Glass, oxide, chemicals

Genetic toxicity: in vitro

Administrative data

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: other: Cytotoxicity and production of cytokines (Tumor Necrosis Factor-alpha (TNF-alpha))

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 1997 – May 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study. No guideline for the study was identified, but the method and results are described scientifically correct.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1985

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Tumour Necrosis Factor-Alpha (TNF-alpha) is a pleiotropic cytokine involved in inflammatory processes, which is toxic to some cell types and is suspected to be involved in the mechanisms leading to development of hyperplasia and tumours as sequelae to chronic inflammation. The in vitro assay involves testing of the potential of E-glass microfibre (Glass-fibre code 104E) to induce expression of TNF-alpha in lung macrophages as analyzed by a quantitative assay for cytotoxicity of TNF-alpha towards cultures of the L929-cell line, which is sensitive to TNF-alpha.

GLP compliance:

no

Type of assay:

other: In vitro study for production of Tumor Necrosis Factor-alpha (TNF-alpha)

Test material

Test material information

Constituent 1

Reference substance name:

E-glass microfibre

IUPAC Name:

E-glass microfibre

Details on test material:

- Name of test material (as cited in study report): Special purpose glass microfibre code 104E (abbreviated 104E)
- Produced and provided by: Manville Insulation, Mountain Technical Centre, Schuller International Inc. Littleton, Colorado, USA.
- Substance type: Man-made vitreous fibre
- Physical state: Solid, respirable fibre material (dust)
- Composition of test material, percentage of components: See Table 1.
- Fibre geometric dimensions (length, diameter and surface area, mean values): See Table 2
- Lot/batch No.: NA.
- Stability under test conditions: Stable

- Preparation of test samples: Test material provided in a bale. Respirable fibres were created by chopping in a Manesty rotary chopper, followed by milling in a Retsch Pin mill (Type ZM1) fitted with a 1 mm mesh. Samples of respirable fibres were weighed and suspended.

Method

Target gene:

Expression of TNF-alpha by alveolar macrophages

Species / strain

Species / strain 1

Species / strain / cell type:

primary culture, other: Alveolar macrophages from rat, obtained by bronchoalveolar lavage

Details on mammalian cell type (if applicable):

- Type and identity of media: F-10 medium containing 0.2% Bovine serum albumin
- Primary culture
- cell concentration: 1*10^6 cells per well

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

mammalian cell line, other: L929 cell line

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM medium containing 5% fetal calf serum and 1 µg/ml actinomycin D. The cell line was kept in 96 well plates during the test.
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

other: TNF-alpha is toxic to the L929 cell line.

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

8.2 * 10^6 fibres (L > 5 µm) added to each well of a 24-well culture plate (volume of medium not mentioned).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test fibres were added suspended in an aqueous medium (not specified), and mixed with the F-10 cell medium.
- Justification for choice of solvent/vehicle: Not specified

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Recombinant human TNF-alpha was used for establishing a standard curve

Positive control substance:

other: 10 other fibre types were tested in the assay concomitantly.

Details on test system and experimental conditions:

Alveolar macrophages were harvested from rat lungs by bronchoalveolar lavage and cultured in F-10 medium containing 0.2% bovine serum albumin (BSA) at a cell concentration of 10^6 cells per well. To each well an aliquot of 8.2*10^6 fibres (Length > 5 µm) were added in an aqueous suspension. After 24 hours the supernatants were harvested, centrifuged to remove cells and fibres and stored at -70°C untill analysis. Serial dilutions of macrophage supernatants were made and added to cultures of the L929-cell line, which is sensitive to TNF-alpha. After one day of incubation the surviving L292-cells were stained using crystal violet and the staining intensity was quantified at 540 nm using a microplate reader. Each dilution of macrophage supernatant was analysed in triplicate, and for creation of a standard curve dilutions of recombinant human TNF-alpha was used. The results were expressed in international units (IU) per 10^6 macrophages. The complete experiment was repeated three times to obtain the data presented in the results section.

Results and discussion

Test results

Species / strain:

primary culture, other: Macrophages from rat lung

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Supernatant from fibre-treated macrophage cultures added to cultures of L929 cells

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

E-glass microfibre (Glass fibre code 104E) has an intermediate activity in relation to production of TNF-alpha in lung macrophages. See results in Table 2 below.

Remarks on result:

other: other: Production of TNF-alpha by rat lung macrophages

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2. TNF-alpha production by alveolar macrophages exposed in vitro to equal fibre numbers. Table contains means with estimated standard errors in italics
Fibre type	TNF-alpha Units per 10^6cells
	Mean	Standard error
No fibre	37	10
104E	71	19

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The E-glass microfibre has an activity intermediate between the most active fibres- silicon carbide fibres and asbestos fibres, and the relatively inactive man-made vitreous fibres (MMVF's) and refractory ceramic fibres (RCF's). In conclusion, E-glass microfibre holds the potential to induce the production of the cytokine TNF-alpha by lung macrophages.

Executive summary:

The potential of E-glass microfibre to induce expression of TNF-alpha in lung macrophages was tested in vitro. Tumour Necrosis Factor-Alpha (TNF-alpha) is a cytokine which is toxic to some cell types, e.g. the L929 cell line used in this study, and is suspected to be involved in the mechanisms leading to development of hyperplasia and tumours in connection to chronic inflammation.

E-glass microfibre has an intermediate activity in relation to induce TNF-alpha production in lung macrophages. In conclusion E-glass microfibre holds the potential to induce the production of the cytokine TNF-alpha by lung macrophages.