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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Many genotoxicity tests, covering various endpoints of mutagenicity/genotoxicity, were performed with melamine. 19 are reported here. The predominant result is negative.

In vitro standard tests:

- Eight reverse mutation assays, i.e. Ames tests or Ames-test like assays, were negative.

- Two chromosome aberration tests were negative.

- Two cell gene mutation tests were negative.

- Three unscheduled DNA synthesis assays were negative.

- One in vitro micronucleus test was negative.

Other in vitro tests:

- Three sister chromatid exchange assays were performed, 2 were negative, one was equivocal.

- One lambda prophage induction test was positive.

- One bioluminescence assay was negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: NTP studies are considered as high quality studies, even if GLP was formally not stated and some standard methods were not applied. The studies are peer reviewed.
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Modification of the preincubation test of Yahagi et al., Cancer Letters 1, 91-96, 1975.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his- (S. typhimurium)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (Rat and Hamster)
Test concentrations with justification for top dose:
0, 3.3, 10, 33, 100, 111, 333, 1000, 1111, 3333, 5550 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Positive controls:
yes
Remarks:
see below
Details on test system and experimental conditions:
All chemicals were tested using the preincubation procedure of the Salmonella assay as described by Yahagi et al., Cancer Letters 1, 91-96, 1975.. Briefly, 0.5 ml of S-9 mix or 0.1 M P04 buffer was dispensed into an appropriate number of 13 x 100 mm culture tubes maintained at 37°C in a dry-bath. Then 0.05 ml of cells and 0.05 ml of solvent or chemical dilution were added to each tube. The mixture was vortexed and allowed to incubate at 37°C. Following the preincubation period 2.0 ml of molten top agar (45°C) supplemented with 0.5 mM L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose bottom agar in a 15 x 100-mm plastic petri dish. After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h.
At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer. Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.

In the standard protocol (preincubation) for conducting the Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium (in this test TA1535, TA100, TA98, TA1537) plus S9 mix (10 % induced male Sprague Dawley rat liver S9 or 10 % induced male Syrian hamster liver S9) or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical.
The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine-manufacturing gene. The number of colonies is usually counted after 2 days.
Positive control chemicals used in NTP Ames tests:
For strains tested in the absence of S9:
TA98, 2-nitrofluorene or alternatively, TA98 and TA1538, 4-nitro-o-phenylenediamine
TA100 and TA1535, sodium azide
TA97 and TA1537, 9-aminoacridine
For strains tested with S9:
All strains, 2-aminoanthracene (or occasionally, sterigmatocystin)

Evaluation criteria:
If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain of bacteria. The positive control plates are also counted, and the number of mutant colonies appearing on them must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.
If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Cytotoxicity at 3333 µg/plate without metabolisation; no cytotoxicity up to 5550 µg/plate with hamster S9; cytotoxicity at 5550 µg/plate with rat S9
Additional information on results:
No toxicity of the test substance to the bacteria was observed up to 5550 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 four Salmonella tester strains under the conditions of the test.
Based on the results of the Preincubation 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 or hamster liver S-9 metabolic activation system.
Conclusions:
Interpretation of results (migrated information):
negative

No mutagenicity of Melamine to the bacteria used in this test was observed.
Executive summary:

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

Four mutant strains of Salmonella typhimurium, TA98, TA100, TA1535 and TA1537, which require histidine for growth, were used as the indicator cells. Incorporation in the test of an S-9 microsomal fraction prepared from a homogenate of Aroclor 1254-induced rat or hamster liver, permits detection of a wide range of mutagens that require mammalian metabolic activation. Several dose levels of Melamine (0, 3.3, 10, 33, 100, 111, 333, 1000, 1111, 3333, 5550 µg/plate) were tested.

No toxicity of the test substance to the bacteria was observed up to 5550 µg per plate with metabolisation using hamster S9 -Mix, cytotoxicity was noted at 5550 µg/plate with metabolisation using rat S9 -Mix and at 3333 µg/plate in the samples without metabolisation.

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

Based on the results of the Preincubation 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 or hamster liver S-9 metabolic activation system.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only the IUCLID4 entry could be seen.
Principles of method if other than guideline:
Method: other: Matsushima T. et al.: "Short Test Systems for Detecting Carcinogens", Springer Verlag, NY, 237-285
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium strains TA 100, TA 98, TA 97, and TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
up to 5000 µg/plate
Details on test system and experimental conditions:
IUCLID4 Type: Ames test
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
other: other: Salmonella typhimurium strains TA 100, TA 98, TA 97, and TA 102
Remarks:
Migrated from field 'Test system'.
Conclusions:
Melamine was negative in this reverse mutation assay.
Executive summary:

Melamine was negative in this reverse mutation assay with and without an external metabolizing system, and using Salmonella typhimurium strains TA 100, TA 98, TA 97, and TA 102.

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
Reason / purpose for cross-reference:
reference to same study
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
Target gene:
his- (S. typhimurium)
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.
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'.
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.

Endpoint:
in vitro gene mutation study in mammalian cells
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: Acceptable, well-documented publication/study report which meets basic scientific principles
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
This specific-locus mutagenicity assay system utilizes the X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. Hypoxanthine-guanine phosphoribosyl transferase is a "salvage enzyme" which permits cells to use exogenous hypoxanthine and guanine in their DNA synthesis. This enzyme allows the cells to circumvent the folate-dependent de novo synthesis of purines. The assay can be used to measure the induction, by mutagenic agents, of a forward mutation in the HGPRT locus from HGPRT+ to HGPRT-. Since the HGPRT- mutant has little or no HGPRT enzyme activity, the mutant can be selected by growing the cells in medium containing a purine analogue such as 6-thioguanine (TG). Incorporation by the HGPRT+ cells of TG into their DNA is lethal. The mutant HGPRT cells, which lack HGPRT enzyme activity, are not capable of utilizing the TG in their DNA synthesis and therefore, survive and grow in the selection medium containing TG.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
see below
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomes from Aroclor 1254 treated rats
Test concentrations with justification for top dose:
0.6 to 1.0 mg/mL
Vehicle / solvent:
The Melamine was prepared as a 50x concentrate stock solution in DMSO (Mallinckrodt Inc., Paris, KY).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethylnitrosamine (with S9), Ethyl methanesulfonate (without S9)
Details on test system and experimental conditions:
The indicator cells used in this assay are a subclone of Kao and Puck’s Chinese Hamster Ovary Cells, clone K1. They were obtained from the laboratory of Dr. A. Hsie at the Oak Ridge National Laboratory, Oak Ridge, Tennessee. Cells are examined periodically for mycoplasma infection.
The assay was performed according to Raltech Standard Operating Procedure OP-TOX 806. This procedure is based on that described by O'Neill et al (O'Neill J.P., P.A. Brimer, R. Machanoff, G. P. Hirsch and A. A. Hsie (1977), A Quantitative Assay of Mutation Induction at the Hypoxanthine-Guanine Phosphoribosyl Transferase Locus in Chinese Hamster Ovary Cells (CHO/HGPRT System): Development and Definition of the System, Mutation Research, 45: 91-101).

Preparation of Media and Reagents
Culture media and reagents were prepared according to Raltech Standard Operating Procedure OP-TOX 806. The base medium was Ham's F12 medium supplemented with penicillin, streptomycin, and Fungizone. The selection medium consisted of Ham's F12 medium without hypoxanthine with 5 % dialyzed new born calf serum containing 1.34 x 10E-5 M 6-thioguanine (TG) (Sigma Chemical Company). The 100x concentrate stock solution of 6-thioguanine used in the preparation of this medium was prepared in 10E-3 N NaOH.

Stock Treatment Solutions
Test Article. The Melamine was prepared as a 50x concentrate stock solution in DMSO (Mallinckrodt Inc., Paris, KY).
Positive Controls: Dimethylnitrosamine (DMN) (Aldrich Chemical Company, Milwaukee, MI) is a positive control mutagen requiring metabolic activation. DMS was prepared as a 50x concentrate stock solution in DMSO.
Ethyl methanesulfonate (EMS) (Eastman Chemicals, Rochester, NY) is a positive control mutagen not requiring metabolic activation. EMS was prepared as a 50x concentrate stock solution in DMSO.
Negative Control: The solvent control was 2 % (v/v) DMSO in Ham's F12 medium. The solvent control was tested with and without S-9 metabolic activation.
S-9 Metabolic Activation System: The S-9 Activation system was prepared just before use according to Raltech 5tandard Operating Procedure OP-TOX 806 and was kept in an ice bath until used. The Aroclor 1254-induced rat liver S-9 microsomal protein preparation was obtained from Litton Bionetics, Kensington, MD.

Evaluation criteria:
The following criteria must be met for the assay to be considered valid:
• Cloning efficiency of the negative controls must be at least 50 %.
• The mean background mutation frequency in the negative controls must not be greater than 35/10E6 surviving cells.
• Positive controls must show a significant (p < 0.05) increase in their mean mutation frequency compared with the mean of the comparable negative controls.

Statistics:
Determination of statistical significance was performed according to the method described by Irr and Snee (Irr, J.D. and R.D. Snee (1979), Statistical Evaluation of Mutagenicity In the CHO/HGPRT System, Banbury Report 2, eds., A. Hsie, J.P. O'Neill and V., McElheny, Cold Spring Harbor Laboratory, pp 263-275.). Dunnett's test for the comparison of treatments with a control mean was used.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See attached file "Finch 1981c HGPRT Tables.pdf", Table 1, for results.

Conclusions:
Interpretation of results (migrated information):
negative

The results presented in this report indicate that melamine is not mutagenic for the CHO/HGPRT Chinese Hamster ovary cells under the conditions of the assay.
Executive summary:

This assay meets all of the criteria for a valid assay. The results presented in this report indicate that the Melamine is not mutagenic for the CHO/HGPRT Chinese Hamster ovary cells under the conditions of the assay. The absence of a positive mutagenic effect on the indicator cells is demonstrated by the absence of a significant (p 0.05) increase in the mutation frequency in cells exposed to the test article, as well as the absence of significant (p 0.05) positive dose-response relationships over the dose range tested, either in the presence or absence of the S-9 activation system.

A comparison of the cell survival at the dose levels tested with the corresponding solvent controls, indicated no appreciable cytotoxicity of the test article for the indicator cells, either in presence or absence of the S-9 activation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: NTP studies are considered as high quality studies, even if GLP was formally not stated. The studies are peer reviewed. Basic data given. However, no details are reported.
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Method: Galloway S. et al.: Environ. Mutagen., 7, 1
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
other: CHO cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix: 15 µL/mL liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254, 2.4 mg/mL NADP, and 4.5 mg/mL isocitric acid in serum-free medium
Test concentrations with justification for top dose:
0; 240; 270; 300 µg/mL
Vehicle / solvent:
unknown
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: triethylenemelamine. Mitomycin C or cyclophosphamide
Details on test system and experimental conditions:
Test system: Cloned Chinese hamster ovary cells (CHO-W-BI)
Medium: Mc Coy's 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics.

Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The S9 mix consisted of 15 µL/mL liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 4.5 mg/mL isocitric acid in serum-free medium. Test chemicals were supplied under code by the National Toxicology Program chemical repository (Radian Corp., Austin, TX) and were dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.
Evaluation criteria:
All slides were scored blind and those from a single test were read by the same person. One hundred or two hundred first-division metaphase cells were scored at each dose level.
Statistics:
For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used. The P values were adjusted by Dunnett's method to take into account the multiple dose comparisons. For data analysis, the "total" aberration category was used, and the criterion for a positive response was that the adjusted P value be <= 0.05.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: other: CHO cells
Remarks:
Migrated from field 'Test system'.

For detailed results please see the attachment.

Conclusions:
Interpretation of results (migrated information):
negative

The aberration test was negative for Melamine under the test conditions used. No toxicity was observed.
Executive summary:

Melamine was tested for its potential to induce chromosome aberrations with and without a metabolic activation system (S9mix) in CHO cells. No statistically significant increase in aberration frequency was noted up to the highest tested concentration.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientific publication with appropriate description of the method.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 2 strains used.
Principles of method if other than guideline:
Ames test improved by Kubo et al. with the strains TA98 and TA100. Comparative testing of 255 substances.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium: TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver S9 mix induced with phenobarbital and 5,6-benzoflavone.
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
other: other: Salmonella typhimurium: TA98, TA100
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Melamine was negative in this reverse mutation assay.
Executive summary:

An Ames test like study with the strains TA98 and TA100 was performed with and without external metabolising system. Melamine was negative.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: NTP studies are considered as high quality studies, even if GLP was formally not stated. The studies are peer reviewed. Basic data given: comparable to standards.
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Method: Clive and Spector (1975); Clive et al. (1979) with modifications.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus
Species / strain / cell type:
other: Mouse lymphoma cells L5178Y
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0; 10; 20; 40; 80; 100; 120; 140; 160
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: and 3-methylcholanthrene
Details on test system and experimental conditions:
The tk+/tk- -3.7.2C heterozygote of L5178Y mouse lymphoma cells was obtained from Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709, and stored in liquid nitrogen. Thawed samples were cultured and used for up to 3 months, then discarded. Laboratory cultures were confirmed as free from mycoplasma by cultivating or Hoechst staining techniques and maintained in Fischer's medium at 37°C on gyratory tables. Fischer's medium (designated F0) was supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated F10P). On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk-/tk- mutants by exposure for 1 day to F1OP containing THMG (thymidine, 6 mg/mL hypoxanthine, 5 mg/mL, glycine, 7.5 µg/mL and methotrexate, 0.1 µg/mL), then for 3 days to FlOP containing THG only, (i.e., THMG without methotrexate).
Supplementary Activation Conditions
Post-mitochondrial supernatant fractions of liver homogenates (S9) were prepared from male, 200 g, Fischer 344 rats, obtained from the Frederick Cancer Research Center, MD. To prepare induced S9 (RLI), the rats were injected intraperitoneally with Aroclor 1254 (500 mg/kg) in corn oil 5 days before they were killed. Uninduced S9 (RLN) was prepared from untreated rats. Livers were removed and washed in cold 0.15 M KCl, then homogenized in 3 volumes 0.15 M KCl, using a Potter-Elvejhem homogenizer. The homogenate was centrifuged for 10 min at 10,000 rpm (9,000g.av.) in a Beckman L-25 centrifuge. The supernatant fluid was mixed, distributed in 5 mL volumes to gamma-irradiated plastic tubes, then stored in liquid nitrogen (-196 °C) until used. S9 mix was prepared by dissolving preweighed cofactors in Fischer's medium containing 5% heat-inactivated horse serum (F5P), 9 parts of this solution mixed with 1 part S9. The concentrations of the cofactors in S9 mix were NADP, 4 mM and glucose-6-phosphate, 25 mM.
If required, S9 mix was added to constitute 10% of the incubation mixture, i.e., the S9 concentration in the final incubation mixture was 10 µL/mL. This mixture is different from that used by Clive and Spector [1975] but is justified by specific studies on the activation mixture in this laboratory (manuscript in preparation).

Controls
Appropriate vehicle control and positive control groups were incubated in parallel with the test groups. The positive control treatments were 1) without S9 mix, 250 µg EMS/mL; and 2) with RLI and RLN, 2.5 mg 3-Methylcholanthrene (3-MC)/mL.

Evaluation criteria:
Compliance with predetermined quality control criteria was required before the response of a cellular population to the test chemical was evaluated. The basis for this choice of criteria is described elsewhere] and is the outcome of evaluation of historical data in two laboratories (Litton Bionetics, Inc., and SRI International). Four response categories were defined. Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of consensus of all valid experimental results. See also below (any other information).
Statistics:
Calculations. Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG), which is defined as
RTG = (total suspension growth X cloning efficiency) in dosed culture / (total suspension growth X cloning efficiency) in control culture.

The statistical analysis was based upon the mathematical model proposed for this system and consisted of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control. Where a statistically significant response occurred, the lowest observed effective dose (LOED) was noted.
Mutant fraction (MF) was calculated as follows:
MF = 200 x mutant clones per plate (usually a mean of 3) / total clones per plate (usually a mean of 3) = mutants/10E6 clonable cells
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: Mouse lymphoma cells L5178Y
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Testing of melamine was limited to 160 µg/mL by its solubility in the exposure medium. In four experiments, there were no indications of a mutagenic response.
Executive summary:

Specific locus mutation tests with mammalian cells in vitro can be used to demonstrate and quantitate genetic damage, thereby confirming or extending the data obtained in the more widely used bacterial cell tests. The objective of the study was to evaluate the ability of chemicals to induce forward mutations at the thymidine kinase locus (tk) in a mammalian cell, as assayed by colony growth of L5178Y clone 3.7.2C mouse lymphoma cells in the presence of 5-trifluorothymidine (TIT).

Testing of melamine was limited to 160 µg/mL by its solubility in the exposure medium. In four experiments, there were no indications of a mutagenic response.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Documentation is insufficient for validation.
Principles of method if other than guideline:
No data
GLP compliance:
not specified
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
other: rat hepatocytes (male F-344 rat)
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-acetyl-aminofluorene, dimethylnitrosamine
Details on test system and experimental conditions:
Unscheduled DNA synthesis
Metabolic activation:
not applicable
Genotoxicity:
negative
Remarks on result:
other: other: rat hepatocytes (male F-344 rat)
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Melamine is negative in this UDS assay in hepatocytes.
Executive summary:

Melamine is negative in this UDS assay in hepatocytes.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1 Apr 1982 to 16 Apr 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication/study report which meets basic scientific principles
Qualifier:
no guideline followed
Principles of method if other than guideline:
To evaluate the ability of a chemical to interact with DNA by exposing rat liver cells to the chemical and tritiated thymidine. The amount of radioactivity incorporated into the nucleus of the exposed cells is measured and compared to unexposed cells to determine the extent of repair occurring in the DNA.
GLP compliance:
yes
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
not applicable
Species / strain / cell type:
hepatocytes: rat, primary cultures
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: not applicable
Metabolic activation:
not applicable
Metabolic activation system:
not applicable
Test concentrations with justification for top dose:
0.06, 0.2, 0.6, 2.0 and 6.0 mg/mL
Vehicle / solvent:
distilled water
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Remarks:
not applicable
Positive controls:
yes
Positive control substance:
other: 2-Acetoamidofluorene (2AAF)
Details on test system and experimental conditions:
Test system:
Species, strain, sex: rat, Fischer, male
Weight: 150 – 300 g
Supplier: Charles River Breeding Laboratories, Wilmington, Massachusetts

Housing and Feeding conditions:
Caging: individually, stainless steel wire mesh cages, sanitization every two weeks
Light/dark cycle: 12hrs
Temperature/Humidity: Every attempt was made to maintain a temperature of 22 +- 3 °C, and a humidity of 30 to 70 %.
Food: Wayne Lab Blox, ad lib.
Water: fresh tap water, fit for human consumption, ad lib., using an automatic watering system, analysed annually by the Pennsylvania Gas and Water Company

Perfusion medium:
Medium A: 0.5 mM ethylene-glycol-bis-(ß-aminoethylether)-N-N’ – tetra acetic acid (EGTA) in Ca++Mg++ free Hank’s balanced salt solution buffered with 10 mM Hepes, pH adjusted to 7.35, sterilized (0.2 µm filter), maintained at 37 °C
Medium B: Collagenase (Sigma Type I), 100 U/mL prepared in serumless Williams’ medium E (GIBCO), buffered with 10 mM Hepes, pH adjusted to 7.35. Gentamicin was added to the final volume of 0.05 mg/mL. Sterilized (0.45 µm filter) and placed in a 37 °C water bath before and during perfusion.

Surgical and perfusion procedure:
A rat was anaesthetized with 50 mg/kg of sodium pentobarbital (ip injection) and the ventral cavity was opened. The hepatic portal vein was cannulated to allow entrance of the perfusion medium (21 gauge butterfly needle). Perfusion (Manostat Ministaltic pump) until blanching just commenced. Blanching was regulated by adjusting the flow at a very low flow rate. At this point, the infra hepatic inferior vena cava was severed. The speed of the pump was increased to 40 mL/min for 4 min. The diaphragm was cut open and the thoracic vena cava severed. The infra superior vena cava was clamped above the cut keeping the flow rate continuous at 40 mL/min. The liver was then perfused with 250 mL of medium B at a flow rate of 20 mL/min for 10 min. To maintain the perfusion temperature, a 40 W bulb was positioned 6 cm above the liver during perfusion.

Dissociation of hepatocytes:
The liver was excised and placed in 50 mL of serumless WME in a sterile culture dish. After trimming the liver from excessive fat and connective tissue and any sections still showing signs of blood the liver was transferred to a clean, sterile culture dish with 50 mL of medium B. The capsular membrane of the liver was opened. Cells were detached by gently combing the liver with an aluminium comb. The cells were then aliquotted into 50 mL centrifuge tubes by pipetting with a 5 mL pipet. The volume of each tube was brought to 50 mL with WMES. The tubes were allowed to stand in a vertical position for 10 minutes to pellet the hepatocytes. Viability of the resuspended hepatocytes was measured by a trypan blue dye exclusion.

Test protocol:
Cell exposure: 5x10E5 viable hepatocytes were inoculated into 35 x 10 mm culture dishes containing 25 mm2 round Thermanox plastic cover slips in WMES containing 10 % calf serum. The hepatocytes were allowed to attach for 2 hours in a 37 °C CO2 incubator. The cultures were rinsed and reefed with serum-free medium containing test compound and 10 µC/mL 3H-thymidine (New England Nuclear, specific activity 5080 µC/mM). Eighteen to 20 hours after exposure, the cultures were washed five times in each of three successive 100 mL washes of WME.
Cell fixation: The cells on cover slips were swelled in 1 % sodium citrate for 10-15 minutes and fixed in three 30-min changes of 100 % ethanol: glacial acetic acid (3:1). The coverslips were air dried and mounted cell surface up on glass slides with permount. Slides were dipped in NTB-2 photographic emulsion (Eastman Kodak) in the dark, allowed to dry overnight and stored at 4 °C in light-proof slide boxes containing anhydrous calcium sulfate as a desiccant.
Staining: After seven days of exposure time, autoradiographs were developed in D19 (Eastman Kodak) for 4 min at 4 °C, washed in distilled water with 5 mL glacial acetic acid for 30 sec, immersed in Fixer (Eastman Kodak) for 20 minutes, washed in running tap water for 5 min, dried, stained in Harris Alum hematoxylin (Scientific Products), followed by rinsing in tap and ammonium H2O, then dipped into eosin, followed by two separate dips into 95 % ethanol, and 4 more dips in 95 % ethanol, air dried, and coverslipped in permount.
Evaluation criteria:
Unscheduled DNA “repair” synthesis, evidenced by a net increase in black silver grains over the nucleus was quantified by determining nuclear and background grain counts of 20 cells using an automated colony counter with microscopic attachment. A correction coefficient was calculated by the following method. An area/grain ration was obtained by visually scoring an area of each slide containing 3-5 nuclear grains and then obtaining an object area count by the Artek Model 880 Colony Counter. Five such areas were scored, the differences summed and the mean obtained. Background grain count was quantified by randomly selecting the highest of three nuclear-sized areas adjacent to each nucleus. Both nuclei of binucleated cells are recorded separately. Replicative DNA synthesis was evidenced by nuclei blackened with grains too numerous to count. The data of HPC/DNA Repair Assay were reported as mean grain/nucleus from the triplicate cultures.
Species / strain:
hepatocytes: rat, primary cultures
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: hepatocytes
Remarks:
Migrated from field 'Test system'.

Treatment

Concentration

Net Nuclear Grains,
mean +/- sd

Untreated

-

0.33 +/- 0.24

Distilled water

-

0.40 +/- 0.23

2AAF

1 x 10E-4 M

46.90 +/- 26.01 *

Melamine

6.0 mg/mL

1.42 +/- 0.35

Melamine

2.0 mg/mL

0.90 +/- 0.26

Melamine

0.6 mg/mL

1.02 +/- 0.08

Melamine

0.2 mg/mL

0.60 +/- 0.38

Melamine

0.06 mg/mL

0.75 +/- 0.33

* positive finding

Conclusions:
Interpretation of results (migrated information):
negative

The result for the test article were negative in the Rat Hepatocyte Primary Culture/DNA Repair Test under conditions of this assay. These findings are based upon the inability of Melamine to produce a mean grain count of five or greater than the untreated mean grain count at any level of concentration.
Executive summary:

A Rat Hepatocyte Primary Culture/DNA Repair Test was performed at test substance concentrations of 0.06, 0.2, 0.6, 2.0 and 6.0 mg/mL medium. The result for the test article were negative. These findings are based upon the inability of Melamine to produce a mean grain count of five or greater than the untreated mean grain count at any level of concentration.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Secondary literature, no further data are provided, except for the statement under "Executive summary".
Principles of method if other than guideline:
Not reported.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium strains: his G 46; TA1530; TA1531; TA1532; TA1534
Metabolic activation:
not specified
Test concentrations with justification for top dose:
no data
Details on test system and experimental conditions:
IUCLID4 Type: Ames test
Metabolic activation:
not specified
Genotoxicity:
negative
Remarks on result:
other: other: Salmonella typhimurium strains: his G 46; TA1530; TA1531; TA1532; TA1534
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Melamine was negative in this reverse mutation assay.
Executive summary:

5 Salmonella typhimurium strains: his G 46; TA1530; TA1531; TA1532; TA1534 were exposed to the test substance. Two controls were run on each group, the standard control giving the rate of spontaneous mutations of the bacterial strains used, and a positive control with known mutagens demonstrating the mutagenic responsiveness of this particular strain. Melamine was found to be not mutagenic to the strains under the given test conditions.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Documentation is insufficient for assessment
Principles of method if other than guideline:
An in vitro flow cytometric DNA repair assay has been developed and validated by comparison to conventional autoradiography. Both assays measure unscheduled DNA synthesis (UDS).
GLP compliance:
not specified
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
not applicable
Species / strain / cell type:
other: rat hepatocytes (male CRCD Cr1:CD (SD)BR rats)
Test concentrations with justification for top dose:
63-630 ng/ml (0.5-5 x10-3 mM)
Details on test system and experimental conditions:
Unscheduled DNA synthesis
Genotoxicity:
negative
Remarks on result:
other: other: rat hepatocytes (male CRCD Cr1:CD (SD)BR rats)
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Melamine was negative in both methods to determine unscheduled DNA synthesis.
Executive summary:

The main focus of the paper is on the validation of an in vitro flow cytometric DNA repair assay by comparison to conventional autoradiography. Both assays measure unscheduled DNA synthesis (UDS). Melamine was negative in both assays.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Not well-documented publication. No GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
other: National standards of China
Deviations:
not specified
Principles of method if other than guideline:
An in vitro mammalian chromosome aberration test with CHO cells and with and without S-9 mix was performed.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster ovary cell line CHO were obtained from the Type Culture Collection of the Chinese Academy of Science (Shanghai. China) and used from passage 6 to 30.
Metabolic activation:
with and without
Metabolic activation system:
Fractions of rat liver homogenates S-9 were prepared stored at -80 °C. The working concentration of S-9 mixture was 3% obtained by mixing with 5 mM glucose-6-phosphate and 2.5 mM NADP.
Test concentrations with justification for top dose:
0.16, 0.8 and 4 mM.
Vehicle / solvent:
0.85 % saline.
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C in the presence of 5% CO2.
Ten milliliters of CHO cells (6.5 x 10E4/mL) were treated with melamine for 24 and 48 h, respectively, and then treated with 0.2 µg/mL colchicine for 4 h. Cells were then harvested to prepare slides according to the conventional protocol. Cells at metaphase and with well-dispersed chromosomes were examined using single-blind method. Number of cells with chromosome aberrations including chromosome or chromatid fracture, deletion and other types of structural abnormalities in 100 cells per experiment was counted. Each experiment was performed in triplicate.
Statistics:
Distortion rate was calculated and statistically analyzed using Chi2 test with SPSS 11.5 software.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Cell survival rates were 60% and 100% when treated with 4 mM and 0.16 mM melamine, respectively.

Chi square test indicates that the aberration number and distortion rate of chromosome in all melamine groups with and without S-9 were not significantly different from those of the negative control group, indicating that melamine has no mutagenic effect on mammalian cells in vitro.

Conclusions:
Interpretation of results (migrated information):
negative

Melamine has no mutagenic effect on mammalian cells in vitro.
Executive summary:

An in vitro mammalian chromosome aberration test with CHO cells and with and without S-9 mix was performed.

Cell survival rates were 60% and 100% when treated with 4 mM and 0.16 mM melamine, respectively.

Chi square test indicates that the aberration number and distortion rate of chromosome in all melamine groups with and without S-9 were not significantly different from those of the negative control group, indicating that melamine has no mutagenic effect on mammalian cells in vitro.

Endpoint:
genetic toxicity in vitro, other
Remarks:
Ames like test and in vitro cytokinesis-block micronucleus assay (CBMN).
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
<=2014
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Scientific investigation only partly meeting standards as in guideline studies.
Principles of method if other than guideline:
Ames like test and in vitro cytokinesis-block micronucleus assay (CBMN).
GLP compliance:
not specified
Type of assay:
other: Ames like test and in vitro cytokinesis-block micronucleus assay (CBMN).
Species / strain:
S. typhimurium, other:
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Additional information on results:
In the bacterial reverse mutation assay no evidence of toxicity or mutagenicity was observed at any dose tested.
In the in vitro CBMN assay no positive increases in the number of cells containing micronuclei were seen.
Conclusions:
In the bacterial reverse mutation assay no evidence of toxicity or mutagenicity was observed at any dose tested.
In the in vitro CBMN assay no positive increases in the number of cells containing micronuclei were seen.
Executive summary:

The genotoxicity of melamine was evaluated with the bacterial reverse mutation assay, and the in vitro cytokinesis-block micronucleus assay (CBMN). In the bacterial reverse mutation assay, melamine was tested from 62.5 to 1000 µg/plate in tester strains TA97a, TA98, TA100, TA 102, and TA1535, with and without metabolic activation. In the in vitro CBMN assay, in Chinese hamster ovary (CHO) cells, melamine was tested (75, 150, and 300 µg/mL) in the presence and absence of 59 mix.

In the bacterial reverse mutation assay no evidence of toxicity or mutagenicity was observed at any dose tested.

In the in vitro CBMN assay no positive increases in the number of cells containing micronuclei were seen.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Many mutagenicity tests, covering various endpoints of mutagenicity/genotoxicity, were performed with melamine. 9 are reported here. The predominant result is negative.

In vivo standard tests:

- One chromosome aberration assay was negative.

- 4 micronucleus tests were negative.

Other in vivo tests:

- One sister chromatid exchange assay was equivocal.

- Two Drosophila tests were performed, one was equivocal the other was negative.

- The Comet assay in the urinary bladder of rats was negative (Wada 2014), that with sperms was positive (Zhang 2011c).Wada pointed out that a false positive Comet assay can be the result of cell death. The positive outcome in the test by Zhang is considered to be a false positive on.

- Two types of Pig-a assays: the red blood cell (RBC) Pig-a assay, which uses RBCs, and the PIGRET assay, which uses reticulocytes were performed and were negative.

- A further Pig-a assay with RBC and RET of rats was negative.

There is enough weight of evidence that melamine is not genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
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:
other: NTP studies are considered as high quality studies, even if GLP was formally not stated. The studies are peer reviewed. The available data are only very condensed ones.
Qualifier:
according to guideline
Guideline:
other: NTP standards
Principles of method if other than guideline:
The chemical to be examined is administered to the test animals. Bone marrow is taken from the femur and the chromosomes in metaphase are examined microscopically.
GLP compliance:
not specified
Type of assay:
chromosome aberration assay
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
No data
Route of administration:
intraperitoneal
Vehicle:
Corn oil.
Details on exposure:
No data
Frequency of treatment:
1
Post exposure period:
Sampling time: 36 h p.a.
Remarks:
Doses / Concentrations:
0 - 150 - 300 - 600 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
8 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
DMBA (25 mg/kg)
Tissues and cell types examined:
Bone marrow
Sex:
male
Genotoxicity:
negative
Remarks:
only the mid dose is positive
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The number of cells with aberrations was significantly increased only in the mid dose, but not in the low or high doses, compared to the control. The overall trend was negative. Therefore an overall negative result is concluded.
Conclusions:
Interpretation of results (migrated information): negative Negative for the low and high dose, only the mid dose is positive.
Executive summary:

Melamine was administered to mice. Bone marrow was taken from the femur and the chromosomes in metaphase were analysed microscopically. The number of cells with aberrations was significantly increased only in the mid dose, but not in the low or high doses, compared to the control. The overall trend was negative. Therefore an overall negative result is concluded.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed similar to guideline studies, and with GLP.
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
Animals were sacrificed at 30 and 48 hours after a single dose and at 48 and 72 hours after the first dose in the multiple dose groups. Positive controls (0.5mg/kg, IP, triethylenemelamine) were sacrificed 30 hours after dosing and negative controls (water) 48 hours after dosing. At sacrifice femur marrow was harvested, slides prepared and scored for numbers of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: Charles River Breeding Laboratories, Wilmington, Massachusetts
Age at initiation: 7 to 12 weeks
Acclimation period: 5 days
Animal rooms: separate isolation by test system, light cycle - 12 hours light, 12 hours dark; temperature: 22°C ± 3°C ; humidity: 30 to 70%
Housing: mice were housed 4 per box, according to sex, in plastic tote boxes measuring 11-1/4" x 6-3/4" x 5-1/4", fitted with 1/4" galvanized mesh lids.
Bedding: Beta Chip(R) Bedding, North Eastern Products, Warrnsburg, New York. Bedding amount used was sufficient to provide sanitary conditions for 3 days.
Food: Wayne Lab Box(R), ad libitum, ad libitum, checked daily and added or replaced as needed.
Water: availability - fresh tap water, fit for human consumption, ad libitum, in sanitized 8 ounce glass bottles with rubber stopper and stainless steel sipper tube.
Route of administration:
oral: gavage
Vehicle:
distilled water
Details on exposure:
Test article and negative control were administered by oral gavage at 10 ml/kg of body weight.
Groups of mice were treated orally with a single dose of melamine or by two doses separated by 24 hours.
Frequency of treatment:
Once in the positive control and single dose groups.
Two doses separated by 24 hours in the multiple dose groups, and the negative control.
Remarks:
Doses / Concentrations:
1000 mg/kg body weight; single or twice
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
10 ml/kg body weight (test article and negative control)
Basis:
no data
Remarks:
Doses / Concentrations:
20ml/kg of body weight (test article and positive control)
Basis:
no data
No. of animals per sex per dose:
Eight - four males and four females
Positive control: dose = 0.5 mg/kg; Negative control: dose = 2 x vehicle (distilled water); Single Dose Group I: dose = 1000 mg/kg; Single Dose Group II: dose = 1000 mg/kg; Multiple Dose Group I: dose = 2 x 1000 mg/kg; Multiple Dose Group II: dose = 2 x 1000 mg/kg
Control animals:
yes
Positive control(s):
Positive Control was administered by the intraperitoneal route at 20 ml/kg of body weight.
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
All animals were sacrificed by cervical dislocation and their femurs were removed. The femurs were removed by leaving them intact on the knee and pelvis ends. The bone was opened carefully at the proximal end with a scissors until a small opening to the marrow canal became visible. A 1ml tuberculin syringe filled with 0.2 ml fetal calf serum was inserted into the bone and the bone marrow was gently flushed (to assure maximum dispersion) into 1.0 ml of fetal calf serum in a 3 ml conical centrifuge tube. The femur was flushed with fetal calf serum until all the marrow was out and the bone appeared almost transparent. If necessary, the distal end was opened and flushed. The suspension was centrifuged at 1000 rpm for five minutes in a HS-4 Sorvall Centrifuge leaving a small amount of fetal calf serum with the remaining cell button. The cells remained in a fetal calf serum for no longer than 15 minutes. The button was mixed with a pasteur pipette to assure a homogenous mixture. A small drop of the mixture was immediately placed near the frosted end of a glass microcop slide previously cleaned in absolute ethanol and pulled behind a clean slide at a 45º angle. The slides were quick dried on a slide warmer set at 56º.Following preparation of the smears, they were dipped in absolute methanol and allowed to air dry overnight.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
The result for the test article, Melamine were negative in the Micronucleus Test (MNT), at a dose of 1000 mg/kg in the single dose groups and at 2 x 1000 mg/kg administered in a split dose regimen to the multiple dose groups. This is based upon the inability of the chemical to produce a statistically significant increase in the number of micronuclei per 1000 polychromatic erythrocytes in the treated versus the control group.
Conclusions:
Interpretation of results (migrated information): negative
Melamine was negative in the Micronucleus Test (MNT), at a dose of 1000 mg/kg in the single dose groups and at 2 x 1000 mg/kg administered in a split dose regimen to the multiple dose groups.
Executive summary:

The mean number of micronuclei/1000 PCE was 30.38 in the positive controls, 0.50 in the negative controls and ranged from 0.50 to 1.12 in the melamine treated groups. These data indicate an inability of melamine to break chromosomes as measured by the lack of a significant increase in the number of micronuclei/1000 PCE.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: NTP studies are considered as high quality studies, even if GLP was formally not stated. The studies are peer reviewed.
Qualifier:
according to guideline
Guideline:
other: NTP-standards
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
Male B6C3F1 mice (obtained from the National Toxicology Program production facility at Taconic Farms) of a common age between 9 and 14 weeks and weighing within a 2 g range of a mean weight between 25 and 33 g were used. Additional information on animal husbandry can be found in Tice et al. [1990a].
Route of administration:
intraperitoneal
Vehicle:
water; corn oil
Duration of treatment / exposure:
one injection/day on 3 consecutive days
Remarks:
Doses / Concentrations:
0; 500; 1000; 2000 mg/kg
Basis:
no data
No. of animals per sex per dose:
Groups of 5 mice were administered the test chemical by IP injection (at a volume of 0.4 ml per mouse) on three consecutive days.
Control animals:
yes
Positive control(s):
DMBA in corn oil or MMC in PBS.
When corn oil solvent control data were compared (two-tailed t-test) between laboratories, there was no statistically significant differences. But for PBS, the mean of ILS was significantly higher than that for EHRT (P = 0.009). For the positive control data, there were no significant differences between laboratories for either DMBA or Mitomycin C.
Tissues and cell types examined:
Bone marrow ; peripheral blood
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The initial test (bone marrow) gave a positive trend (P=0.003) from 2.1 in the control to 3.8 in the high dose (2000 mg/kg bw/day). Peripheral blood smears from the dose determination test were scored and were negative as was a repeat bone marrow  test using doses of 1000 and 2000 mg/kg bw/day.
Conclusions:
Interpretation of results (migrated information): negative
The primary purposes of the present study were, first, to determine the micronucleus-including ability of 49 chemical carcinogens and non-carcinogens in a mouse bone marrow micronucleus test protocol and second, to help determine what role the test might fulfil in a process of identifying genotoxic chemicals that present a cancer hazard.
Executive summary:

Forty-nine chemicals were tested in a mouse bone marrow micronucleus test that employed 3 daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure.

Melamine was negative.

Endpoint:
genetic toxicity in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Not well-documented publication. No GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
other: Standard of China.
Deviations:
not specified
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: NIH
Sex:
male/female
Details on test animals or test system and environmental conditions:
Specific pathogen-free male NIH mice, aged 4-5 weeks or 7-8 weeks, were used.
Route of administration:
intraperitoneal
Vehicle:
0.85 % saline.
Duration of treatment / exposure:
Two injections, 24 h apart.
Frequency of treatment:
/
Post exposure period:
6 h after the second exposure.
Remarks:
Doses / Concentrations:
400, 800 and 1600 mg/kg bw
Basis:
no data
No. of animals per sex per dose:
5 per sex and group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide.
Tissues and cell types examined:
Bone marrow.
Details of tissue and slide preparation:
Mice were sacrificed and their bone marrows were smeared on slides, fixed with methanol for 15 min, stained with Giemsa dye, pH 6.8, for 20 min, rinsed with water and air-dried. Those slides with well-dispersed cells were examined under microscope at 1000x magnification. Number of micronucleated polychromatic erythrocytes (PCE), in total 1000 PCE per mouse and number of micronucleated normochromatic erythrocytes (NCE) in 200 NCE were counted.
Evaluation criteria:
Micronucleus rate was calculated as the ratio of PCE/NCE.
Statistics:
Differences of micronucleus rates among different groups were analyzed by Chi2 test using SPSS 11.5 software.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
During the experimental period, all animals in melamine groups did not show toxic manifestations.
Chi square test indicates that the micronucleus rate in all experiment groups were not significantly different from that of the negative control group, and the ratios of PCE/NCE in all experimental groups were between 1.10 and 1.28, the results above suggesting that melamine has no bone marrow toxicity.
Conclusions:
Interpretation of results (migrated information): negative
Melamine at doses of 400 to 1600 mg/kg bw did not increased the number of micronucleated polychromatic erythrocytes.
Executive summary:

A micronucleus test was performed with mice and intraperitoneal doses of 400, 800 and 1600 mg/kg bw, giving twice, 24 h apart. Polychromatic and normochromatic erythrocytes in the bone marrow were analysed for micronuclei.

The micronucleus rate in all experiment groups were found to be not significantly different from that of the negative control group.

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
<=2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable scientific publication. No GLP.
Principles of method if other than guideline:
The Pig-a assay is a new in vivo genotoxicity test for detecting mutagens in the bodies of animals, using the endogenous Pig-a gene as the target. There are two types of Pig-a assays: the red blood cell (RBC) Pig-a assay, which uses RBCs, and the PIGRET assay, which uses reticulocytes. The test was performed to investigate the usefulness of the assay.
GLP compliance:
not specified
Type of assay:
other: RBC Pig-a assay and PIGRET assay.
Specific details on test material used for the study:
Melamine (CAS No. 108-78-1) was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, japan).
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Male Sprague-Dawley rats (Cri:CD (SD)) were supplied by Charles River Laboratories japan, Inc. (Kanagawa, Japan).
Sex:
male
Details on test animals or test system and environmental conditions:
Rats were eight weeks old at the start of the experiment.
Rats were maintained on a 12-h light/dark cycle at 22.0 ± 2.0 'C and were fed a standard diet (MF Powder, Oriental Yeast Co., Ltd., Tokyo, japan) and water ad libitum.
Route of administration:
oral: gavage
Vehicle:
0.5 % CMC.
Frequency of treatment:
Once.
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6m / group.
Control animals:
yes, concurrent vehicle
Positive control(s):
N-nitroso-N-ethylurea (ENU) at 40 mg/kg. N = 3.
Tissues and cell types examined:
For all animals, blood was collected from the tail vein two days before administration and at 7 and 14 days after administration, and blood was collected from the post-cava at 28 days after administration. The blood was promptly transferred to micro blood collection tubes coated with di-potassium ethylenediaminetetraacetic acid (K2-EDTA), and the blood samples were mixed well and refrigerated. The blood samples were used within one week of collection.
Details of tissue and slide preparation:
Blood processing for the RBC Pig-a assay
The sample preparation and gating strategy for the RBC Pig-a assay followed the JEMS-MMS collaborative study protocol based on previous papers. Cells were labeled with an APC-conjugated HIS49 antibody and a FITC-conjugated CD59 antibody. The samples were then incubated for 1 h in the dark at room temperature. Then, the samples were centrifuged for 5 min at 1700 x g and resuspended in 1 mL of PBS.
 
Blood processing for the PIGRETassay
Aliquots of blood were suspended in PBS containing PE-conjugated CD71 antibody, and the suspension was incubated in the dark at 4 °C for 15 min. The cells were then mixed with BD IMag™ PE Particles Plus-DM (BD Biosciences) and incubated for 15 min in the dark at 4 °C. To enrich the CD71-positive cells, 1 mL of 1 x BD IMag™ Buffer was added to the samples, which were then allowed to sit for 6 min in a BD IMagnet™ magnetic stand (BD Biosciences). Then, the Buffer was removed from the samples and another fresh 1-mL aliquot of Buffer was added. The samples were left to sit for 2 min. This procedure was repeated twice. The samples were then suspended in PBS. These samples were labeled with APC-conjugated HIS49 antibody and FITC-conjugated CD59 antibody and incubated for 30 min in the dark at room temperature. These samples were centrifuged and resuspended in PBS.
Statistics:
Was performed.
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No abnormal clinical signs were observed in the rats after the administration of melamine. Significant changes in body weight were not observed in any of the melamine groups or in the ENU group.

The RBC Pig-a assay
A significant decrease in the RBC Pig-a MF was found in the 500 mg/kg melamine group on day 7 after administration. No significant changes in the Pig-a MF were found in the other melamine groups. In the ENU group, a significant increase in RBC Pig-a MFs was found on days 7 and 14 after administration.

The PlGRET assay
No significant changes in the RET Pig-a MFs were found in any of the melamine groups throughout the experimental period. In the ENU group, there was a significant increase in the RET Pig-a MFs found on days 7 and 14 after administration.

The %RET analysis
No significant changes were found for any of the melamine groups throughout the experimental period. There were also no significant changes in the %RET found for the ENU group. In the control and treated groups on day 7, the %RET values were slightly higher than the %RET values for those groups on day -2. However, because there were no differences in %RET between the control and the treated groups on day 7, it was reasoned that these changes were not caused by treatment, but by a flow cytometer setting.

The RBC Pig-a assay demonstrated that there was a significant decrease in Pig-a MF in the 500 mgfkg melamine group on day 7. The Pig-a MFs on days -2 and 7 were the same, and the Pig-a MFs on days 14 and 28 were slightly higher than those on day 7 in the 500 mg/kg melamine group. However, there was no similar increase with time for the groups receiving the higher dose of 1000 or 2000 mgfkg melamine. Therefore, it was considered that the increases in Pig-a MFs in the SOOmgfkg group on days 14 and 28 were not due to mutagenicity and that the significant decrease in Pig-a MF on day 7 was incidental. Neither the RBC Pig-a assay nor the PIGREf assay found any significant increase in the Pig-a MFs in any of the melamine groups, indicating that melamine was not mutagenic.

On the other hand, a significant increase in Pig-a MF was found in the ENU group.

Conclusions:
The results show that both the RBC Pig-a and the PIGRET assays can detect mutagenicity after a single administration. It is shown that melamine does
not have mutagenicity.
Executive summary:

The Pig-a assay is a new in vivo genotoxicity test for detecting mutagens in the bodies of animals, using the endogenous Pig-a gene as the target. There are two types of Pig-a assays: the red blood cell (RBC) Pig-a assay, which uses RBCs, and the PIGRET assay, which uses reticulocytes. The Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group collaborative study of the Pig-a assay was carried out to investigate the usefulness of the PIGRET assay. The mutagenicity of melamine was evaluated as part of this study.

Melamine was shown to be non-mutagenic.

Endpoint:
genetic toxicity in vivo, other
Remarks:
In vivo Pig-a mutation / micronucleus assay
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
<=2014
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Scientific investigation not meeting standards as in guideline studies.
Qualifier:
no guideline followed
Principles of method if other than guideline:
An in vivo Pig-a mutation / micronucleus assay was performed to detect gene mutations.
A flow cytometric Pig-a mutation assay for measuring gene mutation in the RBCs and RETs of rats or mice has been developed. These assays have advantageous characteristics and may constitute an effective tool for in viva mutation and hazard assessment. For example, the Pig-a gene is highly conserved, a feature that facilitates studies across species of toxicological interest. Also, mutant cells accumulate with repeat dosing, so that even weak mutagens or exposures might be detected, and the assay is attractive for integration into repeat-dose toxicology studies. These and other strengths make the Pig-a assay a promising tool for regulatory safety assessments.
GLP compliance:
not specified
Type of assay:
other: In vivo Pig-a mutation / micronucleus assay to detect gene mutations.
Specific details on test material used for the study:
Purchased from Sigma-Aldrich, USA.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Route of administration:
oral: gavage
Vehicle:
sesame oil
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes. N-ethyl-N-nitrosourea.
Statistics:
Was performed.
Sex:
male
Genotoxicity:
negative
Remarks:
Pig-a assay
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
micronucleus assay
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No treatment-related clinical observations were noted during the study. Rats receiving melamine gained appreciably less weight than vehicle controls. Mean body weights treated at 1000 and 2000 mg/kg/day on day 4 were less than their day 1 body weights, demonstrating severe body weight loss on day 4.

The mean RET frequencies (%RETs), as measured during the MN analyses, were reduced in a dose-dependent manner for melamine-treated groups on day 4, and the reductions in %RETs for the 1000 and 2000 mg/kg/day treatment group were statistically significant (p <0.05 ), indicating that melamine was reaching the bone marrow and inhibiting the hematopoietic system shortly after the treatment. However, %RETs of melamine-treated groups on days -1, 15, 29, and 60, measured during the Pig-a analyses, were not statistically significant different from the concurrent vehicle control group.

Over the study period, RBCCD59- or RETCD59- frequencies in the vehicle control group were ranged from 0.5 to 1.3 x 10E-6 and 1.5 to 2.9 x 10E-6, respectively. No significant increases in RBCCD59- or RETCD59- frequencies were observed for melamine treated group at any of the time points studied. However, animals treated with the positive control produced highly significant increases above vehicle control at all time points in RBCCD59- or RETCD59- frequencies.

No significant increases in %MN-RET frequencies were observed on day 4 in melamine-treated groups compared to the concurrent vehicle control group. In contrast, a significant increase in %MN-RETs frequency over the vehicle control group was detected in rats treated with positive control.
Conclusions:
Melamine was negative in the in vivo Pig-a assay to detect gene mutations and also negative in the micronucleus test.
Executive summary:

The genotoxicity of melamine was evaluated with the combined Pig-a mutation/micronucleus assay, the bacterial reverse mutation assay, and the in vitro cytokinesis-block micronucleus assay (CBMN). Only the combined Pig-a mutation/micronucleus assay is reported here. Five groups of rats were given three daily doses of vehicle control (100% pure sesame oil), melamine (500, 1000, and 2000 mg/kg) or positive control (N-ethyl-N-nitrosourea, ENU by oral gavage. Peripheral blood was sampled pre-dose (day -1) and at time points up to day 60. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBCCD59-and RETCD59-frequencies, on days -1, 15, 29 and 60, and micronucleus frequencies were measured in RETs on day 4.

No significant increases in RBCCD59-and RETCD59-frequencies were observed for the melamine-treated group at any of the time points studied.

Similar results were obtained in the micronucleus assay. Melamine did not induce statistically significant increases in %MNRET.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
<2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientific publication with appropriate description of the method. No GLP.
Qualifier:
no guideline followed
Qualifier:
according to guideline
Guideline:
other: JaCVAM validation study protocol, v14.2.
Principles of method if other than guideline:
It was examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells. Seven chemicals related to urinary bladder carcinogenesis in rodents were used, i.a. melamine. Melamine is Ames test-negative with metabolic activation but is considered to have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3 h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity.
GLP compliance:
not specified
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc
- Age at study initiation: 7 w
- Assigned to test groups randomly: yes
- Housing: 5 per cage
- Diet: Standard laboratory pellet chow MF, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12 h
Route of administration:
oral: gavage
Vehicle:
0.5 % methylcellulose.
Duration of treatment / exposure:
2 doses, 21 h apart.
Frequency of treatment:
2 doses, 21 h apart.
Post exposure period:
3 h after the second exposure.
Remarks:
Doses / Concentrations:
1000 and 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes.
Tissues and cell types examined:
Urinary bladder.
Details of tissue and slide preparation:
Comet assay:
The urinary bladder was cut in half, and one half was processed by the mincing method. A portion of the left lateral lobe of the liver was cut out.
The comet assay was conducted under alkaline conditions. The obtained cell suspension was mixed with 0.5% low-melting-point agarose at a ratio of 1:9. The mixture was dropped onto a bottom layer of 1.0% normal-melting-point agarose on glass slides.Two slides were prepared for each sample: they were coded, and the agarose was allowed to solidify at 4 °C for 10min. Then, the slides were immersed in lysis solution at 4 °C for 1 or 2 days. Next, the slides were rinsed with chilled pure water to remove residual detergent and salts and placed in electrophoresis solution for 20min at 4 °C to allow the DNA to unwind . Electrophoresis was conducted in a horizontal electrophoresis platform in chilled electrophoresis solution for 20 min. The amperage was set to 300 mA and the voltage was 25 V (0.7V/cm). The slides were neutralized with Tris-HCI buffer (pH 7.5) for 5 min, dehydrated by immersion in absolute ethanol for 5 min, and stained with SYBR gold. Cells (100 per tissue) with duplicate slides were analyzed with an Olympus fluorescence microscope equipped with an automatic digital analysis system (Komet 5.5; Andor Technology, Belfast, UK).
Evaluation criteria:
The percentage of DNA in the tail (% tail DNA), which is the most suitable measurement parameter of DNA-break frequency, was calculated. "Hedgehogs" that showed large diffuse tails containing 90 % or more of the DNA were excluded from data collection: however, their frequency was measured as a reference for severe DNA damage per sample based on the visual scoring of 200 cells per tissue with duplicate slides.
Statistics:
To evaluate DNA damage, the mean% tail DNA in each treatment group was compared with the corresponding vehicle control group. Dunnett's test was used to compare multiple groups (P<0.05).
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid

Body weight gain or loss and clinical signs of toxicity:

Animals treated with melamine showed decreased body weight.

Gross hematuria was detected in the urine of rats treated with the low and high doses of melamine, as evidenced by an occult blood-positive test using urine test papers.

 

Comet assay:

DNA damage (% tail DNA) in the urinary bladder or liver did not differ significantly between the melamine treated groups and the vehicle control group.

No increase in hedgehog frequency was observed in either the urinary bladder or liver in any of the treatments groups.

 

Necropsy findings:

At necropsy, no white solids were observed in the urinary bladder, but the color of the urinary bladders from rats treated with low and high doses of melamine turned red. In the liver, there were no abnormal necropsy findings.

 

Histopathology:

Several histopathological changes were observed in the melamine treated groups. The changes that were seen in three or more rats per group were as follows: urothelial cell hyperplasia and mitosis in the melamine-treated group. Other minor changes were also detected, such as hemorrhage, neutrophil infiltration and erosion of the urothelium.

Conclusions:
Interpretation of results (migrated information): negative
No DNA damage was induced in the urinary bladders of rats treated with melamine under conditions where some histopathological changes were observed.
Executive summary:

It was examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells. Seven chemicals related to urinary bladder carcinogenesis in rodents were used, i.a. melamine. Melamine is Ames test-negative with metabolic activation but is considered to have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3 h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats, treated with melamine, no DNA damage was induced under conditions where some histopathological changes were observed.

The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Not well-documented publication. No GLP.
Qualifier:
no guideline followed
Principles of method if other than guideline:
A single cell electrophoresis assay was performed with mice and 3 doses of melamine.
GLP compliance:
not specified
Type of assay:
mammalian comet assay
Species:
mouse
Strain:
other: NIH
Sex:
male
Details on test animals or test system and environmental conditions:
Specific pathogen-free male NIH mice, aged 4-5 weeks or 7-8 weeks, were used.
Route of administration:
intraperitoneal
Vehicle:
0.85 % saline.
Duration of treatment / exposure:
5 days.
Frequency of treatment:
Daily.
Post exposure period:
7 days.
Remarks:
Doses / Concentrations:
400, 800 and 1600 mg/kg bw
Basis:
no data
No. of animals per sex per dose:
10.
Control animals:
yes, concurrent vehicle
Positive control(s):
MMS.
Tissues and cell types examined:
Cell from epididymises.
Details of tissue and slide preparation:
The abdominal cavities were opened and bilateral epididymises were obtained and cut into pieces in phosphate buffer. After 5 min of static stay, the mixtures were filtered and prepared as 5 x 10E6 sperm/mL PBS suspension. Fifteen microliters of cell suspension was mixed with 110µL of 0.65% low melting point agarose (LAM) that had been cooled down to 42°C, transferred into a penicillin bottle, stayed statically at 10°C for 5 min to solidify. Cells in penicillin bottle were lysed by incubation with 2 ml of fresh prepared cell lysis buffer at 10°C for 1 h. Proteins were removed by discarding residue liquid and incubating with 0.5 mg/ml, proteinase K in 2 mL of digestion buffer at 55 °C for 6 h. Sperm-LAM mixture were placed on the top of a 20 x 20 mm coverslip on a glass slide pre-coated with 1% normal melting point agarose (NMA) prepared in Ca2+/Mg2+-free PBS, incubated at room temperature for 10 min to solidify, and covered with 20 x 20 mm coverslip. The whole slide was then soaked in fresh prepared running buffer at 4°C for 30 min and then subjected to electrophoresis under constant voltage of 15 V at 4 °C for 60 min. During the electrophoresis, the current was maintained at 130 mA by adjusting the liquid level. After electrophoresis, the slide was stained with 40 µL of 15 µg/mL ethidium bromide for 5 min, examined under a fluorescent microscope with excitation wavelength at 580 nm at magnification of 400x.
Evaluation criteria:
One hundred cells per slide and seven slides per group were randomly examined. Positive cells were bright, comet-like in dark background under fluorescent microscope. DNA content in the tail of sperm cells, and the length and area of comet tail were quantified using image analysis software.
Statistics:
Single-factor variance analysis.
Sex:
male
Genotoxicity:
other: A DNA-damage was observed.
Positive controls validity:
not specified

Treatment with melamine at different dosages significantly increased the DNA content in comet tails, and tail length and area compared with those of control group. Analysis indicated significant differences between any two of melamine groups with different dosages.

Conclusions:
Interpretation of results (migrated information): other: A DNA damage was observed.
Melamine significantly increased the DNA content in comet tails, and tail length and area compared with those of control group, indicating a damage to the DNA of sperms in epididymis.
The result is possibly a false positive one. Wada et al. 2014, see the record in section 7.6.2, pointed to the false-positive results obtained with the comet assay, due to non-specific DNA damage associated with cell death. Cell deaths of sperms or precursors were observed for melamine, see Yin 2013 and Chang 2014 in section 7.8.3, and are of relevance for the genotoxicity interpretation of the finding.
Executive summary:

A possible DNA damage in sperms in the epididymis was investigated by the Comet Assay after dosing mice at 400, 800 and 1600 mg/kg bw/d for 5 days.

Melamine significantly increased the DNA content in comet tails, and tail length and area compared with those of control group.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

19 in vitro and 9 in vivo tests were reported for melamine. For comparison of test methods or for the development of new methods, melamine is an attractive substance, as it is considered to be a non-genotoxic carcinogen in rats. Results of standard tests and also of not so well known or not - yet - accepted tests were reported.

Justification for classification or non-classification

The standard tests were all negative. Therefore no classification of melamine as to genotoxicity is required.