N,N-bis(2-hydroxyethyl)glycine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2016 - 17 January 2017

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)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-hydroxyethyl)glycine

EC Number:

205-755-1

EC Name:

N,N-bis(2-hydroxyethyl)glycine

Cas Number:

150-25-4

Molecular formula:

C6H13NO4

IUPAC Name:

N,N-bis(2-hydroxyethyl)glycine

impurity 1

Reference substance name:

N-(2-hydroxyethyl)ethylenediaminetriacetic acid

EC Number:

205-759-3

EC Name:

N-(2-hydroxyethyl)ethylenediaminetriacetic acid

Cas Number:

150-39-0

Molecular formula:

C10H18N2O7

IUPAC Name:

N-{2-[bis(carboxymethyl)amino]ethyl}-N-(2-hydroxyethyl)glycine

impurity 2

Reference substance name:

Not specified

IUPAC Name:

Not specified

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid: crystalline

Details on test material:

Appearance: white crystalline powder
Storage conditions: at room temperature protected from light

Specific details on test material used for the study:

Stability at higher temperatures: stable, maximum temperature: 40 °C, maximum duration: 2 days
pH: 4-5 (10% solution)

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µl of a 20% (w/v) solution of test substance prepared in physiological saline per cornea

NEGATIVE CONTROL
- Amount applied: 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount applied: 750 µl 20% (w/v) Imidazole solution prepared in physiological saline

Duration of treatment / exposure:

240 ± 10 minutes

Number of animals or in vitro replicates:

Test item: 3
Negative control: 3
Positive control: 3

Details on study design:

TEST SITE
- Isolated bovine cornea, obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

REMOVAL OF TEST SUBSTANCE
- Washing: yes, three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Life Technologies)
- Time after start of exposure: 240 minutes

SCORING SYSTEM:
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

TOOL USED TO ASSESS SCORE:
- opacitymeter (OP-KIT) and microplate reader (TECAN Infinite® M200 Pro Plate Reader).

DATA EVALUATION:
A test substance that induces an IVIS = 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgegory 1);
For a test substance that induces an IVIS >3 and = 55, no prediction on irritant potency can be made.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

Mean of 3 replicates

Run / experiment:

1st experiment

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(2.4)

Positive controls validity:

valid

Remarks:

(159.7)

Remarks on result:

other: Individual scores were spread over 2 categories

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

Mean of 3 replicates

Run / experiment:

2nd experiment

Value:

3.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(0.2)

Positive controls validity:

valid

Remarks:

(129.9)

Remarks on result:

other: Individual scores were spread over 2 categories

Other effects / acceptance of results:

- In the first experiment the individual IVIS for the negative controls were 2.0-2.8. The 3 cornea were slightly translucent but since all opacity and permeability values were within the historical data control range this had no effect on the study. Individual IVIS of the positive control were 143-184, the corneas were turbid after 240 minutes of treatment. The corneas treated with test item showed opacity scores of 1.6-6.4 and permeability values of -0.030 - 0.061. The corneas were clear after 240 minutes of treatment. A pH effect of the test item was observed on the rinsing medium. Therefore the corneas were rinsed until no colour change of the medium was observed. Hence, the IVIS ranged from 1.3-5.9 after 240 minutes of treatment. Because the individual IVIS were spread over 2 categories, the test was inconclusive and a second experiment was performed.
- In the second experiment the individual IVIS for the negative controls were -0.6-0.9 and for the positive control 106-175. The corneas treated with the positive control were turbid after 240 minutes of treatment. The corneas treated with test item showed opacity values of 1.0-5.0 and permeability values of 0.000-0.013. The corneas were slightly translucent after the 240 minutes of treatment. In addition, two corneas showed a spot. a pH effect of the test item was observed. Hence, the IVIS ranged from 1.2 to 5.2 after 240 minutes if treatment. Since individual IVIS were spread over 2 categories, the test was inconslusive.
- Proper functioning of the test system in both experiments was shown by the results from the positive and negative controls, which were within the historical data range. Therefore, the acceptability criteria were met and the test was considered to be valid.
- Since both experiments provided inconclusive results, no conclusion can be drawn for eye irritation or serious eye damage.

Any other information on results incl. tables

Table 1 First experiment: Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	1.8	0.037	2.4
Positive control	135.9	1.588	159.7
Bicin	3.2	0.001	3.3

¹Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

²In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Table 2 Second experiment: Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	0.1	0.004	0.2
Positive control	109.3	1.379	129.9
Bicin	3.2	0.009	3.4

¹Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

²In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Applicant's summary and conclusion

Interpretation of results:

other: Results were spread in two categories and were therefore inconclusive.

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline and GLP principles, no conclusion can be drawn for eye irritation of the substance. The mean IVIS in two experiments were 3.3 and 3.4, but individual scores were spread over two categories.