1,3,5-triazine-2,4,6(1H,3H,5H)-trione, compound with 1,3,5-triazine-2,4,6-triamine (1:1)

Endpoint summary

Description of key information

Ingestion of melamine cyanurate causes kidney toxicity via crystal formation as shown in a 7 -day feeding study in rats with a NOEL of 20 mg/kg bw for appearance
of crystals in the kidney and a NOAEL for 66 mg/kg bw for systemic toxicity (BASF 2010). In a follow-up 28 -day feeding study in rats, no adverse effects were observed at the highest tested dose of 450 ppm (mean 28.6 mg/kg bw for females and mean 23.8 mg/kg bw for males). This is consistent with various experimental data on co-exposure to melamine and cyanuric acid (Gamboa da Costa 2010 and 2011, Kobayashi 2010, Stine 2011, Gamboa da Costa 2012). In a 28 -day feeding study in weanling pigs a NOAEL of 2 mg/kg bw was established (Stine 2011).

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This is a publication of a GLP-compliant non guidline study specifically designed to investigate kidney toxicity.

Qualifier:

no guideline available

Principles of method if other than guideline:

28-day feeding study with weanling pigs for investigation of melamine cyanurate kidney toxicity

GLP compliance:

not specified

Remarks:

Indicated to be done according to GLP, but adress of the test institute not given

Limit test:

no

Specific details on test material used for the study:

Melamine (; 99% pure; Sigma–Aldrich Co., St. Louis, MO,USA)
cyanuric acid (98% pure; Sigma–Aldrich Co., St.Louis, MO, USA)

Species:

pig

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

Weanling pigs were obtained from local farms

Ear-tagged pigs were housed in individual indoor pens at the USFDA’s Center for Veterinary Medicine, Office of Research in Laurel, MD .
Acclimatization: for at least 2 weeks.
Health Check: A standard health check was performed by the attending veterinarian upon the pigs’ arrival to our facility.
Diet: ad libitum (standard corn and soybean diet (18% crude protein) prepared at the facility
Water: ad libitum

Route of administration:

oral: feed

Vehicle:

other: chocolate pudding

Details on oral exposure:

Melamine (MEL; 99% pure; Sigma–Aldrich Co., St. Louis, MO, USA) and/or cyanuric acid (CYA; 98% pure; Sigma–Aldrich Co., St.
Louis, MO, USA) were mixed with retail chocolate pudding in amounts tabulated from daily weight measurements to ensure
accurate dosing. Pudding also tested negative for melamine and cyanuric acid. All pigs ate all pudding each dosing day and thus received
complete test doses.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days
7 days (range-finding study)

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 1 and 3.3 mg/kg bw each M and C
Basis:
nominal in diet

No. of animals per sex per dose:

8

Control animals:

yes, plain diet

Details on study design:

Pigs were given the test compound(s) in the morning (approximately 23–25 h apart)
Body weight: 24 ± 2 kg bw
Origin: obtained from local farms

Positive control:

not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: No data

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 data


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 29 (24h after the last dose)
- Animals fasted: No data
- How many animals: all
- Parameters checked: BUN and creatinine

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters: Total urine was tested for glucose, bilirubin, ketones, specific gravity, traces of blood, pH, proteins, urobilinogen, nitrite and leukocytes
with Siemens Multistix 10 SG (Bayer HealthCare LLC, Elkhart, IN, USA).


The bladder was excised and total urine was taken by emptying the bladder into a bottle. A microscope slide was scraped along the fundus and observed under at least 20 magnification for crystals. Portions of the bladder were fixed in NBF and 70% ethanol. The urethra was removed and palpated for the presence of stones. Samples of the urethra were also fixed in 10% NBF. As previous studies in fish showed intestinal tract crystals (Reimschuessel et al., 2008), portions of stomach, duodenum, jejunum, and ileum, were taken and fixed in 10% NBF. The ureter and penis were examined in several animals. Routine samples were limited to the urogenital and gastrointestinal tracts as previous laboratory studies have shown these tissues to be the target organs in MEL and CYA toxicity and this study was designed to focus on the adverse effect on which to base the NOAEL (Dobson et al., 2008; Puschner et al., 2007).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy included visual examination for any external or internal lesions (including kidney cysts, soft masses and cartilage pathology). If any lesions were present they were recorded and samples were fixed in 10% neutral buffered formalin (NBF) for routine histopathology.

HISTOPATHOLOGY: Yes
Kidneys were excised whole and weighed. One kidney was cut in
half sagitally and thin slices of medulla and cortex were taken for
wet mount observation of typical melamine–cyanurate spherulite
crystals. Fresh tissue was placed on a microscope slide, overlaid with
a cover slip, and compressed with another slide (Reimschuessel
et al., 2008). The slides were observed on an inverted microscope
and ranked on a subjective scale from 0 to 5 as follows: 0 – no crystals
seen; 1 – only 1 crystal in an entire section; 2 – few crystals with
scattered distribution; 3 – moderate numbers of crystals seen
throughout section; 4 – large numbers of crystals seen immediately;
and 5 – extensive numbers of crystals obliterating the regular tissue
architecture. Medulla and cortex samples were also preserved in
10% NBF, 70% ethanol, as well as frozen at -80 °C for archival purposes.
All NBF samples were processed for routine histology.

Other examinations:

All feeds, including those from the local producer, were tested for the presence of MEL and CYA by liquid chromatography tandem mass spectrometry and no peaks were detected above the limit of quantification, 0.5 mg/kg dry weight.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

BUN and CREA levels were normal for all pigs in the 28 day study. BUN ranged from 5 to 15 mg/dl and CREA ranged from 0.9 to 1.6 mg/dl.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

One small cluster of crystals was detected in one of eight pigs at 6.6 mg/kg bw MC

Histopathological findings: neoplastic:

not examined

Details on results:

No crystals were found in the urine sediments of any pigs fed at 0, 1.0 or 3.3 mg/kg bw/day of MEL + CYA (6.6 mg/kg bw MC). One of eight pigs given 3.3 mg/kg bw/day of MEL + CYA had a small cluster of crystals with morphology typical of melamine–cyanurate crystals in the kidney wet mount. The NOAEL based upon formation of renal crystals after exposure for 28 days was 2 mg/kg bw/day.

Urine pH ranged from 5.0 to 8.5 and was not associated with dose. Specific gravity values were normal and ranged from 1.005 to 1.030. All other parameters on the test strip (glucose, bilirubin, ketones, blood, protein, urobilinogen, nitrite and leukocytes) were either negative or within normal limits.

No typical melamine–cyanurate crystals were observed in any tissues from the pigs. The cartilage sample taken from the defect in the tibia tarsal bone showed frayed cartilage en section most likely related to trauma unrelated to study parameters. Several kidneys had incidental renal cysts in both controls and experimental animals. Lesions in skin and the leg were consistent with minor trauma. No other significant lesions were noted in the histologic evaluation of other tissues.

Pigs entered the experimental period at 19.2–28.0 kg and were 35.0–49.0 kg at necropsy. The percent body weight gain over the course of the study of all experimental groups (15.8–21.8 kg) was comparable to that of the controls (15.0– 21.0 kg).

Average kidney weights of the dose groups ranged from 71 to 121 g and were not associated with dose. Average kidney weight/body weight remained constant (range 1.72–2.69 g/kg) and was not associated with dose.

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day (nominal)

Based on:

other: Melamine (1mg)+Canuric acid (1mg)

Basis for effect level:

other: No presence of crystals in kidneys

Critical effects observed:

not specified

In the range-finding study, each two weaniling pigs were treated for 7 days. Doses were 1, 3.3, 10, 33 and 100 mg/kg bw of each M and C. One and a few scattered crystals were observed in the two weanlings given 10 mg/kg bw of each melamine and cyanuric acid. BUN and creatinine were not affected. Body weight gain, relative and absolute kidney weights were also not affected.

No mortality occurred in the range-finder study. A dose-dependent increase in the number of crystals, BUN, creatinine and absolute and relative kidney weights were recorded. BUN values were 58 and 115 mg/dL at the highest dose group, 23 and 15 mg/dL at next lower dose group and 11 and 14 mg/dL at the control group.

Executive summary:

In a 28day study, 36 weanling pigs were fed 0, 1, or 3.3 mg/kgbw/day of melamine and cyanuric acid or 200mg/kgbw/day melamine only. Only one of the 3.3 mg/kg melamine and cyanuric acid pig kidneys contained crystals. The no-observed-adverse-effect level (NOAEL) for pigs fed melamine and cyanuric acid for 28days was concluded to be 1 mg/kgbw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Non GLP, but well documented and performed in a GLP-laboratory. Design adequate for the established effect of kidney toxicity.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Parameters related to kidney toxicity examined

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 14 weeks
- Weight at study initiation: see tables 1 and 2
- Fasting period before study: not applicable
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 06:00 h to 18:00 h; 2 hours darkness from
18:00 h to 06:00 h

:

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test-substance preparations were prepared once at the start of the administration period and stored deep frozen. The food was changed weekly.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

150 ppm

Remarks:

for females: 10.1 mg/kg bw (mean)
for males: 8.1 mg/kg bw (mean)

Dose / conc.:

450 ppm

Remarks:

for females: 28.6 mg/kg bw (mean)
for males: 23.5 mg/kg bw (mean)

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: 7 day feeding study (BASF 2010)

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes /
- Time schedule: twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.


DETAILED CLINICAL OBSERVATIONS: Yes (daily)


BODY WEIGHT: Yes
- Time schedule for examinations: determined before the start of the administration period in order to randomize the rats. During the administration period the body weight was determined on study day 0 (start of administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
determined weekly and calculated as mean food consumption in grams per animal and day.

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 AND COMPOUND INTAKE: Yes
- Time schedule for examinations: determined weekly and calculated as mean water consumption in grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: all
- Parameters checked below were examined:
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11. Preparation of blood smears (only evaluated blood smears will be archived)
12. Prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters checked below were examined.
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum y-glutamyl transferase
5. Sodium
6. Potassium
7. Chloride
8. lnorg. phosphate
9. Calcium
10. Urea
11. Creatinine
12. Glucose
13. Total bilirubin
14. Total protein
15. Albumin
16. Globulins
17. Triglycerides
18. Cholesterol
19. Bile acids



URINALYSIS: Yes
- Time schedule for collection of urine: at the end of treamtent: On the afternoon preceding the day fixed for urinalysis, the animals were transferred individually into metabolism cages (no food or drinking water provided).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked below were examined.


1. Volume
2 . Color
3. Turbidity
4. pH value
5. Protein
6. Glucose
7. Ketones
8. Urobilinogen
9. Bilirubin
10. Blood
11. Specific gravity
12. Microscopy of sediment


NEUROBEHAVIOURAL EXAMINATION: No


IMMUNOLOGY: No

Other examinations:

Blood samples were taken at the end of the administration period from fasted animals by puncturing the retro-orbital venous plexus under isoflurane anesthesia. BUN and creatinine were measured.

Statistics:

DUNNETT's (Body weight and body weight change)
KRUSKAL-WALLIS-H and WILCOXON test (absolute and relative organ weights)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Dose descriptor:

NOEL

Effect level:

450 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Critical effects observed:

no

Table 1: ABSOLUTE WEIGHTS - MEAN VALUES FOR MALES

Group	0	150 ppm	450 ppm
Terminal body weight [g]	369.76	359.5	357.8
%dev	100	97	97
SD	25.015	11.728	12.755
n	5	5	5
Adrenal glands [mg]	59.2	58.6	59.2
%dev	100	99	100
SD	10.159	11.127	8.468
n	5	5	5
Kidneys [g]	2.126	2.08	2.106
%dev	100	98	99
SD	0.183	0.222	0 .14
n	5	5	5
Liver [g]	7.726	7.202	7.412
% dev	100	93	96
SD	1.178	0.332	0.257
n	5	5	5
Spleen [g]	0.56	0.568	0.538
%dev	100	101	96
SD	0.065	0.028	0.083
n	5	5	5

*:P<= 0.05,**:P<= 0.01

Kruskal-Wallis H and Wilcoxon test, twosided

Table 2: ABSOLUTE WEIGHTS - MEAN VALUES FOR FEMALES

Group	0	1	2
Terminalbody weight [g]	226.48	226.26	228.06
%dev	100	100	101
SD	11.747	9.438	7.54
n	5	5	5
Adrenalglands [mg]	87.4	75.6	79
%dev	100	86	90
SD	9.423	8 .112	14.195
n	5	5	5
Kidneys [g]	1.382	1.362	1.532
%dev	100	99	111
SD	0.127	0.156	0.178
n	5	5	5
Liver [g]	5.056	5.14	5.282
% dev	100	102	104
SD	0.268	0.419	0.513
n	5	5	5
Spleen [g]	0.39	0.414	0.46
%dev	100	106	118
SD	0.072	0.057	0.074
n	5	5	5

*:P<=  0.05,**:P<=0.01

Kruskal-Wallis H and Wilcoxon test, two sided

Table 3: Incidence of gross lesions

Sex Group	M 0	1	2	F 0	1	2
Animals in selected group	5	5	5	5	5	5
No abnormalities	5	5	5	5	5	5

Table 4 Food consumption of males (d = days)

		0 / M 0 ppm	1 / M 150 ppm	2 / M 450 ppm
d 0 -> 7	Mean [g]	21.1	20.1	21.2
	S.d.
	N	1	1	1
	Deviation Vs Control		-4.7	0.5
d 7 -> 14	Mean [g]	21.1	22.7	20.2
	S.d.
	N	1	1	1
	Deviation Vs Control		7.6	-4.3
d 14 -> 21	Mean [g]	20.7	20.8	19.1
	S.d.
	N	1	1	1
	Deviation Vs Control		0.5	-7.7
d 21 -> 28	Mean [g]	18.2	17.5	17.6
	S.d.
	N	1	1	1
	Deviation Vs Control		-3.8	-3.3

Table 5: Food consumption of females (d = days)

		0 / F 0 ppm	1 / F 150 ppm	2 / F 450 ppm
d 0 -> 7	Mean [g]	16.6	15.8	15.6
	S.d.
	N	1	1	1
	Deviation Vs Control		-4.8	-6.0
d 7 -> 14	Mean [g]	15.5	15.1	15.4
	S.d.
	N	1	1	1
	Deviation Vs Control		-2.6	-0.6
d 14 -> 21	Mean [g]	15.8	15.1	15.7
	S.d.
	N	1	1	1
	Deviation Vs Control		-4.4	-0.6
d 21 -> 28	Mean [g]	15.0	19.6	15.2
	S.d.
	N	1	1	1
	Deviation Vs Control		30.7	1.3

Further tables can be found in the attachment.

Conclusions:

No adverse effects indicating kidney toxicity in male and female rats were observed upon feed application of 150 and 450 ppm.

Executive summary:

The objective of the study was to assess whether the animals tolerate the test substance at the offered concentrations in the diet after 4 weeks of oral administration in order to select dose levels for subsequent studies. Furthermore, the toxicological profile of the test substance including the target organs should be determined.

The test material was administered via the diet to groups of each 5 male and 5 female animals. The concentrations were 0 ppm (test group 0), 150 ppm (test group 1) and 450 ppm (test group 2).   Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period, the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change. Food and drinking water consumption were determined weekly and calculated as mean food or water consumption in g per animal and day. All animals were checked daily for any abnormal clinically signs. Abnormalities and changes were documented for each animal. Clinicochemical and hematological examinations were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examination of the kidneys.   The following test substance-related, relevant findings were noted:    Test group 2: 450 ppm (24 mg/kg bw/d in males and 29 mg/kg bw/d in females)

Clinical examinations, Clinical Pathology and Pathology:

• No test substance-related, adverse findings were observed    Test group 1: 150 ppm (8.1 mg/kg bw/d in males and 10 mg/kg bw/d in females)

Clinical examinations, Clinical Pathology and Pathology:

• No test substance-related, adverse findings were observed   Conclusively, the administration of the test material via the diet male and female Wistar rats for about 4 weeks did not cause significant signs of toxicity up to a concentration in the diet of 450 ppm.

Reference 3

Endpoint:

repeated dose toxicity: oral

Remarks:

other: 7-day feeding study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Non GLP, but well documented and performed in a GLP-laboratory. Design adequate for the established effect of kidney toxicity.

Principles of method if other than guideline:

7-day feeding study to assess kidney toxicity

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services,
Sulzfeld, Germany
- Age at study initiation: 41-43 days
- Weight at study initiation:
- Fasting period before study: not applicable
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 06:00 h to 18:00 h; 2 hours darkness from
18:00 h to 06:00 h

IN-LIFE DATES: From: To:

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test substance preparations were prepared once before the beginning of the study.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
66, 200, 666, 2000, 4000 and 8000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
ca 6.6, 20, 66.6, 200, 400 and 800 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Positive control:

not applicable

Observations and examinations performed and frequency:

A check for moribund and dead animals were made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.
All animals were checked daily for any abnormal clinically signs.
Food consumption was determined on study days 3 and 7 and calculated as mean food consumption in g per animal and day.
Drinking water consumption was determined on study days 3 and 7 and calculated as mean water consumption in g per animal and day.
Body weights were determined on days 0, 3 and 7. The difference between the body weight on the respective day of weighing and the body weight on day 0 were calculated as body weight change.

Sacrifice and pathology:

Gross pathology was performed.
Histopathology was performed on kidneys and all gross lesions

Organ weights were determined for Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Prostate, Seminal vesicles with coagulation glands, Spleen, Testes, Thymus, Thyroid glands

Other examinations:

Blood samples were taken at the end of the administration period from fasted animals by puncturing the retro-orbital venous plexus under isoflurane anesthesia. BUN and creatinine were measured.

Statistics:

DUNNETT's (Body weight and body weight change)
KRUSKAL-WALLIS-H and WILCOXON test (absolute and relative organ weights)

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

Test group 6 (8000 ppm)
• Two of 5 animals died on study day 3
• Three of 5 animals were sacrificed moribund on study day 3
• Three of 5 animals showed reduced nutritional condition
• All animals showed piloerection
• Food consumption was reduced by -89% on study day 3
• Water consumption was reduced by -62% on study day 3
• Body weight loss was observed on study day 3
• Four of 5 animals showed enlarged and 3 of 5 animals discolored kidneys
• Two of 5 animals showed discoloration of urinary bladder contents
• One of 5 animal showed a focus within the heart muscle
• Up to moderate subacute multifocal pyelonephritis with tubular dilation and
degeneration in kidneys of all animals
• Varying number of intratubular (proximale and distale tubules, Henle’s loop) greenbrownish
crystals in kidneys of all animals

Test group 5 (4000 ppm)
• All animals were sacrificed moribund on study day 3
• All animals showed reduced nutritional condition
• One of 5 animals showed piloerection
• Food consumption was reduced by -75% on study day 3
• Water consumption was reduced by -37% on study day 3
• Body weight loss was observed on study day 3
• All animals showed enlarged and discolored kidneys
• Four of 5 animals showed discoloration of urinary bladder contents
• One of 5 animal showed a focus within the heart muscle
• Up to severe subacute multifocal pyelonephritis with tubular dilation and degeneration
in in kidneys of all animals
• Varying number of intratubular (proximale and distale tubules, Henle’s loop) greenbrownish
crystals in kidneys of all animals
• Multifocal necrotizing myocarditis in one animal

Test group 4 (2000 ppm)
• One of 5 animals died on study day 2
• Four of 5 animals were sacrificed moribund on study day 3
• All animals showed reduced nutritional condition
• One of 5 animals showed piloerection
• Food consumption was reduced by -79% on study day 3
• Water consumption was reduced by -42% on study day 3
• Body weight loss was observed on study day 3
• All animals showed enlarged and discolored kidneys
• One of 5 animals showed discoloration of urinary bladder contents
• Three of 5 animals showed a focus within the heart muscle
• Up to severe subacute multifocal pyelonephritis with tubular dilation and degeneration
in kidneys of all animals
• Varying number of intratubular (proximale and distale tubules, Henle’s loop) greenbrownish
crystals in kidneys of all animals
• Multifocal necrotizing myocarditis in 3 of 5 animals

Test group 3 (660 ppm)
• Slightly increased plasma levels of urea and creatinin (not statistically significant)
• A slight pyelonephritis was observed in kidneys of 2 of 5 animals (with intratubular crystals in 1 animal).

Test group 2 (200 ppm)
• No test substance-related findings were detected

Test group 1 (66 ppm)
• No test substance-related findings were detected

Dose descriptor:

NOEL

Effect level:

ca. 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: absence of kidney histopathology findings (including crystals) and changes in BUN and urea (7-day exposure)

Dose descriptor:

NOEL

Effect level:

200 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: BUN and Creatinine increased at 660 ppm; severe kidney toxicity at 2000 ppm and above (7-day exposure)

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

Table 1: Urea and Creatinine levels after 7 -day feed exposure to melamine cyanurate (mean of five rats)

dose	Urea	Urea	Creatinine	Creatinine
ppm	mM	SD	mM	SD
0	5.64	0.86	41.4	1.9
66	6.31	1.03	40.1	3.6
200	5.85	0.98	40.8	2.5
660	8.14	2.95	45.3	6.7
2000	nd		nd
4000	nd		nd
8000	nd		nd

No statistical significance observed based o Kruskal-Wallis test and two-sided Wilcoxon-test

Conclusions:

Melaminecyanurate causes severe kidney toxicity by formation of precipatates in the kidney upon application in the feed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Study duration:

subacute

Species:

pig

System:

other: kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Melamine cyanurate is a 1:1 complex of melamine and cyanurate, held togehter by hydrogen bonds. The substance is poorly soluble at neutral pH (2.7 mg/L in water), but soluble both under acidid or basic conditions (interference of hydrogen bonding, dissociation into melamine and cyanurate which are both of higher solubility in water).

Due to the poor solubility, concentration of the substance in the primary urine in kidney results in formation of melamine cyanurate precipitates in the kidneys. The crystals block the tubules and result in kidney damage and at high concentrations in renal failure. Melamine cyanurate crystal formation in kidneys has been shown for pigs, cats, monkeys, rats and mice and is plausible for humans.

Additional information

Melamine cyanurate causes kidney toxicity via precipitation in the form of poorly soluble melamine cyanurate crystals in the kidneys. Kidney toxicity is not or only mildly observed for either melamine or cyanurate alone. It is also not observed at dose levels which do not result in exceedence of the solubility limit in kidneys. Melamine cyanurate shows a NOEL of 200 ppm (ca 20 mg/kg bw) for crystal formation and a NOAEL of 66 mg/kg bw (660 ppm) for systemic toxicity in a 7-day feeding study (BASF, 2010) and a NOEL of 450 ppm in a 28 -day feeding study in rats (BASF 2016a) and a NOEL of 220 ppm and a NOAEL of 660 ppm in the reproductive screening study in rats (BASF 2017). In the latter study, females were treated for 51 days and males were treated for 28 days.

The difference between the NOEL and NOAEL is based on the increase of kidney weights without histopathological corrlate and of a tendency to slightly increased creatinine concentrations without statistical relevance.

Whereas a dose of 660 ppm given for up to 51 days only results in effects on kidney weights, a dose of 2000 ppm results in kidney-failure and mortality within 2 - 5 days. The dose of 450 ppm given for 28 days caused no effects.

The critical concentration resulting in precipitation in the kidney and blockage of tubules of rats is somewhere between a feed concentation of >660 and 2000 ppm.

The DNEL is derived from the NOEL of 450 ppm for subacute oral dosing.

Kidney toxicity observed in the feeding studies is consistent with the very limited information regarding industrial hygiene investigations with melamine cyanurate, including a 120 -day inhalation study in rats, that were published in several occupation hygiene journals in russian language between 1984 and 1986. A NOEC of 1.15 mg/m3 air for 120 -day inhalation by rats (probably 4h daily, probably whole body exposure, unknown particle size) was published. The amount of ingestion via grooming cannot be accounted for. Information is also available from co-exposure of melamine and cyanuric acid. For the combined 7 -day feed exposure (melamine and cyanuric acid), identifcal effects (crystal formation and kidney toxicity), albeit with a lower NOEL of 8.6 mg/kg bw was obtained (Gamboa da Costa, 2010). The lower NOAEL of the mixture is consistent with the lower uptake of the preformed complex observed in toxicokinetic investigations (Dominguez-Estevez et al 2010). A 28-day feeding study was performed with weanling pigs from local farms (Stine 2011) with doses of 0, 1 and 3.3 mg/kg bw each of melamine and cyanuric acid and eight pigs per dose group. Melamine and cyanuric acid were mixed with retail chocolate pudding in amounts tabulated from daily weight measurements to ensure accurate dosing. All pigs ate all pudding on the morning of each dosing day and thus received complete test doses. Doses were derived from a 7-day dose –range-finding study which showed that doses of each 33 mg/kg bw and higher resulted in an increase in BUN and creatinine. On day 29, 24 hours after the last dose, blood was collected from pigs to measure BUN and creatinine. Total urine was tested for glucose, bilirubin, ketones, specific gravity, blood, pH, proteins, urobilinogen, nitrite and leukocytes. Kidneys were excised whole and weighed. One kidney was cut in half sagitally and thin slices of medulla and cortex were taken for wet mount observation of typical melamine–cyanurate spherulite crystals. Gross necropsy included visual examination for any external or internal lesions. Pigs entered the experimental period at 19.2–28.0 kg and were 35.0–49.0 kg at necropsy. The percent body weight gain over the course of the study of all experimental groups was comparable to that of the controls. Also the absolute and relative weights of the kidneys were not affected by treatment. Urine pH ranged from 5.0 to 8.5 and was not associated with dose. Specific gravity values were normal. All other parameters (glucose, bilirubin, ketones, blood, protein, urobilinogen, nitrite and leukocytes) were either negative or within normal limits.One of eight pigs given 3.3 mg/kg bw/day of M+C had a small cluster of crystals with morphology typical of melamine–cyanurate crystals in the kidney wet mount. No crystals were found in the urine sediments of any pigs.The NOAEL for weanling pigs based upon formation of renal crystals after exposure for 28 days was (1+1 =) 2 mg/kg bw/day. Upon 14 -day gavage application, a NOEL of 2.4 mg/kg bw (1.2 mg/kg bw melamine plus 1.2 mg/kg bw cyanuric acid) was defined based on absence of crystals in kidneys and serum and urine biochemistry (Kobayashi 2010), the next higher concentration that caused crystal formation in kidney was 24 mg/kg bw. A 28 -day feeding study with a 1:1 combination of melamine and cyanuric acid resulted in a NOAEL of equal or less than 10 mg/kg bw (Gamboa da Costa 2011). Gavage dosing leads to more hazardous effects than continuous doing (Sprando 2013). Results from a 90 -day gavage study with co-dosing of melamine and cyanuric acid have been published in form of a poster presentation (Gamboa da Costa 2012). Cyanuric acid gavage was within 1 minutes after melamine gavage. Doses for each melamine and cyanuric acid were 0, 0.63, 1.25, 2.5, 5 or 10 mg/kg/bw. 0.1% aqueous carboxymethylcellulose was used as vehicle. Each 12 rats per dose group were treated for 90 days once daily. At study termination (or upon early removal), the animals were euthanized and a complete necropsy was performed. The kidneys underwent wet‐mount analysis for detection of melamine cyanurate crystals. The terminal blood underwent clinical chemistry analysis for blood urea nitrogen and serum creatinine. Blood urea nitrogen was significantly elevated in both males and females in the 20 (10M + 10C) mg/kg bw/day group and in males in the 10 (5M + 5C) 5 mg/kg bw/day group relative to the control group. Serum creatinine was significantly elevated in both male and female rats in the 20 ( 10M + 10C) mg/kg bw/day group relative to the control group. The lowest dose that evidenced the formation of melamine cyanurate spherulites was 2.5 (1.25M + 1.25C) mg/kg bw/day (14/48 rats), resulting in a NOAEL of 0.1.25 (0.63M + 0.63 C) mg/kg bw/day. Target organs other than kidney have not been identified for melamine and cyanurate in repeated dose oral studies (NTP 1983, MHLW 1997). Therefore, melamine cyanurate is not expected to have other adverse effects than those that are arise from crystal formation in the kidneys. Recent publications, considered as supporting information, assessed the effects of repeated oral exposure to a mixture of melamine and cyanuric acid (1:1) on kidneys (Reimschuessel et al., 2008; Kim et al., 2014; Lv et al., 2013b), ovaries (Sun et al., 2016), visceral organs (Yin et al., 2013a; Chang et al., 2015), testes (You et al., 2012; Yin et al., 2013b; Lv et al., 2013a) and spleen lymphocytes (Yin et al., 2014). The study results generally confirm the appropriateness of the NOEL of the key study for DNEL derivation. Of note, histopathological changes in the testes were reported for low dose levels (0.6 mg/kg bw/d melamine and cyanuric acid (1:1) according to You et al., 2012; 2 mg/kg bw/d melamine and cyanuric acid (1:1) [1mg melamine plus 1 mg cyanuric acid] according to Yin et al., 2013b and LV et al., 2013a), but due to methodological and reporting deficiencies a reliable assessment of a potential toxicological relevance of these findings is not possible (deficiencies in histopathological methodology which is critical for findings in testes, no information on incidences and or severity grades).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

Based on the kidney toxicity observed in the 7-day feeding study, classification for repeated dose target organ toxicity (kidney) is clearly indicated. Based on the results of the 28 -day feeding study (NOEL >= 450 ppm, 29 mg/kg bw for females and 23 g/kg bw for males), the criteria for STOT RE Cat 1 (serious effects upon subacute dosing of <30 mg/kg bw) are not met. Melamine cyanurate fulfills the criteria for STOT RE category 2 (target organ kidney). The data on inhalation toxicity given in the Russian publication is not reliable and suitable and therefore not taken into account for classification.