bis[C11-14-(branched and linear)-alkyl]aminium nonadecaoxohexatungstate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22.12.2011 - 06.04.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted according to OECD Guideline under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 22 July 2010).

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate

IUPAC Name:

reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate

Test material form:

liquid: viscous

Details on test material:

- Molecular formula (if other than submission substance): [R2NH2]2[W6O19];R=C11-C14alkyl (branched and linear) * 4R2NH
- Physical state: Clear yellow viscous liquid (determined at NOTOX)
- Analytical purity: UVCB
- Lot/batch No.: PB-39-131
- Expiration date of the lot/batch: 01 December 2012 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

other: adult human-derived epidermal keratinocytes.

Details on test animals or test system and environmental conditions:

Not applicable

Test system

Controls:

other: Not applicable

Amount / concentration applied:

Not applicable

Duration of treatment / exposure:

Not applicable

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

Test substance preparation
The test substance has a sticky consistence and was applied using a cotton swab directly on top of the skin tissue. The test substance was spread to match the size of the tissue.

Reference substances

Negative control:
Phosphate buffered saline (PBS, Invitrogen Corporation,, The Netherlands).
 
Positive control:
5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands) [CAS Number 151-21-3] in PBS.

Test system

EPISKIN Small Model (TM) (EPISKIN-SM(TM), 0.38 cm2, Batch no.: 12-Ekin-011).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Source: SkinEthic Laboratories, Lyon, France.

Preparation and preincubation

Tissue
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37 degrees Centigrade. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Nice, France.

Killed tissue (EPISKIN-SM)TM), 0.38 cm2, Lot no.: 12 Ekin-007, received: 14 February, 2012)
Living epidermis was transferred to 12-well plates and incubated with 2 ml Milli-Q for 48 +/- 1 hours. After incubation, killed epidermis was stored at <= 15 degrees Centigrade. Killed tissues were thawed by placing them for 1 hour at room temperature in 12-well plateson 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.

MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10 x) in Assay medium (final concentration of 0.3 mg/ml).

Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 75 - 97%), containing 5.0 +/- 0.5% CO2 in air in the dark at 37.0 ± 1.0 degrees Centigrade (actual range 35.2 - 37.6 degrees Centigrade). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.2 - 36.0 degrees Centigrade) and humidity (with a maximum of 5%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Study design

Test for reduction of MTT by the test substance
The test substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 21 mg of the test substance was added to a 12 well plate filled with 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37 degrees Centigrade. A negative control, sterile Milli-Q water was tested concurrently.

In case the test substance reacts with the MTT medium in addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues must be used for the cytotoxicity evaluation with MTT.

Application/Treatment of the test substance
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The undiluted test substance was added (using a cotton swab) into 12-well plates on top of the skin tissues. Due to the sticky consistence of the test substance, dead tissue was separated from the collagen matrix and the skin was damaged. Three tissues were treated with 25 microL PBS (negative control) and 3 tissues with 25 microL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 44 hours at 37 degrees Centigrade.

Cell viability measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37 degrees Centigrade. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: tissue viability

Value:

125

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 100.0. Remarks: scores as percentage relative to negative control (100%). (migrated information)

In vivo

Irritant / corrosive response data:

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that the test substance did interact with MTT.

In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test substance was 5% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

Any other information on results incl. tables

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 125%. Since the mean relative tissue viability for the test substance was above 50% the test substance is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 5%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Mean tissue viability with test substance

	Mean tissue viability (percentage of control)
Negative control	100
Test substance	125
Positive control	5

Individual OD measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	0.950 0.989	0.983 1.010	1.012 1.043
Test substance on viable tissue OD570measurement 1 OD570measurement 2	0.987 1.539	1.116 1.346	1.264 1.483
Test substance on killed tissue OD570measurement 1 OD570measurement 2	0.104 0.204	0.130 0.127	0.116 0.156
Non treated killed tissue OD570measurement 1 OD570measurement 2	0.073 0.104	0.072 0.113	0.075 0.085
Positive control OD570measurement 1 OD570measurement 2	0.087 0.093	0.081 0.107	0.084 0.099

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an OECD 439 study (In Vitro Skin Irritation: Reconstituted Human Epidermis Test Method), conducted according to GLP, reaction mass of bis(C11-14-alkyl, branched and linear)amine nonadecaoxo hexatungstate (the surrogate substance) is a non-irritant (Notox B.V., 2012).