Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-12-05 to 2012-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
5-day inhalation study according to GLP and OECD/EU guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
7 September 2009
Deviations:
yes
Remarks:
only 5 day exposure
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
only 5 day exposure
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-propylheptyl) phthalate
EC Number:
258-469-4
EC Name:
Bis(2-propylheptyl) phthalate
Cas Number:
53306-54-0
Molecular formula:
C28H46O4
IUPAC Name:
1,2-bis(2-propylheptyl) benzene-1,2-dicarboxylate
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material: Palatinol 10-P
- Analytical purity: 99.4 corr. area-%
- Batch No.: B4504 v. 2.8.2012
- Expiry date: Not reported
- Appearance: Colourless, liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; SandhoferWeg 7, 97633 Sulzfeld
- Age at study initiation: about 8 weeks
- Fasting period before study: no data
- Housing: Polysulfon cages (H-Temp [PSU]), floor area about 2065 cm2 (610x435x215 mm); supplied by TECNIPLAST, Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- No. of animals per cage: up to 5 animals
- Diet: ad libitum, not during exposure
- Water: ad libitum, not during exposure
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30-70 %
- Air changes: 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12/12 from 06.00 a.m. - 06.00 p.m.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.4 - <= 2.2 µm
Remarks on MMAD:
MMAD / GSD:
50 mg/m3: 1.4 / 2.8 µm and 1.4 / 2.6 µm
250 mg/m3: 1.4 / 2.8 µm and 1.4 / 2.9 µm
1000 mg/m3: 2.2 / 2.1 µm and 1.6 / 2.6 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps KP 2000 (DESAGA; SARSTED AG & Co, Nümbrecht, Germany), Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)
- Exposure chamber volume: 90 L
- Method of holding animals in test chamber: The rats were restrained in exposure tubes, their snouts projecting into the inhalation chamberto inhale the atmosphere.
- System of generating aerosols: For each concentration, a respective constant amount of the test substance was supplied to a two-component atomizer by means of a metering pump and sprayed with compressed air into a glass mixing stage. The so generated aerosol was mixed with conditioned air and passed into the inhalation system.
- Method of particle size determination: Gravimetrical Marple 298 cascade impactor measurements
- Treatment of exhaust air: The exhaust air system connected to the exposure systems was adjusted in such a way that the amount of exhaust air is lower than the supply air (positive pressure). Thus the test atmosphere was not diluted with laboratory air in the breathing zones ofthe animals.
- Temperature, humidity, pressure in air chamber:
The air flow rates of supply and exhaust air, relative humidities and temperatures in the inhalation systems were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated.

TEST ATMOSPHERE
- Brief description of analytical method used: The aerosol concentration was determined by gas chromatography of absorption samples.
- Samples taken from breathing zone: yes
- Sampling frequency: three samples per concentration and exposure

TEST ATMOSPHERE
- Particle size distribution:
Particle Size determinations of the test atmospheres was made with the TSI APS 3321. Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) was obtained directly by the piece of equipment used (TSI APS 3321).
- Frequency: two times during the exposure period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The aerosol concentration was determined by gas chromatography of absorption samples.
Duration of treatment / exposure:
5 consecutive days
Frequency of treatment:
Once a day for 6 hours
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/m³ air
Dose / conc.:
250 mg/m³ air
Dose / conc.:
1 000 mg/m³ air
No. of animals per sex per dose:
10 male animals per concentration
Control animals:
yes, concurrent vehicle
Details on study design:
- Duration of observation period following administration: one day
- Fasting period: animals were fasted over night before blood sampling
- Necropsy of survivors performed: yes
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until the day prior to gross necropsy

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.1 were examined.

ACUTE PHASE PROTEINS
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.3 were examined.

PALMITOYL CoA OXIDATION
- Tissue: liver
- How many animals: all animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see table No. 4
HISTOPATHOLOGY: Yes, see table No. 5
Other examinations:
not applicable
Statistics:
Body weights, body weight change: Dunnett's test
Clinical pathology examinations: Kruskal-Wallis test and Wilcoxon test
Weight of anesthetized animals and absolute and relative organ weight: Kruskal-Wallis test and Wilcoxon test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
During the whole study period, the animals showed no clinical signs and findings different from normal.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of all test groups were not statistically significant different from the control groups. However, the mean body weight change of test group 3 (1000 mg/m3) was slightly, though significantly lower than in the control.

The following significant body weight changes were observed:
- Test group 1 (50 mg/m³), from study day 0 to 4 (-1.9 g, p< 0.05)
- Test group 3 (1000 mg/m³) from study day 0 to 4 (-5.1 g, p< 0.01)
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:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In rat of test group 3 (1000 mg/m³), relative eosinophil counts were higher compared to controls, but the values were within historical control ranges (relative eosinophil counts 1.12.8 %). Therefore, this alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of test group 3 (1000 mg/m3), globulin and total protein levels were decreased, whereas cholesterol levels were increased. These effects were considered test-substance related and adverse.

In the mentioned test group, total bilirubin levels were decreased. In the absence of an anemia this decrease was most probably due to an increased conjugation rate of bilirubin coupled with a greater excretion of bilirubin via the bile. This effect was regarded as treatment-related, but not adverse.

In the same test group calcium levels were lower compared to controls, but the mean was within the historical control range. In rats of test group 2 (300 mg/m3) potassium levels were low, but this decrease was not dose-dependent. Therefore, the lower calcium levels in rats of test group 3 as well as the lower potassium levels in test group 2 were regarded as incidental and not treatment-related.

In males of test groups 2 and 3 (250 and 1000 mg/m3), cyanide sensitive Palmitoyl CoA oxidation (Pal CoA) in liver tissue was higher compared to controls. However the mean in test group 2 was within the historical control range (4.30 – 7.69 mU/mg protein) and it was not accompanied with an alteration of any other liver parameter in serum. Therefore, the PalCoA in test group 2 (250 mg/m3) was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
A minimal to moderate granulomatous inflammation was observed in the lungs of two males in test group 2 (250 mg/m3) and in nine males of test group 3 (1000 mg/m3). showed very few to multiple granulomatous foci, often nearby the bronchi, bronchioli and terminal bronchioli. The granulomatous foci were characterized by a large number of intraalveolar macrophages, granulocytes, lymphocytes, desquamated epithelial cells and single multinucleated giant cells.

A minimal to moderate lympho-reticulocellular hyperplasia of the tracheobronchial and mediastinal lymph nodes was noted in six males of test group 3 (1000 mg/m³), that was regarded to be treatment-related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/m³:
- Increased absolute and relative weight of the lungs (+37%).
- Increased absolute and relative weight of the liver (+30%).

250 mg/m³:
- Increased absolute weight of the lungs (+9%).
- Increased relative weight of the lungs (+7%).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross lesions reported in the following organs (control, group 1, group 2, group 3):
- Epididymides (3, 1, 1, 3)
- Liver (0, 0, 0, 1)
- Mediastinal lymph node (0, 0, 0, 1)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nasal cavity:
Single or few mucous cells occurred in the maxillary sinus (nasal cavity, level III) in all treated males. In the septal window (nasal cavity, level III), a minimal increased number of mucous cells was observed in four (out of 10) males in test group 3 (1000 mg/m³). Seven males in test group 3 (1000 mg/m³) showed a minimal increased number or hypertrophy of mucous cells in the nasopharyngeal duct (nasal cavity, level IV). The epithelium in the respective areas was regular and signs of inflammation or degenerative changes were completely missing in the whole nasal cavity. The occurrence, increase in number, and hypertrophy of mucous cells were regarded to be treatment-related.

Trachea:
A minimal to moderate multifocal hypertrophy or hyperplasia of the respiratory epithelium including mucous cells was seen in the trachea of eight males in test group 3 (1000 mg/m3). The occurrence of hypertrophy/ hyperplasia of the respiratory epithelium including mucous cells was considered to be treatment-related.

Lungs:
A minimal to moderate granulomatous inflammation was observed in the lungs of two males in test group 2 (250 mg/m3) and in nine males of test group 3 (1000 mg/m3). These males showed very few to multiple granulomatous foci, often nearby the bronchi, bronchioli and terminal bronchioli. The granulomatous foci were characterized by a large number of intraalveolar macrophages, granulocytes, lymphocytes, desquamated epithelial cells and single multinucleated giant cells. Partly, the alveolar walls were lined by large cuboidal cells with vacuolated cytoplasm and prominent nuclei (type II pneumocyte hyperplasia). Some of these males showed in addition areas with muco-purulent features.

Alveolar histiocytosis, characterized by focal accumulation of some histiocytes (alveolar macrophages) in few alveoli, frequently occurs spontaneously. This type of alveolar histiocytosis was observed in four control males as well as in three males of test group 1 (50 mg/m³). In these males, one or few foci of alveolar histiocytosis occurred in one or two lobes of the lungs (minimal, grade 1) and is therefore interpreted as spontaneous lesion. However, in test groups 2 (250 mg/m³) and 3 (1000 mg/m³) all males were affected and the alveolar histiocytosis was often seen in the same areas as the inflammation, distributed over the whole lung. The occurrence of granulomatous inflammation and the increased incidence and severity of alveolar histiocytosis in males of test groups 2 (250 mg/m³) and 3 (1000 mg/m³) correlated very well with the increased lung weights in these test groups and was related to treatment.

In the epithelium of bronchi, bronchioles, and/ or terminal bronchioles, a concentration-related minimal to moderate hypertrophy/ hyperplasia, partly accompanied by an increase of mucous cells, was observed in all males of test groups 2 (250 mg/m³) and 3 (1000 mg/m3). The occurrence of the epithelial hypertrophy/ hyperplasia in bronchi, bronchioles, and/ or terminal bronchioles was regarded to be treatment-related.

Liver:
A slight diffuse hepatocellular hypertrophy was observed in all males of test group 3 (1000 mg/m³). The diffuse hepatocellular hypertrophy correlated with the increased absolute and relative liver weights in this test group and was regarded to be treatment-related.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Remarks:
(systemic toxicity)
Effect level:
250 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
NOAEC
Remarks:
(local effects)
Effect level:
50 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Respiratory irritation

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion