2-(2-aminoethylamino)ethanol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Reference 2

Reference Type:

publication

Title:

Malformations of the Great Vessels in the Neonatal Rat Induced by N-(2-Aminoethyl)ethanolamine

Author:

Schneider et al.

Year:

2012

Bibliographic source:

Birth Defects Research (Part B) 95:95–106 (2012)

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-aminoethylamino)ethanol

EC Number:

203-867-5

EC Name:

2-(2-aminoethylamino)ethanol

Cas Number:

111-41-1

Molecular formula:

C4H12N2O

IUPAC Name:

2-[(2-aminoethyl)amino]ethan-1-ol

Details on test material:

- Physical state: liquid (colorless clear)
- Analytical purity: 99.8 %, had been verified by reanalysis prior to the study commencing
- Lot/batch No.: continuous operation
- Stability: The storage stability under storage conditions was confirmed by reanalysis (analytical report 05L00199).
- Storage condition of test material: stored at room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (former name: Charles River Laboratories, Germany)
- Age at study initiation: 11 - 12 weeks old
- Weight at study initiation: 242 - 295 g (males), 176 - 218 g (females)
- Housing: singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions: for the overnight mating the females were put into the cages of the males; from day 18 p.c. until day 4 p.p. the pregnant animals and their litters were housed in Makrolon type M III cages (floor area about 800 cm²). The M III cages were also supplied by Becker & Co.. Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air-conditioned, 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

IN-LIFE DATES: From: 18 October 2005 To: 13 December 2005

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration solutions the test substance was weighed in a graduated measuring flask depending on the dose group, topped up with doubly distilled water and subsequently thoroughly mixed using a magnetic stirrer. The test substance solutions were prepared at the beginning of the administration period and thereafter at intervals that took into account the stability of the test substance preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in drinking water (“Frankenthaler Leitungswasser”) for a period of 10 days at room temperature were carried out before the study was initiated. Given that AEEA is completely miscible with water, solutions were considered to be homogenous without further analysis. Samples of the aqueous test substance solutions were sent to the analytical laboratory twice during the study period for verification of the concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight at least 13 days after start of treatment
- Verification of same strain and source of both sexes: yes. According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: male animal No. 87 of dose group 3 (5 mg/kg bw/day) was mated with two female animals of dose group 3 (Nos. 287 and 288), because of the preterminal death of one male animal No. 88 of dose group 3).

Duration of treatment / exposure:

38 days (males), 54 days (females)

Frequency of treatment:

7 times/week

Duration of test:

54 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.2, 1, 5, 50 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The following dose levels were selected:
0.2 and 1 mg/kg bw/day: as doses covering a 2-fold range below NOAEL in low power probe study in effort to find NOAEL given increased statistical power of present study
5 mg/kg bw/day: NOAEL in low statistical power probe study
50 mg/kg bw/day: bridging dose where effect of interest was seen in other studies.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: at least daily

BODY WEIGHT:
- Time schedule for examinations: In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on days 0, 7, 14 and 20 post coitum. Females with litter were weighed on days 0 and 4 post partum. Females without litter, waiting for necropsy, were weighed weekly.

FOOD CONSUMPTION
- Time schedule: Generally, food consumption was determined once a week for parental animals, with the following exceptions: food consumption was not determined during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on days 0, 7, 14 and 20 post coitum. Food consumption of F0 females, which gave birth to a litter was determined on days 0 and 4 post partum.

POST-MORTEM EXAMINATIONS:
Females were allowed to litter and rear their pups until day 4 p.p.; thereafter the parental females were sacrificed.

Fetal examinations:

not applicable

Statistics:

Weight parameters were analyzed using the two-sided Kruskal-Wallis test, followed by the Wilcoxon test if the p-value was =/< 0.05.
Proportions of affected pups per litter with necropsy observations was analyzed using the one-sided Wilcoxon test.

Indices:

Male mating and fertility, and female mating, fertility and gestation indices were determined. Live birth index and post implantation loss were assessed.

Historical control data:

Historical control data were included in the report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Parental clinical examinations: Mortality: there was no substance-related mortality in any of the male and female animals in any of the dose groups. Clinical signs: there were no changes noted in male or female animals. Clinical signs during gestation (females): there were no substance-related clinical observations. There were several sperm-positive rats of the treated groups which did not deliver F1 pups (1/1/3 in groups at 0.2/5/50 mg/kg bw/day, respectively). This was not considered to be associated with the test compound. Clinical signs during lactation (females): there were no substance-related clinical observations. Food consumption: there was no difference between treated and control male and female groups. Body weight: there was no difference between treated and control male and female groups. Male fertility index: one low dose male and two high dose males did not generate pups. Thus the male fertility index was 100/96/100/100/92 % in the groups at 0/0.2/1/5/50 mg/kg bw/day, respectively, which was within the normal range of biological variation. Sperm parameters: no treatment-related effects were noted for the sperm parameters, examined at or after the sacrifice of the F0 parental males. The number of homogenization resistant testicular spermatids or caudal epididymal sperm, the percentages of abnormal and normal sperm and sperm motility data were similar between the examined test substance-treated groups and the concurrent control group (0, 0.2, 1, 5 and 50 mg/kg bw/day) and did not show any biologically or statistically significant differences.

The female fertility index was 100/96/100/100/92 % in the groups at 0/0.2/1/5/50 mg/kg bw/day, respectively, which was within the range of historical control data. The duration of gestation was similar in all test groups, i.e. 21.9 to 22.0 days. The gestation index was 100/100/100/96/96 % in the groups at 0/0.2/1/5/50 mg/kg bw/day, respectively. Implantation was not affected as the number of implantation sites was comparable between all control and treated groups. There was no indication of intrauterine embryo-fetolethality, as the postimplantation loss showed no statistically significant differences between the groups. The rate of liveborn pups was 99 % in the control and test groups at 0.2 to 5 mg/kg bw/day and 100 % in the treatment group at 50 mg/kg bw/day. The number of stillborn pups was comparable between the groups.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The mean number of delivered pups per dam was comparable between all groups. The viability index on day 4 after birth (post parturition, p.p.) was 99 % in all groups. There was no biologically relevant difference in the sex ratio between control and test groups. There were no clinical signs noted on day 4 p.p. in any group. There was no statistically significant difference in pup body weight between control and test groups, including the number of stunted pups. The macroscopic examination of all pups at necropsy revealed a number of findings in the pericardial vessels, which were considered to be test substance-induced (see "Remarks on results including tables and figures"). Additionally, a few of the examined F1 pups showed some spontaneous findings at necropsy (e.g. post mortem autolysis, hemorrhagic thymus, short innominate, dilated innominate, long innominate, dextrocardia, enlarged atrial chamber, absent atrial chamber) scattered throughout the test substance-treated groups and the control. These findings occurred without a clear relation to dosing and/or most of it can be found in the historical control data at comparable or even higher incidences. The histopathological examination revealed aneurysms and focal hemorrhages of the major pericardial blood vessels. Aneurysms occurred at all tested dose levels (see "Remarks on results including tables and figures").

The female fertility index was 100/96/100/100/92 % in the groups at 0/0.2/1/5/50 mg/kg bw/day, respectively, which was within the range of historical control data. The duration of gestation was similar in all test groups, i.e. 21.9 to 22.0 days. The gestation index was 100/100/100/96/96 % in the groups at 0/0.2/1/5/50 mg/kg bw/day, respectively. Implantation was not affected as the number of implantation sites was comparable between all control and treated groups. There was no indication of intrauterine embryo-fetolethality, as the postimplantation loss showed no statistically significant differences between the groups. The rate of liveborn pups was 99 % in the control and test groups at 0.2 to 5 mg/kg bw/day and 100% in the treatment group at 50 mg/kg bw/day. The number of stillborn pups was comparable between the groups.

Effect levels (fetuses)

Dose descriptor:

LOAEL

Effect level:

0.2 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Developmental toxicity was noted at dose levels without maternal toxicity.

INCIDENCES OF SELECTED PERICARDIAL VESSEL FINDINGS IN PUPS
Finding	Dose Group [mg/kg bw/day]
	0	0.2	1	5	50
All animals
Animals [n]	304	298	319	288	270
Dissecting aneurysm^#[n]	0	2	1	1	60
(Multi) focal hemorrhage [n]	1	1	2	3	4
Increased thickness of adventitial layer [n]	0	0	0	0	5
Males
Animals [n]	156	148	159	132	125
Dissecting aneurysm^#[n]	0	0	0	0	26
(Multi) focal hemorrhage [n]	1	0	0	2	2
Increased thickness of adventitial layer [n]	0	0	0	0	0
Females
Animals [n]	148	150	160	156	145
Dissecting aneurysm^#[n]	0	2	1	1	34
(Multi) focal hemorrhage [n]	0	1	2	1	2
Increased thickness of adventitial layer [n]	0	0	0	0	5
^#: Summary of animals with one or more aneurysms of major pericardial blood vessel locations (aorta, pulmonary trunk, ductus arteriosus, innominate arteria).

OCCURRENCE OF PERICARDIAL VESSEL FINDINGS IN PUPS^#
Finding	Dose Group [mg/kg bw/day]
[mean % of affected pups/litter]	0.2	1	5	50
Dilated descending aorta	0	0	0	9.8**
Aneurysm of descending aorta	0	0	0	4.4**
Dilated pulmonary trunk	0	0	0	0.3
Aneurysm of ductus arteriosus	0	0.4	0.3	8.1**
Aneurysm of pulmonary trunk	0	0	0	2.8**
Malpositioned subclavial branch	0.3	0	0	2.3**
Aneurysm of aortic arch	0	0	0	1.7**
Dilated aortic arch	0	0	0	0.3
^#: The incidence of all these findings was 0 in the control group; **: p < 0.01

Applicant's summary and conclusion

Executive summary:

In this developmental toxicity study, N-(2 -aminoethyl)ethanolamine (AEEA) was administered orally by gavage to groups of 25 male and 25 female Wistar rats at doses of 0.2, 1, 5 or 50 mg/kg bw/day in order to detect possible effects of the test substance on the great pericardial blood vessels of both sexes when exposed during prenatal and postnatal development. Control animals were dosed daily with the vehicle (doubly distilled water). The duration of treatment covered a 2 week premating period and mating in both sexes, approximately 2 weeks post-mating in males, as well as entire gestation and 4 days of lactation period in females. The NOAEL for general, systemic toxicity of the test substance was 50 mg/kg bw/day for the parental animals. The NOAEL for reproductive performance and fertility was 50 mg/kg bw/day for the F0 parental rats. A NOAEL for developmental toxicity in the F1 progeny was not determined because aneurysms of the pericardial vessels occurred at all tested dose levels. The LOAEL was therefore 0.2 mg/kg bw/day under the conditions of this reproduction/developmental screening test. Further, hemorrhages of the pericardial blood vessels were seen in all dose groups including the controls (one male affected). Hemorrhages are regarded to be precursor lesions of aneurysms, which can also develop in untreated animals, which was demonstrated by the observed case. The observation of blood vessel lesions in untreated animals is important, because this prevents the determination of NOAEL or LOAEL values. The pericardial blood vessels are not routinely examined in repeated dose studies such as OECD TG 421/422 studies. Consequently, there is no historical control database which would allow to judge whether the effects seen in the treated groups are within or above the historical control range. Therefore, a treatment-related NOAEL for developmental toxicity in the F1 progeny could not be derived in this study. In the absence of a reliable historical control database the same problem arises regarding the setting of a LOAEL, because there was one case of hemorrhage, thought to be a precursor of aneurysm, in an untreated animal. It is therefore necessary to establish a historical control database. It should be further mentioned that the dose-response curve was rather low in the range from 0 to 5 mg/kg bw/day and that there was a steep increase at 50 mg/kg bw/day, based on the cumulated percentage of animals showing either aneurysms, hemorrhage, or an increased thickness of the adventitial layers (0.33/1.01/1.57/1.39/25.5% in groups at 0/0.2/1/5/50 mg/kg bw/day, respectively). Overall, developmental toxicity was noted at all dose levels without maternal toxicity at any dose level. A clearly increased incidence of blood vessel lesions was seen at 50 mg/kg bw/day, whereas it cannot be determined whether the incidence of lesions observed in the dose range from 0.2 to 5 mg/kg bw/day is comparable or increased compared to untreated control animals.

This developmental toxicity study in the rat with a special focus on damage to vessels mainly in the form of aneurysms (best examined shortly after birth) is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3550; OECD 421) in rats.