(2-hydroxyethyl)ammonium nitrate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

other: two-generation reproductive toxicity study

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 416 (Two-generation reproduction toxicity study)

Deviations:

yes

Remarks:

Food consumption was not determined between days 14 and 21 after parturition

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.35 (Two-Generation Reproduction Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxyethylammonium chloride

EC Number:

217-900-6

EC Name:

2-hydroxyethylammonium chloride

Cas Number:

2002-24-6

IUPAC Name:

2-hydroxyethanaminium chloride

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 or test system 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 oral 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of 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 towards 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

Frequency of treatment:

continuous

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

Examinations

Observations and examinations performed and frequency:

For parental animals:

CAGE SIDE OBSERVATIONS
- Time schedule: twice daily on working days and once daily on weekends

BODY WEIGHT
- 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
- 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 test substance concentration.

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.

Parameters examined in [all/P/F1] male parental generations:
motility, sperm head count, morphology

Sacrifice and pathology:

For 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 epididymidis, 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 to be analyzed for Choline concentration.

Statistics:

See "Any other information on materials and methods incl. tables" below

Results and discussion

Results of examinations

Details on results:

Clinical examinations revealed no test substance-related adverse effects for F0 and F1 parental animals of low and mid dose levels (100 and 300 mg/kg bw/day). The test substance adversely affected food consumption of the high dose F0 females (1000 mg/kg bw/day) during lactation. Also, the body weight gain and, for the F0 generation, body weights of the high dose dams were statistically significantly less compared to the control group during gestation, which was likely secondary to an increased post-implantation loss in these animals.

Estrous cycle data and sexual organ weights and morphology were comparable between the F0 and F1 dams of all test groups and the corresponding controls and ranged within the historical control data of the test facility.
In the high dose F0 and F1 males the test substance administration led to a decrease of absolute and relative organ weights of cauda epididymidis and epididymides. Furthermore, prostate weight and the number of homogenization resistant caudal epididymal sperm was slightly, but significantly decreased in the F0 males. These findings were considered to be treatment-related effects, whereas histomorphological correlates were missing.

A statistically significant increase of absolute and relative kidney weights was noted in male and female F1 animals of the mid and high dose groups (300 and 1000 mg/kg bw/day). Because no histomorphological correlate was detected, the treatment-related weight increase was considered to be of no toxicological concern. As compared to control animals, the kidneys of low, mid and high dose male and female animals revealed a low incidence of basophilic tubules in a slightly higher number of animals. The severity (minimal to slight) was comparable between controls and treated animals and a clear dose-response relationship was missing. Thus, this finding was considered to have no toxicological relevance.

Effect levels

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Based on reduced food consumption and/or body weight gain and slight weight reductions in internal male genital organs without concomitant histopathological changes noted at 1000 mg/kg bw/day.

Target system / organ toxicity

Critical 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 %).

Test item plasma concentrations 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 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.

 

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 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 epididymis	99 %	102 %	88 %**
Epididymidis	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, epididymidis, and prostate in male high dose animals (1000 mg/kg bw/day) was considered as treatment-related effect. The decrease of brain weights in top 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 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 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 in male and female animals of the 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.