Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (Animal arrival) 22 May 2019
Experimental completion date (Fetal Pathology) 19 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyldimethylamine
EC Number:
209-940-8
EC Name:
Ethyldimethylamine
Cas Number:
598-56-1
Molecular formula:
C4H11N
IUPAC Name:
ethyldimethylamine
Test material form:
liquid
Details on test material:
Test item: Ethyldimethylamine
Test item identity (including alternative names): Dimethylethylamine
DMEA
N,N-Dimethylethylamine
CAS number: 598-56-1
Intended use: Industrial chemical
Appearance: Clear-colorless liquid
Storage conditions: In a dry, cool and well-ventilated place. Storage in hermetically closed bottle in a refrigerator (2-8°C)
Supplier: Sponsor
Batch number: SAMP181373
Expiry date: 17 May 2019
Purity: 99.57%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 mL representative sample was taken, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.
Specific details on test material used for the study:
Test item: Ethyldimethylamine.
Test item identity (including alternative names): Dimethylethylamine
DMEA
N,N-Dimethylethylamine
CAS number: 598-56-1
Intended use: Industrial chemical.
Appearance: Clear-colorless liquid.
Storage conditions: In a dry, cool and well-ventilated place. Storage in hermetically closed bottle in a refrigerator (2-8¿).
Supplier: Sponsor.
Batch number: SAMP181373
Expiry date: 17 May 2020
Purity: 99.57%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Strain/Species New Zealand White rabbit.
Supplier Envigo RMS UK
Number of animals ordered 96 females
Duration of acclimatization 19 days.
Age of the animals at the start of the study (Day 0 of gestation) 18 to 29 weeks old.
Weight range of the animals at the start of the study (Day 0 of gestation) 2.13 to 4.34 kg.

Animal Care and Husbandry
Environmental Control
Multispecies facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 15-21°C and 45-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 14 hours light : 10 hours dark.
Alarm systems Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also fitted with a plastic resting platform.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.

Number of animals per cage
Acclimatization one female.
During mating one stock male and one female.
Gestation one female.

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Stainless steel key ring Attached to the cage.
Cage paper Provided to each cage from Day 20 after mating and replaced as necessary.

Diet Supply
Diet Teklad 2930 Diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Restricted (initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter).

In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are provided by the water supplier.

Certificates of analysis were also received from the suppliers of the Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Allocation and Identification
Allocation On the day of mating. Where possible only females mating at least twice were allocated; the following animals were allocated to study after a single mating, all were confirmed to be pregnant:
• Group 1 nos. 2 and 6
• Group 4 no. 75

Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.
Identification of animals Each animal was assigned a number and identified uniquely within the study using a microchip.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Method of preparation The vehicle was chilled in an ice bath/fridge prior to starting formulation.
The required amount of test item was weighed into the smallest practical sealed container ensuring it was kept cold. 50% of the final volume of chilled vehicle was measured in a cylinder and placed to one side. Approximately 10% of the final volume of vehicle was measured into a separate cylinder. The test item was added to the vehicle. The weigh container was rinsed with vehicle which was added to the measuring cylinder and made to 50% of the final volume with vehicle. The mixture was transferred to a sealed container. The measuring cylinder was rinsed with the premeasured chilled purified water previously weighed and this was added to the mixing container. The mixing container was placed in an ice bath and magnetically stirred to mix. The mixture was then split into the final containers, via syringe. All containers were flushed with Nitrogen.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item in order of ascending concentration.

Frequency of preparation Weekly.

Storage of formulation Refrigerated (2-8°C).

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Homogeneity and stability The suitability of the proposed mixing procedure was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix (Covance Study No. CT68FN). In that study, formulations in the concentration range 5 to 200 mg/mL were confirmed to be stable for:
• two hours stored at ambient temperature (15 to 25°C)
• 14 days stored refrigerated (2 to 8°C).
Stability could not be established for formulation at less than 5 mg/mL.

Achieved concentration Samples of each of the first and last formulations prepared for administration were analyzed for achieved concentration of the test item.

Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose Groups 1, 3 and 4: 3 mL/kg/day
Group 2: 1 mL/kg/day
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as Groups 3 and 4.
Frequency Females were treated from Day 6 to Day 28 (inclusive) after mating, once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Procedure
The samples were analyzed in accordance with the validated Covance Analytical Procedure
(DFA/M103/18).

The analytical method involved extraction and dilution in acetone followed by liquid
chromatographic analysis with mass spectrometric detection (LC-MS/MS). Sample
concentrations were determined with reference to single bracketing standards (2000 ng/mL).

Procedural recovery samples were prepared concurrently with samples and results were
corrected for the mean recovery value at each level at analysis.

Due to analytical issues experienced in a related study (CT68FN), the chromatographic
conditions were altered and the instrumentation was optimized in order for the analytical
sequences to run. These included changes to the column length, run time and source
temperature.

¿ Phenomenex Kinetex F5, 2.6 µm, 100 × 3 mm column was used from the original
column of Phenomenex Kinetex F5, 2.6 µm, 150 × 3 mm column.
¿ Due to a shift in the retention time, the run time was extended to 10 minutes from the
original run time of 5 minutes to ensure the test item peak was fully integrated.
¿ The source temperature was changed to 400ºC from the original source temperature of
500ºC.

All changes were made in order to improve the chromatography, repeatability, peak area
responses and overall increased sensitivity of the instrumentation. Therefore, this is
considered to have no impact on the integrity of the results.

Concentration of Dose Formulations
The formulations for the First formulation and Last formulation were sampled. For all
groups, 4 × 1 mL (accurately weighed) was sampled from the middle of the formulation by
Pharmacy personnel.

Two samples from each group were analyzed in accordance with the analytical
procedure. For the First formulation, Groups 2, 3 and 4 re-dilutions in duplicate and
contingency samples were analyzed as the original results were out of specification. The
remaining samples were retained for contingency. Samples were disposed of once
satisfactory results were achieved or confirmatory results were obtained.

Major Computerized Systems
Chromatography data handling: Analyst
Sample handling system: Sample Registry System, Covance
Test item management: Pristima, Xybion Medical Systems Corporation
Version numbers of the systems are maintained by Covance.

RESULTS AND CONCLUSION
The mean concentrations of Ethyldimethylamine in test formulations analyzed during the
study and the deviation of the mean result from the nominal value are detailed in Table 1.
During the First formulation, Groups 2 to 4 had low relative mean error (RME) values.
Re-dilutions and contingency analysis was performed. The re-dilutions confirmed the original
results. Therefore, the contingency results have been reported alongside the original results.
The RME values for the four results for Groups 2, 3 and 4 were -32.1%, -39.5% and -39.1%
respectively.

The mean concentrations for the Last formulation were within the applied limits of +10/-15%
of the nominal concentration, confirming the accuracy of formulation, with the exception of
Group 2 where the RME was -15.5%. Rework was not performed in error.

The difference from mean and coefficient of variation for all samples remained within 4%,
confirming precise analysis, with the exception of the First formulation, Groups 2 to 4 and
the Last formulation, Group 2.

The procedural recoveries remained within the validated range, confirming the continued
accuracy of the analytical methodology, with the exception of one recovery prepared during
the First formulation analysis and one recovery prepared during the Last formulation
analysis. As 2 out of 3 recoveries were acceptable for each occasion, the recoveries can be
excluded in line with the SOP
Details on mating procedure:
Male/female ratio 1:1 using identified stock New Zealand White bucks.

Checks Natural mating observed.

After mating Each female was injected intravenously with 25 i.u. luteinizing hormone.
Day 0 of gestation On the day of mating.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females Day 6 to 28 after mating
Frequency of treatment:
Daily
Duration of test:
Day 0-6: Mating
Day 6-28: Treatment
Day 29: Necropsy
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Remarks:
volume dose for Group 2 was adjusted to ensure that concentrations were within the established stability range
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
Purpose
The purpose of this study was to assess the influence of ethyldimethylamine on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of the New Zealand White Rabbit.

Animal Model
The rabbit was chosen as the test species because it is accepted by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Rationale for Dose Level Selection
The doses used in this study (0, 8, 25 and 70 mg/kg/day) were selected in conjunction with the Sponsor.

Dose levels of 15, 45 and 70 mg/kg/day were selected in conjunction with the Sponsor based on the findings in a pilot rabbit study (Study no. FD84BQ) and a preliminary embryo-fetal study in the rabbit (Study no. XG69WJ).

• In the pilot study with non-pregnant rabbits a dose level of 100 mg/kg/day (administered as 33.3 mg/mL at 3mL/kg) was tolerated for the 14 day treatment period however all animals showed reduced food consumption, one throughout treatment, one during the first 3 days of treatment and one during the last six days of treatment. Macroscopic examination revealed severe stomach lesions in all three animals at necropsy. At 50 mg/kg/day (administered as 16.7 mg/mL at 3mL/kg) all animals showed periods (< 5 days) of low food consumption and one out of the three had stomach findings at necropsy -> one female with macroscopic stomach findings namely dark areas in the non-glandular mucosa and depressions in the glandular mucosa.

• In the preliminary embryo-fetal rabbit study the high dose of 45 mg/kg/day (administered as 15 mg/mL at 3mL/kg) was well tolerated with no sign of maternal toxicity or effects on embryo-fetal survival/development.

It was therefore decided to use a higher dose than the 45 mg/kg/day high dose-level used in the preliminary embryo-fetal study; 70 mg/kg/day was selected as the high dose, with the aim to induce some maternal toxicity, as no effects were evident at 45 mg/kg/day. Low and intermediate dose levels of 8 and 25 mg/kg/day were selected to provide an approximate 3 fold dose interval, provide information on dose-response relationship and threshold for any adverse toxicological effect observed .

Examinations

Maternal examinations:
Serial Observations
Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.


Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage trays were inspected daily for evidence of animal ill-health amongst the occupant. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:

Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 23 and 29 after mating to monitor general health.

Body Weight
The weight of each adult was recorded weekly during acclimatization, on the day of mating and on Days 3 and 6 to 29 after mating.

Food Consumption
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.

Terminal Investigations
Method of Kill
Method of kill for all adult animals Intravenous injection of sodium pentobarbitone.
Method of kill for fetuses Subcutaneous injection of sodium pentobarbitone.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 29 after mating.
Sequence To allow satisfactory inter-group comparison.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Stomach All adult females.
Routine staining Sections were stained with hematoxylin and eosin.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).
Apparently non pregnant animals and for apparently empty uterine horns The absence or number of uterine implantation sites was confirmed.
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus.
Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fixation Nominally one half of fetuses were decapitated; heads were initially stored in Bouin’s fluid.

Remaining fetuses and torsos were eviscerated and fixed in Industrial Methylated Spirit.

Processing Bouin’s fixed fetal heads were subject to free-hand serial sectioning.
Industrial Methylated Spirit fixed fetuses and torsos were processed and double stained with Alizarin Red and Alician Blue.

Fetal Pathology Examination
Bouin’s fixed heads Serial sections were examined for soft tissue abnormalities.
Alizarin Red and Alician Blue stained fetuses and torsos Assessed for skeletal and cartilage development and abnormalities.
Statistics:
Please refer to "Any other information on materials and methods" below
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations)/ Number of corpora lutea) x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations - Number of live fetuses)/ Number of implantations) x 100


All group values and SD were calculated from the individual litter values.
Historical control data:
PLEASE REFER TO THE ATTACHED ANNEX - HISTORICAL CONTROL DATA

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or signs associated with doisng that were considered to be related to treatment at dose levels up to and including 70 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were four early decedents in this study. One control female (No. 6) was killed for welfare reasons on GD (gestation day) 28 due to red staining in the cage tray, uterine examination revealed two late resorptions and 6 live fetuses but there were no macroscopic or microscopic abnormalitiesand the cause of death was undetermined. One female (No. 41) that received 8 mg/kg/day was found dead on GD 23; additionally, females Nos. 40 (8 mg/kg/day) and 45 (25 mg/kg/day) were killed for welfare reasons on GD 12 and 11, respectively. Macroscopic examination of these animals revealed the presence of adhesions in the thoracic cavity involving multiple organs, dark area on the oesophagus (No. 41) and perforated oesophagus (Nos. 40 and 45). The cause of death of these animals was therefore considered accidental and attributed to the dosing procedure (oral gavage).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For females receiving 70 mg/kg/day mean body weight loss of 80 g (0.08 kg) was recorded between Days 6 and 8 of gestation (p<0.01) compared with mean body weight stasis in Controls. Thereafter, body weight change from Day 8 of gestation was generally similar to Controls but overall the weight gain (Day 6 to 29 of gestation) was 25 % lower than Controls (p<0.05).

There was no effect of treatment on body weight change at 8 mg/kg/day.

There was no conclusive effect of treatment on mean gravid uterine weight. Mean maternal body weight loss following adjustment for the gravid uterine weight was greater at 25 and 70 mg/kg/day compared with Controls (p<0.05 and p<0.01 respectively); a dose response was apparent .

PLEASE REFER TO ATTACHED TABLE "BODYWEIGHT AND BODYWEIGHT CHANGES - GROUP MEAN VALUES"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 70 mg/kg/day, mean food consumption was moderately lower than Controls on Days 6-9 of gestation (p<0.01) and slightly/marginally low on Days 23-29 of gestation (p<0.05 or p<0.01).
Food consumption at 8 or 25 mg/kg/day was considered to be unaffected by treatment.
PLEASE REFER TO THE ATTACHED TABLE"FOOD CONSUMTPION - GROUP MEAN VALUES"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic examination performed on GD 29 after 3 weeks of treatment revealed the following changes in the stomach.
Stomach
Dark and/or raised areas were observed on the stomach wall of animals that received Ethyldimethylamine at 70 mg/kg/day.

The incidence and distribution of all other findings were considered to be unrelated to treatment.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with Ethyldimethylamine were seen in the stomach.
Stomach
Microscopic changes findings consistent with edema, hemorrhage, mucosal ulceration, vascular necrosis and inflammatory cell infiltrate in the mucosa and/or submucosa of the fundic region of the stomach or the antrum were observed in animals that received
70 mg/kg/day. Dilated glands of the glandular mucosa (fundic mucosa or antrum) were also observed at higher incidence and severity in animals at 70 mg/kg/day.

Summary of treatment related findings in the stomach
Group/sex 1F 2F 3F 4F
Dose (mg/kg/day) 0 8 25 70
Dilation, Glands
Minimal 1 1 1 7
Slight 0 0 0 1
Moderate 0 0 0 1
Total 1 1 1 9
Edema
Minimal 0 0 0 2
Slight 0 0 0 2
Moderate 0 0 0 3
Total 0 0 0 7
Hemorrhage
Minimal 0 0 0 1
Slight 0 0 0 10
Total 0 0 0 11
Infiltrate, Inflammatory Cell, Mucosa/Submucosa
Minimal 0 0 0 1
Slight 0 0 0 1
Total 0 0 0 2
Necrosis, Vascular, Mucosa/Submucosa
Slight 0 0 0 1
Total 0 0 0 1
Ulceration
Slight 0 0 0 1
Total 0 0 0 1
Number Examined 21 20 21 22

Incidental Findings
The incidence and distribution of all other findings were considered to be unrelated to treatment.

PLEASE ALSO REFER TO THE ATTACHED TABLE "HISTOPATHOLOGY - GROP DISTRIBUTION FOR FEMALES ON DAY 28"
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean numbers of implantations or live fetuses, pre and post implantation losses or sex ratio at dose levels up to and including 70 mg/kg/day.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean numbers of implantations or live fetuses, pre and post implantation losses or sex ratio at dose levels up to and including 70 mg/kg/day.
PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean numbers of implantations or live fetuses, pre and post implantation losses or sex ratio at dose levels up to and including 70 mg/kg/day.
PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
gross pathology

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Stomach
Description (incidence and severity):
dark and/or raised areas were observed on the stomach wall of animals that received Ethyldimethylamine at 70 mg/kg/day and microscopic examination of the stomach revealed microscopic changes including edema, hemorrhage, mucosal ulceration, vascular necrosis and inflammatory cell infiltrate in the mucosa and/or submucosa of the fundic region of the stomach or the antrum.

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 70 mg/kg/day, mean placental and fetal weights were slightly lower than in Controls with the differences for female fetal weights and overall fetal weights attaining statistical significance (p<0.05); these differences were considered to be related to the slightly higher live litter size in this group rather than an effect of treatment.

Placental and fetal weights at 8 and 25 mg/kg/day showed no adverse effects of maternal treatment.
PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
slightly higher live litter size at 70 mg/kg/day
PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no major abnormalities considered to be related to treatment: in all treated groups there were a small number of major abnormalities, they were of low incidence, several are within historical control data (HCD) range and there was no treatment related effect for any particular abnormality.
At 70 mg/kg/day there was a slight increase in incidence of full supernumerary 13th rib with associated 20 thoracolumbar vertebrae and unilateral shift of pelvic girdle compared to concurrent control (all parameters just outside of fetal HCD range but within litter HCD range).

This indicates a very slight shift in rib/vertebral configuration, which, as variants are not considered adverse, but may be associated with treatment with Ethyldimethylamine.
There was also a slight increase in incidence of incompletely ossified metacarpals/phalanges (forepaw digits) compared to concurrent control but within the HCD range and therefore considered unrelated to treatment. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at this dose level.


PLEASE REFER TO THE ATTACHED TABLES: "FETAL EXAMINATIONS - MAJOR ABNORMALITIES" AND "FETAL EXAMINATIONS - MINOR SKELETAL ABNORMALITIES"
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Formulation Analysis


Groups 2 to 4 in the first formulation had low relative mean error (RME) values.  Re-dilutions and contingency analysis was performed. The re-dilutions confirmed the original results. Therefore, the contingency results have been reported alongside the original results.  The RME values for the four results for Groups 2, 3 and 4 were -32.1%, -39.5% and -39.1% respectively. 


The mean concentrations for the last formulation were within the applied limits of +10/-15% of the nominal concentration, confirming the accuracy of formulation, with the exception of Group 2 where the RME was -15.5%. Rework was not performed in error. 


The difference from mean and coefficient of variation for all samples remained within 4%, confirming precise analysis, with the exception of the first formulation, Groups 2 to 4 and the last formulation, Group 2.


The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical methodology, with the exception of one recovery prepared during the First formulation analysis and one recovery prepared during the Last formulation analysis. As 2 out of 3 recoveries were acceptable for each occasion, the recoveries can be excluded in line with the SOP.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the no observed adverse effect level (NOAEL) for maternal toxicity is 25 mg/kg/day and the high dose of 70 mg/kg/day is the NOAEL for embryo-fetal survival, growth and development.
Executive summary:

Summary


The purpose of this study was to assess the influence of ethyldimethylamine on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of the New Zealand White Rabbit.


Three groups of 22 females received ethyldimethylamine at doses of 8, 25 or 70 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating.  A similarly constituted Control group received the vehicle, purified water, at a volume dose of 3 mL/kg throughout the same duration.  Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight and food consumption were recorded.  Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight was recorded.  Microscopic investigations were also undertaken.  All fetuses were examined macroscopically (internal and external) at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.


Results


There were no deaths, clinical signs or dosing signs considered to be related to treatment.


Treatment at 70 mg/kg/day was associated with mean body weight loss of 80 g (0.08 kg) between Days 6 and 8 of gestation compared with mean body weight stasis in Controls (body weight gain during treatment was appproximately 75 % of Controls), greater adjusted mean maternal body weight loss following adjustmnet for the gravid uterine weight and  moderately low food consumption.


In addition, dark and/or raised areas were observed on the stomach wall of animals that received Ethyldimethylamine at 70 mg/kg/day and microscopic examination of the stomach revealed microscopic changes findings including edema, hemorrhage, mucosal ulceration, vascular necrosis and inflammatory cell infiltrate in the mucosa and/or submucosa of the fundic region of the stomach or the antrum.  Dilated glands of the glandular mucosa (fundic mucosa or antrum) were also observed at higher incidence and severity in animals at 70 mg/kg/day.


Treatment at 25 mg/kg/day was associated with greater adjusted mean maternal body weight loss when compared with Controls; there was no maternal response to treatment at 8 mg/kg/day.


Embryo-fetal survival was unaffected by treatment and developmnet was not considered to be adversley affected at dose levels up to and including 70 mg/kg/day.


Conclusion


Based on the results of this study it is concluded that the no observed adverse effect level (NOAEL) for maternal toxicity is 25 mg/kg/day and the high dose of 70 mg/kg/day is the NOAEL for embryo-fetal survival, growth and development.