Ethyltris(2-hydroxyethyl)ammonium ethyl sulphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltris(2-hydroxyethyl)ammonium ethyl sulphate

EC Number:

250-796-0

EC Name:

Ethyltris(2-hydroxyethyl)ammonium ethyl sulphate

Cas Number:

31774-90-0

Molecular formula:

C6H15NO3 (TEA) C16H37NO7S (Triethoxy-TEA ethylsulfate)

IUPAC Name:

ethyltris(2-hydroxyethyl)azanium ethyl sulfate

impurity 1

Reference substance name:

2,2',2''-nitrilotriethanol

EC Number:

203-049-8

EC Name:

2,2',2''-nitrilotriethanol

Cas Number:

102-71-6

Molecular formula:

C6H15NO3

IUPAC Name:

2,2',2''-nitrilotriethanol

Test material form:

solid: bulk

Details on test material:

Name: Ethyltris(2-hydroxyethyl)ammonium ethyl sulphate
Product Description: Triethanolamine DES Quat
CAS No.: 31774-90-0
Physical state: white solid at 20 °C
Batch No.: PFS-755-175
Re-certification date of batch: 21 April 2018
Purity: 100 % (mono constituent substance, water content 0.33 % (w/w))
pH, 5% in water 7.85
Acid Value , mg KOH/g 38.45
Moisture, % 0.33
Total Amine, mg/g 33.10
Viscosity,cps, #4@60,25C 1580
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Specific details on test material used for the study:

Name: Ethyltris(2-hydroxyethyl)ammonium ethyl sulphate
Product Description: Triethanolamine DES Quat
CAS No.: 31774-90-0
Physical state: colourless to yellow viscous liquid at 20 °C
Batch No.: PFS-755-175
Re-certification date of batch: 21 April 2018
Purity: 100 % (UVCB, water content 0.33 % (w/w))
pH, 5% in water 7.85
Acid Value , mg KOH/g 38.45
Moisture, % 0.33
Total Amine, mg/g 33.10
Viscosity,cps, #4@60,25C 1580
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Preparation of the 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.

Test Groups:

- 3 corneas for the test item
- 3 corneas as negative controls treated with physiological saline 0.9% NaCl
- 3 corneas as positive controls treated with ethanol 100%

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of the test substance or the control substance was introduced into the anterior chamber. As the viscosity of the test item was relatively high, it was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment. After 10 minutes incubation at 32 +- 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

Duration of treatment / exposure:

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. 750 µL of the test substance or the control substance was introduced into the anterior chamber. As the viscosity of the test item was relatively high, it was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment. After 10 minutes incubation at 32 +- 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber expect of cornea no. 8 was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 +- 1 °C. Also, each cornea was observed visually and pertinent observations were recorded.

Duration of post- treatment incubation (in vitro):

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 4 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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

2.29

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Validity

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.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Any other information on results incl. tables

Results

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay. The test item was tested as provided by the sponsor. None of the 3 corneas treated with the test item showed any opacity of the tissue. The anterior chamber of cornea no. 8 was not refilled with complete RPMI between the first and second measurement of the opacity. Certainly the scores of the opacity measurement as well of the permeability measurement of cornea no. 8 are comparable with the scores of the other corneas. The following mean in vitro irritation score was calculated: 2.29

Therefore the test item was classified into UN GHS No Category.

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.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Table 1: In Vitro Irritation Score

Cornea No.	Test item	Corrected opacity	Corrected OD490 value	IVIS
1	Negative control	0.07	0.027	-
2	Negative control	-1.55	0.018	-
3	Negative control	-0.37	0.002	-
MV	Negative control	-0.61	0.016	-0.38
4	Positive control	27.88	1.227	-
5	Positive control	26.10	1.829	-
6	Positive control	27.04	2.249	-
MV	Positive control	27.01	1.769	53.54
7	Test item	1.86	-0.005	-
8	Test item	1.64	-0.007	-
9	Test item	3.58	-0.003	-
MV	Test item	2.36	-0.005	2.29

MV = mean value

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to the evaluation criteria the test item is classified into UN GHS No Category.

Executive summary:

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay. The following mean in vitro irritation score was calculated to 2.29.

Therefore the test item was classified into UN GHS No Category.