Neutralisation product of iron oxide hydroxide, phosphoric acid, phosphonic acid, lithium hydroxide or lithium carbonate, D-Glucose, 4-O-β-D-galactopyranosyl-, hydrate (1:1) and amylopectin

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jul 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: part of a testing strategy

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jul 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: part of a testing Strategy

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Rheinland-Pfalz

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: 011021P040BB1
- Purity: 100% UVCB (Substance of unknown or variable composition, complex reaction products or biological materials)
- pH-value: Ca. 6 (undiluted test substance, moistened with water; determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Form of application: The test substance was applied undiluted, therefore no preparation of the test substance in a vehicle was performed.

Species:

human

Strain:

other: normal human derived epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

Description of the cell system used: Reconstructed human cornea-like epithelium
- Model used: EpiOcular™ model (OCL-200)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue batch number: 21559
- Certificate of authenticity: provided by the supplier
- Verification of tissue functionality and quality (performed by the supplier):
Tissue viability: acceptance criteria met
Barrier function: acceptance criteria met
Sterility: no contamination

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): ca. 50 μL bulk volume (ca. 16 mg) of the undiluted test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL, undiluted

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were immersed and swiveled three times in each of three beakers filled with sterile PBS.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control.

NUMBER OF REPLICATE TISSUES PER TEST CHEMICAL AND CONTROLS: 2

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm, without reference filter

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to the eye if the mean relative tissue viability with a test material is ≤ 60%.
- The test substance is considered to be non- irritant to the eye if the mean relative tissue viability with a test material is > 60%.

ACCEPTANCE CRITERIA
- Negative control: Tissue viability is acceptable if the mean OD570 of the negative control (NC) is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
- Positive control: Methyl acetate used as positive control (PC) usually leads to a tissue viability of approx. 25%. A viability of < 60% is acceptable.
- Variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.

Irritation parameter:

other: mean viability [%]

Remarks:

mean of 2 replicate tissues

Value:

8.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes (demonstrated by historical control values of negative and positive controls, gathered over an appropriate time period)

Table 1: Individual and mean OD₅₇₀values, individual and mean viability values and inter-tissuevariability

 

Test substance		tissue 1	tissue 2	mean	Inter-tissue variability [%]
NC	mean OD570	1.476	1.518	1.497
	viability [% of NC]	98.6	101.4	100.0	2.8
Test substance	mean OD570	0.171	0.097	0.134
	viability [% of NC]	11.4	6.4	8.9	5.0
PC	mean OD570	0.296	0.352	0.324
	viability [% of NC]	19.7	23.5	21.6	3.8

 

Minimal compound residues remained on tissue 2 after the washing procedure.

NC: negative control

PC: positive control

Executive summary:

The objective was to assess the eye irritating potential of the test substance. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular™ Eye Irritation Test.

EpiOcular™

The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (ca. 16 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by an 18-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues was compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ eye irritation assay:

The test substance was not able to reduce MTT directly. The mean viability of the test substance treated tissues was 8.9%. Based on these results the test substance was identified as irritant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Rheinland-Pfalz

Test material

Test material information

Constituent 1

Reference substance name:

Neutralisation product of iron oxide hydroxide, phosphoric acid, phosphonic acid, lithium hydroxide or lithium carbonate, D-Glucose, 4-O-β-D-galactopyranosyl-, hydrate (1:1) and amylopectin

EC Number:

942-575-9

Molecular formula:

not available

IUPAC Name:

Neutralisation product of iron oxide hydroxide, phosphoric acid, phosphonic acid, lithium hydroxide or lithium carbonate, D-Glucose, 4-O-β-D-galactopyranosyl-, hydrate (1:1) and amylopectin

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: 011021P040BB1
- Purity: 100% UVCB (Substance of unknown or variable composition, complex reaction products or biological materials)
- pH-value: Ca. 6 (undiluted test substance, moistened with water; determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a high speed homogenizer and a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.
- Form of application: 20% (w/v) solution in de-ionized water

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle; supplier: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: age of the animals: minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: Corneas free of defects (opacity, scratches, pigmentation etc.)

Test system

Vehicle:

water

Remarks:

de-ionized

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/v) solution in de-ionized water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL of de-ionized water

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 μL of 20% (w/v) solution of imidazole in de-ionized water

Duration of treatment / exposure:

approx. 4 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour. After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer.

QUALITY CHECK OF THE ISOLATED CORNEAS: Any corneas that showed macroscopic tissue damage or an opacity value < 542 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.(According to OECD TG 437, corneas that have an opacity value >7 or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period are to be discarded. In the test facility´s opacitometer in combination with the used cornea holder set 2013-22, the maximal initial opacity of >7 arises from I= 542 lux with Io= 632 lux).

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: Application of 750 µL of the 20% (w/v) test-substance preparation, 4 hours exposure time

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times (positive and negative control). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Yes (with an opacitometer)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader: Yes, OD490, SunriseTM Absorbance Reader

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS: < 3, Prediction: No classification for eye irritation.*
IVIS: > 3, ≤ 55, Prediction: No prediction can be made for eye irritation, further testing with another suitable method is required.
IVIS: > 55, Prediction: Ocular corrosive or severe irritant.
* further testing required to establish a definitive classification

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean of 3 single corneas

Value:

3

Vehicle controls validity:

other: vehicle control = negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of severe eye damage

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes (demonstrated by historical control values of negative and positive controls, gathered over an appropriate time period)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1: In Vitro Irritancy score (IVIS) of the test substance, negative control (NC) and positive control (PC)

 

Test substance	Cornea- No.	Opacity per cornea	Permeability per cornea	per cornea	IVIS per group
					mean	SD
	16	6.4	0.009	6.5
Test substance	17	1.9	0.005	2.0	3.0	3.1
	18	0.6	0.002	0.6
	1	6.3	0.004	6.3
NC	2	4.6	0.004	4.7	7.3	3.3
	3	10.9	0.006	11.0
	4	96.1	3.492	148.5
PC	5	69.2	3.052	115.0	115.1	33.3
	6	45.4	2.432	81.8

Applicant's summary and conclusion

Executive summary:

The objective was to assess the eye irritating potential of the test substance. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular™ Eye Irritation Test.

BCOP Test

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% test-substance preparation in de-ionized water to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC; 20% imidazole in de-ionized water) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score (IVIS) of the test substance.

The mean IVIS of the test-substance treated corneas was 3.0.

Based on this the BCOP identified the test substance as not corrosive or severe irritant to the eyes. However, to establish a definitive classification a further test on the eye irritating potential is required.