Hexadecyl hydrogen maleate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: data retrieved from literature

Data source

Reference

Reference Type:

publication

Title:

Nonmutagenicity of maleic acid and its sodium salts in Salmonella assays

Author:

R.S. Lake, G.L. De Vito, R.J. Szot and E.Schwartz

Year:

1988

Bibliographic source:

Mutation Research, 207 (1988) 1-5

Materials and methods

Principles of method if other than guideline:

The determination of the the potential of the substance per se or its indirect pH effects to influence his + revertant rates in Ames Salmonella plate incorporation mutagenicity assays

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Maleic acid

EC Number:

203-742-5

EC Name:

Maleic acid

Cas Number:

110-16-7

Molecular formula:

C4H4O4

IUPAC Name:

(2Z)-2-Butenedioic acid

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98, TA 100.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

938, 1875, 3750 and 7500 µg/plate

Vehicle / solvent:

Not specified

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

not specified

Details on test system and experimental conditions:

The Ames Salmonella tester strains TA1535, TA1537, TA1538, TA98 and TA100 were used in the standard plate incorporation assay. Tester strains were maintained and checked for retention of properties. Metabolic activation was provided by adding Aroclor-1254-induced male rat liver microsomal enzymes (S9, Organon Teknika, Charleston, SC) supplemented with cofactors. The S9 mix consisted of: 100 mM sodium phosphate, pH 7.4, 8 mM MgC12, 33 mM KC1, 4 mM Na NADP, 5 mM glucose 6-phosphate, and S9 fraction at a concentration of 0.1 ml/ml of mix.
3 plates per dose point were used for treated and control groups. Revertant colonies were quantitated after 2 days of incubation at 37°C on an automatic colony counter. Solutions of test substance were prepared and diluted in Oxoid nutrient broth No. 2.

pH determinations
Individual top agar solutions containing all components except agar were prepared separately for pH determinations. Each reconstructed top agar consisted of 2.0 ml of 0.5% NaC1 (pH 6.18); 0.5 ml 0.2 M NaPO4 (pH 7.50) or S9 mix (pH 7.10); 0.1 ml 20-h nutrient broth culture (pH 8.05); and 0.1 ml of test item stock diluted in nutrient broth.
pH was determined at room temperature using a Corning pH/ion 135 meter equipped with a combination electrode. This measurement was considered initial pH with recognition that pHof the actual plate incorporation top agar would be further buffered by base agar during the incubation at 37°C.

For positive control: TA1535 NaAz 5.00 µg/plate - S9,2-AA 2.5 µg/plate +S9; TA100 NaAz 5.00 µg/plate - S9, 2-AA 2.5 µg/plate + S9; TA1537 NaAz 75.00 µg/plate -S9, 2-AA 2.5 µg/plate + S9; TA1537 2-NF 5.00/µg/plate -S9, 2-AA 2.5 µg/plate + S9; TA98 2-NF 5.00 µg/plate -S9, 2-AA 2.5 µg/plate +S9.

Statistics:

Each mutagenesis trial was analyzed for treatment-related effects and dose-response trends using the statistical model of Snee and Irr (1984).
Snee, R.D., and J.D. Irr (1984) A procedure for the statistical evaluation of Ames Salmonella assay results, Comparison of results among 4 laboratories, Mutation Res., 128, 115-125

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The substance tself, with a pKa of 1.83, depressed initial pH to as low as 2.4 in the top agar containing S9 mix at 5555 µg/ml. The pH of all top agars containing S9 mix was consistently lower than without S9 mix due to the acidic contribution of NADP.

Applicant's summary and conclusion

Conclusions:

The test substance is not mutagenic in the Salmonella/mammalian microsome assay and that background reversion is not sensitive to lowered pH.

Executive summary:

The test substance was examined in plate incorporation assays at toxicity limited dose levels with the routine tester strains TA1535, TA1537, TA1538, TA98 and TA100. Initial pH of top agar was monitored at each dose level.

The substance failed to induce any significant increases in revertant count in any of the strains tested. At doses as high as 7500 / µg/plate where the initial pH was as low as 2.4, neither phase showed increases which were dose-related or statistically significant. Test substance dose levels above 7500 µg/plate were highly cytotoxic resulting in microcolonies too numerous to count.