Potassium trifluorozincate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtseweg 48 3704 HE Zeist, the Netherlands

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Potassium trifluorozincate

EC Number:

237-537-7

EC Name:

Potassium trifluorozincate

Cas Number:

13827-02-6

Molecular formula:

F3Zn.K

IUPAC Name:

potassium trifluorozincuide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Nocoloc Zn Flux
- Chemical name: Potassium trifluorozincate
- Physical state: white powder
- Analytical purity: ≥99%
- Lot/batch No.: BWF10118
- Expiration date of the lot/batch: 31 December 2011
- Storage condition of test material: ambient temperature

Method

Target gene:

thymidine kinase (TK) locus on chromosome 11

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y tk +/- 3.7.2C line

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver homogenate

Test concentrations with justification for top dose:

- In the absence of S9-mix: 0.32, 0.65, 1.3, 2.6, 3.7, 5.3, 7.5, 8.9, 10, 12, 14, 17, and 20 µg/mL.
- In the presence of S9-mix: 0.33, 0.67, 1.3, 2.7, 5.3, 7.6, 11, 16, 22, 26, 31, 36, 42, and 50 µg/mL.

Vehicle / solvent:

dimethylsulphoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with S9-mix

Details on test system and experimental conditions:

Single cultures were exposed for 24 hours in the absence and 4 hours in the presence of S9-mix to 13 concentrations of the test substance ranging from 0.32 to 20 μg/mL and from 0.33 to 50 μg/mL, respectively. In both cases, 9 concentrations were evaluated for mutagenicity.
- Cell culturing: The L5178Y cells were grown in culture medium consisting of RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10 % v/v for growing in flasks, and 20 % for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin. The cells were cultured in a humidified incubator at ca. 37°C in air containing ca. 5 % CO2. Five to seven days prior to treatment, the cells were generated from a frozen stock culture by seeding them in sterile, screw-capped tissue culture flasks (about 10,000,000 cells per flask: area ± 75 cm²) containing 50 mL culture medium (with 10 % horse serum). Fresh cultures of L5178Y cells were harvested from a number of culture flasks and suspended in culture medium (with 10 % horse serum), and the number of cells were counted. For the cytotoxicity and gene mutation tests portions of ca. 3,000,000 and 5,000,000 L5178Y cells were used per culture in the absence and presence of S9-mix, respectively.
- Preparation of the test substance solution: Just before use, the test substance was suspended in dimethylsulphoxide (DMSO) at a concentration of 5 mg/mL. A homogeneous, slightly turbid suspension was obtained. From this stock solution serial dilutions in DMSO were made and from each of these 0.1 mL was added to a final volume of 10 mL culture medium. The actual concentrations of the test substance in the test solutions were not determined. The concentrations quoted in this report are therefore nominal concentrations.
- Cell treatment without metabolic activation: In the assay without metabolic activation the cells were exposed to the test substance according to the following procedure; 100 μL test substance solution, positive control or negative control and 4.9 mL culture medium without serum were added to ca. 3,000,000 L5178Y cells in 5 mL culture medium (with 10 % horse serum) to a final volume of 10 mL. Two cultures treated with the vehicle (DMSO) were used as negative controls; one single culture treated with MMS was used as positive control substance at a final concentration of 0.1 mmol/L. Single cultures were used for each concentration of the test substance. The cells were exposed for 24 h at ca. 37°C and ca. 5 % CO2 in a humidified incubator. The dose levels of the test substance used ranged from 0.32 to 20 μg/mL. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.
- Cell treatment with metabolic activation: In the assay with metabolic activation the cells were exposed to the test substance according to the following procedure; 100 µL test substance solution, positive control or negative control and 3.9 mL culture medium without serum were added to 1 mL 20% (v/v) S9-mix and 5 mL culture medium (with 10% horse serum) containing ca. 5,000,000 L5178Y cells to a final volume of 10 mL. Two cultures treated with the vehicle (DMSO) were used as negative controls; one single culture treated with MCA was used as positive control substance at a final concentration of 10 μg/mL. Single cultures were used for each concentration of the test substance. The cells were exposed for 4 h at ca. 37°C and ca. 5 % CO2 in a humidified incubator. The dose levels of the test substance used ranged from 0.33 to 50 μg/mL. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.
- Assessment of cytotoxicity: The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG). The relative initial cell yield is the ratio of the number of cells after treatment to that of the vehicle control and is a measure for growth during treatment. The RSG is a measure for the cumulative growth rate of the cells 24 h and 48 h after treatment compared to untreated control cultures. The RTG is the product of the relative initial cell yield, the RSG and the relative colony-forming ability (‘cloning efficiency’) of the cells 48 h after treatment compared with negative control cultures, and is a measure for cytotoxicity that occurs in all phases of the assay. After the treatment period, the cultures were checked for visibly aberrant effects (eg. flocculation/precipitation of the test substance and lysed cells), and the viability of the cells treated with the higher concentrations of test substance was checked. The medium containing the test substance, negative control or positive control was removed and the cells were washed twice with culture medium (with 10% horse serum). Finally, the cells were resuspended in culture medium (with 10% horse serum) and the number of cells was counted. The cell suspensions were diluted to 200,000 cells per ml and the cultures were incubated for about 44-48 h at ca. 37°C and ca. 5% CO2 in a humidified incubator to allow near-optimal phenotypic expression of induced mutations After 20-24 h and 44-48 h the number of cells of all remaining cultures was counted. After 20-24 h the cell suspensions were diluted, if required, to 200,000 cells per mL and further incubated at ca. 37°C and ca. 5% CO2 as described above. After 44- 48 h a portion of the cells was diluted in culture medium (with 20% horse serum) to 10 cells per mL for determining the cloning efficiency. The remaining cells were used for determining the frequency of TFT-resistant mutants. Portions (200 μL) of each dilution at 10 cells per mL were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-14 days at ca. 37°C and ca. 5% CO2 in a humidified incubator. After this period the number of wells without growth of cells was counted and the cloning efficiency was determined using the zero term of the Poisson distribution. The ratio of the cloning efficiency of cells treated with the test substance or the positive control to that of the vehicle control yields the relative cloning efficiency (RCE). The ratio of the suspension growth (SG) of treated cells to that of the vehicle control yields the relative suspension growth (RSG). The relative total growth (RTG) is adjusted for growth during treatment to obtain a measure for cytotoxicity that occurs in all phases of the assay. Reduction of the cell count after treatment, or of the RSG and of the RTG is a measure for the cytotoxicity of the test substance.
- Gene mutation analysis: The frequency of TFT-resistant mutants and the cloning efficiency of the cells were determined 2 days after starting the test. The number of cells was counted and the cloning efficiency of the cells was determined. To determine the frequency of TFT-resistant mutants, the cell suspensions were diluted to a density of 10,000 cells per mL in culture medium (with 20 % horse serum) containing 4 μg TFT per mL. Portions (200 μL) of each dilution were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-14 days at ca. 37°C and ca. 5 % CO2 in a humidified incubator. After this period the number of wells without growth of cells was counted and the cloning efficiency in the TFT plates (Mutant cloning efficiency) was calculated. The mutant colonies of the negative and positive controls were scored using the criteria of small and large colonies. In case the test substance induced a positive response, colony sizing was also performed at concentrations causing a positive response. The following definitions were used for colony sizing: large colony: covers >25% of the well area and the edge consists of one cell layer, small colony: covers <25% of the well area, the edge consists of more than one cell layer, and the diameter was 1/10 or more of the well.

Evaluation criteria:

- The average cloning efficiency of the negative controls should not be less than 60 % or more than 140 %.
- The average suspension growth of the negative controls should be between 8 and 32.
- The average mutant frequency of the negative controls should fall within the range of 40-300 TFT-resistant mutants per 1,000,000 clonable cells.
- The mutant frequency of the positive controls should be higher than 400 TFTresistant mutants per 1,000,000 clonable cells, and should be at least twice that of the corresponding negative control.
- Unless the test substance shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH and osmolar effects), the highest
concentration should result in a clear cytotoxic response. The RTG value of one of the data points should be between 10 and 20%, or one data point
between 1 and 10% and another between 20 and 30%.

- A response was considered to be positive if the induced mutant frequency was more than 126 mutants per 1,000,000 clonable cells. A response was considered to be equivocal if the induced mutant frequency was more than 88 mutants (but smaller than 126 mutants) per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity and with no evidence of mutagenicity at RTG > 10%, was considered to be an artefact and not indicative of genotoxicity.
- The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.
- The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test substance concentrations.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range-finding test:
- In the dose range finding test single cultures were treated for 24 hours in the absence of S9-mix with 5 concentrations of the test substance ranging from 6.2 to 100 μg/mL. After 4, 24 and 48 hours aliquots were taken to assess the viability and/or cell yield. After 4 hours treatment at a concentration of 100 μg/mL the viability was more than 90%. After 24 hours, at and above 25 μg/mL, the viability was below 40% and the cell yield was below 25%. At 12.5 μg/mL, the viability was about 70-90% and the cell yield about 30%. After 48 hours, the cell yield was about 50 and 96% at 12 and 6.2 μg/mL, respectively. Based on these results, the maximum concentrations in the main test were 20 μg/mL and 50 μg/mL in the absence and presence of S9-mix, respectively.
Main test:
- Dose levels and visual observations before and after treatment: In the absence and presence of S9-mix, the dose levels of the test substance used ranged from 0.32 to 20 μg/mL and from 0.33 to 50 μg/mL, respectively. At the start of the treatment in the absence of S9-mix no abnormalities were observed. At the end of the treatment slight discolouring of the medium was observed at and above 17 μg/mL due to reduced cell growth. At the end of the treatment period, the viability of the cells at the highest two concentrations (17 and 20 μg/mL) was 20-30% and 40-50%, respectively. At 14 μg/mL the viability of the cells was 80-90% and at and below 12 μg/mL the viability of the cells was above 90%. At the start and the end of the treatment in the presence of S9-mix slight discolouring of the medium was observed at 50 μg/mL. At the end of the treatment period, the viability of the cells at and above 11 μg/mL could not be determined; at and below 7.6 μg/mL the viability of the cells was above 90%.
- Cytotoxicity: In the absence of S9-mix the test substance was toxic to the cells. The initial cell yield and/or relative suspension growth (RSG) and relative total growth (RTG) were reduced by more than 10% at and above 7.5 μg/mL. The two highest dose levels of the test substance evaluated for mutagenicity were 17 and 14 μg/mL; the RTG at these doses were 2% and 12% respectively. In the presence of S9-mix the test substance was also toxic to the cells. The initial cell yield and/or RSG and RTG were reduced at and above 16 μg/mL. The two highest dose levels evaluated for mutagenicity were 31 and 26 μg/mL; the RTG at these dose levels were 5% and 14%, respectively.
- Mutagenicity:
In the absence of S9-mix, after 24 hours treatment, at a concentration of 17 and 10 μg/mL, the mutant frequency was significantly increased by 205 and 96 mutants per 1,000,000 clonable cells compared to the negative control, respectively. Since the increase at 17 μg/mL was observed at a concentration causing more than 90% cytotoxicity (RTG 2%) it was considered to be an artefact and not biological relevant. The increase at 10 μg/mL was considered to be equivocal. At all other concentrations no increase in mutant frequency was observed. In the presence of S9-mix, dose related significant increases in mutant frequency were observed; at 16, 22, 26 and 31 μg/mL the MF was increased by 251, 371, 446 and 707 mutants per 1,000,000 clonable cells compared to the negative control. The RTG were 42%, 39%, 14% and 5%, respectively.
- Colony sizing: At concentrations causing an increase in mutant frequency the mutant colonies were scored using the criteria of small and large colonies.
In presence of S9-mix relatively more small then large colonies were formed. The mean percentage of small colonies at 16, 22, 26 and 31 μg/mL (positive responses in MF) was 71% compared to 29% of large colonies. In the absence of S9-mix also relatively more small then large colonies were formed. The mean percentage of small colonies at 14 and 17 μg/mL was 79% compared to 21% of large colonies. In comparison, in the positive (clastogenic) control (MMS) the percentage of small colonies was 75%.
- Positive and negative controls: Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively; Dimethylsulphoxide (DMSO) served as negative control. The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

Nocolok Zn Flux is mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence of metabolic activation. However, since relatively more small than large colonies were formed it might be concluded that the mechanism of mutation induction is clastogenicity than mutagenicity. In the absence of S9-mix equivocal results were obtained.

Executive summary:

In a GLP compliant gene mutation test performed according to OECD guideline 476, Nocoloc Zn flux was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). One test was conducted. In this test 13 and 14 single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively. The test substance was dissolved in dimethylsuphoxide (DMSO). The highest concentrations of Nocolok Zn Flux evaluated for mutagenicity were 17 μg/mL and 31 μg/mL in the absence and presence of S9-mix, respectively, based on cytotoxicity. In both the absence and presence of S9-mix the test substance was toxic to the cells resulting in a reduction in initial cell yield and suspension growth. The relative total growth (RTG) at the highest concentration evaluated in the absence (17 μg/mL) and presence of S9-mix (31 μg/mL) was 2% and 5%, respectively. In the absence of S9-mix, at a concentration of 17 and 10 μg/mL, the mutant frequency was increased by 205 and 96 mutants per 1,000,000 clonable cells compared to the negative control, respectively. Since the increase at 17 μg/mL was observed at a concentration causing more than 90% cytotoxicity (RTG 2%) it was considered to be an artefact and not biological relevant. The increase at 10 μg/mL was considered to be equivocal. At all other concentrations no increase in mutant frequency (MF) was observed. In the presence of S9-mix, dose related significant increases in mutant

frequency were observed; at 16, 22, 26 and 31 μg/ml the MF was increased by 251, 371, 446 and 707 mutants per 1,000,000 clonable cells compared to the negative control. The RTG were 42%, 39%, 14% and 5%, respectively. In the presence of S9-mix at the concentrations causing an increase in mutant frequency, more small then large colonies were formed. The mean percentage of small colonies formed was 71%. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix,

respectively; DMSO served as negative control. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls. It is concluded that under the conditions used in this study, the test substance Nocolok Zn Flux is mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence of metabolic activation (S9-mix). However, since relatively more small than large colonies were formed it might be concluded that the mechanism of mutation induction is clastogenicity than mutagenicity. In the absence of S9-mix equivocal results were obtained.