Quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl)trimethyl, chlorides

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the studies with a structurally similar substance, the registered substance is not considered to be genotoxic. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From 21 August, 2006 to 01 November, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I (with and without S9 mix): 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/plate
Experiment II (with and without S9 mix): 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: Deionised water was chosen because of its solubility properties and its low toxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

10 µg/plate for TA 1535 and TA 100 (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

other:

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 10 µg/plate for TA 98, 50 µg/plate for TA 1537 (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

3 µL/plate for WP2 uvrA (Without metabolic activation)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2.5 µg/plate for TA 1535, TA 1537, TA 98, TA 100 and 10 µg/plate for WP2 uvrA (With metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicates

Evaluation criteria:

A test item is considered as a mutagen if there is a biologically relevant increase in the number of revertants exceeding the threshold by twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control.

A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in the number of revertants observed in the test groups (i.e., at 100 – 333, 3 – 333 µg/plate with and without S9 mix respectively in experiment I; 100 – 333, 3 – 333 µg/plate respectively with and without S9 mix in experiment II)

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the study conditions, the source substance was non-mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvrA in the reverse mutation assay with and without metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of source substance, C16 TMAC (25% active in water) to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were treated with the source substance using the Ames plate incorporation (pre-experiment and experiment I) and pre-incubation methods (experiment II). The assay was performed in three independent experiments both with and without metabolic activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The concentrations of the source substance ranged from 3 to 5000 µg/plate in the pre-experiment, from 0.1 to 333 µg/plate in experiment I and from 0.01 to 333 µg/plate in experiment II. The plates incubated with the source substance showed reduced background growth in all strains with and without metabolic activation in all experiments. Strong cytotoxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers in any of the five tester strains was observed following treatment with the source substance either in the presence or absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. All positive control showed a distinct increase in the number of revertant colonies. Under the study conditions, the source substance was determined to be non-mutagenic with and without metabolic activation in the reverse mutation assay (Sokolowski, 2006). Based on the results of the source study, similar non- mutagenic potential can be expected for the target substance.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From August 30, 2006 to March 12, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4E

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) supplemented with 10% foetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I: without S9 mix: 0.2, 0.4, 0.8, 1.5 and 2.3 µg/mL, with S9 mix: 1.6, 3.1, 6.3, 12.5 and 18.8 µg/mL
Experiment II:without S9 mix: 0.4, 0.8, 1.6, 3.1 and 4.7 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water

Untreated negative controls:

yes

Remarks:

Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

other: Untreated cells were cultivated without interruption throughout the assay and without addition of test item, in order to obtain the initial spontaneous mutation rate at the beginning of the experiments.

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Remarks:

Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

other: Untreated cells were cultivated without interruption throughout the assay and without addition of test item, in order to obtain the initial spontaneous mutation rate at the beginning of the experiments.

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Seeded into plastic culture flasks
DURATION
- Preincubation period: 24h
- Exposure duration: 4h (in experiment I; with and without S9), 24h (in experiment II; without S9)
- Expression time (cells in growth medium): 7d
- Selection time (if incubation with a selection agent): Day 7

SELECTION AGENT (mutation assays): Thioguanine (6TG)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The test item was considered positive if (a) It reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment. (b) There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants. (c) Survival of the responding dose group is at least 30%. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects
- Precipitation: No precipitation or phase separation of the test substance was observed up to the maximum concentration in both main experiments.

RANGE-FINDING/SCREENING STUDIES: Two range finding pre-tests were performed in the presence (4h treatment) and absence (4h and 24h treatment) of S9. In the first pre-test test substance concentrations between 25 and 3200 µg/mL (active substance) were used to evaluate toxicity. In this first pretest strong toxic effects were noted at all concentrations with and without metabolic activation. Therefore, a second pre-test was performed using concentrations of 0.2 to 25 µg/mL (active substance).
Following 4h treatment without S9 mix strong toxicity occurred at 1.58 µg/mL. The cell growth was completely stopped at the next higher concentration of 3.13 µg/mL and above. In the presence of S9 mix (4h treatment) strong toxicity was determined at the highest concentration of 25 µg/mL. After 24h of treatment a relevant toxic effect occurred at 3.13 µg/mL. At all higher concentrations the cell growth was also completely inhibited.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

The sensitivity of the test system and efficacy of the S9 mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

Mutation results:

Main experiment:

- No relevant and reproducible increase of the mutation frequency occurred at any concentration with and without metabolic activation. All mutant frequencies remained well within the historical range of negative and solvent controls.

- A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in both cultures of experiment I with metabolic activation. However, a small increase of the mutation frequency at toxic concentrations is common in this assay system and does not indicate a possible mutagenic potential provided that the mutation frequency does not exceed the threshold of 3 times above the corresponding solvent control. Since the mutation frequency neither exceeded the historical range of negative and solvent controls nor the threshold as indicated above, the statistical results were considered as biologically irrelevant.

Conclusions:

Under the study conditions, the source substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of source substance, TMAC C16 (25% active in water) to induce gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. The assay was performed in two independent experiments. The cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The concentrations of the source substance in experiment I ranged from 1.6 to 5 µg a.i./mL and 0.2 to 3 µg a.i./mL with and without S9 mix respectively and from 0.4 to 6.3 µg a.i./mL without S9 mix in experiment II. All positive controls showed a distinct increase in the number of mutant colonies. No substantial and reproducible concentration-dependent increases in the mutation frequency at the HPRT locus, was seen in source substance-treatment cells either with or without metabolic activation. Under the study conditions, the source substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Wollny, 2007). Based on the results of the source study, target test substance does not show mutagenic activity in mouse lymphoma assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From April 17, 1989 to September 15, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Information on the category justification can be found in the Quaternary ammonium salts (QAS) category and section 13.2 of IUCLID.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Toxicity test guideline, Japan 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium supplemented with 10% foetal calf Serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

without S9 mix: (7 hours: 1.0 µg/mL; 18 hours: 0.3, 1.0 and 3.0 µg/mL; 28 hours: 3.0 µg/mL)
with S9 mix: (7 hours: 10.0 µg/mL; 18 hours: 1.0, 3.0 and 10.0 µg/mL; 28 hours: 10.0 µg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Fixation time (start of exposure up to fixation or harvest of cells): 7h (high dose), 18h (low, medium and high dose), and 28h (high dose)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (approx. 0.2 µg/mL/culture medium)

STAIN (for cytogenetic assays): Giemsa stains

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 100 cells of each cell culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

- A test article is classified as clastogenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
- A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-clastogenic in this system.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- In the pre-experiments for toxicity the colony forming ability of the V79 cells was totally reduced after treatment with 6.0 µg/mL. Accordingly, one (for 7 and 28h time point) and three concentrations (for 18h time point) were selected to evaluate metaphases for cytogenetic damage. Mitotic index was reduced after treatment with the highest dose levels in the absence and presence of S9 mix, in the main test.
- Mutation results:
There was no relevant increase in cells with structural aberrations after treatment with the test substance at any fixation interval either without or with metabolic activation by S9 mix. Positive controls showed distinct increases in cells with structural chromosome aberrations. The sensitivity of the test system and efficacy of the S9 mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
(COMPARISON WITH HISTORICAL CONTROL DATA: Yes)

Conclusions:

Under the study conditions, the source substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of source substance, TMAC C16 (24 -26% active in water) to induce chromosome aberrations in V79 Chinese hamster lung cells, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The concentration range of the source substance was determined in a pre-experiment using the plating efficiency assay as an indicator of toxicity response. Cells were exposed for 7, 18 or 28 h at concentrations levels of 0.3 to 10.0 µg a.i./mL source substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg a.i./mL completely reduced the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix. Positive controls showed a distinct increase in the number of cells with structural chromosome aberrations. There was no relevant increase in cells with structural aberrations after treatment with the source substance at any fixation interval either without or with S9 mix. Under the study conditions; the source substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Heidemann, 1989).Based on the results of the source study, the target substance is not clastrogenic in V79 chinese hamster lung cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1: An in vitro study was conducted to investigate the potential of source substance, TMAC C16 (25% active in water) to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA were treated with the source substance using the Ames plate incorporation (pre-experiment and experiment I) and pre-incubation methods (experiment II). The assay was performed in three independent experiments both with and without metabolic activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The concentrations of the source substance ranged from 3 to 5000 µg/plate in the pre-experiment, from 0.1 to 333 µg/plate in experiment I and from 0.01 to 333 µg/plate in experiment II. The plates incubated with the source substance showed reduced background growth in all strains with and without metabolic activation in all experiments. Strong cytotoxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers in any of the five tester strains was observed following treatment with the source substance either in the presence or absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. All positive control showed a distinct increase in the number of revertant colonies. Under the study conditions, the source substance was determined to be non-mutagenic with and without metabolic activation in the reverse mutation assay (Sokolowski, 2006).

 

Study 2: An in vitro study was conducted to investigate the potential of source substance, TMAC C16 (24 -26% active in water) to induce chromosome aberrations in V79 Chinese hamster lung cells, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The concentration range of the source substance was determined in a pre-experiment using the plating efficiency assay as an indicator of toxicity response. Cells were exposed for 7, 18 or 28 h at concentrations levels of 0.3 to 10.0 µg a.i./mL source substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg a.i./mL completely reduced the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix. Positive controls showed a distinct increase in the number of cells with structural chromosome aberrations. There was no relevant increase in cells with structural aberrations after treatment with the source substance at any fixation interval either without or with S9 mix. Under the study conditions; the source substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Heidemann, 1989).

 

Study 3: An in vitro study was conducted to investigate the potential of source substance, TMAC C16 (25% active in water) to induce gene mutations at the HPRT locus in V79 Chinese hamster cells, according to OECD Guideline 476, in compliance with GLP. The assay was performed in two independent experiments. The cells were exposed to the source substance for 4 h in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The concentrations of the source substance in experiment I ranged from 1.6 to 5 µg a.i./mL and 0.2 to 3 µg a.i./mL with and without S9 mix respectively and from 0.4 to 6.3 µg a.i./mL without S9 mix in experiment II. All positive controls showed a distinct increase in the number of mutant colonies. No substantial and reproducible concentration-dependent increases in the mutation frequency at the HPRT locus, was seen in source substance-treatment cells either with or without metabolic activation. Under the study conditions, the source substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Wollny, 2007).

 

Based on the results of the studies with a structurally similar substance, similar non-mutagenic and clastogenic potential can be expected for the registered substance.

Justification for classification or non-classification

Based on the results of the studies with a structurally similar substance, the registered substance does not warrant a classification for genotoxicity according to the EU CLP criteria (Regulation 1272/2008/EC).​