Dipotassium hydrogen citrate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Clastogenic effects of food additive citric acid in human peripheral lymphocytes

Author:

Yilmaz S, Uenal F, Yuebasioglu D

Year:

2008

Bibliographic source:

Cytotechnology 56: 137-144

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Citric acid

EC Number:

201-069-1

EC Name:

Citric acid

Cas Number:

77-92-9

Molecular formula:

C6H8O7

IUPAC Name:

citric acid

Method

Species / strain

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

Controls

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

Results and discussion

Test results

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

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.