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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Meets generally accepted scientific standards, well documented and acceptable for assessment, performed with melamine (Restrictions: no examination of blood (hematology and clinical biochemistry, no ophthalmologicaol examinations were performed, no information about the determination of organ weights is given).

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1983
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 Type:
secondary source
Title:
Joint Assessment of Commodity Chemicals, No. 1 Melamine CAS: 108-78-1
Author:
unknown
Year:
1983
Bibliographic source:
ECETOC
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

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
-Analytical purity: >= 97%

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 6000, 9000, 12000, 15000, 18000 ppm (estimated melamine consumption according to NTP study report: males 0, 560, 850, 1100, 100, 1700 mg/kg/day; females 0, 560, 880, 1200, 1400, 1600 mg/kg/day; in the OECD SIDS a conversion factor of 12 was applied)
Basis:
other: 1 st study
Remarks:
Doses / Concentrations:
0, 750, 1500, 3000, 6000, 12000 ppm (estimated melamine consumption according to NTP study report: males 0, 72, 150, 300, 590, 1300 mg/kg/day; females 0, 84, 150, 300, 600, 1300 mg/kg/day; In the OECD SIDS a conversion factor of 12 was applied)
Basis:
other: 2nd study
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 (if feeding study): 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

Details on results:
1st study
CLINICAL SIGNS AND MORTALITY
One male rat receiving 18,000 ppm and two males receiving 6,000 ppm died (cf. Table 1)

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.

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/HISTOPATHOLOGY
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.
Histopathologic evaluations were performed on 10 animals of either sex from the high-dose (18,000 ppm), low-dose(6,OOO 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.

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

open allclose all
Dose descriptor:
LOAEL
Effect level:
72 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Urinary bladder stones in males (refers to 750 ppm)
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)
Dose descriptor:
LOAEL
Effect level:
72 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: urinary bladder stones observed in the second study (refers to 750 ppm)

Target system / organ toxicity

Critical effects observed:
not specified

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

Applicant's summary and conclusion