Chlorphenesin

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 22nd, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

2013

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Chlorphenesin

EC Number:

203-192-6

EC Name:

Chlorphenesin

Cas Number:

104-29-0

Molecular formula:

C9H11ClO3

IUPAC Name:

3-(4-chlorophenoxy)propane-1,2-diol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir as a by-product from freshly slaughtered animals.
- Number of animals: not specified.
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle (typically 12 to 60 months old).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were excised by an abattoir employee after slaughter, and were placed in Hanks' Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/ml and streptomycin at 100 µg/ml). They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: the corneas were prepared immediately on arrival at the laboratory.

Test system

Vehicle:

physiological saline

Controls:

other: positive and negative controls

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): The test item was prepared as a 20 % w/v solution in 0.9 % w/v sodium chloride solution.

VEHICLE: 0.9% w/v sodium chloride solution

Duration of treatment / exposure:

240 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

3 replicates for test item, positive control and negative control (total 9 treatments).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle's minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.
The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES: 3 replicates for test item, positive control and negative control (total 9 treatments).

NEGATIVE CONTROL USED: 0.9% wlv sodium chloride solution

POSITIVE CONTROL USED: Imidazole. The positive control was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

APPLICATION DOSE AND EXPOSURE TIME:
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 ml of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 240 minutes.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at the end of the exposure period the test item preparation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM.
- POST-EXPOSURE INCUBATION: not applicable

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader: the medium from the anterior chamber was removed and replaced with 1 ml of sodium fluorescein solution (5 mg/ml). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes. 360 µl of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS):
IVIS = mean opacity value + (15 x mean OD492 value)

DECISION CRITERIA:
IVIS ≤ 3; Classification:No category. Not requiring classification to UN GHS or EU CLP
3 < IVIS ≤ 55 Classification: No prediction of eye irritation can be made
IVIS > 55 Classification: Category 1. UN GHS or EU CLP Causes serious eye damage

EVALUATION OF RESULTS
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
- Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

ACCEPTANCE CRITERIA
- Acceptance criteria met for negative control: the test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2013 for bovine corneas treated with the respective negative control. When testing solids the negative control range for opacity should be ≤ 4.7 and for permeability ≤ 0.085.
- Acceptance criteria met for positive control: the test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2013 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 73.8 - 103.6.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

test item

Value:

ca. 25.2

Vehicle controls validity:

valid

Remarks:

see negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean of 3 replicates

Other effects / acceptance of results:

RESULTS:
In vitro irritancy score for test item was 25.2%.

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2013 for bovine corneas treated with the respective negative control. When testing solids the negative control range for opacity should be ≤ 4.7 and for permeability ≤ 0.085. Mean opacity of negative control = 2.9 ≤ 4.7 and mean permeability of negative control = 0.058 ≤ 0.085. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: the test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2013 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 73.8 - 103.6. Mean IVIS positive control = 75.0 inside 73.8 - 103.6. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Results

Treatment	Cornea Number	Opacity				Permeability(OD)		IVIS
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre-treatment	Corrected value		Corrected value
Negative Control	10	2	5	3		0.046
	11	2	3	1		0.071
	12	2	4	2		0.058
				2.0 (mean)		0.058 (mean)		2.9
Positive Control	1	3	51	48	46.0	1.558	1.500
	2	1	60	59	57.0	1.224	1.166
	3	3	68	65	63.0	1.322	1.264
					55.3(mean)		1.310 (mean)	75.0
Test item	7	1	26	25	23.0	0.177	0.119
	8	1	21	20	18.0	0.337	0.279
	9	1	29	28	26.0	0.236	0.178
					22.3(mean)		0.192 (mean)	25.2

Corneal epithelium condition

Treatment	Cornea number	Observation
		Post treatment
Negative control	10	clear
	11	clear
	12	clear
Positive control	1	cloudy
	2	cloudy
	3	cloudy
Test item	7	cloudy
	8	cloudy
	9	cloudy

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The study can use as WoE with other studies to classification purpose.

Conclusions:

Based on the result of the test no prediction of eye irritation can be made (IVIS test item: 25.2).
The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and bioitem function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

Method

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Interpretation

The test item is classified according to the prediction model below:

IVIS ≤ 3;              Classification: No category. Not requiring classification to UN GHS or EU CLP

3 < IVIS ≤ 55       Classification: No prediction of eye irritation can be made

IVIS > 55             Classification: Category 1. UN GHS or EU CLP Causes serious eye damage

 

Results

Test item: IVIS = 25.2

Negative control: IVIS = 2.9

Positive control: IVIS = 75.0

 

Conclusion

No prediction of eye irritation can be made.

The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.