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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity of zinc lactate can be understood in terms of the genotoxicity of lactic acid and zinc(II). Lactic acid is not genotoxic as tested in a battery of in vitro genotoxicity tests. Zinc(II) salts are not genotoxic as tested also in a battery of in vitro genotoxicity tests.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable

Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, 9-aminoacridine hydrochloride monohydrate, N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

NUMBER OF REPLICATIONS: Triplicate
Statistics:
Not available
Species / strain:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
none

None

Conclusions:
The test material was considered to be non-mutagenic for all the used bacterial strains (Salmonella typhimurium as well as Escherichia coli) with as well as without metabolic activation.
Executive summary:

A study was conducted to determine the potential mutagenicity of the test material using bacterial reverse mutation assay (e.g. Ames test). Four indicator Salmonella typhimurium strains TA98, TA100, TA1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were treated with the test material suspended in dimethylsulfoxide using the plate incorporation method at doses of 50 - 5000 µg/plate). No significant increases in the frequency of revertant colonies were recorded at any dose level. Based on the results, the test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Principles of method if other than guideline:
Cells deficient in thymidine kinase (TK) due to the mutation TK+/- to TK-/- are resistant to the cytotoxic effects of trifluorothymidine (TFT). Thymidine kinase proficient cells (TK+/-) are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not able to proliferate.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus/TK +/-

Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Metabolic activation system:
No data
Test concentrations with justification for top dose:
1.21-12.13 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Normal saline (1 %)
- Justification for choice of solvent/vehicle: Not reported
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
None
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: Not reported
- Exposure duration: 3 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 7 d (at 37 °C in 5 % C02-95 % humidified air)

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT) - 4 μg/mL

NUMBER OF REPLICATIONS: Duplicate cell culture for test material treatment while triplicate plates were prepared for both survival and mutation frequency determinations for each of the 2 replicate cultures

NUMBER OF CELLS EVALUATED: Cells densities were 30000/mL (1 x 100,000/plate) for mutant selection and 15/mL (500/plate) for viability detrmination

DETERMINATION OF CYTOTOXICITY
- Method: Cell survival for each culture was the product of growth in suspension culture and cloning efficiency in soft-agar medium, each relative to solvent controls

OTHER: Cell culture contained 6 x 100,000 cells each in 10 mL test medium
Light exposure was minimal during treatment of cells.
Test conducted at 37 °C
For the recovery and mutant expression, all cells were maintained at 37 °C for 48 h in log phase growth after treatment with test material
Evaluation criteria:
Colonies growing in the presence of triflurothymidine (TFT resistant) or its absence (viable count colonies) were counted. TFT Resistant colonies which were equivalent in size to colonies growing in the solvent control viable count plates ie., large, were scored as mutants.
Statistics:
Not reported
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Not reported
Conclusions:
The test material was found to be non-mutagenic under the test conditions.
Executive summary:

A study was conducted to assess the potential mutagenicity of test material in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The mouse lymphoma cells (TK+/-) were treated with test material at 1.21-12.13 µg/mL for 3 h. 48 h after treatment, cells were treated with 4 µg/mL trifluorothymidine (TFT) for 7 d. Colonies growing in the presence of triflurothymidine (TFT resistant) or its absence (viable count colonies) were counted. TFT resistant colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants.

The test material was found to be non-mutagenic under the test conditions.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chromosome aberrations were studied in human lymphocyte cultures using three subtoxic doses of test material added to 48 and 72 h cultures at 0 and 24 h after initiation. Metaphases from each culture were evaluated for the presence of numerical and structural aberrations.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F 10 media
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Metabolic activation system:
No data
Test concentrations with justification for top dose:
3 X 10-3, 1.5 X 10-3, 3 X 10-5 and 3 X 10-4 M
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER: Test material was added to 48 and 72 h cultures at 0 and 24 h after initiation.

Evaluation criteria:
100 well-spread metaphases from each culture were evaluated for the presence of numerical and structural aberrations.
Statistics:
Chi-square test was used to compare the incidence of dicentrics in the cells treated with zinc and controls.
Species / strain:
lymphocytes: Human
Metabolic activation:
not applicable
Genotoxicity:
ambiguous
Remarks:
Chromosome aberration observed only at the lowest concentration tested, since higher concentrations were unsuitable for the analysis due to cytotoxicity. However, the findings were not statistically significant when compared to the controls only.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
None

Table 1: Chromosome analysis of leucocyte cultures contaminated by zinc chloride

Duration of the culture (h) Dose of salt administered (M) Interval of time between initiation of the culture and salt administration (h) Number of cells analysed  Numerical aberrations Structural aberrations Type and number of structural aberrations
Aneuploid cells Cells in endoreduplication Chromatid aberrations Chromosome aberrations
Gap Gap Fragment Dicentric
48 0  - 100 3 0 1 1      
3 X 10-4 0 100 1 0 2 2      
24 100 4 0 4 2   2  
3 X 10-5 0 100 4 0 3 3     1
24 100 4 0 4 2   2  
72 0  - 100 2 0 3 3      
3 X 10-4 0 100 3 0 0        
24 100 6 0 4 3   1  
3 X 10-5 0 100 4 0 5 3     2
24 100 2 1 3 2 1    
Conclusions:
Chromosome aberrations (dicentric chromosomes) were observed at the lowest concentration of 3 X 10-5 M of the test material . However, the results were found to be insignificant when compared to only controls in chi-square analysis.
Executive summary:

A chromosome aberration test was conducted on human lymphocyte cultures to determine the mutagenic potential of zinc chloride. The concentration of zinc chloride inhibiting mitotic activity was found to be 3 X 10-3 M. Three subtoxic doses i.e., 1.5 X 10-3, 3 X 10 -5 and 3 X 10-4 M were taken for the study. Human lymphocytes were obtained from a healthy donor and cultured for 48 or 72 h in Ham's F 10 medium. The test material was added to 48 and 72 h cultures at 0 and 24 h after initiation. Chromosome preparations were prepared and 100 well-spread metaphases from each culture were evaluated for the presence of numerical and structural aberrations. Chromosome aberrations (dicentric chromosomes) were observed at the lowest concentration of 3 X 10-5 M of the test material . However, the results were found to be insignificant when compared to only controls in chi-square analysis.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The assessment of the genotoxic potential of zinc lactate follows a weight-of-evidence approach. Lactic acid is a ubiquitous and essential molecule of life in all higher organisms and many micro-organisms. It is found in a multitude of food components. For such a molecule the determination of any genotoxic effect is irrelevant, since the internal exposure, and to a lesser extent the external exposure to this molecule is an unavoidable and necessary element of real life.

Genotoxicity of zinc lactate can be understood in terms of the genotoxicity of lactic acid and zinc(II). Lactic acid is not genotoxic as shown and tested in a battery of in vitro genotoxicity tests; in view of the ubiquitousness and essentiality of lactic acid, this result is expected and consequential. Zinc(II) salts are not genotoxic as tested in a battery of in vitro genotoxicity tests.

Justification for classification or non-classification

No genotoxic effects were observed in any of the test systems. Therefore, based on the available data from suitable read-across partners in a weight-of-evidence approach, no classification of zinc lactate for genotoxicity is warranted.