Butane-1,2,3,4-tetracarboxylic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Absence of mutagenic activity of acidity regulators in the Ames Salmonella/microsome test

Author:

Farouk Y. Al-Ani and Salah K. Al-Lami

Year:

1988

Bibliographic source:

Mutation Research, 206 (1988) 467-470

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to determine the mutagenic nature of malic acid

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxybutanedioic acid

Cas Number:

6915-15-7

Molecular formula:

C4H6O5

IUPAC Name:

2-hydroxybutanedioic acid

Details on test material:

- Name of test material: Malic acid- IUPAC name: 2-hydroxybutanedioic acid- Molecular formula: C4H6O5- Molecular weight: 134.0864 g/mol- Substance type: Inorganic- Physical state: No data- Purity: No data- Impurities (identity and concentrations): No data

Specific details on test material used for the study:

- Name of test material: Malic acid- IUPAC name: 2-hydroxybutanedioic acid- Molecular formula: C4H6O5- Molecular weight: 134.0864 g/mol- Substance type: Inorganic- Physical state: No data- Purity: No data- Impurities (identity and concentrations): No data

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100 and TA104

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate S9 fraction was prepared by induction of cytochrome P-450 in male CD-COBS rats

Test concentrations with justification for top dose:

0, 1100, 1500 or 2000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water- Justification for choice of solvent/vehicle: The chemical was soluble in distilled water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2- aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: No data- Exposure duration: No data- Expression time (cells in growth medium): No data- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: TriplicateNUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for a dose dependent increase in the number of revertants/plate

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA97, TA98, TA100 and TA104

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: No dataCOMPARISON WITH HISTORICAL CONTROL DATA:ADDITIONAL INFORMATION ON CYTOTOXICITY: Routine examination of the bacterial background lawn indicates the absence of toxicity associated with the doses of the compound tested

Any other information on results incl. tables

Table: His ± Revertants/Plate Induced By Various Concentrations of Malic Acid in the Presence and Absence of S9

Dose (µL/plate)	S. typhimurium strains
	TA97		TA98		TA100		TA104
	±S9	-S9	±S9	-S9	±S9	-S9	±S9	-S9
0	61.6±4.16	33.0±2.64	69.3±2.08	41.6±3.51	152.0±4.35	136.6±5.77	470.0±4.00	333.0±8.54
0.5	43.0±2.00	42.3±2.08	54.0±3.60	36.6±2.08	127.6±2.08	123.3±3.51	494.6±3.05	396.6±4.16
1.00	48.3±5.85	52.0±2.64	73.0 ±2.64	35.6±3.05	127.3±2.30	125.0±3.00	571.0±3.60	448.3±4.72
2.00	70.3±6.11	55.0±5.00	65.3±4.04	52.0±2.00	130.3±2.30	124.3±3.60	449.0±3.60	425.3±5.03
2-AA	428.3±7.63	68.3±1.52	481.3±6.02	56.6±1.15	592.6±4.16	150.6±6.42	946.6±7.57	619.3±3.05

Data represents mean of 3 plates

Applicant's summary and conclusion

Conclusions:

Malic acid did not induce gene mutation in Salmonella typhimurium strain TA97, TA98, TA100 and TA104 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of malic acid. The study was performed by the standard plate incorporation method. The chemical was dissolved in distilled water as the solvent and used at dose levels of 0, 1100, 1500 or 2000µg/plate using Salmonella typhimurium strainTA97, TA98, TA100 and TA104 with and without S9 metabolic activation system. The spontaneous reversions of the tester strains were within the acceptable range and a decreased spontaneous reversion frequency of TA97 was noted. In the presence or absence of S9 mix, the numbers of revertants in all tester strains for all concentrations of the acids tested were not significantly different from the respective negative controls.Malic did not induce gene mutation in Salmonella typhimurium strainTA97, TA98, TA100 and TA104 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.