Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Nephrotoxic Potential and Toxicokinetics of Melamine Combined with Cyanuric Acid in Rats
Author:
Kim GH, Kang MJ, Noh K, Oh DG, Kang W, Jeong HG, Lee KY, Kim H, Kim HS, Jeong TC
Year:
2014
Bibliographic source:
Journal of Toxicology and Environmental Health, Part A, 77:1346–1358

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
7-day oral toxicity study in rats, conducted to examine the effects of melamine (MEL) and cyanuric acid (CYA) (1:1) on kidneys
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid
Details on test material:
different mixtures of 108-78-1 and 108-80-5 from 1:1 to 3:1

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Co., Seoul, Korea
- Age at study initiation: 7 weeks old
- Weight at study initiation: approximately 200 g
- Housing: 2 or 3 rats per cage
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3 °C
- Humidity: 50 ± 10 %
- Photoperiod: 12 hours light / 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.3 % Tween 80 and 0.1 % CMC-Na in saline with 0.25 % 1 N HCl, pH 4.5
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- MEL and CYA were each dissolved in the vehicle.
- Combination: MEL and CYA in half of the volume of vehicle were separately prepared and mixed 1:1.

VEHICLE
- Concentration in vehicle: 10 mg/mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
7 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
4 mg/kg bw/day (nominal)
Remarks:
MEL and CYA (1:1)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
MEL and CYA (1:1)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
MEL and CYA (1:1)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
MEL
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
CYA
No. of animals per sex per dose:
3 to 5 rats
Control animals:
no
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
MORTALITY: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: daily

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before each dosing, on Day 0, 1, 3, 7
- Anaesthetic used for blood collection: not specified
- Animals fasted: not specified
- How many animals: 3-5 rats per dose group and sampling timepoint
- Parameters examined: BUN, creatinine, total protein

TOXICOKINETIC INVESTIGATIONS: Yes
- Time schedule for collection of blood: before and 24, 48, 72, 96, 120, 144, and 168 hours after treatment start
- Concentrations of MEL and CYA were determined in serum, urine, and kidney tissues by means of liquid chromatography–mass spectrometry

URINALYSIS: Yes
- Time schedule for collection of urine: one day prior to treatment start and immediately after final dosing, urine was collected for 24 hours.
- Metabolism cages used for collection of urine: yes (1 rat per cage)
- Animals fasted: not specified
- Parameters examined: baseline exposure to MEL and/or CYA, urinary excretion, urine volume
Sacrifice and pathology:
ORGAN WEIGHTS: Yes
Kidney, spleen, liver

HISTOPATHOLOGY: Yes
Kidney
Other examinations:
DETERMINATION OF MEL AND CYA IN KIDNEYS: Yes
Determination of accumulated MEL and CYA was performed by LC-MS/MS.
Statistics:
Mean ± standard error (SE)
Statistical significance:
- ANOVA followed by Dunnett’s t-test (significance at p < 0.05)
- Duncan’s multiple range tests, where applicable (significance at p < 0.05)

Results and discussion

Results of examinations

Details on results:
MORTALITY:
Two rats treated with 100 mg/kg bw/d MEL and CYA (1:1) were found dead (one rat on day 2, one rat on day 3).

BODY WEIGHTS:
Body weights were statistically significantly reduced at 20 and 100 mg/kg bw/d MEL and CYA (1:1).

CLINICAL CHEMISTRY:
BUN and creatinine were statistically significantly increased and total protein was statistically significantly reduced at 20 and 100 mg/kg bw/d MEL and CYA (1:1).

URINALYSIS:
After 7 days of dosing, MEL and CYA were present only in small amounts in urine of animals treated at 100 mg/kg bw/d MEL and CYA (1:1) as compared to levels determined in animals treated with either 100 mg/kg bw/d MEL or CYA. The combined dose of MEL and CYA (1:1) led to accumulation.

ORGAN WEIGHTS:
Kidney weights were statistically significantly increased at 20 and 100 mg/kg bw/d MEL and CYA (1:1).
The weights of spleen and liver were significantly decreased in these groups.

HISTOPATHOLOGY:
Renal histopathologic findings: Tubular dilatation, crystal deposition, granulomatous tubulo-interstitial inflammation, and tubular necrosis with regeneration were noted at 20 and 100 mg/kg bw/d MEL and CYA (1:1). There were no relevant microscopic findings in kidneys of animals treated at 4 mg/kg bw/d MEL and CYA (1:1) or at 100 mg/kg bw/d MEL. Minimal signs of crystal deposits were noted in 1/5 rats at 100 mg/kg bw/d CYA, together with tubular dilatation, tubular necrosis and pelvic epithelial hyperplasia.

TOXICOKINETIC INVESTIGATION:
MEL or CYA alone were eliminated almost completely within 24 hours after dosing showing no accumulation in kidney.
MEL and CYA (1:1) treatment produced marked accumulation of MEL and CYA in blood.

DETERMINATION OF MEL AND CYA IN KIDNEYS
After 7 days of treatment, significant amounts of MEL and CYA were accumulated in the kidneys.

Effect levels

Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day (nominal)
Based on:
other: MEL and CYA (1:1)
Sex:
male
Basis for effect level:
other: crystals of melamine cyanurate in kidneys
Remarks on result:
other: No dose descriptor was derived by the study authors

Any other information on results incl. tables

MEL and CYA (1:1) exposure produced renal toxicity, apparently due to significant chemical accumulation in kidney accompanied by low excretion.

Applicant's summary and conclusion

Executive summary:

In a 7 day repeated dose toxicity study (Kim et al., 2014), groups of male rats were treated by oral gavage with a 1:1 mixture of melamine (MEL) and cyanuric acid (CYA). Additional groups received 100 mg/kg bw/d MEL or 100 mg/kg w/d CYA. A concurrent control group was treated with the vehicle (aqueous CMC/Tween 80) alone. Mortality, body weight, and clinical chemistry, urinalysis and toxicokinetic parameters were assessed. After scheduled necropsy selected organ weights were determined and the kidneys were examined microscopically. MEL and CYA contents were determined in homogenized kidneys. Only the combined exposure to MEL and CYA (1:1) induced nephrotoxicity. According to the results of this study, combined dosing at high dose levels might produce renal toxicity due to significant chemical accumulation in kidney accompanied by low excretion. Based on crystal formation in kidneys with associated nephrotoxicity and secondary changes in investigated parameters at 20 and 100 mg/kg bw/d MEL and CYA (1:1), a NOAEL of 4 mg/kg bw/d can be deemed for exposure to MEL and CYA (1:1) under the conditions of this study (no NOAEL was set by the study authors).