Dimethylamine-borane (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-24 to 2017-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylamine-borane (1:1)

EC Number:

200-823-7

EC Name:

Dimethylamine-borane (1:1)

Cas Number:

74-94-2

Molecular formula:

C2H10BN

IUPAC Name:

N-Methylmethanamine-borane (1:1)

Test material form:

solid: crystalline

Details on test material:

CAS: 74-94-2
Description: white crystalline solid
Batch No.: 1609024
Purity: 99.33 %
Expiry Date: 14 Sep 2017

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +5 °C, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test item was suspended with physiological saline 0.9% NaCl (Alleman Pharma, lot no. 609709, expiry date: 2017-03-03) to give a 20% concentration, afterwards it was vortexed prior to administration.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES:
- The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20%

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): physiological saline 0.9% NaCl
- Lot/batch no. (if required): Alleman Pharma, lot no. 609709

Duration of treatment / exposure:

4 hours ± 5 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

Three corneas each for the test item, negative control (physiological saline 0.9% NaCl) and positive control (imidazole 20% in physiological saline 0.9% NaCl).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32  1 °C.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Washing with MEM containing phenol red until the medium was free of test substance (at least 3 times), then completely rinsed with RPMI without phenol red.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. After the 4 hours incubation period and subsequent washing the anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490):
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 +/-1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in Table 1 (see "Any other information on materials and methods").
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.
For this purpose further testing with another suitable method is required

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

101.13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid

Any other information on results incl. tables

Table 2: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control Physiological Saline	2.01	2.46	0.54
2		2.23	2.62	0.38
3		1.75	3.32	1.58
MV		2.00	2.80	0.80
4	Positive Control 20% Imidazol in physiological saline	2.54	105.91	103.36	102.56
5		2.54	101.87	99.33	98.52
6		2.66	84.64	81.98	81.18
MV		2.58	97.47	94.89	94.09
7	Test Item dimethylamine-borane (20% in physiological saline)	1.60	24.25	22.65	21.84
8		0.32	60.75	60.43	59.63
9		0.88	42.62	41.74	40.94
MV		0.93	42.54	41.61	40.80

Table 3: Permeabilty

Cornea No.	Test Item	OD490	Corrected OD490
1	Negative Control Physiological Saline	0,007
2		0,009
3		0,018
MV		0,011
4	Positive Control 20% Imidazol in physiological saline	3,060	3,049
5		4,660	4,649
6		4,235	4,224
MV		3,985	3,974
7	Test Item dimethylamine-borane (20% in physiological saline)	0,000	-0,011
8		5,610	5,599
9		6,490	6,479
MV		4,033	4,022

Table 4: In vitro Irritation Score (IVIS)

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control Physiological Saline	0.45	0.007	0.97
2		0.38	0.009
3		1.58	0.018
MV		0.80	0.011
4	Positive Control 20% Imidazol in physiological saline	102.56	3.049	153.69
5		98.52	4.649
6		81.18	4.224
MV		94.09	3.974
7	Test Item dimethylamine-borane (20% in physiological saline)	21.84	-0.011	101.13
8		59.63	5.599
9		40.94	6.479
MV		40.80	4.022

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In conclusion, based on the mean in vitro irritation score of 101.13 obtained in the bovine corneal opacity and permeability assay (OECD 437), the target substance is classified for irreversible effects on the eye (Category 1, H318).

Executive summary:

The eye irritation potential of dimethylamine-borane (DMAB) (> 99% purity) was investigated in the bovine corneal opacity and permeability assay (OECD 437). The test item was suspended with physiological saline 0.9 % NaCl to gain a 20 % concentration. A mean in vitro irritation score (IVIS) of 101.13 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. Based on the results, classification for irreversible effects on the eye (Category 1, H318) is warranted.