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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16.08.1993-15.12.1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996
Reference Type:
publication
Title:
The Inhalation toxicity of di- and triethanolamine upon repeated exposure
Author:
Gamer AO, et al
Year:
2008
Bibliographic source:
Food and Chemical Toxicology 46, 2173–2183

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Principles of method if other than guideline:
The study was carried out according to the OECD guidelines method 413 [OECD, 1981], the EU guidelines 87/302/EEC [EEC 1988] and the EPA guidelines [EPA 1984] . Neurotoxicity was tested following the EPA test guideline [EPA 1985/87] .
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Details on test material:
- Name of test material (as cited in study report): Diethanolamine; 2,2'-Iminobisethanol; Substance No. 93/75
- Physical state: clear liquid
- Analytical purity: 99.4%
- Lot/batch No.: storage tank B 810
- Stability under storage conditions: ensured
- Storage condition of test material: Room temperature excluded from humidity, air and covered with nitrogen
- Homogeneity: homogeneous

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr . K . Thomae GmbH, Biberach, Germany
- Age at study initiation: about 7 weeks
- Weight at study initiation: males: about 218 g; females: about 166 g
- Housing: in wire cages type MD III
- Diet: KLIBA rat/mouse/hamster laboratory diet 24-343-4, 10 mm pellets, Klingentalmuehle AG, Kaiseraugst, Switzerland ad libitum
- Water: tap water ad libitum


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

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
ADMINISTRATION / EXPOSURE
Male and female Wistar rats were exposed to an aerosol of Diethanolamine (DEA) in head/nose exposure systems for 6 hours each working day for a total of 90 days (65 exposures). A control group was exposed under similar conditions to clean air only. Concentrations: Target: 0; 15; 150; 400 mg/m³ (analytical determined: 0; 15; 152; 410 mg/m³). The test substance was supplied to a atomizer at a constant rate by means of a metric pump. An aerosol was generated by means of compressed air and passed into the inhalation system.

Generator system
The aerosol was generated using a diaphragm metering pump Prominent (CfG), a two-component atomizer mod. 970 (Schlick) and a glass cyclone separator (BASF AG). The head-nose exposure technique was preferably selected for this inhalation study to minimize fur contamination of the animals with the test substance, which cannot be avoided during whole-body exposure. The aerosol was passed into an aerodynamic exposure apparatus (INA 60, volume V = 90 1, BASF AG). The apparatus consists of a cylindrical inhalation chamber of stainless steel sheeting and  cone-shaped outlets and inlets. The rats were restrained in exposure tubes (glass tubes). Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The apparatus was also connected to an exhaust air system. A slight positive pressure was maintained by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air. In order to accustom the animals to the exposure conditions they were exposed to supply air in headnose only exposure systems under comparable flow conditions on 5 days before the exposure period (preflow period). Then all test groups were exposed for 6 hours on each workday over a time period of 15 days (14-day test). The number of exposure days was 10. The animals did not have access to water or feed during the exposure.

Measurement of the operation conditions (air flows, relative humidity, temperature and pressure conditions) of the exposure systems 
The amounts  of test substance set at the metering pumps were recorded once daily.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days (total of 65 exposures)
Frequency of treatment:
6 h/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
15; 150; 400 mg/m³
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
15.2; 153.4; 409.7 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
13 males, 13 females
Control animals:
yes, sham-exposed
Details on study design:
Post-exposure period: none
Positive control:
not done

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily on working days

BODY WEIGHT: Yes
- Time schedule for examinations: once per week, starting on day of the preflow period (day -5)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the beginning of the preflow period and at the end of the study
- Dose groups that were examined: in control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: daily, 10 animals (randomized sequence used)
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- Parameter investigated: volume, color, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, billirubin, blood, specific gravity, sediment, leukocyte count, erythrocyte count, hemoglobin,  hematocrit, MCV, MCH, MCHC, platelet count, differential cell count, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: daily 10 animals (randomized sequence used)
- Animals fasted: Yes

CLINICAL PATHOLOGY: YES
- Time schedule for collection of blood:
- Parameter investigated: ALT, AST, ALP, GGT, Na, K, Cl, INP, Ca, UREA, CREA, GLUC, TBIL, TPROT, ALB, GLOB, TRIG, CHOL, Mg, thromboplastin time, Quick test for coagulation time

URINALYSIS: Yes
- Time schedule for collection of urine: daily, 10 animals (randomized sequence used)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- parameter investigated: volume, color, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, billirubin, blood, specific gravity, sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: daily on working days, at least 3 times on exosure days
- Dose groups that were examined: 20 animals in total consisting of animals all dosing groups
- Functional observation battery (FOB) included: home cage observation, open field observation and motor activity (MA) measurement in 10 animals per group and sex before start of exposure and 4 times during exposure period (about every 3 week): general appearance, tremors, convulsion, piloerection, lacrimation/secretion of pigmented tears, salivation, pupil size, diarrhea, vocalization, paresis, paralysis, ataxia, body tone, posture, animal body (appearance), locomotor activity, respiration, urination, skin color, righting reflex, behavior, grip strength, papillary reflex, winking reflex, vision,  audition, olfaction, sensitivity of body surface, pain perception, tail  pinch, toe pinch, visual placing response, all other clinical signs.
Sacrifice and pathology:
The animals were sacrificed at the end of the study. A complete necropsy  including weight of selected organs and gross pathology evaluation was  
performed in 10 animals per group and sex. 
Histopathology was performed  in several organs and tissues in these animals. Additionally, 3 animals  per group and sex were sacrificed by perfusion fixation and underwent  neuropathological examinations. -Organ weights: adrenals, testes, heart, kidneys, brain, liver, ovaries -Histopathology: Samples of the following tissues: All gross lesions and tumors, skin, brain (cerebral cortex, medulla/pons, cerebellum), pituitary, thyroid and parathyroid, thymus, lung and trachea, heart, femur with bone marrow, salivary glands, liver, spleen, kidney, adrenal, pancreas, testes, accessory genital organs and uterus, female mammary gland, muscle, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, intestinal lymph nodes, sciatic nerve, eyes and aorta
Statistics:
- ANOVA and Dunnett test (two sided): clinical data, hematology (except differential blood count), clinical pathology parameter
- Fisher's exact test: urinalysis
- Kruskal Wallis test (two-sided) and MANN-WHITNEY U Test: neurofunctional tests

Results and discussion

Results of examinations

Details on results:
- Mortalities: No deaths
- Clinical findings: No treatment related effects
- Body weight: Slightly decreased body weight in males at 400 mg/m³
- Functional observation battery: Home cage observation: No treatment related effects Open filed observation: No treatment related effects Sensorimotor tests/reflexes: No treatment related effects Quantitative observations: No treatment related effects Home cage observation: No treatment related effects Open filed observation: No treatment related effects Sensorimotor tests/reflexes: No treatment related effects Quantitative observations: No treatment related effects
- Motor activity: No treatment related effects
- Ophthalmoscopy: No treatment related effects
- Hematology: at 400 mg/m³ significant decrease in red blood cells, hemoglobin, hematocrit and mean corpuscular volume in male and female rats; morphological examination revealed only a marginal increase in anisocytosis in the respective males; no treatment related effect in the white or differential blood count was found.
- Enzymes: increased ALP activities at 0.15 and 0.4 mg/l in males and  females; reduced ALT 150 and 400 mg/m³ in males.
- Blood chemistry: increased Ca, total protein (TPROT), ALB, GLOB at 0.15 and 0.4 mg/l in  females; increased TPROT and ALB in males as a trend at 150 and 400 mg/m³.
- Urinalysis: increase in blood at 0.4 mg/l in males and females; increase  excretion in renal tubular epithelium cells including casts at 150 and 400 mg/m³ in males
- Organ weights: increased relative and absolute liver and kidney weights in both sexes at 150 and 400 mg/m³.
- Gross and histopathology: Histopathology revealed diffuse testicular atrophy accompanied by oligozoospermia in the epididymides and slight atrophy of prostate in some males of the high concentration group only. Occurrence of minimal or slight tubular hyperplasia in the kidneys of some female rats as well as of intratubular lithiasis in increased numbers (male rats at 400 mg/m³). Local irritant effects to the upper respiratory system were observed. The most sensitive location for irritation was the larynx, where some epithelial damage was observed in all concentrations. In the mid and high concentration focal inflammation at the tracheal bifurcation occurred additionally.
- Neuropathology: no treatment related effects.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
15 mg/m³ air
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEC
Remarks:
local toxicity
Effect level:
15 mg/m³ air
Sex:
male/female
Basis for effect level:
other: No NOAEC for sequelae of local irritation in the upper respiratory tract could be established, since in the larynx in the low concentration squamous metaplasia of parts of the epithelial surface was seen regularly .

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Detailed results:

- Concentration measurements in the exposure system:

The following mean (± SD) concentrations were obtained for the entire study:

Target concentration

Measured concentration

MMAD

% aerosol

15 mg/m³

15.2 ± 2.3 mg/m³

1.1-1.9 µm

95%

150 mg/m³

153.4 ± 20 mg/m³

1.0 µm

94%

400 mg/m³

409.7 ± 41.3 mg/m³

0.6-0.9 µm

92%

MMAD = Mass Median Aerodynamic Diameter

- Mortalities: No deaths

- Clinical findings: No treatment related effects

- Body weight: Slightly decreased body weight in males at 400 mg/m³

dosing group (mg/m³)             

0

15

150

400

Body weights (g)

males day 0

280

271

271

269

males terminal

433

407

396

376**

weight gain

153

136

125

107

females day 0

200

202

201

206

females terminal

266

264

253

269

weight gain

66

62

52

63

** p< 0.01

- Functional observation battery:
Home cage observation: No treatment related effects
Open filed observation: No treatment related effects
Sensorimotor tests/reflexes: No treatment related effects
Quantitative observations: No treatment related effects
Home cage observation: No treatment related effects
Open filed observation: No treatment related effects
Sensorimotor tests/reflexes: No treatment related effects
Quantitative observations: No treatment related effects

- Motor activity: No treatment related effects

- Ophthalmoscopy: No treatment related effects

- Hematology:
 at 400 mg/m³ significant decrease in red blood cells, hemoglobin, hematocrit and mean corpuscular volume in male and female rats; morphological examination revealed only a marginal increase in anisocytosis in the respective males; no treatment related effect in the white or differential blood count was found.

dose (mg/m³)

0

15

150

400

RBC

male

8.6

8.69

8.56        

8.11**

female

7.72        

8.0        

7.62        

7.1**

HGB

male

9.7        

9.6        

9.5        

8.8**

female

9.0        

9.3        

8.7        

7.9**

HCT

male

0.457

0.452

0.450

0.414**

female

0.409

0.424

0.396

0.367**

MCV

male

53.1        

52

52.6        

51.0*

female

53  

53  

52.1

51.7*

data given as means of 10 animals; * p< 0.05; ** p< 0.01
RBC: red blood cell count

HGB: haemoglobin

HCT: haematocrit

MCV: mean corpuscular volume

- Clinical pathology:
- Enzymes: increased ALP activities at 0.15 and 0.4 mg/l in males and
  females; reduced ALT 150 and 400 mg/m³ in males 

- Blood chemistry: increased Ca, total protein (TPROT), ALB, GLOB at 0.15 and 0.4 mg/l in
  females; increased TPROT and ALB in males as a trend at 150 and 400 mg/m³

- Urinalysis: increase in blood at 0.4 mg/l in males and females; increase
  excretion in renal tubular epithelium cells including casts at 150 and 400 mg/m³ in males

- Organ weights: increased relative and absolute liver and kidney weights in both sexes at
 150 and 400 mg/m³.

Selected relative organ weights (mg/100 g) in male and female rats

dose (mg/m³)                         

0

15

150

400

Liver

male

2834

2793

2814

3077*

female

3171

3191

3488**

3771**

Kidneys

male

652

674

716*

734**

female

795

811

891**

922**

Lungs

male

319

323

321

332

female

451

410

419

418

*p<0.05

** p <0.01

-Gross and histopathology:
Histopathology revealed diffuse testicular atrophy accompanied by
 oligozoospermia in the epididymides and slight atrophy of prostate in some males of the high concentration group only. 

Occurrence of minimal or slight tubular hyperplasia in the kidneys of
 some female rats as well as of intratubular lithiasis in increased numbers (male rats at 400 mg/m³).

Local irritant effects to the upper respiratory system were observed. The
 most sensitive location for irritation was the larynx, where some epithelial damage was observed in all concentrations. In the mid and high concentration focal inflammation at the tracheal bifurcation occurred additionally. 

Incidence and severity of changes of larynx following DEA exposure (males/females):
                    

dose (mg/m³)

15

150

400

Metaplasia, squamous                          

Level#1

10/10

10/10

10/10

Level#2

-

-

-

Hyperplasia, squamous

Grade 1

-

3/1

0/1

Grade 2

-

3/6

2/7

Grade 3

-

-

4/1

Grade 4

-

-

2/0

Inflammatory cells

Grade 1

-

-

-

Grade 2

1/3

-

-

Grade 3

-

-

-

Chronic inflammation                         

Grade 2

-

1/4

2/1

Grade 3

-

9/6

5/8

Grade 4

-

-

3/1

n = 10 per sex (level = anatomical localisation, grade = severity description)

- Neuropathology: no treatment related effects

Applicant's summary and conclusion