Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

Repeated dose toxicity: oral

Administrative data

Endpoint:

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

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

GLP compliance:

not specified

Limit test:

no

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
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: males: mean: 120 g, females: mean: 105 g
- Fasting period before study: no
- Housing: Rats were housed five per cage in polycarbonate cages covered with nonwoven polyester filter sheets.
- Diet: ad libitum, Purina Laboratory Chow
- Water: ad libitum, tap water (acidified with hydrochloric acid to pH 2.5).
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with Purina B Lab Chow: 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 minutes in a Patterson-Kelly@ twinshell blender equipped with an intensifier bar.
- Storage temperature of food: Test diets were stored at -20 °C 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 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:

103 weeks

Frequency of treatment:

continuously

Dose / conc.:

2 250 ppm

Remarks:

only males; corresponds to 126 mg/kg bw/day

Dose / conc.:

4 500 ppm

Remarks:

for males and females; corresponds to 263 and 262 mg/kg bw/day for males and females, respectively

Dose / conc.:

9 000 ppm

Remarks:

only females; corresponds to 542 mg/kg bw/day

No. of animals per sex per dose:

50 (except for only 49 control male rats)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on preliminary short-term study
- Post-exposure recovery period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were not further described.

DETAILED CLINICAL OBSERVATIONS: Clinical signs were recorded monthly.

BODY WEIGHT: Yes
- Time schedule for examinations: at study initiation, once weekly for the first 13 weeks, monthly until week 91, and every 2 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: feed consumption data collected every 4 weeks

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes: First study: gross lesions, skin with mammary gland, mandibular lymph nodes, salivary gland, sternum with bone marrow, larynx or anterior trachea, esophagus, thyroid, parathyroid, lungs with mainstream bronchi, heart, stomach (glandular and nonglandular), duodenum, large intestine, liver, pancreas, spleen, kidneys, adrenal glands, urinary bladder, gonads, prostate or uterus, brain, and pituitary gland

Statistics:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

No compound-related clinical signs observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

The survival of the high-dose group of male rats was significantly reduced when compared with that of the controls (p=0.03). No significant differences were observed between any other groups of either sex (see figure 2 in the attachment). In male rats, 30/49 (61%) of controls, 30/50 (60%) of the low-dose group, and 1/50 (38%) of the high-dose group lived to the termination period of the study at 105 weeks. 30/50 (60%) of the high-dose group of male rats survived 92 weeks of the study. In female rats, 34/50 (68%) of the controls, 30/50 (60%) of the low-dose, and 27/50 (54%) of the high-dose group lived to the termination period of the study at 103 weeks.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

After week 20, mean body weights of dosed rats of each sex were lower than those of the controls (see table 10 and figure 1 in the attachment)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The average daily feed consumption per rat by low- and high-dose rats was 97% and 99% that of the controls for males and 99% and 99% for females (see table H1 in the attachment)

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:

effects observed, treatment-related

Description (incidence and severity):

Urinary bladder: Stones were found in 10/50 (20%) high-dose males, 1/50 (2%) low-dose males and none in the control group. No stones were found in female animals of any group. The transitional-cell carcinomas (8/49 male rats from high-dose group) were visualized grossly as 1- to 1.5 cm masses attached to the mucosal surface of the urinary bladder. 7/8 high-dose male rats had bladder stones (calculi).
Kidney: pitted or roughend renal cortical surfaces in female rats of high-dose group

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidney: Chronic inflammation was observed in significantly (<=0.01) increased incidence in dosed female rats (table 11 in the attachment). The dose relationship and intensity of the increased interstitial lymphoplasmocytic infiltrates and cortical fibrosis clearly set these changes apart from the minor inflammatory component that may accompany the progessive nephropathy normally encounterd in aging F344/N rats. The changes in the high-dose females were often observed grossly as pitted or roughened renal cortical surfaces. Chronic inflammation of the kidney was not significant in dosed male rats. In those animals in which this lesion was observed there was no correlation with urinary bladder stones.

Uterus: Endometrial stromal polyps were observed in statistically significant (p<=0.017) negative trend (controls 11/50 (22%), low-dose 7/50 (14%), high-dose 2/50 (4%) and in decreased incidence (p<=0.022) in the high-dose group in a pairwise comparison with the controls. The combined incidence of endometrial stromal polyps and sarcomas was statistically significant (p<=0.017) in the negative direction (controls 14/50 (28%), low-dose 7/50 (14%), high-dose 4/50 (8%). A significantly lower (p<=0.029) incidence in the high-dose group was observed in the pairwise comparisons with the controls. The combined incidence of endometrial stromal polyps and sarcomas in the high-dose group was not significantly different from the historical rate of this tumor in untreated female F344/N rats at the same laboratory (117/759 (15.4%). (see table J3 n the attachment).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Urinary bladder: Transitional-cell carcinomas in the urinary bladder of male rats occurred with a statistically significant (p<=0.002) positive trend (controls 0/45, low-dose: 0/50, high-dose 8/49 (16%)) and the incidence in the high-dose group was significantly higher (p<=0.016) than that in the controls (table 11 and 12 in the attachment). The combined incidence of transitional-cell carcinomas and papillomas showed a statistically significant (p<0.001) positive trend (controls 0/45, low-dose 0/50, high dose 9/49 (18%)) and the incidence in high-dose rats was significantly higher (p<=0.008) than that in the controls. These tumors were not observed in statistically significant proportions in female rats (0/49, 1/49, 1/47). Microscopically, most of the carcinomas had transitional-like cells that formed protrusions into the bladder lumens. Some had papillary areas. The carcinomas had moderate numbers of mitoses and some nuclear pleomorphism. Discrete invasions through the bladder wall occurred in one male rat, but no metastases were evident in the lungs or other tissues.

Pancreas: Pacreatic islet-cell carcinomas in male rats occurred with a statistically significant (p=0.034) negative trend (control 3/44 (7%), low-dose 0/48, high-dose 0/45) by the Cochran Armitage test. the incidences were not significant in pairwise comparisons between the dosed groups and the controls, and these tumors were not observed in female rats. Total pancreatic islet-cell tumors (adenomas and carcinomas) were not significantly different for either sex of rats.

Thyroid: C-cell carcinomas in the thyroid were observed in female rats with a statistically significant (p<=0.038) positive trend (controls 0/50, low-dose 0/49, high-dose 3/50 (6%)). Neither the pairwise comparisons of the high-dose group with the controls nor the combination of C-cell adenomas or carcinomas was statistically significant in any the tests. These tumors were not observed in statistically significant proportions in male rats.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

2 250 ppm

Based on:

test mat.

Remarks:

corresponds to 126 mg/kg bw/day

Sex:

male

Basis for effect level:

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Effect level:

4 500 ppm

Based on:

test mat.

Remarks:

corresponds to 262 mg/kg bw/day

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 500 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Melamine was administered in the diet to F344 rats for 103 weeks (chronic) to determine its toxicological profile. The dose levels of melamine in this chronic study were 2250 and 4500 ppm for males and 4500 and 9000 ppm for females. In this study, compound-related lesions were observed in the urinary tract. The urinary bladder was the primary site affected in male rats, and is considered to be the primary target organ for melamine. Transitional-cell carcinoma of the urinary bladder occurred in male rats with a statistically significant positive trend, and the incidence in the high-dose group was signficantly higher than that compared in the controls. Also an increased incidence of urinary bladder stones occurred in high-dose male rats. Thus, a NOAEL of 2250 ppm corresponding to 126 mg/kg bw/day for male rats was determined.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

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

Principles of method if other than guideline:

Three sub-chronic studies were performed. After in the first study effects on urinary tract were observed the second study was done in order to determine a NOAEL. The third study was performed in order to investigate the effect of ammonium chloride on urinary stone formation in rats.

GLP compliance:

not specified

Limit test:

no

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
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: first study: males: 87 - 92 g, females: 79 - 87 g; second study: males: 119 - 120 g, females: 93 - 95 g; third study: males: 100 - 120 g, females: 93 - 95 g
- Fasting period before study: no
- Housing: Rats were housed four per cage in polycarbonate cages covered with nonwoven polyester filter sheets.
- Diet: ad libitum, Purina Laboratory Chow
- Water: ad libitum, tap water (acidified with hydrochloric acid to pH 2.5).
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 -26
- Humidity (%): 30-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with Purina B Lab Chow: 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 15 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

Dose / conc.:

6 000 ppm

Remarks:

first study (estimated: 560 and 850 mg/kg bw/day for males and females, respectively)

Dose / conc.:

9 000 ppm

Remarks:

first study (estimated: 850 and 880 mg/kg bw/day for males and females, respectively)

Dose / conc.:

12 000 ppm

Remarks:

first study (estimated: 1100 and 1200 mg/kg bw/day for males and females, respectively)

Dose / conc.:

15 000 ppm

Remarks:

first study (estimated: 1400 mg/kg bw/day for males and females, respectively)

Dose / conc.:

18 000 ppm

Remarks:

first study (estimated: 1700 and 1600 mg/kg bw/day for males and females, respectively)

Dose / conc.:

750 ppm

Remarks:

second study (estimated: 72 and 84 mg/kg bw/day for males and females, respectively)

Dose / conc.:

1 500 ppm

Remarks:

second study (estimated: 150 and 150 mg/kg bw/day for males and females, respectively)

Dose / conc.:

3 000 ppm

Remarks:

second study (estimated: 300 and 300 mg/kg bw/day for males and females, respectively)

Dose / conc.:

6 000 ppm

Remarks:

second study (estimated: 590 and 600 mg/kg bw/day for males and females, respectively)

Dose / conc.:

12 000 ppm

Remarks:

second study (estimated: 1300 mg/kg bw/day for males and females, respectively)

Dose / conc.:

10 000 ppm

Remarks:

third study

Dose / conc.:

18 000 ppm

Remarks:

third study

No. of animals per sex per dose:

12 for first study
10 for second and third study, respectively

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on preliminary short-term study
Two additional 13-week studies were conducted to find a no-effect level for urinary bladder stone formation (second study) and to determine the effect of ammonium chloride in the drinking water on stone formation (third study).
- Post-exposure recovery period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were not further described.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at study initiation, once weekly, and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: once weekly by cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Yes: only for the second study: 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.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes for all studies

HISTOPATHOLOGY: Yes: First study: gross lesions, tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, sciatic nerve, bone marrow, costochondral junction (rib), thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles/ prostate/ testes or ovaries/ uterus, nasal cavity, brain, and pituitary gland. For lowest dose group only kidney and urinary bladder were examined microscopically.
Second study: kidney and urinary bladder
Third study: no histopathology was performed

Statistics:

not specified

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

First study: One male rat receiving 18,000 ppm and two males receiving 6000 ppm died (see table 4 in the attachment)

Second study: No mortality was observed (see table 6 in the attachment).

Third study: No mortality was observed (see table 9 in the attachment).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

First 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 (see table 4 in the attachment)

Second study: Mean body weight gain was depressed by more than 10% when compared with controls for male rats receiving 6000 and 12,000 ppm, but no depression was observed in any group of dosed females (see table 6 in the attachment).

Third study: 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 (see table 9 in the attachment).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

First study: Feed consumption by rats receiving 18,000 ppm was approximately 80%-90% that of controls (see table 4 in the attachment)

Second study: Feed consumption was not affected by incorporation of melamine in the feed (see table 6 in the attachment).

Third study: not specified

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:

no effects observed

Description (incidence and severity):

Second study: There were no differences in the urine samples that could be attributed to the presence of melamine in the feed (see table 8 in the attachment). Microscopic examination of the urine did not provide any evidence of melamine crystalluria.

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):

First study: Stones were found in the urinary bladders of most dosed male rats, and the incidence was dose related (0/12 in 0 ppm; 6/12 in 6,000 ppm; 8/12 in 9,000 ppm; 12/12 in 12,000 ppm; 10/12 in 15,000 ppm and 12/12 in 18,000 ppm). Twenty-five percent (3/12 in 15,000 ppm and 5/12 in 18,000 ppm) females in the two highest dosed groups had stones (see table 5 in the attachment).

Second study: 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 (1/10 in 0 ppm; 2/10 in 750 ppm; 5/10 in 1500 ppm; 7/10 in 3,000 ppm; 9/10 in 6000 ppm and 9/9 in 12,000 ppm; see table 7 in the attachment).

Third study: 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 (see table 9 in the attachment). No other compound-related effects were observed at necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

First study: Histopathologic evaluations were performed on 10 animals of either sex from the high-dose (18,000 ppm), low-dose (6000 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 6000 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.

Second study: 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 3000 ppm, in 3/10 receiving 6000 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 1500 ppm, 10/ 10 receiving 3000 ppm, 8/ 10 receiving 6000 ppm, and 10/ 10 receiving 12,000 ppm melamine).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

750 ppm

Based on:

test mat.

Remarks:

corresponds to 72 and 84 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

gross pathology

other: formation of urinary bladder stones

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:

not specified

Conclusions:

Melamine was administered in the diet to F344 rats for 13 weeks (subchronic) to determine its toxicological profile. The dose levels of melamine in the subchronic studies ranged from 750 to 18,000 ppm. 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. Increased incidences of urinary bladder stones and hyperplasia of the bladder epithelium were observed at 13 weeks in male rats fed diets containing melamine.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

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

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Center, Frederick, MD
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: males: mean: 19 g, females: 15 - 16 g
- Fasting period before study: no
- Housing: Mice were housed five per cage in polycarbonate cages covered with nonwoven polyester filter sheets.
- Diet: ad libitum, Purina Laboratory Chow
- Water: ad libitum, tap water (acidified with hydrochloric acid to pH 2.5).
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with Purina B Lab Chow: 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 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

Dose / conc.:

6 000 ppm

Remarks:

estimated: 1400 and 1800 mg/kg bw/day for males and females, respectively

Dose / conc.:

9 000 ppm

Remarks:

estimated: 2000 and 2700 mg/kg bw/day for males and females, respectively

Dose / conc.:

12 000 ppm

Remarks:

estimated: 2800 and 3500 mg/kg bw/day for males and females, respectively

Dose / conc.:

15 000 ppm

Remarks:

estimated: 3900 and 4800 mg/kg bw/day for males and females, respectively

Dose / conc.:

18 000 ppm

Remarks:

estimated: 4700 and 5900 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on preliminary short-term study
- Post-exposure recovery period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were not further described.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at study initiation, once weekly, and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: once weekly by cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes: gross lesions, tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, sciatic nerve, bone marrow, costochondral junction (rib), thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, gallbladder, pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles/ prostate/ testes or ovaries/ uterus, nasal cavity, brain, and pituitary gland. For lowest dose group only kidney and urinary bladder were examined microscopically.

Statistics:

not specified

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female mouse receiving 9,000 ppm died. (see table 16 in the attachment)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain relative to controls was depressed by 9% or more in all dosed groups. (see table 16 in the attachment)

Description (incidence and severity):

Feed consumption data were not considered reliable because the mice scattered the contents of their feeders.

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:

effects observed, treatment-related

Description (incidence and severity):

The incidence of mice with bladder stones, observed at necropsy was dose-related and was greater in males (0/10 in 0 ppm, 6000 and 9000 ppm; 6/10 in 12,000 ppm; 9/10 in 15,000 ppm and 7/10 in 18,000) than in females ((0/10 in 0 ppm, 6000 and 9000 ppm; 1/10 in 12,000 ppm; 3/10 in 15,000 ppm and 7/10 in 18,000) (see table 17 in the attachment).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidence of mice with bladder stones was dose related and was greater in males than in females. Ulceration of the urinary bladder epithelium was also dose related (see table 17 in the attachment). 60% of the mice having bladder ulcers also had urinary bladder stones; 40% did not. Bladder ulcers were multifocal or associated with inflammation (cystitis). These results do not provide evidence for an association between ulceration and bladder stones in either sex. Epithelial hyperplasia in the bladder was observed in 2/10 males fed 18,000 ppm melamine. Both animals also had bladder stones. Epithelial cell atypia was seen in 2/10 males fed 9000 ppm melamine. Because of epithelial changes in the bladder in males fed 9000 ppm melamine, doses of 2250 and 4500 ppm melamine in feed were selected for mice in the two-year-study.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

6 000 ppm

Based on:

test mat.

Remarks:

corresponds to 1400 and 1800 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

9 000 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Melamine was administered in the diet to B6C3F1 mice for 13 weeks (subchronic) to determine its toxicological profile. The dose levels of melamine in the subchronic studies ranged from 6000 to 18,000 ppm. 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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

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

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:

Substance name in report: melamine
Purity: > 95%

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Center, Frederick, MD
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: males: mean: 22 g, females: mean: 17 g
- Fasting period before study: no
- Housing: Rats were housed five per cage in polycarbonate cages covered with nonwoven polyester filter sheets.
- Diet: ad libitum, Purina Laboratory Chow
- Water: ad libitum, tap water (acidified with hydrochloric acid to pH 2.5).
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with Purina B Lab Chow: 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 minutes in a Patterson-Kelly@ twinshell blender equipped with an intensifier bar.
- Storage temperature of food: Test diets were stored at -20 °C 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 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:

103 weeks

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 250 ppm

Remarks:

corresponding to 327 and 523 mg/kg bw/day for males and females, respectivley

Doses / concentrations 2

Dose / conc.:

4 500 ppm

Remarks:

corresponding to 688 and 1065 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

50 (except for only 49 control male mice)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on preliminary short-term study
- Post-exposure recovery period: none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were not further described.

DETAILED CLINICAL OBSERVATIONS: Clinical signs were recorded monthly.

BODY WEIGHT: Yes
- Time schedule for examinations: at study initiation, once weekly for the first 13 weeks, monthly until week 91, and every 2 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: feed consumption data collected every 4 weeks

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes: First study: gross lesions, skin with mammary gland, mandibular lymph nodes, salivary gland, sternum with bone marrow, larynx or anterior trachea, esophagus, thyroid, parathyroid, lungs with mainstream bronchi, heart, stomach (glandular and nonglandular), duodenum, large intestine, liver, gall bladder, pancreas, spleen, kidneys, adrenal glands, urinary bladder, gonads, prostate or uterus, brain, and pituitary gland

Statistics:

not specified

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No compound-related clinical signs observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

The survival of the high-dose group of male mice was significantly reduced when compared with that of the controls (p=0.013). No other significant differences in survival were observed between any groups of either sex. In male mice, 39/49 (80%) of the controls, 36/50 (72%) of the low-dose, and 28/50 (56%) of the high dose group lived to the termination period of the study at 103 weeks. In female mice, 37/50 (74%) of the controls, 43/50 (86%) of the low-dose, and 41/50 (82%) of the high-dose group lived to the termination period of the study at 103 weeks. (see figure 4 in the attachment)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights of high-dose male mice were slightly lower than those of the controls after week 50 of the study. Mean body weights of dosed and control female mice were comparable throughout the study (see figure 3 and table 18 in the attachment).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The average daily feed consumption per mouse by low- and high-dose mice was 93% and 95% that of the controls for males and 88% and 87% for females (see table H2 in the attachment).

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:

effects observed, treatment-related

Description (incidence and severity):

Stones in the urinary bladder were found in male mice increasing by dose (2/49 (4%) in controls, 40/50 (85%) in low-dose and 41/50 (93%) in high-dose) whereas in females only in the high-dose group 4/50 (8%) showed stones (calculi) (see table 19 in the attachment). Most of the stones were green and multiple.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Urinary bladder: Epithelial hyperplasia was found to be increased over the dosing (2/49 (4%) in male controls, 11/50 (23%) in low-dose males, 13/50 (30%) in high-dose males and 0/50 in control and low-dose females and 4/50 (8%) in high-dose females; see table 19 in the attachment). Although epithelial hyperplasia occurred at higher incidences in males and females fed diets containing melamine, there was no evidence, either in histopathologic features or incidence of neoplasia in the bladder, to suggest that this change was preneoplastic. Hyperplasia was generally very mild. Acute and chronic inflammation was observed in 0/49 male controls, 25/50 (53%) male low-dose and 24/50 (55%) in male high-dose mice wheras no acute and chronic inflammation was observed in control and low-dose females and 4/50 (8%) in high-dose females. Looking at chronic inflammation alone the following results were observed: 2/49 (4%) male controls, 10/50 (21%) male low-dose, 14/50 (32%) male high-dose and 0/50 in control and low-dose females and 2/50 (4%) in high-dose females.

Lung: The lungs of three high-dose females were diffusely infiltrated with structures morphologically compatible with the protozoan organism Pneumcystis carinii. Similar involvment was not present in other groups of mice. P. carinii can occur as a latent, inapparent infection in laboratory rodents. Under conditions of stress (e.g. immunosupression), the organism is capable of being pathogenic. Two of the affected mice killed at the end of the study had a neoplasm (chromophobe adenoma or malignant lymphoma). The other mouse died with no evidence of generalized systemic disease. Other than the presence of generalised neoplasia in two of the affected mice, there was no direct morphologic evidence of systemic immunosupression in these animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Lung: Alveolar/bronchiolar adenoms in female mice were observed in decreased (p<=0.025) incidence in the low-dose group compared with that of the controls (controls 5/44 (11%), low-dose 0/48). The incidence in the high-dose group (2/50 (4%)) was not significantly different from that in the controls, and the combined incidence of alveolar/bronchiolar adenomas and carcinomas was not significantly different between any groups of either sex of mice. In addition, the low-dose incidence of alveolar/bronchiolar adenomas is not significantly different from the historical rate of this tumor in untreated female B6C3F1 mice at the same laboratory (25/502, 5%).(see table J4 in the attachment)

Other effects:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

2 250 ppm

Based on:

test mat.

Remarks:

corresponds to 523 mg/kg bw/day

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

LOAEL

Effect level:

2 250 ppm

Based on:

test mat.

Remarks:

corresponding to 327 mg/kg bw/day

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 250 ppm

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Melamine was administered in the diet to B6C3F1 for 103 weeks (chronic) to determine its toxicological profile. The dose levels of melamine in this chronic study were 2250 and 4500 ppm for males and females. In this study, compound-related hyperplasia and stones in the urinary bladder were observed. The urinary bladder was the primary site affected in male mice, and is considered to be the primary target organ for melamine. As a high incidence of stones was found in the low-dose group in males no NOAEL for male mice could be determined. For female mice a NOAEL of 2250 ppm corresponding to 523 mg/kg bw/day was determined.