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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November 1994 to 15 April 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Melamine (mixture of Melamine from Agrolinz and BASF at a ratio of 1:1)
- Analytical purity: about 100 % (method: potentiographic titration; analytical report from November 1994)
- Purity test date: November 1994
- Lot/batch No.: "Musterpalette U24 vom 05.01.94" + Melamine AGROLINZ
- Stability under test conditions: proven by reanalysis (method: potentiographic titration and infra-red spectroscopy; analytical report from March, 1995)
- Storage condition of test material: room temperature

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations: 1500; 4500; 15000 ppm
Basis: nominal in diet
Remarks:
Doses / Concentrations: corresponding to ca. 136; 400; 1060 mg/kg bw/day
Basis: other: calculated
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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
See also the attached Tables.
Test group 3 (15000 ppm = about 1060 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 (15000 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 1500 and 4500 ppm . Thus a clear relation to dosing is missing. Furthermore, the highest mean number of live fetuses occurred at 15000 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 (1500 and 4500 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).

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
See above.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes
See also the attached Tables.
Details on maternal toxic effects:
Test group 3 (15000 ppm = about 1060 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 (15000 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 1500 and 4500 ppm . Thus a clear relation to dosing is missing. Furthermore, the highest mean number of live fetuses occurred at 15000 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 (1500 and 4500 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).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 400 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
urinalysis

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
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 .
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External examination of the fetuses:
There were two external malformations which occurred in 3 low dose fetuses (1500 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 (1500 ppm) and 3 high dose fetuses (15000 ppm) . These findings are considered to be spontaneous in nature, because they appeared without a clear dose-response relationship.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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 1500 ppm group, in 6 out of 149 (= 4 .0%) fetuses (in 5 out of 23 litters (= 22%)) of the 4500 ppm group and in 10 out of 158 (= 6 .3%) fetuses (in 10 out of 23 litters (= 43%)) of the 15000 ppm group. These malformations were related to the vertebral column (thoracic/lumbar vertebral body/bodies dumbbell-shaped (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 (15000 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 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.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Details on embryotoxic / teratogenic effects:
See the attached Tables.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 060 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
See above and the attached Tables.

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
yes

Applicant's summary and conclusion

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 1500 ; 4500 and 15000 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 (15000 ppm = about 1060 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.

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

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

Test group 1 (1500 ppm= 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.