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

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

no examination of blood (hematology and clinical biochemistry), no ophthalmological examinations were performed, no information about the determination of organ weights is given

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1983

Reference 2

Reference Type:

publication

Title:

Urolithiasis and bladder carcinogenicity of melamine in rodents

Author:

Melnick R.L. et al.

Year:

1984

Bibliographic source:

Toxicol. Appl. Pharmacol., 72, 292-303

Reference 3

Reference Type:

secondary source

Title:

Joint Assessment of Commodity Chemicals, No. 1 Melamine CAS: 108-78-1

Author:

unknown

Year:

1983

Bibliographic source:

ECETOC

Reference 4

Reference Type:

secondary source

Title:

Melamine

Author:

Trochimowicz H. J. et al.

Year:

2001

Bibliographic source:

Patty´s Toxicology, 5th Edition, Vol.4

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

NTP Thirteen-Week toxicity study.
Two studies using different dose levels were described. The second study was conducted to determine a no effect level for the formation of bladder stones.

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

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
- Age at study initiation: 5-6 weeks
- Weight at study initiation: Animals assigned to cages such that the average cage weights were approximately the same
- Housing: 4 rats/cage
- Water (e.g. ad libitum): ad libitum (acidified with hydrochloric acid to pH 2.5)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 -26 °C
- Humidity (%): 30 - 70%

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
Test diets were prepared by first mixing a small amount of PurinaB 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.
Test diets were stored in the freezer for no longer than 3 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prepared diets containing 100 000 ppm melamine were found to be stable for 2 weeks at temperatures up to 45°C; samples were extraceted with boiling water and analyzed by HPLC

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

estimated melamine consumption, 1 st study

Doses / concentrations 2

Dose / conc.:

6 000 ppm

Remarks:

estimated melamine consumption, 1 st study
males/females: 560 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

9 000 ppm

Remarks:

estimated melamine consumption, 1 st study
males: 850 mg/kg bw/day
females: 880 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

12 000 ppm

Remarks:

estimated melamine consumption, 1 st study
males: 1100 mg/kg bw/day
females: 1200 mg/kg bw/day

Doses / concentrations 5

Dose / conc.:

15 000 ppm

Remarks:

estimated melamine consumption, 1 st study
females: 1400 mg/kg bw/day

Doses / concentrations 6

Dose / conc.:

18 000 ppm

Remarks:

estimated melamine consumption, 1 st study
males: 1700 mg/kg bw/day
females: 1600 mg/kg bw/day

Doses / concentrations 7

Dose / conc.:

0 ppm

Remarks:

estimated melamine consumption, 2nd study

Doses / concentrations 8

Dose / conc.:

750 ppm

Remarks:

estimated melamine consumption, 2nd study
males: 72 mg/kg bw/day
females: 84 mg/kg bw/day

Doses / concentrations 9

Dose / conc.:

1 500 ppm

Remarks:

estimated melamine consumption, 2nd study
males/females: 150 mg/kg bw/day

Doses / concentrations 10

Dose / conc.:

3 000 ppm

Remarks:

estimated melamine consumption, 2nd study
males/females: 300 mg/kg bw/day

Doses / concentrations 11

Dose / conc.:

6 000 ppm

Remarks:

estimated melamine consumption, 2nd study
males: 590 mg/kg bw/day
females: 600 mg/kg bw/day

Doses / concentrations 12

Dose / conc.:

12 000 ppm

Remarks:

estimated melamine consumption, 2nd study
males/females: 1300 mg/kg bw/day

No. of animals per sex per dose:

1st study: 12
2nd study: 10

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (weekly)

2nd study only: Urine samples collected from each cage were centrifuged and the sediment fractions were examined microscopically.
(At day 65, 5 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 cetnrifuged and the dediment fractions were examined microscopically)

Sacrifice and pathology:

GROSS PATHOLOGY/HISTOPATHOLOGY:
1st study:
necropsies performed on all animals;
following tissues examined histologically in controls and animals in highest dosed groups:
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, esophagus, stomach,
duodenum, jejenum, ileum, colon, mesenteric lymph nodes, liver, gallbladder (mice), pancreas, spleen, kidneys, adrenals, bladder, seminal vesicles/ prostate/ testes or ovaries/ uterus, nasal cavity, brain, and pituitary. For lowest dose group, only kidney and urinary bladder examined.
2nd study:
necropsoies performed on all animals;
Kidney and urinary bladder of all animals examined microscopically

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

1st study: One male rat receiving 18,000 ppm and two males receiving 6,000 ppm died (cf. Table 1)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1st study: Mean body weight gain in males and females receiving 12,000 ppm or more was depressed by more than 8% when compared with controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1st study: Feed consumption by rats receiving 18,000 ppm was approximately 80%-90% that of controls.

Food efficiency:

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:

effects observed, treatment-related

Description (incidence and severity):

1st study: Stones were found in the urinary bladders of most dosed male rats, and the incidence was dose related (cf. Table 2). Twenty-five percent (3/ 12) or more females in the two highest dosed groups had stones.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1st study: 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.
18,000 ppm: Diffuse epithelial hyperplasia of the urinary bladder was found in 8/ 10 males and 2/ 10 females
6,000 ppm: 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.

Histopathological findings: neoplastic:

not examined

Details on results:

Results for the 2nd Study:
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 (Table 3).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Feed consumption was not affected by incorporation of melamine in the feed.
GROSS PATHOLOGY/HISTOPATHOLOGY
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 (Table 4). Stones were present even in the male group receiving 750 ppm.
Males:
750 ppm: Bladder stones
1,500 ppm: Bladder stones
3,000 ppm: Bladder stones, Hyperplasia of the transitional epithelium of the bladder was present in 1/10 male rats
6,000 ppm: Bladder stones, Hyperplasia of the transitional epithelium of the bladder was present in 3/10 male rats
12,000 ppm: Bladder stones, Hyperplasia of the transitional epithelium of the bladder was present in 9/9 male rats
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.
Females:
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:
0 ppm: 2/10
750 ppm: 3/ 10
1,500 ppm: 4/ 10
12,000 ppm: l0/ 10

URINANALYSIS:
Urine samples were analyzed from male and female rats receiving 750 ppm melamine and compared with urine samples from control rats (Table 5).
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.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

72 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: urinary bladder stones/Hyperplasia of bladder epithelium in males

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 400 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: based on the formation of urinary bladder stones and reduced body weights observed in the first study (refers to 15000 ppm)

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

Table 1: 1st study: Survival, mean body weights and feed consumption of rats

		Mean Body Weights (g)
Dose (ppm)	Survival (a) (Week of death)	Initial	Final	Gain	Weight Change relative to controls (%) (b)	Feed consumption (% of control)
Males
0	12/12	88	297	209
6,000	10/12 (8, 10)	87	287	200	-4	96
9,000	12/12	90	288	198	-5	92
12,000	12/12	91	276	185	-11	94
15,000	12/12	92	259	167	-20	0
18,000	11/12 (7)	87	255	168	-20	88
Females
0	12/12	87	191	104
6,000	12/12	79	181	102	-2	87
9,000	12/12	83	187	104	0	94
12,000	12/12	83	179	96	-8	94
15,000	12/12	82	174	92	-12	84
18,000	12/12	81	165	84	-19	78

(a) Number surviving/number per group

(b) ((Weight change dosed group – weight change control group)/weight change control group) X 100

Table 2: 1st study: Incidence of rats with urinary bladder stones or granular material

	Dose (ppm)	No of rats with urinary bladder stones
Males
	0	0/12
	6,000	6/12
	9,000	8/12
	12,000	12/12
	15,000	10/12
	18,000	12/12
Females
	0	0/12
	6,000	0/12
	9,000	0/12
	12,000	0/12
	15,000	3/12
	18,000	5/12

Table 3: 2nd study: Survival, mean body weights and feed consumption of rats

		Mean Body Weights (g)
Dose (ppm)	Survival (a) (Week of death)	Initial	Final (b)	Gain	Weight Change relative to controls (%) (c)	Feed consumption (% of control)
Males
0	10/10	120	312	192
750	10/10	120	302	182	-5	86
1,500	10/10	119	302	183	-5	87
3,000	10/10	120	299	179	-7	89
6,000	10/10	119	290	171	-11	84
12,000	10/10	119	276	157	-18	86
Females
0	10/10	95	176	81
750	10/10	94	179	85	+5	100
1,500	10/10	95	179	84	+4	91
3,000	10/10	93	175	82	+1	87
6,000	10/10	95	176	81	0	89
12,000	10/10	93	173	80	-1	92

(a) Number surviving/number per group

(b) Weight at day 84

(c) ((Weight change dosed group – weight change control group)/weight change control group) X 100

Table 4: 2nd study: Incidence of rats with urinary bladder stones and hyperplasia of the bladder epithelium

Dose (ppm)	No of rats with urinary bladder stones Dose (ppm)	Hyperplasia of the Bladder Epithelium
Males
0	1/10	0/10
750	2/10	0/10
1,500	5/10	0/10
3,000	7/10	1/10
6,000	9/10	3/10
12,000	9/9	9/9
Females
0	0/10	0/10
750	0/10	0/10
1,500	0/10	0/10
3,000	0/10	0/10
6,000	0/10	0/10
12,000	0/10	0/10