Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test substance: Monoethanol amine (2-Aminoethanol)
Test substance No.: 08/0924-1
Batch-Identification: MEOA K 540
Purity: 99.93 area % (assessed by the manufacturer)
Homogeneity: the test substance was homogenous on account of the high purity.
Storage stability: the stability under storage conditions over the exposure period was guaranteed by the manufacturer
Physical state/ appearance: liquid, colorless, clear
Storage conditions: ambient

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 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion