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

Endpoint summary

Description of key information

Under the conditions of the studies performed the following outcomes were determined for SABoTBA:
Skin irritation: predicted to be non-irritant to the skin
Eye irritation: predicted to be  non-corrosive/non-severe irritant to the eye

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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)

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.

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.

Irritation / corrosion parameter:

% tissue viability

Value:

97.2

Remarks on result:

no indication of irritation

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-16 May 2013 (in-life dates)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline (OECD 437) compliant study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The bovine eyes, supplied by Joseph Morris Abattoir, were excised by an abattoir employee and collected as soon after slaughter as possible (excised at 12.40 hours, 14 May 2013). Instructions were given to avoid damaging the corneas during excision. Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) penicillin/streptomycin solution, to cover all the eyes in the receptacle. The eyes were used within 4 hours of slaughter (incubation of mounted corneas commenced at 14.41 hours, 14 May 2013).

Vehicle:

physiological saline

Controls:

no

Amount / concentration applied:

750 µL of a 20% w/w solution in 0.9% sodium chloride

Duration of treatment / exposure:

4h (± 5 min)

Number of animals or in vitro replicates:

3 corneas per treatment group

Details on study design:

Preparation of corneas
All eyes were carefully examined, macroscopically, for defects (opacity, scratches, pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea dissected leaving approximately 2 to 3 mm of sclera present around the cornea. The isolated corneas were stored in a petri dish containing HBSS plus 1% penicillin/streptomycin solution until all the corneas had been dissected. Once all the corneas had been dissected, they were rinsed in fresh HBSS plus 1% penicillin/streptomycin solution prior to mounting.
The corneas were mounted in the cornea holders with the endothelial side against the O-ring of the posterior half of the holder. Each cornea was gently flattened over the O-ring and holder surface with a wetted, gloved finger to expel any air. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with HBSS plus 1% penicillin/streptomycin, using a syringe. The posterior compartment was always filled first to return the cornea to its natural shape. Care was taken to ensure no air bubbles were present within the holders. The holders were then plugged and incubated, in an upright position, overnight at room temperature (approximately 20°C).
After overnight incubation at room temperature (approximately 20°C) the HBSS plus 1% penicillin/streptomycin was removed from both chambers. Both chambers were filled with cMEM (posterior chamber first), taking care to exclude air bubbles and plugged. The corneas were then incubated in the upright position for 60 minutes ± 5 minutes at 32°C± 1°C in a waterbath. The waterbath temperature remained within the limits of 32°C ± 1°C throughout the experiment.
At the end of the 60 minute incubation period, the medium was removed from both the anterior and posterior compartments using a pipette tip attached to a vacuum pump. The compartments were refilled with fresh cMEM (posterior chamber first). Again care was taken to ensure no air bubbles were present within the holders. The posterior compartment was then plugged and the basal opacity measurements performed.

Opacity measurement
The opacitometer measured the light transmission through the centre of each mounted cornea, displaying a numerical opacity value (arbitrary unit). The opacity of each cornea was measured by reading each holder in the right-hand chamber of the calibrated opacitometer. Corneas with an opacity value greater than 7 units were discarded. The mean basal opacity value of the corneas was then calculated and three corneas with opacity values close to the mean value were chosen for use as negative control corneas.

Treatment groups
Corneas were treated in triplicate with either the test substance, positive control (imidazole) or negative control (0.9% sodium chloride solution).

Treatment of corneas
The test substance, SABoTBA, a white crystalline powder, and the positive control material (imidazole) were tested at 20% (w/w) in 0.9% sodium chloride solution. The test substance was mixed during preparation using a mortar and pestle. The 0.9% sodium chloride solution was added to achieve the desired concentration.
Immediately prior to treatment, the medium was removed from the anterior compartment of the holder using a pipette tip attached to a vacuum pump, taking extra care to ensure all excess liquid had been removed. Seven hundred and fifty µL (750 µL) of test substance, negative control (0.9% sodium chloride solution) or positive control material (imidazole 20% w/w solution) was introduced in to the anterior part of the holder. The test substance at 20% w/w formed a white suspension and was added to the anterior compartment using a 1 mL syringe via the top port. Following application the anterior compartment was plugged. The holder was slightly rotated to ensure uniform distribution of the test substance over the surface of the cornea. Each holder was incubated in a horizontal position at 32°C ± 1°C for 4 hours ± 5 minutes in a waterbath.
Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the cornea washed, at least three times or until the wash medium (EMEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on the top of the holder. After each wash, the wash medium was removed using a pipette tip attached to a vacuum pump. The corneas were gently rinsed with the wash medium until the medium was clear and the colour unchanged. The corneas treated with the test substance required 7 washes. Following completion of the wash step, the medium was removed from both compartments and re-filled with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score.
Throughout the assay the corneas were examined for opaque spots or other irregularities.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Summary of results

Sample	Opacity ± SD	Permeability ± SD	In vitro irritancy Score ± SD	In vitro classification
SABoTBA	1.000 ± 1.000	0.025 ± 0.038	1.4 ± 1.5	Non-corrosive/Non-severe irritant
Imidazole	126.333 ± 24.583	3.212 ± 0.563	174.5 ± 17.6	Corrosive/Severe irritant
0.9% saline	0.000 ± 0.000	0.078 ± 0.004	NA	NA

   

Assay validity

The positive control, imidazole,elicited an In Vitro Irritancy Score of 174.5. This value was within the historical range (mean ± 2 x SD 126.0 – 185.4) for the assays performed to date. 

 The negative control, 0.9% saline, opacity mean change value was 0.000 which was below the maximum acceptance value of 3.0. The permeability mean of the negative control was 0.078 which was below the maximum acceptance value of 0.1.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance, SABoTBA, elicited an In Vitro Irritancy Score of 1.4 ± 1.5 and was predicted to be a non-corrosive/non-severe eye irritant.

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular irritancy potential in vitro of the test substance, SABoTBA. Imidazole was tested in parallel as a positive control.

The assay used isolated bovine corneas to assess the ocular corrosivity or severe irritancy potential of the test substance in vitro. The isolated corneas were obtained as a by-product of the meat production industry. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score which was used to classify the test substance as potential eye irritants according to OECD Guideline 437.

The test substance,SABoTBA, elicited an In Vitro Irritancy Score of 1.4 ± 1.5 and was predicted to be a non-corrosive/non-severe eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation

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.

Eye irritation

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular irritancy potential in vitro of the test substance, SABoTBA. Imidazole was tested in parallel as a positive control.

The assay used isolated bovine corneas to assess the ocular corrosivity or severe irritancy potential of the test substance in vitro. The isolated corneas were obtained as a by-product of the meat production industry. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score which was used to classify the test substance as potential eye irritants according to OECD Guideline 437.

The test substance,SABoTBA, elicited an In Vitro Irritancy Score of 1.4 ± 1.5 and was predicted to be a non-corrosive/non-severe eye irritant

Justification for selection of skin irritation / corrosion endpoint:
Key study, GLP/Guideline compliant study

Justification for selection of eye irritation endpoint:
Key study, GLP/Guideline compliant study

Justification for classification or non-classification

In accordance with EU CLP (Regulation (EC) No. 1272/2008), classification is not required for skin/eye irritation based on the available data.