2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol hydrochloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-06-22

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

08 December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

EC Number:

230-237-7

EC Name:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Cas Number:

6976-37-0

Molecular formula:

C8H19NO5

IUPAC Name:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Test material form:

liquid

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.4 ºC to 20.2 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Time interval prior to initiating testing: 2 h
- Indication of any existing defects or lesions in ocular tissue samples: After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g (per eye)

CONTROLS
- Amount applied: 0.03 g imidazole; 30 µL NaCl (in saline)

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse (minor variations +/- 5 minutes were considered acceptable).
Cornea thickness and opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and at 30 min.

Duration of post- treatment incubation (in vitro):

4 hours as observation period

Number of animals or in vitro replicates:

3 eyes (test substance and positive control)
1 eye (negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES:
With the eyes still in the chicken heads, eyelids were carefully cut away with scissors from the chicken eyes. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and then rinsed off with 20 mL isotonic saline. The fluorescein-treated cornea was examined with a hand-held slit lamp or a slit lamp microscope. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye, which may lead to corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box to maintain the appropriate humidity.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a stell clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head), again avoiding too much pressure on the eye. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approx. 3 to 5 drops/min. The door of the chamber was closed, except for manipulations and examination, to maintain temperature and humidity.
Eyes selected for testing were again examined with the slit lamp microscope to ensure that they were in good condition. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Thorough rinsing with approx. 20 mL saline solution at ambient temperature after 10 seconds of treatment. Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30, 75 and 120 of observation. All test item treated eyes were totally cleared at 180 minutes after the post-treatment rinse.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity scores ranging from 0 (no opacity) to 4 (complete opacity; iris invisible) according to the test guideline were measured at all time points
- Damage to epithelium based on fluorescein retention: Fluorescein retention scores ranging from 0 - 3 according to the test guideline were measured at 0 and 30 min.
- Swelling: Cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Strei BQ 900) with the slit-width set at 9.5, i.e. 0.095mm.
- Macroscopic morphological damage to the surface: not applicable
- Others (e.g, histopathology): not performed

SCORING SYSTEM:
Following scores were measured accoring to test guideline
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: Decision criteria as indicated in the TG were used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

mean (240 min)

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

28

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

4

Results 3

Irritation parameter:

fluorescein leakage

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

3

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Positive and negative control values were within the corresponding historical control data ranges.

Any other information on results incl. tables

Table 1: Results for the test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	1%	I
Mean maximum corneal swelling at up to 240 min	3%	I
Mean maximum corneal opacity	1.0	II
Mean fluoresin retention	1.0	II
Other Observations	None
Overall ICE class	1xI, 2xII

 

Table 2: Results for positive control Imidazole

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	21%	III
Mean maximum corneal swelling at up to 240 min	28%	III
Mean maximum corneal opacity	4.0	IV
Mean fluoresin retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE class	1xIII, 2xIV

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1. 

 

Table 3: Results for the negative control NaCl (9 g/L saline)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I
Mean maximum corneal opacity	0.0	I
Mean fluoresin retention	0.0	I
Other Observations	None
Overall ICE class	3xI

 

Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Positive and negative control values were within the corresponding historical control data ranges.

 

Table 4: Historical control data of Positive control (Period of 2011 - 2015)

IMIDAZOLE HISTORICAL CONTROL Dose level: 30 mg / eye

n=168	Relative obobservation time (m(min)	Corneal thickness					Corneal opacity score						Fluorescein retention
		30	75	120	180	240		30	75	120	180	240		∆FR
Maximium	swelling (%):	34	45	49	54	44	Max. OS:	4.0	4.0	4.0	4.0	4.0	Max. FR:	3.0
Minimum swelling (%):		3	9	12	14	15	Min. OS:	2.8	3.3	3.5	3.5	3.5	Min. FR:	2.7
Average:		19	27	31	35	37	Average:	3.7	3.9	3.9	3.9	3.9	Average:	3.0

 

Table 5: Historical control data of Negative control (Period of 2011 - 2015)

NaCl (9 g/L saline) SOLUTION HISTORICAL CONTROL Dose level: 30 µL / eye

n=120	Relative obobservation time (m (min)	Corneal thickness					Corneal opacity score						Fluorescein retention
		30	75	120	180	240		30	75	120	180	240		∆FR
Maximum	swelling (%):	3	3	3	4	3	Max. OS:	0.5	0.5	0.5	0.5	0.5	Max. FR:	0.5
Minimum swelling (%):		0	0	0	0	0	Min. OS:	0	0	0	0	0	Min. FR:	0.0
Average:		0.1	0.3	0.3	0.3	0.3	Average:	0.0	0.0	0.0	0.0	0.0	Average:	0.0

Remark:

n = number of examined eyes

∆FR = Difference between fluorescein retention and fluorescein retention reference value

OS = Opacity score

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xI, 2xII.

Executive summary:

A Isolated Chicken Eye Test (ICET) according to OECD 438 was conducted to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points. Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 2xII.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

According to the guideline OECD 438, the test items overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorized as “No prediction can be made”.