6-chlorohexan-2-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 2nd to February 25th, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-chlorohexan-2-one

EC Number:

233-546-5

EC Name:

6-chlorohexan-2-one

Cas Number:

10226-30-9

Molecular formula:

C6H11ClO

IUPAC Name:

6-chlorohexan-2-one

Test material form:

liquid

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Characteristics of donor animals (e.g. age, sex, weight): healthy approximately 7-week-old chickens used for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Chicken heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container.
- Time interval prior to initiating testing: The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes.
- indication of any existing defects or lesions in ocular tissue samples: the eyeball surface was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. Only eyeballs without damage were analyzed (fluorescein retention and opacity profusion scores fell below 0.5). Eyeballs with corneal thickness deviating more than 10% from the mean value for all eyeballs were also replaced.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL of test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

No post-treatment incubation was performed.

Number of animals or in vitro replicates:

Three in vitro replicates.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After a careful excision of the eyelids so as not to damage the cornea, the eyeball surface was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED (HAAG STREIT) to ensure that the cornea was undamaged. Only eyeballs without damage were analyzed (fluorescein retention and opacity profusion scores fell below 0.5).
The enucleated eyeball was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to the superfusion apparatus so that the entire cornea was supplied with the physiological salt drip.
After the insertion of the eyeballs to the apparatus, another evaluation of corneal opacity and swelling was performed. Eyeballs with the corneal opacity and/or fluorescein retention score higher than 0.5 or some additional signs of damage were replaced. Eyeballs with corneal thickness deviating more than 10% from the mean value for all eyeballs were also replaced. The corneal thickness was determinated using the depth measuring device no. 1 on the slit-lamp microscope and an SP-100 pachymeter (TOMEY).

EQUILIBRATION AND BASELINE RECORDINGS
Prior to the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32°C ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. During the incubation, the eyeballs were continuously supplied with physiological salt at constant temperature of 32 ± 1.5°C and in the average volume of 0.10-0.15 mL/min.
After that, a zero reference measurement for corneal thickness and opacity was recorded. It served as a baseline (i.e. time = 0). The fluorescein score determined at dissection served as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological salt.

POSITIVE CONTROL USED: 10 % acetic acid.

APPLICATION DOSE AND EXPOSURE TIME: 0.03 mL of test ítem, negative and positive control applied for 10 seconds each.

OBSERVATION PERIOD
The corneas treated with the test item and the control items were evaluated pretreatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse.
At all observation times points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only at 30 minutes after the end of the exposure.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The corneal surface were rinsed from the eye with 20 mL of physiological salt at ambient temperature. Additional rinsing was not necessary.
- Indicate any deviation from test procedure in the Guideline: No deviations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit-lamp microscope BP 900 LED (HAAG STREIT)
- Damage to epithelium based on fluorescein retention: slit-lamp microscope BP 900 LED (HAAG STREIT)
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: slit-lamp microscope and an SP-100 pachymeter (TOMEY)
- Macroscopic morphological damage to the surface: slit-lamp microscope BP 900 LED (HAAG STREIT)
- Others (e.g, histopathology): Histopathology evaluations: the eyeballs were fixed in a 4% solution of formaldehyde. Next, specimens were collected (one specimen in the plane including the cornea, lens, and optic nerve). The tissue material was dehydrated and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using Hematoxylin and Eosin

SCORING SYSTEM:
- Mean corneal swelling (%):
0 to 5 ICE Class I
>5 to 12 ICE Class II
>12 to 18 (>75 min after treatment) ICE Class II
>12 to 18 ( <75 min after treatment) ICE Class III
>18 to 26 ICE Class III
>26 to 32 ( >75 min after treatment) ICE Class III
>26 to 32 ( <75 min after treatment) ICE Class IV
>32 ICE Class IV

- Mean maximum opacity score:
0.0 – 0.5 ICE Class I
0.6 – 1.5 ICE Class II
1.6 – 2.5 ICE Class III
2.6 – 4.0 ICE Class IV

- Mean fluorescein retention score at 30 minutes post-treatment:
0.0 – 0.5 ICE Class I
0.6 – 1.5 ICE Class II
1.6 – 2.5 ICE Class III
2.6 – 3.0 ICE Class IV

DECISION CRITERIA: The decision criteria indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

ca. 1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Remarks:

at up to 240 min

Run / experiment:

mean

Value:

ca. 1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

mean

Value:

ca. 7

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: Not observed
- Gross examination: eyeballs treated with the test item did not exhibit any changes of the corneal surface.
- Histopathological evaluation: Corneas treated with the test item revealed: Exfoliation (eyeball no. 2), slight vacuolation of the anterior corneal epithelium (eyeballs no. 1, no. 2, no. 3), coagulation, slight exfoliation (eyeballs no. 1, no. 3), very slight erosions (eyeball no. 1), slight erosions of the superficial layer of the anterior corneal epithelium (eyeballs no. 2, no. 3).



ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control Physiological saline was classified as non-irritating, UN GHS Classification: Non-classified
- Acceptance criteria met for positive control: The positive control (acetic acid) was classified as severely irritating, UN GHS Classification: Category 1
This experiment was considered to be valid on the basis of the OECD guideline used

Any other information on results incl. tables

Table1. Fluorescein retention

observation after time t (minutes)	test item			positive control 10 % acetic acid			negative control physiological saline
	eyeball no.			eyeball no.			eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	1.0	2.0	2.0	3.0	3.0	3.0	0.0	0.0	0.0

Table2. Corneal opacity

observation after time t (minutes)	test item			positive control 10 % acetic acid			negative control physiological saline
	eyeball no.			eyeball no.			eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	0.5	0.5	1.0	4.0	4.0	4.0	0.0	0.0	0.0
75	0.5	1.0	1.0	4.0	4.0	4.0	0.0	0.0	0.0
120	0.5	1.0	1.0	4.0	4.0	4.0	0.0	0.0	0.0
180	1.0	1.0	1.0	4.0	4.0	4.0	0.0	0.0	0.0
240	1.0	1.0	2.0	4.0	4.0	4.0	0.0	0.0	0.0

Table3. Corneal swelling (%)

observation after time t (minutes)	test item			positive control 10 % acetic acid			negative control physiological saline
	eyeball no.			eyeball no.			eyeball no.
	1	2	3	4	5	6	7	8	9
0	-	-	-	-	-	-	-	-	-
30	-0.3	0.5	-0.3	6.7	14.4	14.7	-0.8	-2.0	-3.2
75	-1.9	-0.2	3.5	8.7	12.7	18.4	-1.4	-1.9	-5.0
120	2.8	2.4	9.5	11.4	7.7	27.0	-3.7	-1.1	-3.0
180	3.1	3.4	9.0	21.1	16.0	37.8	-3.2	-1.5	-4.6
240	5.9	2.9	12.3	30.9	28.8	49.9	-3.8	-1.5	-8.5

“-”= decrease in corneal thickness, no swelling

Table4. Gross evaluation of the treated corneas

observation after time t (minutes)	test item			positive control 10 % acetic acid			negative control physiological saline
	eyeball no.			eyeball no.			eyeball no.
	1	2	3	4	5	6	7	8	9
30	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
75	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
120	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
180	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
240	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC

NC = no changes

SIGNS = roughening of the corneal surface

Table5. Evaluation of fluorescein retention

observation after time t (minutes)	test item		positive control 10 % acetic acid		negative control physiological saline
	average	ICE class	average	ICE class	average	ICE class
30	1.7	III	3.0	IV	0.0	I

Table6. Evaluation of corneal opacity

observation after time t (minutes)	test item		positive control 10 % acetic acid		negative control physiological saline
	average	ICE class	average	ICE class	average	ICE class
30	0.7	II	4.0	IV	0.0	I
75	0.8	II	4.0	IV	0.0	I
120	0.8	II	4.0	IV	0.0	I
180	1.0	II	4.0	IV	0.0	I
240	1.3	II	4.0	IV	0.0	I

Table7. Evaluation of corneal swelling (%)

observation after time t (minutes)	test item		positive control 10 % acetic acid		negative control physiological saline
	average	ICE class	average	ICE class	average	ICE class
30	-0.1	I	11.9	II	-2.0	I
75	0.5	I	13.2	III	-2.8	I
120	4.9	I	15.4	II	-2.6	I
180	5.2	II	25.0	III	-3.1	I
240	7.0	II	36.5	IV	-4.6	I

“-”           - percentage of corneal thickness decrease, no swelling

Applicant's summary and conclusion

Interpretation of results:

other: the test item is not classified as a severe irritant and not classified as non-irritant

Conclusions:

Based on this in vitro eye irritation study, the test item is not classified as a severe irritant and not classified as non-irritant.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes according to the OECD No. 438 guideline with GLP. After equilibration of the test system and zero reference measurements, the test item, the positive control (10 % acetic acid) and the negative control (physiological salt) were applied to the corneal surface for 10 seconds in a volume of 0.03 mL. Three test item treated eyes, three positive and three negative control treated eyes were examined. After exposure time, they were rinsed from the eye with 20 mL of physiological salt at ambient temperature. Corneal opacity and increased thickness (swelling) were evualuated and morphological changes recorded pretreatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. Fluorescein retention was performed only after 30 minutes of the exposure. At the end of those observations, histopathological examinations were conducted. The results for the test item treated eyes showed a mean fluorescein retention of 1.7 (ICE class III), mean corneal opacity from 0.7 (ICE class II) to 1.3 (II), and a maximal mean corneal swelling of 7.0 % (ICE class II). These results were accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations did not exhibit any changes of the treated test ítem corneal surface. Histopathological examinations of the corneas treated with the test item revealed: exfoliation (eyeball no. 2), slight vacuolation of the anterior corneal epithelium (eyeballs no. 1, no. 2, no. 3), coagulation, slight exfoliation (eyeballs no. 1, no. 3), very slight erosions (eyeball no. 1) and slight erosions of the superficial layer of the anterior corneal epithelium (eyeballs no. 2, no. 3). Based on this in vitro eye irritation study, the test item is not classified as a severe irritant and not classified as non-irritant since the overall ICE Class was 2xII 1xIII, although it can be concluded, according to the histopathological findings, that the test item has a negative effect on the chicken cornea in the ICE test.