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EC number: 253-575-7 | CAS number: 37640-57-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
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- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
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- Endpoint summary
- Stability
- Biodegradation
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data

Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Remarks:
- According to NTP study protocol
Data source
Reference
- Reference Type:
- publication
- Title:
- Dose–response assessment of nephrotoxicity from a twenty-eight-day combined-exposure to melamine and cyanuric acid in F344 rats
- Author:
- Gonçalo Gamboa da Costa, Cristina C. Jacob, Linda S. Von Tungeln, Nicholas R. Hasbrouck,
Greg R. Olson, David G. Hattan, Renate Reimschuessel, Frederick A. Beland - Year:
- 2 012
- Bibliographic source:
- Toxicology and Applied Pharmacology
Volume 262, Issue 2, 15 July 2012, Pages 99-106
Materials and methods
- Principles of method if other than guideline:
- NTP study protocol: Specifications for the conduct of studies to evaluate the toxic and carcinogenic potential of chemical, biological and physical agents in laboratory animals for the National Toxicology Program (NTP, 2011)
- GLP compliance:
- yes
- Remarks:
- as stated in the NTP protocol (NTP, 2011): The NTP requires that studies be conducted in compliance with FDA GLP regulations as specified in Part 58 "Good Laboratory Practices for Non-clinical Laboratory Studies"
- Limit test:
- no
Test material
- Reference substance name:
- Cyanuric acid
- EC Number:
- 203-618-0
- EC Name:
- Cyanuric acid
- Cas Number:
- 108-80-5
- Molecular formula:
- C3H3N3O3
- IUPAC Name:
- 1,3,5-triazine-2,4,6-triol
- Reference substance name:
- Melamine
- EC Number:
- 203-615-4
- EC Name:
- Melamine
- Cas Number:
- 108-78-1
- Molecular formula:
- C3H6N6
- IUPAC Name:
- 1,3,5-triazine-2,4,6-triamine
Constituent 1
Constituent 2
- Specific details on test material used for the study:
- Melamine source: Aldrich, stated purity 99%
Cyanuric acid: Fluka, anhydrous, stated purity>98%
Test animals
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- 10-weeks old F344 rats were obtained from the breeding colony at the NCTR, weight-ranked (acceptable weight: ±20% of the mean body weight), and randomly assigned to treatment groups
that were fed ad libitum for 28 days with NIH-41 irradiated meal (control animals) or with NIH-41 irradiated meal containing melamine and cyanuric acid (treatment groups).
The animals were housed in individual polycarbonate cages with hardwood chip bedding and
the room environmental controls were set to maintain a relative humidity of 40–70%, with 10–15 air changes per hour, a temperature of 22± 4 °C, and a 12-hour light/day cycle.
Administration / exposure
- Route of administration:
- oral: feed
- Details on oral exposure:
- Melamine and cyanuric acid were individually pulverized to fine powders in a mechanical ball mill
and incorporated in NIH-41 irradiated meal using a 2.5 cubic feet twin shell blender to afford the concentrations of 0 (control), 30, 60, 120, 180, 240, or 360 ppm each of melamine and cyanuric acid.
These concentrations were selected taking into consideration the average National Center for Toxicological Research (NCTR) breeding colony historical body weight and feed consumption data for 10-week-old male and female F344 rats and aimed to afford the exposures of, respectively, 0, 2.5, 5, 10, 15, 20, or 30 mg/kg bw/day each of melamine and cyanuric acid. The control feed underwent the same mechanical blending procedure as the dosed feed preparations. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The dosed feed formulations were analyzed by isotopic dilution mass spectrometry. The analyses revealed that the melamine and cyanuric acid in the feed formulations were on average, respectively, at 100.0±1.6% and 100.6±2.9% of the target concentrations.
- Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- daily; through feed
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 ppm
- Remarks:
- Control
- Dose / conc.:
- 30 ppm
- Remarks:
- corresponding to ca. 2.5 mg/kg bw/d Melamine or Cyanuric acid; 5 mg/kg bw/d MC (1:1)
- Dose / conc.:
- 60 ppm
- Remarks:
- corresponding to ca. 5 mg/kg bw/d Melamine or Cyanuric acid; 10 mg/kg bw/d MC (1:1)
- Dose / conc.:
- 120 ppm
- Remarks:
- corresponding to ca. 10 mg/kg bw/d Melamine or Cyanuric acid; 20 mg/kg bw/d MC (1:1)
- Dose / conc.:
- 180 ppm
- Remarks:
- corresponding to ca. 15 mg/kg bw/d Melamine or Cyanuric acid; 30 mg/kg bw/d MC (1:1)
- Dose / conc.:
- 240 ppm
- Remarks:
- corresponding to ca. 20 mg/kg bw/d Melamine or Cyanuric acid; 40 mg/kg bw/d MC (1:1)
- Dose / conc.:
- 360 ppm
- Remarks:
- corresponding to ca. 30 mg/kg bw/d Melamine or Cyanuric acid; 60 mg/kg bw/d MC (1:1)
- No. of animals per sex per dose:
- 12
- Control animals:
- yes, plain diet
- Details on study design:
- Study performed according to the NTP protocol (NTP, 2011)
Examinations
- Observations and examinations performed and frequency:
- Study performed according to the NTP protocol (NTP, 2011)
The NTP protocol states that individual animal body weights shall be determined on all animals on day one on study prior to initial treatment, weekly and at necropsy. Food and water consumption shall be measured weekly (for a 7 day period) for control and treated animals for test articles administered in feed or drinking water. Animals shall be observed two times daily (once in the early morning and once in the late afternoon at least six hours apart, and no later than 10:00 AM in the morning and no earlier than 2:00 PM in the afternoon, including holidays and weekends) for moribundity and death. Formal clinical signs shall be recorded daily by animal number. Animals whose condition makes it unlikely that they will survive until the next observation, based upon criteria established by the Laboratory Animal Veterinarian in concert with the Study Director, shall be sacrificed immediately, necropsied, and tissues retained in NBF for possible histopathological evaluation. At terminal sacrifice, complete necropsies shall be performed on all animals. Histopathological evaluation shall be performed in accordance with the histopathology requirements in Section VII.E. Generally, histopathological evaluation is conducted on all organs showing evidence of gross lesions, plus corresponding organs from control animals. Organ weights are generally taken from all animals surviving to the end of the study. The publication refers to the NTP protocol; however, details are not given in the publication. - Sacrifice and pathology:
- At the end of the 28-day exposure period, the animals were anesthetized by carbon dioxide inhalation and blood was withdrawn by cardiac puncture until exsanguination. All tissues listed in the National Toxicology Program (NTP) specifications for histopathology in 28-day studies (NTP, 2011) were examined grossly, removed, and preserved in 10% neutral buffered formalin (NBF), with the exception of the eyes and testes, which were fixed in modified Davidson's fixative.
- Other examinations:
- The following special procedures were conducted on the kidneys:
Left kidney — sectioned longitudinally; one half was flash frozen for wet-mount analysis; the other half was sectioned transversally, ¼ of the kidney was fixed in NBF for 2 h, and the other ¼ was fixed in 70% ethanol. Right kidney — sectioned transversally; one half was frozen for residue analysis; the other half was sectioned longitudinally, ¼ of the kidney was fixed in NBF for 2 h, and the other ¼ was fixed in 70% ethanol. There were no appreciable differences between the two fixation methods.
The fixed tissues were trimmed, processed, and embedded in Formula R, sectioned at approximately
5 μm, and stained with hematoxylin and eosin (H&E). Lesions were graded for severity as 1(minimal), 2 (mild), 3 (moderate), or 4 (marked).
Wet-mount procedures:
The wet-mount analyses were conducted by pressing approximately 2-mm-thick sections (approximately 50–100 mg) of thawed flash-frozen tissue between two microscope slides and observation under bright field and polarized light microscopy. The presence of crystals was scored on a subjective scale from 0 to 5, with 0 = no crystals seen, 1 = one crystal in entire section, 2 = a few crystals with a scattered distribution, 3 = a moderate num-
ber of crystals seen throughout the section, 4 = large numbers, seen immediately when viewing under the microscope, and 5 = extensive numbers of crystals, obliterating the regular architecture of kidney.
Clinical chemistry:
Terminal blood samples obtained by cardiac puncture were allowed to clot and then centrifuged. The serum was removed and frozen at −80 °C until clinical chemistry analysis. Blood urea nitrogen (BUN) and creatinine levels were analyzed on an Alfa Wassermann ALERA analyzer. The instrument was calibrated daily and two levels of assayed controls were included in daily analyses as internal controls.
Hematology:
Terminal blood samples obtained by cardiac puncture were collected in tubes with EDTA and the analyses were performed on the same day of collection. Complete blood counts were determined on an ABX Pentra 60 C+ analyzer using ABX reagents. Maintenance and calibration were done according to the manufacturer's recommendations. Three levels of assayed controls were included in daily analyses as internal controls.
Melamine and cyanuric acid residue analysis in the kidneys:
Melamine and cyanuric acid residues in the kidneys were quantified by liquid chromatography coupled with isotopic dilution mass spectrometry using a system comprised of an Acquity ultraperformance liquid chromatograph (UPLC) interfaced with a Waters Quattro Premier XE tandem mass spectrometer equipped with an electrospray ionization probe. The methodology was validated by analyzing kidney tissue samples obtained from untreated rats spiked with 10, 1000, or 5000 μg/g of melamine cyanurate on two separate days. - Statistics:
- Body weight data were analyzed with a repeated measures design in SAS, using the general linear model, with day as the repeated factor. Pairwise comparisons to the control group were conducted with Tukey's Studentized Range test. Kidney weights and clinical chemistry and hematology measurements were analyzed by one-way analysis of variance (ANOVA),
with pairwise comparisons to the control group conducted by Dunnett's test. When necessary, the data were transformed by taking the natural logarithm to obtain a more normal data distribution or
equal variance. In instances where there was still an unsatisfactory data distribution or variance, the data were analyzed by a Kruskal-Wallis one-way ANOVA on Ranks, with pairwise comparisons to the control group conducted by Dunn's method.
Histopathology results were analyzed by a one-tailed Fisher's Exact test.
Benchmark doses (BMD) and the lower (BMDL) 95% confidence limits were calculated using Environmental Protection Agency Benchmark Dose Software (version 2.1.1; http://www.epa.gov/ncea/bmds). Hill and exponential models (EFSA, 2009) were used to fit continuous variables (the mean ± standard deviation of kidney weights, BUN, creatinine, and kidney levels of melamine and cyanuric acid) and the amount of melamine and cyanuric acid consumed during the first day of the study. The BMD for continuous variables, except the
kidney levels of melamine and cyanuric acid, were defined as the dose corresponding to a change in the mean response equal to one control standard deviation from the control mean. For the kidney levels of melamine and cyanuric acid, the BMD were defined as the dose corresponding to a change in the mean response equal to one control standard deviation from the mean lowest administered dose of melamine or cyanuric acid.
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical symptoms of the animals that were removed from the experiment consisted of emaciation, abnormal body posture, and hematuria.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- All animals in the 360 ppm melamine and cyanuric acid group were removed on days 3–5; 15 of the 24 rats in the 240 ppm melamine and cyanuric acid group were removed between days 4 and 25 of treatment (median = 16 days). One male rat in the 180 ppm melamine and cyanuric acid group was removed on day 14. One female rat in the 120 ppm MC group was removed on day 4 due to evidence of hematuria. The necropsy of this rat revealed the presence of a large stone in the urinary bladder. This stone was described as not related to treatment. A single male rat in the 60 ppm melamine and cyanuric acid group was removed on day 1 of treatment due to abdominal swelling. The necropsy of this animal failed to reveal any cause for the swelling.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Significant treatment-related trends in body weights were observed in male and female rats at the mid- and high dose.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- BUN and creatinine levels were significantly increased in both sexes of rats administered the two highest doses (240 and 360 ppm). Alanine aminotransferase (ALT) was significantly decreased at the two highest doses in both sexes. Alkaline phosphatase was significantly decreased at the three highest doses in male rats and at the two highest doses in female rats.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Male and female rats administered the two highest doses of melamine and cyanuric acid (240 and 360 ppm) had significant increases in kidney weights.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- A significant increase in melamine cyanurate crystals was observed in both sexes of rats administered the four highest doses (120, 180, 240, and 360 ppm) of melamine and cyanuric acid.
Male rats administered the three highest doses (180, 240, and 360 ppm) of melamine and
cyanuric acid had a significant increase in renal tubule dilatation, renal tubule epithelium necrosis, renal polymorphonuclear cellular infiltration, renal lymphocyte cellular infiltration, and pelvis transitional epithelium hyperplasia. In female rats, a significant increase in renal tubule dilatation, renal tubule epithelium necrosis, renal lymphocyte cellular infiltration, and pelvis transitional epithelium hyperplasia occurred at the two highest doses (240 and 360 ppm). Renal fibrosis was observed at the 180 and 240 ppm doses in both sexes of rats but not at the highest dose (360 ppm).
The lack of renal fibrosis in the highest dose group may be due to the fact that these rats were removed from the study after only 3–5 days of treatment.
A full necropsy conducted on all rats failed to reveal the occurrence of treatment-related lesions
in any other organ or tissue. - Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Wet-mount evaluation of the kidneys has been performed:
Kidneys of the control groups had normal appearing renal tubules.
Wet-mount sections revealed golden brown crystals in the renal tubules of 100% of the rats exposed to 120, 180, 240, or 360 ppm of melamine and cyanuric acid. Even the female rat removed from the study at 4 days in the 120 ppm dose group was already positive for crystal presence. Crystal spherulites were noted in 1 out of 24 rats exposed to 30 ppm and 3 out of 23 rats exposed to 60 ppm of melamine and cyanuric acid.
Higher doses resulted in higher crystal intensity. Crystals in the 240 ppm dose group had a significant impact in the health of the animals so that six of these animals had to be removed from the study by day 5, with most being removed by day 17. A comparison of male and female intensity grades shows that grades of 1–3 were found in 39% of females compared to 24% of males, while the higher scores (4 and 5) were found in only 21% of the females versus 35% of the males. Thus, overall, it appears that the males tend to have more crystals observed by wet mount analysis.
Effect levels
open allclose all
- Dose descriptor:
- NOAEL
- Effect level:
- < 2.6 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: crystal formation in the kidney
- Remarks on result:
- other: Corresponding to < 5.2 mg/kg bw melamine and cyanuric acid (1:1)
- Dose descriptor:
- NOAEL
- Effect level:
- 2.1 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: crystal formation in the kidney
- Remarks on result:
- other: Corresponding to 4.2 mg/kg bw melamine and cyanuric acid (1:1)
Target system / organ toxicity
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 30 ppm
- System:
- urinary
- Organ:
- kidney
- Treatment related:
- yes
- Dose response relationship:
- yes
Applicant's summary and conclusion
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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