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EC number: 253-575-7
CAS number: 37640-57-6
Dose level (mg/kg bw/day)
Females on study
- Females that aborted or delivered
- Females that died
- Females that were euthanized
Females examined at scheduled necropsy
Mean number of corpora lutea
Mean number of implantation sites
(total number and % per litter)
Dams with stillbirths, resorptions only and/or dead fetuses
Pre-implantation loss (number and %)
Post-implantation loss (number and % per litter)
Mean body weight on day 29 pc (g)
Mean body weight gain day 6-29 pc (%)
Gravid uterine weight (g)
Bodyweight corrected for gravid uterine weight (g)
Mean live offspring (number and %)
Mean fetal body weight (males)
Mean fetal body weight (females)
Mean fetal body weight (sexes combined)
Malformations (including runts) (number and % per litter)
Variations (% per litter)
**: significantly different from the control group at 0.01
For description and incidences of malformations and main variations see attached background material.
SUMMARY DOSE RANGE FINDER
A dose range finder was conducted to select dose levels for the Prenatal Developmental Toxicity Study. No guidelines were applicable as this study was intended for dose level selection purposes only.
If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. Dosing of the DRF was initiated on 28 Aug 2018. The in-life phase of the DRF was completed on 20 Sep 2018.
Preparations were visually inspected for homogeneity prior to use and all preparations were used within 6 hours after preparation of the formulation.
On 23 Aug 2018, time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 17-19 weeks old at mating/arrival and weighed between 3092 and 4405 g at the initiation of dosing. The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days prior to the commencement of dosing. The actual daily mean temperature during the study period was 21°C with an actual daily mean relative humidity of 49 to 96%.
0 mg/kg bw/day
120 mg/kg bw/day
250 mg/kg bw/day
400 mg/kg bw/day
The test item and vehicle were administered to the appropriate animals (6 females/group) by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive. The dose volume (5 mL/kg bw) for each animal was based on the most recent body weight measurement (Deviation: The dose volume for one animal in the 250 mg/kg bw/day and the 400 mg/kg bw/day group was not based on the most recent body weight measurement. As a result, on Days 16 and 17 post-coitum, one animal recieved received 2.2% less volume (245 mg/kg bw instead of 250 mg/kg bw) and on Day 20 post-coitum, one animal received 1.4% less volume (395 mg/kg bw instead of 400 mg/kg bw). Evaluation: Both deviations in dose volume were slight and incidental and were, therefore, considered not to have an impact on the evaluation of the study results. In addition, the animal in the 250 mg/kg bw/day group, although receiving a slightly less dose for 2 consecutive days, was sacrificed in extremis in Day 22 post-coitum due to clear signs of toxicity.)
Laboratory Evaluations (Clinical Pathology)
Blood of F0-animals (except for animals which were sacrificed in extremis) was collected from the ear artery using vacutainers on the day of scheduled necropsy. Samples were analyzed for hematology, coagulation and clinical chemistry parameters.
Organ weights – F0-Generation
Liver, spleen and adrenal gland (paierd examination) were collected and weighed. No histopathological examination was performed.
Terminal Procedures – F1-Generation
There was no examination of uterine content; number of implantations sites, corpora lutea, distribution of life and dead fetuses and/or embryo-fetal deaths was not recorded. Fetuses and/or implantation sites in development, fetuses of animals sacrificed before planned necropsy were not examined. Only the number of fetuses was counted for registration of laboratory animal welfare reasons. The number of fetuses is not reported.
Mortality was observed at dose levels of 250 and 400 mg/kg bw/day. One female each at 250 and 400 mg/kg bw/day were sacrificed in extremis for animal welfare reasons on Day 22 and 16 post-coitum, respectively. They presented with persistent body weight loss from start of treatment (7-10%) together with absent/severely reduced food consumption from Day 9 postcoitum onwards. Clinical signs observed in these preterm sacrificed animals were: hunched posture and/or piloerection, yellow particles in the urine, and up to severely reduced faeces production. Red fluid on the manure tray (slight) was additionally observed in the preterm sacrificed female at 250 mg/kg/day on Day 21 post-coitum. No macroscopic findings were observed at necropsy for these 2 animals.
From Day 14 post-coitum onwards, yellow particles in the urine10 were observed in 2/5 and 4/5 surviving animals at 250 and 400 mg/kg/day, respectively, and red fluid on the manure tray (moderate) was observed in one animal at 120 mg/kg bw/day at the end of the dosing period.
On average, treatment at 250 and 400 mg/kg bw/day resulted in absent/very low body weight gain from start of treatment until Day 15 post-coitum. Afterwards, mean body weight gain started to partially recover with a recurrent absent mean body weight gain at the end of the treatment period (more severely at 400 mg/kg bw/day), resulting in 5% lower mean body weight vs control on Day 29 post-coitum in both groups.
At 250 and 400 mg/kg bw/day, mean food consumption (before and after allowance for body weight) was increasingly reduced vs control from start of treatment until Day 15 post-coitum, resulting in up to 43% lower mean relative food intake vs control in both groups. Afterwards, mean food consumption partially recovered, with a recurrent drop at 400 mg/kg bw/day and a complete recovery at 250 mg/kg bw/day at the end of the treatment period.
No relevant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. clinical chemistry, haematology and coagulation, macroscopic examination, and organ weights).
Due to the mortality observed at 250 and 400 mg/kg bw/day, dose levels of 0, 15, 50 and 150 mg/kg bw/day were selected for the main study (high-dose level slightly above the half-lethal dose).
See also attached background material.
RESULTS ANALYTICAL VERIFICATION OF FORMULATIONS
It was demonstrated that the analytical method was adequate for the determination of the test item in the study samples. The mean accuracies of the QC samples were within the criterion range of 85-115% (mean accuracy of the 1 mg/g and 50 mg/g QC samples was 94% and 88%, respectively (n=2).
In the 0 mg/kg formulation, no test item was detected.
The concentrations analyzed in the formulations of the 15 mg/kg bw/day, 50 mg/kg bw/day and 150 mg/kg bw/day groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%; accuracy was 91% (n=6), 96% (n=2) and 96% (n=6) for the 15, 50 and 150 mg/kg bw/day groups, respectively).
The formulations of the 50 mg/kg bw/day and 150 mg/kg bw/day groups were homogeneous (i.e. coefficient of variation ≤ 10%; Coefficent of variation were 2.4% and 1.6%, respectively (n=6)).
Analysis of the 50 mg/kg bw/day group and 150 mg/kg bw/day group formulations after storage yielded a relative difference of ≤ 10% (0.6% and -3.4%, respectively (n=2)). The formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.
A prenatal developmental toxicity study was performed in rabbits
according to OECD/EC guidelines and in accorance with GLP principles.
Time-mated New Zealand White rabbits were exposed orally by gavage to
melamine from Day 6 to 28 post-coitum, inclusive. The dose levels were
selected to be 0, 15, 50, 150 mg/kg bw/day, based on the results of the
dose range finder, in which mortality occurred at 250 mg/kg bw/day.
The following parameters and end points were evaluated in this study for
the F0-generation: mortality/moribundity, clinical signs, body weights,
food consumption, gross necropsy findings, organ weights (kidneys),
number of corpora lutea, uterus weight and uterine contents. In
addition, the following parameters were determined for the
F1-generation: the number of live and dead fetuses, early and late
resorptions, total implantations, fetal body weights, sex ratio, and
external, visceral and skeletal malformations and developmental
Formulation analyses confirmed that formulations of test item in 1%
Aqueous carboxymethyl cellulose were prepared accurately and
homogenously and were stable over at least 6 hours at room temperature
and under normal laboratory light conditions.
No mortality occurred during the study period that was considered to be
related to treatment with the test item.
In total, there were 2 preterm decedents over the study period: One
animal at 150 mg/kg bw/day died after dosing on Day 10 post-coitum
likely related to an oral gavage incident. One animal at 50 mg/kg bw/day
was sacrificed for animal welfare reasons on Day 18 post-coitum as it
was noted with severe clinical signs, body weight loss (14% vs start of
dosing) and absent food consumption over 2 consecutive periods. This
female sacrificed in extremis was considered unrelated to treatment as
it occurred only in a single mid dose animal and no dose-related
response was observed. In addition, one control animal had an early
delivery on Day 28 post-coitum that was considered unrelated to
treatment with the test item as occurred only in a single female of the
At 150 mg/kg bw/day, a treatment-related decrease in food consumption
(before and after allowance for body weight) was observed at the
beginning of the treatment period (over Days 6-12 post-coitum), followed
by a recovery afterwards. This decrease in mean food consumption
resulted in differences up to 19% and 17% vs control in absolute and
relative food intake, respectively, over Days 6-12 post-coitum, and
resulted in a slight decrease in body weight gain (2% vs control) on Day
12 post-coitum. As mean values remained within the historical control
range, effects on body weight gain were marginal, and complete recovery
was observed from Day 12-15 post-coitum onwards, these findings were
considered non-adverse. Reduced faeces production was also noted with a
slightly higher persistence and severity at 150 mg/kg bw/day that was
regarded as non-adverse as it did not noticeably affect the general
health status of the animals.
No treatment-related changes were noted in any of the remaining maternal
parameters investigated in this study (i.e. macroscopic examination and
The apparent dose-related higher fetal body weights observed in this
study were not considered toxicologically relevant as they could be
attributed to the lower mean fetal weight in the concurrent control
group (below the 5th percentile of the historical control data), and as
mean fetal body weight values in all treated groups were well within the
historical control range. The lower fetal weights observed in the
control group could be partly explained by the slightly larger litter
size observed in this group and were in line with the slightly higher
litter incidence of ossification-related parameters (i.e. sternebra(e) 5
and/or 6 unossified, unossified metacarpals and/or metatarsals and
unossified tarsals) observed in the concurrent controls.
No treatment-related changes were noted in any of the other
developmental parameters investigated in this study (i.e. litter size,
post-implantation loss, sex ratio, external, visceral and skeletal
malformations and developmental variations).
In conclusion, based on the results in this prenatal developmental
toxicity study a maternal and developmental No Observed Adverse Effect
Level (NOAEL) for Melamine of at least 150 mg/kg bw/day was established.
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