Melamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study report, comparable to guideline studies, under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Reference 2

Reference Type:

publication

Title:

Mutagenicity testing of melamine

Author:

Mast RW, Friedman MA, Finch RA

Year:

1982

Bibliographic source:

Toxicologist 2, 172, (1982)

Materials and methods

Principles of method if other than guideline:

Method: other: Ames et al.: Mut. Res. 31, 347-364, (1975)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Melamine

EC Number:

203-615-4

EC Name:

Melamine

Cas Number:

108-78-1

Molecular formula:

C3H6N6

IUPAC Name:

1,3,5-triazine-2,4,6-triamine

Method

Target gene:

his- (S. typhimurium)

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium; TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

50, 100, 500, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

Six dose levels of Melamine (5000, 2500, 1000, 500, 100, and 50 µg/plate) were tested. All of the dose levels were tested on duplicate plates, in the presence and absence of an Aroclor 1254-induced rat liver S-9 activation system. The test utilized the five standard Salmonella tester strains: TA98, TA100, TA1535, TA1537, and TA1538. Overnight cultures of the tester strains, started from Master Plates, furnished the bacteria used in the test.
Both positive and solvent controls, as well as sterility controls were performed on triplicate plates concurrently with the test plates. The positive control mutagens used in the test consisted of the following:
TA98: 4-Nitroquinoline-N-oxide (4-NQO), 1 µg/Plate
TA100: Benzo(a)pyrene (BP), 5 µg/Plate
TA1535: N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4 µg/Plate
TA1537: 9-Aminoacridine (9-AA), 100 µg/Plate
TA1538: 2-Aminofluorene (2-AF), 10 µg/Plate

Tests of Benzo(a)pyrene and 2-Aminofluorene were performed in the presence of the S-9 activation system and tests of the other mutagens were performed in the absence of metabolic activation.
The solvent control for the test material and positive control mutagens consisted of the addition of 0.1 mL of DMSO to the top agar. The solvent controls were performed both with and without the S-9 activation system.
The sterility of the S-9 activation system, the solvent and the test article stock solution was determined by mixing the same volume of each actually used in the mutagenicity test with 2 mL of top agar and spreading the mixtures on the surface of separate minimal glucose agar plates. The sterility of the top agar was determined by spreading 2 mL of the melted (45°C) top agar on the surface of minimal glucose agar plates. The sterility plates were incubated
for 2 days at 37°C and then examined for colony growth. The number of colonies, if any, arising from contaminating organisms, was recorded on the data sheet.

Plate Incorporation Technique:
The following were added (in order) to 2 mL molten (45°C) top agar in a 20 x 125-mm sterile screw-cap glass tube: 0.1 mL (approximately 2 to 4 x 108 cells) of an overnight culture of the appropriate Salmonella tester strain, 0.1 mL of appropriately diluted solution of the test article, or an appropriate volume of solvent or positive mutagen control solution, 0.5 mL of the S-9 activation system in those cultures to be run with metabolic activation. The contents of the tubes were quickly mixed on a Vortex mixer and immediately poured onto the surface of minimal glucose agar plates.
Uniform distribution of the top agar on the surface of the plate agar was accomplished by gently tilting and rotating the uncovered plate. The top agar was allowed to harden for approximately 1/2 hour before the plates were placed in a dark 37°C incubator.

Revertant Colony Counts
After 48 hours incubation at 37°C, the colonies (revertants to histidine prototrophy) on all test and control plates were counted, and the presence of the light background growth (background lawn), due to the trace amounts of histidine in the agar, was confirmed. The test plates were observed for the presence of a precipitate or cytotoxic effects. If they were present, they were noted on the data sheet. Counting of the colonies of the plates was accomplished with the aid of a Quebec Colony Counter and a Biotran II Automated Colony Counter. The Biotran II Automated Colony Counter was used to count the colonies on plates having more than 200 colonies/plate.
The number of revertant colonies on each plate was entered in the appropriate space on the data sheet. The average number of revertant colonies/plate was calculated and entered on the data sheet.

Evaluation criteria:

The following criteria must be met for the assay to be considered valid:
• The average number of revertant colonies present on the solvent control plates should be within the normal historical limits of variability.
• The average total number of revertant colonies present on the positive control plates should be at least three times greater than the average number of revertant colonies on the corresponding solvent control plates.

The following criteria must be met in evaluating the results of the test as positive or negative:
For a test article to have a negative result in the test, at least 5 mg/plate (or the maximum nontoxic or non-precipitating dose level, or the maximum dose level based on other considerations such as usage level, etc.) must be tested. Also, the average total number of revertant colonies/plate on the test-article plates must be less than two times the average number of spontaneous (background) revertant colonies/plate on the corresponding solvent control plates and/or there is no significant (p >0.05) positive dose-response relationship. The data were analyzed as a trans format ion of the raw data in order to normalize the variance.
The transformation used was:
TCNT = (CNT)0.2, where TCNT is the transformed number of revertant colonies/plate, and CNT is the actual number of revertant colonies/plate. The transformed data were analyzed within tester strain and within S-9 system groups using One-Way Analysis of Variance. If significant (p <0.05) differences were indicated among treatment groups, a one-side Dunnett's test was performed to determine if colony counts are increased over the solvent control values.

Statistics:

The transformed data were analyzed within tester strain and within S-9 system groups using a linear regression analysis to determine whether a significant (p <0.05) dose-response relationship exists.

Results and discussion

Test results

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All of the positive mutagen controls showed greater than three-fold increases in the mean number of revertant colonies/plate compared with their corresponding solvent (DMSO) controls. All of the mean positive control values were significantly greater (p <0.05) than those of the corresponding solvent controls. The mean number of revertant colonies on the solvent (DMSO) control plates fell within the normal historical limits of variability except for tester strain TA98 both in the presence and absence of the S-9 activation system. These solvent controls had mean numbers of revertant colonies/plate of 101 and 95, respectively, compared with the normal historical range of 20-50/plate. For this reason, the test was repeated with the tester strain TA98 both in the presence and absence of the S-9 activation system.
Tester strains were exposed to Melamine at dose levels ranging from 50 µg/plate to 5 mg/plate. Significantly greater (p < 0.05) numbers of revertant colonies were induced only in tester strain T98 at a dose level of 100 µg/plate in the absence of the S-9 activation system, compared with the corresponding solvent control. Since higher do se levels did not induce significantly greater (p > 0.05) number of revertant colonies in tester strain TA98 in the absence of the S-9 activation system, it was concluded that this result had no biological significance. Cytotoxicity was not observed in any of the tester strains at a dose level of 5000 µg/plate, the highest dose level tested. The results presented in this report indicate that the test article, Melamine, produced no positive mutagenic effect on any of the five Salmonella tester strains under the conditions of the test.

Remarks on result:

other: other: Salmonella typhimurium; TA98, TA100, TA1535, TA1537, TA1538

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of the Plate Incorporation Method of the Ames Salmonella/Microsome Mutagenicity Test, it is concluded that the test article, Melamine, under the conditions of the test, could not be shown to be mutagenic for any of the Salmonella tester strains used in the test, in the presence and absence of an Aroclor 1254-induced rat liver S-9 metabolic activation system.

Executive summary:

The Ames Salmonella/Microsome Mutagenicity Test is a bacterial screening test for chemical mutagens causing gene mutations. It is applicable to pure chemicals, complex mixtures and extracts that are soluble in water or organic solvents.

The objective of this study was to determine the mutagenic potential of Melamine. The Plate Incorporation Method of the Ames Salmonella/Microsome Mutagenicity Test was used in this assessment.

Five mutant strains of Salmonella typhimurium, TA98, TA100, TA1535, TA1537 and TA1538, which require histidine for growth, were used as the indicator cells. When exposed to mutagens, one or more of these strains will revert from a histidine-requiring auxotrophic state back to prototrophy (wild type) which do not require histidine, thus indicating the mutagenic activity of the test article. Incorporation in the test of an S-9 microsomal fraction prepared from a homogenate of Aroclor 1254-induced rat liver, permits detection of a wide range of mutagens that require mammalian metabolic activation. Six dose levels of Melamine (5000, 2500, 1000, 500, 100, and 50 µg/plate) were tested.

No toxicity of the test substance to the bacteria was observed up to 5000 µg per plate in the experiment.

The results of this study indicate that the test article, Melamine, produced no positive mutagenic effect on any of the five Salmonella tester strains under the conditions of the test.

Based on the results of the Plate Incorporation Method of the Ames Salmonella/Microsome Mutagenicity Test, it is concluded that the test article, Melamine, under the conditions of the test, could not be shown to be mutagenic for any of the Salmonella tester strains used in the test, in the presence and absence of an Aroclor 1254-induced rat liver S-9 metabolic activation system.