Potassium carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reliable studies on in vitro gene mutagenicity in bacteria and on in vitro cytogenicity in mammalian cells on potassium carbonate are available. There was no evidence for an intrinsic genotoxic activity of potassium carbonate.
Additionally, reliable in vitro data on mutagenicity in bacteria, mammalian cell gene mutation studies in mouse lymphoma cells are available from the read-across substance potassium chloride. The results of these assays indicate, that potassium chloride did not induce gene mutations.
In conclusion, there is no evidence for an intrinsic genotoxic activity relevant to humans.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

test performed in governmental laboratory

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only strains TA 97 and TA 102 tested

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA97, TA102

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0, 100, 500, 1000, 5000, 10000 µg/plate

Vehicle / solvent:

Destilled water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

mitomycin C

other: 2-Aminoanthracene 5 µg/plate for all strains with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes

NUMBER OF REPLICATIONS:
- 3 plates for each concentration

The test was performed according to the procedures described by Ames et al. 1975

Species / strain:

S. typhimurium, other: Salmonella typhimurium TA97, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not reported

Conclusions:

Interpretation of results:
negative

The test substance Potassium carbonate anhydrous was considered to be negative with and without metabolic activation in this study.

Executive summary:

In a reverse gene mutation assay in bacteria according to the method of Ames et al. (1975), S. typhimurium strains TA 97 and TA 102 of were exposed to Potassium carbonate anhydrous at concentrations of 100, 500, 1000, 5000 and 10 000 µg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method. 

 

Potassium carbonate anhydrous was tested far beyond the limit concentration of 5000 µg/plate. Cytotoxicity was not reported in this study.

There was no evidence of induced mutant colonies over background in any of the tested S. typhimurium strain up to and including the highest dose tested.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

test performed in governmental laboratory

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Principles of method if other than guideline:

According to the publication of Ames, McCann and Yamasaki (1975)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His deficient

Species / strain / cell type:

other: Salmonella typhimurium TA 92, TA 94, TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

up to and including 10 mg/plate, 6 concentrations were tested

Vehicle / solvent:

phosphate buffer

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

The test was carried out according to the method of Ames, McCann and Yamasaki (1975)
METHOD OF APPLICATION: in agar (plate incorporation); preincubation / 2 plates per concentration

DURATION
- Preincubation period: 20 minutes
- Exposure duration: plates were scored after incubation at 37 °C for 2 days

Metabolic activation system: S9-mix from livers of Fisher rats pretreated 5 days before with polychlorinated biphenyls (500mg/kg bw of Kanechlor KC-400 in olive oil, i.p.)

Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated)

Statistics:

no data

Species / strain:

other: Salmonella typhimurium TA 92, TA 94, TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not reported

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

The test substance Potassium carbonate anhydrous was considered to be negative with and without metabolic activation in this study.

Executive summary:

In a reverse gene mutation assay in bacteria similar to OECD guideline 471 (1983), strains TA 92, TA 94, TA 98, TA 100, TA 1535, TA 1537 of S. typhimurium were exposed to Potassium carbonate anhydrous, purity 99.8 % at 6 concentrations (not further specified) up to and including 10 000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver S9-mix) using the pre-incubation method. 

Potassium carbonate anhydrous was tested far beyond the limit concentration of 5000 µg/plate. Cytotoxicity was not reported in this study.

There was no evidence of induced mutant colonies over background in any of the tested S. typhimurium strain up to and including the highest dose tested.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

test performed in governmental laboratory

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

; test performance only without metabolic activation

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster fibroblast cell line CHL

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM GIBCO supplemented by 10 % calf serum

Metabolic activation:

without

Test concentrations with justification for top dose:

3 concentrations (not further specified) including the highest non-cytotoxic concentration of 1000 µg/mL were tested

Vehicle / solvent:

physiological saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

The Chinese hamster fibroblast cell line (CHL) was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo, Japan, and was maintained by 4-day passages in minimum essential medium (MEM, GIBCO) supplemented by 10% calf serum. The doubling time was approximately 15 hours.

Three concentrations including the 50% inhibition dose determined in a preliminary test were used, the highest tested non-cytotoxic concentration was 1 mg/ml (further tested concentrations not reported)
- Number of replicates: When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels.
- Exposure time: 24 and 48 hours

- Chromosome preparation: Colcemid (final concentration 0.2 µg/ml) was added to the culture 2 hours before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCl solution for 13 minutes at room temperature. After centrifugation, the cells were fixed with acetic acid-methanol and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 minutes.
- Number of metaphases analyzed: 100 well-spread per sample

When no reasonalbe dose-response relationship were found, additional experiments were carried out at similar dose levels.

Evaluation criteria:

A result was considered positive if the incidence of cells with aberrations (including gaps) was 10.0 % or more, equivocal if the incidence was between 5.0 and 9.9 %, and negative if the incidence was 4.9 % or less.

Species / strain:

other: Chinese hamster fibroblast cell line CHL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Highest non-cytotoxic concentration 1000 µg/mL

Additional information on results:

Untreated cells and solvent treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0 %

Conclusions:

Interpretation of results:
negative without metabolic activation not tested with metabolic activation

There was no evidence of Chromosome aberration induced over background, when potassium carbonate anhydrous was exposed to Chinese hamster fibroblast without metabolic activation.

Executive summary:

In a mammalian cell cytogenetics assay similar to OECD guideline 473 Chinese hamster fibroblast CHL cell cultures were exposed to potassium carbonate anhydrous, purity 99.8 % at 3 concentrations (not further specified) including the highest non-cytotoxic concentration of 1000 µg/mL without metabolic activation.

2 % polyploid cells and 3 % cells with structural chromosomal aberrations were detected, the incidence of chromosomal aberrations were < 4.9 % the limit for a negative result and therefore the test was considered to be negative.

There was no evidence of Chromosome aberration induced over background by potassium carbonate.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on transformation of the target and source substances to common compounds (scenario 1 of the Read-Across Assessment Framework (RAAF), ECHA, March 2017 - transformation to common compounds). The target substance potassium carbonate as well as the source substances potassium hydrogencarbonate and potassium chloride dissociate in aqueous media to potassium and the respective anion.

For further details, please refer to the Justification for Read-Across attached in Iuclid Chapter 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to the Justification for Read-Across attached in Iuclid Chapter 13.

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the Justification for Read-Across attached in Iuclid Chapter 13.

4. DATA MATRIX
Please refer to the Justification for Read-Across attached in Iuclid Chapter 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

This test system was designed to assess whether a substance induces forward mutations at the thymidine kinase (TK) locus.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer´s powdered medium for leukemic cells of mice (GIBCO) mixed with purified deionized water , supplemented with Donor horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes; thawing new ampules of the original cultures approx. every 2 months

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Fisher rat liver, induced with Aroclor 1254)

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 - 12 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)


NUMBER OF REPLICATIONS: three independent experiments, with two plate per concentration in experiment 1and 2 and three plates in experiment 3


NUMBER OF CELLS EVALUATED: 3 x 10E6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth


Colonies were counted using an automatic colony counter with a zero size setting.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

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:

Experiment without S9
Potassium chloride was tested to 5000 µg/mL in three experiments in the absence of S9. No mutagenicity and only slight cytotoxicity were observed
in the first experiment, in which potassium chloride was nontoxic and the response was negative. A similar, slight depression in RTG was observed in the second experiment, but mutant frequency was increased approximately 2.5- fold over a concentration range of 1049 – 3200 µg/mL. Although
the increase was not concentration-related, this experiment was evaluated as positive. Cytotoxicity, with no mutagenicity, was observed in the third
experiment, which was evaluated as negative. Because the positive response in the second experiment in the absence of S9 was not confirmed, the
test was evaluated as negative.

Experiments with S9
Potassium chloride was also tested to high concentrations in three experiments in the presence on induced S9. In the first, RGT decreased with
increasing concentrations to 4000 µg/mL, but no mutagenicity was observed; therefore, this experiment yielded a negative response. The second
experiment yield a marginally positive mutagenic response, with an approximately twofold increase at 4000 µg/mL and a 2.5- fold increase at
5000 µg/mL. A third experiment yielded a concentration-dependent increase in mutant frequency, over the solvent control value, of about twofold
at 3645 µg/mL, about threefold at 4050 µg/mL, and about fourfold at 4500 µg/mL. Therefore, this test and the assay were evaluated as positive, but the results were considerd to be ambiuous res. false positive due to the impact on osmolarity.

These responses indicate that high salt concentrations, which affect the ionic balance and somatic pressure of the medium, can induce mutations in
cells surviving the treatment. Therefore, testing with high salt concentrations has been considered to be invalid resulting in false positive outcomes.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The results of this mammalian cell gene mutation assay indicate that, potassium chloride did not cause a positive response in the non-activated system. In the S9-activated systems no mutagen response up to 4000 µg/ml were observed. This outcome is applicable also to the target substance Potassium carbonate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The mutagenicity of potassium carbonate was tested in two in vitro gene mutation studies in bacteria and in an in vitro cytogeneticity study in mammalian cells. Although, these tests were not performed according to standard guidelines they are well performed and documented and give adequate and reliable information on the mutagenic activity. All these tests were negative and gave no indications on an intrinsic genotoxic activity of potassium carbonate.

Reliable and adequate in vitro data on mammalian cell gene mutation studies in mouse lymphoma cells are available from the read-across substance potassium chloride. The genotoxic effect of potassium chloride in a mammalian cell gene mutation assay with cell L5178Y, TK+/- was assessed in two independent laboratories. Potassium chloride was evaluated by the study author as non-mutagenic. The reported weak mutagen effect of potassium chloride are found only at high levels of osmolarity (> 4000 µg/mL) with metabolic activation and in one experiment without activation at dose levels of ≥7000 µg/mL. The reported weak mutagenic effect of KCl most probably results from a disruption of osmotic balance of cells with a subsequent interference with chromosomal stability. This may result in the clastogenic effects (DNA breakage and chromosome structural instability) due to increased osmotic pressure and to K+ effects on sequestering of Mg2+ ions required for normal maintenance of chromatin integrity. However, the genotoxic effects has been attributed by the authors to the changed physical environment of the cells rather than to a direct genotoxic effect. Other chemicals may also exert such effect.” quotation from SIDS Initial Assessment Report, KCl, 2001.

 

The impact of the high osmolarity has been discussed by several authors, Scott et al. 1991 considered the relationship between genotoxicity and concentration and concluded “that, at high concentrations of non-DNA reactive chemicals, artefactual genotoxicity could arise because of osmolarity changes in the culture medium and that such effects do not occur at lower concentrations i.e. there is a true threshold response. We also concluded that such effects are unlikely to be relevant to human exposure or human risk.” The Scientific Committee on Health and Environmental Risks (SCHER, 2009) discussed that “chromosomes are complex, dynamic structures made up of DNA and proteins, and their structural integrity depends on a large number of factors including the maintenance of a physiological intracellular milieu, such as a proper osmolarity and pH.”

 

In addition, the study authors Myhr and Caspary 1988 states“ Any proposal for indirect mutagenesis has immediate implications for placing some limits on the routine screening of ionic substances, because it does not seem desirable to label such materials as mutagens. Fischer´s culture medium contains 400 µg/mL KCL and 8000 µg/mL NaCl as well as many other ionic materials (plus additional ions from S9 mix). Clearly, when 5000 µg/mL of some salt is added, both ionic balance and osmotic pressure are changed, and if excessive perturbation of one or both of these factors influences the stability of the TK locus, some physiological limits to the testing of ionic materials will need to be defined.”

 

In conclusion, there was no evidence for an intrinsic mutagenic or genotoxic activity of potassium carbonate relevant to human exposure or human risk. The evaluated studies cover the full set of in vitro tests required by REACH Regulation Annexes VII and VIII. Substances that are negative in the full set of in vitro tests specified in REACH Regulation Annexes VII and VIII are considered to be non-genotoxic (Guidance on information requirements and chemical safety assessment chapter 7.7.1). Therefore, additional testing is not required.

A justification for read-across is attached to Iuclid section 13.

Justification for classification or non-classification

There is no evidence for a clastogenic or mutagenic potential of potassium carbonate relevant to humans obtained from the results of a chromosome aberration study and bacterial mutation assays with potassium carbonate and two mammalian cell gene mutation studies from the read-across substance potassium chloride.

Therefore, no classification is required for genetic toxicity according to CLP, EU GHS (Regulation (EC) No 1272/2008) and directive 67/548 EEC.