Dipotassium hydrogen citrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available studies dipotassium hydrogen citrate can be stated as non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP.

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

without

Test concentrations with justification for top dose:

nominal concentrations: 50, 100, 200, 3000 µg/ml

Vehicle / solvent:

water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Application: in water
Incubation duration: 72 h
Exposure duration: 24 h and 48 h

Actin polymerisation inhibitor cytochalasin B (cytoB) 5.2 µg/ml was added after 48 hours.
STAIN (for cytogenetic assays): Giesma

replications: duplicate cultures (two donors: healthy non-smokers, 27 years, 1 male, 1 female)

number of cells evaluated: 1000 binucleate cells/donor (micronucleus analysis), 500/donor, 2 donors

determination of cytotoxicity: Cytokinesis-Block Proliferation Index

Statistics:

difference in % abnormal cells: z-test; dose-response relationship: correlation and regression coefficient

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at a concentration of 3000 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at a concentration of 3000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

The chromosome aberrations in cultured human lymphocytes treated with citric acid

Test substance	Treatment		Structural Aberrations					Numerical Aberrations		Abnormal cell ± SE (%)	CA/cell ± SE
	Period (h)	Dose (µg/mL)	scu	ctb	dic	csb	cte	p	er
Negative control	24	0	2	-	-	-	-	-	-	2.50 ± 0.70	0.025 ± 0.01
Positive control	24	0.1	18	25	7	3	12	3	-	30.00 ± 3.24	0.325 ± 0.03
Citric acid	24	50	15	4	5	1	-	1	1	12.50 ± 2.34*	0.135 ± 0.02*
Citric acid	24	100	25	5	7	1	-	5	-	19.00 ± 2.77*	0.190 ± 0.03*
Citric acid	24	200	35	5	10	-	-	5	-	20.00 ± 2.83*	0.250 ± 0.03*
Citric acid	24	3000	-	-	-	-	-	-	-	toxic	toxic
Negative control	48	0	3	1	-	-	-	-	-	2.00 ± 0.98	0.020 ± 0.01
Positive control	48	0.1	42	7	6	-	31	3	-	34.00 ± 3.35	0.430 ± 0.035
Citric acid	48	50	34	2	2	-	-	1	-	19.00 ± 2.77*	0.070 ± 0.03*
Citric acid	48	100	27	9	-	-	1	1	-	18.50 ± 2.75*	0.100 ± 0.03*
Citric acid	48	200	30	2	3	1	1	4	-	18.50 ± 2.75*	0.105 ± 0.03*
Citric acid	48	3000	-	-	-	-	-	-	-	toxic	toxic

Scu, Sister chromatid union; ctb, chromatid break; dic, dicentric; csb, chromosome break; cte, chromatid exchange; p, polyploidy; er, endoreduplication; CA/cell, (Chromosome aberrations/cell)

200 Metaphases were scored for each treatment

*Significant from the control P\0.001 (z test)

Induction of micronuclei in cultured human lymphocytes treated with citric acid

Test substance	Treatment		Distribution of BN cells according to the no. of MN				MN(%)	CBPI
	Period (h)	Dose (µg/mL)	(1)	(2)	(3)	(4)
Negative control	48	0	6	0	0	0	0.30 ± 0.12	1.84 ± 0.30
Positive control	48	0.1	220	20	0	0	13.0 ± 0.75	1.30 ± 0.25
Citric acid	48	50	33	0	0	0	1.65 ± 0.28*	1.43 ± 0.27
Citric acid	48	100	45	1	0	0	2.35 ± 0.34*	1.41 ± 0.26
Citric acid	48	200	48	0	0	1	2.60 ± 0.36*	1.34 ± 0.26
Citric acid	48	3000	-	-	-	-	toxic	toxic

BN, Binucleate; MN, Micronucleus; CBPI, Cytokinesis-block proliferation index

*Significant from the control P\0.001 (z-test)

Conclusions:

Citric acid significantly increased the frequency of chromosomal aberrations, Sister chromatid exchanges (except 50 µg/mL for 24 h) and micronucleus in all treatment groups as compared to their negative controls without changing the pH of the medium. Citric acid induced five types of structural aberration in lymphocytes.

It is concluded that the test substance is positive for the induction of micronuclei in cultured human lymphocytes under the conditions of this study.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Potassium citrate salts are known to show a very high solubility in water and dissociate rapidly.
Potassium in known to be non-toxic. The toxicity is determined by the citrate moiety of the substance.
Because of that a read-across to citric acid is suitable for the determination of the toxicity.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP.

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

without

Test concentrations with justification for top dose:

nominal concentrations: 50, 100, 200, 3000 µg/ml

Vehicle / solvent:

water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Application: in water
Incubation duration: 72 h
Exposure duration: 24 h and 48 h

Actin polymerisation inhibitor cytochalasin B (cytoB) 5.2 µg/ml was added after 48 hours.
STAIN (for cytogenetic assays): Giesma

replications: duplicate cultures (two donors: healthy non-smokers, 27 years, 1 male, 1 female)

number of cells evaluated: 1000 binucleate cells/donor (micronucleus analysis), 500/donor, 2 donors

determination of cytotoxicity: Cytokinesis-Block Proliferation Index

Statistics:

difference in % abnormal cells: z-test; dose-response relationship: correlation and regression coefficient

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at a concentration of 3000 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at a concentration of 3000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

The chromosome aberrations in cultured human lymphocytes treated with citric acid

Test substance	Treatment		Structural Aberrations					Numerical Aberrations		Abnormal cell ± SE (%)	CA/cell ± SE
	Period (h)	Dose (µg/mL)	scu	ctb	dic	csb	cte	p	er
Negative control	24	0	2	-	-	-	-	-	-	2.50 ± 0.70	0.025 ± 0.01
Positive control	24	0.1	18	25	7	3	12	3	-	30.00 ± 3.24	0.325 ± 0.03
Citric acid	24	50	15	4	5	1	-	1	1	12.50 ± 2.34*	0.135 ± 0.02*
Citric acid	24	100	25	5	7	1	-	5	-	19.00 ± 2.77*	0.190 ± 0.03*
Citric acid	24	200	35	5	10	-	-	5	-	20.00 ± 2.83*	0.250 ± 0.03*
Citric acid	24	3000	-	-	-	-	-	-	-	toxic	toxic
Negative control	48	0	3	1	-	-	-	-	-	2.00 ± 0.98	0.020 ± 0.01
Positive control	48	0.1	42	7	6	-	31	3	-	34.00 ± 3.35	0.430 ± 0.035
Citric acid	48	50	34	2	2	-	-	1	-	19.00 ± 2.77*	0.070 ± 0.03*
Citric acid	48	100	27	9	-	-	1	1	-	18.50 ± 2.75*	0.100 ± 0.03*
Citric acid	48	200	30	2	3	1	1	4	-	18.50 ± 2.75*	0.105 ± 0.03*
Citric acid	48	3000	-	-	-	-	-	-	-	toxic	toxic

Scu, Sister chromatid union; ctb, chromatid break; dic, dicentric; csb, chromosome break; cte, chromatid exchange; p, polyploidy; er, endoreduplication; CA/cell, (Chromosome aberrations/cell)

200 Metaphases were scored for each treatment

*Significant from the control P\0.001 (z test)

Induction of micronuclei in cultured human lymphocytes treated with citric acid

Test substance	Treatment		Distribution of BN cells according to the no. of MN				MN(%)	CBPI
	Period (h)	Dose (µg/mL)	(1)	(2)	(3)	(4)
Negative control	48	0	6	0	0	0	0.30 ± 0.12	1.84 ± 0.30
Positive control	48	0.1	220	20	0	0	13.0 ± 0.75	1.30 ± 0.25
Citric acid	48	50	33	0	0	0	1.65 ± 0.28*	1.43 ± 0.27
Citric acid	48	100	45	1	0	0	2.35 ± 0.34*	1.41 ± 0.26
Citric acid	48	200	48	0	0	1	2.60 ± 0.36*	1.34 ± 0.26
Citric acid	48	3000	-	-	-	-	toxic	toxic

BN, Binucleate; MN, Micronucleus; CBPI, Cytokinesis-block proliferation index

*Significant from the control P\0.001 (z-test)

Conclusions:

Citric acid significantly increased the frequency of chromosomal aberrations, Sister chromatid exchanges (except 50 µg/mL for 24 h) and micronucleus in all treatment groups as compared to their negative controls without changing the pH of the medium. Citric acid induced five types of structural aberration in lymphocytes.

It is concluded that the test substance is positive for the induction of micronuclei in cultured human lymphocytes under the conditions of this study.

Reference 3

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP. Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium, other: TA92

Species / strain / cell type:

S. typhimurium, other: TA94

Metabolic activation:

with and without

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: L cysteine monohydrochloride

Details on test system and experimental conditions:

- Preincubation period: 20 min
- Exposure duration: 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

duplicate plates were used

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). If no reasonable dose-response was found, the experiment was repeated using different doses.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium, other: TA92

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium, other: TA94

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Conclusions:

The test substance showed negative results on all strains with and without metabolic activation.
No increase in revertant colonies was detected. It was concluded that citric acid is not mutagenic under the conditions of this test.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Potassium citrate salts are known to show a very high solubility in water and dissociate rapidly.
Potassium in known to be non-toxic. The toxicity is determined by the citrate moiety of the substance.
Because of that a read-across to citric acid is suitable for the determination of the toxicity.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP. Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium, other: TA92

Species / strain / cell type:

S. typhimurium, other: TA94

Metabolic activation:

with and without

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: L cysteine monohydrochloride

Details on test system and experimental conditions:

- Preincubation period: 20 min
- Exposure duration: 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

duplicate plates were used

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). If no reasonable dose-response was found, the experiment was repeated using different doses.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium, other: TA92

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Key result

Species / strain:

S. typhimurium, other: TA94

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Test was performed with several substances, positive results were obtained with some substances tested without performing a positive test with a reference substance.

Conclusions:

The test substance showed negative results on all strains with and without metabolic activation.
No increase in revertant colonies was detected. It was concluded that citric acid and its soluble salts are not mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)