2-(2-aminoethylamino)ethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a toxicokinetic study with AEEA in female Wistar rats concerning the distribution into the late gestation fetus and into milk (Moore N.P. et al., 2012) it was shown that rat offsprings were exposed to AEEA both in utero and during lactation at toxicologically relevant dose levels.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

not specified

GLP compliance:

yes

Remarks:

Exception: the additional histopathological examination of pups (see "Cross-reference to same study") was not performed under GLP

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 76 - 83 days
- Weight at study initiation: Males: mean 279 g; Females: mean 192 g
- Housing: during the study period, individually in type DKIII stainless steel wire mesh cages supplied by Becker & Co, Castrop-Rauxel, Germany, with the following exceptions: for the overnight mating the females were put into the cages of the males; from day 18 p.c. until sacrifice, the pregnant animals and their litters were housed in Makrolon type MIII cages also supplied by Becker & Co.
- Diet: Kliba laboratory diet rat/mouse/hamster (Provimi Kliba, Kaiseraugst, Switzerland), ad libitum
- Water: drinking water from bottles, ad libitum

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

IN-LIFE DATES: From: 09 April 2002 To: 03 June 2002

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING 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 10 day intervals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for max. 2 weeks
- Proof of pregnancy: vaginal smear referred to as day 0 p.c. (post coitum)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity and correctness of the prepared concentrations were analysed.

Duration of treatment / exposure:

Exposure period: 53 days (dams)
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 50, 250 and 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen on the basis of the outcome of a Japanese 4-week oral toxicity study. In this repeated dose study slight signs of systemic toxicity were observed at 1000 mg/kg bw/day (i .e. slightly changed hematology and clinical chemistry parameters, histopathological findings in kidneys and stomach) and similar, even less severe findings were seen at a dose level of 250 mg/kg bw/day. A NOAEL of 60 mg/kg bw/day was determined.

A constant dose volume of 10 mL/kg bw was used. Dosing was adjusted weekly, based on the animals' most recent body weights.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality (once daily during the week-end and on holidays), daily for clinical sings, nesting, littering and lactation behaviour

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: generally, once a week. Body weight changes were calculated from these records.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: generally, once a week

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Litter size, number of live born and stillborn pups was recorded as soon as possible. The viability index was calculated from the number of live pups at the day of birth and day 4 after birth. The sex distribution was calculated at day 0 and day 4 after birth from the numbers of live male and female pups. The pups were examined daily for clinical symptoms. They were weighed on day 1 and 4 after birth, and the data were used to calculate body weight gain and to identify "runts" (pups with body weights more than 25 % below  the mean body weight of the respective control pups).

GROSS EXAMINATION OF DEAD PUPS:
Please refer to "Postmortem examinations (Offspring)".

Postmortem examinations (parental animals):

All parental animals that died or were sacrificed were necropsied and subjected to a gross postmortem examination. The anesthesized animals and reproductive tissues (testes, epididymides, ovaries) were weighed. Selected organs (vagina, cervix uteri, uterus, ovaries, oviducts, testes, epididymides, seminal vesicles, coagulating glands, prostate gland, pituitary gland, and all gross lesions) were fixed in 4 % formaldehyde or in Bouin's solution. Testes, epididymides and both ovaries were embedded. All gross lesions and all reproductive organs of the control and high dose animals were examined by light microscopy. A Differential Ovarian Follicle Count (DOFC) was performed on all control and high dose females, and on the non-pregnant animals. 10 to 13 serial sections of embedded ovaries were obtained, depending of the size of the ovaries. Ten hematoxylin and eosin stained slices per animal were selected for follicle count on "primordial follicles", "growing follicles", "primordial and growing follicles", and "antral follicles" according to the definitions given by Plowchalk et al. (1993). The number of corpora lutea was additionally determined on the 5th slide. The findings were assessed on the 5th slide and recorded in EXCEL tables for ovary 1 and 2 of every animal on any slide, giving in summary the incidences of all types of follicles for all 10 animals per group. The number of corpora lutea was obtained accordingly.

Postmortem examinations (offspring):

All stillborn, dead and surviving (after sacrifice by means of carbon dioxide on day 4 after birth) pups were examined externally, eviscerated and their organs were assessed macroscopically. Additional examinations of affected pups were performed if there were notable findings. A modified method of Kimmel and Trammel (1981) was used to examine skeletal findings, and/or pups were fixed in Harrison's Fluid and examined according to Barrow and Taylor (1969) for any visceral findings. The stained skeleton was examined under a stereomicroscope or a magnifying glass.

Statistics:

For the clinical examinations, Dunnett's test, Fisher's exact test, and the Wilcoxon test were used. The Kruskal-Wallis test followed by the Wilcoxon test was used to analyze the pathology data. The follicle data were analyzed using the Wilcoxon test.

Reproductive indices:

For the males, the mating and fertility indices were calculated.
For the females, the mating, fertility and gestation indices were calculated.

Offspring viability indices:

Live birth index and post implantation loss were determined.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no substance-related mortalities in any of the male and female animals. Salivation after treatment was noted in all high dose males (1000 mg/kg bw/day) from study day 4 onwards, and in all high dose females from study week 0 onwards. Incidences ranged between 1 and 10 of 10 animals. Regular care on fur appeared to be impaired in individual males and females on several occasions during the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Statistically significantly reduced food consumption in F0 males was noted during the first study week (by approximately 21 %; -9 % compared to control if calculated for the whole study period (weeks 0-4)) and in females (-12 % during premating weeks 0-2, -10 % during gestation days 0-20 post coitum). At study week 4, the body weight of males was 6 % lower than controls and body weight gain was lowered by 30 % (if calculated for weeks 0-4). Female body weight was 24 % lower than controls at gestation day 20 and body weight gain was lowered by 72 %. Please refer to "Remarks on results including tables and figures".

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The fertility index was reduced to 60 % in males and females, though the mating index was unchanged. No live pups were delivered by the high dose dams. Postimplantation loss was 100 % (see table in "Remarks on results including tables and figures").

ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weights were not affected by the treatment with the test substance.
Absolute weights of the epididymides and ovaries of high dose group animals (1000 mg/kg bw/day) were significantly reduced when compared with control group. No other mean absolute weight parameter showed any significant differences to control. Please refer to "Remarks on results including tables and figures".

GROSS PATHOLOGY (PARENTAL ANIMALS)
Gross lesions occurred only once per group and were not related to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related findings were noted in the reproductive organs of the male and the female rats.

OTHER FINDINGS (PARENTAL ANIMALS)
The differential ovarian follicle count (DOFC) did not detect depletions of the various follicle populations. Instead, compared to controls increases were noted in the high dose animals of the primodial follicles (110 %), growing follicles (151 %**), and primodial and growing follicles (116 %), and antral follicles (125 %). The number of corpora lutea was also increased (145 %**) (** = p < 0.01).

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Clinical signs; body weight; food consumption; fertility index; gestation index; number of implantation sites; post implantation loss

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

VIABILITY (OFFSPRING)
No pups were delivered by the high dose dams. In the other dose groups, the number of stillborn pups and pup survival (days 0-4) was unaffected at 50 mg/kg bw/day, whereas at 250 mg/kg bw/day an increased number of stillborn pups and a significantly reduced viability index were noted (see "Remarks on results including tables and figures").

CLINICAL SIGNS (OFFSPRING)
None of the pups showed any clinical signs.

BODY WEIGHT (OFFSPRING)
No group differences were noted in the pup body weights and in the number of "runts".

GROSS PATHOLOGY (OFFSPRING)
Observation of adverse pup necropsy findings (such as pericardial vessels) was 48 % pups in 100 % of the litters at 50 mg/kg bw/day. The ratio of affected pups per litter was 48.4 % (versus 0 % in the controls).
Observation of adverse pup necropsy findings was 89 % pups in 100 % of the litters at 250 mg/kg bw/day. The ratio of affected pups per litter was 87.8 % (versus 0 % in the controls). For the occurrence of findings of the pericardial vessels please refer to the table in "Remarks on results including tables and figures".

HISTOPATHOLOGY (OFFSPRING)
See "Overall remarks"

Dose descriptor:

LOEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No NOAEL could be established due to malformations (abnormalities of the pericardial vessels) at the low est dose level of 50 mg/kg bw/day.

Reproductive effects observed:

not specified

Test substance analysis:

The stability of AEEA in tap water over a period of up to 10 days at room temperature was proven. Concentration control analyses revealed that the target concentrations were met (98 % - 104 %).

PARENTAL DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	250	1000
Food consumption
Males	-	-	-	Trans. red. *
Females	-	-	-	Trans. red. */**
Body weight
Males	-	-	-	Trans. red. **
Females	-	-	-	Trans. red. */**
-: no effect, Trans. red.: transiently reduced, *: p < 0.05, **: p < 0.01

REPRODUCTION INDICES AND DELIVERY DATA OF PARENTAL ANIMALS
Endpoint	Dose Group [mg/kg bw/day]
	0	50	250	1000
Males
Mating index [%]	100	100	90	100
Fertility index [%]	90	90	90	60
Females
Mating index [%]	100	100	90	100
Fertility index [%]	90	90	100	60
Gestation index [%]	89	100	100	0**
Post implantation loss [%]	4.7	5.5	6.4	100**
Liveborn index [%]	99	99	95	0**
*: p < 0.05, **: p < 0.01

ORGAN WEIGHTS OF PARENTAL ANIMALS
Endpoint	Dose Group [mg/kg bw/day]
	50	250	1000
Absolute weights
Epididymides [%]	-	-	-14**
Ovaries [%]	-	-	-16**
Other weights [%]	-	-	-
Relative weights	-	-	-
-: no effect, *: p < 0.05, **: p < 0.01

OFFSPRING DATA
Endpoint	Dose Group [mg/kg bw/day]
	0	50	250	1000
Delivered pups [n]	82	107	93	0
Stillborn pups [n]	1	1	5	n.a.
Viability index (days 0-4) [%]	99	96	80*	n.a.
n.a. not applicable, *: p < 0.05

MACROSCOPICAL FINDINGS IN PERICARDIAL VESSELS (OFFSPRING)
Endpoint	Dose Group [mg/kg bw/day]
	50	250
Aneurysms, mean pups/litter [%]
Aorta	17.7**	55.2**
Pulmonary trunk	16.3**	44.5**
Carotid artery	0	5.0
Ductus arteriosus	1.0	1.0
Dilatations, mean pups/litter [%]
Carotid artery	18.8**	35.8**
Descending aorta	21.1**	55.2**
Pulmonary trunk	4.6	0
Other findings, mean pups/litter [%]
Abnormal course of carotis	10.3	21.9**
High aortic arch	0	2.8
Pericardium filled with blood	0	1.1
n.a. not applicable, **: p < 0.01

Executive summary:

In a reproduction/developmental toxicity screening test AEEA (99.8 %) was administered to Wistar rats (10 rats/sex/dose level) by oral gavage at dose levels of 0, 50, 250 or 1000 mg/kg bw/day. No substance-related mortality was noted. Clinical signs of toxicity were only seen at a dose of 1000 mg/kg bw/day and included salivation and impairment of the regular care on fur. The food consumption was significantly reduced in F0 males of the 1000 mg/kg bw/day group, particularly during the first week of treatment. In the F0 females of the same group, food consumption was also reduced, particularly during the premating and the gestation periods. For both males and females of the 1000 mg/kg bw/day group, the body weight gain was less than in controls. In females, body weight gain during gestation was 72 % less than in control females. Of the fertility and reproductive parameters, the mating index was unaffected by the treatment, whereas fertility was reduced in animals at 1000 mg/kg bw/day. Implantations per dam were reduced in the 1000 mg/kg bw/day group compared to control. Post implantation loss was 100 %, i.e. none of the females had live pups. Therefore, the gestation index was 0 %. Necropsy of F0 generation revealed no test substance related (histo)pathological abnormalities, including reproductive organs. Pups: No live pups were delivered within the 1000 mg/kg bw/day group. In the group treated with 250 mg/kg bw/day, the number of stillborn pups was increased in comparison to control. The viability index was lowered. Sex distribution was not affected. Pup necropsy revealed high incidences of abnormalities especially affecting the pericardial vessels in terms of aneurysms, dilatations, and abnormal course in pups from dams at 50 and 250 mg/kg bw/day. 48 % and 89 %, respectively, of the pups were affected in 100 % of the litters. Because of the malformations seen in the pups at both 50 and 250 mg/kg bw/day doses, no NOAEL value could be established for the endpoint developmental toxicity in this study. The NOAELs were as follows: Parental animals: Systemic toxicity, reproductive performance and fertility: 250 mg/kg bw/day. Progeny: no NOAEL established.

This study is acceptable and satisfies the guideline requirement for a reproduction/developmental toxicity screening test (OECD 421) in rats.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and Guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a reproduction/developmental toxicity screening test (BASF AG Department of Experimental Toxicology and Ecology, 2003)

AEEA (99.8 %) was administered to Wistar rats (10 rats/sex/dose level) by oral gavage at dose levels of 0, 50, 250 or 1000 mg/kg bw/day. No substance-related mortality was noted. Clinical signs of toxicity were only seen at a dose of 1000 mg/kg bw/day and included salivation and impairment of the regular care on fur. The food consumption was significantly reduced in F0 males of the 1000 mg/kg bw/day group, particularly during the first week of treatment. In the F0 females of the same group, food consumption was also reduced, particularly during the premating and the gestation periods. For both males and females of the 1000 mg/kg bw/day group, the body weight gain was less than in controls. In females, body weight gain during gestation was 72 % less than in control females. Of the fertility and reproductive parameters, the mating index was unaffected by the treatment, whereas fertility was reduced in animals at 1000 mg/kg bw/day. Implantations per dam were reduced in the 1000 mg/kg bw/day group compared to control. Post implantation loss was 100 %, i.e. none of the females had live pups. Therefore, the gestation index was 0 %. Necropsy of F0 generation revealed no test substance related (histo)pathological abnormalities, including reproductive organs. Pups: No live pups were delivered within the 1000 mg/kg bw/day group. In the group treated with 250 mg/kg bw/day, the number of stillborn pups was increased in comparison to control. The viability index was lowered. Sex distribution was not affected. Pup necropsy revealed high incidences of abnormalities especially affecting the pericardial vessels in terms of aneurysms, dilatations, and abnormal course in pups from dams at 50 and 250 mg/kg bw/day. 48 % and 89 %, respectively, of the pups were affected in 100 % of the litters. Because of the malformations seen in the pups at both 50 and 250 mg/kg bw/day doses, no NOAEL value could be established for the endpoint developmental toxicity in this study. The NOAELs were as follows: Parental animals: Systemic toxicity, reproductive performance and fertility: 250 mg/kg bw/day. Progeny: no NOAEL established.

This study is acceptable and satisfies the guideline requirement for a reproduction/developmental toxicity screening test (OECD 421) in rats.

 

In an OECD 421 reproductive/developmental toxicity screening study (BASF SE, Experimental Toxicology and Ecology, 2008) 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 (see section below). 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. 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 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 parental rats.

Short description of key information:
The NOAEL for systemic, reproduction and fertility performance was established to be 250 mg/kg bw/day from the results of OECD Guideline 421 study (BASF AG Department of Experimental Toxicology and Ecology, 2003). Female fertility was reduced in animals at 1000 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
The Key study (BASF AG, 2003) was selected.

Effects on developmental toxicity

Description of key information

N-(2 -aminoethyl)ethanolamine (AEEA) caused aneurysms of the pericardial vessels in rat pups and has to be regarded as a developmental toxicant. A NOAEL for developmental toxicity in the F1 progeny could not be determined because aneurysms of the pericardial vessels occurred at all tested dose levels. 

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

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

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

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.

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.

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.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

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.

Dose descriptor:

LOAEL

Effect level:

0.2 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

0.2 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and Guideline study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a developmental toxicity study (BASF AG Department of Experimental Toxicology and Ecology, 2003), AEEA (99.8 area %) was administered to female Wistar rats (25 time-mated females/group) by oral gavage at dose levels of 0, 0.5, 2, 10 or 50 mg/kg bw/day from days 6 through 19 of gestation. The NOAEL for maternal and prenatal developmental toxicity was 50 mg/kg bw/day. There was no indication of teratogenicity, in particular there were no effects on the fetal great vessels, which had been noted in a preceding reproduction/developmental toxicity screening test with AEEA at 50 and 250 mg/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats.

 

In an OECD 421 reproductive/developmental toxicity screening study (BASF SE, Experimental Toxicology and Ecology, 2008),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. 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.

 

The damage to major vessels (predominately aneurysms) has been also seen in the OECD Guideline 421 study from 2003 which is reported in the section above ("effects on fertility") and in a small scale developmental toxicity study (BASF AG, Department of Experimental Toxicology and Ecology, 2005).

In a developmental toxicity study (The Dow Chemical Company, 2007), N-(2 -aminoethyl) ethanolamine (AEEA) was administered orally by gavage to groups of 5 female Wistar Han rats at a dose level of 250 mg/kg bw/day in order to determine if in utero exposure alone is sufficient to induce aneurysms and other great vessel malformations in the offspring of AEEA-exposed dams as seen in previous studies.

Three groups of five time-mated female animals were dosed on gestation day (GD) 6 -19, necropsied on GD 21; or on GD 6-19, necropsied on lactation day (LD) 4 or on GD 6 -LD 3, necropsied on LD 4. In Group 1, the incidence of malformations was 91.1%, with the two most prevalent malformations being high aortic arch (78.9%) and abnormal course of the carotids (75.5%). There was only one fetus that had an aneurysm (aortic). In Group 2, 100 % of fetuses were malformed. The most prevalent malformations in this group were abnormal course of the carotids (93.8 %) and aneurysms (58.6 %) primarily affecting the aorta. The incidence of high aortic arch was much lower (5.7 %) in Group 2 relative to Group 1. In Group 3, 100 % of the fetuses had malformations, with their type and incidence generally similar to that of Group 2, except that aneurysms were much more prevalent (97.7 %). These data clearly show that exposure to AEEA during gestation alone is sufficient to induce great vessel aneurisms and malformations.

In Xu et al., 2014, AEEA was administered to pregnant sprague-Dawley rats on GD 14 to 20, daily. A preliminary experiment was performed with daily intraperitoneal injections of AEEA. The dose groups were 1 (n=1), 10 (n=3), 25 (n=3), 50 (n=5), 75 (n=1), and 100 mg/kg (n=1), and a control group (saline)(n=2). General signs of toxicity including lethargy and weight loss at were observed in dams exposed to 50, 75, and 100 mg/kg AEEA. The dams from the 75 and 100 mg/kg dose groups died after 5-6 doses. On PND 1 morphological studies were performed on the delivered pups of the surviving dams. AEEA caused an increase in still born pups and dead retained fetuses. Dissecting Aortic Aneurysm (DAA) ratio in live pups was 30, 66, and 78% in 10, 25, and 50 mg/kg dose group, respectively. In the main experiment rats were dosed with AEEA by oral gavage. The dose groups were 10 (n=2), 50 (n=4), mg/kg (n=4), and 150 mg/kg (n=12), and a control group (saline)(n=4). There were no signs of toxicity in dams after gavage administration and there was no increase in retained fetuses, stillborns, or a decrease in litter number or mean pup weights observed. On PND 1, morphological studies were performed on the delivered pups of the surviving dams. The DAA lesion ratio in live pups was 30%, 88%,100%, and 100% in the 10, 50, 100, and 150 mg/kg dose group, respectively. Additionally, the lesions were graded and the higher the dose the higher the grade lesion present in the dose group. This supports a direct dose-response relationship. An additional experiment was performed exposing rats to 50, 100, and 150 mg/kg bw AEEA and rats were necropsied on GD20. After exposure to 100 and 150 mg/kg, immunohistochemistry showed a decrease collagen type 1 in both the ascending and descending aorta in 70% of the fetuses. In the controls, collagen type 3 was mainly observed in adventitia of both ascending and descending aorta, with some positive staining sparsely in media of the vessels. In the two highest dose groups collagen type 3 was hardly detected in the aorta's. In the 150 mg/kg dose group western blot showed a decreased protein expression in the aortas compared to the control (no other dose levels were investigated). The collagen type 1 and 3 expression decreased significantly (p< 0.05) 34% and 30% respectively, compared to the control. In parallel the expression of collagen type 1 and 3 in the skin of these fetuses was also examined, but no differences were found in collagen type 1 and 3 expression compared to the control. This indicated that the defective expression of collagen type 1 and 3 is specific for the blood vessels.

Justification for selection of Effect on developmental toxicity: via oral route:
The key study (BASF AG, 2008) was selected.

Toxicity to reproduction: other studies

Additional information

Please refer to IUCLID chapter 7.1. toxicokinetics.

Justification for classification or non-classification

N-(2 -aminoethyl)ethanolamine (AEEA) caused aneurysms of the pericardial vessels in rat pups at low dose levels and impaired the fertility of the female rat at 1000 mg/kg bw/d. Rat offsprings are exposed to AEEA both in utero and during lactation. The substance is present at potentially toxic levels in breast milk.

Based on these results AEEA is classified as a Repr. Category 2, R61 (may cause harm to the unborn child), Repr. Category 3 and R62 (possible risk of impaired fertility) according to Directive 67/548/EEC and as Reproductive Toxicant Cat 1B with the hazard statement H360Df (may damage the unborn child, suspected of damaging fertility) according to Regulation (EC) 1272/2008.

Additionally AEEA is classified with R64 (may cause harm to breastfed babies) and with H362 (may cause harm to breast-fed children) according to Directive 67/548/EEC and Regulation (EC) 1272/2008, respectively.

This classification differs from the current entry in Annex VI of Regulation (EC) 1272/2008 (Repr. 1B, H360FD and H361 (Table 3.1) and Repr. Cat. 3, R62; Repr. Cat. 2, R61 (Table 3.2)).