Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental 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:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
lower number of litters per dose group
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
Melamine and cyanuric acid were purchased from Sigma-Aldrich (St. Louis, MO).
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Samtako, Osan, Korea
- Age at delivery: 10 weeks old
- Housing: Mating: two females with one male rat together overnight; mated females: individually in clear polycarbonate cages (stainless steel
wire lids)
- Diet: commercial rodent chow (Samtako), ad libitum
- Water: sterilized tap water, ad libitum
- Acclimation period: 1week

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3 °C
- Air changes: 13 to 18 changes per hour
- Humidity: 50 ± 10 %
- Photoperiod: 12 hours light / 12 hours dark
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
aqueous solution of 1% CMC
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Melamine (MEL) and cyanuric acid (CYA) (1:1) were dissolved in a solution of 1% CMC.and mixed into a single formulation.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1M/2F
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (GD 0)
Duration of treatment / exposure:
GD 6 -19
Frequency of treatment:
once daily
Dose / conc.:
6 mg/kg bw/day (nominal)
Remarks:
MEL and CYA (1:1) [3 mg/kg bw/d MEL, 3 mg/kg bw/d CYA]
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
MEL and CYA (1:1)
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
MEL and CYA (1:1)
No. of animals per sex per dose:
12 inseminated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a dose range-finding toxicity study.
Maternal examinations:
MORTALITY: Yes

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6, 9, 12, 15, 18, 20

FOOD CONSUMPTION: Yes
- Time schedule: GD 0, 6, 9, 12, 15, 18, 20

CLINICAL CHEMISTRY:
- On GD 20, blood was collected for determination of blood urea nitrogen (BUN) and creatinine.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 20 with macroscopic examination
- Selected organ weights were recorded: thymus, lung, heart, liver, spleen, kidneys, adrenal glands, gravid uterus, and ovaries
- Histopathological examination: left kidneys of all dams

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses
Fetal examinations:
All live fetuses were weighed, sexed and examined for morphological abnormalities.
- External examinations: Yes [all fetuses]
- Soft tissue examinations: Yes [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data

OTHER:
- Histopathological examination: left kidneys of half of the fetuses per litter
Statistics:
Units for statistical measurements: the pregnant female or the litter;
ANOVA - maternal body weight, food consumption, fetal body weight, placental weight, and serum biochemical values, Scheffe’s multiple comparison test when difference was significant
Kruskal–Wallis nonparametric ANOVA followed by the Mann–Whitney U test, where appropriate - number of corpora lutea, total implantations, live and dead fetuses, and gender ratio
Fisher’s exact probability test - clinical signs, gross findings, histopathological findings, and the proportions of litters with malformations and developmental variations
Details on results:
MORTALITY:
Three of 11 dams at 60 mg/kg bw/d MEL and CYA (1:1) died on GD 9.

CLINICAL OBSERVATIONS:
Treatment-related clinical signs were noted at 20 and 60 mg/kg bw/d MEL and CYA (1:1). These clinical signs included languor, decreased activity, piloerection, rough fur, nasal discharge, soft stool, dragging of hindlimbs, shivering, crouching position, lacrimation, hematuria and polyuria.

BODY WEIGHT:
Maternal body weights on GD 12-20 in the 60 mg/kg bw/d MEL and CYA (1:1) group were statistically significantly lower than those in the control group. Maternal body weight gain, corrected body weight and gravid uterine weight also decreased statistically significantly, as compared to control.

FOOD CONSUMPTION:
There were statistically significant reductions in food consumtion at 20 mg/kg bw/d (GD 9) and 60 mg/kg bw/d MEL and CYA (1:1) (GD 9-15).

CLINICAL CHEMISTRY:
Statistically significant increases in serum BUN and creatinine levels were observed at 60 mg/kg bw/d MEL and CYA (1:1), as compared to control.

ORGAN WEIGHTS:
Absolute and relative weights of thymus and absolute liver weights decreased statistically significantly, whereas absolute and relative weights of the kidneys and relative weights of the lung and heart increased significantly at 60 mg/kg bw/d MEL and CYA (1:1), as compared to control. A statisticaly significant increase in relative heart weight was observed at 20 mg/kg bw/d MEL and CYA (1:1), as compared to control. A statistically significant decrease in the relative and absolute weights of the liver was observed at 6 mg/kg bw/d MEL and CYA (1:1), as compared to control.

GROSS PATHOLOGY:
All dams at 60 mg/kg bw/d MEL and CYA (1:1) had enlarged kidneys and greenish colour changes of the kidneys. One case of enlargement and three cases of greenish colour change were also found at 20 mg/kg bw/d MEL and CYA (1:1).

HISTOPATHOLOGY:
Of the histopathological findings observed, the incidences of congestion, tubular necrosis/degeneration, exfoliation of tubular epithelium, crystals, casts, mineralization, inflammatory cells in tubules, tubular dilation and atrophy of glomeruli were significantly higher in the 60 mg/kg bw/d MEL and CYA (1:1) group than those in the control group. These findings were also observed at 20 mg/kg bw/d MEL and CYA (1:1), but the incidence and severity of the changes did not increase statistically significantly, as compared to control.
Details on maternal toxic effects:
EXAMINATIONS OF OVARIES AND UTERINE CONTENTS:
None of the treatment groups had completely resorbed litters. No treatment-related effects were observed for the number of corpora lutea, implantations, pre- and postimplantation loss rates, fetal deaths, litter size or gender ratios of the live fetuses in any of the treatment groups. However, placental weights in the 60 mg/kg bw/d MEL and CYA (1:1) group were significantly lower than those in the control group.
Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day (nominal)
Based on:
other: MEL and CYA (1:1)
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Remarks on result:
other: The NOAEL was derived by the study author
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
FETAL BODY WEIGHTS:
Fetal body weights in the 60 mg/kg bw/d MEL and CYA (1:1) group were statistically significantly lower than those in the control group.

EXTERNAL AND VISCERAL ALTERATIONS:
Two fetuses at 20 mg/kg bw/d MEL and CYA (1:1) displayed absent renal papilla. Although there were some types of visceral variations, including misshapen thymus, dilated renal pelvis, dilated ureter in treated fetuses, no statistically significant differences were observed in the number of fetuses with visceral variations or in the number of litters with affected fetuses between groups.

SKELETAL ALTERATIONS:
Fetuses with skeletal malformations were not observed in any groups. Although there were some types of skeletal variations, no statistically significant differences were detected in the number of fetuses with developmental variations or in the number of litters with affected fetuses between the groups. Some evidence of treatment-related reductions in the ossification of the fetal skeleton was found. The fetal ossification centers of the sternebra, and sacral and caudal vertebra at 60 mg/kg bw/d MEL and CYA (1:1) were statistically significantly lower than those in the control, but no statistically significant decreases in fetal ossification were observed at 6 and 20 mg/kg bw/d MEL and CYA (1:1).

HISTOPATHOLOGY:
No histopathological changes were observed in the fetal kidneys at any dose tested.
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
other: MEL and CYA (1:1)
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: delay in fetal ossification
Remarks on result:
other: The NOAEL was derived by the study author
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: Body weights and changes [g] in pregnant dams

Dose mg/kg bw (M/C) 0  3/3   10/10  30/30
No. of mated rats 12 12 12 12
No. of pregnant rats 12 12 12  7a
Gestational day 0 248.7 ±10.57b 248.7 ±10.30 248.3 ±10.08 249.9 ±9.78
Gestational day 6 278.7 ±12.15 278.7 ±11.11 277.2 ±12.25 285.9 ±12.69
Gestational day 9 293.3 ±18.28 293.7 ±14.20 284.0 ±19.64 276.5 ±23.57
Gestational day 12 313.7 ±16.98 311.3±15.76 297.6 ±26.25 261.1 ±32.48**
Gestational day 15 334.8 ±19.72 330.8 ±16.71 318.1 ±22.31 262.5 ±41.77**
Gestational day 18 372.9 ±23.46 367.3 ±20.35 358.6 ±21.35 295.5 ±46.54**
Gestational day 20 407.5 ±28.68 398.7 ±19.77 391.3 ±23.28 337.5 ±38.59**
Body weight gain during pregnancy 158.8 ±25.20 150.0 ±15.08 143.0 ±19.08 87.6 ±39.06**
Pretreatment period (GD 0–6) 30.0 ±6.70 30.0 ±6.75 28.8 ±7.37 36.0 ±6.68
Treatment period (GD 6–20) 128.9 ±22.95 120.0 ±14.32 114.1±18.93 51.6 ±39.02**
Corrected body weightc 326.5 ±20.64 317.1 ±16.15 314.4 ±22.23 279.6 ±33.53**
Gravid uterine weight 81.0 ±10.78 81.6±10.11 77.0 ±13.79 65.8 ±10.33*

aThree dams died on GD 9 due to severe intoxication; two other dams were not pregnant.

bValues are presented as means±SD (gm).

cBody weight on GD 20gravid uterine weight.

*Significant difference at p<0.05 level compared with the control group.

**Significant difference at p<0.01 level compared with the control group.

Table 2: Maternal kidney toxicity parameters

Dose mg/kg bw (M/C) 0  3/3   10/10  30/30
No. of pregnant rats 12 12 12 7
Blood urea nitrogen (mg/dl) 20.3 ±4.10a 18.1 ±1.98 19.9 ±4.29 48.4±10.11**
Creatinine (mg/dl) 0.3 ±0.05 0.3 ±0.04 0.3 ±0.04 0.7 ±0.15**

aValues are presented as means±SD.

**Significant difference atp<0.01 level compared with the control group

Table 3: External and visceral vlterations in the fetuses

0  3/3   10/10  30/30 normal range
External alterations
Fetuses examined 158 161 154 99
Litters examined 12 12 12 7
Fetuses with malformations 0 0 0 0 0.00–1.37
Litters affected 0 0 0 0 0.00–16.0
Visceral alterations
Fetuses examined 76 76 74 49
Litters examined 12 12 12 7
Malformations
Fetuses with malformations (%)b 0 0 2 (2.7) 0 0.00–0.66
Litters affected (%)c 0 0 1 (8.3) 0 0.00–4.55
Absent renal papilla 0 0 2 0 0.00–0.60
Variations
Fetuses with variations (%)b 13 (17.1) 26 (34.2) 18 (24.3) 14 (28.6) 1.40–20.2
Litters affected (%)c 7 (58.3) 12 (100) 8 (66.7) 7 (100) 0.00–60.9
Misshapen thymus 2 7 3 7 0.00–11.5
Dilated renal pelvis 0 0 1 0 0.00–7.74
Dilated ureter 6 14 14 10 0.00–12.0
Convoluted ureter 5 8 6 3
Executive summary:

In a developmental toxicity similar to OECD 414 (Kim et al., 2013), groups of pregnant female rats were treated by oral gavage with a 1:1 mixture of melamine (MEL) and cyanuric acid (CYA) in 1% aqueous CMC at 6, 20 or 60 mg/kg bw/d from GD 6 through GD 19 and necropsied on GD 20. The dose of 6 mg/kg bw/d MEL and CYA (1:1) corresponded to 3 mg/kg bw/d MEL plus 3 mg/kg bw/d CYA. Concurrent control animals were treated with the vehicle only. Maternal assessments included mortality, clinical observations, body weight, food consumption, clinical chemistry parameters (BUN, creatinine), gross pathology and examinations of ovaries and uterine contents. Fetal examinations included determination of body weight, sex and assessment of morphological abnormalities (external, visceral, skeletal). Histopathological examinations were conducted on the kidneys of the dams and their fetuses. At 20 mg/kg bw/d MEL and CYA (1:1) and above there were treatment-related clinical signs and histopathological changes in the kidneys including crystal deposition. Fetal kidney did not show any histopathological changes. At the maternal toxic dose of 60 mg/kg bw/d MEL and CYA (1:1), fetal body weight was statistically significantly reduced as compared to control, and there was delayed fetal ossification. Under the conditions of this study, the NOAEL for maternal toxicity was 6 mg/kg bw/d MEL and CYA (1:1) and the NOAEL for developmental effects and fetotoxicity was 20 mg/kg bw/d MEL and CYA (1:1). MEL and CYA (1:1) was not teratogenic.

Reason / purpose:
read-across source
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Limited detail given in publication
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
Sodium control groups received sodium hippurate at doses of 1,118 and 5,590 mg/kg/day.
Analytical verification of doses or concentrations:
not specified
Frequency of treatment:
Days 6-15 of gestation
Duration of test:
20 days
No. of animals per sex per dose:
no data
Control animals:
other: Sodium control groups received sodium hippurate at doses of 1,118 and 5,590 mg/kg/day.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no treatment-related effects on maternal appearance,
behavior and body weight gain in all groups treated with sodium
isocyanurate.
Dose descriptor:
NOAEL
Effect level:
>= 5 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No teratogenic effects were observed in all groups treated with sodium isocyanurate.
Dose descriptor:
NOAEL
Effect level:
>= 5 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
no effects observed
Developmental effects observed:
no
Reason / purpose:
read-across source
Reference
Endpoint:
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
Principles of method if other than guideline:
Treatment of pregnant rats between gestation days 10 and 20; fetal and uterine investigations (embryo- and fetotoxicity, but no skeletal examinations)
GLP compliance:
not specified
Limit test:
yes
Specific details on test material used for the study:
cyanuric acid (98% pure; Sigma–Aldrich, St. Louis, MO, USA)
Species:
rat
Strain:
other: CD IGS VAF/+
Details on test animals and environmental conditions:
8–10 weeks of age when purchased
single caging
temperatre 18–26 C
humidity 40–70%
light cycle 12/12h
food and water supply ad libitum
acclimatization period 3 days
Route of administration:
oral: gavage
Vehicle:
other: 1% aquaeous carboxymethylcellulose
Details on exposure:
Concentration in vehicle: 10%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the dosing solutions was confirmed by a contract laboratory (Eurofins, New Orleans, LA, USA) using LC–MS/MS.
Details on mating procedure:
animals purchased time-mated
Duration of treatment / exposure:
10 days
Frequency of treatment:
daily
Duration of test:
Gestation days 10 - 20
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Maternal examinations:
Fluid and feed consumption was measured every 3 and 7 days, respectively.

Clinical observations were made at least twice on the first day of dosing and at least once daily thereafter throughout the gestational period. Observations included, but were not limited to, changes in skin, fur, eyes, and mucous membranes. Respiratory, circulatory, autonomic, and central nervous systems were observed, in addition to somatomotor activity and behavior patterns. Attention was directed toward observations of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma. Animals taken off study early were considered equivocal to animals that died.
Ovaries and uterine content:
The number of corpora lutea, the number of implantation sites, and the number and position of resorption sites and fetuses (dead or alive) were noted. The gravid uterus was removed in toto and weighed.
Fetal examinations:
Each viable fetus was removed from the uterus and examined individually and records were kept as to its uterine
position, sex, weight, and crown-rump length, as well as any externally visible abnormalities. Runts, defined as an animal whose body weight is <70% of the average weight of the control litter averages by gender, were identified and their position noted.
Statistics:
included
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
four rats with renal crystals had slighly elevated levels of BUN, creatinine and increased renal weight.


Reproductive indces control 1000 mg/kg bw Cyanuric acid
No. corpea lutea (mean ± SEM) 14.8 ± 0.6 15.1 ± 0.5
No. implants (mean ± SEM) 13.4 ± 0.6 13.6 ± 0.5
Implant efficiency (mean% ± SEM) 90.4 ± 2.2 90.7 ± 3.0
No. fetuses alive (mean ± SEM) 12.5 ± 0.7 13.2 ± 0.6
Percent alive both genders (mean% ± SEM) 93.7 ± 2.4 96.7 ± 1.6
Percent live males (mean% ± SEM) 59 ± 3.5 59.3 ± 3.8
Percent live females (mean% ± SEM) 41 ± 3.5 40.7 ± 3.8
Early deaths (mean% of implants ± SEM) 3.4 ± 1.2 1.3 ± 0.9
Late deaths (mean% of implants ± SEM) 3.5 ± 1.6 2.0 ± 1.6
Early and late deaths (mean% of implants ± SEM) 3.5 ± 1.6 2.0 ± 1.6

Litter size (mean ± SEM) 12.5 ± 0.7 13.2 ± 0.6







6.9 ± 2.5 3.4 ± 1.6




Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:

control 1000 mg/kg cyanuric acid
Average fetal body weight (g ± SEM) 4.00 ± 0.08 4.06 ± 0.05
Runts (mean% ± SEM) 3.8 ± 3.0 0 ± 0
Average crown rump length (mean cm ± SEM) 4.09 ± 0.03 4.10 ± 0.02
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Developmental effects observed:
no

In this study, there was another separate group of pregnant rats treated with 1000 mg/kg bw of melamine. These results are not presented since 7 of the 13 animals were sacrificed in moribund condition and the criteria for a maximum tolerated dose are clearly exceeded.

Conclusions:
Cyanuric acid is does not cause visible malformations if rats are treated with high doses between gestation days 10 and 20.
Reason / purpose:
read-across source
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21 November 1994 to 15 April 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
87/302/EEC
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Analytical purity: about 100 %
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. THOMAE GmbH, Biberach an der Riss, FRG
- Age at study initiation:
- Weight at study initiation:
- Housing: Single caging in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, FRG (floor area about 800 square cm). The cages were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (ad libitum): ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by KLINGENTALMÜHLE AG, Kaiseraugst, Switzerland
- Water (ad libitum): drinking water of tap water quality from water bottles.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ° C.
- Humidity (%): 30 - 70 %, relative
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

IN-LIFE DATES: From: 21 November 1994 To: 15 December 1994
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): no data.
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by KLINGENTALMÜHLE AG, Kaiseraugst, Switzerland.
- Storage temperature of food: no data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out at the Analytical Department of BASF AG.
Analyses of the stability of MELAMINE in the diet up to 32 days at room temperature (and additional 4 days in which the mixture was suspended in water prior to analysis) were carried out before the start of this study . The homogeneity and the correctness of the concentrations of the test substance in the maintenance diet were analytically investigated at the beginning of this study .
Details on mating procedure:
- Mating procedure: cohoused.
- If cohoused:
- M/F ratio per cage: 1:2.
- Length of cohabitation: from 4.00 p.m. to about 7.30 a.m. on the following day.
- Further matings after two unsuccessful attempts: no data.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- Any other deviations from standard protocol: no data.
Duration of treatment / exposure:
during post coitum Days 6 - 16 (= 11 days)
Frequency of treatment:
continuous
Duration of test:
20 days
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent no treatment
Details on study design:
Sex: female
Duration of test: until Day 20 post coitum
- Dose selection rationale: The selection of doses for the present prenatal toxicity study was based on the overall results of 10 feeding studies (Melamine, "BUA-Stoffbericht 105", from June 1992) performed with non pregnant rats . The 40 dosages administered in these studies ranged from 100 to 30,000 ppm (6 .7 up to about 2,500 mg/kg body weight/day), the administration periods were between 14 days and two years .
The most relevant studies for dose selection for the present investigation were as follows :
In a 14-day feeding study Fischer-344 rats received doses of 5,000 ; 10,000 ; 20,000 and 30,000 ppm (417 - 2,500 mg/kg body weight/day) . All male and female rats receiving 15,000 ppm and more had mean body weight depressions when compared to the controls or even lost weight . Hard crystalline solids were found in the urinary bladder of most animals fed 10,000 ppm or more . The kidneys of two high dose males were pale and pitted . Apart from the urinary tract, no compound-related effects were observed in the other organs.
In a 4-week study with the same strain of rats and doses of 2,000 ; 4,000 ; 7,000 ; 13,000 ; 16,000 and 19,000 ppm (133 - 1267 mg/kg body weight/day), significant dose-related depression in body weight gain, elevated water intake and altered food pattern were observed . In addition, a dose-dependent incidence of urinary bladder calculi and urinary bladder hyperplasia occurred .
Taking this into consideration, the following doses were chosen with agreement of AGROLINZ Melamin, Agrarchemikalien GmbH, Linz, Austria, for the present full-scale prenatal toxicity study in Wistar rats with MELAMINE :
1,500 ppm: as the expected no observed adverse effect level
4,500 ppm : as the intermediate dose level
15,000 ppm: as the dose level at which some overt signs of maternal toxicity (e .g . impaired food consumption and body weight gains) were expected and adverse effects on the fetuses could not be ruled out.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day on working days or once a day (Saturday, Sunday or on public holidays) (Days 0 - 20 p.c.).
- Cage side observations checked in table [No.?] were included.

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c. The body weight change of the animals was calculated from these results.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20.
- Organs examined: no data.
- Organ weights:
- liver (absolute and relative weights)
- kidneys (absolute and relative weights)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead fetuses: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: Approx. half per litter
- Skeletal examinations: Yes: Approx. half per litter
- Head examinations: Yes: No
Statistics:
The DUNNETT-Test (Dunnett, 1955/1964) was used for a simultaneous comparison of several dose groups with the control . The hypothesis of equal means was tested . This test was performed two-sided and was used for the statistical evaluation of the following parameters :
Food consumption+, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the unopened uterus, weight of liver and kidneys, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses ; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter ; litter mean fetal body weight and litter mean placental weight .
FISHER's Exact Test (Siegel, - 1956) was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions . This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings .
The WILCOXON-Test (Nijenhuis and Wilf, 1978 ; 10 Hettmansperger, 1984) was used for a comparison of each dose group with the control for the hypothesis of equal medians . This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter . If the results of these tests were significant, labels (* for p < 0 .05, ** for p < 0 .01) were printed in the Summary Tables .

Note : For the parameter food consumption the "mean of means" was calculated and can be found in the relevant Summary Tables . The "mean of means" values allow a rough estimation of the total food consumption during the different time intervals (pretreatment, treatment and posttreatment period) ; they are not exactly precise values, because the size of the intervals taken for calculation differs . For the "mean of means" values no statistical analysis was performed .
Indices:
- Reproduction data of dams
- Sex distribution of fetuses
- Weight of placentae
- Weight of fetuses
- External malformations of the fetuses
- Soft tissue variations
- Malformations of the fetal skeletons
Historical control data:
Yes, but no access to the data in the report.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Test group 3 (15,000 ppm = about 1 , 060 mg/kg body weight/day) :
- statistically significantly decreased food consumption between days 6 and 16 p.c . (about 26 % less than the concurrent control group )
- statistically significantly reduced body weights from day 10 to day 17 p .c .
- statistically significant body weight loss between days 6-10 P .C . and significantly decreased body weight gain between days 10 and 15 p.c .
- statistically significantly lower carcass weight and decreased corrected body weight gain (about 53 % less than in the concurrent control group )
- hematuria in nearly all and indrawn flanks in 7 out of 25 animals between days 8 and 17 p .c .; piloerection in one female between days 8 and 15 p.c.
- Weight of placentae:
The mean placental weights in test group 3 (15,000 ppm) were statistically significantly decreased (about 7 % less weight than the control) . This is, however, not considered to represent a substance related effect, because the respective values are fully within the historical control range and the values from the concurrent control group are lower than the values at 1,50 0 and 4,500 ppm . Thus a clear relation to dosing is missing. Furthermore, the highest mean number of live fetuses occurred at 15,000 ppm ; this might be a possible explanation for the observed decrease in placental weights at this dose level . There were no statistically significant differences in the placental weights between the controls and test groups 1 and 2 (1, 5 00 and 4,500 ppm) .
- no test substance related effects on dams gestational parameters in test group 1 (1500 ppm = about 136 mg/kg b. w./day) and test group 2 (4500 ppm = about 400 mg/kg b. w./day).
Dose descriptor:
NOAEL
Effect level:
ca. 400 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Weight of fetuses:
The mean fetal body weights in test group 1, 2 and 3 (1,500 ; 4,500 and 15,000 ppm) were not influenced by the test substance administration and were similar to the control values .
External examination of the fetuses:
There were two external malformations which occurred in 3 low dose fetuses (1,500 ppm). For these fetuses anasarca (No . 2 of dam No . 28) and cleft palate (Nos. 9 and 15 of dam No . 47) were recorded . These malformations are considered random because they are not dose-related and can be also found at a low frequency in the historical control data). The external examination of the fetuses revealed no external variations in any group . So-called unclassified observations (e .g . placenta(e) fused/necrobiotic, amniotic fluid increased, extended abdomen and protruding tongue) were recorded for one control fetus, 7 low dose fetuses (1,500 ppm) and 3 high dose fetuses (15,000 ppm) . These findings are considered to be spontaneous in nature, because they appeared without a clear dose-response relationship .
Tissue examination of the fetuses:
The examination of the organs of the fetuses revealed several malformations in only one low dose litter. In 4 out of a total of 16* fetuses (Nos . 3, 4, 11 and 15) of dam No . 47 cardiomegaly and hyperplasia of liver and/or kidney(s) occurred . Because these soft tissue malformations were only found in some fetuses of one low dose litter and no dose-response relationship exists, these findings are assessed to be spontaneous in nature and not related to the test substance administration .
Soft tissue variations (dilated renal pelvis and/or hydroureter) were detected in all groups including the controls . Both soft tissue variations occurred
without any statistically significant and biologically relevant differences between the groups . Both findings are very common ones in the rat strain used and all respective values are fully in the range of biological variation . No so-called unclassified observations (like bloody imbibition of kidney(s)) were recorded during the soft tissue examination.
* 9 of these fetuses were for soft tissue and 7 for skeletal examinations.
Skeletal examination of the fetuses:
Malformations of the fetal skeletons were seen in 6 out of 153 (= 3 .9%) fetuses (in 5 out of 23 litters (= 22%)) of the control, in 8 out of 148 (= 5 .4%)
fetuses (in 7 out of 24 litters (= 29%)) of the 1,500 ppm group, in 6 out of 149 (= 4 .0%) fetuses (in 5 out of 23 litters (= 22%)) of the 4,500 ppm group and in 10 out of 158 (= 6 .3%) fetuses (in 10 out of 23 litters (= 43%)) of the 15,000 ppm group . These malformations were related to the vertebral column (thoracic/lumbar vertebral body/bodies dumbbellshaped (asymmetrical) or bipartite (asymmetrical); lumbar vertebral arches absent ; different lumbar vertebral bodies and/or arches severely malformed ; sacral vertebrae fused and/or of irregular shape) and the sternum (sternebra(e) bipartit, ossification centers dislocated) .
If compared to the concurrent control group or the historical control values it becomes obvious, that all skeletal malformations except the finding "sternebra(e) bipartite, ossification centers dislocated" occurred without a clear relation to dosing and/or are within the biological range of variation . The statistically significantly increased rate of high dose fetuses (15,000 ppm) concerning bipartite sternebra(e) with dislocated ossification centers has
to be discussed in more detail, because the relevant values for fetal and litter incidence for this finding are outside the historical control range.

If the overall rate of fetuses/litter with skeletal malformations, however, is taken into account, it can be seen, that no dose-response relationship exists and that the values are fully within the historical control range . Therefore, the increased rate of high to dose fetuses showing bipartite sternebra(e) with dislocated ossification centers - as the only statistical significant deviation concerning fetal skeletal malformations - is considered coincidental and not biologically significant. The skeletal variations elicited were related to the ribs (shortened or absent 13th, accessory 14th or rudimentary cervical rib(s)), the sternum (accessory sternebra, sternebra(e) of irregular shape or bipartite) or the skull (epactal bone between parietal and interparietal bones or between nasal and frontal bones) and were found in all groups without any relation to dosing . The differences observed in comparison to the control fetuses were without statistical significance and/or are fully in the historical control range.
In all groups signs of skeletal retardations occurred substantiated by incomplete or missing ossification of skull bones, vertebral column, pelvic girdle, sternebra(e) and metatarsal bones . The observable differences between the groups concerning fetal skeletal retardations are without any biological relevance, because no relation to dosing is given and because the respective values are fully in the range of the historical control values.
Dose descriptor:
NOAEL
Effect level:
ca. 1 060 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
There were no substance-related findings on the gestational parameters and no signs of developmental toxicity up to and including the highest dose level (15 000 ppm). Especially no indications of teratogenicity were found.
The no observed adverse effect level (NOAEL) for the dams is 4 500 ppm (about 400 mg/kg body weight/day), but 15 000 ppm (about 1 060 mg/kg body weight/day) for the fetal organism.
Executive summary:

Melamine was tested for its prenatal toxicity in Wistar rats. The test substance was administered as a constant homogeneous addition to the food to 23 – 24 pregnant female Wistar rats/group at concentrations of 1,500 ; 4,500 and 15,000 ppm on day 6 through day 16 post coitum (p.c.). The control group, consisting of 23 dams, was dosed with the food only. Food consumption and body weights of the animals were recorded regularly throughout the study period.The state of health of the animals was checked each day. On day 20 post coitum, all females were sacrificed and assessed by gross pathology (including weight determination of the terminal body weight, the unopened uterus, the liver and the kidneys). The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and/or skeletal findings. The following findings were obtained and assessed as substance-related: 

Test group 3 (15000ppm = about 1060 mg/kgbody weight/day):

-statistically significantly decreased food consumption between days 6 and 16 p.c. (about 26% less than the concurrent control group )

-statistically significantly reduced body weights from day 10 to day 17 p .c .

-statistically significant body weight loss between days 6-10 p.c. and significantly decreased body weight gain between days 10 and 15 p.c.

-statistically significantly lower carcass weight and decreased corrected body weight gain (about 53 % less than in the concurrent control group )

-hematuria in nearly all and indrawn flanks in 7 out of 25 animals between days 8 and 17 p.c.; piloerection in one female between days 8 and 15 p.c.

Test group 2 (4500ppm = about 400 mg/kg body weight/day):

-no substance-related effects on dams, gestational parameters or fetuses.

Test group 1 (1500ppm= about 136 mg/kg body weight/day) :

-no substance-related effects on dams, gestational parameters or fetuses.

 

Thus, under the conditions of this study, the administration of MELAMINE to pregnant female Wistar rats during organogenesis elicited signs of maternal toxicity at 15000 ppm, but induced no substance related effects in the dams at 1500 or 4500 ppm. Maternal toxicity was substantiated in this full scale prenatal toxicity study by reduced food consumption, impairments in body weight / body weight gain, decreased corrected body weight gain and clinical symptoms like hematuria, indrawn flanks and piloerection at 15000 ppm (about 1060 mg/kg body weight/day) .

Nearly all signs of maternal toxicity proved to be fully reversible after cessation of the test substance administration. The carcass weight and the corrected body weight gain, however, showed still some impairments at terminal sacrifice .

There were no substance-related findings on the gestational parameters and no signs of developmental toxicity up to and including the highest dose level (15000ppm). Especially no indications of teratogenicity were found.

Based on the results of this full-scale prenatal toxicity study in Wistar rats, the no observed adverse effect level (NOAEL) for the dams is 4500 ppm(about 400 mg/kg body weight/day), but 15000 ppm (about 1060 mg/kg body weight/day) for the fetal organism.

Justification for using the oral route:

The chosen oral route in this study is considered to be appropriate, even if it is not the most likely route of human exposure. Justifications are: There is apparently no metabolism of melamine in the organism and by this no first pass effect. A rapid and near to complete absorption was found after the oral route in rats. The same can be assumed for the inhalation route, whereas a low dermal absorption was estimated for melamine. Effects after oral exposure can stand therefore for effects after inhalation exposure and are on the worst case side of effects after dermal exposure.

It is therefore justified to estimate the possible toxic effects after inhalation or dermal exposure, based on the outcome of the oral experiments.

Data source

Materials and methods

Test material

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

Test animals

Species:
rat

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: kidney toxicity
Remarks on result:
other: mortality of some dams at 60 mg/kg bw

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Value taken from co-exposure melamine and cyanuric acid.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Melamine cyanurate is predicted to be non teratogenic based on experimental data on the 1:1 mixture of melamine and cyanuric acid as well as on experimental data on melamine and cyanuric acid/sodium cyanurate..
Melamine cyanurate shows significant general toxicity related to kidney failure after accumulation of melamine cyanurate crystals in the kidney.