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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (animal arrival) 02 September 2020
Experimental completion date (fetal pathology) 16 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

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:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
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:
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:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Duration of acclimatization Five days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 69 to 75 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 226 to 276 g.


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 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:
other: 1% w/v methylcellulose plus 0.1% Tween 80.
Details on exposure:
Method of preparation The required amount of test item was weighed and approximately 50% of the required volume of vehicle was added and it was magnetically stirred until it was uniformly mixed. The pH was adjusted with 37% HCl to pH 7.0-8.0. It was then made up to the required volume with vehicle and magnetically stirred for a minimum of 20 minutes.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation Weekly.
Storage of formulation Refrigerated (2 to 8°C) for up to eight days and ambient (15 to 25°C) for 24 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Stability and homogeneity The homogeneity and stability of formulations during storage were confirmed as part of another study, Covance Study Number LB21BK.
Achieved concentration Samples of each of the first, second and last formulation preparations weeks were analyzed for achieved concentration of the test item.
Sample handling Samples taken following formulations in the second and last weeks were added directly into volumetric containers containing water.

Liquid chromatograph: Waters Acquity UPLC
Mass spectrometer: Waters TQC
Ion source: Positive electrospray
Analytical column: Waters Acquity UPLC BEH C18 1.7 µm, (5 cm ¿ 2.1 mm)
Column temperature: 45°C
Mobile phase A: Water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01 M ammonium formate

Gradient (isocratic): Time flow (mL/min) %A
(mins)
0 0.4 100
4.0 0.4 100

Injection volume: 20 µL (full loop)
Strong injector wash : Water/methanol/acetonitrile/isopropanol 25/25/25/25 v/v/v/v
Weak injector wash : Water/methanol 90/10 v/v

Details on mating procedure:
Mating
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.

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 occupants.
Duration of treatment / exposure:
Day 6 to 19 of mating
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Duration of test:
Day 0-6: Mating
Day 6-19: Treatment
Day 20: Necropsy
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control - vehicle only
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Animal Model
The rat (virgin) 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 dose levels selected for investigation in this main study for effects on embryo-fetal development (0, 10, 30 and 80 mg/kg/day) were selected in conjunction with the Sponsor and were based on the results of a preliminary study for effects on embryo-fetal development study (Covance Study No. 8434891). In that study, pregnant female Crl:CD(SD) rats received Ethyldiisopropylamine at doses of 25, 50 and 100 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.

In the preliminary study, clinical signs were generally evident for females that received 100 mg/kg/day comprising decreased activity, rales, piloerection, partially closed eyelids, prominent eyes, uncoordinated gait and whole body pallor. There were no treatment related clinical signs for females that received 25 or 50 mg/kg/day. Furthermore, body weight losses were evident in the 100 mg/kg/day group and group mean body weight gain for this group was low when compared to the controls. For females that received 25 or 50 mg/kg/day, there was a suggestion of slightly reduced body weight gain when compared to the controls. Group mean food intake was reduced for females that received 100 or 50 mg/kg/day when compared to the controls and there was no effect of treatment on food intake for females that received 25 mg/kg/day.

For females that received 100 or 50 mg/kg/day, there were no abnormalities of the dams or fetuses at the macroscopic examination.

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 to 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 after mating inclusive.

Thyroid Hormone Analysis
Blood samples were collected at the following occasion:
Occasion Animals
At termination All adult females

Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)
Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane.
Anticoagulant None.
Tubes Greiner Minicollect tubes with clotting activator.
Blood volume 1.0 mL.
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Number of aliquots Two per animal. Aliquot 1: 0.2 mL serum for T3/T4
Aliquot 2: residual serum for TSH
Final storage conditions Deep frozen (approximately -60°C to -80¿C) pending analysis.
Fate of samples Aliquot 1 (T3 and T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2: dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.
T3 and T4 Performed by the Department of Department of Bioanalysis, Covance.
The method of analysis and results are presented in Attachment 13.3.
TSH Performed by the Department of Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.

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.

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin.

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.

Thyroid and abnormalities All adult females.
Routine staining Sections were stained with hematoxylin and eosin.

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill All animals from all groups. Thyroid gland only.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Ovaries and uterine content:
The following were recorded for all animals:
Uterus Gravid uterine weight (including cervix and ovaries).

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

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. Fetuses examined externally with abnormalities recorded. The sex and ano-genital distance 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 stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.

Fetal, Litter and Placental Weights
Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.

Group mean values and SD were calculated using individual litter mean values.

Ano-Genital Distance
Ano-genital distance were presented both as absolute/unadjusted and adjusted for fetal body weight, using the weight recorded at necropsy.

Detailed Fetal Examination
Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.

Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences.

Findings observed were classified, according to severity and incidence, as:

Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.

Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.

Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

Observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.
Statistics:
Please refer to "Any other information 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 were calculated from the individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs associated with treatment were evident from Day 6 of gestation, at 30 or 80 mg/kg/day. There were no signs associated with dosing at 10 mg/kg/day.

Partially closed eyelids, with or without piloerection were observed in several animals at 1 to 2 hours after dosing at 30 or 80 mg/kg/day that persisted in some animals at the end of the day and were evident in some animals at each dose before dosing on Days 17 19. The incidence of these signs tended to increase during the treatment period. In addition, underactive behaviour was evident on Day 6 at one to two hours after dosing in one animal (No. 54) at 30 mg/kg/day and one animal (No. 72) at 80 mg/kg/day and on Day 7 in one animal (No. 65) at 80 mg/kg/day. One animal (No. 79) at 80 mg/kg/day had hunched posture on Day 14.
Mortality:
no mortality observed
Description (incidence):
There were no premature deaths throughout the duration of study
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A marked and biologically relevant mean body weight loss of -16g was evident during the first two days of treatment (Days 6 to 8 of gestation) at 80 mg/kg/day (individual body weight losses were -7 g to -32 g). Subsequent mean body weight gain was similar to Control from Day 8. Overall body weight gain (Days 6-20) at 80 mg/kg/day was slightly, but statistically significantly low (82% of Control (p<0.01)).
Body weight gain in females receiving 10 or 30 mg/kg/day was unaffected by treatment.

Gravid uterine weight was unaffected by treatment at 10, 30 or 80 mg/kg/day.
Adjusted body weight change was low at 10 or 30 mg/kg/day (both 65% of Control); adjusted body weight change was markedly low and adverse at 80 mg/kg/day (47% of Control); statistical significance (all p<0.01) was attained by all groups.

PLEASE REFER TO THE ATTACHED TABLES: "BODY WEIGHT AND BODY WEIGHT CHANGES - GROUP MEAN VALUES DURING GESTATION"
"GRAVID UTERINE WEIGHT, BODY WEIGHT AND ADJUSTED BODY WEIGHT CHANGE - GROUP MEAN VALUES ON DAY 20 OF GESTATION"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall food consumption was statistically significantly low at 80 mg/kg/day (84% of Control (p<0.01)) during the period of treatment (measured Days 6-20 of gestation); the greatest reduction in food intake was seen during Days 6 to 10 of gestation (57% of Control) that was considered adverse, after which food intake improved.
Food consumption at 30 mg/kg/day was marginally, but statistically significantly low during Days 6 to 10 (p<0.01) and Days 18 to 20 (p<0.05) of gestation (91% and 93% of Control, respectively) the small difference from Control was considered not to be adverse.
Food consumption at 10 mg/kg/day was unaffected by treatment.
Food efficiency:
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:
no effects observed
Description (incidence and severity):
Absolute and adjusted thyroid weight was unaffected by treatment at 10, 30 or 80 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related macroscopic findings in adults at 10, 30 or 80 mg/kg/day.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or were as expected for Sprague Dawley rats of this age; therefore, they were considered not Ethyldiisopropylamine-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related microscopic findings in the thyroid at 10, 30 or 80 mg/kg/day.
All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or their severity was as expected for Sprague Dawley rats of this age. Consequently, they were considered not Ethyldiisopropylamine-related.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Rats do not abort
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
30 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):
overall fetal weights were unaffected by treatment at 10, 30 or 80 mg/kg/day.

PLEASE REFER TO THE ATTCHED TABLE: "PLACENTAL, LITTER AND FETAL WEIGH - GROUP MEAN VALUES ON DAY 20 OF GESTATION"
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
All animals surviving to scheduled termination were pregnant. The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
All animals surviving to scheduled termination were pregnant. The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
All animals surviving to scheduled termination were pregnant. The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.

PLEASE REFER TO THE ATTCHED TABLE: "PLACENTAL, LITTER AND FETAL WEIGH - GROUP MEAN VALUES ON DAY 20 OF GESTATION"
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
When compared with Control, anogenital distance was unaffected by maternal treatment at 10, 30 or 80 mg/kg/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment at 10, 30 or 80 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLE: FETAL EXAMINATIONS - MAJOR ABNORMALITY FINDINGS - GROUP INCIDENCES"
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment at 10, 30 or 80 mg/kg/day.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed on embryo fetal development

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
80 mg/kg bw/day (actual dose received)
Treatment related:
no

Any other information on results incl. tables

Formulation Analysis


The mean achieved concentrations for Ethyldiisopropylamine in formulations prepared for the first week of treatment were low, ranging from 8.4% (Group 2 (10 mg/kg/day) to 91.6% (Group 4) of nominal. It was considered that the test item evaporated, due to its high volatility into the headspace above the 1 mL sample taken for analysis in a 20 mL vial. 


To prevent potential losses due to headspace in the sample vial, the remaining formulations prepared for the study were sampled directly into volumetric flasks, containing diluent water. The mean analyzed concentrations of the 2nd and 3rd formulations were within -15%/+10% of nominal concentrations, confirming the accuracy of formulation. It was therefore concluded that formulations had been prepared accurately on the study.


The difference from mean remained within 3.3%, confirming precise analysis.


Analyzed Concentrations for Ethyldiisopropylamine in 1% Methylcellulose plus 0.1% Tween 80























































































































































Occasion



Group



Nominal concentration (mg/mL)



Analyzed concentration


(mg/mL)



RME (%)



Difference from mean (%)



Procedural recoveries (%)



Analysis 1



Analysis 2



Mean



At analysis



Mean



14Sep2020



1



0



ND



ND



ND



-



-



-



-



2



5 1



0.429



0.406



0.418



-91.6



±2.75



98.2



99.2



3



15 1



7.32



7.06



7.19



-52.1



±1.81



102.0



4



40 1



33.0



32.2



32.6



-18.5



±1.23



97.3



17Sep2020



1



0



ND



ND



ND



-



-



-



-



2



5 2



4.45



4.41



4.43



-11.4



±0.45



99.0



101.3



3



15 2



15.7



16.7



16.2



+8.0



±3.09



101.3



4



40 2



39.4



39.5



39.5



-.1.3



±0.13



103.5



23Sep2020



1



0



ND



ND



ND



-



-



-



-



2



5 2



4.43



4.51



4.47



-10.6



±0.89



97.8



94.0



3



15 2



13.7



14.3



14.0



-6.7



±2.14



95.3



4



40 2



35.8



38.2



37.0



-7.5



±3.24



88.8



RME      Relative mean error, representing the deviation from nominal


ND          Not detected


1              Samples (1 mL) supplied in 20 mL glass vials.  It is thought that the low analyzed concentrations were due to losses of Ethyldiisopropylamine to the headspace in the vials


2              Samples (1 mL) transferred directly into volumetric flasks containing diluent water, in order to prevent losses to headspace


Mean analyzed concentrations were calculated using unrounded figures.


 Thyroid Hormone Analysis


Attachment 13.3, Attachment 13.4, Attachment 13.6


On Day 20 of gestation, mean serum T3 and T4 and TSH concentrations were statistically significantly low (81%, 81% and 67% of Control, respectively) at 80 mg/kg/day. These were considered non-adverse (See Section 7).


Summary of adult thyroid hormone results on Day 20 of gestation and the HCD ranges





















































Group



1



2



3



4



Dose (mg/kg bw/day)



0



10



30



80



Triiodothyronine (pg/mL)



383



352



374



310**



HCD (pg/mL)



290 - 643



Thyroxine (pg/mL)



12100



11100



11300



9790*



HCD (pg/mL)



8945 - 21790



Thyroid Stimulating Hormone (pg/mL)



1140



928



881



762*



HCD (pg/mL)



374 - 3054



*  p<0.05


**  p<0.01

Applicant's summary and conclusion

Conclusions:
The maternal No-Observed-Adverse-Effect-Level (NOAEL) was 30 mg/kg bw/day, based on the adverse loss in maternal body weight during the first two days of treatment (Days 6 and 7 of gestation), the adverse low adjusted body weight change and the low food consumption during Days 6-10 of gestation at 80 mg/kg/day. The embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 80 mg/kg bw/day.

Executive summary:

SUMMARY


The purpose of this study was to assess the influence of Ethyldiisopropylamine (an industrial chemical), on embryo-fetal survival and development in the Sprague Dawley rat, when administered during the organogenesis and fetal growth phases of pregnancy. Three groups of 20 females received Ethyldiisopropylamine at doses of 10, 30 or 80 mg/kg/day by oral gavage administration at a volume dose of 2 mL/kg bwt, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1% w/v methylcellulose plus 0.1% Tween 80 at the same volume dose as the 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. Gravid uterine and thyroid weights were recorded. A microscopic pathology investigation was performed on the thyroid from all pregnant animals. The ano-genital distance, individual body weight and placental weights were recorded for all fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. Blood samples were taken at necropsy for subsequent thyroid hormone analysis in all pregnant animals.


Results


There were no premature deaths throughout the duration of study.


All animals remained in good clinical condition throughout the study period at 10, 30 or 80 mg/kg/day.


At 30 or 80 mg/kg/day, partially closed eyelids, with or without piloerection were evident from the first day of treatment and persisted in some animals at the end of the day and, at the end of the treatment period (Days 17-19) in some animals before dosing the next day. The incidence of these signs increased during the treatment period. One or two animals at 30 or 80 mg/kg/day were underactive during the first two days of dosing and one animal at 80 mg/kg/day had hunched posture on Day 14 of gestation. There were no signs associated with dosing at 10 mg/kg/day.


A marked and biologically relevant mean body weight loss of -16 g was evident following the first two doses at 80 mg/kg/day (Days 6 and 7 of gestation); overall body weight gain (Days 6-20) was low.


Adjusted body weight change was low at 10 or 30 mg/kg/day and was markedly low and adverse at 80 mg/kg/day; gravid uterine weight was unaffected by treatment at 10, 30 or 80 mg/kg/day.


Food consumption at 80 mg/kg/day was adversely low during Days 6-10 of gestation (57% of Control) and remained slightly low during Days 10-20 of gestation.


Body weight gain in females receiving 10 or 30 mg/kg/day was unaffected by treatment.


Reproductive performance (numbers of resorptions, implantation losses and live young, the ratio of male to female fetuses, placental, total litter or overall fetal weights) and fetal ano-genital distance was unaffected by treatment at 10, 30 or 80 mg/kg/day.


The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment at 10, 30 or 80 mg/kg/day.


On Day 20 of gestation, maternal serum concentrations of Triiodothyronine, Thyroxine and Thyroid Stimulating Hormone were slightly, but statistically significantly low at 80 mg/kg/day, but as absolute and adjusted maternal thyroid weight was unaffected and there were no treatment related microscopic findings in the thyroid at 10, 30 or 80 mg/kg/day and, in the absence of embryo-fetal developmental changes, the findings were considered non-adverse.


Conclusion


The maternal No-Observed-Adverse-Effect-Level (NOAEL) was 30 mg/kg bw/day, based on the adverse loss in maternal body weight during the first two days of treatment (Days 6 and 7 of gestation), the adverse low adjusted body weight change and the low food consumption during Days 6-10 of gestation at 80 mg/kg/day. The embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 80 mg/kg bw/day.