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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Ethylenediamine, propoxylated
IUPAC Name:
Ethylenediamine, propoxylated
Specific details on test material used for the study:
Test item: Ethylenediamine, propoxylated
Test item identity (including alternative names):
EDA-PO
1,2-Ethanediamine, polymer with methyloxirane
Voranol RA 640 polyol
Intended use: Industrial Chemical
Appearance: Colorless to yellow liquid
Storage conditions: At ambient temperature (15 to 25#C), protected from moisture.
Supplier: Sponsor
Batch number: F359KAPL12
Expiry date: 25 October 2022
Purity: 100% (UVCB substance)
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan®:WIST rat.
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan®; WIST) 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:
Animal Information:
Strain/Species: RccHan®:WIST rat.
Supplier: Envigo RMS (UK)
Number of animals ordered: 44 males and 48 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization:
Males: seven days before commencement of treatment.
Females: 21 days before commencement of treatment.
Age of the animals at the start of treatment:
Males: 85 to 91 days old.
Females: 99 to 105 days old.
Weight range of the animals at the start of treatment:
Males: 321 to 359 g.
Females: 206 to 243 g.
Allocation and Identification:
Allocation:
On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management.The groups were adjusted to reduce inter /intra-group variation.
Identification of animals:
Each adult animal was assigned a number and identified uniquely within the study by a microchip
before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of
age, using a toe tattoo.
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 Replacement:
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation:
Body weight range extremes: three males and two females
Irregular cycle: one female
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 degrees 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 appr
opriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
Cage distribution: The cages were distributed on the racking to equalize, as far as possible, en
vironmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage:
Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter
Diet Supply:
Diet SDS VRF1 Certified pelleted diet.
A sample (250 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30 degrees C) until finalization of the report. Samples will be discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted (removed during the period of urine collection during Week 5 only).
Water Supply:
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted (removed during the period of urine collection during Week 5 only).

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Route of Administration:
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure and the preferred route of exposure specified in the OECD 422 guideline.
Vehicle:
water
Details on oral exposure:
Method of preparation:
Starting with the highest concentration, the required amount of test item was weighed and sufficient vehicle was added to obtain a solution. The mixture was transferred and dissolved and mixed using a magnetic stirrer. The test item was fully washed from any containers a minimum of three times or until visibly clean. The pH was measured and adjusted to pH 3-9 using HCl/NaOH. The remaining vehicle was added to achieve the required volume. The formulation was then stirred for a minimum of 20 minutes using a magnetic stirrer resulting in a solution. The stirring was continued. For further concentrations, the required volume was used from the relevant higher concentration and the same method was used.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2 to 8°C).
Test item accounting: Detailed records of compound usage were maintained. The amount of test
item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis:
Stability and homogeneity: The stability of the test item in the vehicle was determined in Covance Study Number 8450936. Stability was confirmed at ambient temperature (15 to 25°C) for 24 hours and at refrigerated temperature (2 to 8°C) for 15 days.

Achieved concentration: Samples of each formulation prepared for administration for the first preparation of treatment, in Week 5 and for the final preparation of treatment were analyzed for achieved concentration of the test item
Duration of treatment / exposure:
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route: Oral gavage using a suitably graduated syringe and a flexible cannula inserted via the mouth.
Prior to the dosing of each individual animal, the cannula was dipped into a container filled with 5% glucose solution to aid intubation.
Treated at: Constant doses in mg/kg/day.
Volume dose : 10 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as treated groups.
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.
Duration of Treatment:
Males: Two weeks pre-pairing up to necropsy after minimum of four weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Animals of the F1 generation were not dosed.
Frequency of treatment:
Frequency: Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection:
The dose levels for investigation were selected in conjunction with the Sponsor and based on the
results of a preliminary toxicity study Labcorp Study No. 8451122. In that study the systemic toxic potential of EDA-PO was assessed over a period of 14 days in Han Wistar rats at dose levels of 250, 500 and 1000 mg/kg/day.
During the preliminary study, there were no premature deaths and no treatment-related clinical signs or post-dose observations. There was no adverse effect of treatment on body weight gain and no effect of treatment on food or visual water consumption. Liver weights were slightly increased in males at 1000 mg/kg/day. There were no treatment-related macroscopic findings in either sex at any dose level investigated.
Based on the results of the preliminary study, 1000 mg/kg/day was selected as the high dose for
investigation in this main OECD 422 study. Low and intermediate dose levels were set at 100 and 300 mg/kg/day, respectively, in order to fulfill the 2-fold to 4-fold dosing interval as specified in the test guideline.

Examinations

Observations and examinations performed and frequency:
Parental animals: Observations and examinations
Clinical and Behavioral 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 performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males:
Week 1 - daily
Week 2 to 4 - twice weekly
Week 5 onwards - once each week
F0 females:
Week 1 - daily
Week 2 - twice during the week
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded 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.

Detailed physical examination and arena observations:
Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength:
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. At Days 7-9 of lactation, females were moved into individual cages prior to transport to the testing room. Animals were removed and returned to their home cages by an assistant, such that the observer was unaware of the treatment group. The cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

The following measurements, reflexes and responses were recorded:
Approach response:
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers).

Pinna reflex:
The inside of one ear was touched lightly with a nylon filament.

Auditory startle reflex:
The animal’s response to a sudden sharp noise was assessed.

Tail pinch response:
The animal’s tail was pinched sharply with forceps approximately one third from the tip.

Grip strength:
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

Motor activity:
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Labcorp.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body Weight:
The weight of animals was recorded as follows:
F0 males:
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.
F0 females:
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7, and 13 of lactation.
On the day of necropsy.

Food Consumption:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals:
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-7, 7-14 and 14-20 after mating
Days 1-4, 4-7 and 7-13 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Parturition Observations and Gestation Length:
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Urinalysis:
Animals were placed in an individual metabolism cage, (without food or water for Week 5 only). Urine samples were collected overnight at the following occasions:
Occasion/Animals
Timed urine collection - immediately following the first dose/The two lowest numbered males per group.
Week 5 of treatment*/The five lowest numbered males per group.
Timed urine collection - immediately following the last dose/The two lowest numbered males per
group.
* At least two days before the timed urine collection following the last dose

Oestrous cyclicity (parental animals)
Estrous Cycle:
Dry and wet smears were taken as follows:
Dry smears: For 15 days before pairing using cotton swabs.
Wet smears:
Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles were not allocated to study.
After pairing until mating.
For four days before scheduled termination (nominally Days 10 to 13 of lactation).

Litter observations:
Records Made During Littering Phase:
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7, 11 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.
Sacrifice and pathology:
Postmortem examinations (parental animals)
Terminal Investigations:
Method of Sacrifice:
All adult animals Carbon dioxide asphyxiation with subsequent exsanguination (No exposure to carbon dioxide occurred until after the completion of blood sampling for thyroid hormone assays).
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age (Decapitation).
Offspring - not selected for thyroid hormone sampling (Intraperitoneal injection of sodium pen
tobarbitone).
Sequence: To allow satisfactory inter-group comparison.

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.

Time of Necropsy:
F0 males: After final investigations completed (after at least four weeks of treatment).
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
F1 offspring: Day 13 of age.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1 and Table 2 for F0 animals.

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 with the exception of those detailed below:
Testes: In modified Davidson’s fluid.
Eyes: In Davidson’s fluid.
Females:
The following were recorded:
Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection.

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.
Full List: All animals killed prematurely.
The five lowest numbered surviving males and the first five lactating females with a surviving litter in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light Microscopy:
Tissues preserved for examination were examined as follows:
Category/Animals/Tissues
Premature deaths/F0 males and F0 females in Groups 1 and 4 only/Tissues specified in Pathology Procedures Tables.
Scheduled kill/Five lowest numbered F0 males and first five lactating females with a surviving litter in Groups 1 and 4/Tissues specified in Pathology Procedures Tables.
Scheduled kill/All F0 animals/Abnormalities 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.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Hematology, Peripheral Blood:
Blood samples were collected at the following occasion:
Occasion/Animals:
At termination/The five lowest numbered males per group.
At termination/The first five lactating females with a surviving litter per group.

Blood Chemistry:
Blood samples were collected at the following occasion:
Occasion/Animals:
At termination/The five lowest numbered males per group.
At termination/The first five lactating females with a surviving litter per group.

Thyroid Hormone Analysis:
Blood samples were obtained as follows:
Occasion/Animals
At termination/All surviving F0 males.
At termination/All surviving F0 females (no samples were obtained from animals which failed to litter).

Toxicokinetics:
Blood samples were obtained from study animals on Study Day 1.
Blood samples were obtained from study animals one hour after dosing on Study Day 7 (males and females), Study Day 28 (males only) and Day 17 after mating (females only).
See the Toxicokinetic Sampling Schedule attachment for details.

Postmortem examinations (offspring)
Offspring:
Premature deaths: Where possible, a fresh external macroscopic examination with an assessment of stomach for milk content was performed. Grossly externally abnormal pups were retained.
F1 offspring on Day 4 of age:
Blood sampling required.
Externally normal offspring discarded without examination.
Externally abnormal offspring identified on despatch to necropsy; examined externally, and retained pending possible future examination.
Offspring at scheduled termination:
Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in appropriate fixative.
Thyroid glands were preserved from one male and one female in each litter, where possible.
Thyroid Hormone Analysis:
Blood samples were obtained as follows:
Day 4 of age Offspring: up to two females per litter (where possible; male pups were reserved for nipple retention evaluation):
• one for T4 (serum)#
• one for TSH (serum)
# Priority given to T4 sample.
No offspring were allocated to these procedures on Day 4 of age if:
• the resultant live litter size would fall below eight offspring
• the resultant number of female pups would fall below three offspring
• If only four female offspring were available within a litter but the overall litter size was >eight, one female was selected with priority given to the T4 sample.
Day 13 of age F1 offspring, two males and two females per litter (where possible)
• two for T4 (serum); where possible one male and one female#
• two for TSH (serum); where possible one male and one female
# Priority given to T4 sample.

Note:
Thyroid hormone analysis:
Performed by the Department of Bioanalysis, Labcorp.
Samples from offspring on Day 13 of age and F0 males were assessed for levels of Thyroxine (T4).
As there was considered no effect of treatment upon serum T4 concentration, no analysis of T4 samples from F0 females and offspring on Day 4 of age or any TSH samples was required.
Optional endpoint(s):
Reproductive indices:
Pre-Coital Interval:
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Mating Performance and Fertility:
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = Number of animals mating/Animals paired x 100

Conception rate (%) = Number of animals achieving pregnancy/Animals mated x 100
Fertility index (%) = Number of animals achieving pregnancy/Animals paired x 100

Gestation Length and Index:
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = Number of live litters born/Number pregnant x 100

Offspring viability indices:
Litter Size:
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling) and 13 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival Indices:
The following were calculated for each litter:
Post-implantation survival index (%) = Total number of offspring born/Total number of uterine im
plantation sites x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering/Total number of offspring born x 100
Viability index (%) = Number of live offspring on Day 4 (before blood sampling)/Number live offspring on Day 1 after littering x 100

Lactation index (%) = Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after blood sampling) x 100

Group mean values were calculated from individual litter values.

Offspring Sex Ratio:
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.
Percentage males = Number of males in litter/Total number of offspring in litter x 100

Group mean values were calculated from individual litter values
Statistics:
Statistical Analysis:
Statistical analyses were performed on the majority of data presented and results of these tests,
whether significant or non significant, were determined. For some parameters, including estrous cycles before treatment, estrous cycles during treatment, pre coital interval, mating performance, gestation length and index and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food
consumption (except during gestation and lactation) were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs or signs observed following dose administration for either sex.
Transient signs of salivation and chin rubbing were observed post-dose in one male that received 1000 mg/kg/day on Day 31 of treatment, however, this is a common finding following the administration of an industrial chemical via oral gavage, usually due to poor palatability, and not considered to be related to EDA-PO administration. Signs of partially closed eyelids were observed post-dose in two males that received 1000 mg/kg/day, one on Day 1 of treatment and the other on Day 36 of treatment, however, these observations were transient in nature.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female that received 1000 mg/kg/day was euthanized for welfare reasons due to being in general poor clinical condition on Day 24 of gestation. She had given birth to one pup but her clinical condition was deteriorating; with clinical signs of irritable behaviour and piloerection and in addition, she had failed to complete parturition after several checks. Macroscopic examination of this female revealed abnormal dark contents in the cecum, ileum and jejunum and abnormal contents (pup material) in the stomach as well as 6 dead fetuses in the uterus. Microscopic examination of this female revealed slight extramedullary hemopoiesis in the spleen, moderate diffuse atrophy of the thymus and slight decreased glycogen in the liver. Since this was an isolated occurrence, and in the absence of any effect on reproductive performance, this premature death was not considered to be treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following the commencement of treatment, male body weight gain at 1000 mg/kg/day was slightly low between Weeks 0-1 and was statistically significantly lower than Control during Weeks 2-3. This was reflected in the overall male body weight gain (Weeks 1-5) which was slightly low compared to control, however statistical significance was not attained.
Body weight change was low for males that received 100 mg/kg/day between Weeks 2-3 and Weeks 4-5 and for males that received 300 mg/kg/day between Weeks 4-5 but overall body weight gain (Weeks 1-5) for males at 300 or 100 mg/kg/day was similar to Control.
There was no adverse effect of treatment on body weight gain for females prior to pairing or during gestation at any dose level investigated. It was noted that at 1000 mg/kg/day, female body weight gain was high, compared to Control, during Days 7-20 of gestation such that overall body weight change at 1000 mg/kg/day (Days 0-20 of gestation) was statistically significantly higher than Control.
During lactation, body weight gain for all treated groups of females was low during Days 1-4 and remained low during Days 4-7 at 1000 or 300 mg/kg/day, however, there was no clear effect of treatment on overall weight gain during lactation (Days 1-13 of lactation) at either dose level.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on male food intake at any dose level investigated.
For females at 1000 mg/kg/day, food intake was slightly reduced, compared to control, during Week 1 prior to pairing, however, statistical significance was not attained. Food intake for females at 1000 mg/kg/day during gestation and lactation was not adversely affected by treatment. It was noted that food intake for females at 1000 mg/kg/day during Days 7-20 of gestation was high, compared to Control. There was no effect of treatment on female food intake at 300 or 100 mg/kg/day.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematology investigations at scheduled termination of males and females revealed no conclusive treatment-related effects.
In males, reticulocyte count was statistically significantly higher than Control at 1000 mg/kg/day,
however, no similar change was observed in females.
In females, red cell distribution width (%) was statistically significantly higher than Control at 1000 mg/kg/day, however no similar change was observed in males.
All other differences from Control, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related consequently were considered to represent normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry investigations at scheduled termination of males and females revealed that bile acid concentration was higher than Control at 1000 or 300 mg/kg/day in both sexes, however, statistical significance was not attained at either dose level in either sex.
In males, chloride concentration was statistically significantly lower than Control at 1000 mg/kg/day, however, no similar change was observed in females.
In males, calcium concentration was statistical significantly higher than Control for all treated groups, however, no dose response was apparent and no similar change was observed in females.
In females, cholesterol concentration was statistically significantly higher than Control at 1000 mg/kg/day, however, no similar change was observed in males.
In females, albumin globulin ratio was statistically significantly higher than Control in all treated groups, however, no dose response was apparent and no similar change was observed in males.
All other differences from Control, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related consequently were considered to represent normal biological variation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinalysis investigations for males during Week 5 of treatment revealed that urinary pH was statistically significantly higher than Control for all groups of treated males, however, no dose response was apparent.
Urinary specific gravity was statistically significantly higher than Control for males at 1000 mg/kg/day.
Urinary chloride concentration for males at 1000 mg/kg/day was statistically significantly higher than Control. A review of the individual data revealed this was predominantly due to one male (4M 22) with an atypically high urinary chloride concentration.
All other differences from Control, including those that attained statistical significance, were generally small or the magnitudes were not dose-related consequently were considered to represent normal biological variation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and body weight relative mean liver weights of F0 males that received 1000 or 300 mg/kg/day and F0 females that received 1000 mg/kg/day were high when compared with Controls. The body weight relative mean liver weight of F0 females that received 300 mg/kg/day was also high when compared with Controls.
All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Description (incidence and severity):
Sensory Reactivity Observations and Grip Strength:
No treatment-related effects were observed for sensory reactivity and grip strength assessment during Week 5 of treatment for males or during Days 7-9 of lactation for females.
Motor Activity:
Motor activity assessment of males during Week 5 of treatment and females during Days 7-9 of lactation revealed no treatment-related effects.
Group mean activity scores for both treated males and females showed some inter-group variation and several individual occasions of statistical significances were achieved, however, there was no dose-relationship and no clear evidence of a treatment-related effect.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related findings at microscopic examination of the F0 animals. All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Han Wistar rats of this age. Therefore, they were considered not test item-related.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Oral gavage administration of the test material at dose levels of 100, 300 or 1000 mg/kg/day was well tolerated. Based on the results of this study, the NOAEL for systemic and reproductive/developmental toxicity was 1000 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Formulation Analysis:
The mean concentrations of EDA-PO in formulations were within 9% of the nominal concentration,confirming the accuracy of formulation. The difference from mean remained within 3%, confirmingprecise analysis. This is with the exception of last preparation for Group 3 where the difference frommean was 8.33%.



Thyroid Hormone Analysis:


The purpose of this phase of the study was to measure total Thyroxine (T4) concentrations in rat serum using liquid chromatography with tandem mass spectrometry detection (LC-MS/MS).


Serum concentrations of T4 were measured using a surrogate matrix calibration range of 700 to 70000 pg/mL.


As per study plan, 120 serum samples from main phase F0 males; and F1 offspring (male and female) in Groups 1-4, were expected to be received of which 112 samples were received and analyzed to measure T4 concentrations. All 112 samples received were analyzed within their storage stability period (T4 storage stability in rat serum was established for 387 days at ‑70ºC in the validation study). The samples were extracted within 114 days following sample collection.


The accuracy of the bioanalytical method, as assessed by the results of calibration standards and Quality Control (QC) samples analyzed with the batch of test samples, was satisfactory.


The integrity and quality of the bioanalytical results generated in this phase of the study are assured and confirmed.


All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.


Treated groups were compared with a Control group using Williams’ test.


No statistically significant difference (p-value > 0.01) was observed between the F0 Adult Terminal Males Treatment Groups 2 - 4 and control group (Group 1) using a Williams test (two tailed).


No statistically significant difference (p-value > 0.01) was observed between the F1 Male offspring Treatment Groups 2 - 4 and control group (Group 1) using a Williams test (two tailed).


No statistically significant difference (p-value > 0.01) was observed between the F1 Female offspring Treatment Groups 2 - 4 and control group (Group 1) using a Williams test (two tailed).


No unexpected events or deviations occurred in the T4 analysis of serum samples.

Applicant's summary and conclusion

Conclusions:
Oral gavage administration of Ethylenediamine, propoxylated (EDA-PO) to Han Wistar rats at dose levels of 100, 300 or 1000 mg/kg/day was generally well tolerated. Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity and for reproductive/developmental toxicity was concluded to be 1000 mg/kg/day.
Executive summary:

The purpose of this study was to assess the general systemic toxic potential of Ethylenediamine,propoxylated (EDA-PO) in Han Wistar rats, including a screen for reproductive/developmental effectsand assessment of endocrine disruptor relevant endpoints, with administration of EDA-PO (an industrialchemical) by oral gavage administration for at least four weeks.
Three groups of ten male and ten female rats received EDA-PO at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration at a dose volume of 10 mL/kg bw/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated
daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, purified water, over the same treatment period and at
the same volume dose as treated groups.
During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.
The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.


 


Results:
The mean concentrations of Ethylenediamine, propoxylated (EDA-PO) in formulations were within 9% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis.



Thyroxine (T4) - No statistically significant difference (p-value > 0.01) was observed between the F0 Adult Terminal Males Treatment Groups 2 - 4 and control group (Group 1) using a Williams test (two tailed). No statistically significant difference (p-value > 0.01) was observed between the F1 Male offspring Treatment Groups 2 - 4 and control group (Group 1) using a Williams test (two tailed). No statistically significant difference (p-value > 0.01) was observed between the F1 Female offspring Treatment Groups 2 - 4 and control group (Group 1) using a Williams test (two tailed).



Among the F0 adults there were no treatment-related premature deaths, no treatment-related signs were observed following dose administration or during detailed physical examination and arena observation
and there was no effect of treatment on sensory reactivity, grip strength or motor activity. Following the commencement of treatment, male body weight gain at 1000 mg/kg/day was slightly low between Weeks 0-1 and was statistically significantly lower than Control during Weeks 2-3. This was reflected in the overall male body weight gain (Weeks 1-5) which was low compared to control, however
statistical significance was not attained. Body weight change was low for males that received 100 mg/kg/day between Weeks 2-3 and Weeks 4-5 and for males that received 300 mg/kg/day between Weeks 4-5 but overall body weight gain (Weeks 1-5) for males at 300 or 100 mg/kg/day was similar to Control.
There was no adverse effect of treatment on body weight gain for females prior to pairing or during gestation at any dose level investigated. During lactation, body weight gain for all treated groups of females was low during Days 1-4 and remained low during Days 4-7 at 1000 or 300 mg/kg/day, however, there was no clear effect of treatment on overall weight gain during lactation (Days 1-13 of lactation) at either dose level.
There was no effect of treatment on male food intake at any dose level investigated. For females at 1000 mg/kg/day, food intake was slightly reduced, compared to control, during Week 1 prior to pairing, however, statistical significance was not attained. Food intake for females at 1000 mg/kg/day during
gestation and lactation was not adversely affected by treatment. There was no effect of treatment on female food intake at 300 or 100 mg/kg/day.
There was no effect of treatment on estrous cycles, pre-coital interval, mating performance and fertility, gestation length or gestation index at any dose level investigated.
Haematology examination of the peripheral blood at scheduled termination of the adult males and females revealed no toxicologically significant differences from Control.
Blood chemistry investigation at scheduled termination of males and females revealed that bile acid concentration was higher than Control at 1000 or 300 mg/kg/day in both sexes. In males, chloride concentration was statistically significantly lower than Control at 1000 mg/kg/day and calcium concentration was statistical significantly higher than Control for all treated groups. In females,
cholesterol concentration was statistically significantly higher than Control at 1000 mg/kg/day and albumin globulin ratio was statistically significantly higher than Control in all treated groups.
Urinalysis investigations for males during Week 5 of treatment revealed that urinary pH was statistically significantly higher than Control for all treated groups, urinary specific gravity was statistically significantly higher than Control for males at 1000 mg/kg/day and urinary chloride concentration for males at 1000 mg/kg/day was statistically significantly higher than Control.
Mean absolute and body weight relative liver weights of F0 males that received 1000 or 300 mg/kg/day and F0 females that received 1000 mg/kg/day were high when compared with Controls. The body weight relative mean liver weight of F0 females that received 300 mg/kg/day was also high when compared with Controls.
There were no treatment-related findings at macroscopic or microscopic examination of the F0 animals.