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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Multiconstituent aluminium potassium fluoride did not induce increased mutation frequency in two GLP-compliant guideline tests with prokaryotes and eukaryotes, with and without metabolic activations, up to limit concentrations. In a GLP-compliant in vitro micronucleus test, performed according to OECD guideline 487, no significant increase in the number of binucleated cells containing micronuclei was observed using a pulse treatment (exposure duration 4 hr). However, in the continuous treatment test (exposure duration 20 hr) a clear dose-related and statistically significant increase (p<0.001) in the number of binucleated cells containing micronuclei was observed at dose levels of 200 μg/ml and above, with and without metabolic activation.

No data are available for the target substance but read across is proposed for this endpoint.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
Justification for type of information:
The proposed analogue read-across approach has been assessed according to the RAAF and is concluded to be scientifically plausible and adequately justified by the reasoning and data presented in the read across justification uploaded in IUCLID section 13.2.
Reason / purpose:
read-across source
Target gene:
his
Species / strain / cell type:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix)
Test concentrations with justification for top dose:
62, 185, 556, 1667 and 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide, 9-aminoacridine, 2-nitrofluorene and N-ethyl-N-nitrosourea; with S9: 2-aminoanthracene and benzo(a)pyrene
Details on test system and experimental conditions:
Set up
The plate-incorporation method with the histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98 and TA 100 and the tryptophan-requiring Escherichia coli mutant WP2 uvrA as indicator strains was applied. A preliminary test to assess the toxicity of the test substance was not performed. Therefore the toxicity test was incorporated in the mutagenicity assay.
One bacterial reverse mutation test was performed. The test substance was suspended in DMSO at a concentration of 50 mg/ml based on a purity of 99%. A homogeneous, turbid, white suspension was obtained. Serial dilutions in DMSO were made. Five concentrations were tested, ranging from 62 to 5000 µg/plate.
Negative controls (DMSO) and positive controls were run simultaneously with the test substance.
The actual concentrations of the test substance in the test solutions were not determined. Therefore, the concentrations reported are nominal concentrations.

Mutation analysis
Fresh bacterial cultures were prepared by inoculation of nutrient broth with a thawed aliquot of the stock culture and subsequent incubation for approximately 10-16 h at 37°C while shaking. Briefly, the mutagenicity assay was carried out as follows. To 2 ml molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin or 0.05 mM tryptophane for the S. typhimurium strains, and E. coli WP2 uvrA strain, respectively), maintained at ca. 46 oC, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain, 0.1 ml of the test substance or of the negative control or of the positive control substance solution, and 0.5 ml S9-mix for the experiments with metabolic activation or 0.5 ml sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at ca. 37 oC for approximately 48-72 hours. Subsequently, the his+ and trp+ revertants were counted. Toxicity is defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control.
Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the vehicle control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, if no more than 5% of the plates are lost through contamination or other unforeseen events, and if at least 3 doses are non toxic.
A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increased in a concentration-related manner or if a two-fold or greater increase is observed compared to the negative control plates. A clear positive response does not need to be verified. Marginally or weakly positive results should be verified by additional testing.
A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.
Omission of a second test under these conditions is acceptable as a single test does not, or hardly ever results in false negative conclusions (TNO historical data and Kirkland and Dean, 1994).
Both numerical significance and biological relevance are considered together in the evaluation.
Statistics:
No statistical analysis was performed.
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed.
In both the absence and presence of S9-mix in all strains, NOCOLOK FLUX did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.
It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that NOCOLOK FLUX is not mutagenic under the conditions employed in this study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
Justification for type of information:
The proposed analogue read-across approach has been assessed according to the RAAF and is concluded to be scientifically plausible and adequately justified by the reasoning and data presented in the read across justification uploaded in IUCLID section 13.2.
Reason / purpose:
read-across source
Species / strain / cell type:
lymphocytes: cultured binucleated human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix)
Test concentrations with justification for top dose:
first test: 1500, 800, 400, 200, 100, 50, 25, 12.5, 6.25 and 3.13 µg/mL
second test: 1500, 1200, 1000, 800, 600, 500, 400, 250, 200, 100 and 50 µg/mL

The concentrations of the test substance were not determined analytically; the concentrations were therefore nominal concentrations.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, mitomycin C and vinblastine sulphate
Details on test system and experimental conditions:
The human lymphocytes were obtained as follows: blood samples were obtained by venapuncture from young (34-35 years old) healthy, non-smoking males with no known recent exposures to genotoxic chemicals or radiation. The blood was collected in sterile, heparinized vacutainer tubes and gently mixed before use to prevent clotting. A different donor was used for each in vitro micronucleus test. The cultures were set up within 1 hour after withdrawal of the blood.

To avoid physiological effects as a result of treatment with the test substance, the osmolality of the maximum final concentrations were determined. The mean measured osmolality of the final concentrations (1500, 800 and 400 Fg/ml) were 452, 448 and 448 mOsmol/kg, respectively. These values were normal when compared to the mean osmolality of culture medium with 1% DMSO (431 mOsmol/kg) and not expected to induce artifactual micronuclei formation.
Based on the observations during the solubility test, it was decided to use 1500 μg/ml (final concentration in the culture medium), as highest dose level for the first test, in both the absence and presence of a metabolic activation system (S9-mix).

First in vitro micronucleus test
In the presence of (PHA-L), aliquots of 0.5 ml of whole blood in 4.5 ml culture medium, were incubated for 48 hours at 37oC in humidified air containing 5% CO2. The incubation was carried out in sterile screw-capped (loose) centrifuge tubes. After this incubation period, the cells were exposed to different concentrations of the test substance, in both the presence and absence of the S9-mix. In all instances duplicate cultures were used. In both the absence and presence of S9-mix, the treatment time was 4 hours (pulse treatment) and the recovery period of the cells was 20 hours after the end of treatment.

Second in vitro micronucleus test
The second in vitro micronucleus test consisted of pulse and continuous treatment groups. The pulse treatment groups were repeated to further clarify the results obtained in the first test. In pulse treatment group, with and without metabolic activation, the treatment time was 4 hours and the recovery period of the cells was 20 hours after the end of treatment. In continuous treatment group, the cells were treated continuously for 20 hours without metabolic activation (S9-mix) and the recovery period of the cells was 28 hours after the end of treatment. The intervals were smaller between the dose levels in both treatment groups (pulse and continuous).
In the presence of phytohaemagglutinin, aliquots of 0.5 ml of whole blood in 4.5 ml culture medium were incubated for 48 hours at 37oC in humidified air containing 5%CO2. The incubation was carried out in sterile screw-capped (loose) centrifuge tubes. After this incubation period, the cells were exposed to different concentrations of the test substance. In all instances duplicate cultures were used. The cells were treated continuously for 20 hours and allowed to recover for 28 hours after the end of treatment.

At the end of the total incubation period the cells in each culture were harvested and processed separately. The cells were harvested by low speed centrifugation, shortly treated with a hypotonic solution (0.075 M potassium chloride), fixed three times with a freshly prepared mixture of methanol and acetic acid, spread on clean slides and air dried. All procedures were performed at room temperature. Three slides were prepared from each selected culture of the test substance, the negative controls and positive controls. The slides were coded by a qualified person not involved in scoring the slides, to enable "blind" scoring and thereafter stained with a fluorescence DNA-specific dye (acridin-orange) for analysis. One slide per culture was analysed for Cytokinesis-Block Proliferation index (CBPI) and two slides were analysed for micronucleus formation.

Quantitative evaluation of cytotoxicity was calculated using the Cytokinesis-Block Proliferation Index (CBPI). Based on the evaluation of cytotoxicity, analysis of micronucleus formation was carried out in at three analysable concentrations of the test substance, together with the negative control cultures and the selected.
The frequencies of binucleated cells with micronuclei were used for the evaluation of micronuclei induction. The CBPI was provided for all treated and control cultures as apositive control cultures. Two thousand binucleated cells per concentration (1000 per culture) were examined for the presence of micronuclei.measure of cell cycle delay. In addition cytotoxicity for all treated and solvent control cultures was recorded.
Evaluation criteria:
A response was considered to be positive if a statistically significant concentrationrelated or a reproducible statistically significant increase in the number of binucleated cells containing micronuclei was induced, at any of the test points.
A response was considered to be equivocal if the percentage of binucleated cells containing micronuclei was statistically marginal higher than that of the negative control (0.05A test substance was considered to be negative if it produces neither a statistically significant concentration-related or reproducible statistically significant increase in the number of binucleated cells containing micronuclei, at any of the test points (p>0.1).
Statistics:
The frequencies of micronuclei found in the cultures treated with the test substance and positive control cultures were compared with those of the concurrent solvent control using Fisher's exact probability test (one-sided). The results were considered statistically significant when the p-value of the Fisher’s exact probability test was less than 0.05 (P<0.05).
The statistical method was used as an aid in evaluating the test results but was not the only determining factor for a positive response. Both statistical methods and biological relevance of the test results were considered together in the evaluation.
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first test, in the presence of S9-mix, the two highest dose levels (800 and 1500 µg/ml) were slightly cytotoxic to the cells. Analysis of micronuclei formation was carried out in the cultures of three dose levels (400, 800 and 1500 µg/ml) of the test substance, the cultures of the solvent control and the cultures of the positive control. The highest dose level (1500 µg/ml) induced a marginal higher increase (p=0.0917) in the number of binucleated cells containing micronuclei when compared to the concurrent negative control.
In the first test, in the absence of S9-mix, the test substance was not cytotoxic to the cells at any of the dose levels analyzed. Analysis of micronuclei formation was carried out in the cultures of three dose levels (400, 800 and 1500 µg/ml) of the test substance, the cultures of the solvent control and the cultures of the positive controls. The highest dose level (1500 µg/ml) induced a statistically significant increase (p<0.05) in the number of binucleated cells containing micronuclei when compared to the concurrent negative control.
Both treatment groups were repeated in the second test to clarify outcome observed at the highest dose level (1500 µg/ml) of the test substance in the first test.

In the second test, in the presence of S9-mix (pulse treatment), the highest dose level (1500 µg/ml) was slightly cytotoxic to the cells. Analysis of micronuclei formation was carried out in three dose levels (250, 1000 and 1500 µg/ml) of the test substance, the cultures of the solvent control and the cultures of the positive control. The test substance did not showed a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations analysed.
In the pulse treatment group, in the absence of S9-mix (pulse treatment), the highest three dose levels (1000, 1200 and 1500 µg/ml) were slightly cytotoxic to the cells. Analysis of micronuclei formation was carried out in three dose levels (250, 1000 and 1500 µg/ml) of the test substance, the cultures of the solvent control and the cultures of the positive controls. The test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations analysed when compared to the concurrent negative control.
In the continuous treatment group, in the absence of S9-mix, all dose levels used were slightly cytotoxic to the cells with the exception of the lowest dose level. Analysis of micronuclei formation was carried out in three dose levels (200, 800 and 1500 µg/ml) of the test substance, the cultures of the solvent control and the cultures of the positive controls. The test substance induced a clear dose-related and statistically significant increase (p<0.001) in the number of binucleated cells containing micronuclei, at all dose levels analysed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 15 Dec 2009 to 29 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The proposed analogue read-across approach has been assessed according to the RAAF and is concluded to be scientifically plausible and adequately justified by the reasoning and data presented in the read across justification uploaded in IUCLID section 13.2.
Reason / purpose:
read-across source
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix)
Test concentrations with justification for top dose:
1.2, 2.5, 4.9, 7 and 10 mmol/L
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: Methyl methanesulphonate (MMS); with S9: 3-methylcholanthrene (MCA)
Details on test system and experimental conditions:
Prior to the main study a dose range finding test was performed; Single cultures were exposed for 24 hours in the absence of S9-mix to 5 concentrations of NOCOLOK FLUX ranging from 0.6 to 10 mmol/l.
In the main study duplicate cultures were exposed for 24 hours in the absence and 4 hours in the presence of S9-mix to 7 concentrations of NOCOLOK FLUX ranging from 0.31 to 10 mmol/l. Finally, 5 concentrations were evaluated for mutagenicity.

- 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. Double cultures were used for each concentration of the test substance. The cells were exposed for 24 h at ca. 37 oC and ca. 5% CO2 in a humidified incubator.
The dose levels of the test substance used ranged from 0.31 to 10 mmol/l NOCOLOK FLUX. 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 (§4.3) 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. Double cultures were used for each concentration of the test substance. The cells were exposed for 4 h at ca. 37 oC and ca. 5% CO2 in a humidified incubator.
The dose levels of the test substance ranged from 0.31 to 10 mmol/l NOCOLOK FLUX. 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).

- 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. 37oC 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 frequency (MF) per 1,000,000 clonable cells was finally calculated as follows:
Mutant frequency (MF) = Mutant Cloning efficiency (MCE) / Cloning efficiency (CE) * 1,000,000

The cloning efficiency of the cells was calculated from the total number of negative wells on the microtiter plates and the number of cells seeded per well. To assess the cytotoxic effects of the test substance or the positive controls on the cells, the initial cell yield after the treatment period, the relative suspension growth and the relative total growth to that of the vehicle negative controls were calculated. The cloning efficiency of the cells was used, together with the cloning efficiency on the TFT-containing plates, to calculate the mutant frequency. The mutant frequency was expressed as the number of TFT-resistant mutants per 1,000,000 clonable cells.
Evaluation criteria:
A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 126 mutants per 1,000,000 clonable cells (Aaron et al, 1994; Clive et al., 1995). 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 sub¬stance causing more than 90% cytotoxicity was considered to be an artefact and not indica¬tive 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.
Both numerical significance and biological relevance were conside¬red together in the evaluation.
Statistics:
No statistical analysis was performed.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
NOCOLOK FLUX was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix cytotoxicity, resulting in a reduction in initial cell yield and suspension growth at and above 1.2 mmol/l. The relative total growth (RTG) value at the highest concentration tested and evaluated for mutagenicity (10 mmol/l) was 29% (mean of duplicate cultures).
In the presence of S9-mix cytotoxicity was observed at and above 2.5 mmol/l; the RTG at the highest concentration tested and evaluated for mutagenicity (10 mmol/l) was 61% (mean of duplicate cultures).
In both the absence and presence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. All data were within the range of the negative control and the historical background.
The negative controls were within historical background ranges and treatment with the positive controls yielded the expected significant increases in mutant frequency compared to the negative controls.
It is concluded that under the conditions used in this study, the test substance NOCOLOK FLUX is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
It is concluded that under the conditions used in this study, the test substance multiconstituent aluminium potassium fluoride is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.
Executive summary:

The test substance multiconstituent aluminium potassium fluoride 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 assay was conducted in which 7 duplicate cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively. The test substance was suspended in dimethyl sulfoxide (DMSO) prior to testing. The highest concentration tested and evaluated for mutagenicity was 10 mmol/l in both the absence and presence of S9-mix.

The test substance was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix cytotoxicity, resulting in a reduction in initial cell yield and suspension growth at and above 1.2 mmol/l. The relative total growth (RTG) value at the highest concentration tested and evaluated for mutagenicity (10 mmol/l) was 29% (mean of duplicate cultures). In the presence of S9-mix cytotoxicity was observed at and above 2.5 mmol/l; the RTG at the highest concentration tested and evaluated for mutagenicity (10 mmol/l) was 61% (mean of duplicate cultures).

In both the absence and presence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. All data were within the range of the negative control and the historical background. 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 controls yielded the expected significant increases in mutant frequency compared to the negative controls.

It is concluded that under the conditions used in this study, the test substance multiconstituent aluminium potassium fluoride is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in vivo data on multiconstituent target substance are available; however, two negative in vivo bone marrow chromosome aberrations assays are available on a structural analogue, cryolite (trisodium hexafluoroaluminate). Taking into account the structural similarity of two substances and their comparable toxicological profiles, it is considered acceptable to derive information on in vivo genotoxicity by read-across from cryolite. Consequently, it is concluded that the substance is not genotoxic in vivo.

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

Additional information

In vitro genotoxicity

Multiconstituent aluminium potassium fluoride was examined for mutagenic activity in the Ames test (OECD guideline 471 and GLP compliant) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA, in the absence and presence of metabolic activation (TNO, 2010a). One bacterial reverse mutation test was performed. All strains were treated with five concentrations of the test substance, ranging from 62 to 5000 µg/plate. Negative controls and positive controls were run simultaneously with the test substance. The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed. In both the absence and presence of S9-mix in all strains, aluminium potassium fluoride did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. 

In a study according to OECD guideline 476 and in compliance with GLP, multiconstituent aluminium potassium fluoride was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells (TNO, 2010b). One assay was conducted in which 7 duplicate cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively. The test substance was suspended in dimethyl sulfoxide (DMSO) prior to testing. The highest concentration of aluminium potassium fluoride tested and evaluated for mutagenicity was 10 mmol/L in both the absence and presence of S9-mix. Aluminium potassium fluoride was cytotoxic in both the absence and presence of S9-mix. In the absence of S9-mix cytotoxicity resulted in a reduction in initial cell yield and suspension growth at and above 1.2 mmol/L. The relative total growth (RTG) value at the highest concentration tested and evaluated for mutagenicity (10 mmol/L) was 29% (mean of duplicate cultures).In the presence of S9-mix cytotoxicity was observed at and above 2.5 mmol/L; the RTG at the highest concentration tested and evaluated for mutagenicity (10 mmol/L) was 61% (mean of duplicate cultures). In both the absence and presence of S9 -mix no increase in mutant frequency was observed at any test substance concentration evaluated. Treatment with the positive controls yielded the expected significant increases in mutant frequency compared to the negative controls.

Multiconstituent aluminium potassium fluoride was also tested in an in vitro micronucleus test (OECD guideline 487 and GLP compliant) with cultured human lymphocytes (TNO, 2010c). Two separate in vitro micronucleus tests were conducted for which blood was obtained from two different donors. Dimethylsulfoxide (DMSO) was used as solvent for the test substance. Dose levels, ranging from 3.13 to 1500 µg/ml were tested in the culture medium. In the first test, in the presence and absence of metabolic activation (S9-mix) the treatment / recovery times was 4/20 hours. In the second test, concentration spacing was modified.In the presence and absence of S9-mix, the treatment / recovery times were 4/20 hours (pulse treatment). In the continuous treatment group, the treatment / recovery times were 20/28 hours. In the first test, in the presence of S9-mix, the two highest dose levels (800 and 1500 µg/ml) were slightly cytotoxic to the cells. The highest dose level (1500 µg/ml) induced a marginal higher increase (p=0.0917) in the number of binucleated cells containing micronuclei when compared to the concurrent negative control. In the first test, in the absence of S9-mix, the test substance was not cytotoxic to the cells at any of the dose levels analysed. The highest dose level (1500 µg/ml) induced a statistically significant increase (p<0.05) in the number of binucleated cells containing micronuclei when compared to the concurrent negative control. In the second test, in the presence of S9-mix (pulse treatment), the highest dose level (1500 µg/ml) was slightly cytotoxic to the cells. The test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei. In the pulse treatment group, in the absence of S9-mix (pulse treatment), the highest three dose levels (1000, 1200 and 1500 µg/ml) were slightly cytotoxic to the cells. The test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei. In the continuous treatment group, in the absence of S9-mix, all dose levels used were slightly cytotoxic to the cells with the exception of the lowest dose level. The test substance induced a clear dose-related and statistically significant increase (p<0.001) in the number of binucleated cells containing micronuclei, at all dose levels (200, 800 and 1500 µg/ml) analysed.In the first and second test, the negative controls and positive controls yielded the expected results. This demonstrates the validity of the study. It is concluded that, under the conditions used in this study, the test substance aluminium potassium fluoride was clastogenic and/or aneugenic to cultured human lymphocytes.

In vivo genotoxicity

In accordance with Column 2 of REACH Annex 8, appropriate in vivo genotoxicity studies should be considered in case of a positive result observed in any of in vitro assay. However, Article 13 of REACH states also that lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. In vivo data addressing clastogenicity are available for a structural analogue of aluminium potassium fluoride, cryolite (trisodium hexafluoroaluminate). The two substances are structural analogues of each other, being complex salts of two homological fluoroaluminic acids and differing primarily in the alkali metal cation, namely potassium vs. sodium. Their human toxicological profiles are expected to be mostly governed by the presence of fluoride anions formed upon dissociation of the fluoroaluminate moieties. As sodium and potassium cations are essential constituents and two of the most abundant ions in all humans, as well as in all animal species, and are not genotoxic in vivo, it is considered acceptable to derive the lacking data on in vivo genotoxicity of aluminium potassium fluoride by read-across from cryolite.

Two in vivo cytogenicity tests were available for cryolite. One bone marrow chromosome aberration test, in which male Crl:CD BR Sprague-Dawley rats were exposed by inhalation to 4.6 mg/m3cryolite for 13 weeks, was run within the 13-week study on repeated dose toxicity (Bayer AG, 1997a). Increased inorganic fluoride concentrations in urine, bones, and teeth were evident, indicating that the test substance has reached the bone marrow. No effect on mitotic activity was observed. Furthermore, no evidence of clastogenicity was observed in the other in vivo bone marrow chromosomal aberration test, in which male and female Crl:CD BR Sprague-Dawley rats were exposed by snout-only inhalation to 2130 mg/m3 cryolite for 6 hours (Huntingdon Life Sciences Ltd., 1997 / Bayer AG, 1997b). Bone marrow cells were sampled after recovery periods of 16, 24 and 48 hours. No clinical signs or mortalities were induced. All experimental parts were run in compliance with GLP and according to OECD guideline 475.

In summary, the available data provide conclusive evidence that cryolite does not induce chromosome aberrations in vivo. Taking into account the structural similarity and comparable toxicological profiles of cryolite and aluminium potassium fluoride, it is concluded that aluminium potassium fluoride should also be considered non-clastogenic in vivo. As also no increased gene mutation frequencies were observed in the available in vitro studies with aluminium potassium fluoride, it is concluded that aluminium potassium fluoride should be regarded as non-genotoxic.


Justification for classification or non-classification

Based on the available data and in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for mutagenicity.