N,N-dibutylbutan-1-aminium bis[2-(hydroxy-kO)benzoato(2-)-kO]borate(1-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 to 13 May 2013 (in-life dates)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline (OECD 439) compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-dioxo-4H,4'H-2,2'-spirobi[[1,3,2]benzodioxaborinin]-2-uide; tributylazanium

EC Number:

700-906-2

Cas Number:

22450-96-0

Molecular formula:

C26H36BNO6

IUPAC Name:

4,4'-dioxo-4H,4'H-2,2'-spirobi[[1,3,2]benzodioxaborinin]-2-uide; tributylazanium

Constituent 2

Reference substance name:

SABoTBA

IUPAC Name:

SABoTBA

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): SABoTBA
- Purity: 98.77%
- Purity test date: 08 April 2014
- Lot/batch No.: 12342
- Expiration date of the lot/batch: Stable under recommended storage conditions
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

human

Strain:

other: human epidermis skin constructs

Details on test animals or test system and environmental conditions:

The 'EPISKIN™ Skin Irritation Test 15 Min - 42 Hours' using human epidermis skin constructs, supplied by SkinEthic Laboratories, Lyon, France, has been accepted as a replacement to the in vivo test, Draize Skin Irritation Test, by the European Centre for the Validation of Alternative Methods (ECVAM, 2007). The test was conducted in accordance with the Standard Operating Procedure, In Vitro Skin Irritation Test: Human Epidermis Model (L’Oreal 2009), supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM) and the OECD 439 Guideline For The Testing of Chemicals: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (OECD 439).
The test involves the application of the test substance for 15 minutes to the EPISKIN™ three-dimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38cm2. The EPISKIN™ kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.
The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell death in the cell layers. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model (OECD 439) uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

Test system

Type of coverage:

open

Preparation of test site:

other: surface wetted prior to application

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2mg was dispensed over each tissue

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 µL of purified water prior to application of the test substance.

Duration of treatment / exposure:

15 min (±0.5min) contact time with the test substance, followed by rinsing and then 42 h (±1 h) incubation time at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

Details on study design:

Tissue
The tissues are received as a kit, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 13 May 2013). The maintenance medium was pre-warmed to 37ºC. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

Application
A weight of 10 ± 2mg was dispensed over each tissue, the tissues were wetted with 5 µL of purified water prior to application of the test substance. The positive (5% Sodium Dodecyl Sulphate (SDS) in purified water) and negative (sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium) controls were in liquid form and were applied by dispensing a volume of 10 µL over each tissue using a positive displacement pipette.

Procedure
After incubation for at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance, negative or positive control at room temperature. The test substance was spread over the surface of the tissues with a curved spatula. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 µL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes (actual time 8 minutes) application time.
After 15 ± 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbecco’s Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT ((3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide)) and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
At the end of 3 hours ± 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube.
When all tissues had been punched, the tissues were vortexed with 500 µL of acidic isopropanol (0.04 N HCl final concentration).
The tissues were extracted by storing at 2-8 ºC, protected from light, for 48 - 70 hours.
After formazan extraction, duplicate 200 µL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

97.2

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table 1: EPISKIN study data

Sample	Tissue replicate	Optical Density (OD)	OD - blank	% Negative Control
Negative control	a	1.071 1.054	0.927 0.910	100.6
	b	1.059 1.018	0.915 0.874	97.9
	c	1.087 1.055	0.942 0.911	101.5
		Mean SD	0.913 0.023	100.0 1.8
Positive control	a	0.569 0.542	0.425 0.398	45.0
	b	0.431 0.445	0.286 0.301	32.2
	c	0.510 0.470	0.366 0.326	37.9
		Mean SD	0.350 0.055	38.4 6.5
SABoTBA	a	1.072 1.027	0.928 0.883	99.1
	b	1.014 0.952	0.870 0.808	91.9
	c	1.093 1.033	0.949 0.888	100.6
		Mean SD	0.887 0.049	97.2 4.7
Blank		0.135 0.149 0.150 0.140 0.145 0.146
	Mean SD	0.144 0.006

The mean Optical Density (OD) for the six replicate blanks was subtracted from the individual substance and control tissues OD. 

The viability of each tissue was expressed as a percentage of the mean negative control value.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that the test substance, SABoTBA, with a mean tissue viability of 97.2 ± 4.7%, was predicted as non-irritant to the skin.

Executive summary:

The study was performed to assess the skin irritation potential, in vitro, of the test substance,SABoTBA in accordance with the OECD Guideline 439.

The test substance was applied to EPISKIN^™human epidermis skin constructs. The constructs consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5‑diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model (OECD Guideline 439) uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

The test substance,SABoTBA, elicited a mean tissue viability of 97.2 ± 4.7% and was predicted as non-irritant to the skin.