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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Sep 2005 - 17 Feb 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-phenoxyethanol
- Physical state: liquid/ colourless, clear
- Analytical purity: > 99.9 core peak-area% (GC)
- Lot/batch No.: 41183068E0
- Expiration date of the lot/batch:
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor and the sponsor holds the responsibility
- Storage condition of test material: room temperature, under N2, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: about 7 weeks
- Weight at study initiation: males: ca. 227 g, females: ca. 163 g
- Housing: animals were housed singly in makrolon-wire cages (type MD III, Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm²)
- Diet (e.g. ad libitum): milled mouse/rat laboratory diet “GLP”, (Provimi Kliba SA, Kaiseraugst, Basel Switzerland)
- Water (e.g. ad libitum): tap water
- Acclimation period: 8 days

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

IN-LIFE DATES: From: 06 Sep 2005 To: 28 Sep 2005

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: see "Details on inhalation exposure"
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Generator systems:
- Continuous infusion pumps PERFUSOR (B. Braun) for test group 1
- Piston metering pumps (Sarstedt DESAGA) for test group 2 and 3
- Two-component atomizers (stainless steel, Schlick mod. 970)
- Glass mixing stages (BASF)
- Glass cyclonic separators (BASF)

Generation procedure:
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 in a mixing stage with conditioned dilution air and passed via the cyclonic separator into the inhalation system. Due to the vapor pressure of the test substance, in all test groups mixtures of vapor and liquid aerosol are tested. The theoretical vapor concentration is up to 40 mg/m3.

TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.)
The target concentration of 40 mg/m3 in test group 1 was the saturation vapor concentration. At this concentration, part of the test substance might condensate in the atmosphere, thus liquid aerosols might be formed. To demonstrate the aerosol formation, two cascade impactor measurements were carried out. These resulted in MMADs between 2.9 and 3.7 μm with GSDs of 4.3 and 4.5. The amount of test substance that was trapped by the cascade impactor was approximately 20 % of that measured in the atmosphere, which was in the expected range for condensation effect. Due to the long sampling time (150 min), the condensation on the cascade stages during the sampling period might be much more pronounced in this group than in the other groups. Therefore, the particle size measurements in test group 1 were considered not to reflect the real particle size distribution, but to describe the liquid aerosol formation in the test atmosphere.
Cascade impactor measurements in the test groups 2 and 3 resulted in MMADs between 0.5 and 1.3 μm that were well within the respirable range. The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 72.0 and 85.2%. The EACD (effective aerodynamic cut-off diameter 50%) of the last impactor stage is 1.2 μm. MMAD values below 1.2 μm are gained by extrapolation outside the effective measuring range of the impactor. Therefore the real value may lie between the calculated one and 1.2 μm.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days per week for 14 days (10 exposures)
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/m³ air (nominal)
Dose / conc.:
200 mg/m³ air (nominal)
Dose / conc.:
1 000 mg/m³ air (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice each working day and once on weekends and holidays
- Cage side observations checked: evident signs of toxicity or mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On exposure days clinical examination was performed before, during and after exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the animals was determined at the start of the preflow (day-2), at the start of the exposure period (day 0) and then on day 7 as well as one day prior to gross necropsy (day 13).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the study (with EDTA-K3 as anticoagulant)
- Anaesthetic used for blood collection: Yes (Isoflurane (Essex GmbH Munich, Germany))
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes, prothrombin time (Hepato Quick's test)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the study
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-γ-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, at termination of the study.
Organ weights: liver, kidneys, adrenals, testes, thymus, spleen and lung

HISTOPATHOLOGY: Yes, at termination of the study.

Gross pathological examination was accompanied by the histopathological examination of following selected organs: nasal cavities, larynx, trachea, lungs, mediastinal lymph nodes, thymus, liver, kidneys, spleen, adrenal glands, heart, stomach (fore- and glandular stomach), and esophagus.

Results and discussion

Results of examinations

Description (incidence and severity):
In males of the mid and top concentration the absolute lung weight (up to 20.4%) and in males of the top concentration the relative lung weight (up to 19.3%) were statistically significantly increased. This is regarded as a substance-related finding although there was no histopathologic correlate that could explain this increased weight. In the lungs there was an increase in thickness of small and terminal bronchi and increased numbers of mucous cells in larger bronchi of mid and top concentration males and females. This was also regarded to be a substance-related finding and is judged to be most likely adaptive. In conclusion, the upper respiratory tract showed mild signs of reaction (degeneration, metaplasia, inflammatory cell infiltration) to the test substance; most sensitive seemed to be the respiratory epithelium of the nose, especially in the anterior septum area. The epithelial lining other sites, e.g. respiratory epithelium of lungs, transitional epithelium larynx was also affected but appeared to be less sensitive.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical pathological examinations revealed no treatment-related changes in either males or females. Morphological changes indicating irritation potential of the test compound were found in nasal cavity, larynx and lung of male and female mid- and high concentration animals. In forestomach no morphological changes were noted.
Details on results:
In none of the test groups clinical signs of toxicity were observed. Treatment related influence on body weight development or food consumption was found in the high concentration group on study day 7. Clinical pathology examinations revealed no treatment-related changes in either males or females. 14 days of aerosol inhalation of the test substance led to histopathologic findings in the respiratory tract. The respiratory epithelium in the nasal cavity was the target tissue in high and mid concentration males and females. In the very anterior part signs of degeneration and metaplasia were noted, whereas in the more posterior parts hyperplasia was observed. Inflammatory cell infiltrates were seen in the mid and top concentration groups as a reaction upon treatment. These findings are thought to be substance-related and the top concentration groups were slightly more affected. In the larynx (level I) in the area of the very sensitive epithelium at the base of the epiglottis, three male animals of the high concentration group showed a minimal to slight hyperplasia of the respiratory epithelium. This finding is also considered to be substance-related. According o the 1st International ESTP expert workshop --“Larynx squamous metaplasia”. A re-consideration of morphology and meaning in rodent studies and its relevance for humans - held on November 13-14, 2006, at Fraunhofer Institute ITEM Hannover, Germany, minimal, focal epithelial changes of the larynx epithelium predominantly occurring at the base of the epiglottis should be given the descriptive term of an “epithelial alteration” as the morphological criteria of a “laryngeal squamous metaplasia” are not completely met. Those lesions are regarded as “adaptive” and non-adverse in character. Further, inhalative exposure to compounds that do not exert systemic or local genotoxic effects may cause “laryngeal squamous metaplasia”, but no tumor formation is reported in long-term studies known to the workshop experts. For non-genotoxic-compounds, the workshop experts do not regard “laryngeal squamous metaplasia” as a precancerous lesion. The workshop outcome from the ESTP meeting has meanwhile be published in a peer-reviewed journal (Kaufmann W. et al. (2009). Experimental and Toxicological Pathology 61, 591-603).
In males of the mid and top concentration the absolute lung weight (up to 20.4%) and in males of the top concentration the relative lung weight (up to 19.3%) were statistically significantly increased. This is regarded as a substance-related finding although there was no histopathologic correlate that could explain this increased weight. In the lungs there was an increase in thickness of small and terminal bronchi and increased numbers of mucous cells in larger bronchi of mid and top concentration males and females. This was also regarded to be a substance-related finding and is judged to be most likely adaptive. In conclusion, the upper respiratory tract showed mild signs of reaction (degeneration, metaplasia, inflammatory cell infiltration) to the test substance; most sensitive seemed to be the respiratory epithelium of the nose, especially in the anterior septum area. The epithelial lining other sites, e.g. respiratory epithelium of lungs, transitional epithelium larynx was also affected but appeared to be less sensitive.
Clinical pathological examinations revealed no treatment-related changes in either males or females. Morphological changes indicating irritation potential of the test compound were found in nasal cavity, larynx and lung of male and female mid- and high concentration animals. In forestomach no morphological changes were noted.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
48.2 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
LOAEC
Effect level:
246 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion