1,4:3,6-dianhydro-2,5-bis-O-(diphenoxyphosphoryl)-D-glucitol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-01-31 - 2011-05-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals No. 431, April 13, 2004 (“In vitro Skin Corrosion: Human Skin Model Test”)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals No. 439, July 22, 2010 (“In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,4:3,6-dianhydro-2,5-bis-O-(diphenoxyphosphoryl)-D-glucitol

EC Number:

935-211-5

Cas Number:

1305113-15-8

Molecular formula:

C30H28O10P2

IUPAC Name:

1,4:3,6-dianhydro-2,5-bis-O-(diphenoxyphosphoryl)-D-glucitol

Details on test material:

- Name of test material (as cited in study report): Isosorbid-O,O'-bis(diphenylphosphorsäureester); Lab test item number: 10/0230-3
- Physical state: solid / white
- Analytical purity: 99.7 mol% (NMR-spectroscopy)
- Lot/batch No.: 10264/10/006
- Stability under test conditions: the stability under storage conditions over the study period was guaranteed.
- Storage condition of test material: room temperature
- Other: the test item was homogeneous by visual inspection
- Other: pH-value: ca. 4 (undiluted test substance, moistened with water).

Test animals

Species:

other: not applicable; in vitro test

Strain:

other: not applicable; in vitro test

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- The test system (target tissue): three dimensional human epidermis model.
The EpiDerm™ model (Epi-200) 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 EpiDem™ 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.
- Supplier: MatTek Corp., Ashland MA, USA.

Test system

Type of coverage:

open

Preparation of test site:

other: in vitro test (direct application)

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro test; 50 µL highly de-ionized water (corrosion test); 50 µL PBS, sterile (irrritation test) were used as negative controls (NC)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): first, 25 μL highly de-ionized water was applied. Thereafter, a bulk volume of 25 μL of the solid test material (ca. 19 mg) was applied with a sharp spoon and homogeneously distributed with the water.

Duration of treatment / exposure:

3 min or 1 h (corrosion test); 1 h (irritation test)

Observation period:

until all tissues per application time were dosed (corrosion test); 24 ± 2 h [incubation] + 18 ± 2 h [postincubation] (irritation test)

Number of animals:

2 tissues (corrosion test) or 3 tissues (irritation test) were used per treatment, the test substance, the negative control (NC) and the positive control (PC).

Details on study design:

TEST PROCEDURE
- Direct MTT reduction: the test substance was added to the MTT solution, and the mixture was incubated in the dark at about 37°C for 55 to 65 minutes. A negative control (highly de-ionized water) was tested concurrently. If direct MTT reduction occurred, one freeze-killed control tissues per exposure time was treated with, each, the test article and the negative control, in the same way as described below, additionally.
- Pre-incubation of the tissues: 1-1.5 h (corrosion test) or 18 ± 3 h (irritation test) in assay medium at 37°C.

1) Corrosion test
- Pretreatment of the tissues and treatments: after the pre-incubation the tissues were pre-treated with 25 μL highly de-ionized water to wet the tissue surface. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water. Control tissues were concurrently applied with 50 μL of highly de-ionized water (NC) or with 50 μL of 8-n potassium hydroxide (PC).
- Removal of the test substance and post-incubation period: 3 min or 1 hour thereafter, the residual test items were washed out with PBS and 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.

2) Irritation test
- Pretreatment of the tissues and treatments: after the pre-incubation the tissues were pre-treated with 25 μL highly de-ionized water to wet the tissue surface. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water. Control tissues were concurrently applied with 30 μL of PBS (NC) or with 30 μL of 5% SDS (PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
- Removal of the test substance and post-incubation period: 1 hour thereafter, the residual test items were washed out with PBS and rinsed tissues were kept in 24-well plates at room temperature on assay medium, incubated for 24 ± 2 h at 37°C, transferred to fresh medium and further incubated at room temperature for 18 ± 2 h (postincubation).

3) Corrosion and irritation tests
- MTT incubation: the assay medium was replaced by MTT solution and the tissues were incubated for 3 hours. The tissues were then washed with PBS to stop the MTT-incubation.
- Determination of optical density: 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 6 microtiter wells filled with isopropanol for each microtiter plate.

DATA EVALUATION
- 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 treated in the same way is calculated.
- Tissue viability: the quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
- The absolute OD570 of the NC tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory 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.
- For corrosion test potassium hydroxide as 8-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%. For 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.
- For every treatment 2 tissues (corrosion test) or 3 tissues (irritation test) were 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).

EVALUATION CRITERIA:
- In the corrosion test 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%.
- In the irritation test a chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

POSITIVE CONTROL
- 8-n potassium hydroxide solution for the corrosion test;
- 5% (w/v) sodium dodecyl sulfate (SDS) in highly de-ionized water, sterile for the irritation test.

HISTORICAL CONTROL DATA
Historical control values of negative and positive controls, gathered over an appropriate time period, were available. These data demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria for the test system.

Results and discussion

In vivo

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria, the test substance does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Any other information on results incl. tables

Table 1: Summary of findings

Test item		Corrosion test (individual values)		Irritation test ± SD
		3 min exposure	1 h exposure	1 h exposure
Negative control	Mean OD570	1.804 (1.787, 1.821)	1.680 (1.648, 1.712)	1.890 ± 0.118
	Mean viability (% of NC)	100 (99.1, 100.9)	100 (98.1, 101.9)	100 ± 6.24
Test substance	Mean OD570	1.862 (1.858, 1.866)	1.789 (1.794, 1.783)	1.888 ± 0.140
	Mean viability (% of NC)	103 (103.0, 103.4)	106 (106.8, 106.1)	100 ± 7.40
Positive control substance	Mean OD570	0.368 (0.397, 0.339)	0.162 (0.161, 0.163)	0.123 ± 0.011
	Mean viability (% of NC)	20 (22.0, 18.8)	10 (9.6, 9.7)	7 ± 0.59
SD: standard deviation

 

- All data were in the range of the historical control data.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS