Flue dust, portland cement

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.5.2010 to 23.10.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

no guideline followed

Version / remarks:

In vitro micronucleus test in cultured human bronchial epithelial BEAS-2B cells

Principles of method if other than guideline:

In order to analyze if chemicals have aneugenic or clastogenic potential, the in vitro
micronucleus assay was conducted using human bronchial epithelial BEAS-28 cells. Because
inhalation is the anticipated main route of exposure (inhalation of dust represents the main
hazard in Flue Dust T-fine (REACH) processing), the human bronchial epithelial (BEAS-2B)
cell line was considered to be more relevant for the hazard identification of the test substance.
By employing the actin polymerization inhibitor cytochalasin B, proliferating cells can be
identified as binucleated cells. If chemicals induce the formation of chromosome fragments,
or acentric chromosomes, these can be visualized as micronuclei in proliferating (binucleated)
cells.

GLP compliance:

yes

Type of assay:

other: In vitro micronucleus test in cultured human bronchial epithelial BEAS-2B cells

Test material

Test material information

Constituent 1

Reference substance name:

Flue dust, portland cement

EC Number:

270-659-9

EC Name:

Flue dust, portland cement

Cas Number:

68475-76-3

Molecular formula:

It is a UVCB.

IUPAC Name:

Flue dust from Portland cement clinker production

Details on test material:

The test substance was supplied by the sponsor. Two containers each containing 800 g Flue dust T (REACH) were received in good condition on 4 March 2010. Gross weights were 870.95 g and 875.67 g, respectively. This batch was given TNO Dispense Reference No. 10005B.

The following characteristics were provided by the sponsor:

Name : Flue dust T (REACH)
Other names : Flue dust, Portland cement (EC number 270-659-9); Cement kiln dust
Colour / appearance : Beige/grey powder
CAS reg. number : 68475-76-3
Purity : UVC substance
Storage conditions : Ambient temperature
Batch number : 12-2009
Expiry date : 1 December 2010

The certificate of analysis pertaining to the batch of test substance used during this study was
provided by the sponsor.

Method

Species / strain

Species / strain / cell type:

other: cultured human bronchial epithelial BEAS-28

Results and discussion

Test results

Key result

Species / strain:

other: human ephitelal cells

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results obtained, the highest analyzable dose level of the test
substance Flue Dust T-fine (REACH) induced a statistically significant increase (***p<0.001)
in the number of binucleated cells containing micronuclei, compared to the negative particle
control Silica with an indication of a dose response relationship. This increase is considered to
be an indication for a clastogenic and/or aneugenic effect, under the conditions used in this
study.

Executive summary:

Summary The test substance Flue Dust T-fine (REACH) was examined for its ability to induce micronuclei in cultured human bronchial epithelial BEAS-28 cells. The complete study consisted of two in vitro micronucleus tests. In the first test, the toxicity curve of the test substance Flue Dust T-fine (REACH) was very steep and acceptable toxicity levels could not be reached. Based on the results obtained in the first test it was decided to perform a second micronucleus test, in which the test substance Flue Dust T-fine (REACH) was tested in a narrower dose range. The choice of dose levels for the second test was based on the toxicity data obtained in the first test. Culture medium containing l%o fetal calf serum (FCS) was included as solvent control. Silica was included as negative particle control. Fullerene C60 was included as positive particle control. Mitomycin C, a known clastogenic compound, was included as positive non-particle control. Statistical significance of micronuclei induction by the test substance Flue Dust T-fine (REACH) and the positive particle control Fullerene C60 was tested against the negative particle control Silica. Statistical significance of micronuclei induction by Silica and the positive nonparticle control Mitomycin C was tested against the solvent control. Evaluation of cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI) and tested against the solvent control. Prior to the tests, the test substance Flue Dust T-fine (REACH) and the two reference materials Silica particles and Fullerene C60 were dispersed in culture medium, autoclaved and sonificated. In the first test, duplicate cultures of the human bronchial epithelial BEAS-2B cells were exposed to the solvent control, l3 dose levels of the test substance Flue Dust Tfine (REACH), ranging from 0.25 to 1000 pg/cmt at a two-fold interval, 2 dose levels of the negative particle control Silica (250 and 500 þglcm'),4 dose levels of the positive particle control Fullerene C60, ranging from 30 to 240 pg/cmt at a two-fold interval, or three dose levels of the non-particle positive control Mitomycin C, ranging from 0.005 to 0.05 pgiml) at a two-fold interval. Exposure to the test substance Flue Dust T-fine (REACH) resulted in high levels of toxicity (only dead cells observed) at the four highest dose levels used (125, 250, 500 and 1000 þg/cm'). These high toxicity levels were most likely the result of suffocation of the cells due to the presence of a layer of test substance particles. The toxicity curve was very steep because at the highest analyzable dose level (62.5 pglcm2) only slight toxicity (26%) was observed. The highest analyzable dose level (62.5 ¡tg/cm2) and one lower dose level (31.3 pglcm2) were evaluated for micronucleus formation and did not induce a statistically significant increase in the number of binucleated cells containing micronuclei compared to the numbers found in the concurrent negative particle control silica. Exposure of the cells to the negative particle control Silica resulted in severe toxicity at the highest dose level of 500 þúcm', which was most likely the result of suffocation of the cells due to the presence of a layer of Silica particles. The toxicity curve was very steep because the lowest dose level of 250 þúcm' induced only 60/o toxicity. The only available dose level of 250 ltglcm'was evaluated for micronuclei formation and was comparable, when compared to the negative solvent control. Exposure of the cells to the positive particle control Fullerene C60 resulted in high levels of toxicity (only dead cells observed) at the three highest dose levels used (60, 120 and 240 p!cm2). The toxicity curve was very steep because exposure to the lowest dose level of 30 pglcm2 resulted in a low toxicity levels of 4o/o. The lowest dose level was evaluated for micronucleus formation analysis and induced a clear statistically significant increase (p