Carbonic acid, compound with 2-aminoethanol (1:2)

Endpoint summary

Description of key information

Repeat dose toxicity of the registered substance is read across from repeat dose (oral and inhalation) studies on the analogue substance, monoethanolamine, which establish NOAELs and NOECs as detailed above which are directly applicable to the registered substance due to the expectation that the substance will decompose to monoethanolamine in aqueous media, thus exhibiting the same toxicity profile systemically. 

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, other

Remarks:

other: two-generation reproductive toxicity study

Type of 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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

The target substance carbonic acid, compound with 2-aminoethanol (1:2) is formed from the reaction of 2-aminoethanol and carbon dioxide, at a 2:1 molar ratio. The hypothesis of the analogue approach is that the systemic toxicity of the target substance will be determined by the fact that in aqueous conditions of a mammalian system, it rapidly degrades to 2-aminoethanol, and therefore reading across to 2-aminoethanol provides a reasonable worst-case scenario.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source substance: 2-aminoethanol
Target substance: carbonic acid, compound with 2-aminoethanol (1:2)

For further information on purity and impurities please see the full read across justification attached in Section 13 of IUCLID.

3. ANALOGUE APPROACH JUSTIFICATION

The target substance is expected to break down to form the source substance in mammalian systems; both target and source substances would be expected to have the same mechanism of action and therefore similar systemic toxicity. The target substance is essentially a neutralised form of the source substance. Therefore, the target substance would not be expected to display the topical irritancy and/or corrosive properties of the source substance. However, for systemic effects read across is appropriate as the target substance will degrade rapidly to produce the source substance under physiological conditions.

For a full justification of the read across approach see the read across justification attached in Section 13 IUCLID.

Reason / purpose for cross-reference:

read-across source

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han)

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) Males: 162.1 (142.5 – 186.5) g ; Females: 126.2 (110.6 – 145.1) g;
- Fasting period before study: none
- Housing: housed individually in type DK III stainless steel wire mesh cages
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland ad libitum
- Water: ad libitum
- Acclimation period: 16 days


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

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test substance (ethanolamine hydrochloride, EAH) was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.

Duration of treatment / exposure:

semichronic duration (> 75 days)

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

For parental animals:

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

DETAILED CLINICAL OBSERVATIONS: Yes

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

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

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

Serum concentrations of the test substance:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of Ethanolamine hydrochloride

Estrous 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, epididmides. Cauda epididymis, prostate, seminal vesicles, ovaries, uterus, spleen, brain, pituitary gland and thyroid glands (with parathyroids) were weighed and the vagina, cervix uterie, uterus, ovaries, oviducts, left testis, left epididymis, seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroids (with parathyroids)and all gross lesions were fixed in an appropriate fixative, histologically processed and examined by light microscopy. From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated for numbers of primordial and growing follicles.
As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration.

Statistics:

See below

Details on results:

Clinical examinations revealed no test substance-related adverse effects for F0 and F1 parental animals of low (100 mg/kg bw/dayand mid (300 mg/kg bw/daylevels. The test compound did adversely affect food consumption of the high dose F0 females (1000 mg/kg bw/day) during lactation. Also, the body weight gain and, for F0 generation, body weights of the high-dose dams were statistically significantly less compared to the control group during gestation, which was likely be 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 top-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 (300 mg/kg bw/day) and top-dose (1000 mg/kg bw/day) groups. 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 top-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: reduced food consumption and/or body weight gain, as well as organ weight changes unaccompanied by histopathological findings

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%).

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

Toxicokinetic dataof 2 -aminoethanol (calculated as 2 -aminoethanol hydrochloride)fromthis two-generation reproduction toxicity studyshow a dose dependency of the plasma levels of 2 -aminoethanol in the experimental animals and there with prove the bioavailability of 2 -aminoethanol hydrochloride in principle.

 

Tables

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

	Test group 01 (100 mg/kg bw/d)	Test group 02 (300 mg/kg bw/d)	Test group 03 (1000 mg/kg bw/d)
F0 males	94.3 (72.4 / 102.5)	283.2 (218.4 / 309.4)	943.3 (716.7 / 1032.6)
F0 females (premating)	96.7 (80.5 / 100.7)	289.6 (241.2 / 304.9)	964.4 (792.4 / 1017.8)
F0 females (F1 litter) - gestation period - lactation period*	103.5 (92.6 / 111.6) 99.2 (81.6 / 120.2)	315.2 (284.8 / 337.9) 306.7 (249.7 / 370.3)	1043.2 (989.4 / 1084.7) 866.0 (668.6 / 1053.9)

* = Days 1–14 p.p. only

Absolute organ weights (P-generation)

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

	Male animals			Female animals
Group	01 100 mg/kg bw/d	02 300 mg/kg bw/d	03 1000 mg/kg bw/d	01 100 mg/kg bw/d	02 300 mg/kg bw/d	03 1000 mg/kg bw/d
Brain	99%	100%	97%*
Cauda epididymis	99%	102%	88%**
Epididymides	100%	101%	92%**
Prostate	92%	99%	86%**
Spleen				105%*	107%	97%

 

*: p≤0.05; **: p≤0.01

 

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

 

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

 

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

Absolute organ weights (F1 generation)

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

 

	Male animals			Female animals
Group	11 100 mg/kg bw/d	12 300 mg/kg bw/d	13 1000 mg/kg bw day	11 100 mg/kg bw/d	12 300 mg/kg bw/d	13 1000 mg/kg bw/d
Cauda epididymis	96%	99%	88%**
Epididymides	100%	101%	91%**
Kidneys	99%	106%*	111%**	103%	106%**	115%**
Spleen	99%	103%	92%*
Thyroid glands	106%	99%	109%*	110%	118%**	111%*

 

*: p≤0.05; **: p≤0.01

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

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

 

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

 

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

Conclusions:

As indicated in the read across justification document, if ingested, the registered substance is expected to decompose to form monoethanolamine and therefore, it is reasonable to assume that the effects reported in this study are a worst-case for the registered substance. As such, exposure of rats over 32 d in the diet to monoethanolamine established a NOAEL of 300 mg/kg bw/day based on reduced food consumption, and/or body weight gain, as well as organ weight changes unaccompanied by histological findings. This NOAEL can be directly applied to the registered substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of 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.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

The target substance carbonic acid, compound with 2-aminoethanol (1:2) is formed from the reaction of 2-aminoethanol and carbon dioxide, at a 2:1 molar ratio. The hypothesis of the analogue approach is that the systemic toxicity of the target substance will be determined by the fact that in aqueous conditions of a mammalian system, it rapidly degrades to 2-aminoethanol, and therefore reading across to 2-aminoethanol provides a reasonable worst-case scenario.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source substance: 2-aminoethanol
Target substance: carbonic acid, compound with 2-aminoethanol (1:2)

For further information on purity and impurities please see the full read across justification attached in Section 13 of IUCLID.

3. ANALOGUE APPROACH JUSTIFICATION

The target substance is expected to break down to form the source substance in mammalian systems; both target and source substances would be expected to have the same mechanism of action and therefore similar systemic toxicity. The target substance is essentially a neutralised form of the source substance. Therefore, the target substance would not be expected to display the topical irritancy and/or corrosive properties of the source substance. However, for systemic effects read across is appropriate as the target substance will degrade rapidly to produce the source substance under physiological conditions.

For a full justification of the read across approach see the read across justification attached in Section 13 IUCLID.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar rats, Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation: male ± 228 g; female ± 165 g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust free bedding. For enrichment wooden gnawing blocks (Typ NGM E-022), were added.
- Diet (e.g. ad libitum): mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

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

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 resulted in MMADs of 1.1 and 1.2 µm with a GSD of 5.3and 6.4.
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 the inhalation atmospheres
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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres were analysed by a gas chromatography analysis (GC) in all test groups including control. The vapor and liquid aerosol concentration 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 these groups, the constancy of concentrations in the inhalation systems in the chambers were continuously monitored using scattered light photometers.
In the control group (test group 0) one sample was analysed over the study period.

The particle size analysis was carried out with a cascade impactor. In test group 3, particle size distribution was determined two times during the exposure period. In this test group significant amount liquid aerosol was found and the concentration was high enough for this measurement. In test group 2, due to the low aerosol concentration, 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, 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 and 150 mg/m3
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
10.2±2.7, 49.1±8.3 and 155.9±23.4 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

The concentrations to be tested in this study were selected based on the results of the 5-day range finding study.
Summarizing the results, inhalation exposure to MEA for 6 hours per day on 5 consecutive days 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 in the submucosa of the ventral meatus in level I of the nasal cavity. In addition, four of the five 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, four of the five animals revealed minimal to mild squamous metaplasia of the respiratory epithelium. In level II of the nasal cavity three of the five 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 could still be observed.
In the carina (trachea) respiratory epithelium hyperplasia and degeneration intermingled with inflammatory cell infiltrates 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 less incidence and severity. At 20 mg/m3, no adverse effects were observed.
The observed effects seem 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 effect. The mid concentration for the main study should be 50 mg/m3 because this concentration was around the saturated vapor concentration in the inhalation system. The low concentration should be 10 mg/m³, as the expected No Observed Adverse Effect Concentration (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: 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.

Clinical observations: 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 data: The body weight of the animals 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: 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, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only one cage per sex, no statistical evaluation of food consumption is possible.

Ophthalmology: Before the start of the exposure period (day -3) the eyes of all animals, and at the end of the study (day 26) the eyes of all animals 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:

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: The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
3.10.3. 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: Fixation was followed by histotechnical processing and examination by light microscopy and assessment of findings according to the list below: Organs and tissues of main group animals designated for histological processing and light microscopic 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
A correlation 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 DUNNETT's test (twosided) for the hypothesis of equal means.

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

Weight parameters: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal 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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality: No deaths were recorded throughout the study.

Clinical observations: 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 and findings different from normal. During the exposure period a few animals crossbench all test groups showed salivation after exposure.

Body weight: The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.

Body weight change: The mean body weight changes of the test substance exposed groups were not statistically significantly different from the control group 0.

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

Ophthalmology: The ophthalmologic 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 dose group 2 and 3 (50 mg/m3 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 is 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 dose groups the creatinine values were higher compared to controls, whereas in females of dose group 1 (10 mg/m3) the urea levels were lower compared to controls. The values were not changed dose-dependently. Therefore, they are regarded as incidental and not treatment-related. In male rats of dose 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, this decreased triglyceride concentrations were regarded as not adverse (ECETOC Technical Report No. 85, 2002).

Absolute weights: All mean absolute weight parameters did not show significant differences when compared with the control group 0.

Relative organ weights: When compared with control group 0 (=100%), 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 0. The decrease of mean liver weights in treated males was not concentration dependent and there were no histopathological correlates. Therefore, the reduced liver weights in males of all treatment groups were regarded to be incidental and not related to treatment.

Gross lesions:
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 one 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 one female of test group 2 (50 mg/m3) as well as in one male and two 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.

Histopathology 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 one 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 of inflammation in males of test group 3 (150 mg/m3) was related to treatment. In females, a minimal focal inflammation was only seen in one control animal.

Histopathology 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.

Histopathology rest: All other findings occurred either individually or were biologically 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: concentration-related lesions in larynx, trachea and lung

Dose descriptor:

NOEC

Remarks:

systemic effects

Effect level:

150 mg/m³ air

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects were reported

Critical effects observed:

not specified

Conclusions:

Exposure over 28d to up to 150 mg/m3 of monoethanolamine via inhalation exposure to aerosol with vapour fraction was not shown to cause statistically significant systemic adverse effects. Local effects such as on mucous cell are considered to be treatment related at 150 mg/m3.
These data are considered directly applicable to the registered substance, as a very worst-case estimate of the effects of the registered substance, given the low vapour pressure of the registered substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

150 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of 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.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

The target substance carbonic acid, compound with 2-aminoethanol (1:2) is formed from the reaction of 2-aminoethanol and carbon dioxide, at a 2:1 molar ratio. The hypothesis of the analogue approach is that the systemic toxicity of the target substance will be determined by the fact that in aqueous conditions of a mammalian system, it rapidly degrades to 2-aminoethanol, and therefore reading across to 2-aminoethanol provides a reasonable worst-case scenario.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source substance: 2-aminoethanol
Target substance: carbonic acid, compound with 2-aminoethanol (1:2)

For further information on purity and impurities please see the full read across justification attached in Section 13 of IUCLID.

3. ANALOGUE APPROACH JUSTIFICATION

The target substance is expected to break down to form the source substance in mammalian systems; both target and source substances would be expected to have the same mechanism of action and therefore similar systemic toxicity. The target substance is essentially a neutralised form of the source substance. Therefore, the target substance would not be expected to display the topical irritancy and/or corrosive properties of the source substance. However, for systemic effects read across is appropriate as the target substance will degrade rapidly to produce the source substance under physiological conditions.

For a full justification of the read across approach see the read across justification attached in Section 13 IUCLID.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar rats, Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation: male ± 228 g; female ± 165 g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust free bedding. For enrichment wooden gnawing blocks (Typ NGM E-022), were added.
- Diet (e.g. ad libitum): mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

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

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 resulted in MMADs of 1.1 and 1.2 µm with a GSD of 5.3and 6.4.
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 the inhalation atmospheres
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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres were analysed by a gas chromatography analysis (GC) in all test groups including control. The vapor and liquid aerosol concentration 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 these groups, the constancy of concentrations in the inhalation systems in the chambers were continuously monitored using scattered light photometers.
In the control group (test group 0) one sample was analysed over the study period.

The particle size analysis was carried out with a cascade impactor. In test group 3, particle size distribution was determined two times during the exposure period. In this test group significant amount liquid aerosol was found and the concentration was high enough for this measurement. In test group 2, due to the low aerosol concentration, 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, 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 and 150 mg/m3
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
10.2±2.7, 49.1±8.3 and 155.9±23.4 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

The concentrations to be tested in this study were selected based on the results of the 5-day range finding study.
Summarizing the results, inhalation exposure to MEA for 6 hours per day on 5 consecutive days 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 in the submucosa of the ventral meatus in level I of the nasal cavity. In addition, four of the five 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, four of the five animals revealed minimal to mild squamous metaplasia of the respiratory epithelium. In level II of the nasal cavity three of the five 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 could still be observed.
In the carina (trachea) respiratory epithelium hyperplasia and degeneration intermingled with inflammatory cell infiltrates 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 less incidence and severity. At 20 mg/m3, no adverse effects were observed.
The observed effects seem 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 effect. The mid concentration for the main study should be 50 mg/m3 because this concentration was around the saturated vapor concentration in the inhalation system. The low concentration should be 10 mg/m³, as the expected No Observed Adverse Effect Concentration (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: 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.

Clinical observations: 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 data: The body weight of the animals 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: 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, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only one cage per sex, no statistical evaluation of food consumption is possible.

Ophthalmology: Before the start of the exposure period (day -3) the eyes of all animals, and at the end of the study (day 26) the eyes of all animals 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:

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: The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
3.10.3. 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: Fixation was followed by histotechnical processing and examination by light microscopy and assessment of findings according to the list below: Organs and tissues of main group animals designated for histological processing and light microscopic 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
A correlation 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 DUNNETT's test (twosided) for the hypothesis of equal means.

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

Weight parameters: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal 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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality: No deaths were recorded throughout the study.

Clinical observations: 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 and findings different from normal. During the exposure period a few animals crossbench all test groups showed salivation after exposure.

Body weight: The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.

Body weight change: The mean body weight changes of the test substance exposed groups were not statistically significantly different from the control group 0.

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

Ophthalmology: The ophthalmologic 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 dose group 2 and 3 (50 mg/m3 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 is 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 dose groups the creatinine values were higher compared to controls, whereas in females of dose group 1 (10 mg/m3) the urea levels were lower compared to controls. The values were not changed dose-dependently. Therefore, they are regarded as incidental and not treatment-related. In male rats of dose 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, this decreased triglyceride concentrations were regarded as not adverse (ECETOC Technical Report No. 85, 2002).

Absolute weights: All mean absolute weight parameters did not show significant differences when compared with the control group 0.

Relative organ weights: When compared with control group 0 (=100%), 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 0. The decrease of mean liver weights in treated males was not concentration dependent and there were no histopathological correlates. Therefore, the reduced liver weights in males of all treatment groups were regarded to be incidental and not related to treatment.

Gross lesions:
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 one 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 one female of test group 2 (50 mg/m3) as well as in one male and two 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.

Histopathology 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 one 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 of inflammation in males of test group 3 (150 mg/m3) was related to treatment. In females, a minimal focal inflammation was only seen in one control animal.

Histopathology 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.

Histopathology rest: All other findings occurred either individually or were biologically 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: concentration-related lesions in larynx, trachea and lung

Dose descriptor:

NOEC

Remarks:

systemic effects

Effect level:

150 mg/m³ air

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects were reported

Critical effects observed:

not specified

Conclusions:

Exposure over 28d to up to 150 mg/m3 of monoethanolamine via inhalation exposure to aerosol with vapour fraction was not shown to cause statistically significant systemic adverse effects. Local effects such as on mucous cell are considered to be treatment related at 150 mg/m3.
These data are considered directly applicable to the registered substance, as a very worst-case estimate of the effects of the registered substance, given the low vapour pressure of the registered substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10 mg/m³

Study duration:

subacute

Species:

rat

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

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Adequate study on the analogue substance which is expected to represent a reasonable worst case for the repeat dose oral toxicity of the registered substance. Findings at the limit dose (1000 mg/kg bw/day) were: yellow discoloured urine, decreased weight of male accessory sex organs, but with no histopathological change.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Reliable study on the analogue substance which is expected to represent a reasonable worst case for the repeat dose inhalation toxicity of the registered substance. No systemic effects at the highest tested dose.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Reliable study on the analogue substance which is expected to represent a reasonable worst case for the repeat dose inhalation toxicity of the registered substance. Local effects reported were concentration-related lesions in larynx, trachea and lung.