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Diss Factsheets

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:
Animal arrival 02 January 2019
Necropsy 29 January to 01 February 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:
N,N-dimethylethanamine
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°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.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) rat.
Details on test animals or test system and environmental conditions:
Animals
Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd.
Number of animals 90 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Six days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 70 to 76 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 210 to 293 g.

Allocation and Identification
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

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 treatment group.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.

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(s).

Animal Care and Husbandry
Environmental Control
Rodent 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 20-24ºC and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light : 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.

Cage distribution The cages constituting each group were blocked by group and mounted in batteries.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization Up to four animals
During pairing 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.

Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted.

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

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and 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.

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, and may have been prepared in advance of the first day of dosing.

Storage of formulation Refrigerated (2-8°C) for up to 14 days and ambient (15 25¿C) for up to two hours.

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
Stability and homogeneity Before commencement of treatment 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. Formulations in the concentration range 1 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).
Achieved concentration Samples of each of the first and last formulation prepared 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 4 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 treated groups.

Frequency Females were treated from Day 6 to Day 19 (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:
Preparation of Calibration Standards
A primary standard solution (500000 ng/mL) was prepared by dissolving an accurately
weighed quantity (ca. 50 mg) of Ethyldimethylamine in acetone (100 mL). A secondary
standard solution (50000 µg/mL) was prepared by appropriate dilution of the primary
standard (1 mL) using acetone (10 mL). A tertiary standard solution (5000 µg/mL) was
prepared by appropriate dilution of the secondary standard (1 mL) using acetone (10 mL).

Solutions for instrument calibration were prepared by appropriate dilution of the tertiary
standard using acetone and contained Ethyldimethylamine at nominal concentrations of
50 ng/mL, 100 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 400 ng/mL and 500 ng/mL.

Calibration solutions were injected onto the LC-MS, at the beginning of each sample analysis
sequence as a minimum with a bracketing standard (200 ng/mL) injected in duplicate after
every three samples, using the conditions detailed in the chromatographic section.

Due to a change in response from the LC-MS/MS part way through the study the calibration
range was increased for some runs. Where this happened the calibration range was prepared
at 500 ng/mL, 1000 ng/mL, 2000 ng/mL, 3000 ng/mL, 4000 ng/mL and 5000 ng/mL, these
were based on the peak areas and responses obtained for these concentrations.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using
ultrasonic vibration in a suitable volume of acetone. The extract was diluted using acetone,
where necessary, to provide a solution containing Ethyldimethylamine at an expected
concentration of 200 ng/mL. Where the calibration concentration range was increased
extracts were diluted to an expected concentration of 2000 ng/mL.

The concentration of Ethyldimethylamine in the final solution was quantified by LC-MS as
detailed in the chromatographic section.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (purified
water) with known amounts of Ethyldimethylamine. The prepared procedural recoveries
were analyzed in accordance with the analytical procedure.

Instrumentation Parameters
These conditions were optimised depending on which instrument was in use.
High performance liquid chromatograph
(HPLC) and mass spectrometer
(LC-MS/MS):
Shimadzu LC-10AD pump, Shimadzu SIL-HTAautosampler, and a Sciex API 3000 equivalent
mass spectrometer
Column: Phenomenex Kinetex F5, 2.6 µm, 150 × 3 mm
Column temperature: 30°C
Sample temperature: Ambient
Mobile Phase A: 0.05% Acetic acid (aq)
Mobile Phase B: 0.05% Acetic acid in acetonitrile
Isocratic conditions: 50% A/50% B
Flow rate: 0.35 mL/minute
Needle wash: ACN/water 50/50 v/v
Ionization: Turboionspray – positive ion mode
Source temperature: +500°C
Collision gas: Nitrogen
Collision energy: 25 eV
Dwell time: 200 ms
Pause time: 5 ms
Ions to be monitored: m/z 74.16 to m/z 46.10 (used for quantitation)
Injection volume: 20 µL
Run time: 5 minutes
Approximate retention time: 2.91 minutes
Details on mating procedure:
Male/female ratio 1:1 with identified stock males.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.

Day 0 of gestation When positive evidence of mating was detected.

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 19 after mating
Frequency of treatment:
Daily
Duration of test:
Day 0-6: Mating
Day 6-19: Treatment
Day 20: Necorpsy
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
Purpose
The purpose of this study was to assess the influence of Ethyldimethylamine (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis phase and fetal growth phase of pregnancy in the Sprague-Dawley rat.

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at 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, 20, 60 and 180 mg/kg/day) were selected in conjunction with the Sponsor.

A high dose level of 180 mg/kg/day was selected based on the results of a preliminary embryo-fetal study in the female rat conducted at these laboratories (Covance No. MK81TG).
In that study animals received daily administration of Ethyldimethylamine over Days 6-19 of gestation at 50, 180 and 360 mg/kg/day.

Females receiving 360 mg/kg/day started showing signs of decreased activity, partially closed eyelids, flattened or hunched posture, shallow breathing, cold to touch and piloerection on Day 8 or 9 of gestation. Two females were euthanised for welfare reasons, both on Day 9 of gestation, after more than 15% bodyweight loss, which reached or exceed the moderate severity limit defined in the Home Office project license; other terminal signs included including partially closed eyelids, gasping and piloerection. The high dose level was subsequently reduced to 270 mg/kg/day for the remaining four females.

The females dosed at 270 mg/kg/day were prostrate at 30 minutes post dosing and underactive, hunched with piloerection and had abnormal breathing at 1 hour post dose. Prior to dosing on Day 7 of gestation at the reduced high dose, one female was found dead and a second female had lost body weight, exceeding the moderate severity limit. As the dose level was not tolerated, the remaining females were euthanised for welfare reasons on Day 9 or 10 of gestation.

Treatment at 180 mg/kg/day was tolerated and was associated with reduced weight gain (86% of Controls), reduced adjusted maternal weight gain (60% of Controls) and a slight reduction in food consumption (90% of Controls). One female had depressions on the nonglandular region of the stomach but remained in good clinical condition. Embryo-fetal survival was unaffected by treatment and mean fetal weight was low when compared with the concurrent Control animals.

Therefore a high dose of 180 mg/kg/day was considered suitable as the high dose with 60 and 20 mg/kg/day chosen as the intermediate and low dose levels to allow assessment of any dose response.

Examinations

Maternal examinations:
Serial Observations
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). 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, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

Terminal Investigations
Method of Kill
Method of kill for all adult animals Carbon dioxide asphyxiation.
Method of kill for fetuses Chilling on a cool plate (approximately 0¿C)

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 20 after mating.
Sequence To allow satisfactory inter-group comparison.
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

For each ovary/uterine horn
Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

For apparently empty uterine horns The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski, E, 1964)].

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. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter Sexed internally and eviscerated.

Fixation Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid.

Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and double stained with Alizarin and Alician Blue.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Double stained with Alizarin Red and Alician Blue fetuses, Assessed for skeletal and cartilage development and abnormalities.

Statistics:
Please refer to "Any other comments on materials and methods"
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 (as appropriate) 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:
effects observed, treatment-related
Description (incidence and severity):
Signs associated with dose administration were restricted to the animals receiving 180 mg/kg/day, with 4/20 exhibiting a low incidence of rales (no. 61 on one occasion; no. 65 on two occasions; no. 67 on two occasions; no 71 on three occasions) and 4/20 females exhibiting increased salivation each on a single occasion.
Signs at routine physical examination were limited to two females at 180 mg/kg/day (nos. 65 and 71) with rales; there were other signs that could be related to treatment.
The clinical condition of animals receiving 20 or 60 mg/kg/day was unaffected by treatment.
PLEASE REFER TO THE ATTACHED TABLES - "SIGNS ASSOCIATED WITH DOSING - GROUP DISTRIBUTION" AND "CLINICAL SIGNS - GROUP DISTRIBUTION"
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females (nos 61 and 67) receiving 180 mg/kg/day were killed for welfare reasons; female no 61 was euthanised on GD14 and female no.67 was euthanised on GD12. Terminal signs for both of these females comprised of respiratory distress (gasping) and body weight loss, however no macroscopic abnormality was apparent at necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
From GD9 (Day 4 of treatment) mean body weight gain for females receiving 180 mg/kg/day was low with the difference attaining statistical significance (p<0.01) and overall (GD6-20) the mean body weight change at 180 mg/kg/day was approximately 14% lower than Controls (p<0.01). The resultant mean bodyweight on GD20 for females receiving 180 mg/kg/day was approximately 6% lower than Controls (p<0.05).

Group Body weight GD20 Body weight change GD6-20
(g) % difference from Controls (g) % difference from Controls
1 411 122
2 401 -2 117 -4
3 402 -2 118 -3
4 388* -6 105** -14
(*- p<0.005; ** p<0.01)

The mean gravid uterine weight was essentially similar across the groups. However the maternal weight gain following adjustment for the gravid uterine was statistically significantly low at 180 mg/kg/day, approximately 40% lower than Controls (p<0.01) and the maternal mean weight on GD20 was approximately 6% lower than Controls (p<0.05).

Both the absolute body weight gain and adjusted maternal weight gain of females receiving 20 or 60 mg/kg/day was unaffected by treatment.

PLEASE REFER TO THE ATTACHED TABLE - "BODYWEIGHT AND BODYWEIGHT CHANGE - GROUP MEAN VALUES"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 180 mg/kg/day mean food consumption was low when compared with Controls with the difference attaining statistical significance for GD10-17 (p<0.01; 86-88% of Controls) and GD18 19 (p<0.05; 93 % of Controls). Overall the total food consumption during the treatment period (GD 6 -19) was approximately 10% lower than Controls.

Group Total Food consumption GD6-19
(g) % difference from Controls
1 366
2 354 -3
3 356 -3
4 330 -10

Food consumption at 20 or 60 mg/kg/day was unaffected by treatment.

PLEASE REFER TO THE ATTACHED TABLE - "FOOD CONSUMPTION - 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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females on GD20 did not reveal any findings that could be attributed to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A higher incidence of post-implantation loss was observed at 180 mg/kg/day (9.3% versus 2.3% in Controls), however the incidence was within the historical control range and there was no impact on the live litter size. This difference was therefore not considered to be related to treatment.
PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of implantations, resorptions, live young and sex ratio and the extent of the pre- and post-implantation loss was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Early or late resorptions:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of implantations, resorptions, live young and sex ratio and the extent of the pre- and post-implantation loss was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Dead fetuses:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of implantations, resorptions, live young and sex ratio and the extent of the pre- and post-implantation loss was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.
PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
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:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Litter, placental and fetal weight was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.
PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter, placental and fetal weight was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.
PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
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):
At 60 mg/kg/day and 180 mg/kg/day there was a slightly higher incidence of incompletely ossified 1st to 4th sternebrae, compared with concurrent control (12/11 fetuses in 8 litters per group versus 4 fetuses in 4 Control litters); the incidences were within the historical control range of the litters, which is the principal unit of evaluation, with the exception of fetuses at 60 mg/kg/day which exceeded the fetal historical control range. Incomplete ossification is a transient stage in development and is therefore not considered to be adverse.

PLEASE REFER TO THE ATTACHED TABES - "FETAL EXAMINATIONS - MAJOR ABNORMALITY FINDINGS" 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:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Embryo-fetal survival, growth and development showed no adverse effects of maternal treatment at all dose levels tested.

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: sternum
Description (incidence and severity):
At 60 mg/kg/day and 180 mg/kg/day there was a slightly higher incidence of incompletely ossified 1st to 4th sternebrae, compared with concurrent control (12/11 fetuses in 8 litters per group versus 4 fetuses in 4 Control litters); the incidences were within the historical control range of the litters, which is the principal unit of evaluation, with the exception of fetuses at 60 mg/kg/day which exceeded the fetal historical control range. Incomplete ossification is a transient stage in development and is therefore not considered to be adverse.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Formulation analysis


For the First dose there were significant instrument problems meaning that the samples could not be injected until a substantial amount of time had passed since extraction.  The results for this occasion are all low and this is considered to be a consequence of the time that elapsed between extraction and injection.  These results are reported for information only and it is considered unlikely that they are a true reflection of the accuracy of the dose prepared.


The Last dose samples were also injected some time after extraction, although the time period was shorter than that for the first dose.  Both the analyzed concentration and the procedural recoveries were outside of acceptance criteria for Groups 2 and 3.  When the analyzed concentration was corrected for the appropriate procedural recovery all results were within acceptable limits.  It is thought that the time between extraction and injection meant that the samples had started to degrade.  As the procedural recoveries had also aged at the same rate as the samples this correction was considered an appropriate action.  After correction the mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation.  The difference from mean for Groups 2 and 3 remained within 2%, confirming precise analysis. The difference from mean for Group 4 was ±7.24%.  Due to the factors mentioned above this was considered acceptable.


 

Applicant's summary and conclusion

Conclusions:
It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo fetal survival and development the NOAEL was considered to be 180 mg/kg/day.
Executive summary:

Summary


The purpose of this study was to assess the influence of Ethyldimethylamine (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis phase and fetal growth phase of pregnancy in the Sprague-Dawley rat.


Three groups of 20 females received Ethyldimethylamine at doses of 20, 60 or 180 mg/kg/day by oral (gavage) administration, from Day 6 to 19 after mating.  A similarly constituted Control group received the vehicle, purified water, at the same volume dose as treated groups.  Animals were killed on Day 20 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 20 after mating and the gravid uterine weight recorded.  All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.


Results


Two females receiving 180 mg/kg/day were killed for welfare reasons on GD12/14.  Terminal signs for both of these females comprised of respiratory distress (gasping) and body weight loss, however no macroscopic abnormality was apparent at necropsy. 


At the high dose (180 mg/kg/day) there was a low incidence of rales and increased salivation that were attributed to treatment. The high dose animals also exhibited low absolute body weight gain, low food consumption and low maternal weight gain following adjustment for the gravid uterine weight at approximately 86%, 90% and 60% of Controls, respectively. 


Clinical condition, body weight and food consumption for animals receiving 20 or 60 mg/kg/day were unaffected by treatment with Ethyldimethylamine.


A higher incidence of post-implantation loss was observed in the high dose group at caesarian section (9.3% versus 2.3% in Controls) but was within the historical control range. Litter data, fetal and placental weights were unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.


Macroscopic examination of females at necropsy did not reveal any abnormality that could be related to treatment.


The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.  At 60 mg/kg/day and 180 mg/kg/day there was a slightly higher increase in incompletely ossified 1st to 4th sternebrae; the incidences were within the historical control range of the litters with the exception of fetuses at 60 mg/kg/day in which outside the fetal historical control range.


 


Conclusion


Oral administration of Ethyldimethylamine to pregnant Sprague-Dawley rats during organogenesis and the fetal growth phase at 20, 60 and 180 mg/kg/day was not tolerated at the high dose with two animals killed for welfare reasons on Gestation Days 12/14.  Terminal signs included respiratory distress (gasping) and body weight loss; no macroscopic abnormality was apparent at necropsy. These deaths were not considered to be incidental and are attributed to administration of Ethyldimethylamine.


At 180 mg/kg/day females that survived to scheduled termination showed a low incidence of rales and increased salivation with low maternal weight gain and low food consumption; these parameters were unaffected by treatment at either 20 or 60 mg/kg/day and no maternal macroscopic abnormality was apparent at dose levels up to and including 180 mg/kg/day.


Embryo-fetal survival, growth and development showed no adverse effects of maternal treatment for all dose levels tested.


It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo-fetal survival and development the NOAEL was considered to be 180 mg/kg/day.