Zinc Chelate Complex

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- odour: odourless
- color: colourless
- expiration date: 16 October 2020

Specific details on test material used for the study:

- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)

Method

Target gene:

Salmonella typhimurium
Strains - Genotype - Type of mutations indicated
TA1537 - his C 3076; rfa-; uvrB-: - frame shift mutations
TA98 - his D 3052; rfa-; uvrB-;R-factor - frame shift mutations
TA1535 - his G 46; rfa-; uvrB-: - base-pair substitutions
TA100 - his G 46; rfa-; uvrB-;R-factor - base-pair substitutions

Escherichia coli
Strains - Genotype - Type of mutations indicated
WP2uvrA - trp-; uvrA-: - base-pair substitution

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 fractions prepared from the livers of phenobarbital/beta-naphthoflavone-induced rats

Test concentrations with justification for top dose:

- Assay 1: 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate
- Assay 2: 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate

Vehicle / solvent:

N,N-Dimethylformamide (DMF)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; 4-nitro-1,2-phenylene-diamine

Details on test system and experimental conditions:

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/-naphthoflavone-induced rats. The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 (Plate Incorporation Method) and an Assay 2 (Pre-Incubation Method). Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. B

Evaluation criteria:

A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

Criteria for a Negative Response:
- A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Statistics:

No statistical analysis

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the maximum test concentration was 1581 μg test item/plate due to cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the maximum test concentration was 1581 μg test item/plate due to cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the maximum test concentration was 1581 μg test item/plate due to cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the maximum test concentration was 1581 μg test item/plate due to cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the maximum test concentration was 1581 μg test item/plate due to cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

Applicant's summary and conclusion

Conclusions:

The test item had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 (Plate Incorporation Method), an Assay 2 (Pre-Incubation Method) and an Assay 3 (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test item was dissolved in DMSO. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate. Based on the results of the Assay 2, the examined test concentrations in Assay 3 were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

No precipitate was detected on the plates in the main tests. Inhibitory, cytotoxic effect of the test item was observed in the main tests in all examined bacterial strains with and without metabolic activation at higher concentrations.

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.