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Toxicological information

Toxicity to reproduction

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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:
2009

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Monoethanolamin-hydrochlorid (2-Aminoethanol hydrochloride)
- 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 study termination)
- Storage condition of test material: room temperature, under nitrogen

Test animals

Species:
rat
Strain:
Wistar
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) mean: 162.1 g (142.5–186.5 g) males, mean: 126.2 g (110.6 – 145.1 g) females
- Fasting period before study: none
- Housing: 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: 20-24 °C
- Humidity: 30-70 %
- Air changes: 10-15 changes/hour
- Photoperiod: 12 hours dark / 12 hours light


Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: plain diet (feed admixture)
Details on exposure:
DIET PREPARATION
The test substance 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: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance 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.
Duration of treatment / exposure:
>75 days (F0 generation parental animals)
Frequency of treatment:
continuous
Details on study schedule:
- F1 parental animals were not mated until 11 weeks after selection from the F1 litters.
- Selection of parents from F1 generation was conducted when the pups were 4 days of age.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
25 rats
Control animals:
yes, plain diet
Positive control:
not done

Examinations

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

BODY WEIGHT
- Time schedule: 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
- 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).

Plasma 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 2-Aminoethanol hydrochloride.
Estrous cyclicity (parental animals):
Estrous cycle data were evaluated for F0 and F1 generation females over a 3 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
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were killed and discarded.

GROSS EXAMINATION OF DEAD PUPS
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
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, epididymides, cauda epididymidis, 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 of F1 female animals (control and high dose), 5 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 for analysis of 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 Carbon dioxide exposure. The spleen and thymus of 1 pup/sex/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 "Any other information on materials and methods incl. tables" below
Reproductive indices:
Male and female mating indices, male and female fertility indices, gestation index
Offspring viability indices:
Live birth index, viability index, lactation index

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/day
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/day
F0 or F1 parental animals: No test substance-related adverse effects


100 mg/kg bw/day
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: Based on effects on fertility and reproductive performance as well as systemic toxicity noted at 1000 mg/kg bw/day

Results: F1 generation

Details on results (F1)

No test-substance related adverse effect was noted at any dose level.

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: There was no pre-and/or 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: There was no pre-and/or postnatal developmental toxicity.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Test substance stability

The stability of the 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 present 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 bw for all control animals, <3 -4 mg/kg bw for the low dose animals, 8 -11 mg/kg bw for the mid dose animals and 60 -81 mg/kg bw for the high dose animals.

Toxicokinetic data of the test substance showed a dose dependency of the plasma levels of 2 -Aminoethanol in the experimental animals and therewith demonstrated 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 indicated that the administration of the test substance 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/day. 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/day), absolute and relative weights of epididymides and cauda epididymidis were decreased and, in the F0 generation only, the number of homogenization resistant caudal epididymidal sperm was slightly, but statistically reduced. However, histomorphological correlates for these findings were missing.

In the high dose F0 and F1 generation females (1000 mg/kg bw/day), decreased numbers of implants and increased resorption rates resulted in significantly smaller litters, giving evidence for an adverse effect of the test substance on fertility and/or reproductive performance at high doses. It has to be noted that the dose level of 1000 mg/kg bw/day 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/day. 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 was 300 mg/kg bw/day.

 

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

Tables

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

 

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

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

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

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/day

02

300 mg/kg bw/day

03

1000 mg/kg bw/day

01

100 mg/kg bw/day

02

300 mg/kg bw/day

03

1000 mg/kg bw/day

Brain

99%

100%

97%*

 

 

 

Cauda epididymidis

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 epididymidis, epididymides and prostate in male high dose animals (1000 mg/kg bw/day) were considered as treatment-related effects. The decrease of brain weights in high dose males (1000 mg/kg bw/day) as well as the increase of spleen weights in low dose females (100 mg/kg bw/day) 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 were significantly changed (printed in bold):

 

 

Male animals

Female animals

Group

11

100 mg/kg bw/day

12

300 mg/kg bw/day

13

1000 mg/kg bw day

11

100 mg/kg bw/day

12

300 mg/kg bw/day

13

1000 mg/kg bw/day

Cauda epididymidis

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 organ weight parameters did not show significant differences compared to the control groups. The decrease of absolute weights of cauda epididymidis and epididymidis in male high dose animals (1000 mg/kg bw/day) was considered to be treatment-related. The increase of absolute kidney weights of male and female animals in mid and high dose groups (300 and 1000 mg/kg bw/day) was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase was less likely. The decrease of spleen weights in high dose males as well as the increase of thyroid glands in high dose males and mid and high dose females was considered incidental and not treatment-related due to a missing dose-response relationship.

Applicant's summary and conclusion