Melamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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 and some standard methods were not applied. The studies are peer reviewed. The test substance was analysed extensively.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and carcinogenicity

Type of information:

other: re-evaluation of data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Studies for NTP are usually of high standard. The study has one specific aim, therefore not meeting the full requirements of a guideline study.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline required

Principles of method if other than guideline:

The histopathologic changes induced in F344 rat kidney by oral administration of melamine for 13-week and 2-year periods in studies conducted by the National Toxicology Program, NIH, from 1976 to 1983 (see "NTP 1983" under sections 7.5.1 and 7.7) have been re-evaluated and described in detail.

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Route of administration:

oral: feed

Details on oral exposure:

As in "NTP 1983".

Observations and examinations performed and frequency:

Histopathologic changes have been re-evaluated.

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The reflux nephropathy was observed in the high dosed (12000 and 18000 ppm) animals of the two 13-week studies with one exception in the 6000 ppm group. It was observed at 2250 ppm and higher in the 2-years study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

A constellation of tubule changes extending from papilla to cortex consistently included tubule dilatation and tubule basophilia as salient features at the subchronic time point. By 2 years, these lesions had usually resolved into fibrotic scars, in which tubule loss and collagen deposition were prominent, running from superficial cortex into the medulla. These fibrotic lesions required discrimination from chronic scars resulting from infarcts and foci of chronic progressive nephropathy (CPN). A case is presented here for interpreting the constellation of histologic changes induced in rats by melamine as representing an ascending form of nephropathy. The term retrograde nephropathy is considered to be appropriate nomenclature for both the acute and chronic lesions. The cause for the reflux, emanating from the lower urinary tract, appeared not to be infection as an inflammatory response was not prominent.
The reflux nephropathy was observed in the high dosed (12000 and 18000 ppm) animals of the two 13-week studies with one exception in the 6000 ppm group. It was observed at 2250 ppm and higher in the 2-years study.

Key result

Dose descriptor:

LOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: For the two 13-weeks studies.

Key result

Dose descriptor:

LOAEL

Effect level:

2 250 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: For the 2-years study.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 250 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Kidney lesions, detected in the tissues of the NTP 1983 study at higher doses (compared to uroliths), are described.

Executive summary:

The histopathologic changes induced in F344 rat kidney by oral administration of melamine for 13 -week and 2 -year periods in studies conducted by the National Toxicology Program, NIH, from 1976 to 1983 have been re-evaluated and described in detail.

A constellation of tubule changes extending from papilla to cortex consistently included tubule dilatation and tubule basophilia as salient features at the subchronic time point. By 2 years, these lesions had usually resolved into fibrotic scars, in which tubule loss and collagen deposition were prominent, running from superficial cortex into the medulla. These fibrotic lesions required discrimination from chronic scars resulting from infarcts and foci of chronic progressive nephropathy (CPN). A case is presented here for interpreting the constellation of histologic changes induced in rats by melamine as representing an ascending form of nephropathy. The term retrograde nephropathy is considered to be appropriate nomenclature for both the acute and chronic lesions. The cause for the reflux, emanating from the lower urinary tract, appeared not to be infection as an inflammatory response was not prominent. It can be speculated that melamine precipitation in the lower urinary tract created pressure effects through transient obstruction leading to the renal changes. These changes were different from those involved in a major US outbreak of renal disease and death in cats and dogs associated with triazine-contaminated pet food, in which crystalluria from insoluble melamine/cyanuric acid complexes occurred in the kidney. However, the rat findings may be relevant to melamine-associated kidney disease recently reported in infants in China.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1983

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1984

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: NTP standards

Deviations:

yes

Remarks:

No haematology and clinical chemistry investigations were performed. No statistical analysis was reported or performed.

Principles of method if other than guideline:

Three 13 weeks toxicity studies were performed:
In the first 13-week study, diets containing 0, 6000, 9000, 12000, 15000, or 18000 ppm melamine were fed to groups of 12 male and 12 female rats and to groups of 10 male and 10 female mice for 13 weeks.

Two additional 13-week studies were conducted to find a no-effect level for urinary bladder stone formation and to determine the effect of ammonium chloride in the drinking water on stone formation.
In the second 13-week study, groups of 10 rats of either sex were fed diets containing 0, 750, 1500, 3000, 6000, or 12000 ppm melamine for 13 weeks; At day 65, five rats of either sex fed 750 ppm melamine and two control rats of each sex were placed in metabolism cages and fasted overnight. Urine samples collected from each cage were centrifuged and the sediment fractions were examined microscopically.

In a third 13-week study, groups of 10 rats of either sex were fed diets containing 0, 10000, or 18000 ppm melamine in the presence and absence of 1 % ammonium chloride in the drinking water.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Melamine

EC Number:

203-615-4

EC Name:

Melamine

Cas Number:

108-78-1

Molecular formula:

C3H6N6

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): melamine.
- Analytical purity: Results of purity and identity analyses conducted at Midwest Research Institute (MRI) indicated that both lots were similar in purity in all respects.
Results of elemental analyses for both lots agreed with the theoretical values. Titration of one amino group indicated that both lots were approximately 100 % ± 1 % pure. For both lots, only one spot was detected by thin-layer chromatography.
- Impurities (identity and concentrations): Several minor impurities were detected in both lots by high-performance liquid chromatography (HPLC). The impurities were not further characterized, but 6-amino-s-triazine-2,4-diol and 4,6-diamino-s-triazine- 2-ol are reported to be common impurities in melamine.
The differences in purity determined by HPLC and titration could be due to impurities with titratable amine groups, such as the impurities mentioned above, that would result in an apparently high titration value. In addition, the detector response in HPLC is highly dependent upon the absorbance of the substance at the detector wavelength used. The values reported are absolute areas expressed as percentages of the area of the major peak and do not take into account the different values of the compound and its impurities at 254 nm. Therefore, the relative areas determined by HPLC do not necessarily reflect the actual weight percentages of the impurities in the sample.
- The infrared and ultraviolet spectra of both lots were consistent with the literature spectra.
- Lot/batch No.: Technical-grade melamine was obtained in two batches from American Cyanamid Company (Bound Brook, NJ).
Lot No. A7-11-75 was used for the single-dose and prechronic studies and for the first 14 months of the chronic study.
Lot No. A13179 was used for the remainder of the chronic studies.
- Stability under test conditions: The chemical was stable at 60 °C for 2 weeks and therefore required no special storage temperature.
- Storage condition of test material: The chemical was stored at 4 °C throughout the study, and periodic analyses by thin-layer chromatography and infrared spectroscopy indicated that no significant degradation occurred over the lifetime of the study.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Center, Frederick, MD
- Age at study initiation: 5 - 6 weeks.
- Housing: Rats were housed four per cage in polycarbonate cages covered with nonwoven polyester filter sheets. Racks and filters were changed once every 2 weeks. Cages, bedding, and glass water bottles (equipped with stainless steel sipper tubes) were replaced twice per week.
- Diet: Purina Laboratory Chow. Ralston Purina Co. Stainless steel feed containers were changed once per week.
- Water:
First and second studies: Tap water (acidified with hydrochloric acid to pH 2.5).
Third study: ± 1 % ammonium chloride in drinking water.
Test diets, control diets, and tap water were available ad libitum.
- Bedding: Absorb-Dri heat-treated hardwood chips, changed twice per week.
- Acclimation: 2 weeks
- Randomization: Animals assigned to cages by species and sex such that the cage weighs were approximately the same.

ENVIRONMENTAL CONDITIONS
- Temperature, humidity: The temperature in the animal rooms was 22 °- 26 °C and the relative humidity was 30 %-70 %.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with: Test diets were prepared by first mixing a small amount of Purina B Lab Chow and the required amount of melamine with a mortar and pestle and then adding this premix to the required amount of animal meal and mixing for 10 to 30 minutes in a Patterson-Kelly@ twinshell blender equipped with an intensifier bar.
- Storage temperature of food: Test diets were stored at 4 °C for no longer than 2 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Prepared diets containing 100 000 ppm melamine were analyzed and were found to be stable for 2 weeks at temperatures up to 45 °C.
Control animals were fed Purina Lab Chow.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Remarks:

First study: 0, 6000, 9000, 12000, 15000 or 18000 ppm melamine in the diet for rats of bothe sexes. Basis: nominal in diet. The estimated melamine consumption in mg/ kg/ day for each animal was: 0, 560, 850, 1100, 1400, or 1700 for male rats; 0, 560, 880, 1200, 1400 or 1600 for female rats; according to Table 1 of NTP 1983.

Doses / concentrations 2

Remarks:

Second study: 0, 750, 1500, 3000, 6000 or 12000 ppm for bothe sexes. Basis: nominal in diet
The estimated melamine consumption in mg/ kg/ day for each animal was: 0, 72, 150, 300, 590, or 1300 for male rats and 0, 84, 150, 300, 600, or 1300 for female rats, according to Table 1 of NTP 1983.

Doses / concentrations 3

Remarks:

Third study: 0, 10000 or 18000 ppm. Basis: nominal in diet.

No. of animals per sex per dose:

FIRST STUDY
- 12 males/12 females
SECOND STUDY
- 10 males/10 females
THIRD STUDY
- 10 males/10 females

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for mortality and signs of morbidity.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: weekly.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

URINALYSIS: Yes (only study No. 2)
-control animals: 1 male, 2 females.
- low dosed animals (750 ppm): all animals.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY (study No. 1 - 3): Yes (all animals)

HISTOPATHOLOGY (study No. 1): Yes (all animals control group (0 ppm) and highest dosed group (18000 ppm).
-gross lesions, tissue masses
-abnormal lymph nodes
-skin
-mandibular lymph nodes
-mammary glands
-salivary gland
-thigh muscle
-sciatic nerve
-bone marrow
-costochondral junction (rib)
-thymus
-larynx
-trachea
-lungs and bronchi
-heart
-thyroid
-parathyroid
-oesophagus
-stomach
-duodenum
-jejunum
-colon
-mesenteric lymph nodes
-liver
-pancreas
-spleen
-kidneys
-adrenals
-urinary bladder
-seminal vesicles
-prostate
-testes
-ovaries
-uterus
-nasal cavity
-brain
-pituitary

all animals low dose (6000 ppm) group:
-kidney
-urinary bladder

HISTOPATHOLOGY (study No. 2): Yes (all animals)
-kidney
-urinary bladder

HISTOPATHOLOGY (study No. 3): No

Statistics:

not reported.

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Study 1: One male rat receiving 18,000 ppm and two males receiving 6,000 ppm died.
Study 2: None of the rats died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study 1: Mean body weight gain in males and females receiving 12000 ppm or more was depressed by more than 8% when compared with controls.
Study 2: Mean body weight gain was depressed by more than 10% when compared with controls for male rats receiving 6,000 and 12,000 ppm, but no depression was observed in any group of dosed females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Study 1: Feed consumption by rats receiving 18000 ppm was approximately 80%-90% that of controls.
Study 2: Feed consumption was not affected by incorporation of melamine in the feed.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

Study 2: There were no differences in the urine samples that could be attributed to the presence of melamine in the feed. Microscopic examination of the urine did not provide any evidence of melamine crystalluria.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Study 1: Stones were found in the urinary bladders of most dosed male rats, and the incidence was dose related. Twenty-five percent (3/ 12) or more females in the two highest dosed groups had stones.
Study 2: Other than stones in the bladder of dosed male rats, no compound-related effects were observed at necropsy. The incidence of stones in the urinary bladder of male rats was dose related.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Study 1: Histopathologic evaluations were performed on 10 animals of either sex from the high-dose (18,000 ppm), low-dose (6,000 ppm), and control groups. Diffuse epithelial hyperplasia of the urinary bladder was found in 8/ 10 males and 2/ 10 females receiving 18,000 ppm melamine, while in animals receiving 6,000 ppm melamine, focal epithelial hyperplasia was observed in only 1 / 10 males and in none of the females. The urinary bladders of animals from other dosed groups were not examined microscopically. No other compound-related histopathologic effects were observed.
Study 2: Stones were present even in the male group receiving 750 ppm. Hyperplasia of the transitional epithelium of the bladder was present in 1/10 male rats receiving 3,000 ppm, in 3/10 receiving 6,000 ppm, and in 919 receiving 12,000 ppm melamine. The hyperplastic epithelial changes, which were found only in male rats that had bladder stones, were accompanied by prominent capillaries and occasional edema and scattered mast cells in the submucosa. Kidney changes in male rats were minimal. There was no evidence of urinary bladder stones or hyperplasia of the bladder epithelium in any groups of dosed female rats, but dose-related calcareous deposits were observed in the straight segments of the proximal tubules in female rats (2/10 controls, 3/ 10 receiving 750 ppm, 4/ 10 receiving 1,500 ppm, 10/ 10 receiving 3,000 ppm, 8/ 10 receiving 6,000 ppm, and 10/ 10 receiving 12,000 ppm melamine).

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

STUDY No. 1:
CLINICAL SIGNS AND MORTALITY
One male rat receiving 18,000 ppm and two males receiving 6,000 ppm died .
(See Table 4)
BODY WEIGHT AND WEIGHT GAIN
Mean body weight gain in males and females receiving 12,000 ppm or more was depressed by more than 8% when compared with controls.
(See Table 4)
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Feed consumption by rats receiving 18,000 ppm was approximately 80%-90% that of controls.
GROSS PATHOLOGY
Stones were found in the urinary bladders of most dosed male rats, and the incidence was dose related. Twenty-five percent (3/ 12) or more females in the two highest dosed groups had stones.
HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathologic evaluations were performed on 10 animals of either sex from the high-dose (18,000 ppm), low-dose (6,000 ppm), and control groups. Diffuse epithelial hyperplasia of the urinary bladder was found in 8/ 10 males and 2/ 10 females receiving 18,000 ppm melamine, while in animals receiving 6,000 ppm melamine, focal epithelial hyperplasia was observed in only 1 / 10 males and in none of the females. The urinary bladders of animals from other dosed groups were not examined microscopically. No other compound-related histopathologic effects were observed.

STUDY No. 2:
CLINICAL SIGNS AND MORTALITY
None of the rats died.
BODY WEIGHT AND WEIGHT GAIN
Mean body weight gain was depressed by more than 10% when compared with controls for male rats receiving 6,000 and 12,000 ppm, but no depression was observed in any group of dosed females.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Feed consumption was not affected by incorporation of melamine in the feed.
URINALYSIS
There were no differences in the urine samples that could be attributed to the presence of melamine in the feed. Microscopic examination of the urine did not provide any evidence of melamine crystalluria.
GROSS PATHOLOGY
Other than stones in the bladder of dosed male rats, no compound-related effects were observed at necropsy. The incidence of stones in the urinary bladder of male rats was dose related.
HISTOPATHOLOGY: NON-NEOPLASTIC
Stones were present even in the male group receiving 750 ppm. Hyperplasia of the transitional epithelium of the bladder was present in 1/10 male rats receiving 3,000 ppm, in 3/10 receiving 6,000 ppm, and in 919 receiving 12,000 ppm melamine. The hyperplastic epithelial changes, which were found only in male rats that had bladder stones, were accompanied by prominent capillaries and occasional edema and scattered mast cells in the submucosa. Kidney changes in male rats were minimal. There was no evidence of urinary bladder stones or hyperplasia of the bladder epithelium in any groups of dosed female rats, but dose-related calcareous deposits were observed in the straight segments of the proximal tubules in female rats (2/10 controls, 3/ 10 receiving 750 ppm, 4/ 10 receiving 1,500 ppm, 10/ 10 receiving 3,000 ppm, 8/ 10 receiving 6,000 ppm, and 10/ 10 receiving 12,000 ppm melamine).

STUDY No. 3:
CLINICAL SIGNS AND MORTALITY
None of the rats died.
BODY WEIGHT AND WEIGHT GAIN
Rats fed diets containing 18,000 ppm melamine plus 1% ammonium chloride in the drinking water had decreased weight gains relative to groups receiving drinking water acidified with hydrochloric acid.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no effects.
GROSS PATHOLOGY
The addition of ammonium chloride in the drinking water had no apparent effect on the incidence of urinary bladder stones in male or female rats. Urinary bladder stones were seen in 8/8 males and 3/9 females in the group that received 18,000 ppm melamine in feed plus 1% ammonium chloride in drinking water, compared with 10/ 10 males and 3/10 females in the groups administered 18,000 ppm melamine in feed without 1% ammonium chloride in the water. No other compound-related effects were observed at necropsy.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

750 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

other: 2 urinary bladder stones were detected compared to 1 in the control group. Statistically, 750 ppm is the NOAEL.

2

Dose descriptor:

NOAEL

Effect level:

72 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: 2 urinary bladder stones were detected compared to 1 in the control group. Statistically, 750 ppm is the NOAEL.

3

Key result

Dose descriptor:

NOAEL

Effect level:

6 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

other: body weight

4

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: body weight

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

The relevant results in the tables of the study are assembled in the attachment.

Applicant's summary and conclusion

Conclusions:

Most noticeable was the development of uroliths (urinary bladder stones) at 750 ppm and higher doses, mainly in males.

Executive summary:

Melamine (2,4,6-triamino-s-triazine) was administered in the diet to F344 rats or B6C3F1 mice for 13 weeks (subchronic) to determine its toxicologic profile. The dose levels of melamine in the subchronic studies ranged from 750 to 18000 ppm for rats, and 6000 to 18000 ppm for mice. In these studies, compound-related lesions were observed in the urinary tract. Most noticeable was the development of uroliths (urinary bladder stones), which occurred at a greater frequency in males than females of either species. Increased incidences of urinary bladder stones and hyperplasia of the bladder epithelium were observed at 13 weeks in male rats fed diets containing melamine.