Reaction mass of 2-amino-2-methyl-1-propanol hydrochlorid (3207-12-3) and 2-amino-2-methyl-1-propanol (124-68-5)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2018 - 07 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

2017

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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

(2-hydroxy-1,1-dimethylethyl)ammonium chloride

EC Number:

221-713-5

EC Name:

(2-hydroxy-1,1-dimethylethyl)ammonium chloride

Cas Number:

3207-12-3

Molecular formula:

C4H11NO.ClH

IUPAC Name:

1-hydroxy-2-methylpropan-2-aminium chloride

Test material form:

liquid

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: poultry slaughterhouse
- Number of animals: 5
- Characteristics of donor animals: 7-8 weeks old
- Storage, temperature and transport conditions of ocular tissue: The intact heads were transported from the slaughterhouse at ambient temperature (typically between 18°C and 25°C) in plastic boxes humidified with tissues moistened with isotonic saline.
- Time interval prior to initiating testing: max. 2 hours
- Indication of any existing defects or lesions in ocular tissue samples: none

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

VEHICLE
- Amount applied: 30 µL
- Concentration: 0.9 % (w/v) Sodium Chloride solution

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control eyes and one negative control eye were used in this study.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES:
After confirming that the eyes were undamaged, the eyes were further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles was cut with a bent, blunt-tipped scissor. All necessary precautions were taken to aviod any corneal damage due to excessive pressure (i.e, compression artifacts). The visible portion of the optic nerve was left attached to the eye when it was removed from the orbit. Immediately after removing the eye from the orbit, the eye was placed on an absorbent pad and the nictitaing membrane and other connective tissue were removed.

EQUILIBRATION AND BASELINE RECORDINGS
Immediately after examination and approval of all eyes, they were incubated for 55 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline values (i.e., time=0). Additionally, the fluorescein retention score was also recorded at 0 hr as baseline measurment value.

NUMBER OF REPLICATES: 3 replicates per test item and positive control, 1 replicate per negative control

NEGATIVE CONTROL USED: 0.9 % (w/v) Sodium Chloride solution

POSITIVE CONTROL USED: 5 % Benzalkonium chloride (in saline solution; 9 g NaCl/L)

APPLICATION DOSE AND EXPOSURE TIME: 30 µL, 10 sec

OBSERVATION PERIOD: The treated corneas were evaluated prior to treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The test item, negative control and positive control were applied for 10 seconds and then rinsed from the eye with isotonic saline (approximately 20 mL) at ambient temperature.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: determined using the area of the cornea that was most densely opacified for scoring
- Damage to epithelium based on fluorescein retention: none
- Swelling: measured with depth measuring device (Pachymeter)
- Macroscopic morphological damage to the surface: none

SCORING SYSTEM:
- 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 was used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1-3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The overall ICE class was 2xI and 1xII.

Any other information on results incl. tables

TABLE 1.     Corneal swelling/Thickness and isolated chicken eye (ICE) Classification

Treatment	Eye No. and Sw%	Corneal Thickness in Instrument Units (µm) at t (Minutes)							ICE Class
		Selection	0	30	75	120	180	240
Negative Control	1	285	285	288	286	298	299	299	I
	Sw%	NA	NA	1.05	0.35	4.56	4.91	4.91
Positive control	2	302	301	337	466	480	539	562	IV
	Sw%	NA	NA	11.96	54.82	59.47	79.07	86.71
	3	304	306	336	425	465	545	513
	Sw%	NA	NA	9.80	38.89	51.96	78.10	67.65
	4	291	294	344	481	480	536	538
	Sw%	NA	NA	17.01	63.61	63.27	82.31	82.99
	Mean sw% ±SD	NA	NA	12.92 ±3.70	52.44 ±12.53	58.23 ±5.75	79.83 ±2.20	79.12 ±10.11
Test Item	5	296	291	324	312	318	317	320	II
	Sw%	NA	NA	11.34	7.22	9.28	8.93	9.97
	6	276	287	318	300	318	314	311
	Sw%	NA	NA	10.80	4.53	10.80	9.41	8.36
	7	302	301	322	314	302	315	301
	Sw%	NA	NA	6.98	4.32	0.33	4.65	0.00
	Mean sw% ±SD	NA	NA	9.71 ±2.38	5.36 ±1.62	6.80 ±5.66	7.66 ±2.62	6.11 ±5.35

Sw%: Corneal Swelling percentage, SD: Standard deviation, t: time.

      

TABLE 2.     Corneal opacity SCORES and isolated chicken eye (ICE) Classification

Treatment	Eye No.	Corneal Opacity Scores at t (Minutes)							ICE Class
		Selection	0	30	75	120	180	240
Negative Control	1	0	0	0	0	0	0	0	I
Positive Control	2	0	0	2	2	3	3	4	IV
	3	0	0	2	2	3	3	4
	4	0	0	2	2	3	3	4
	Mean	0	0.0	2.0	2.0	3.0	3.0	4.0
Test Item	5	0	0	0.5	0.5	0.5	0.5	0.5	I
	6	0	0	0.5	0.5	0.5	0.5	0.5
	7	0	0	0.5	0.5	0.5	0.5	0.5
	Mean	0	0.0	0.5	0.5	0.5	0.5	0.5

SD: Standard deviation, t: time.

TABLE 3.     Fluorescein retention, Morphological effects andisolated chicken eye (ICE) Classification

Treatment	Eye No.	Fluorescein retention at t (Minutes)			Morphological effects	ICE Class
		Selection	0	30
Negative Control	1	0	0	0	No morphological effects observed	I
Positive Control	2	0	0	1	Loosening of epithelium	II
	3	0	0	1	Loosening of epithelium
	4	0	0	1	Loosening of epithelium
	Mean	0	0.0	1.0	NA
Test Item	5	0	0	0.5	No morphological effects observed	I
	6	0	0	0	No morphological effects observed
	7	0	0	0	No morphological effects observed
	Mean	0	0.0	0.2	NA

SD: Standard deviation, t: time.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the percentage of corneal swelling, corneal opacity score, fluroscein retention score and mophologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end points the test item was identified as UN GHS No Category.

Executive summary:

The test item was evaluated in the “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” according to OECD Guideline for the Testing of Chemicals, Section 4, No. 438, adopted on 9th October 2017. Heads of chickens approximately 7 to 8 weeks old and weighing around 1.5 to 2.5 kg were collected at a poultry slaughterhouse. Within 2 hours of killing, enucleated eyes were placed in a susperfusion apparatus at 32±1.5 °C. Before dosing, the eyes were incubated for 55 minutes to equilibrate them to the test system prior to treatment. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e., time=0). Additionally, the fluorescein retention score was also recorded at 0 hour as baseline measurement value. Eyes with corneal opacity <0.5 and fluorescein retention score <0.5 were selected. 30 µL of test item and Benzalkonium chloride [5% in saline solution (9 g NaCl/L)] was applied onto the cornea of three eyes for 10 seconds, respectively. Similalry, 30 µL normal saline, 0.9 % w/v was used as negative control and was applied onto the cornea of a eye for 10 seconds. The control and test eyes were examined for corneal thickness and corneal opacity at 0, 30, 75, 120, 180 and 240 min after treatment and results were recorded according to a fixed scoring system. Fluorescein retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes treated with negative control, positive control and test item were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter.

The in vitro classification for a test item was assessed by reading the UN GHS classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity and fluorescein retention. For the test item the combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 2 x I and 1 x II (ICE class of I observed in two endpoints and Class II in one endpoint). Based on the percentage of corneal swelling, corneal opacity score, fluroscein retention score and mophologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end points the test item was identified as UN GHS No Category. The test is considered acceptable as the concurrent negative control and the positive control were identified as UN GHS Non-Classified and GHS Category 1, respectively.