Flue dust, portland cement

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-04-2010 to 10-08-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, according to the OECD draft technical guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, Version 4, 11 December 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

Test substance information
Date of receipt : 4 March 2010
Test material name : Flue Dust T (REACH)
Other names : Flue dust, Portland cement clinker (EC number 270-659-
9), Cement kiln dust
Batch number : 12-2009
CAS Reg. no. : 68475-76-3
Storage conditions : ambient temperature
Appearance : beige/grey powder
Expiry date : 1 December 2010
TNO dispense number : 10005B

Test animals

Species:

other: in vitro test

Test system

Details on study design:

The EpiDerm EPI-200 skin model consists of normal human epidermal keratinocytes (NHEK) from one single donor, derived from neonatal-foreskin tissue. The keratinocytes are plated on chemically modified, collagen-coated, 8 mm ID cell culture inserts (surface area 0.5 cm2). The skin models are commercially available and were obtained form MatTek Corporation, USA. Upon arrival at TNO Quality of Life on 28 April, the EPI-200 skin models were pre-incubated in 0.9 mL assay medium (lot. 042310TTE) provided with the kit for 60 ± 5 min (at ca. 37ºC and ca. 5% CO 2 ). At the end of the first pre-incubation period, the tissues were pre-incubated overnight for 6 ± 0.5 h (at ca. 37ºC and ca. 5% CO2 .

Preliminary tests
The tissue staining potential of the test substance was investigated by incubating 25±1 mg of the test substance in 300 μL demineralised water for 1 h (at ca. 37ºC). At the end of the exposure time the presence and intensity of the staining (if any) was evaluated. The test substance did not change the colour solution. Therefore, it was concluded that the test substance did not have the potential to stain the tissue.
Some chemicals are known to non-specifically reduce MTT, resulting in a blue precipitate. Therefore, prior to the start of the study, 25±1 mg of the test substance was incubated in 1 mL 1 mg/mL MTT solution for 3 h (at ca. 37ºC) and the formation of a blue formazan product was assessed. During incubation the solution turned blue/purple and it was concluded that the test substance reduces MTT. Therefore, an additional functional check was performed using freeze-killed tissues (frozen controls), i.e. the test substance was applied to two frozen controls in the main study (60 min exposure only) and data were corrected for MTT reduction of the test substance, if applicable.

Exposure to the study substances
After overnight pre-incubation, the skin membranes were exposed to 25±1 mg of the test substance and 30 μL of the positive and negative controls in triplicate for 60±5 min. Prior to application of the test substance, the tissue surface was moistened with 25 μL PBS. Only for the negative and positive controls, immediately after application a nylon mesh (provided with the EPI-200 kit) was placed on the tissue surface to facilitate an equal distribution.
Exposure was performed at ambient temperature in the flow cabin. After dosing the last tissue, all tissues were transferred to a humidified incubator (ca. 37ºC and ca. 5% CO 2 ). After 35±1 min, the tissues were removed from the incubator and placed in the flow cabin until the period of 60 min was completed for the first dosed tissue. When the exposure period was completed, the inserts were removed from the well and the skin surface was carefully washed using excess of phosphate buffered saline (PBS). Subsequently, the insert was blotted dry and the skin membranes were carefully dried
using a sterile cotton swab. The insert was then transferred to clean 6-well plate containing fresh medium (0.9 mL/well). Medium was refreshed after 22 h. Following an additional 18 h incubation period at ca. 37 °C and ca. 5 % CO 2 in a humidified incubator, viability was determined using the MTT assay.

MTT assay
An MTT solution of 1 mg/mL was prepared by diluting MTT concentrate 5 times in MTT diluent (both provided with the kit). The bottom of the inserts was blotted dry and the inserts placed in a 24-well plate containing 300 μL of MTT medium/well. The plates were placed in an incubator at ca. 37 ºC and ca. 5% CO 2 . After 180±5 min, the skin membranes were rinsed with PBS and placed in an empty 24-well plate. The formazan product was extracted from the tissue using 2 mL MTT extractant (lot. 022210RMA) (provided with the kit). Extraction was performed in the dark for 3 days at 2-10 ºC.
After completion of the extraction period, the skin membrane was pierced with a needle to allow the extract to run into the well from which the membrane was taken. The optical density was measured in triplicate in 200 µL sub-fractions using a spectrophotometer set at 570 nm. Extractant solvent alone was used as blank. The mean optical density was calculated and expressed as the percentage viability compared to the negative control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 26

In vivo

Irritant / corrosive response data:

Flue Dust T (REACH) was examined for its in vitro skin corrosive potential using EpiDerm™ skin membranes. Two in vitro skin corrosion tests with EpiDerm™ skin membranes were performed.

The results of the MTT assay of the first test, following a 3-minute and 60-minute exposure to Flue Dust T (REACH) is presented in Table 1. The negative control (demineralised water) and positive control (8M KOH) correctly indicated non corrosivity and corrosivity, respectively (individual data are presented in Appendix 1).

Following application, the test substance hardened on the skin surface and was difficult to rinse from the skin surface after the exposure period. This was more difficult after 60 min exposure compared to 3 min exposure. Consequently, a more aggressive rinsing procedure than normal was used and the tissues may therefore have suffered from mechanical stress and it was not possible to completely remove all the test substance from the skin.

Frozen controls treated with Flue Dust T (REACH) showed no MTT conversion notably higher than the non-treated controls. Therefore, it was concluded that no interference of the test substance with the MTT formation (which may cause a false indication of tissue viability) had occurred.

In the first test, two replicates were used (according to OECD guideline 431) and the standard deviation between these tissues was 34% of the mean of the tissues. In general lower variations are observed between two replicates. This variation was most likely due to the problems with removal of the test substance. Because the %viability (A 570 % of control) after 60 min exposure was the closest value to the border for classification as corrosive, a second in vitro skin corrosion test was performed, including four replicates for the test substance group.

The results of the MTT assay of the second test, following a 3-minute and 60-minute exposure to Flue Dust T (REACH) is presented in Table 1. The negative control (demineralised water) and positive control (8M KOH) correctly indicated non corrosivity and corrosivity, respectively (individual data are presented in Appendix 1).

In the second test again considerable variation was observed between the tissues; the standard deviation between these tissues was 38% of the mean of the tissues.
If the A 570 % of control values are calculated for each individual replicate in the second test separately, the %viabilities are respectively 21%, 33%, 15% and 35%. In the first test the %viabilities of the individual replicates are then respectively 11% and 17%. The overall mean of the viability is 22% ± 10%.

Based on the results of Tables 1 and 2, i.e. results of 6 replicate tissues divided over 2 independent tests, the test substance Flue Dust T (REACH) is indicated as not corrosive. However, it should be taken into account that considerable variation between replicates was observed.

Any other information on results incl. tables

Table 1: Formazon production in EpiDerm skin membranes exposed to the test substance and negative and positive controls for 3 minutes and 60 minutes (first test)

Test group	Study substance	A570 (% of control) after 3 minutes exposure
NC	Negative control (PBS)	100
A	Flue Dust T (REACH)	104
PC	Positive control	16

Test group	Study substance	A570 (% of control)after 60 minutes exposure
NC	Negative control (PBS)	100
A	Flue Dust T (REACH)	14
PC	Positive control	5

Table 2: Formazon production in EpiDerm skin membranes exposed to the test substance and negative and positive controls for 3 minutes and 60 minutes (second test)

Test group	Study substance	A570 (% of control)after 3 minutes exposure
NC	Negative control (PBS)	100
A	Flue Dust T (REACH)	119
PC	Positive control	16

Test group	Study substance	A570 (% of control)after 60 minutes exposure
NC	Negative control (PBS)	100
A	Flue Dust T (REACH)	26
PC	Positive control	8

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Based on the study results, i.e. results of 6 replicate tissues divided over 2 independent tests, the test substance Flue Dust T (REACH) is indicated as not corrosive. However, it should be taken into account that considerable variation between replicates was observed.

Executive summary:

Flue Dust T (REACH) was examined for its in vitro skin corrosive potential. Two in vitro skin corrosion tests in EpiDerm™ skin models were performed. The EpiDerm™ Skin Model (EPI-200) was exposed to the test substance for 3 minutes and 60 minutes, followed by assessment of the viability of the EpiDerm™ skin membranes by measuring the reduction of MTT to a purple formazan precipitate by mitochondrial dehydrogenase activity. The test substance was tested undiluted.

According to the present in vitro tests according to the OECD Guideline 431, Flue Dust T (REACH) was classified as non-corrosive. However, it should be taken into account that considerable variation in viability of the tissues was observed after exposure to the test substance related to the fact that the test substance formed a solid plaque on the skin surface that was difficult to remove without evoking damage to the skin membranes.