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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Specific details on test material used for the study:
- Source and lot/batch number of test material: SAMP18373
- Purity. 99.57%

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River (UK) Ltd
- Age at study initiation: 70 to 76 days old
- Weight at study initiation: 210 to 293 g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet (ad libitum)
- Water (e.g. ad libitum): tap water (ad libitum)
- Acclimation period: Six days before commencement of pairing

- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
purified water
Analytical verification of doses or concentrations:
Details on mating procedure:
Male/female ratio: 1:1 with identified stock males.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation: When positive evidence of mating was detected.
Duration of treatment / exposure:
GD 6-19
Frequency of treatment:
once daily
Duration of test:
20 d
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
180 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The high dose level of 180 mg/kg/day was selected based on the results of a preliminary embryo-fetal study in the female rat conducted at these laboratories.
In that study animals received daily administration of Ethyldimethylamine over Days 6-19 of gestation at 50, 180 and 360 mg/kg/day.

Females receiving 360 mg/kg/day started showing signs of decreased activity, partially closed eyelids, flattened or hunched posture, shallow breathing, cold to touch and piloerection on Day 8 or 9 of gestation. Two females were euthanised for welfare reasons, both on Day 9 of gestation, after more than 15% bodyweight loss, which reached or exceed the moderate severity limit. The high dose level was
subsequently reduced to 270 mg/kg/day for the remaining four females. These females were prostrate at 30 minutes post dosing and underactive, hunched with piloerection and had abnormal breathing at 1 hour post dose. Prior to dosing on Day 7 of gestation at the reduced high dose, one female was found dead and a second female had lost body weight, exceeding the moderate severity limit. As the dose level
was not tolerated, the remaining females were euthanised for welfare reasons on Day 9 or 10 of gestation.

Treatment at 180 mg/kg/day was tolerated, but was associated with reduced weight gain (86% of Controls), reduced adjusted maternal weight gain (60% of Controls) and a slight reduction in food consumption (90% of Controls). One female had depressions on the nonglandular region of the stomach but remained in good clinical condition.

Therefore a high dose of 180 mg/kg/day was considered suitable as the high dose with 60 and 20 mg/kg/day chosen as the intermediate and low dose levels to allow assessment of any dose response.


Maternal examinations:
Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing:
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
Pre-dose observation. One to two hours after completion of dosing. As late as possible in the working day.

Clinical Signs:
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight:
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption:
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Schedule: Animals surviving until the end of the scheduled study period were killed on Day 20 after mating.
Sequence: To allow satisfactory inter-group comparison.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus: Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals
For each ovary/uterine horn: Number of: Corpora lutea. Implantation sites. Resorption sites (classified as early or late). Fetuses (live and dead).
For apparently empty uterine horns: The number of uterine implantation sites were checked after staining with ammonium sulphide
Fetal examinations:
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with
abnormalities recorded. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.

Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.

Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning. IMS fixed fetuses were processed and double stained with Alizarin and Alician Blue.

Fetal Pathology Examination
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Double stained with Alizarin Red and Alician Blue fetuses: Assessed for skeletal and cartilage development and abnormalities.
Different test methods were used: Bartlett`s test; Williams`test; Dummet`s test; Kruskal-Wallis`test; Shirley`s test, Steel`s test

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs associated with dose administration were restricted to the animals receiving 180 mg/kg/day, with 4/20 exhibiting a low incidence of rales (no. 61 on one occasion; no. 65 on two occasions; no. 67 on two occasions; no 71 on three occasions) and 4/20 females exhibiting increased salivation each on a single occasion. Signs at routine physical examination were limited to two females at 180 mg/kg/day (nos. 65 and 71) with rales; there were other signs that could be related to treatment. The clinical condition of animals receiving 20 or 60 mg/kg/day was unaffected by treatment.
Dermal irritation (if dermal study):
not examined
mortality observed, treatment-related
Description (incidence):
Two females (nos 61 and 67) receiving 180 mg/kg/day were killed for welfare reasons; female no 61 was euthanised on GD14 and female no.67 was euthanised on GD12. Terminal signs for both of these females comprised of respiratory distress (gasping) and body weight loss, however no macroscopic abnormality was apparent at necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
From GD9 (Day 4 of treatment) mean body weight gain for females receiving 180 mg/kg/day was low with the difference attaining statistical significance (p<0.01) and overall (GD6-20) the mean body weight change at 180 mg/kg/day was approximately 14% lower than Controls (p<0.01). The resultant mean bodyweight on GD20 for females receiving 180 mg/kg/day was approximately 6% lower than Controls (p<0.05).
The mean gravid uterine weight was essentially similar across the groups. However the maternal weight gain following adjustment for the gravid uterine was statistically significantly low at 180 mg/kg/day, approximately 40% lower than Controls (p<0.01) and the maternal mean weight on GD20 was approximately 6% lower than Controls (p<0.05). Both the absolute body weight gain and adjusted maternal weight gain of females receiving 20 or 60 mg/kg/day was unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 180 mg/kg/day mean food consumption was low when compared with Controls with the difference attaining statistical significance for GD10-17 (p<0.01; 86-88% of Controls) and GD18-19 (p<0.05; 93 % of Controls). Overall the total food consumption during the treatment period (GD 6 -19) was approximately 10% lower than Controls.
Food consumption at 20 or 60 mg/kg/day was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females on GD20 did not reveal any findings that could be attributed to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A higher incidence of post-implantation loss was observed at 180 mg/kg/day (9.3% versus 2.3% in Controls), however the incidence was within the historical control range and there was no impact on the live litter size. This difference was therefore not considered to be related to treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 60 mg/kg/day and 180 mg/kg/day there was a slightly higher incidence of incompletely ossified 1st to 4th sternebrae, compared with concurrent control (12/11 fetuses in 8 litters per group versus 4 fetuses in 4 Control litters); the incidences were within the historical control range of the litters, which is the principal unit of evaluation, with the exception of fetuses at 60 mg/kg/day which exceeded the fetal historical control range. Incomplete ossification is a transient stage in development and is therefore not considered to be adverse.

Effect levels (fetuses)

Dose descriptor:
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

no effects observed

Overall developmental toxicity

Developmental effects observed:

Applicant's summary and conclusion

It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo-fetal survival and development the NOAEL was considered to be 180 mg/kg/day.