Ethylenediamine, propoxylated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Unaudited Draft Report: Received on 12/6/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethylenediamine, propoxylated

EC Number:

500-035-6

EC Name:

Ethylenediamine, propoxylated

Cas Number:

25214-63-5

Molecular formula:

(C3H6O)n (C3H60)n (C3H6O)n(C3H6O)n C2H8N2 sum of n: >1 - 8.5

IUPAC Name:

Ethylenediamine, propoxylated

Specific details on test material used for the study:

Test Item: Ethylenediamine, propoxylated
CAS Number: 25214-63-5
Batch number: F359KAPL12
Purity: 100% (UVCB substance)
Appearance: Colorless to yellow liquid
Expiry date: 25 October 2022
Storage conditions: Room temperature protected from moisture

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

Animal Specifications and Acclimation:
Eighty six time mated Crl:WI(Han) rats were received on 02 and 04 June 2022 from Charles River Laboratories, Kingston, New York. Animals acclimated for 3 or 4 days.
At initiation of dosing, animals were 11 to 12 weeks old, and body weights ranged from 176 to 231 g.

Environmental Conditions, Diet, and Water:
Housing:
Animals were individually housed in polycarbonate cages with Diamond Soft® bedding.
Water:
Water was provided ad libitum.
Diet:
Animals were offered Certified Rodent Diet #2016C (Envigo RMS, Inc.) ad libitum.
Environment:
Environmental controls were set to maintain a temperature range of 20 to 26C, a relative humidity range of 30 to 70%, 10 or greater air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions are maintained in the raw data and had no effect on the outcome of the study.
Environmental Enrichment:
Animals were given various cage-enrichment devices as environmental enrichment.

Animal Identification and Assignment to Study:
Mating was performed at the supplier. Males of the same strain were used for mating. Mating records were provided by the supplier and were maintained in the study record; males used for breeding were each only used one time. Gestation Day 0 was designated as the day mating was confirmed. Females were shipped prior to GD 4. Animals were eliminated from consideration for study selection based on data collected during acclimation (pre-dose phase). Animals were assigned to the study using a computerized procedure designed to achieve body weight balance with respect to subgroup assignment. Prior to group assignment, animals were excluded from the selection pool to produce minimal variation. After subgroup assignment, it was established that homogeneity of variance was achieved at the 5.0% probability level, as indicated by Levene’s test for heterogeneity of variance. Additionally, the mean body weight for each subgroup/sex was not statistically different at the 5.0% probability level, as indicated by analysis of variance F probability. Animals not used on study were removed from the study room.
Following randomization, performed prior to GD 4, each animal was assigned a unique number by means of an implantable microchip identification device, cage card and/or tail mark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified Water

Details on exposure:

Vehicle Control Item: The vehicle control item was Purified Water stored at room temperature.

Test Item Formulation:
Test item formulations were prepared three times by Labcorp according to the mixing procedure and were apportioned for daily use. Group 4 was re-formulated due to over quantum status.
Starting with the highest concentration, a stock formulation was made by weighing the required amount of test item and adding a portion of the vehicle control item. The pH was measured and adjusted to pH 3 to 9 using HCl/NaOH. The remaining vehicle control item was added to achieve the required volume and the formulation stirred for a minimum of twenty minutes using a magnetic stirrer resulting in a solution. Serial dilutions were then performed. Each formulation had the pH adjusted 3 to 9 with HCl/NaOH and mixed for a minimum of 20 minutes prior to diluting to the next concentration.
Dose formulations were stored in a refrigerator, set to maintain 2 to 8°C, until removed for dosing.

Vehicle Control Item Formulation:
Vehicle control item formulations were dispensed three times by Labcorp. Vehicle control item was stored in a refrigerator, set to maintain 2 to 8°C, until removed for dosing.

Stability:
Stability of the test item prepared in vehicle control item at concentrations of 10, 30, and 100 mg/mL have been evaluated under room temperature conditions up to 24 hours and under refrigerated (set to maintain 2 to 8°C) conditions up to 15 days under Labcorp Study 8450936.

Test Item Administration:
Dose formulations were stirred at room temperature for at least 30 minutes before and throughout dosing.
Animals were dosed a dose volume of 10 mL/kg once daily by oral gavage beginning on GD 6 and continuing through GD 20. Doses were based on the most recently recorded scheduled body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Handling:
Homogeneity:
The following samples were collected from the formulations prepared for administration on the first day of dosing.
Group No. 2 (10 mg/ml)
Group No. 4 (100 mg/ml)
Samples were protected from light in a refrigerator, set to maintain 2 to 8C, until shipped to the test site for analysis.

Concentration Verification:
The following samples were collected from the formulations prepared for administration on the first and last days of dosing.
Group No. 1 (0 mg/ml)
Group No. 2 (10 mg/ml)
Group No. 3 (30 mg/ml)
Group No. 4 (100 mg/ml)
Samples were protected from light in a refrigerator, set to maintain 2 to 8°C, until shipped to the test site for analysis.

Sample Analysis and Disposition:
After each collection interval, samples were shipped on cold packs by Marken Shipping to the test site for analysis. Primary and backup samples were shipped on separate days.
Dose formulations were analyzed by Labcorp using a method previously validated by Labcorp Method DFA/M126/20.
For homogeneity, one set of duplicate samples (from each stratum) were analyzed for test item content. The second set was retained as backup.
For concentration, one control group sample was analyzed to confirm the absence of test item. The second sample was retained as backup. One set of triplicate samples were analyzed for test item content. The second set was retained as backup.
Remaining samples were discarded after Study Director approval.

Details on mating procedure:

Mating was performed at the supplier. Males of the same strain were used for mating. Mating records were provided by the supplier and were maintained in the study record; males used for breeding were each only used one time.

Duration of treatment / exposure:

Animals were dosed a dose volume of 10 mL/kg once daily by oral gavage beginning on GD 6 and continuing through GD 20.

Frequency of treatment:

Daily from GD 6 and continuing through GD 20.

Duration of test:

Animals were dosed daily by oral gavage beginning on GD 6 and continuing through GD 20. Cesarean sections/necropsies were performed on GD 21 for surviving animals.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day

No. of animals per sex per dose:

21 females/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Species Selection and Dose Administration Rationale:
Rats were chosen for this study because they are sensitive to a number of agents known to cause reproductive or developmental toxicity. Rats historically have been used in safety evaluation studies of this type and are recommended by appropriate regulatory agencies.
In a previous OECD 422 study, four groups of 10 male and 10 female rats were administered 0, 100, 300 or 1000 mg/kg/day EDA-PO in purified water by oral gavage administration at a dose volume of 10 mL/kg. Males were dosed daily for 2 weeks before pairing, up to necropsy after a minimum of 4 consecutive weeks. Females were treated daily for 2 weeks before pairing, throughout pairing and gestation, until Day 12 of lactation. Dose levels of up to 1000 mg/kg/day were well tolerated. Based on these results, the no observed adverse effect level (NOAEL) for systemic toxicity and for reproductive/developmental toxicity was concluded to be 1000 mg/kg/day; therefore, the dose levels for this study were 0, 100, 300, and 1000 mg/kg/day as well.
The oral gavage route of administration was chosen as it is the preferred route of exposure specified in the OECD 414 guideline.

Examinations

Maternal examinations:

Clinical Observations:
Animals were checked twice daily (a.m. and p.m.) for mortality, abnormalities, and signs of pain or distress. Abnormal findings were recorded.
Cageside observations were made daily during the dosing period predose and approximately 2 to 3 hours postdose, based on the last animal dosed in each group. Abnormal findings were recorded.
Detailed observations were made on GD 4, 6, 12, 18, and 21. Abnormal findings or an indication of normal were recorded.
Unscheduled observations were recorded as they were observed.

Body Weights:
Animals were weighed daily prior to dosing beginning on GD 4 through 20, and on 21. The animal supplier provided GD 0 body weights for entry into the computer data collection system.

Food Consumption:
Food consumption was measured daily beginning on GD 4.
Spilled or contaminated feeders were recorded in the data and reported as such.

Ovaries and uterine content:

Terminal Procedures:
Animals were sacrificed by isoflurane inhalation, followed by exsanguination. All necropsies were conducted blinded to animal group numbers and all data were recorded using blinded numbers.
Animal R0307 (Group 4) was sacrificed on GD 19 at an unscheduled interval following Veterinarian directive. Pregnancy status was determined. An examination of the external features of the carcass; external body orifices; abdominal, thoracic, pelvic, and oral cavities; organs; and tissues performed, and any macroscopic abnormalities were noted.

Maternal Necropsy:
Cesarean sections/necropsies were performed on GD 21 for surviving animals. An examination of the external features of the carcass; external body orifices; abdominal, thoracic, cranial, pelvic, and oral cavities; organs; and tissues were performed, and any macroscopic abnormalities were noted. Pregnancy status was determined. If no fetuses were in utero and if implantation sites were not apparent when pressing the uteri between glass plates, then the uterus was placed in ammonium sulfide solution.
Fetuses were taken immediately by cesarean section, and uterine contents were examined. The placenta and amniotic sac were examined for abnormalities. The uterus from each pregnant animal was excised, weighed, and examined for the number and placement of live and dead fetuses, the number of early or late resorptions, and any abnormalities. The right and left ovaries from each pregnant female were examined for the number of corpora lutea. To the extent possible, animals that delivered on GD 21 were treated as indicated previously. Each fetus received an Ano-Genital Distance measurement. Each fetus was measured, and the value in millimeters (mm) to two decimal places were recorded.

Blood sampling:

Clinical Pathology Procedures:
Thyroid Hormone - Triiodothyronine (T3), Thyroxine (T4), and Thyroid Stimulating Hormone (TSH):
At least 4 mL of blood for thyroid hormone analysis was collected from all surviving animals via vena cava into serum separator tubes at time of scheduled necropsy). Sampling was performed at a similar time on each occasion (between 09:00 a.m. and 1:00 p.m. local time) to allow for accurate comparison between groups. Several animals were bled prior to 09:00 a.m. due to beginning parturition.
Samples were allowed to clot for at least 30 minutes at room temperature prior to centrifugation. Samples were centrifuged at 4°C for 10 minutes at 2000g within 1 hour of collection. Samples were processed and aliquoted for Triiodothyronine (T3), Thyroxine (T4), and Thyroid-stimulating hormone (TSH) by the Greenfield Specimen Processing department with priority given to T4 analysis and Triiodothyronine (T3). At least 0.2mL of serum was aliquoted for additional T3 and T4 analysis (if required). Residual serum was aliquoted into two serum samples of approximately equal volumes for T3.
Samples were stored in a freezer, set to maintain -60 to -80°C, until shipped to the test site for analysis.
After the final scheduled collection interval, serum samples were shipped frozen on dry ice via Marken carrier to the test site for analysis. An electronic sample manifest was included.

Triiodothyronine (T3) and Thyroxine (T4) Analysis:
All serum samples were analyzed using LC-MS/MS (method number BM/2016/0632); this method was validated under Labcorp Study No. FF58YR. At least 75% of the calibration standards were within ±20% of the nominal concentration (±25% at LLOQ).
Samples were analyzed for both T3 and T4. All samples were analyzed together for each occasion. The actual storage time between sampling and analysis were recorded in the raw data and included in the final report.

Fetal examinations:

Fetal Examinations:
Fetal examinations were conducted blinded to treatment groups.
Each fetus (live or dead) was sexed, weighed, and examined for external abnormalities. Live fetuses were sacrificed via injection with an appropriate barbiturate.
Approximately one-half of all fetuses from each litter were selected by a computer. Their heads were removed, stored frozen on dry ice, and cross-sectioned using the Wilson’s sectioning technique (Astroff et al., 2002). Internal organs of the thoracic and abdominal cavities were examined in the fresh state using modified Staples’ technique (Stuckhardt and Poppe, 1984). Fetuses identified for modified Staples’ examination (visceral examination) were then discarded.
The remaining fetuses were eviscerated and processed for skeletal examination using the Alizarin Red S staining method (Dawson, 1926). This evaluation included examination of the skull, vertebral column, rib cage, pectoral and pelvic girdles, long bones, and extremities. Bone alignment and degree of ossification were assessed. Fetuses that were processed for skeletal evaluation were retained in glycerin, with thymol added as a preservative.
Findings were classified as variations or malformations. Malformations are developmental deviations which are gross structural changes, incompatible with life, or may affect the quality of life. Variations are structural deviations thought to have no effect on body conformity or the well-being of an animal.
Representative photographs of visceral fetal malformations were taken and included in the study records following directive given from the Study Director.

Statistics:

Data Evaluation and Statistical Analysis:
Various models of calculators, computers, and computer programs were used to analyze data in this study. Values in some tables (e.g., means, standard deviations, or individual values) may differ slightly from those in other tables, from individually calculated data, or from statistical analysis data because different models round off or truncate numbers differently. Neither the integrity nor the interpretation of the data was affected by these differences.
Only data collected on or after the first day of dosing was analyzed statistically.
Data from non-pregnant animals were excluded from statistical analysis.
The pairwise comparisons of interest are:
• Group 1 versus Groups 2, 3, and 4

Means and standard deviations were calculated. All statistical tests were evaluated at the 5.0 1.0, and 0.1% probability levels.
Due to system limitations, additional statistical analyses were run but were not reported or used to interpret study data.

See "Any other information on materials and methods" section for further information on statistical analysis.

Indices:

Calculations:
Mean % Preimplantation Loss
Mean % Postimplantation Loss
Sex Ratios
Corrected Body Weight (Carcass Weight)
Corrected Weight Change
Total Weight Change (Weight Change)
Fetal Weight Dam Mean

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Remarks on result:

other: Data pending

Results (fetuses)

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No EDA-PO-related effects were seen in anogenital distance of male or female fetuses.

External malformations:

no effects observed

Description (incidence and severity):

No EDA-PO-related external variations or malformations were noted in animals at any dose level. All external observations were considered spurious and not related to EDA PO.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in unossified phalanx was noted in fetuses whose dams were administered 300 or 1000 mg/kg/day. This decrease was considered not related to EDA-PO due to a lack of dose response. All other skeletal observations were considered spurious and not related to EDA-PO.

Visceral malformations:

no effects observed

Description (incidence and severity):

No EDA-PO-related visceral variations or malformations were noted in animals at any dose level. All visceral observations were considered spurious and not related to EDA PO.

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No effects on fetal development were noted at any dose level up to the limit dose of 1000 mg/kg/day.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

The required definitive OECD 414 developmental toxicity study in the first species with EDA PO was commissioned in September 2021 immediately following availability of dose selection data from the OECD 422 study with EDA PO (necropsies conducted mid-August 2021).

At this time the conducting laboratory indicated the following dates would be met for the EDA PO definitive OECD 414 study in the rat, which allowed for meeting the 8 December 2022 deadline given by ECHA for this study:

• Study initiation:  15 February 2022


• Draft audited report: 15 May 2022

This laboratory and test site were chosen as they were the only testing facility able to meet the 8 December 2022 deadline outlined by ECHA’s final decision for the OECD 414 in the first species.

Due to subsequent laboratory capacity and animal availability limitations, the conducting laboratory was not able to initiate the EDA PO definitive OECD 414 study in the rat until June 2022.  An unaudited draft report of available results (which includes developmental and fetal results but not maternal endpoints) has been provided by the laboratory.  These draft results have been included in the study record.

The conducting laboratory has invited ECHA to contact them if further details regarding the reasons for the delayed finalization of the study report are desired.

Applicant's summary and conclusion

Conclusions:

Unaudited draft report:
Pregnant Han-Wistar rats were administered purified water control or 100, 300, or 1000 mg/kg/day Ethylenediamine, propoxylated (EDA-PO) daily by oral gavage during the period of gestation (GD 6 through 20). No effects on fetal development were noted at any dose level up to the limit dose of 1000 mg/kg/day.

Executive summary:

Unaudited draft report:

Pregnant Han-Wistar rats were administered purified water control or 100, 300, or 1000 mg/kg/day Ethylenediamine, propoxylated (EDA-PO) daily by oral gavage during the period of gestation (GD 6 through 20). No effects on fetal development were noted at any dose level up to the limit dose of 1000 mg/kg/day.