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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well reported guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diethylene glykol mono-n-butyl ether
- Physical state: clear liquid
- Analytical purity: 99.6%
- Lot/batch No.: ex tank 63, test substance no 87/30
- Stability under test conditions: confirmed stable
- Storage condition of test material: room temperature, dark, oxygen excluded
- Other: date of manufacture 20/1/87

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr K Thomae GmbH, Biberach
- Weight at study initiation: Male: approx 236g. female approx 171g
- Housing: in same sex pairs, wire cages. Cages also used for exposure period (2 per chamber)
- Diet : KLIBA rat/mouse laboratory diet 24-343-4 10 mm pellets, Klingentalmuehle AG, CH-4303 Kaiseraugst, Switzerland; ad libitum (during exposure food was withdrawn). Checked for contaminants
- Water: ad libitum except during exposure. Checked for contaminants.
- Acclimation period: Animals sham exposed for 5 days before exposure period

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


IN-LIFE DATES: From: 25/7/89 To: 6/12/89

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION:
- Exposure apparatus: Glass/steel exposure chamber volume ~1.1m3
- Method of conditioning air: Glass heat exchanger. Heated glass evaporator for solvent vapour plus two component atomiser with compressed air and counterstreamwise to supply air. The exhaust air system was set lower than the supply air system (positive pressure). This ensured that no laboratory air reached the control animals. The exhaust air system was set higher than the supply air system (negative pressure). This ensured that the laboratory was not contaminated as the result of any leakages from the inhalation chambers.
- Air flow rate: 21-23 m3/h
- Temperature, humidity, pressure in air chamber: 24-26C, 35-45% RH, -10Pa differential pressure to atmosphere (+10Pa for control)
- Method of particle size determination: HIgh dose group checked on days 10 and 66 for evidence of aerosols in test atmosphere using Polytec analyser. - Method of particle size determination: The following equipment was used: particle size analyzer HC 15 (Polytec), particle counter unit (BASF), millivolt writer (Kipp & Zonen), vacuum pump (Millipore), sampling probe (glass) internal diameter 4 mm.

TEST ATMOSPHERE
- Brief description of analytical method used: Daily by total hydrocarbon analysers and gas chromatography.
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations in the inhalation chambers of test groups 2 (6 ppm) and 3 (14 ppm) and partially of test group 1 (2 ppm) were monitored by means of total hydrocarbon analyzers. The total hydrocarbon analyzers were calibrated with mixtures of the test substance in air that were generated in the inhalation chambers not containing animals and analyzed by gas chromatography. The concentration of test group 1 (2 ppm) was analyzed partially by gas chromatography after absorption of DGBE in propanol-2. Sampling period was 20 minutes. For measured concentrations, see remarks below.
Duration of treatment / exposure:
90 days plus satellite recovery group (4 weeks)
Frequency of treatment:
6hrs per day.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2, 6 and 14 ppm
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
0.013, 0.04, 0.094mg/l
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: 4 weeks
- Dose selection rationale: 2 week range finder (reported in separate record. Top dose is saturated vapour pressure at max temperature recommended in guideline for exposure conditions, mid and low dose as previously used in Gushow study (included as separate record) where liver effects reported at mid dose.
- Post-exposure recovery period in satellite groups: 4 weeks, all dose groups
Positive control:
no

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during exposure (3x time) and recovery period (1x day). A groupwise observation of clinical signs was performed during exposure; clinical signs and findings were also recorded after exposure, before exposure and during the preflow and post-exposure observation period.

BODY WEIGHT: Yes
- Time schedule for examinations: Before acclimation, before exposure then weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before acclimation and at end of study
- Dose groups that were examined: Top dose and control using handslit lamp
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At end of study, from retro-orbital venous plexus
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- Blood was taken from all animals (main groups) at the end of the exposure period.
- The following parameters were determined using a particle counter (S Plus model, by Coulter, Krefeld, FRG):
- leukocytes
- erythrocytes
- hemoglobin
- hematocrit
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelets
- differential cell count
- thromboplastin time
- reticulocytes
The data obtained were transferred to a computer (VAX 11/780; supplied by DEC, Munich, FRG).
The differential blood count was evaluated visually. The reticulocytes were counted using an automatic differential system (HEMATRAK 480 model by Geometric Data, Munich, FRG). The data were transferred to the computer.
CLINICAL CHEMISTRY: Yes
- Numerous clinicochemical parameters and various enzyme activities were measured and a clotting time analysis was performed.
- Time schedule for collection of blood: At end of study, from retro-orbital venous plexus
- Animals fasted: Yes
- The following parameters were determined:
1. Enzymes
- alanine aminotransferase
- aspartate aminotransferase
- alkaline phosphatase
2. Blood chemistry
- sodium
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: At end of study, overnight collection
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
The following examinations were carried out:
- nitrite
- pH
- protein
- glucose
- ketones
- urobilinogen
- bilirubin
- blood
- sediment
The sediment was evaluated microscopically.
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Sacrifice by exsanguination from the abdominal aorta/vena cava under anaesthesia. Moribund animals sacrificed and necropsied as soon as possible. A complete necropsy of all animals including weighing of certain organs and a gross-pathologic evaluation was performed. Selected organs/tissues were examined histopathologically.

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Organs examined for control and top dose group: Heart, liver, kidneys, testes, ovaries, skin, jejenum, rectum, mediastinal & mandibular lymph node, musculature, sternum, extraorbital lachrimal glands, nasal cavity & paranasal sinus, brain, thymus, aorta, spleen, adrenals, prostate, seminal vesicle, esophagus, ileum, urinary bladder, nervus ischiadicus, femur/knee joint/bone marrow, pituitary gland, trachea, salivary glands, pancreas, stomach, cecum, eyes, mammary gland, cervical/thoracic/lumbar cord, thyroid, lungs, uterus, duodenum, colon plus all gross lesions
Organs examined for mid and low dose group: lungs, nasal cavity, salivary (mandibular/sublingual) glands, liver, mandibular lymph nodes plus all gross lesions.
Organs examined for recovery group animals: salivary (mandibular/sublingual) glands, liver, mandibular lymph nodes plus all gross lesions.
Other examinations:
none
Statistics:
The statistical evaluation of the data was carried out on the computer systems of the Department of Toxicology.

Clinical examinations:
Means and standard deviation were calculated for the variables (body weight and body weight change) of the animals of each test group for the statistical evaluation of the study and printed in tables together with the individual values (body weight). Statistical relevance was established using methods of ANOVA (2), DUNNETT (3, 4).
Significances were shown in the tables (* for p <= 0.05 and ** for p <= 0.01) .

Clinical chemistry, hematology and urinalysis:
- Clinical chemistry and hematology:
Means and standard deviations have been calculated for each test group and tabulated together with the individual values. In order to test if the results of the individual dose groups differed statistically significantly from the results of the control group, the means for the dose groups, excepting the differential blood count, were compared with those for the control group using the analysis of variance (ANOVA and DUNNETT's test 2, 3, 4).
Significances resulting from the statistical comparison have been indicated in the tables on means.
- Urinalyses
The assessment to whether certain characteristics differed in degree in the control and test groups was carried out using the chi2 test in appropriate two by two contingency tables. Significances which resulted from this chi2 test have been indicated in the tables (* for S >= 95%, ** for S >= 99%).

Results and discussion

Results of examinations

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: Isolated spontaneous findings unrelated to exposure. No mortality. During the preflow period and the post-exposure observation period the animals showed no clinical signs and findings different from normal. Animal No. 4 (male) from the control group showed on day 84 swellings in the region of the neck, and the anogenital region was smeared with urine, and the animal showed intermittent breathing. Animal No. 97 (male) from test group (2 ppm) showed on day 84 swellings in the region of the neck. Animal No. 39 (male; 14 ppm) showed on day 3 and on day 7 reddish discharge from the right eye and both eyes, respectively, after exposure (blood test positive). Animal No. 116 (male, 14 ppm) showed on days 0 - 3 and on day 6 after exposure reddish discharge from the left eye (blood test positive) as well as on days 1 - 3 before exposure reddish crusts on the eye margins (blood test positive) and on days 1 - 3 crusts on the eye margins during exposure. Animal No. 153 (female; 14 ppm) showed on day 8 after exposure reddish discharge from the left eye (blood test positive) and on day 9 before, during, and after exposure reddish crusts on the margin of the left eye (blood test positive). No deaths were recorded during the post-exposure observation period.The animals No. 4 (male) from control group and animal No. 97 (male) from satellite group (2 ppm) were sacrificed in a moribund state on day 84. Due to the results of histopathologic examinations of these animals the moribund state of animals No. 97 and No. 4 was not caused by the treatment with the substance.
BODY WEIGHT: There were occasionally reported significant differences between dose groups and controls but these appeared to be random occasional occurences with no consistent pattern and therefore deemed not biologically significant. The body weight of the males of satellite group (6 ppm) during the exposure period had arithmetically significantly increased on day 14 and that of the males of satellite group (14 ppm) on day 49 when compared to the control group (p
BODY WEIGHT CHANGE: During exposure period: In the males of test group 3 (14 ppm) there was on day -7 an arithmetically significant influence (p HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS: Some random effects across all treatment groups observed but not attributed to treatment.

ORGAN WEIGHTS: No changes in main dose groups: In recovery group: High dose group absolute and relative liver weight increase (15 and 8% respectively, males only) and relative testes and female lung weight increase (both ~10%). These changes were not thought to be treatment related.

Effect levels

Dose descriptor:
NOAEL
Effect level:
94 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOAEL was 14ppm (94mg/m3) This is the maximum concentration that can be tested (effective saturated vapour pressure for a dynamic atmosphere)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Measured concentrations in atmosphere.

 Test group

 Measured concentration (ppm)

 Target concentration (ppm)

 Low

2.0 +/- 0.11 

 2

Mid

 6.0 +/- 0.92

 6

 High

 14.0 +/- 0.53

 14

Aerosol particles were not detected in the inhalation atmosphere.

Applicant's summary and conclusion

Conclusions:
The organ weight changes in the high dose animals were not deemed biologically significant as they were sex specific and not seen in the main study animals (ie only seen in the recovery group animals.)
Executive summary:

In a 90 day guideline and GLP inhalation study rats were exposed in 3 dose groups up to the maximum saturated vapour pressure of 2 -(2 -butoxyethoxyethanol). Satellite recovery groups were also included for all 3 dose groups and the controls. No adverse effects were seen in any dose group. The NOAEL was therefore 14ppm (94mg/m3), the maximum concentration tested.