Registration Dossier

Administrative data

Description of key information

Based on key study results conducted with relevant source chemicals and following correction of mass proportions (read-across), the NOAEL/NOAEC for repeated oral/inhalation exposure to (2-Hydroxyethyl)ammonium nitrate was set at 382 mg/kg bw/day and 305 mg/m³, respectively. A repeated dose dermal toxicity study is not required for a reliable hazard assessment of systemic effects. No study is available for the reliable assessment of local effects associated with repeated exposure to (2-Hydroxyethyl)ammonium nitrate by the oral, inhalation or dermal route of exposure.
Please refer also to the read-across and weight-of-evidence statement provided in section 13.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Reference:
Composition 0
Qualifier:
according to
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
Qualifier:
according to
Guideline:
other: EU Method B.35 (Two-Generation Reproduction Toxicity Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
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


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
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
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
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.
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.
Critical effects observed:
not specified

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
382 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
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 compliant, guideline study, no restrictions, fully adequate for assessment
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: about 7 weeks old
- Weight at study initiation: ± 228 g (males), ± 165 g (females)
- Housing: in groups of up to 5 animals/cage, in Polysulfon cages (H-Temp [PSU]), floor area about 2065 cm². Type Lignocel fibres dust free bedding served as bedding material. For enrichment wooden gnawing blocks (Typ NGM E-022) were added.
- Diet: mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 15 changes/hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: From: 10-Nov-2009 To: 17-Dec-2009
Route of administration:
other: inhalation exposure to aerosol with vapour fraction
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: The measurements of particle size in test group 3 (150 mg/m³) resulted in MMADs of 1.1 and 1.2 µm with a GSD of 5.3 and 6.4, respectively. The calculated mass fractions of particles below 3 µm aerodynamic size were 70.0 and 70.3 %. Thus the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Generator systems:
- Continuous infusion pumps PERFUSOR (B. Braun)
- Two-component atomizers (stainless steel, Schlick mod. 970)
Generation procedure:
The test substance was used unchanged.
For each concentration the test substance was supplied to a two-component atomizer at a constant rate by means of a metering pump. The aerosol was generated with compressed air mixed with conditioned dilution air into the inhalation system.
The control group was exposed to conditioned air.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography (GC) analysis
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres were determined by a gas chromatography analysis (GC-analysis) in all test groups including control. The vapor and liquid aerosol concentrations were determined separately. Daily means were calculated based on two measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In the treatment groups, the constancy of concentrations in the inhalation chambers were continuously monitored using scattered light photometers. In the control group one sample was analysed over the study period.

The particle size analysis was carried out with a cascade impactor. In test group 3 (150 mg/m³), particle size distribution was determined two times during the exposure period. In this test group a significant amount liquid aerosol was found and the concentration was high enough for this measurement. In test group 2 (50 mg/m³), due to the low aerosol concentration, a long sampling time was necessary. During ongoing sampling, deposited aerosol would get evaporated again and the measured particle size distribution would not reflect the real size distribution. Therefore, no cascade impactor measurement was performed in this test group. In test group 1 (10 mg/m³), no significant aerosol fraction was determined.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
10, 50, 150 mg/m³
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
10.2 ± 2.7, 49.1 ± 8.3, 155.9 ± 23.4 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
5 rats
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
The concentrations to be tested in this study were selected based on the results of a 5-day range finding study (BASF SE, 2011; 30I0924/08074). In this study, groups of 5 male Wistar rats were head-nose exposed to a dynamic inhalation atmosphere of 2-Aminoethanol for 6 hours on 5 consecutive days. The targeted concentrations were 20, 200 and 500 mg/m³. Summarizing the results, the inhalation exposures caused histological changes all over the respiratory tract. Most pronounced effects were observed in the upper respiratory tract.
At 500 mg/m³, minimal to mild inflammatory cell infiltrates were noted in the submucosa of the ventral meatus in level I of the nasal cavity. In addition, 4/5 animals revealed (multi)focal perivascular hemorrhage in this region. One animal showed necrosis of the squamous epithelium. In the area of the transition from squamous to the respiratory epithelium, 4/5 animals revealed minimal to mild squamous metaplasia of the respiratory epithelium. In level II of the nasal cavity 3/5 animals of the same concentration group showed minimal to moderate inflammatory cell infiltrates. In the larynx, minimal to severe epithelial necrosis, mild to severe inflammatory cell infiltrates, and minimal to moderate squamous metaplasia was observed. In level I of the larynx, inflammation was accompanied by necrosis of the submucosal glands. Moreover, cellular atypia within the metaplastic epithelium was observed in level I and II of the larynx. These findings were less severe in level III. Inflammatory cell infiltrates, focal epithelial necrosis and minimal diffuse epithelial hyperplasia were still observed. In the carina (trachea) respiratory epithelium hyperplasia and degeneration intermingled with inflammatory cell infiltrates were observed in almost all animals of the high concentration group. In the lung minimal to mild hyperplasia of the bronchiolar epithelium in the areas of bifurcation of large bronchi was observed.
At 200 mg/m³ similar findings were noted in the above mentioned organs and tissues with lower incidence and severity. At 20 mg/m³, no adverse effects were observed.
The observed effects seemed to be associated with aerosol exposure. Considering the histological findings in the respiratory tract, 150 mg/m³ was selected as the high concentration for the main study to cause toxic effects. For the mid concentration of the main study 50 mg/m³ was selected, because this concentration was around the saturated vapor concentration in the inhalation system. The low concentration was set at 10 mg/m³, as the expected NOAEC.
150 mg/m³ (61 ppm) as high concentration causing toxic effects
50 mg/m³ (20 ppm) as mid concentration
10 mg/m³ (4 ppm) as low concentration and expected NOAEC
Observations and examinations performed and frequency:
MORTALITY, CLINICAL SIGNS
- Time schedule: the animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays. The clinical condition of the test animals was recorded once during the pre-exposure period and on the post-exposure observation day and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

BODY WEIGHT
- Time schedule: body weight was determined at the start of the pre-exposure, at the start of the exposure period and then, as a rule, once a week as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day.
Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived from the individual differences.

FOOD CONSUMPTION
- Time schedule: food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.
The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, food consumption was determined cagewise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the number of animals per cage. As the animals of each test group were housed in only one cage per sex, no statistical evaluation of food consumption was possible.

OPHTHALMOSCOPIC EXAMINATION
- Time schedule and groups examined: before the start of the exposure period (day -3) and at the end of the study (day 26)
- Dose groups that were examined: all dose groups were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG) after administration of a mydriatic (Mydrum, Chauvin Ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY
In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using Isoflurane (Isoba®, Essex GmbH, Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI). The following examinations were carried out in 5 animals per test group and sex.

HEMATOLOGY
Leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), inorganic Phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), total Bilirubin (TBIL), total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)
Sacrifice and pathology:
GROSS PATHOLOGY, ORGAN WEIGHTS
Necropsy
All animals were sacrificed under Narcoren anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
After determination of the terminal body weight (anesthetized animals), the following organ weights were determined in all animals sacrificed on schedule:
1. Adrenal glands
2. Brain
3. Epididymides
4. Heart
5. Kidneys
6. Liver
7. Lungs
8. Spleen
9. Testes
10. Thymus
11. Thyroid glands

HISTOPATHOLOGY
Organ/tissue fixation
The following organs or tissues were fixed in 4 % buffered Formaldehyde:
1. All gross lesions
2. Adrenal glands
3. Brain with olfactory bulb
4. Bone marrow (femur)
5. Eyes with optic nerve
6. Heart
7. Kidneys
8. Larynx/pharynx
9. Liver
10. Lungs
11. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
12. Nose (nasal cavity)
13. Esophagus
14. Ovaries
15. Seminal vesicle
16. Spinal cord (cervical, thoracic and lumbar cords)
17. Stomach (forestomach and glandular stomach)
18. Spleen
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder
24. Uterus
From the liver, each one slices of the lobus dexter medialis and the lobus sinster lateralis were fixed in Carnoy’s solution and embedded in paraplast.

Histotechnical processing, examination by light microscopy and assessment of findings
The following organs and tissues of main group animals were designated for histological processing and light microscopical examination
1. All gross lesions
2. Nasal cavity (4 levels)
3. Larynx (3 levels)
4. Trachea
5. Lungs (5 lobes)
6. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
7. Adrenal glands
8. Bone marrow (femur)
9. Brain
10. Heart
11. Kidneys
12. Liver
13. Esophagus
14. Ovaries
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cords)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Uterus
An assessment of correlations between gross lesions and histopathological findings was performed.
Statistics:
Body weight, body weight change: a comparison of each group with the control group was performed using the Dunnett's test (two-sided) for the hypothesis of equal means.

Clinical pathology parameters, urine volume, urine specific gravity: a non-parametric one-way analysis using the Kruskal Wallis test (two-sided) was conducted .If the resulting p-value was equal to or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the Wilcoxon-test (two-sided) for equal medians.

Weight parameters: a non-parametric one-way analysis using the Kruskal Wallis test (two-sided) was performed. If the resulting p-value was equal to or less than 0.05, a pair wise comparison of each concentration group with the control group was performed using the Wilcoxon test for the hypothesis of equal medians.
Details on results:
MORTALITY, CLINICAL SIGNS
No deaths were recorded throughout the study. During the pre-exposure period and the post-exposure observation day the animals showed no clinical signs and findings different from normal. During the exposure period the animals of the control group showed no clinical signs or any findings different from normal. During the exposure period a few animals crossbench all test groups showed salivation after exposure.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights of the test substance exposed groups were not statistically significantly different from the control group.The mean body weight changes of the test substance exposed groups were not statistically significantly different from the control group.

FOOD CONSUMPTION
No substance-related changes of food consumption were observed during the whole study period.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmoscopic examinations did not show any impairment of the refracting media. Spontaneous findings such as remainders of the pupillary membrane or corneal stippling, striation of lens and opacity were observed in several animals of all test groups and the control group without any concentration-response relationship.

HEMATOLOGY
No treatment-related, adverse changes among hematological parameters were measured. In male rats of concentration groups 2 and 3 (50 and 150 mg/m³) the mean corpuscular hemoglobin concentration (MCHC) was higher compared to controls. The increase of this calculated parameter was not accompanied by an alteration of any other red blood cell parameter value. Therefore, the MCHC increase was regarded as possibly treatment-related, but not adverse.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were measured. At the end of the study, in male rats of all concentration groups the creatinine values were higher compared to controls, whereas in females of concentration group 1 (10 mg/m³) the urea levels were lower compared to controls. The values were not changed dose-dependently. Therefore, they were regarded as incidental and not treatment-related. In male rats of concentration group 3 (150 mg/m³) the triglyceride values were decreased. This was the only altered clinical chemistry parameter and it was especially not accompanied by any change of protein, glucose or cholesterol levels. Therefore, these decreased triglyceride concentrations were regarded as not adverse (ECETOC Technical Report No. 85, 2002).

ORGAN WEIGHTS
All mean absolute weight parameters did not show significant differences when compared with the control group. When compared with the control group, the mean relative weights of liver in male treatment groups were significantly decreased. All other mean relative weight parameters did not show significant differences when compared with the control group. The decrease of mean liver weights noted in treated males was not concentration dependent and there were no histopathological correlates. Therefore, the reduced relative liver weights in males of all treatment groups were regarded to be incidental and not related to treatment.

GROSS PATHOLOGY
There were no gross lesions in treated male and female animals.

HISTOPATHOLOGY
Larynx: at the base of epiglottis (level I), a submucosal inflammation that was characterized by infiltrates of granulocytes and lymphoid cells occurred in all males and females of test groups 2 (50 mg/m³) and 3 (150 mg/m³). In animals of test group 3 (150 mg/m³), the inflammation was accompanied by degeneration of the submucosal glands. In addition, 4 males and 3 females of test group 3 (150 mg/m³) showed a focal epithelial necrosis at the base of epiglottis. In the same region, a focal squamous cell metaplasia was observed in 3 males and 2 females of test group 2 (50 mg/m³) as well as in all males and females of test group 3 (150 mg/m³). All these findings were related to treatment. The occurrence of a minimal inflammation at the base of epiglottis in one female of test group 1 (10 mg/m³) was considered incidental. A minimal or slight epithelial alteration was observed in 2 males and 3 females of the control group, in 4 males and 1 female of test group 1 (10 mg/m³), as well as in 2 males and 3 females of test group 2 (50 mg/m³). The epithelial alteration was located at the base of epiglottis and was characterized by a slight focal flattening of epithelial cells. The epithelial alteration was regarded as a spontaneous lesion. At the entrance to the ventral pouch (larynx, level II), a minimal (grade 1) focal squamous metaplasia was seen in 1 female of test group 2 (50 mg/m³) as well as in 1 male and 2 females of test group 3 (150 mg/m³). A minimal focal epithelial hyperplasia occurred in all males and in 4 females. A mostly minimal inflammation was observed in 2 males and 3 females of test group 2 (50 mg/m³) as well as in all males and 4 females of test group 3 (150 mg/m³). All findings were considered treatment-related.

Trachea: in males, a minimal or slight focal squamous metaplasia that was located in the area of the carina occurred in 3 animals of test group 3 (150 mg/m³). A minimal or slight inflammation was observed in 1 male of test group 1 (10 mg/m³) and in 4 males of test group 3 (150 mg/m³). The occurrence of squamous metaplasia and inflammation in males of test group 3 (150 mg/m³) was considered to be related to treatment. In females, a minimal focal inflammation was only seen in 1 control animal.

Lungs: a minimal or slight focal or multifocal mucous cell hyperplasia was seen in single or few large bronchi in all males and 2 females of test group 3 (150 mg/m³). In affected bronchi, the number of goblet cells was minimally or slightly increased. The occurrence of mucous cell hyperplasia was regarded as treatment-related.

All other histopathological findings occurred either individually or were equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
10 mg/m³ air
Sex:
male/female
Basis for effect level:
other: The NOAEC was based on concentration-related histopathological lesions in larynx, trachea and lung seen at the two higher concentration levels.
Dose descriptor:
NOAEC
Remarks:
systemic effects
Effect level:
150 mg/m³ air
Sex:
male/female
Basis for effect level:
other: There were no adverse systemic effects up to and including the high concentration level of 150 mg/m³.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
305 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

Repeated dose toxicity: via oral route - systemic effects (read-across)

 

In a read-across assessment, 2 -Aminoethanol was identified as the source chemical displaying the highest toxicity hazard when considering repeated exposure. Since no reliable conventional repeated dose oral toxicity study with 2 -Aminoethanol was available, a 2 -generation repeated dose dietary oral toxicity study with the salt 2 -Aminoethanol hydrochloride (BASF SE, 2009; 75RO372/05055) was identified as key study and starting point for the read-across assessment under the assumption that the observed test substance related effects were entirely attributable to 2 -Aminoethanol (and not to Hydrochloride). 2 -Aminoethanol hydrochloride was administered to groups of 25 male and 25 female healthy young Wistar rats (F0 parental generation) as a homogeneous addition to the food in different concentrations, which were adjusted regularly to obtain target dose levels of 0, 100, 300 and 1000 mg/kg bw/day. At least 75 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). The detailed description of the study results regarding reproduction toxicity is provided in the section on reproductive toxicity. Regarding general repeated dose toxicity, the dose level of 1000 mg/kg bw/day caused systemic toxicity in parental females, as was indicated by reduced food consumption and/or body weight gain during gestation and lactation. In the mid and high dose F1 animals (300 and 1000 mg/kg bw/day) the absolute and relative kidney weights were statistical significantly increased without histopathological correlate findings. In the high dose F0 and F1 males (1000 mg/kg bw/day) 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 epididymidal sperm was slightly, but significantly, decreased in the F0 males. These findings were considered to be treatment-related effects, even though histomorphological correlates were missing. Based on this study with 2 -Aminoethanol hydrochloride, the NOAEL for general systemic toxicity was set at 300 mg/kg bw/day, which - under consideration of mass proportions (62.6 % w/w 2 -Aminoethanol in 2 -Aminoethanol hydrochloride; 49.2 % w/w 2 -Aminoethanol in (2 -Hydroxyethyl)ammonium nitrate) - corresponds to a NOAEL of 188 mg/kg bw/day for 2 -Aminoethanol (300 mg/kg bw/day x 0.626) and a NOAEL of 382 mg/kg bw/day (188 mg/kg bw/day / 0.492) for the target chemical (2 -Hydroxyethyl)ammonium nitrate.

 

In a supporting screening study for reproductive hazards (Pereira et al., 1987), 4 pregnant mice/dose level were exposed to the source chemical 2 -Aminoethanol by oral gavage at 500, 579, 671, 777 or 900 mg/kg bw/day for a period of 9 days (second phase 2 according to the study protocol). The NOAEL was established at 671 mg/kg bw/day based on mortality seen at higher doses levels. No histopathological examination was conducted in this study.

In a reliable supporting subacute toxicity study (Product Safety Laboratories, 2002; 11686), the source chemical Potassium nitrate was shown to display a lower hazard as compared with 2 -Aminoethanol when considering repeated exposure by the oral route. In this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, 5 rats/sex received single daily oral gavage treatments with Potassium nitrate at dose levels of 0, 50, 750 or 1500 mg/kg bw/day for at least 28 days. Mortality, clinical signs, body weight and food consumption were assessed periodically during the treatment period. Haematology and clinical chemistry parameters were determined in blood samples collected on day 28 of the treatment period. Neurobehavioural examinations including assessment of sensory activity, grip strength and motor activity were conducted towards the end of the treatment period. At the end of the treatment period, all surviving animals were sacrificed, examined macroscopically and the weights of selected organs were determined. Tissues and organs were examined histopathologically. Treatment with Potassium nitrate resulted in no deaths and no signs of overt clinical toxicity. There were no substance-related effects on body weight, food consumption, hematology and clinical chemistry parameters and neurobehavioural endpoints. Organ weights were not affected by the treatment with the test substance. There were no macroscopical lesions or microscopical findings attributable to the treatment with the test substance. Under the conditions of this study, the NOAEL for systemic effects was established at the high dose level of 1500 mg/kg bw/day.

 

Repeated dose toxcity: inhalation - systemic effects (read-across)

 

A subacute inhalation toxicity study in rats (BASF SE, 2010; 40I0924/08083) with the source chemical 2 -Aminoethanol was identified as key study for the assessment of the repeated dose inhalation toxicity hazard of the target chemical (2 -Hydroxyethyl)ammonium nitrate by means of a read-across assessment. In this study, groups of 5 Wistar rats/sex/concentration were nose-only exposed to a dynamic atmosphere of 2 -Aminoethanol for 6 hours per day on 5 consecutive days per week for 4 weeks. The target concentrations were 10, 50 and 150 mg/m³. A concurrent control group was exposed to conditioned air. Clinical observations, body weight determinations and food consumption determinations were performed for all animals. Ophthalmological examinations were conducted prior to exposure and towards the end of the exposure in all animals. After the last exposure, blood was sampled for the assessment of relevant hematology and clinical chemistry parameters. At the end of the treatment period, all surviving animals were subjected to gross necropsy (including macroscopic examination of the major internal organs and determination of organ weights). Selected tissues were processed histopathologically and were evaluated by light microscopy. Histological examinations were performed according to standardised methods with particular emphasis on the nasal cavity (4 levels) and the larynx (3 levels). The exposure of rats to 2 -Aminoethanol caused concentration-related lesions in larynx, trachea and lung. At the high concentration (150 mg/m³), submucosal inflammation (levels I, II) in males and females, degeneration of submucosal glands (level I) in males and females, focal epithelial necrosis (level I) in males and females, focal squamous metaplasia (level I) in males and females (level II) in one male and 2 females and focal epithelial hyperplasia (level II) in males and females were observed in the larynx. In the trachea, focal squamous metaplasia (carina) accompanied by inflammation was observed in males. At the mid concentration (50 mg/m³), submucosal inflammation (level I and II) in males and females and squamous metaplasia (level I and II) in few males and females in the larynx was reported. No treatment-related weight changes, gross lesions or microscopic findings were noted at the low concentration (10 mg/m³). No histopathological effects were seen in any other organ outside the respiratory tract. The NOAEC for systemic toxicity was established at the high concentration of 150 mg/m³. The NOAEC for local effects was set at the low concentration of 10 mg/m³ under the conditions of this study. Under consideration of stoichiometry and of the molecular masses of source and target chemicals, the identified NOAEC for systemic effects resulting from repeated inhalation exposure at 150 mg/m³ is extrapolated to a systemic NOAEC for the target chemical (2 -Hydroxyethyl)ammonium nitrate of 305 mg/m³ (150 mg/m³ / 0.492).

 

Repeated dose toxicity: all routes of exposure - local effects

 

No study is available for the reliable assessment of local effects associated with repeated exposure to (2 -Hydroxyethyl)ammonium nitrate by the oral, inhalation or dermal route. A read-across assessment is not considered appropriate for this endpoint as effects are to a large extent triggered by acid/alkaline properties of the source chemicals. However, the source chemical 2 -Aminoethanol is known to display irritant/corrosive effects and is currently classified with C; R34 (Directive 67/548/EEC) and Skin Corr. 1B; H314 (Regulation (EC) No 1272/2008). Upon repeated inhalation exposure, 2 -Aminoethanol induced concentration-related lesions in larynx, trachea and lungs of rats (BASF SE, 2010; 40I0924/08083). Furthermore, irritant/corrosive effects were reported for 2 -Aminoethanol in acute dermal toxicity (Union Carbide Corporation, 1988; 51 -86), skin irritation and eye irritation studies (BASF AG, 1966; XV/305), and eye irritating properties were shown for the source chemical Sodium nitrate (NOTOX B.V., 2010; 494137). On this basis the induction of local effects via repeated exposure to (2 -Hydroxyethyl)ammonium nitrate cannot be excluded.



Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The key study was selected based on the source ion[aq] displaying the highest toxicity hazard for repeated oral exposure among the selected source chemicals (read-across approach), i.e 2-Aminoethanol. Since no reliable conventional repeated dose oral toxicity study with 2-Aminoethanol was available, a 2-generation repeated dose dietary oral toxicity study with the salt 2-Aminoethanol hydrochloride (BASF SE, 2009; 75RO372/05055) was identified as key study and starting point for a read-across assessment. In the context of dietary food admixture, the hazard identified for the test item 2-Aminoethanol hydrochloride in this key study was considered solely attributable to 2-Aminoethanol, which was in turn used for a read-across assessment to the target chemical (read-across from 2-Aminoethanol hydrochloride via 2-Aminoethanol to (2-Hydroxyethyl)ammonium nitrate) under consideration of mass proportions.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The key study was selected based on the source ion[aq] considered to display the highest toxicity hazard for repeated inhalation exposure among the source chemicals (read-across approach), i.e 2-Aminoethanol.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No study is available for the reliable assessment of local effects associated with repeated inhalation exposure to (2-Hydroxyethyl)ammonium nitrate. A read-across assessment is not considered appropriate for this endpoint. However, the key study for repeated dose toxicity by the inhalation route indicated that upon repeated/continuous inhalation exposure 2-Aminoethanol [Cation(aq)] induced concentration-related lesions in larynx, trachea and lungs of rats.

Justification for classification or non-classification

Considering repeated dose toxicity and the source chemicals idenitifed for read-across assessment (section 13), 2 -Aminoethanol is currently classified with STOT SE 3; H335 according to Regulation (EC) No 1272/2008 (CLP/GHS), while the salt used in the key repeated dose oral toxcity study (BASF SE, 2009; 75RO372/05055), 2 -Aminoethanol hydrochloride, is currently not classified in the EU. Sodium nitrate, Potassium nitrate and Ammonium nitrate are not classified for hazards related to repeated exposure.

Based on the NOAEL/NOAEC estimated for (2 -Hydroxyethyl)ammonium nitrate from key studies conducted with the relevant source chemical 2 -Aminoethanol or 2 -Aminoethanol hydrochloride, (2 -Hydroxyethyl)ammonium nitrate is not considered to be subject to classification for repeated dose toxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (GHS/CLP).