Phosphonic acid, P-(3-silylpropyl)-, Si,Si,Si-tris(mixed ethoxy and methoxy) derivs., mixed Et and Me diesters

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD (2014a) Draft Proposal for a New Test Guideline (EpiOcularTM)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

diethyl [3-(trimethoxysilyl)propyl]phosphonate

EC Number:

619-721-2

Cas Number:

1079258-99-3

Molecular formula:

C8H21O6PSi - C13H31O6PSi, C14H35O9PSi2 - C17H41O9PSi2

IUPAC Name:

diethyl [3-(trimethoxysilyl)propyl]phosphonate

Details on test material:

- Name of test material (as cited in study report): Diethylphosphonatopropyltrimethoxysilan
- Physical state: liquid
- Analytical purity: GC column DB1 94.5 area-% and GC column RTX-5-Amine 92.4 area-% For details see analytical report No.: 14S00760.
- Lot/batch No.: 130011
- Expiration date of the lot/batch: October 23, 2015
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals / tissue source

Species:

other: Isolated bovine cornea and reconstructed three dimensional human cornea model

Strain:

not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro controls

Amount / concentration applied:

750 μL of the undiluted test substance (BCOP) and 50 μL of the undiluted test substance (EpiOcular)

Duration of treatment / exposure:

10 minutes (BCOP) or 30 minutes (EpiOcular)

Observation period (in vivo):

after the 2-hours post-incubation period (both BCOP and EpiOcular)

Number of animals or in vitro replicates:

N/A

Details on study design:

OBJECTIVES
The objective of the present study is the determination of a possible eye irritating potential of MNSAO Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of eye irritating potential. Therefore, two assays are part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP) and EpiOcular Eye Irritation Test.
BCOP:
The objective of this in vitro test is to assess the potential of the test substance to cause serious eye damage. According to the current version of the OECD Guideline 437 (adopted July 2013), also substances that do not require classification for eye irritation or serious eye damage can be identified. However, due to insufficient predictivity the latter derivation is not recommended by the test facility.
The test method consists of a topical exposure of the test substance to the epithelial surface of isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements are used to calculate an In Vitro Irritancy Score (IVIS) of the test substance, which is used for the prediction of serious eye damage.
EpiOcular:
The objective of this in vitro test is to assess the eye irritation potential of the test substance using the reconstructed human ocular model EpiOcularTM. The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three dimensional human cornea model EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanolextraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to values from negative control tissues and expressed as relative tissue viability.
TEST SYSTEM
BCOP
Isolated bovine cornea: The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly
slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
Supplier: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim, Germany
EpiOcular
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on especially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

EXPERIMENTAL PROCEDURE

Experimental procedure BCOP Test
The test substance was tested in test no. 175.
Preparation of the bovine corneas and measurement of initial corneal opacity
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 Eagles’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 with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 537 opacity units1 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.
Each corneal holder was uniquely identified with a number on the chambers.

Application of the test substance and washing
Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application, the medium in the anterior chamber was removed using a syringe. The undiluted, viscous test substance could not be applied with a pipette. Therefore 750 μL of the undiluted liquid test substance was applied directly to the epithelial surface of the cornea using a syringe (open chamber method).
For the control tissues 750 μL of de-ionized water (negative control, NC) or 750 μL of 100% ethanol (positive control, PC), were applied into the anterior chamber using a pipette.
The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

Post-exposure incubation for liquid test substances and surfactants
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

Measurement of final corneal opacity
Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

Determination of permeability
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C.
The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.
An aliquot of PC1 was diluted 1:5 in Eagle’MEM (without phenol red) and measured analogously.


Experimental procedure EpiOcular Test
Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested
concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.

Basic procedure
Several test substances were tested in parallel within the present test (test no. 60) using the same control tissues (NC and PC).
Two tissues were treated with each, the test substance, the PC and the NC.
In addition two killed tissues were used for each, the test substance and the NC, in order to detect direct MTT reduction.
There are two separate protocols for liquids and solids, differing in exposure time and postincubation
period. Due to the physical condition of the test substance the protocol for liquids was applied.

Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

Pretreatment of the tissues
After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance
Using a pipette, fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC) or test substance (killed tissue control, KC).
After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.

Removal of the test substance and postincubation period
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS.
Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance.
After 12 minutes (liquids) of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.
Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation period).

MTT incubation
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.

Acceptance criteria BCOP
A study is considered acceptable if the PC gives an IVIS that falls within two standard deviations of the current historic mean.
The NC responses should result in opacity and permeability values that are less than the established upper limits.
Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low.
In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. In this context, a result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
• 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
• 1 of the 3 corneas gave a discordant prediction from the mean of all 3 corneas, AND the discordant result was >10 IVIS units from the cut-off threshold of 55.

Acceptance criteria EpiOcular
Assay acceptance criterion for the NC
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
Acceptance criteria for the PC Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
Assay acceptance criterion for tissue 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%.
Acceptance criteria for the KC
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35. The value for direct MTT-reduction of a test substance should be ≤ 50% of the NC.

EVALUATION OF RESULTS
The evaluation of the eye irritation potential of the test substance uses the results of the BCOP Test and the EpiOcular Test.
If a test substance was not tested in both of the test systems or an inconclusive result was obtained in one of the tests, the test strategy might still lead to an overall evaluation when the other test system result gives a clear prediction. However, if contradictory results are obtained a test evaluation might not be possible.

Evaluation of results BCOP
The following decision criteria apply:
Decision criteria of BCOP
IVIS Prediction
≤ 3 no classification for eye irritation
> 3; ≤ 55 no prediction can be made for eye irritation, further testing with another
suitable method is required
> 55 ocular corrosive or severe irritant

According to the current OECD Guideline 437 (adopted July 2013), this prediction is possible, however, not recommended by the test facility. If the IVIS obtained for the substance tested in this study fell within this range, this aspect is discussed in section 5.
The test method according to the OECD test guideline 437 revised and adopted in 2013 does not allow for the evaluation of eye irritation. I.e., the result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from
another study are needed.

Evaluation of results EpiOcular
The following decision criteria apply:
Decision criteria of EpiOcular
Mean tissue viability (% of negative control) and Prediction
≤ 60 irritant
> 60 non-irritant

Combined assessment of eye irritating potential
If in a top-down approach, the BCOP test resulted in the prediction of ocular corrosive or severe irritant, no EpiOcular was conducted. If in a bottom-up approach EpiOcular resulted in non-irritant, no BCOP test was conducted. In all other cases both assays were necessary for
a final assessment.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: In Vitro Irritancy score (IVIS) of the test substance (BCOP)

Basis:

mean

Time point:

other: end of observation time

Score:

16.2

Remarks on result:

other: Result due to IVIS: no prediction possible

Results 2

Irritation parameter:

other: viability [% of NC] (EpiOcular)

Basis:

mean

Time point:

other: end of the observation time

Score:

53.7

Remarks on result:

other: Result based on mean tissue viability: irritant

Irritant / corrosive response data:

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria, Diethylphosphonatopropyltrimethoxysilan shows an eye
irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU