false-positive results in this and also other assays (Heck et al., 1989). Therefore, the results obtained with acetic acid must be cautiously interpreted. However, acetic acid, which initially gave an increase in SCE in Chinese hamster ovary cells under unphysiological conditions, were later shown to be negative when tested at physiological pH (Morita et al., 1990). As reported in the JECFA assessment of safety (JECFA 1998) acetic acid was also negative in a bacterial reverse mutation assay conducted by Zeiger et al. (1992). The S. typhimurium strains TA100, TA1535, TA97 and TA98 were tested with and without metabolic activation and were shown to be non-mutagenic to all tested strains. Same results were also observed in a primary mutagenicity screening of food additives conducted in Japan. Acetic acid with a purity of 99.9% was tested up to concentrations of 10 mg/plate and was shown to be negative in the gene mutation test in bacteria. The chromosomal aberration test using Chinese hamster fibroblast cells was also conducted. The cells were exposed to acetic acid for 24 and 48 hours and analysed for polyploid cells as well as for structural chromosomal aberrations. The results clearly indicated that acetic acid had no genotoxic effects (Ishidate; M. Jr. et al., 1992).  Further, acetic acid is an essential compound conducting the in vivo as well as in vitro micronucleus test. According to the OECD Guideline No. 474 and No. 487 cells obtained in both assays need to be fixed and stained appropriately. For this purpose, cells obtained are treated in a hypotonic buffer and dropped onto microscope slides, where they are fixed with a mixture of methanol and acetic acid and stained with Giemsa (Toxicological testing handbook, 2nd ed.).  This clearly indicates, that acetic acid is an integral part of two validated assays required for registration. Based on this, positive results in in vivo and in vitro micronucleus tests seems to be highly unlikely.   Beside the fact, that acetic acid has shown negative results in in vitro mutagenicity testing, it is also noteworthy that acetic acid is known to be a part in many biochemical reactions such as protein, carbohydrate and lipid metabolism. Coenzyme A (CoA) is acetylated to acetyl-CoA by the breakdown of carbohydrates through glycolysis and by the breakdown of fatty acids through β-oxidation. Acetyl-CoA then enters the citric acid cycle, where the acetyl group is oxidized to carbon dioxide and water, and the energy released captured in the form of 11 ATP and one GTP per acetyl group. Thus, acetyl-CoA is required for ATP generation necessary for all viable cells and processes like DNA synthesis as well as repair.      

 

The fact, that acetic acid, sodium diacetate, and calcium acetate are permitted food additives that may be added directly to food intended for human consumption without any limitation as well as the crucial role of acetic acid in cellular processes such as the citric acid cycle and the above mentioned in vitro data indicate that acetic acid has no genotoxic effects to human cells. All cells in every organism are highly dependent on acetic acid maintaining their functionality and viability. Thus, it appears highly unlikely that acetic acid exerts any genotoxic or mutagenic effects to human cells. Based on the above existing information on the genotoxicity of acetic acid and the weight of evidence information, acetic acid is considered not genotoxic and further experimental testing is therefore waived using the rules laid down in Annex XI, Section 1.1.2, 1.1.3 and 1.2.    

 

Lactate

Genetic toxicity in vitro

A registration dossier shall contain information on the human health hazard assessment (regulation 1907/2006, Art.10). However, it is considered that the information requirements for lactate/lactic acid as laid down in annex VII to IX can be fulfilled by adaptation of the standard testing regime according to Annex XI, points 1.1. and 1.2. as presented in the following:

Bacterial Reverse Mutation Test: 

As reported in the final report on the safety assessment of lactic acid and other substances (CIR, 1998) negative results were obtained when the mutagenic potential of Lactic Acid, 90.5% pure, in phosphate buffer was assayed in an Ames test using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94, and TA98 with metabolic activation (Ishidate et al., 1984). Duplicate plates of six concentrations < 10.0 mg/plate were examined. Negative results were also obtained in an Ames test for 1000 pg/mL 11 mM Lactic Acid using a clonal subline of Chinese hamster fibroblasts derived from lung tissue in the absence of metabolic activation (Ishidate et al., 1984).

An Ames test was performed to determine the mutagenic potential of Lactic Acid using S. typhimurium strains TA97, TA98, TA100, and TA104 (Al-Am and Al-Lami, 1988). Triplicate plates of 0.5, 1.0, and 2.0 pL/plate Lactic Acid were tested with and without metabolic activation, and negative (medium only) and positive (2-aminoanthracene) controls were used. Lactic Acid was not mutagenic with or without metabolic activation.

In vitro cytogenicity assay:

A chromosomal aberration test was performed using a Chinese hamster fibroblast cell line in which the cells were exposed to three doses 11.0 mg/mL of Lactic Acid, 90.5% pure, in physiological saline for 48 h without metabolic activation (Ishidate et al., 1984). Lactic Acid was negative for chromosomal aberrations.

Lactic Acid was evaluated for its ability to induce clastogenic effects using Chinese hamster ovary (CHO) Kl cells in a chromosomal aberration test (Morita et al., 1990). Doses of 10-16 and 8-14 mM were used without and with metabolic activation, respectively, with initial pH ranging from 6.3 to 5.8 and 6.4 to 5.7, respectively at a dose of 14 mM Lactic Acid without metabolic activation, initial pH 6.0, 22.5% of the cells had aberrations. At a dose of 12 mM with metabolic activation, initial pH 6.0, 35.5% of the cells had aberrations. Initial pHs of 5.8 and 5.7 were toxic without and with metabolic activation, respectively. Neutralization of the media decreased the number of aberrations both without and with metabolic activation. No clastogenic activity was observed when the cultures were first exposed to Lactic Acid and then neutralized to pH 6.4 or 7.2 with sodium hydroxide. With F12 medium supplemented with 34 mM sodium bicarbonate, no clastogenic activity was seen at concentrations ~25 mM Lactic Acid, but approximately 10% of the cells had aberrations at pH I 5.7. The investigators concluded that Lactic Acid was non-clastogenic and that the “pseudo-positive” results were attributable to non-physiological pH.

Overall, based on these results and the fact that lactic acid is a naturally occurring substance and part of the human diet as well as it is formed endogenously as an intermediate in various cellular processes, it is considered that the available data did not raise a concern for genotoxicity of lactic acid and their salts. Therefore, no further testing is required.

Ammonium

A registration dossier shall contain information on the human health hazard assessment (regulation 1907/2006, Art.10). However, it is considered that the information requirements for ammonium as laid down in annex VII to IX can be fulfilled by adaptation of the standard testing regime according to Annex XI, points 1.1. and 1.2. as presented in the following:

Bacterial Reverse Mutation Test: 

As reported by the OECD SIDS for ammonium chloride (2003) two in vitro studies have been available, the study by Hoechst AG (1987a) was considered to be more reliable and identified as the key study. The study was conducted according to OECD TG 471 and in compliance with GLP. Strains used were Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 and Escherichia coli WP2uvrA, and doses were 0, 4, 20, 100, 500, 2,500, 5,000 ug/plate. This test was conducted with and without metabolic activation. No increase of revertants was observed at any dose in any strains, indicating a negative result for this substance. Toxic effects were not observed at any dose. Another study [Ishidate et al., 1984] also gave a negative result.

In vitro cytogenicity assay:

A chromosomal aberration test in Chinese hamster lung cells (CHL/IU) was conducted without metabolic activation at 0, 0.25, 0.3 and 0.4 mg/mL. Positive results were observed at concentrations of 0.3 mg/mL at 24 hrs and at 0.3 mg/mL and 0.4 mg/mL at 48 hrs [Ishidate et al., 1984]. However, this result is ascribable to the acidity of this substance, considering the physico-chemical properties of this substance.

Overall, based on these results it was concluded that ammonium chloride is considered to be not genotoxic. The available data did not raise a concern for genotoxicity of ammonium and their salts. Therefore, no further testing is required.

 

Zirconium, acetate lactate oxo ammonium complexes

Zirconium, acetate lactate oxo ammonium complexes is not expected to be genotoxic, since the assessment entities zirconium, acetate, lactate and ammonium have not shown gene mutation potential in bacteria as well as in vitro clastogenicity. Although tests on in vitro gene mutation in mammalian cells are not provided, the bacterial reverse mutation test covering the same endpoint did not show any sign of mutagenic potential with and without metabolic activation. This data suggests that the moieties acetate, lactate, and ammonium are not genotoxic in vitro and likely not genotoxic in vivo.

Moreover, zirconium did not show gene mutation potential in mammalian cells. Genotoxicity and mutagenicity of acetate and lactate are highly unlikely due to their natural occurrence in physiological process. Thus, zirconium, acetate lactate oxo ammonium complexes is not to be classified according to regulation (EC) 1272/2008 for germ cell mutagenicity. Further testing is not required. For further information on the toxicity of the individual constituents, please refer to the relevant sections in the IUCLID and CSR.

Justification for classification or non-classification

As the assessment entities of zirconium, acetate lactate oxo ammonium complexes do not show genotoxic activity in vitro, it is therefore concluded that the substance zirconium, acetate lactate oxo ammonium complexes has also no potential to induce genotoxic effects.

According to the criteria of regulation (EC) No 1272/2008, zirconium, acetate lactate oxo ammonium complexes does not have to be classified for germ cell mutagenicity.

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