Ashes (residues), nonhazardous municipal solid waste

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.09.2011 - 22.10.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

yes

Remarks:

The experiment was repeated because acceptance criteria (3) was not fulfilled. Standard deviation of individual tissue viabilities was <18%. This deviation had no impact on the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues), nonhazardous municipal solid waste

EC Number:

937-417-0

Molecular formula:

not applicable

IUPAC Name:

Ashes (residues), nonhazardous municipal solid waste

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): Ashes (residues), nonhazardous municipal solid waste
- Substance type: UVCB (inorganic oxides)
- Physical state: solid
- Composition of test material, percentage of components: SiO2 47.71 %; Al2O3 12.24 %; Fe2O3 9.62 %; CaO 15.80 %; Na2O 3.93 %; MgO 1.98 %; SO3 (total) 1.73 %; K2O 1.46 %; TiO2 1.22 %;
- Lot/batch No.: SPRUK-pololetí II/2010

Test animals

Details on test animals or test system and environmental conditions:

TISSUES:
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Ashland, USA);
MTT test: EPI-200, Lot No. 15650, kit A (the second experiment), and EPI-200, Lot No. 15626, kit A (the first experiment).

Test system

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- not used

Duration of treatment / exposure:

60 min

Details on study design:

TEST METHOD
The test methods are described in the guidelines referred in chap. 1.4. Details of routine are available in Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm, Model EPI-200-SIT, MatTek Corporation January 1st, 2010, 1 37.

PRINCIPLE OF THE ASSAY
The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-(4,5-dimethylthiazole-2-yl))-2,5-diphenyltetrazolium bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential.
Evaluation is determined by measuring of optical density of the formazan extracts using a spectrophotometer at 570 nm (OD570). Relative cell viability is calculated for each tissue as % of the mean OD value of the negative control tissues. Skin irritation potential of the test material is predicted if the remaining relative cell viability is below 50 %.

TEST SYSTEM
The reconstructed human epidermal model EpiDerm (EPI-212, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm System is manufactured according to defined quality assurance procedures.
The EpiDerm tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped world-wide as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

MTT TEST
a) Test substance application:
The test substance was previously grinded in mortar with pestle. The test substance (25 mg) was placed directly atop the tissue previously moistened with 25 μl of sterile PBS to improve contact of the tissue surface with the test chemical. The material was spread on the tissue surface.
b) Procedure:
On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After pre-incubation, tissues were topically exposed to the test chemicals for 1 hour. Three tissues were used per test chemical and for the positive control (PC) and negative control (NC). Tissues were then thoroughly rinsed, blotted to remove the test substances, and transferred to fresh medium.
After 24 hour incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropanol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. Detailed procedure was described in SOP M/48/3 (VUOS-CETA, 2011).
c) OD570 measuring:
OD570 was measured on a spectrophotometer Libra S22. Isopropanol served as a blank.

NEGATIVE CONTROL
The absolute OD of the negative control (NC) tissues (treated with sterile PBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 1.0 and ≤ 2.5.

POSITIVE CONTROL
A 5 % SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95±1 % confidence interval of the historical data.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: OD570

Value:

1.796

Remarks on result:

other:

Remarks:

Basis: other: viability of tissue. Time point: 60 min. Max. score: 1.983. Remarks: score: OD570 of tested substance / max. score: OD570 of negative control. (migrated information)

Any other information on results incl. tables

Table 1: Values obtained at the MTT test

Treatment	Parameter	OD570			mean	SD	Average viability
		1	2	3			(% NC)
NC (PBS)	OD570	2.079	1.960	1.911	1.983	0.071
	viability (%) NC)	104.824	98.824	96.353	100.0	3.557	100.0
C2 (Test substance)	OD570	1.565	1.876	1.947	1.796	0.166
	viability (%) NC)	78.908	94.588	98.168	90.6	8.364	90.6
PC (5% SDS)	OD570	0.230	0.205	0.239	0.222	0.017
	viability (%) NC)	11.597	10.336	12.050	11.3	0.725	11.2

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance, Ashes (residues), nonhazardous municipal solid waste, is non-irritating in vitro skin irritation test (EpiDerm model).

Executive summary:

Test substance Ashes (residues), nonhazardous municipal solid waste was assayed for the in vitro skin irritation in human epidermal model EpiDerm. The test was performed according to Method B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDerm Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (see par. 1.4, (1), (3)).

Test substance (25 mg) was placed directly atop the tissue. Length of exposition was 60 minutes. Nine tissues were used for the experiment, three per test substance (TS), three for positive control (PC) and three for negative control (NC).

After rinsing, tissues were post incubated for 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropanol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, average viability of tissues treated by the test substance was 90.6 % of negative control average value (i.e. viability was >50 %). According to the criteria given in Method B.46, the test substance did not fall into any of quoted categories of skin irritation.

The test substance, Ashes (residues), nonhazardous municipal solid waste, is nonirritating in in vitro skin irritation test (EpiDerm model).