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Repeated dose toxicity: oral

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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:
2012
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

Constituent 1
Chemical structure
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
Constituent 2
Chemical structure
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
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