Tricalcium dicitrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated 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:

other: see 'Remark'

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. Read across to the registered substance is considered scientifically justified.

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:

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

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:

2-hydroxypropane-1,2,3-tricarboxylic acid

Details on test material:

- Name of test material (as cited in study report): citric acid

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
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