Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Food consumption was not determined between days 14 and 21 after parturition
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Monoethanolamin-hydrochlorid (2-Aminoethanol) Test substance Nos: 1) 05/0372-2; 2) 05/03723; 3) 05/0372-4
- Analytical purity: >99 %
- Physical state: solid / white
- Date of production: ad 1) 04 Aug 2006; ad 2) 14 Aug 2006; ad 3) 15 Sep 2006
- Lot/batch No.: ad 1) JB116/2+3 (from 09 Aug – 04 Oct 2006); ad 2) JB116/4 (from 04 Oct – 29 Nov 2006); ad 3) JB116/9-17 (from 29 Nov 2006 until the scheduled termination of the in life part of the study)
- Storage condition of test material: Room temperature, under N2

Test animals

Species:
rat
Strain:
other: Crl:WI (Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Age at study initiation: (P) 16 days
- Weight at study initiation: (P) Males: 162.1 (142.5 – 186.5) g ; Females: 126.2 (110.6 – 145.1) g;
- Fasting period before study: none
- Housing: housed individually in type DK III stainless steel wire mesh cages
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland ad libitum
- Water: ad libitum
- Acclimation period: 16 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test substance (ethanolamine hydrochloride, EAH) was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period.

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individual
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Positive control:
not done

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days and once daily on weekends


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: body weights of F0 and F1 parents were determined once weekly; during gestation and lactation F0 and F1 females were weighed on days 0, 7, 14 and 20 of gestation, and on days 1, 4, 7, 14 and 21 after birth.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: once weekly (over a period of at least 6 days each) and weekly during gestation (days 0-7, 7-14, 14-20 post coitum; p.c.) and lactation periods (days 1-4, 4-7, 7-14 post partum; p.p.).

OTHER:
The F1 and F2 pups were sexed on the day of birth (day 0 p.p.) and weighed on days 1, 4, 7, 14, and 21 p.p. Their viability was recorded. At necropsy, all pups were examined macroscopically (including weight determinations of brain, spleen and thymus in one pup/sex/litter).

Serum concentrations of the test substance:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of Ethanolamine hydrochloride
Estrous cyclicity (parental animals):
Estrous cycle data were evaluated for F0 and F1 generation females over a three week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1] male parental generations:
motility, sperm head count, morphology
Postmortem examinations (parental animals):
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs. The liver, kidneys, adrenal glands, testes, epididmides. Cauda epididymis, prostate, seminal vesicles, ovaries, uterus, spleen, brain, pituitary gland and thyroid glands (with parathyroids) were weighed and the vagina, cervix uterie, uterus, ovaries, oviducts, left testis, left epididymis, seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroids (with parathyroids)and all gross lesions were fixed in an appropriate fixative, histologically processed and examined by light microscopy. From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated for numbers of primordial and growing follicles.
As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration.
Postmortem examinations (offspring):
All pups with scheduled sacrifice (i.e. pups, which were culled on day 4 p.p., and pups, which were sacrificed on day 21 p.p. or subsequent days) were killed by means of CO2. The spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
Statistics:
see below

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

The following test substance-related effects/findings were recorded:

1000 mg/kg bw/d
F0 parental animal:

Yellow discolored urine for male and female parental animals
Statistically significantly decreased body weight gain of the dams during gestation, body weight 8% below control on gestation day 20
Statistically significantly decreased food consumption in parental females during lactation
Statistically significantly decreased sperm head count in the cauda epididymidis of males
Statistically significantly decreased absolute and relative weight of epididymides, cauda epididymidis and prostate in males
Statistically significantly less implantation sites
Statistically significantly increased post-implantation loss
Statistically significantly smaller litters


F1 parental animals:
Yellow discolored urine for male and female parental animals
Statistically significantly decreased body weight gain of the dams during gestation
Decreased food consumption in parental females during lactation
Statistically significantly decreased absolute and relative weight of epididymides and cauda epididymidis in males
Statistically significantly less implantation sites
Statistically significantly increased post-implantation loss
Statistically significantly smaller litters


300 mg/kg bw/d
F0 or F1 parental animals: No test substance-related adverse effects


100 mg/kg bw/d
F0 or F1 parental animals: No test substance-related adverse effects

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: fertility, reproductive performance and systemic toxicity

Results: F1 generation

Details on results (F1)

The following test substance-related effects/findings were recorded:

F1 or F2 pups: No test substance-related adverse effects


300 mg/kg bw/d
F1 or F2 pups: No test substance-related adverse effects


100 mg/kg bw/d
F1 or F2 pups: No test substance-related adverse effects

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no pre-and postnatal developmental toxicity

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no pre-and postnatal developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Test substance stability:

The stability of test substance in rat diet was demonstrated for a period of 32 days at room temperature in a different batch of comparable quality, which was not used for the study. The homogeneity of the mixtures was verified. The concentration control analyses of the samples taken revealed that the values were within a range of 90-110% of the nominal concentration in all analyses at all time points, with the exception of one concentration in the feed of the high-dose group (88%).

Plasma concentrations of 2 -aminoethanol were below 3 mg/kg for all control animals, <3 - 4 mg/kg for the low dose animals, 8 - 11 mg/kg for the mid dose animals and 60 – 81 mg/kg for the high dose animals.

Toxicokinetic data of 2 -aminoethanol (calculated as 2 -aminoethanol hydrochloride) from this two-generation reproduction toxicity study show a dose dependency of the plasma levels of 2 -aminoethanol in the experimental animals and there with prove the bioavailability of 2 -aminoethanol hydrochloride in principle.

 

Under these conditions, no test substance-related findings from clinical examinations or gross and histopathology were observed, which indicate that the administration of the test compound via the diet adversely affected the fertility or reproductive performance of the F0 or F1 parental animals up to and including a nominal dose of 300 mg/kg bw/d. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups.

At the high-dose level (1000 mg/kg bw/d), absolute and relative weights of epididymides and cauda epididymidis were decreased and, in the F0 generation only, the number of homogenization resistant caudal epididymal sperm was slightly, but significantly reduced. However, histomorphological correlates for these findings were missing.

 

In the high-dose F0 and F1 generation females (1000 mg/kg bw/d), decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test compound on fertility and/or reproductive performance at high doses. It has to be noted that a dose of 1000 mg/kg bw/d also caused beginning systemic toxicity in these females, as was indicated by reduced food consumption and/or body weight gain during gestation/lactation.

 

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 1000 mg/kg bw/d. The test substance did not adversely influence pup viability, body weight, sex ratio and sexual maturation.

 

Thus, under the conditions of the present two-generation reproduction toxicity study, the NOAEL (no observed adverse effect level) for fertility, reproductive performance and systemic toxicity in parental F0 and F1 Wistar rats is 300 mg/kg bw/d.

 

The NOAEL for pre-and postnatal developmental toxicity in their offspring is 1000 mg/kg bw/d.

Tables

Mean test substance intake (mg/kg bw/d; minimum value / maximum value)

 

Test group 01
(100 mg/kg bw/d)

Test group 02
(300 mg/kg bw/d)

Test group 03
(1000 mg/kg bw/d)

F0 males

94.3 (72.4 / 102.5)

283.2 (218.4 / 309.4)

943.3 (716.7 / 1032.6)

F0 females (premating)

96.7 (80.5 / 100.7)

289.6 (241.2 / 304.9)

964.4 (792.4 / 1017.8)

F0 females
(F1 litter)
- gestation period
- lactation period*



103.5 (92.6 / 111.6)
99.2 (81.6 / 120.2)



315.2 (284.8 / 337.9)
306.7 (249.7 / 370.3)



1043.2 (989.4 / 1084.7)
866.0 (668.6 / 1053.9)

* = Days 1–14 p.p. only

Absolute organ weights (P-generation)

Compared to the controls (= 100%), the following values (in %) were significantly changed (printed in bold):

 

Male animals

Female animals

Group

01

100 mg/kg bw/d

02

300 mg/kg bw/d

03

1000 mg/kg bw/d

01

100 mg/kg bw/d

02

300 mg/kg bw/d

03

1000 mg/kg bw/d

Brain

99%

100%

97%*

 

 

 

Cauda epididymis

99%

102%

88%**

 

 

 

Epididymides

100%

101%

92%**

 

 

 

Prostate

92%

99%

86%**

 

 

 

Spleen

 

 

 

105%*

107%

97%

 

*: p≤0.05; **: p≤0.01

 

All other mean absolute weight parameters did not show significant differences compared to the control groups.

 

The decrease of absolute weights of cauda epididymis, epididymides, and prostate in male top-dose animals (1000 mg/kg bw/d) were considered as treatment-related effects.

 

The decrease of brain weights in top-dose males (1000 mg/kg bw/d) as well as the increase of spleen weights in low-dose females (100 mg/kg bw/d) was considered as incidental and not treatment-related due to a missing dose-response relationship.

Absolute organ weights (F1 generation)

Compared to the controls (= 100%), the following values (in %)were significantly changed (printed in bold):

 

 

Male animals

Female animals

Group

11

100 mg/kg bw/d

12

300 mg/kg bw/d

13

1000 mg/kg bw day

11

100 mg/kg bw/d

12

300 mg/kg bw/d

13

1000 mg/kg bw/d

Cauda epididymis

96%

99%

88%**

 

 

 

Epididymides

100%

101%

91%**

 

 

 

Kidneys

99%

106%*

111%**

103%

106%**

115%**

Spleen

99%

103%

92%*

 

 

 

Thyroid glands

106%

99%

109%*

110%

118%**

111%*

 

*: p≤0.05; **: p≤0.01

All other mean absolute weight parameters did not show significant differences compared to the control groups.

The decrease of absolute weights of cauda epididymis and epididymides in male top-dose animals (1000 mg/kg bw/d) were considered to be treatment-related.

 

The increase of absolute kidney weights of male and female animals in mid- (300 mg/kg bw/d) and top-dose (1000 mg/kg bw/d) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase was less likely.

 

The decrease of spleen weights in top-dose males as well as the increase of thyroid glands in top-dose males and mid- and top-dose females, respectively, is considered incidental and not treatment-related due to a missing dose-response relationship.

Applicant's summary and conclusion