Citric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in all strains tested (similar to OECD TG 471)
Cytogenicity in mammalian cells: positive for induction of micronuclei in cultured human lymphocytes without activation (similar to OECD draft TG 487)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The tests were conducted to generally acceptable scientific standards, with acceptable restrictions, i.e. no mention of cytotoxic concentrations and positive controls; no appropriate 5th strain

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

specific positive controls not included; no appropriate 5th strain included

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 100, TA 98, TA 1537, TA92 and TA 94

Metabolic activation:

with and without

Metabolic activation system:

polychlorinated biphenyl induced rat liver S9

Test concentrations with justification for top dose:

Up to 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffer
- Justification for choice of solvent/vehicle: sample soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

other: control was exposed to appropriate solvent or untreated

True negative controls:

no

Positive controls:

other: specific controls not tested, but positive results obtained with some test substances screened

Positive control substance:

other: include Fast Green FCF (with activation), L cysteine monohydrochloride (with and without activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

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

NUMBER OF REPLICATIONS: duplicate plates were used

DETERMINATION OF CYTOTOXICITY
- Method: other: not described

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.

Statistics:

No statistical analysis is described.

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:

other: positive results were obtained with some substances tested

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:

other: positive results were obtained with some substances tested

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:

other: positive results were obtained with some substances tested

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:

other: positive results were obtained with some substances tested

Key result

Species / strain:

S. typhimurium, other: TA 92

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:

other: positive results were obtained with some substances tested

Key result

Species / strain:

S. typhimurium, other: TA 94

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:

other: positive results were obtained with some substances tested

Conclusions:

Citric acid has been tested in a bacterial reverse mutation assay conducted according to a protocol similar to OECD Test Guideline 471. No significant increase in the number of revertant colonies was detected in any Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA 100, TA 92 and TA 94 at the maximum dose, using the preincubation method with and without metabolic activation. It is concluded that citric acid is negative for mutagenicity to bacteria under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

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

Remarks:

The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP. The method used was similar to the appropriate OECD guideline, with acceptable restrictions. The restrictions were that no activation was used,

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD draft guideline 487 2009

Deviations:

yes

Remarks:

no activation

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: peripheral human

Metabolic activation:

without

Test concentrations with justification for top dose:

50, 100, 200, 3000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 72 hours
- Expression time (cells in growth medium): 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours

CYTOKINESIS INHIBITOR (micronucleus assays): actin polymerisation inhibitor cytochalasin B (cytoB) 5.2 µg/ml added after 48 hours.
STAIN (for cytogenetic assays): Giesma

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

NUMBER OF CELLS EVALUATED: 1000 binucleate (BN) cells/donor (micronucleus analysis), 500/donor (CBPI)

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI)

Evaluation criteria:

None given in report

Statistics:

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

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: reported "without changing the pH of the medium"

RANGE-FINDING/SCREENING STUDIES: not reported

COMPARISON WITH HISTORICAL CONTROL DATA: not compared with historical control data

Table 1 Induction of micronuclei in cultured human lymphocytes treated with citric acid

Test substance	Treatment		BN cells scored	Distribution of BN cells according to the no. of MN				MN (%)	Cytokinesis-block proliferation index (CBPI)
	Period (hour)	Dose (μg ml^−1)		-1	-2	-3	-4
Negative control	48	0	2,000	6	0	0	0	0.30 ± 0.12	1.84 ± 0.30
Positive control	48	0.1	2,000	220	20	0	0	13.0 ± 0.75	1.30 ± 0.25
Citric acid	48	50	2,000	33	0	0	0	1.65 ± 0.28*	1.43 ± 0.27
		100	2,000	45	1	0	0	2.35 ± 0.34*	1.41 ± 0.26
		200	2,000	48	0	0	1	2.60 ± 0.36*	1.34 ± 026
		3,000	–	–	–	–	–	Toxic	Toxic

Conclusions:

Interpretation of results:
positive without metabolic activation

Citric acid has been tested according to a method that is similar to OECD draft guideline 487 (in vitro mammalian cell micronucleus test). A statistically significant, dose-dependent increase in the percentage of binucleate cells with micronuclei was observed. It is concluded that the test substance is positive for the induction of micronuclei in cultured human lymphocytes under the conditions of this study

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mammalian Chromosome Aberration Test in rat (oral gavage administration) (similar to OECD TG 475) negative
Rat dominant lethal Assay (oral gavage administration) (similar to EU 22): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Information is available from a reliable study for mutagenicity to bacteria. There is also information available from studies of lower reliability that citric acid does not cause chromosome aberrations in vitro. Information from reliable in vivo studies indicates that citric acid does not cause chromosome damage in somatic or in germ cells. A recent publication described positive results in mammalian cells, including an in vitro chromosome aberration analysis and an in vitro micronucleus assay. The micronucleus assay was chosen as key, as the method was close to the draft guideline and effects were observed.

It is considered that this positive result does not affect the overall genetic toxicity assessment of this substance as the effects seen in vitro were not observed in vivo and are not considered biologically relevant.

A recent in vitro comet assay concluded that citric acid was positive, but was the least genotoxic of the food additives tested (Yilmaz et. al, 2014). In view of the deficiencies of the study, particularly the absence of evaluation criteria, the lack of historical control data, and the lack of information on pH, this result is not considered significant. There is no OECD guideline available for this assay, it is not part of regulatory testing and it is considered not to add to the standard in vitro studies (Frötschl, R., 2015). Furthermore, citric acid is known for its chelating properties and ability to change pH in in vitro test systems, therefore it is not surprising that in vitro positive results are found. The potential for DNA damage suggested by the result is not confirmed by the in vivo data.

Reference: Frötschl, R., 2015. Experiences with the in vivo and in vitro comet assay in regulatory testing, Mutagenesis, Volume 30, Issue 1, 1 January 2015, Pages 51–57, https://doi.org/10.1093/mutage/geu069

Justification for classification or non-classification

Citric acid (CAS number 77-92-9) has been tested in a number of bacterial assays, all of which gave negative results. There is also information from a lower reliability study that citric acid does not cause chromosome aberrations in vitro: this result does not agree with a recently published study. Evidence for genetic toxicity has been described in published results from an in vitro micronucleus study and an in vitro comet assay. An in vivo chromosome aberration study does not support the conclusion of the recently reported in vitro studies in mammalian cells, and an in vivo rodent dominant lethal assay also showed no evidence of chromosome damage.

Citric acid is negative in in vivo genotoxicity testing, although effects have been observed in some in vitro studies. Moreover, it has been used as a food additive over a long period. In addition, citrate plays a central role in cellular metabolism, so it is considered that classification for mutagenicity is not required according to Regulation (EC) No 1272/2008.