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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Remarks:
Prenatal Developmental Toxicity Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 July 2020 - 05 Nov 2020
Reliability:
1 (reliable without restriction)

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:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
No detailed examinations were performed for Female Nos. 1-6 (control group), 23-27 (Group 2), 45-49 (Group 3) and 67-72 (Group 4) on Day 21 postcoitum (prior to necropsy). These animals were individually handled for blood collection prior to necropsy
Qualifier:
according to guideline
Guideline:
other: EC No 440/2008, B.31 Prenatal Developmental Toxicity Study, 2008.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction products of fatty acids, C14-C18 (branched and linear) and C18 (unsaturated) with tetraethylenepentamine (linear, branched, cyclic)
EC Number:
701-204-9
Cas Number:
68784-17-8
IUPAC Name:
Reaction products of fatty acids, C14-C18 (branched and linear) and C18 (unsaturated) with tetraethylenepentamine (linear, branched, cyclic)
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:1000352370
- Expiration date of the lot/batch:31 March 2022
- Purity test date:NA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions: in vehicle: stability for at least 4 hours at room temperature under normal laboratory light conditions and stability for at least 6 hours in the refrigerator is confirmed over the concentration range 20.0 to 250 mg/mL (suspensions),
- Stability under test conditions: stable

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France. Details will be documented in raw data and report.
- Age at study initiation: Approximately 10-14 weeks.
- Weight at study initiation: Approximately 180 to 230 g.
- Fasting period before study: no
- Housing: On arrival, females will be individually housed in Macrolon plastic cages (MIII type, height 18 cm). The cages will contain appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and will be equipped with water bottles. The housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records. Each cage will be clearly labeled with a color-coded cage card indicating Test Facility Study No., group, and animal number.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light):12-hours light and 12-hours dark (may be interrupted for designated procedures).

IN-LIFE DATES: From: 07 July 2020 To: 30 July 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):The dosing formulations were prepared daily as a suspension and dosed as soon as possible after the formulation was completed, at least within 4 hours after adding the vehicle.
- Mixing appropriate amounts with (Type of food): Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
- Storage temperature of food: Room temperture

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility. Residual samples were discarded after completion of the sample analysis. Analyses were performed using a validated analytical procedure (Test Facility Study No. 20242508).

Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±15% for suspensions of target concentration.
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Stability analyses were performed in conjunction with the method development and validation study (Test Facility Study No. 20242508) to assess whether the test item was stable in the vehicle when prepared and stored at room temperature under normal laboratory light conditions for at least 6 hours or in a refrigerator (2-8°C) for at least 2 days. Stability data was retained in the study records of Test Facility Study No. 20242508. As stability over 6 hours at room temperature or 2 days in a refrigerator was not confirmed during this method development and validation study (Test Facility Study No. 20242508), additional stability analysis was performed in Week 1 of dosing, on duplicate formulations subsamples collected from a low (20 mg/mL) and high (250 mg/mL) formulation (not used for dosing) to demonstrate if the test item was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Samples and remaining formulation were stored in normal glassware causing the samples stored at room temperature exposed to normal laboratory light conditions. Formulations were considered stable if the relative difference before and after storage was ≤ 10%.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral
gavage 7 days a week from Day 6 to Day 20 post-coitum, inclusive.
The dose volume for each animal was based on the most recent body weight measurement.
The doses were given using a plastic feeding tube.
The dosing formulations were stirred continuously during dose administration.
Dose pot identification via Provantis was used as additional check to verify the dosing
procedure according to Standard Operating Procedures.
Frequency of treatment:
Once daily from Day 6 to Day 20 post coitum
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The test item and vehicle were administered to the appropriate animals by once daily oral
gavage 7 days a week from Day 6 to Day 20 post-coitum, inclusive.
The dose volume for each animal was based on the most recent body weight measurement.
The doses were given using a plastic feeding tube.
The dosing formulations were stirred continuously during dose administration.
Dose pot identification via Provantis was used as additional check to verify the dosing
procedure according to Standard Operating Procedures.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage side observations were performed once daily, beginning on Day 6 post-coitum onwards up to the day prior to necropsy. During the dosing period, this observation was performed
postdose. Animals were not removed from the cage during observation, unless necessary for
identification or confirmation of possible findings. Cage debris was examined to detect
premature birth.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were removed from the cage, and a detailed clinical observation was performed weekly, beginning during the Pre-Treatment Period, and on the day of necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21 post coitum
- Organs examined: All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the uterus and thyroid gland) were weighed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead and females that delivered their offspring early) will be collected on the day of scheduled necropsy. Animals will not be fasted overnight. Samples will be collected randomized at the discretion of the Study Director, between 7.00 and 9.00 a.m., from the jugular vein in the animal facility. After collection, samples will be transferred to the appropriate laboratory for processing.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
Any data collected during the (Pre)treatment Period are tabulated, summarized or statistically
analyzed. All statistical analyses were performed within the respective study phase, unless
otherwise noted. Numerical data collected on scheduled occasions were summarized and
statistically analyzed as indicated below according to sex and occasion or by litter.

Means, standard deviations (or % coefficient of variation or standard error, when deemed
appropriate), ratio, percentages, numbers, and/or incidences were reported as appropriate by
dataset.
All statistical tests were conducted at the 5% significance level. All pairwise comparisons
were conducted using two sided tests and were reported at the 1% or 5% levels.

Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Historical control data:
yes
Historical control data for pregnant Wistar Han rats (period 2016-2020)
TSH (mU/L) mean: 0.353, P5-P95: 0.127 – 0.699 (n = 373)
External malformation: Eye(s)- Absent and/or Small: incidence – 6/6890 fetuses (6/1291 litters)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Any clinical signs noted during the treatment period (including salivation, fur loss and skin
scab in the dorsal cervical area), occurred within the range of background findings to be
expected for rats of this age and strain which are housed and treated under the conditions in
this study and did not show any apparent dose-related trend. At the incidence observed, these
were considered to be unrelated to treatment with the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated
animals remained in the same range as controls over the treatment period
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption were recorded.
The statistically significant increase in food consumption observed between Day 18-21 at
1000 mg/kg/day (9.8% above control level) was considered not toxicologically relevant given
the small magnitude and the direction of the response. Additionally, there were no differences
between treatment groups for the entire dosing period (between Day 6-21).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum levels of total triiodothyronine (Total T3), thyroid stimulating hormone (TSH) and
total thyroxine (Total T4) were considered to be unaffected by treatment with the test item up
to 1000 mg/kg/day.
The apparent increase of TSH in all treated groups may have arisen as a result of slightly low
control values (see historical control mean value2) in combination with individual animals
with high levels of TSH compared to group average. Moreover, as all mean values remained
within the historical control range2,
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):
There were no test item-related alterations in thyroid gland weights.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations.
The only macroscopic finding observed was a bilateral small size of the thyroid gland of a
single 450 mg/kg/day group female. This macroscopic finding was considered to be within
the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
The recorded microscopic findings in the thyroid gland of one control and one
1000 mg/kg/day female were within the range of background pathology encountered in rats of
this age and strain.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
No test item-related toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, body weight, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and
postimplantation loss).

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not examined
Other effects:
not specified
Details on maternal toxic effects:
The numbers of pregnant females, corpora lutea and implantation sites, and pre- and postimplantation loss in the control and test groups were similar and in the range of normal biological variation.
All females were gravid and had litters with viable fetuses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
dead fetuses
endocrine findings
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
mortality
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
other:
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Any clinical signs noted during the treatment period (including salivation, fur loss and skin scab in the dorsal cervical area), occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on fetal body weights (both sexes) noted by treatment
up to 1000 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, and pre- and
postimplantation loss in the control and test groups were similar and in the range of normal
biological variation.
All females were gravid and had litters with viable fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 1000 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group.
Mean litter sizes were 11.1, 10.2, 10.3 and 10.8 fetuses/litter for the control, 150, 450 and
1000 mg/kg/day groups, respectively.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no test item-related effects on fetal anogenital distance (both sexes) noted after treatment up to 1000 mg/kg/day.
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
No external malformations and variations were seen in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on skeletal morphology following treatment up to
1000 mg/kg day.
Skeletal malformations occurred in two fetuses at 1000 mg/kg/day and in one fetus each at
the 150 mg/kg/day and control group. At 1000 mg/kg/day, fetus 87-L1 had a bent humerus,
but as also control fetus 2-L4 had bent limb bones (humerus and radius bilaterally), this
malformation was considered a chance finding.
The other fetus at 1000 mg/kg/day, 69-R5, had a malformation affecting thoracic vertebrae
and ribs. Malformations affecting these areas also occurred in 150 mg/kg fetus 30-R12 that
had fused mandibles with a single incisor socket as well. In the absence of a dose response
relationship and based on the single incidence, these findings were considered not to be test
item-related.
All skeletal variations occurred in the absence of a dose-related incidence trend, infrequently
and/or in control fetuses only. Therefore, they were considered to be unrelated to treatment
with the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on visceral morphology following treatment up to
1000 mg/kg/day.
The fresh visceral examination did not reveal any malformations. However, at soft tissue
serial sectioning of heads a small eye was observed in fetus 63-R6 and 77-L4 (450 and
1000 mg/kg/day, respectively). As this malformation occurred singly in two dose groups and
was also observed previously in control fetuses3
, these small eyes were considered chance
findings.
Visceral variations affected the liver (supernumerary lobe), kidney (absent renal papilla) and
ureter (convoluted or dilatated). These findings occurred infrequently and in the absence of a
dose-related incidence trend and therefore, they were considered unrelated to treatment with
the test item.
Description (incidence and severity):
No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).
Details on embryotoxic / teratogenic effects:
No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this prenatal developmental toxicity study in Wistar
Han rats, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for
EC 701-204-9 were established as being at least 1000 mg/kg/day.
Executive summary:

The objectives of this study were to determine the potential of EC 701-204-9 to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.
The dose levels in this study were selected to be 0, 150, 450, 1000 mg/kg/day, in consultation with the Sponsor, based on information provided by the Sponsor: i.e. the results of an OECD 407 (repeated dose 28-day oral toxicity) study and an OECD 421 (reproduction/developmental toxicity screening test) study in Sprague Dawley rats with EC 701-204-9.











































Group n°Test item IDDose level (mg/kg/day)Dose Volume (mL/kg)Dose concentration (mg/mL)Number of females
1Control0 (control)4022
2EC 701-204-9150437,522
34504112,522
41000425022

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. Additionally, formulations prepared at low (20 mg/mL) and high (250 mg/mL) concentrations were assessed for stability over 2, 4 and 6 hours.



The following parameters and end points were evaluated in this study for the F0-generation:
mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss.
In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.
Formulation analyses confirmed that formulations of test item in Corn oil were prepared accurately and homogenously. Additionally, formulations prepared at low (20 mg/mL) and high (250 mg/mL) concentrations (not used for dosing) were considered stable, for at least 4 hours at room temperature and 6 hours in the refrigerator.



No maternal toxicity was observed in the 150, 450 and 1000 mg/kg/day groups.
No developmental toxicity was observed in the 150, 450 and 1000 mg/kg/day groups.


In conclusion, based on the results of this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for EC 701-204-9 of at least 1000 mg/kg/day was established.