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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Experimental start date (Animal arrival) 24 October 2018
Experimental completion date (clinical pathology results) 30 November 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline followed
GLP compliance:
no
Remarks:
The study was not designed to meet any particular regulatory requirements. No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.
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°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 g 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:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because it is accepted by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Number of animals 23 males and 23 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Nine days before commencement of treatment.

Age of the animals at the start of treatment Males: 58 to 64 days.
Females: 79 to 85 days.

Weight range of the animals at the start of treatment Males: 323 to 364 g.
Females: 238 to 272 g.

Allocation and Identification
Allocation Randomly allocated on arrival.

Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.

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 occupants.

Animal Care and Husbandry
Environmental Control
Animal 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 Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.

Cage distribution Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable.

Number of animals per cage Up to three of the same sex, unless reduced by mortality or isolation.

Bedding Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen chew block Provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet SDS VRF1 Certified, pelleted diet.
Availability Non-restricted (removed overnight before blood sampling for hematology or blood chemistry).

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

Certificates of analysis were also received from the suppliers of the wood based 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
Details on route of administration:
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.
Vehicle:
water
Details on oral 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 (2-8oC).

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 The suitability of the proposed mixing procedures 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)
• ten days stored refrigerated (2 to 8°C).

No analysis of formulations was performed as part of this study.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analysis of formulations was performed as part of this study.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle only
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
225 mg/kg bw/day (nominal)
No. of animals per sex per dose:
2 males
2 females
Control animals:
yes, concurrent vehicle
Details on study design:
Purpose
The purpose of this study was to assess the systemic toxic potential of Ethyldimethylamine in a 14 day oral gavage study in Crl:CD(SD) rats, and to aid in the selection of suitable dose levels for future repeat dose studies.

Animal Model
The rat was chosen as the test species because it is accepted by regulatory agencies. The 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, 25, 75 and 225 mg/kg/day) were selected in conjunction with the Sponsor.

The acute oral toxicity of Dimethylethylamine (DMEA) was evaluated in rats according to a protocol similar to the OECD N°401 guideline (Acute Toxic Standard Method) (BASF AG, 1973). Groups of five male and five female Sprague Dawley rats were given a single oral dose of DMEA at doses of 136, 272, 544, 680, 850 and 1088 mg/kg. Following treatment, rats were observed daily and weighted weekly. A gross necropsy examination was performed at the time of scheduled euthanasia (Day 7). 10/10 animals died on Day 1 after administration of 1088 mg/kg (five males and five females). Animals showed congestive hyperemia, acute dilatation of the heart, dilatation of the stomach, gastritis and diffuse reddening of the stomach. 9/10 animals died on Day 1 after administration at 850 mg/kg (four males and five females); the last male that survived died on Day 2. Animals showed adhesion on the stomach, thickened forestomach and irregular folding at fundus. At 680 mg/kg 8/10 animals died on Day 1 (four males and four females). One additional female died on Day 2. At 544 mg/kg 1/10 animal died on Day 1. No death was recorded at doses of 272 or 136 mg/kg. Symptoms observed included accelerated respiration, abdominal position, squatting posture, apathy, atony, dyspnoea, tremor, nose and eye discharge. Between Days 1 and 6 all symptoms reversed in surviving animals. The oral LD50 of DMEA is 594 mg/kg (540-650 mg/kg) in Sprague Dawley rats with 95% confidence interval limits.

A high dose of 225 mg/kg/day was selected, with intermediate and low doses of 75 and 25 mg/kg/day chosen to allow a dose response.

Examinations

Observations and examinations performed and frequency:
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 occupants. 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.

Clinical Observations
Signs are considered in two parts: detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’ and extended changes in condition, classified as ‘clinical signs’.
Clinical observations are presented for each animal that showed signs, providing detail of type of sign, day of occurrence and information on the duration of the sign applicable.


Signs Associated with Dosing
Detailed observations were recorded daily 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 to monitor general health on Day -3, weekly throughout the treatment period and on the day of necropsy.

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.

A complete necropsy was performed in all cases as described in Section 3.7.

Body Weight
The weight of each animal was recorded three days before treatment commenced, on the day that treatment commenced (Day 1) and on Days 4, 8, 11 and 15 (before necropsy).

Group mean weight changes were calculated from the weight changes of individual animals surviving the specified period.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the three days before treatment started and twice weekly throughout the study.

Group mean food consumptions and standard deviations for each period were derived from unrounded cage values. Overall mean food consumption values were calculated for each cage and the mean of these cage means were calculated for each group/sex.
Values presented for the amount of food consumed in each cage in each food consumption period allow for any animal that was killed during that period.


Water Consumption
Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food at the following occasion:
Occasion Animals
At termination All animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*

Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:

Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected after overnight withdrawal of food at the following occasion:
Occasion Animals
At termination All animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:

Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Globulin (Glob)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.
Sacrifice and pathology:
Necropsy
All 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.

The retained tissues were checked before disposal of the carcass.

Schedule Animals were killed following 14 days of treatment.
Sequence To allow satisfactory inter-group comparison.
The organs weighed and tissue samples fixed are detailed as follows:
Tissue and regions examined Necropsy
Weigh Fix
Abnormalities *
Adrenals * *
Heart * *
Kidneys L+R *
Liver * *
Ovaries L+R *
Spleen * *
Stomach *
Testes L+R *
L+R Bilateral organs weighed individually.
* Organs weighed and samples fixed.

This list of tissues preserved was intended to satisfy any possible future requirement for further examination of tissues.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at the scheduled interval.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes Initially in modified Davidson’s fluid.
Statistics:
Please refer to "Any other information on materials and emthods"

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs exhibited related to treatment.
Chin rubbing, salivation, post dose salivation staining and piloerection were observed post dosing in some males at 225 mg/kg/day from Day 7 of treatment and some females showed post-dose salivation staining.
No dosing signs were exhibited in males and females at 25 and 75 mg/kg/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 225 mg/kg/day, Male No. 16 was killed for welfare reasons on Day 8 of treatment. On Days 7 and 8 of treatment chin rubbing, piloerection and rales was observed and in addition on Day 8 of treatment, salivation and gasping were also observed. From Day 4 to 8 of treatment, the male had lost 26g in weight (a 7% reduction in body weight from Day 4). Macroscopic examination revealed gaseous distention of the stomach and jejunum, depressions on the mucosal surfaces of the stomach, thickening of the glandular stomach and partially blocked nasal turbinates.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 225 mg/kg/day showed a lower weight gain over Day 1-15 of treatment when compared with Control (30% lower than Controls).

There was no effect of treatment on bodyweights in males and females at 25 and
75 mg/kg/day or in females at 225 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In males and females at 225 mg/kg/day, food consumption was marginally low over Days 1-8 when compared to Control. From Day 8 of treatment onwards food consumption was comparable with Control.
There was no effect of food consumption in males and females receiving 25 or 75 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological examination of peripheral blood performed after 14 days of treatment revealed statistically significant increases in reticulocyte count and red cell distribution width in males and females receiving 225 mg/kg/day when compared with Control (Males: +49% and +11%; Females: +82% and +13%, respectively).
All other inter-group differences were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical examination of plasma performed after 14 days of treatment for males and females did not reveal any toxicologically significant differences from control.
All inter-group differences were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation.
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:
effects observed, treatment-related
Description (incidence and severity):
When compared with Controls, the adjusted mean adrenal weight was statistically significantly high in males treated at 225 mg/kg/day (22% higher than Comtrols).
All other organ weights were unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings that were attributable to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Dose descriptor:
other: were no adverse findings that would preclude the use of at least 225 mg/kg/day as the highest dose level for future repeat dose studies.
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: were no adverse findings that would preclude the use of at least 225 mg/kg/day as the highest dose level for future repeat dose studies.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
It was therefore concluded that there were no adverse findings that would preclude the use of at least 225 mg/kg/day as the highest dose level for future repeat dose studies.
Executive summary:

 Summary


The purpose of this study was to assess the systemic toxic potential of Ethyldimethylamine in a 14 day oral gavage study in Crl:CD(SD) rats, and to aid in the selection of suitable dose levels for future repeat dose studies.


Three groups, each comprising five male and five female Crl:CD(SD) rats, received Ethyldimethylamine at doses of 25, 75 or 225 mg/kg/day.  A similarly constituted control group received the vehicle, purified water, at the same volume dose as treated groups. 


During the study, clinical condition, body weight, food consumption, water consumption (visual assessment only), hematology (peripheral blood), blood chemistry, organ weight and macropathology investigations were undertaken.


Results


At 225 mg/kg/day, Male No. 16 was killed for welfare reasons on Day 8 of treatment. Terminal signs included increased salivation, chin rubbing, piloerection, rales, gasping and body weight loss.  Macroscopic examination revealed gaseous distention of the stomach and jejunum, depressions on the mucosal surfaces of the stomach, thickening of the glandular stomach and partially blocked nasal turbinates.


There were no clinical signs exhibited related to treatment. Chin rubbing, salivation, post dose salivation staining and piloerection were observed post-dosing in some males at 225 mg/kg/day from Day 7 of treatment and some females showed post dose salivation staining. No dosing signs were exhibited in males and females at 25 and 75 mg/kg/day.


Males receiving 225 mg/kg/day showed a lower weight gain over Day 1-15 of treatment when compared with Control (30% lower than Controls). There was no effect of treatment on bodyweights in males and females at 25 and 75 mg/kg/day or in females at 225 mg/kg/day. In males and females at 225 mg/kg/day, food consumption was marginally low over Days 1-8 when compared to Control. From Day 8 of treatment onwards food consumption was similar to Controls. There was no effect of food consumption in males and females receiving 25 or 75 mg/kg/day.


The hematological examination revealed statistically significant increases in reticulocyte count and red cell distribution width for males and females receiving 225 mg/kg/day when (Males: +49% and +11%; Females: +82% and +13%, respectively).  


When compared with Controls, the adjusted mean adrenal weight was statistically significantly high in males treated at 225 mg/kg/day (approximately 22%higher than Controls).


There were no macroscopic findings that were attributable to treatment.


Conclusion


It was therefore concluded that there were no adverse findings that would preclude the use of at least 225 mg/kg/day as the highest dose level for future repeat dose studies.

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