Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: European Joint Research Centre. (1999) Sub-chronic inhalation toxicity of synthetic mineral fibres in rats (ECB/TM/16 rev. 1). In Bernstein DM, Sintes JMR, editors. EUR 18748 EN. Ispra: European Commission Joint Research Centre.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:
- Physical state:
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.:
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:
- Storage condition of test material:
- Other:

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
strain: Crl:WI(WU)BR

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: See above
Details on inhalation exposure:
6 h/day, 5 days/wk for 3 mo to a fiber aerosol concentration of approximately 150 fibers/ml (fiber length >20 μm). The gravimetric
fiber concentrations for the high dose level was 37 mg/m3 for MMVF21.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
scanning electron microscope (SEM, Leo-430). SEM analysis one filter was sampled per exposure day and group. In wk 1, all 5 filters per exposure group were analyzed, and in the following weeks only 2 filters were analyzed. For 1 of the 2 filter samples in wk 2 to 13, only the concentration of long fibers (L > 20 μm) was analyzed by counting at least 100 long fibers. This procedure is a modification of the one described in protocol ECB/TM/16(97) rev. 1 (1999).
Duration of treatment / exposure:
3 months
Frequency of treatment:
6 h/day, 5 days/wk
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Low dose 76.6 WHO fibers/ml, 17.4 fibers L>20um/ml
Basis:
other: SEM bivariate fiber measurement
Remarks:
Doses / Concentrations:
Medium dose 232.2 WHO fibers/ml, 57.8 fibers L>20um/ml
Basis:
other: SEM bivariate fiber measurement
Remarks:
Doses / Concentrations:
High dose 705.2 WHO fibers/ml, 173.6 fibers L>20um/ml
Basis:
other: SEM bivariate fiber measurement
No. of animals per sex per dose:
45 rats per dose
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
Lung retention of test material 3; 4.5; 6 months after the start of exposure
Bronchoalveolar lavage 3; 4.5; 6 months after the start of exposure
Histology/proliferation test (BrdU) 3; 4.5; 6 months after the start of exposure
Sacrifice and pathology:
All animals were subjected to a full gross necropsy, which include careful examination of the external surface of the body and all orifices, as well as the cranial, thoracic, and abdominal cavities and their contents.
Formalin-fixed lungs were examined for cell proliferation using the sensitive S-phase response method. Proliferating cells were labeled with 5-bromo-2’-deoxyuridine (BrdU; 20 mg/ml in phosphate-buffered saline), which was administered to the animals by a minipump 7 days prior to sacrifice. After 7 days of incubation the rats were sacrificed and lung section slides were prepared according to histological routine procedures. These were stained immunohistochemically following denaturation of the DNA (antibody technique).
The evaluation of the slides was done by analyzing an appropriate number of the airway cells and the cells from the proximal regions of the pulmonary parenchyma for each rat. In addition, pleural cells were analyzed and the unit length proliferation index (1 cm in length of pleura per lung section) was estimated.
The following measurement strategy was used.
Parenchyma Four fields of parenchyma (top, below, right, and left of the bronchiolus; distance of 1 alveolus between the counting frame and the
bronchiolar wall) per evaluated bronchiolus were analyzed. At a minimum, 1000 cells per animals were counted (40× objective lens).
Terminal bronchioles The unit length labeling index (ULLI) method was used. The ULLI of at least 4 terminal bronchioli (longitudinally sectioned)
was determined using light microscopy (40× objective lens) and an image analysis system. The number of BrdU-positive cells per terminal bronchiolus was recorded on a length of approximately 2 × 500 μm of bronchiolar epithelium, counting from the bronchiolo-alveolar transition toward the proximal terminal bronchiole.
Other examinations:
Bronchoalveolar Lavage
The method of Henderson et al. (1987) was used with minor modifications. After having been prepared, the lungs were lavaged twice with 5 ml saline without massage. As further lavage would dilute the noncellular constituents like lactate dehydrogenase (LDH) or cytokines, two lavages were considered optimal. The cell concentration was determined using a counting chamber. The lavagate was centrifuged at 160 × g and the supernatant used for determining the biochemical parameters (LDH, β-glucuronidase, total protein). Cytoslides were prepared for differential cell count.
The lavage fluid was collected in calibrated tubes and, after recording the harvested volume, kept on ice until processing. Using a 20-μl aliquot, the cell concentration was determined with a Fuchs–Rosenthal counting chamber un- der a light microscope at 125× magnification. From each lavage sample two cytoslides were prepared. An optimum coating of cytoslides was achieved using approximately 80,000 cells per slide. Cells were centrifuged onto the slides in a cytocentrifuge running at 1500 rpm (= 250 × g) for 10 min.
Cytoslides were stained with May–Grünwald and Giemsa solution. Under light microscope at 625× magnification, 2 × 100 cells were differentiated per cytoslide. Differentiation included macrophages, neutrophils, eosinophils, epithelial cells, and other cells.
The lavagate was centrifuged at 5°C for 10 min at 1000 rpm (= 209 × g). One milliliter of the cell-free supernatant was taken for the determination of the biochemical parameters LDH, _-glucuronidase, and total protein). These parameters were analyzed according to routine clinical chemistry protocols.

Cytokine Analysis in Lavage Supernatant
Tumor necrosis factor-α (TNF- α ) TNF- α is produced by activated macrophages. It activates epithelial cells, fibroblasts, and granulocytes, which are involved in the development of fibrotic lesions.
The TNF- α concentration in the BALF was determined in a commercially available enzyme-linked immunosorbent assay (ELISA) (R&D Systems [www.rndsystems.com], Quantikine Eliza Rat TNF-alpha) according to the ELISA protocol. Since TNF- α is sensitive to proteases, proteolytic degradation was prevented by adding the protease inhibitor aprotinin.
Interleukin-6 (IL-6) IL-6 is produced by macrophages and other cell types. It is important for the activation and differentiation of B cells (antibody production) and the induction of acute phase proteins in the liver. The IL-6 content of the lavage supernatant was determined using IL-6-dependent 7TD-1 cells and MTT as an indicator system (Mosmann et al., 1986).
Statistics:
Differences between groups were considered to be statistically significant at p < .05. Data were analyzed using analysis of variance. If the group means differed significantly by the analysis of variance, the means of the treated groups were compared with the means of the control group based on Dunnett’s test.

Results and discussion

Results of examinations

Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not examined
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
The following clear dose-dependent effects on MMVF21 exposure were observed as main findings of the study: increase in lung weight, in measured biochemical parameters and polymorphonuclear leukocytes (PMN) in the bronchoalveolar lavage fluid (BALF), in cell proliferation (BrdU-response) of terminal bronchiolar epithelium, and in interstitial fibrosis. The values observed in the proliferation assay on the carcinogenic. In summary, this study demonstrates the importance of fiber dust contamination by granular components.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 

Summary of histopathological findings as number of animals with given score at each postexposure interval

 

Number of animals (out of 5) with indicated lesion for postexposure interval

Group

Groupnumber

 

1 wk

 

 

7 wk

 

 

14 wk

 

 

Veryslight

Slight      Moderate

Total

Veryslight

Slight

Total

Veryslight

Slight

Total

 

Accumulation of fiber/particle-laden macrophages

 

 

 

 

 

 

 

 

Control

1

0

0

0

0

0

0

0

0

0

 

E-glass low

2

5b

0

5b

5b

0

5b

3

2

5b

 

E-glass medium

3

1

4a

5b

3

2

5b

0

5b

5b

 

E-glass high

4

0

5b

5b

1

4a

5b

0

5b

5b

 

MMVF21 low

5

5b

0

5b

4a

0

4a

5b

0

5b

 

MMVF21 medium

6

3

2

5b

3

2

5b

3

2

5b

 

MMVF21 high

7

0

5b

5b

2

3

5b

1

4a

5b

 

CMS

8

2

3

5b

1

4a

5b

0

5b

5b

 

Bronchioloalveolar hyperplasia

 

 

 

 

 

 

 

 

 

 

Control

1

0

0

0

0

0

0

0

0

0

 

E-glass low

2

5b

0

5b

4a

0

4a

2

3

5b

 

E-glass medium

3

4a

2

5b

1

4a

5b

1

4a

5b

 

E-glass high

4

2

3

5b

0

5b

5b

1

4a

5b

 

MMVF21 low

5

4a

1

5b

2

0

2

5b

0

5b

 

MMVF21 medium

6

4a

1

5b

4a

1

5b

2

3

5b

 

MMVF21 high

7

0

5b

5b

0

5b

5b

4a

1

5b

 

CMS

8

3

2

5b

1

4a

5b

1

4a

5b

 

Microgranulomas

 

 

 

 

 

 

 

 

 

 

 

Control

1

0

0         0

0

0

0

0

0

0

0

 

E-glass low

2

5b

0         0

5b

4a

0

4a

3

2

5b

 

E-glass medium

3

1

4a                             0

5b

0

5b

5b

1

4a

5b

 

E-glass high

4

0

4a                           1

5b

0

5b

5b

0

5b

5b

 

MMVF21 low

5

3

1         0

4a

0

0

0

0

0

0

 

MMVF21 medium

6

1

4a                             0

5b

5b

0

5b

2

0

2

 

MMVF21 high

7

0

5         0

5b

0

5b

5b

1

4a

5b

 

CMS

8

4a

1         0

5b

1

4a

5b

0

5b

5b

 

Interstitial fibrosis

 

 

 

 

 

 

 

 

 

 

 

Control

1

0

0

0

0

0

0

1

0

0

 

E-glass low

2

5b

0

5b

4a

0

4a

4a

1

5b

 

E-glass medium

3

5b

0

5b

5b

0

5b

2

3

5b

 

E-glass high

4

1

4a

5b

0

5b

5b

1

4a

5b

 

MMVF21 low

5

5b

0

5b

1

0

1

0

0

0

 

MMVF21 medium

6

5b

0

5b

5b

0

5b

5a

0

5b

 

MMVF21 high

7

2

3

5b

2

3

5b

2

3

5b

 

CMS

8

5b

0

5b

2

3

5b

0

5b

5b

 

Note.Significance of difference in a pairwise Fisher’s test compared to control group given as follows.ap< .05.

bp< .01.

 

 

 

 

Applicant's summary and conclusion

Conclusions:
At mean stone wool (MMVF 21) exposure concentrations of 76.6, 232.2 and 705.2 WHO fibers/ml a dose related increase in lung weight, in the biochemical parameters and in PMNs in the BALF, in proliferation of terminal bronchiolar epithelium, and in interstitial fibrosis were seen. These effects were similar to that seen in animals exposed to CMS fiber which has been shown to be not carcinogenic in a chronic inhalation toxicology study. The authors concluded that unfortunately, due to the large and varying number of particles and short fibers in the different fiber groups, these parameters cannot be associated specifically to fiber concentration, either of long fibers or WHO fibers. Therefore, from this study specific fiber-related endpoints are not established so far.
Executive summary:

At mean stone wool exposure concentrations of 76.6, 232.2 and 705.2 WHO fibers/ml a dose related increase in lung weight, in the biochemical parameters and in PMNs in the BALF, in proliferation of terminal bronchiolar epithelium, and in interstitial fibrosis were seen. These effects were similar to that seen in animals exposed to CMS fiber which has been shown to be not carcinogenic in a chronic inhalation toxicology study. The authors concluded that unfortunately, due to the large and varying number of particles and short fibers in the different fiber groups, these parameters cannot be associated specifically to fiber concentration, either of long fibers or WHO fibers. Therefore, from this study specific fiber-related endpoints are not established so far.