Ashes (residues), nonhazardous municipal solid waste

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24.08.2011 - 25.08.2011

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:

other: Method B.47 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Council Regulation (EC) No.1152/2010, published in O.J. L 324, 2010

Deviations:

no

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 / tissue source

Details on test animals or tissues and environmental conditions:

TISSUES:
Bovine eyes source: Breeding service CHOVSERVIS a.s., division TORO Hlavečník, Hradec Králové, Czech Republic
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. Only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.

SELECTION CRITERIA FOR EYES USED IN BCOP:
The eyes, once they arrive at the laboratory, were carefully examined for defects including increased opacity, scratched, and neovascularisation. Only corneas from eyes free of such defects were used.

Test system

Vehicle:

physiological saline

Amount / concentration applied:

APPLICATION FORM:
The test substance was tested as solution at 20% concentration in a 0.9% sodium chloride solution.
2g of the test substance was dissolved in 10mL of 0.9% sodium chloride.
Applied volume: 750 µL.

Duration of treatment / exposure:

4 h

Details on study design:

TEST PROCEDURE:
20 eyes were chosen (without macroscopic tissue damage - e.g., scratches, pigmentation, neovascularization) from the supply for the test and from this number 5 eyes were eliminated after inductive incubation, because the baseline opacity values were > 7.
Number of corneas per group:
Exposed group - 3 corneas
Positive control group – 3 corneas
Negative control group –3 corneas

PROCEDURE SCHEME:
1. Selection of corneas, mounting in holders,
2. Incubation with Eagle`s Minimum Essential Medium (EMEM) 1hour (32 ± 1°C),
3. Removed EMEM, measurement of baseline opacity,
4. Treatment by positive and negative control substances and test substance (incubation 4 hour),
5. Washing epithelium, measurement of opacity after application,
6. Application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C),
7. Measurement of absorbance (490 nm).

PREPARATION OF THE EYES:
Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium and endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32±1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity > 7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated.
Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

APPLICATION OF THE TEST SUBSTANCE:
The test substance was tested as solution at 20% concentration in a 0.9% sodium chloride solution. 2g of the test substance was dissolved in 10mL of 0.9% sodium chloride.
Closed-chamber method was used, because the test substance was applicable by micropipette. The test substance (750 µL) to cover the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure.
After the exposure period, the test substance, the negative control, or the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

POSITIVE CONTROL:
Imidazole (CAS Number: 288-32-4, Sigma-Aldrich)

NEGATIVE CONTROL:
0.9% sodium chloride solution (Batch Number: 15DF065A1, Braun, Melsungen)

ENDPOINTS MEASURED:
Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale.
Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32±1°C.
The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYS 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

3.93

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Opacity and optical density

	Optical density (490nm)	Mean	Baseline opacity	Opacity after treatment	Opacity difference	Mean
Negative Control (0.9% sodium chloride solution)	6	0.032	7	7	0	0.67
	12		6	8	2
	14		5	5	0
Positive Control (20% imidazole)	17	2.050	7	67	60	63.00
	19		5	72	67
	20		7	69	62
Test Substance	9	0.005	6	8	2	5.00
	10		7	9	2
	13		7	18	11

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The In Vitro Irritancy Score (IVIS) value (3.93) for the test substance Ashes (residues), nonhazardous municipal solid waste was below the threshold (55.1), so that the test substance was not identified as a corrosive or severe irritant.

Executive summary:

The test substance, Ashes (residues), nonhazardous municipal solid waste, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the Method B.47 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Council Regulation (EC) No.1152/2010, published in O.J. L 324, 2010.

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for Ashes (residues), nonhazardous municipal solid waste was 3.93. This is less than the limit value of IVIS (55.1), which means that the test substance was not identified as a corrosive and severe irritant.