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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

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

Species:

other: human reconstructed epidermis

Strain:

not specified

Details on test animals or test system and environmental conditions:

Tissue model: Epi-200
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the EpidermTM human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).

ENVIRONMENTAL CONDITIONS
about 37 °C, 5 % CO2, 90-95 % humidity

Test system

Controls:

other: negative control: human reconstructed epidermis

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): bulk volume of 30 or 50 µL

Duration of treatment / exposure:

Corrosion test: 3 minutes or 1 hour
Irritation test: 1 hour followed by a 42-hours post-incubation period.

Observation period:

Corrosion test: Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.
Irritation test: The tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. 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.

Details on study design:

The objective was to assess the potential for corrosive activity and skin irritation of the test material. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).
The present test is based on the experience that corrosive and irritant chemicals produce cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is designed to predict a skin corrosion or irritation potential of a chemical by using the three dimensional human epidermis model EpiDermTM. After application of the test material to the stratum corneum surface of the EpiDermTM 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 isopropanol-extraction 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 negative control values from tissues and expressed as relative tissue viability.

EXPERIMENTAL PROCEDURE
Mesh compatibility
For liquid test substances a nylon mesh can be used as a spreading support. To exclude a reaction of the test substance with the mesh, the compatibility of the test substance with the nylon mesh was checked in a pretest.
The test substance and the mesh are brought together on a slide and the reaction was observed after 60 minutes exposure, using a microscope. An interaction between test substance and the mesh was not noticed. However, it was judged that the use of a mesh was not necessary for the test substance.
Direct MTT reduction
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.
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below.
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.
In case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time (corrosion test) was treated with, each, the test article and the negative control, in the same way as described in section “Experimental procedure”, additionally.
In the irritation test concurrent testing of killed controls was considered in the case that direct MTT reduction occurred and visible residues of the test substance remained on the tissues after washing, only.

Basic procedure
Corrosion test:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6 - well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. In addition, one killed tissue per exposure time was treated with the test substance and NC, respectively, in order to detect direct MTT reduction.
Fifty microliter (50 μL) of the undiluted liquid test substance was applied using a pipette.
For better handling the highly viscous test substance was heated at ca. 40°C. Before application the test-substance was cooled down to room temperature.
Control tissues were concurrently treated with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 N potassium hydroxide (positive control, PC) or test substance (killed tissue control, KC).
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated 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. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 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 for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively.
In addition, three killed tissues were treated with the test substance and NC, respectively, in order to detect direct MTT reduction.
Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette.
For better handling the highly viscous test substance was heated at ca. 40°C. Before application the test-substance was cooled down to room temperature.
Control tissues were concurrently treated with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC) or test substance (killed tissue control, KC). A nylon mesh was placed carefully onto the tissue surface of the NC and PC controls afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
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. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced using PC based tabular calculation software. The mean and individual data were not always rounded but the significant digits were produced by changing the display format. As a consequence, calculation of mean values using the individual data presented in the report will, in some instances, yield minor variations in value.
Principle:
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues
treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or
not a test material is corrosive or irritant.
Calculation of individual and mean optical densities:
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the
respective individual tissue OD570 value. The mean OD570 for a test group of two tissues (corrosion test) or three tissues
(irritation test) treated in the same way is calculated.
Application of measurements using killed control tissues:
Killed tissues are primarily used for the corrosion test. However, as in the irritation test visible residues of the test
substance remained on the tissues after the washing procedure, a killed control was tested concurrently.
In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues
(KC) will be used to correct the mean OD570 of the testsubstance treated tissues (mean OD570 KC corrected). Since
killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by
subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than 0.1 it
is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected
represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the
test substance.
Tissue viability:
The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if
applicable) divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
Assay acceptance criterion for the negative control (NC):
The absolute OD570 of the negative control tissues in the MTTtest 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 ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
Assay acceptance criterion for the positive control (PC):
Corrosion test: Potassium hydroxide as 8.0 normal ready made solution is used as positive reference. A 3-minute treatment with 8.0 N KOH usually reveals a mean relative tissue viability of ~20%. An assay is acceptable if mean relative tissue viability of the 3 min positive control is ≤ 30%.
Irritation test: 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability
of ≤ 20% is acceptable.
Assay acceptance criterion for tissue variability:
For every treatment, except the killed controls, 2 tissues (corrosion test) or 3 tissues (irritation test) are treated in parallel. The inter-tissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is ≤ 0.3 (corrosion test) or if the SD of %-viability is ≤ 20 (irritation test).
Assay acceptance criterion for killed controls (KC):
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35.

EVALUATION OF RESULTS
The evaluation of the in vitro skin irritation potential of the test substance is based on the results of the Skin Corrosion Test (SCT) and the Skin Irritation Test (SIT).
If a test substance is not tested in both systems or an inconclusive result is obtained in one of the studies, the test strategy may still lead to an overall evaluation, when the result of a single study gives a clear prediction. However, if both studies are inconclusive or contradictory results are obtained a test evaluation may not be possible.
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
Decision criteria for evaluation of results of corrosion test
Mean tissue viability (% of negative control)
3 min: < 50
Prediction: Corrosive Optional Sub-category 1A
3 min: ≥ 50 and 1 hour: < 15
Prediction: Corrosive Optional Sub-category 1B and 1C
3 min: ≥ 50 and 1 hour: ≥ 15 Non-corrosive

According to the current OECD Guideline 431 a sub-categorization is possible based on the results. However, the sub-categorization into 1A is highly over-predictive as stated in the guideline and differentiation into sub-category 1B or 1C is not possible. If the test substance is identified to be corrosive by SCT and a transport classification is needed, the Corrositex® test should be performed, if applicable, to confirm classification as 1A or to differentiate between 1B and 1C.

Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
Decision criteria for evaluation of results of irritation test
Mean tissue viability (% of negative control)
≤ 50
Predition: Irritant
> 50
Prediction: Non-irritant

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: viability [% of NC]

Basis:

mean

Time point:

other: 3 min

Score:

93.4

Remarks on result:

other: corrosion, exposure: 3 min, mean OD570: 1.868

Results 2

Irritation parameter:

other: viability [% of NC]

Basis:

mean

Time point:

other: 1 hour

Score:

96.1

Remarks on result:

other: corrosion, exposure: 1 hour, mean OD570: 1.912

Results 3

Irritation parameter:

other: viability [% of NC]

Basis:

mean

Time point:

other: end of observation

Score:

100.4

Remarks on result:

other: irritation, mean OD570: 2.298

Irritant / corrosive response data:

Based on the observed results and applying the evaluation it was concluded, that Diethylphosphonatopropyltrimethoxysilan does not show a skin
irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU