Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attachment

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attachment

3. ANALOGUE APPROACH JUSTIFICATION
see attachment

4. DATA MATRIX
see attachment
Cross-referenceopen allclose all
Reason / purpose:
read-across source
Reference
Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: summarized iinformation from reliable sources
Principles of method if other than guideline:
Three-generation-study in rats
GLP compliance:
not specified
Remarks:
indicated in OECD SIDS to be GLP-compliant
Limit test:
no
Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
no data
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on exposure:
P0: A minimum of 100 days from 36 days of age to mating
F1 and F2: 120 days after weaning
F3: 4 weeks
Details on mating procedure:
premating procedure: A minimum of 100 days
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Treatment was initiated at 36 days of age for parents and continued for a minimum of 100 days before mating.
Frequency of treatment:
daily
Details on study schedule:
Parents were mated to produce two litters (Fia, Flb).
Weanlings from the Flb litter were randomly selected as parents for the next generation and continued on treatment for an additional
120 days. These animals were subsequently mated to produce 2 litters (F2a, F2b).
Weanlings from the F2b litter were randomly selected as parents for the last generation. These animals were also administered
sodium cyanurate for 120 days and mated to produce one litter (F3a).
Randomly selected F3a progeny continued on cyanurate treatment for an additional 4 weeks and were then sacrificed.
Dose / conc.:
400 mg/L drinking water
Dose / conc.:
1 200 mg/L drinking water
Dose / conc.:
5 375 mg/L drinking water
Remarks:
highest soluble concentration, ca. 370 mg/kg/day for males and 634 mg/kg/day
for females
No. of animals per sex per dose:
12 males
24 females
Control animals:
yes, concurrent vehicle
other: sodium hippurate
Details on study design:
Where possible, all progeny from various matings were given a post mortem examination.
Organ weight measurements and microscopic examination of tissues (including gonads and gross lesions) were carried out for all parental animals
i.e., selected Fib and F2b progeny and F3a offspring that were sacrificed 4 weeks after weaning.
Parental animals: Observations and examinations:
mortality, body weight, food consumption, and gestation length.
Sperm parameters (parental animals):
no data
Litter observations:
mortality, body weights, food consumption litter size, pup survival to weaning, sex ratio, and pup weight.
Postmortem examinations (parental animals):
gross pathology and histopathology
Postmortem examinations (offspring):
gross pathology and histopathology
Statistics:
no data
No compound related changes were observed in mortality, body weight, food consumption, and gestation length. In pathological and histological findings, there were also no changes.
Dose descriptor:
NOAEL
Effect level:
>= 5 375 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see remarks on results
Remarks on result:
other: A few high-dose cyanurate males did exhibit calculi in the urinary bladder accompanied by microscopic evidence of epithelial hyperplasia or chronic cystitis. These histologic changes were attributed to chronic irritation by the calculi.
Critical effects observed:
no
Dose descriptor:
NOAEL
Effect level:
5 375 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See P0 generation
Critical effects observed:
no
No compound-related changes were observed in mortality, body weights, food consumption litter size, pup survival to weaning, sex ratio, and pup weight. In pathological and histological findings, epithelial hyperplasia with chronic cystitis was observed in a few of high-dose treated males in F2 offsprings, which were attributed to chronic irritation by the calculi in the urinary bladder. In other treated groups, there were no changes.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
5 375 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See P0
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
5 375 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see P0 generation
Reproductive effects observed:
no

Sodium hippurate was provided an equivalent amount of sodium administered to high-dose sodium isocyanurate animals as second control. Weanlings from the F1 and F2 litters were randomly selected as parents for the next generation and continued on treatment. Related litters and F3 offsprings were sacrificed 4 weeks after weaning and organ weight measurements and microscopic examination of tissues were carried out.

Reason / purpose:
read-across source
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
purity 99.8%
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
females: 14 days before mating to day 3 of lactation
males: 44 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
10, 40, 150 and 600 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Reproductive indices:
copulation index, fertility index, implantation, implantation index, gestation index, delivery index
Offspring viability indices:
live birth index, viability index, sex ratio
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: kidney toxicity
Remarks on result:
other: effects on kidneys at 600 mg/kg bw
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects
Critical effects observed:
no
Reproductive effects observed:
no

The parental animals exhibited no alteration in reproductive parameters including the copulation index, fertility index, gestation length, numbers of corpora lutea or implantation, implantation index, gestation index, delivery index, and behavior at delivery and lactation. There were no significant differences in offspring parameters including number of offspring or live offspring, the sex ratio, live birth index, viability index and body weight. No external or visceral abnormalities related to the test substance were detected in any of the offspring. Therefore, NOAEL for parental fertility and offsprings was considered to be 600 mg/kg/day. Parental animals sufferend from adverse effects on kidney at 600 mg/kg bw/d.

Reason / purpose:
read-across source
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: repeated dose toxicity study (13 weeks and carcinogenicity study)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-performed investigation with melamine, adresses effects on reproductive organs
Principles of method if other than guideline:
repeated dose toxicity study (13 weeks and carcinogenicity study) with examination of reproductive organs
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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 %.
Route of administration:
oral: feed
Details on 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:
cf. 7.5.1 and 7.7
Frequency of treatment:
cf. 7.5.1 and 7.7
Remarks:
Doses / Concentrations:
0, 6000, 9000, 12000, 15000 or 18000 ppm
Basis:
nominal in diet
first study
Remarks:
Doses / Concentrations:
0, 750, 1500, 3000, 6000 or 12000 ppm
Basis:
nominal in diet
second study
Remarks:
Doses / Concentrations:
0, 10000 or 18000 ppm
Basis:
nominal in diet
third study
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 vehicle
Details on study design:
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
Postmortem examinations (parental animals):
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
No toxicity to reproductive organs were observed. Melamine affected the kidneys at tested doses.
Dose descriptor:
NOAEL
Effect level:
>= 18 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproductive organs. Animals suffered from urinary bladder stones and associated systemic toxicity with a LOAEL of 750 ppm (ca 72 mg/kg bw).
Critical effects observed:
yes
Lowest effective dose / conc.:
18 000 ppm
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Remarks on result:
other: No F1 generation was produced.
Reproductive effects observed:
no
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Study period:
2016-2017
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: 31 Mar 2017 is scheduled experimental completion date (draft report to QAU)
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
28 Jul 2015
Deviations:
yes
Remarks:
additional investigations for kidney toxicity
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Test substance: white solid
Date of manufacture: 28 Oct 2015
Batch no. 52334524U0
Validity: 15 Sep 2020
Species:
rat
Strain:
other: Crl:Wl(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males 10-11 weeks, females 9 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no
- Housing: single caging (except for mating; then 1 female with one male; except for rearing dams, then one dam with up to 13 pups)
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 05-July 2016 To: 30-Aug-2016 (males) and 29-Sept-1016 (females)
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): Ground Kliba Maintenance diet (Provimi Kliba SA, Switzerland)
- Storage temperature of food: room temperature

The stability of the substance in the diet for at least four days was shown experimentally prior to the beginning ot the study.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy

- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individual cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Doses were selected based on the results of a 7-day and a 28-day feeding study with melamine cyanurate in rats.
Duration of treatment / exposure:
28 days (males)
58 days (females)
Frequency of treatment:
daily
Dose / conc.:
660 ppm (analytical)
Dose / conc.:
200 ppm (analytical)
Dose / conc.:
66 ppm (analytical)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
Determination of estrous cycle was done during 14 days after the acclimatization period and prior to the first dosing.

On postnatal day 4, culling will be done so that per litter 4 female and 4 male pups remain until scheduled sacrifice on postnatal day 14.
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days, once daily on weekends and public holidays
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week, and for females on GD 7, 14 and 20, and on PND 4, 7,10 and 13
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Once a week, and for females on GD 7, 14 and 20, and on PND 4, 7,10 and 13

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes



OTHER:
Blood is taken from all males at the end of the in-life phase (day 28). Clinical Chemistry values are determined.
Histopathology of kidneys is included and kidney weights are determined.
WATER CONSUMPTION: Once a week, and for females on GD 7, 14 and 20, and on PND 4, 7,10 and 13
Oestrous cyclicity (parental animals):
yes
Reproductive effects observed:
not specified

Results are not yet available.

Reason / purpose:
read-across: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
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.
Reason / purpose:
reference to same study
Qualifier:
according to
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, 6,000, 9,000, 12,000, 15,000, or 18,000 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, 1,500,3,000,6,000, or 12,000 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, 10,000, or 18,000 ppm melamine in the presence and absence of 1% ammonium chloride in the drinking water.
Three 13-weeks toxicity studies were performed.

GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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 %.
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
Remarks:
Doses / Concentrations:
First study: 0, 6000, 9000, 12000, 15000 or 18000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Second study: 0, 750, 1500, 3000, 6000 or 12000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
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
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.
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.

Dose descriptor:
LOAEL
Effect level:
750 ppm
Sex:
male
Basis for effect level:
other: urinary bladder stones
Dose descriptor:
LOAEL
Effect level:
ca. 72 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: urinary bladder stones
Dose descriptor:
NOAEL
Effect level:
6 000 ppm
Sex:
female
Basis for effect level:
other: body weight
Dose descriptor:
NOAEL
Effect level:
ca. 600 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: body weight
Critical effects observed:
not specified
The relevant results in the tables of the study are assembled in the attachment.
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.

Data source

Materials and methods

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Results and discussion

Results: P0 (first parental animals)

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
450 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
660 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
450 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Derived from NOEL for kidney toxicity in the 28-day study

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
660 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOEL
Generation:
F2
Effect level:
450 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (F2)

Critical effects observed:
yes
Lowest effective dose / conc.:
660 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Melamine canurate does not cause adverse effects on fertility or reproduction.
Melamine cyanurate shows significant general toxicity related to kidney failure after accumulation of melamine cyanurate crystals in the kidney.