Ashes from fluidized Bed combustion coal fired Power stations with and without co-combustion of secondary fuels (biomass; other fuels - to be verified in view of ecotoxicological and toxicological tests)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3.9.2009-4.9.2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with internationally valid GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: MatTek Ocular Irritation Protocol: Neat Method (MTT ET-50), Rev. 1/1/01 (1)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

FBC Ash

IUPAC Name:

FBC Ash

Details on test material:

- Name of test material: Fluidized Bed Combustion (FBC) Bottom Ash
- Substance type: technical product
- Physical state: solid
- Appearance: Light grey solid powder with some small black particle
- Chemical structure: Complex product of oxides
- Main components: SiO2 - 27,78%, Fe2O3 - 5,98%, CaO (total) - 35,65%, Na2O - 0,25%, P2O5 - 0,38%, CO2 - 0,5%, CaO (free) - 19,91%, Al2O3 - 11,16%, TiO2 - 2,65%, MgO - 0,44%, K2O - 0,42%, SO3 (sulphate) - 2,84%
- Impurities: Metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn - sum < 0,1%
- Lot/batch No.: FBC/230309/T2
- Expiration date of the lot/batch: 03/2024
- Stability under test conditions: stable
- Storage: The substance will be stored in PE container at room temperature.

Test system

Amount / concentration applied:

TEST MATERIAL
The test substance was grinded in mortar with pestle before treatment.
As the test substance is partially soluble it could not be applied as a paste. First, tissues were moistened with 100 µL of H2O for injection before layering the test substance on its surface. At application it was assured to the tissues were completely covered by the test substance.

Duration of treatment / exposure:

3 min, 30 min, 60 min

Details on study design:

TEST SYSTEM
MatTek's EpiOcular™ corneal model (OCL-200, MatTek, Ashland, USA) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. The epidermal cells, which are cultured on specially prepared cell culture inserts using serum free medium, differentiate to form a multi-layered structure which closely parallels the corneal epithelium.
The EpiOcular™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped world-wide as kits, containing 24 tissues on shipping agarose together with the necessary amount of culture media.

TEST PROCEDURE
DIRECT MTT REDUCTION
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, before exposure, functional checks for this possibility was performed as follows: 25 mg of the test substance was added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 60 min. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.

PROCEDURE
On the day of receipt, EpiDerm tissues were conditioned for one hour by incubation to release transport stress related compounds and debris. After pre-incubation, tissues were topically exposed to the test chemical for 3, 30 and 60 minutes. Three tissues were used per each time interval three for the positive control (PC) and three for negative control (NC). Tissues are then thoroughly rinsed, blotted to remove the test substances, and transferred to 5 mL of fresh medium. After 10 minutes of soaking the tissues were transferred to 24-well plates containing MTT medium (1 mg/ml). After 3 hr MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted overnight, without shaking with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M/49/1 (VUOS-CETA, 2009).

OD570 MEASURING
OD570 is measured on a spectrophotometer Libra S22. Isopropanol serves as a blank.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

3 min

Value:

1.437

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

30 min

Value:

0.042

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

60 min

Value:

0.025

Any other information on results incl. tables

STUDY RESULTS

Direct MTT reduction

Before the test itself,direct MTT reduction was assayed.

After 60 min incubation of the test substance with MTT medium no changes in colouring were observed compared with concurrent control without test substance. The test substance did not reduce MTT directly.

MTT Test

OD₅₇₀measuring was performed after overnight extraction.

EVALUATION OF RESULTS

The average viabilities of affected tissues are 84.8% after 3 minutes of treatment, 2.5 % after 30 min and 1.5% after 60 min of administration.So, viability does decrease with time and value of ET-50 calculated is 8.85 min. According to the Table 1, it responses to the Draize score 25.1-50.0 and to the Kay and Calandra classification category moderate irritant.

Applicant's summary and conclusion

Interpretation of results:

moderately irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the above-described experimental design. the test substance Fluidized Bed Combustion (FBC) BottomAsh was moderately irritating for EpiOcularTM tissues.

Executive summary:

Test substance Fluidized Bed Combustion (FBC) Bottom Ash was assayed for the in vitro eye irritation in human corneal model EpiOcular^TM. The test was performed according to MatTekOcular Irritation Protocol: Neat Method (MTT ET-50),Rev. 1/1/01 (1).

The test substance was grinded and then it was applied as a powder. The tissue was previously moistend with water. Length of exposition was 3, 30 and 60 minutes. Three tissues were used for each timeinterval, six for positivecontrol (PC) and three for negative control (NC).

After rinsing and soaking in medium, tissues were incubated with MTT for three hours and then extracted overnight without shaking.OD₅₇₀of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each three tissues as % of the mean viability of the negative control. Achart of semi-log scale - the % viability (linear y axis) versus the dosing time (log x axis) was constructed and ET-50 value was calculated from slope of the line obtained. The ET-50 calculated was 8.85 min. It responses to the Draize score 25.1-50.0 and to the Kay and Calandra classification category moderate irritant.

In the experiment arrangement given above, the test substanceFluidized Bed Combustion (FBC) Bottom Ash was moderately irritating in EpiOcular^TMmodel.