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Diss Factsheets

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) 19 February 2020
Experimental completion date (Necropsy) 11 March 2020
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:
equivalent or similar to guideline
Guideline:
other: The Acute oral toxicity of Ethyldiisopropylamine (EDIPA) was evaluated in rats according to a protocol similar to the OECD 423 guideline (Acute Oral Toxicity - Acute Toxic Class Method, 1997)
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:
Ethyldiisopropylamine
EC Number:
230-392-0
EC Name:
Ethyldiisopropylamine
Cas Number:
7087-68-5
Molecular formula:
C8H19N
IUPAC Name:
ethylbis(propan-2-yl)amine
Test material form:
liquid
Details on test material:
Purity= 99.7%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat (virgin) was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD (SD) rat strain was used because it is accepted by regulatory agencies and because of the historical control data available at this laboratory
Sex:
female
Details on test animals or test system and environmental conditions:
Strain/Species Crl:CD(SD) rat.

Supplier Charles River (UK) Ltd.

Number of animals 10 females.

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Seven days before commencement of treatment.

Age of animals at start of treatment 72 days.

Weight range of animals at the start of treatment 206 to 266 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.

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 Replacement
Before the start of treatment checks upon health and body weight were performed. All
animals were considered acceptable for use upon the study and so no replacements were
performed.

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 20-24¿C 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 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 two of the same sex, unless reduced by mortality.

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

Environmental Enrichment
Aspen gnawing material 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

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 gnawing material.

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:
methylcellulose
Remarks:
1% w/v methylcellulose plus 0.1% Tween 80
Details on oral exposure:
Method of preparation
The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.

A series of suspensions at the required concentrations were prepared by dilution of individual weightings of the test item.

The pH of formulations were adjusted with aqueous HCl to pH 8.0

Frequency of preparation Daily.

Storage of formulation Refrigerated (2 to 8°C) for up to 24 hours and ambient (15 to 25°C) for 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 The homogeneity and stability of formulations in the concentration range of 5 to 200 mg/mL during ambient and refrigerated storage were confirmed as part of another study, Covance Study No. LB21BK.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control only
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
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 Ethyldiisopropylamine, an industrial chemical, 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.

Rationale for Dose Level Selection
The doses used in this study (0, 25, 75 and 150 mg/kg/day) were selected in conjunction with the Sponsor.

The Acute oral toxicity of Ethyldiisopropylamine (EDIPA) was evaluated in rats according to a protocol similar to the OECD 423 guideline (Acute Oral Toxicity - Acute Toxic Class Method, 1997) (ECHA registration dossier). Groups of three female Wistar rats were given a single oral dose of EDIPA using dose steps of 2000, 500 and 200 mg/kg bw, with three males also receiving 200 mg/kg bw. Following treatment, rats were observed daily for clinical sings and weighed weekly. A gross necropsy examination was performed at the time of euthanasia (Day 14). 3/3 females died within one hour of receiving EDIPA at 2000 mg/kg bw. Animals showed slight hyperemia of the glandular stomach and moderate erythema of the small intestine. 1/3 females died within one hour of receiving EDIPA at 500 mg/kg bw, the remaining two females died within three hours. Animals showed slight thickening of the fore stomach and glandular stomach walls. No death was observed at the dose of 200 mg/kg bw. Clinical symptoms were observed (poor general state, dysponea, apathy, staggering, tremor, twitching and piloerection) at 500 mg/kg bw up to hour two and at 200 mg/kg bw in all males up to Day 2 and all females between three and five hours post administration.

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.

Signs Associated with Dosing

Detailed observations were recorded daily at the following times in relation to dose administration:

• Pre-dose
• Approximately 30 minutes after dosing#
• One to two hours after dosing
• Three to four hours after dosing#
• As late as possible in the working day

# - Additional dose observations were observed.

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health, for three days prior to the start of treatment and daily throughout the study.

Mortality
A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.

Body Weight
The animals were weighted daily for three days prior to the start of treatment, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

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

Water Consumption
Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.
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 and abnormalities retained and fixed. All animals, thoracic and abdominal cavities opened, cranial cavity opened only if observations indicate possible neurotoxic action. Special attention to be paid to stomach tissue 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.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin.
Statistics:
No statistical analysis was performed on this study.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Between Days 1 to 3 of treatment, female’s No. 7 and 8 that received 150 mg/kg/day showed signs associated with the administration of dose comprising elevated gait, decreased activity, piloerection, closed and/or partially closed eyelids, hunched posture and chromodacryorrhea. Given the persistence of these observations, female’s No. 7 and 8 were prematurely euthanized without being dosed on Day 3 of treatment on animal welfare grounds. From Day 1 to 3 of treatment, female No. 7 had lost a total 8g body weight and female No. 8 had lost a total 12g body weight. Furthermore, food consumption for these animals was markedly reduced when compared to the Control group and 25 or 75 mg/kg/day groups. At the macroscopic examination, there were no abnormalities detected in either female No. 7 or 8.

Between Days 1 to 13 of treatment, female’s No. 5 and 6 receiving 75 mg/kg/day showed signs at the 1 to 2 hour and late in the working day post-dose observation comprising partially closed eyelids. Furthermore, female No. 5 was observed to have unsteady gait on Day 1 of treatment only.

There were no signs recorded for females receiving 25 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLE: "CLINICAL SIGNS - GROUP DISTRIBUTION OF OBSERVATIONS"
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
150 mg/kg/day : From Day 1 to 3 of treatment, female No. 7 had lost a total 8g body weight and female No. 8 had lost a total 12g body weight.

From Day 1 to 15 of study, there was no clear treatment related effect on body weight change for females receiving 25 or 75 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLE: "BODY WEIGHT - GROUP MEAN VALUES"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
150 mg/kg/day: Female No. 7 and female No. 8 - food consumption for these animals was markedly reduced when compared to the Control group and 25 or 75 mg/kg/day groups.

There was a suggestion of a slight reduction in food consumption for females receiving 75 mg/kg/day on Days 1 to 3 of treatment, from Days 3 to 15 of study, there was no clear treatment related effect on food consumption.

There was no clear treatment related effect on food consumption for females receiving 25 mg/kg/day on Days 1 to 15 of study.
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):
There were no macroscopic abnormalities detected at scheduled termination on Day 15 of study.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
other: Considering the adverse clinical signs observed at 150 mg/kg/day and clinical signs observed at 75 mg/kg/day, it was concluded that the selection of 100 mg/kg/day is a suitable high dose for a subsequent preliminary study for effects on embryo-fetal devel
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Considering the adverse clinical signs observed at 150 mg/kg/day and clinical signs observed at 75 mg/kg/day, it was concluded that the selection of 100 mg/kg/day is a suitable high dose for a subsequent preliminary study for effects on embryo-fetal development (Covance Study No. 8434891).
Executive summary:

SUMMARY


The purpose of this study was to assess the systemic toxic potential of Ethyldiisopropylamine, an industrial chemical, 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 two female Crl:CD(SD) rats, received Ethyldiisopropylamine at doses of 25, 75 or 150 mg/kg/day. A similarly constituted control group received the vehicle, 1% w/v methylcellulose plus 0.1% Tween 80, at the same volume dose as control groups.


 


During the study clinical condition, body weight, food consumption and macropathology investigations were undertaken.


Results


From Day 1 to 3 of study, females that received 150 mg/kg/day showed signs comprised of elevated gait, decreased activity, piloerection, closed and/or partially closed eyelids, hunched posture and chromodacryorrhea. Subsequently, this group was entirely prematurely killed on the grounds of animal welfare on Day 3 of study. Macroscopic examination of these females revealed no abnormalities.


Females that received 75 mg/kg/day showed signs of a lesser severity and comprised partially closed eyelids from Day 1 to 13 of treatment and for 1 female, unsteady gait on Day 1 of treatment only.


There were no signs recorded for females receiving 25 mg/kg/day.


Body weight gain from Day 1 to 15 of study was not clearly affected by treatment at 25 or 75 mg/kg/day.


There was a suggestion of slightly reduced food intake from Day 1 to 3 of treatment for females that received 75 mg/kg/day. Food intake was not clearly affected by treatment for females that received 25 mg/kg/day.


There were no macroscopic findings after 14 days of treatment for females that received 25 or 75 mg/kg/day.


Conclusion


Considering the adverse clinical signs observed at 150 mg/kg/day and clinical signs observed at 75 mg/kg/day, it was concluded that the selection of 100 mg/kg/day is a suitable high dose for a subsequent preliminary study for effects on embryo-fetal development (Covance Study No. 8434891).