1-(N,N-bis(2-hydroxyethyl)amino)propan-2-ol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 October 2020 - 04 January 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 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

July 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-(N,N-bis(2-hydroxyethyl)amino)propan-2-ol

EC Number:

229-764-5

EC Name:

1-(N,N-bis(2-hydroxyethyl)amino)propan-2-ol

Cas Number:

6712-98-7

Molecular formula:

C7H17NO3

IUPAC Name:

1-[bis(2-hydroxyethyl)amino]propan-2-ol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sasol, Marl, Germany; Batch no. 200162527.
- Purity: 99.6%.
- Appearance: clear liquid.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature, in the dark.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, UK.
- Age at study initiation: 17 - 22 weeks.
- Weight at study initiation: 2.30 - 4.17 kg.
- Fasting period before study: No.
- Housing: One animal (female) per cage.
- Diet: Commercial diet, 200 g/animal/day. "A small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly."
- Water: Tap water ad libitum.
- Acclimation period: Five days before commencement of treatment (days 1 - 5 after mating).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21 °C.
- Humidity (%): 45 - 70%.
- Air changes (per hr): Not specified. Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 14 hours light / 10 hours dark.

IN-LIFE DATES:
From: Approx. July 2020 (based on age on arrival at testing facility).
To: 24 - 27 November 2020.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Water was added to pre-weighed test material and mixed (for at least 40 minutes) to obtain a homogenous solution at the highest concentration. Lower concentrations were prepared by serial dilution.

VEHICLE
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first and last preparation were analyzed for achieved concentration of the test item.

Details on mating procedure:

Animals were received at the test facility having already been successfully mated. Natural mating with New Zealand white bucks of established fertility at the supplier’s facility. Males and females were not closely related.

Duration of treatment / exposure:

Days 6 - 28 (inclusive) after mating (i.e. 23 days total).

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels selected for this study, were based on the results from a Combined Pilot and Preliminary Study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: At least twice daily.
- Specific cage side observations not listed in study report.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: At least twice daily.

BODY WEIGHT: Yes.
- Time schedule for examinations: On arrival (day 1 after mating), day 3 after mating, and daily on days 6 - 29 after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 29 after mating.
- Organs examined: 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries).
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: Yes.
- Number of late resorptions: Yes.
- Other: Number of foetuses (live and dead).

Fetal examinations:

- External examinations: Yes [all per litter].
- Soft tissue examinations: Yes [all per litter].
- Skeletal examinations: Yes [all per litter].
- Head examinations: Yes [50% per litter].
- Anogenital distance of all live rodent pups: Not applicable.

Statistics:

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Placental, litter and fetal weights

A parametric analysis, using the t-test or F1 approximate test, was performed if Bartlett's test was not significant at the 1% level.

A non-parametric analysis, using Kruskal-Wallis' test, Wilcoxon rank sum tests, or the H1 approximate test, was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.

Historical control data:

Historical control data from 14 studies conducted from January 2020 are included, using New Zealand white rabbits from the same supplier (Envigo RMS) as used in the present study.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal in each of the vehicle control, mid-dose and high-dose groups was killed early for welfare reasons.

In the case of the control and mid-dose animals, the litters had resorbed. "The poor clinical condition of [the mid-dose animal] was attributed to the loss of the litter and not attributable to treatment with DEIPA."

The high-dose animal was killed for welfare reasons on suspicion that the pregnancy had resorbed or aborted. Upon examination, the death of the animal was due to a twisted caecum, and not attributable to treatment with DEIPA.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See Table 4.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was 18% lower than control in the high-dose group. See Table 1.

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

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

not specified

Description (incidence and severity):

Data not available in study report.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

See Table 3.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

not specified

Description (incidence and severity):

Data on stillbirths and/or dead foetuses not available in study report.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Data, including on early deliveries, not available in study report.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

11 animals were not pregnant: 3 in the control group, 2 in the low-dose group, 4 in the mid-dose group and 2 in the high-dose group. See Table 2.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Total litter weight was marginally reduced in the high-dose group, attributed to the marginally smaller litter size.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

"Marginally smaller" litter size at the high dose. See Table 5.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

See Table 5.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

See Table 6.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

See Table 7.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was 1 incidence each of lumbar scoliosis, lumbar hemivertebra, bent clavicle and pointing backwards hindpaw(s). See Table 7.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 2 incidences each of dilated ascending aorta and narrow pulmonary trunk, and 1 each of acephalostomia, truncus arteriosus and septal defect. See Table 7.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of incompletely ossified/unossified epiphyses and metacarpals/phalanges at all treated doses exceeded the concurrent and historical controls ranges. These are a transient stage in foetal development and indicative of foetal immaturity, and therefore non-adverse. See Table 8.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1 - Food consumption during gestation days 6-29

Group	Nominal dose (mg/kg bw/day)	Mean food consumption (g/animal/day)	Standard Deviation
1	0	102	14.6
2	100	100	11.7
3	300	97	14.0
4	1000	84**	14.7

** p < 0.01

 

Table 2 - Number of pregnancies

Group	Nominal dose (mg/kg bw/day)	Number of treated animals	Number of animals that died during study	Number of non-pregnant dams	Number of pregnant dams
1	0	24	1	3	20
2	100	24	0	2	22
3	300	24	1	4	19
4	1000	24	1	2	21

 

Table 3 - Pre- and post-implantation loss - litter data

Group	Nominal dose (mg/kg bw/day)	Number of pregnant dams	Resorptions			Pre-implantation loss (%)	Post-implantation loss (%)
			Early	Late	Total
1	0	20	0.6	0.1	0.7	10.0	8.7
			SD 0.94	SD 0.31	SD 0.92	SD 12.08	SD 11.87
2	100	22	0.7	0.4	1.0	15.3	12.9
			SD 1.36	SD 0.49	SD 1.36	SD 22.51	SD 15.66
3	300	19	0.6	0.4	0.9	8.8	10.3
			SD 1.02	SD 0.76	SD 1.18	SD 12.23	SD 12.05
4	1000	21	0.5	0.3	0.8	13.3	10.8
			SD 0.98	SD 0.64	SD 1.21	SD 18.31	SD 16.00

 

Table 4 - Body and gravid uterine weights

		Group mean values (kg)
Group	Nominal dose (mg/kg bw/day)	Body weight, day 6	Terminal body weight	Body weight change	Gravid uterine weight	Adjusted terminal body weight	Adjusted body weight change
1	0	3.13	3.42	0.29	0.420	3.00	-0.13
		SD 0.415	SD 0.343	SD 0.133	SD 0.0997	SD 0.343	SD 0.154
2	100	3.19	3.45	0.25	0.396	3.05	-0.14
		SD 0.374	SD 0.318	SD 0.145	SD 0.1325	SD 0.222	SD 0.175
3	300	3.13	3.41	0.28	0.408	3.00	-0.13
		SD 0.321	SD 0.301	SD 0.115	SD 0.0718	SD 0.278	SD 0.102
4	1000	3.08	3.36	0.28	0.366	2.99	-0.08
		SD 0.323	SD 0.307	SD 0.174	SD 0.0990	SD 0.274	SD 0.132

 

Table 5 - Live offspring

Group	Nominal dose (mg/kg bw/day)	Mean number of live young			Sex ratio (% male)
		Male	Female	Total
1	0	3.9	3.6	7.5	50.3
		SD 2.13	SD 1.27	SD 2.37	SD 14.27
2	100	4.2	2.8	7.0	62.8
		SD 2.18	SD 2.14	SD 2.60	SD 25.75
3	300	3.1	4.2	7.3	41.5
		SD 1.66	SD 1.65	SD 1.53	SD 21.33
4	1000	3.0	3.7	6.7	43.7
		SD 1.70	SD 1.76	SD 2.19	SD 19.50

 

Table 6 - Litter and foetal weights

Group	Nominal dose (mg/kg bw/day)	Mean total litter weight (g)	Mean foetal weights (g)
			Male	Female	Combined
1	0	279.5	39.2	37.5	38.4
		SD 71.98	SD 6.26	SD 5.97	SD 5.68
2	100	260.6	38.0	36.7	38.0
		SD 96.02	SD 6.86	SD 6.03	SD 5.99
3	300	268.4	36.6	37.3	37.2
		SD 55.61	SD 4.93	SD 4.68	SD 4.71
4	1000	242.9	36.2	36.2	36.6
		SD 74.07	SD 6.06	SD 4.80	SD 4.40

 

Table 7 - Foetal examinations - major abnormality findings

Group			1	2	3	4
Nominal dose (mg/kg bw/day)			0	100	300	1000
Litters examined			20	22	19	21
Total number of litters affected			1	0	0	2
Foetuses examined			150	154	139	141
Total number of foetuses affected			3	0	0	4
Number of foetuses with major abnormalities
Skeletal	Head	Acephalostomia	0	0	0	1
	Cervical/Thoracic	Thoracic scoliosis	1	0	0	0
		Dorsoventral distortion of sternum	1	0	0	0
	Lubmar (and abdominal)/ sacral/ caudal	Sacral/caudal spina bifida	1	0	0	0
		Lumbar scoliosis	0	0	0	1
		Lumbar hemivertebra	0	0	0	1
	Appendicular	Bent clavicle	0	0	0	1
		Pointing backwards hindpaw	0	0	0	1
Visceral	Cervical/Thoracic	Dilated ascending aorta/aortic arch	0	0	0	2
		Dorsally displaced pulmonary trunk	1	0	0	0
		Truncus arteriosus	0	0	0	1
		Membranous ventricular septal defect	1	0	0	1
		Muscular ventricular septal defect	0	0	0	1
		Fistula ascending aorta/pulmonary trunk	1	0	0	0
		Narrow/marked pulmonary trunk	0	0	0	2

 

Table 8 - Foetal examinations - minor abnormality findings

Group		1	2	3	4
Nominal dose (mg/kg bw/day)		0	100	300	1000
Litters examined		20	22	19	21
Foetuses examined		150	154	139	141
Number of foetuses with minor abnormalities
Skeletal	Cranial abnormality - unossified area(s)	0	0	1	0
	Cranial abnormality - sutural bone(s)	1	0	0	0
	Cranial abnormality - bent cornu(a) of hyoid	0	8	3	3
	Vertebral abnormality - thoracic	1	0	0	1
	Vertebral abnormality - lumbar	1	0	0	1
	Rib abnormality - short without costal cartilage	1	0	0	0
	Rib abnormality - misaligned	1	0	0	0
	Rib abnormality - partially fused	1	0	0	0
	Rib abnormality - distally thickened	1	0	0	0
	Rib abnormality - branched with additional costal cartilage	1	0	0	0
	Sternebrae - fused/partially fused	3	3	1	4
	Sternebrae - misaligned hemicentres	0	2	1	0
	Sternebrae - supernumerary site	0	1	0	0
	Sternebrae - wide	1	0	0	0
	Sternebrae - misshapen	2	0	0	0
	Sternebrae - branched 6th	0	1	0	0
	Costal cartilage - partially fused	2	0	0	0
	Costal cartilage - misaligned	0	3	1	2
	Costal cartilage - branched	1	1	0	0
	Costal cartilage - additional	1	0	0	0
	Costal cartilage - 7th not connected to sternum	0	2	0	0
	Appendicular abnormality - absent 1st digit forepaw(s)	0	0	0	1
	Cervical rib abnormality - short supernumerary	1	9	7	2
	Cervical rib abnormality - full supernumerary	1	0	0	0
	Number of 13th ribs - short supernumerary	44	34	37	45
	Number of 13th ribs - full supernumerary	35	32	22	57
	Number of 13th ribs - total	67	61	54	89
	Thoracolumbar vertebrae - 18	1	0	1	0
	Thoracolumbar vertebrae - 20	7	12	4	19
	Pelvic girdle - unilateral cranial shift	2	0	0	0
	Pelvic girdle - unilateral caudal shift	6	2	0	4
Delayed/incomplete ossification	Cranial - large posterior fontanelle	1	0	0	1
	Sternebrae - 5th	20	23	39	24
	Sternebrae - 1st-4th, 6th	9	5	11	13
	Sternebrae - total	28	26	46	32
	Vertebrae - cervical	3	0	0	2
	Vertebrae - thoracic	1	0	0	0
	Appendicular - pubes	0	2	0	1
	Appendicular - epiphyses	11	20	10	14
	Appendicular - talus	1	1	0	0
	Appendicular - metacarpals/phalanges	9	16	15	17
	Appendicular - metatarsals/phalanges	2	7	1	4
Increased ossification	Cranial - partially fused jugal to maxilla	1	1	0	0
	Vertebrae - long lumbar transverse processes	1	0	0	0
Visceral	Eyes - folded retina	3	2	0	0
	Brain - dilated interventricular foramen	1	0	0	0
	Head - supernumerary minor upper incisor	0	1	0	0
	Lungs - abnormal lobation	1	0	0	0
	Gall bladder - bilobed	0	0	1	0
	Ovary - cyst(s)	0	0	1	0
	Head - tongue present	0	0	0	1
	Limb(s) - flexure forepaw(s)	1	1	0	3
	Tail - curled	0	0	0	1
	Tail - kinked	0	2	0	0

Applicant's summary and conclusion

Conclusions:

In a good-quality guideline study to GLP, gavage administration of DEIPA at doses up to 1000 mg/kg bw/day (nominal) to time-mated rabbits on GD 6-28 (inclusive) did not produce any clinical signs of toxicity. Reproductive parameters were not altered and no signs of treatment-related malformations were seen. On this basis, the study NOAELs for both maternal and developmental toxicity were 1000 mg/kg bw/day (nominal), the highest dose tested.

Executive summary:

In a good quality guideline study, conducted to GLP, groups of 24 time-mated female New Zealand white rabbits were administered DEIPA by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day (nominal) on days 6-28 of gestation. Animals were killed on gestation day 29 and examined for gross pathological alterations. Gravid uterine weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead foetuses. All foetuses were weighed and sexed, and examined for external, visceral and (in 50%) skeletal alterations.

 

No clinical signs of toxicity were seen, reproductive parameters were not altered, and there were no treatment-related malformations or other developmental toxicity observed in the foetuses at any dose of DEIPA. On this basis, the no-observed-adverse-effect levels (NOAELs) for maternal toxicity and developmental toxicity were 1000 mg/kg bw/day (nominal), the highest dose tested.