sodium;2-chloroprop-2-enoate;hydrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-13 to 2018-05-28

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

Constituent 1

Reference substance name:

sodium 2-chloroprop-2-enoate

EC Number:

608-818-5

Cas Number:

32997-86-7

Molecular formula:

C3H2ClO2.Na

IUPAC Name:

sodium 2-chloroprop-2-enoate

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: local slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: The intact heads are transported from the slaughterhouse at ambient temperature (typically between 18°C and 25°C) in plastic boxes humidified with tissues moistened with isotonic saline. The procedure involving the collection of chicken heads and placing the eyes in the superfusion chamber following enucleation will be completed within two hours to minimize deterioration and/or bacterial contamination.
- Time interval prior to initiating testing: maximally 2 hours
- indication of any existing defects or lesions in ocular tissue samples: eyes applied in the test were unremarkeble
- Indication of any antibiotics used: no

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES :
Upon receipt of the chicken heads to the laboratory, first the eyelids were carefully excised, taking care not to damage the cornea. Corneal intergrity was asssessed with a drop of 2% (w/v) sodium fluorescein applied to the corneal surface for few seconds, and then rinsed with isotonic saline. Fluorescein-treated eyes were then examined with a slit lamp microscope to ensure that the cornea is undamaged (i.e., fluorescein retention and corneal opacity scores ≤ 0.5).

Only undamaged 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 were 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 was removed.

The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion appratus. The clamps were positioned in the superfusion appratus in such a way that the entire cornea was supplied with the isotonic saline drip (3 to 4 drops per minute or 0.1 to 0.15 mL/min). The temperature of the chambers of the superfusion appratus was maintained at 32 ± 1.5 °C.

After being placed in the superfusion appratus, the eyes were again examined with a slip-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit –lamp microscope. Eyes with (i), a fluorescein retention score of >0.5, (ii) corneal opacity >0.5 or (iii) any additional signs of damage were replaced. Out of the eyes that were not rejected based on the beforementioned criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes of this batch were rejected. During corneal thickness measurements, slit-width of slit-lamp microscope was set at 0.095 mm.

EQUILIBRATION AND BASELINE RECORDINGS : Immediately after examination and approval of all eyes, they were incubated for approximately 45 - 60 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 (i.e., time=0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED : physilogical saline, Sodium Chloride injection IP, 0.9 % w/v

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

APPLICATION DOSE AND EXPOSURE TIME : 30 µL for 10 seconds

OBSERVATION PERIOD : approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse (+/- 5 min)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: isotonic saline (approximately 20 mL) at ambient temperature

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: scroing the area of the cornea that is most densely opacified
- Damage to epithelium based on fluorescein retention: Fluorescein retention was evaluated at the 30 minutes observation time point
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 0.095 mm
- 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: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

test item

Value:

>= 4.51 - <= 17.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

test item

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

test item

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: roughening of the corneal surface observed

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use the technical proficiency of the test method was established by using proficiency chemicals under Bioneeds Study No.: BIO-GT 1046 according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

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 = (mins)							ICE Class
		Selection	0	30	75	120	180	240
Negative Control	1	281	291	288	289	288	290	284	I
	Sw%	NA	NA	-1.03	-0.69	-1.03	-0.34	-2.41
Positive Control	2	289	296	351	352	463	468	481	IV
	Sw%	NA	NA	18.58	18.92	56.42	58.11	62.50
	3	279	289	338	353	465	472	492
	Sw%	NA	NA	16.96	22.15	60.90	63.32	70.24
	4	299	288	335	360	455	477	490
	Sw%	NA	NA	16.32	25.00	57.99	65.63	70.14
	Mean sw% ±SD	NA	NA	17.29 1.17	22.02 3.04	58.43 2.27	62.35 3.85	67.63 4.44
Test Item	5	269	266	292	320	322	328	324	I
	Sw%	NA	NA	9.77	20.30	21.05	23.31	21.80
	6	267	278	295	317	317	326	326
	Sw%	NA	NA	6.12	14.03	14.03	17.27	17.27
	7	279	298	291	316	322	337	328
	Sw%	NA	NA	-2.35	6.04	8.05	13.09	10.07
	Mean Sw% ±SD	NA	NA	4.51 6.22	13.46 7.15	14.38 6.51	17.89 5.14	16.38 5.92

Table 2 Data of corneal opacity scores and isolated chicken eye (ICE) Classification

Treatment	Eye No.	Corneal Opacity Scores at t =							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	4	4	IV
	3	0	0	2	2	3	4	4
	4	0	0	1	2	2	4	4
	Mean	0	0.0	1.7	2.0	2.7	4.0	4.0
Test Item	5	0	0	0	0	0	0	0	I
	6	0	0	0	0	0	0	0
	7	0	0	0	0	0	0	0
	Mean	0	0.0	0.0	0.0	0.0	0.0	0.0

Table 3 Data of fluorescein retention, morphological effects and isolated chicken eye (ICE) classification

Treatment	Eye No.	Fluorescein retention at t = (Min)			Morphological effects	ICE Class
		-50	0	30
Negative Control	1	0	0	0	No morphological effects observed	I
Positive Control	2	0	0	3	Loosening of epithelium	IV
	3	0	0	3	Loosening of epithelium
	4	0	0	3	Loosening of epithelium
	Mean	0	0.0	3.0	NA
Test Item	5	0	0	0	Roughening of the corneal surface	I
	6	0	0	0	Roughening of the corneal surface
	7	0	0	0	Roughening of the corneal surface
	Mean	0	0.0	0.0	NA

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test item is identified to not require classification as eye damaging or eye irritant.

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” as per OECD Guideline for the Testing of Chemicals, Section 4, No. 438, adopted on 9th October 2017.

Heads of chickens approximately 6 to 7 weeks old and weighing around 1.8 to 2.8 kg were collected at poultry slaughterhouse. Within 2 hours of killing, enucleated eyes were placed in a susperfusion apparatus and maintained at 32 ± 1.5oC for 50 minutes. Before dosing, the eyes were incubated for 50 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. Quantity of 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. Similarly, 30 µL of Sodium Chloride solution, 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. Fluorescin retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes treated with vehicle, positive control and test item were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter.

For the test item the combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 3 x I (ICE class of I observed in all 3 endpoints). This test is considered acceptable as the concurrent negative control and the positive control were identified as UN GHS Non-Classified and UN GHS Category 1, respectively.