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Toxicity to reproduction

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003
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

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
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 (yellowish-clear)
- Analytical purity: 99.8 area % (analytical report 01L00492)
- Stability: stable under storage conditions, confirmed by reanalysis
- Storage condition of test material: stored at room temperature
- Source: BASF AG

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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).

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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"

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

 

 

Applicant's summary and conclusion

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.