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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:
23 Aug 2018 - 13 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Melamine
EC Number:
203-615-4
EC Name:
Melamine
Cas Number:
108-78-1
Molecular formula:
C3H6N6
IUPAC Name:
1,3,5-triazine-2,4,6-triamine
Test material form:
solid: particulate/powder

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 17-19 weeks
- Weight at study initiation: 2903 - 4174 g at the initiation of dosing
- Fasting period before study: F0-females were not fasted.
- Housing: On arrival and following randomization females were housed individually in cages with perforated floors (dimensions 67 x 62 x 55 cm). For psychological/environmental enrichment, animals were provided with shelters and wooden sticks.
- Diet: Pelleted diet for rabbits (Global Diet 2030 from Envigo Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period.
- Water: municapl tap water, ad libitum.
Feed and water are considered to contain no known contaminants that would interfere with the objectives of the study
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 2 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): actual 20-21 (target 18 - 24)
- Humidity (%): actual 56-71 (target 40 - 70)
- Air changes (per hr): ten or greater
- Photoperiod (hrs dark / hrs light): 12/12

Deviation: Temporary deviations from the maximum level of target humidity occurred. Evaluation: This study plan deviation was considered not to have affected the integrity of the study because it did not noticeably affect the clinical condition of the animals or the outcome of the study.

IN-LIFE DATES: From: 14 Oct 2018 To: 09 Nov 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 3, 10 and 30 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.
On 14 Oct 2018, formulations were considered visually homogeneous and, therefore, appropriate for dosing. On 15 Oct 2018 mortar was used, and from 16 Oct 2018 onwards blending was added to optimize the formulation process and improve the appearance of the formulations (as few small clumps were observed in Group 3 and 4 formulations).
Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy, homogeneity and stability were determined for formulations prepared for use during treatment.
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 10 or 25 mL. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature under normal laboratory light conditions for 6 hours.
The volumetric flasks were filled up to the mark with 50/50 (v/v) tetrahydrofuran/water and ultrasonicated for 20 minutes, shaken and ultrasonicated for another 10 minutes. The solutions were further diluted with 50/50 (v/v) tetrahydrofuran/water to obtain concentrations within the calibration range. Samples were analyzed using UPLC TUV based on the analytical method validated for the test item in vehicle (for analytical conditions see background information).
For results see 'any other information on results'
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
6-28 days pc
Frequency of treatment:
once daily
Duration of test:
6-29 days pc. Scheduled necropsy were conducted day 29 pc or within 24 h of early delivery.
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
group 2
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
group 3
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
group 4
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of the dose range finder (see Any Other Information on Results), and in consultation with the Sponsor. The dose level of 150 mg/kg bw/day was selected as high dose, since mortality occurred at 250 mg/kg bw/day in the dose-range finding study.

- Rationale for animal assignment: On the day of receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights. Each set of females mated on the same date (i.e. 4 sets) was distributed as evenly as possible over the dose groups with body weights within ± 25% of the mean for each set of animals.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 6 post-coitum and lasting up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum

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, food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 pc
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and female with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

The kidneys were weighed at necropsy for all scheduled euthanasia F0 animals. Organ weights were not recorded for animals found dead or euthanized in extremis and for the female that delivered early. Paired organs were weighed together. Organ to body weight ratio (using the body weight on Day 29 post-coitum) were calculated.
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter were placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. The remaining half per litter were examined by a mid-coronal slice.
Deviation: The head of one control fetus was not examinated by mid-coronal slice. Evaluation: As it was only one control fetus, sufficient data were available for a proper
evaluation of the results. Therefore, this study plan deviation was considered not to have affected the integrity of the study.

For recognizable fetuses or normal implantations in development of females found dead, sacrificed before planned necropsy or that delivered before the day of scheduled necropsy, no examination was performed. Fetuses were counted only.
Deviation: Recognizable fetuses of one control female that delivered early were externally examined. Evaluation: Additional data were obtained and, therefore, this deviation had no impact on the integrity of the study.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, corrected terminal maternal body weight, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Maternal Variables
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.
Corrected terminal maternal body weight:Terminal body weight (on Day 29 post-coitum) minus gravid uterus weight.
Corrected Body Weight Gains: Terminal body weight (on Day 29 post-coitum) minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 29 post-coitum.

Reproduction and Developmental Variables
For each group, the following calculations were performed:
Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites)/number of corpora lutea) x 100
Post-implantation loss (%): ((number of implantation sites - number of live fetuses))/number of implantation sites) x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter)/(number of viable fetuses/litter)) x 100
Historical control data:
See background information.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced faeces production was noted in several animals across all the groups, including controls, with a slightly higher severity and persistence at 150 mg/kg bw/day. Among surviving animals presenting with reduced faeces production, moderate up to severe reduction was observed during several days in 16 out of 18 high dose animals (89%) vs 9 out of 15 control animals (60%). In addition, in 12 out of 18 high dose animals (67%) the reduction in faeces production lasted more than 7 days over the treatment period vs 8 out of 15 animals (53%) in the control group.
The reduced faeces production was regarded as non-adverse as it did not noticeably affect the general health status of the animals.

No other treatment-related clinical signs were noted during the observation period.

One animal at 150 mg/kg bw/day was observed with dark urine (slight) on 2 consecutive days at the end of the pregnancy period (Days 27-28 post-coitum). At the incidence observed (only in a single animal), it was considered to be a chance finding.

After 1-3 days of treatment, 2 animals at 50 mg/kg/day were noted with transient restless behaviour (slight) over Days 7-8 and 9-23 post-coitum, respectively. In addition, one of these animals was observed with an aggressive behaviour (slight) over Days 11-25 post-coitum. At the incidence observed (only in 2 mid dose animals) and in the absence of a dose-related response, these observations were considered to be unrelated to treatment.

One animal at 15 mg/kg bw/day was noted with transient swelling of the vagina (up to moderate) on 4 consecutive days (over Days 15-18 post-coitum). At the incidence observed (only in one low dose animal) and in the absence of a dose-related response, this observation was considered to be unrelated to treatment.

Further incidental findings that were noted included scabs, scars, wounds, transient diarrhoea in one control animal, and alopecia. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In total, there were 2 preterm decedents over the study period: one animal at 50 mg/kg bw/day and another animal at 150 mg/kg bw/day.

At 150 mg/kg bw/day, one animal died after dosing (on Day 10 post-coitum). After dosing, blood was observed in the catheter and she was noted moribund, slightly restless and with breathing problems (gasping). No clinical signs were recorded for this animal the previous days, and normal body weight and food consumption were observed over Days 6-9 post-coitum. No macroscopic findings were noted at necropsy and normal thoracic fluid volume was determined. She presented with gravid uterus (11 normal implantations in development). Based on the observations recorded immediately after dosing, and as any signs of toxicity were observed for this animal the previous days, this spontaneous death was likely related to an oral gavage incident.

One animal at 50 mg/kg bw/day had to be sacrificed in extremis for animal welfare reasons on Day 18 post-coitum as she was noted with severe body weight loss from Day 15 post-coitum onwards (14% on Day 18 post-coitum vs start of treatment) together with absent food consumption over 2 consecutive periods (Days 12-18 post-coitum). Moreover, she presented with severely reduced faeces production for 4 consecutive days, piloerection and lean appearance for 3 consecutive days, and pale skin (slight) on Day 18 post-coitum. No macroscopic findings were observed at necropsy and she presented with gravid uterus (10 normal implantations in development). The single mid dose female sacrificed in extremis was considered unrelated to treatment as no dose-related response was observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At 150 mg/kg bw/day, mean body weight gain was slightly reduced (not significant) when compared with concurrent control mean at the beginning of the treatment period. This slight decrease in body weight gain was partly attributed to three individual females that presented with body weight loss (up to 4%) on Days 9-12 post-coitum and resulted in a slightly lower mean body weight (2%) vs control over that period. Mean values were within the historical control range. From Day 15-18 post-coitum until the end of the treatment period, mean body weight and body weight gain remained in the same range as controls.

No toxicologically relevant changes in mean body weight and body weight gain were noted at 15 and 50 mg/kg bw/day.

No toxicologically relevant changes were observed in body weight gain corrected for gravid uterus by treatment up to 150 mg/kg bw/day. Mean values in all groups remained well within the range of available historical control data. Mean body weight on Day 29 post-coitum corrected for gravid uterus in the treated groups remained in the same range as controls (no statistical analysis was performed).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg bw/day, a decrease in mean food consumption when compared with controls was observed during the early phase of the treatment period (over Days 6-12 post-coitum), reaching statistical significance for absolute and relative values on Days 6-9 post-coitum.
Decreases up to 19% and 17% vs control in absolute and relative food intake, respectively, were noted in the high dose group during that period, followed by a recovery afterwards. Mean relative values remained within the range of available historical control data.
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.

No toxicologically relevant changes in mean food consumption (before or after allowance for body weight) were noted at 15 and 50 mg/kg bw/day.

There was a slightly higher absolute and relative food consumption in all treated groups when compared with control values during the end of the treatment period (over Days 24-29 postcoitum). A dose-related response could not be stablished and statistical significance was only reached for absolute mean value at the low dose level over Days 27-29 post-coitum. This higher food intake over that period (up to 29% in relative values) could be explained by the slightly lower mean food intake observed in the concurrent control group when compared to historical control mean, that was mainly attributed to six control females that presented with low food intake values over Days 24-27 and/or 27-29 post-coitum.

Mean food consumption (before and after allowance for body weight) over the entire treatment period was similar in all groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

One animal at 50 mg/kg bw/day presented with a misshapen placenta. This observation in a mid dose female was considered a chance finding and not related to treatment with the test item as it concerned only one female and it was not noted at 150 mg/kg bw/day.
Black discolouration of the caecum contents was observed in 2 control animals also presenting with very low relative food consumption over the last 5-8 days of the treatment period.
These and other findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rabbits of this age and strain.
Other effects:
no effects observed
Description (incidence and severity):
Kidney weights (absolute and relative to body weight) of treated animals were unaffected by treatment up to 150 mg/kg bw/day.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Litter incidence of post-implantation loss was considered to be unaffected by treatment up to 150 mg/kg bw/day. All mean values were within the range of available historical control data.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 15 mg/kg bw/day was noted with 1 dead fetus (and 8 viable fetuses). At the incidence observed (only one low dose fetus), in the absence of a dose-related response, and as dead fetuses can occasionally be observed in control animals at a similar incidence, this finding was considered to be unrelated to treatment.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
One control animal had an early delivery on Day 28 post-coitum. This animal presented with reduced faeces production (up to severe) from Day 15 post-coitum onwards and red fluid (slight) on the manure tray was observed on Day 28 post-coitum. She presented with a body weight loss of 4% over Days 21-27 post-coitum together with reduced food consumption (up to severe) over Days 12-18 and 21-27 post-coitum. No macroscopic findings were observed for this female at necropsy and she presented with a litter of 13 pups (12 outside and one inside the uterus), consisting of 4 alive and 9 dead fetuses. This early delivery was considered a chance finding and not related to treatment with the test item as occurred only in a single female of the control group.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
In total, 9 females among the surviving animals were not pregnant: 4 control females, one female at 15 mg/kg bw/day, one female at 50 mg/kg bw/day and 3 females at 150 mg/kg bw/day. The incidence of nonpregnancy was considered to be unrelated to treatment with test item as no dose-response was observed. All the other surviving females were pregnant and had litters with viable fetuses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: No adverse effects observed up to and including the highest dose level tested.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in fetal body weight were observed by treatment up to 150 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 36.3, 38.4, 39.4 and 41.2 gram for the control, 15, 50 and 150 mg/kg bw/day groups, respectively.
The apparent dose-related higher fetal body weight observed was not considered toxicologically relevant as could be explained by the lower mean fetal weight in the concurrent control group that was 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 (males: mean = 40.4, P5-P95=36.9-46.8 gram; females: mean = 39.8, P5-P95=36.3-45.8 gram; combined: mean = 40.2, P5-P95=36.8-46.4 gram).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 150 mg/kg bw/day.
Mean sex ratios (males:females) were 46:54, 45:55, 42:58 and 45:55 for the control, 15, 50 and 150 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 9.9, 9.5, 9.4 and 8.9 fetuses/litter for the control, 15, 50 and 150 mg/kg bw/day groups, respectively. All mean values (absolute and % per litter) were well within the historical control range.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 150 mg/kg bw/day.

Two external malformations were observed in this study at scheduled necropsy. Local edema in the neck region was observed in one fetus at 50 mg/kg bw/day and one control fetus
presented with carpal flexure for which no skeletal origin was found. Due to the single occurrence and/or occurrence in a control fetus, these malformations were considered to be chance findings.

External variations were not observed in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on skeletal morphology following treatment up to 150 mg/kg bw/day.

Only two skeletal malformations were observed in this study. One fetus at 150 mg/kg bw/day had a rib anomaly and one fetus at 50 mg/kg bw/day had a vertebral anomaly. Due to the single occurrence of these malformations and as they occurred at incidences that were within the range of available historical control data, these malformations were considered to be chance findings.

Skeletal variations occurred at an incidence of 68.4%, 76.2%, 72.6% and 62.4% per litter in the control, 15, 50 and 150 mg/kg bw/day groups, respectively. All the variations noted were considered not to be treatment related as they occurred infrequently, in the absence of a doserelated trend and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 150 mg/kg bw/day.

Four visceral malformations were observed in this study. Tetralogy of Fallot was observed in one fetus each from the control , low dose and high dose group. One fetus at 15 mg/kg bw/day was noted with both a ventricular septum defect and a small eye; and one fetus at 50 mg/kg bw/day was observed with a cyst attached to a lung lobe. The single occurrence and group distribution of these malformations did not indicate a treatment relationship and, therefore, all were considered to be spontaneous in origin.

Visceral variations occurred at an incidence of 11.0%, 6.1%, 6.9% and 7.9% per litter in the control, 15, 50 and 150 mg/kg bw/day groups, respectively. All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently and/or at incidences that were within the range of available historical control data.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects observed up to and including the highest dose level.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables













































































































































































Dose level (mg/kg bw/day)



0



15



50



150



Females on study



22



22



22



22



- Females that aborted or delivered



1



0



0



0



- Females that died



0



0



0



1 (gravid)



- Females that were euthanized



0



0



1 (gravid)



0



Females examined at scheduled necropsy



21



22



21



21



-  Pregnant



17



21



20



18



- non-pregnant



4



1



1



3



Mean number of corpora lutea



10.4



10.8



10.8



9.8



Mean number of implantation sites



10.2



10.2



10.1



9.3



-early resorptions


-late resorptions


(total number and % per litter)



5 (3.5%)


1 (0.3%)


 



12 (5.6%)


2 (0.8%)


 



8 (4.0%)


7 (3.2%)


 



6 (4.1%)


0 (0.0%)


 



Dams with stillbirths, resorptions only and/or dead fetuses



0



1



0



0



Pre-implantation loss (number and %)



2 (1.2%)



12 (5.7%)



13 (5.1%)



9 (4.8%)



Post-implantation loss (number and % per litter)



6 (3.8%)



15 (7.0%)



15 (7.1%)



6 (4.1%)



Mean body weight on day 29 pc (g)



3838



3928



3916



3843



Mean body weight gain day 6-29 pc (%)



10



12



13



11



Gravid uterine weight (g)



498.8



516.4



527.0



512.2



Bodyweight corrected for gravid uterine weight (g)



3339.5



3411.4



3388.9



3331.0



Mean live offspring (number and %)



9.9 (96.2%)



9.5 (93.0%)



9.4 (92.9%)



8.9 (95.9%)



Mean fetal body weight (males)



36.2



38.8



39.7



40.1



Mean fetal body weight (females)



36.2



38.0



39.1



41.0**



Mean fetal body weight (sexes combined)



36.3



38.4



39.4



41.2**



Malformations (including runts) (number and % per litter)


-external


-soft tissue


-skeletal



2 (1.7%)


   


1 (0.8%)


1 (0.8%)


0 (0%)



2 (1.0%)


 


  0 (0%)


2 (1.0%)


0 (0%)



3 (1.5%)


 


  1 (0.5%)


1 (0.5%)


1 (0.6%)



2 (1.2%)


 


  0 (0%)


1 (0.6%)


1 (0.6%)



Variations (% per litter)


-external


-soft tissue


-skeletal



71.0


0


11.0


68.4



76.6


0


6.1


76.2



74.1


0


6.9


72.6



64.7


0


7.9


62.4



 


**: 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.


 


Dose Formulations


Preparations were visually inspected for homogeneity prior to use and all preparations were used within 6 hours after preparation of the formulation.


 


Test System


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%.


 


Dose levels


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.


 


Results


Maternal findings


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).


 


Conclusion


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.

Applicant's summary and conclusion

Conclusions:
A prenatal developmental toxicity study was performed in rabbits according to OECD/EC guidelines and in accordance with GLP principles. Based the absence of adverse effects up to and including the highest dose level tested, a maternal and developmental NOAEL for Melanine of at least 150 mg/kg bw/day was established.
It should be noted that mortality occured at a slightly higher dose level of 250 mg/kg bw/day in the range-finding study.
Executive summary:

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 variations.

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 control group.

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 organ weights).

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.