Butyl toluene-4-sulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

9 October 2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Butyl toluene-4-sulphonate

EC Number:

212-295-5

EC Name:

Butyl toluene-4-sulphonate

Cas Number:

778-28-9

Molecular formula:

C11H16O3S

IUPAC Name:

butyl 4-methylbenzene-1-sulfonate

Test material form:

liquid

Remarks:

(colourless to slightly yellow)

Details on test material:

- Storage Conditions: room temperature protected from light

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from a slaughterhouse (Vitelco, 's-Hertogenbosch, The Netherlands). The eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

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): 750 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Number of animals or in vitro replicates:

Three corneas were allocated to the test material, three corneas were allocated to the negative control item, and three corneas were allocated to the positive control item.

Details on study design:

PREPARATION AND SELECTION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1 % (v/v) L-glutamine and 1 % (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for a minimum of 1 hour at 32 ± 1 °C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or test material was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or test material over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions were removed and the epithelium was washed with MEM (Earle’s Minimum Essential Medium) with phenol red and thereafter with cMEM. Possible pH effects of the test material on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.

POST-EXPOSURE INCUBATION
Corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS
After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
- Opacity measurement: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to the following equation:
Opacity = [(I0/I) - 0.9894] / 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Application of Sodium Fluorescein: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.
- Permeability Determinations: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

INTERPRETATION
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

The test material was classified according to the following prediction model:
IVIS ≤ 3: No Category (UN GHS)
IVIS >3; ≤ 55: No prediction can be made (UN GHS)
IVIS > 55: Category 1 (UN GHS)

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Test material (mean)

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative control (mean)

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control (mean)

Value:

55

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The corneas treated with the test material showed opacity values ranging from -0.2 to 0.5 and permeability values ranging from 0.006 to 0.018. The corneas were clear after the 10 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.1 to 0.7 after 10 minutes of treatment with the test material. The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.
The individual in vitro irritancy scores for the negative controls ranged from 0.5 to 2.6. One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. The corneas treated with the negative control item were clear after the 10 minutes of treatment.
The individual positive control in vitro irritancy scores ranged from 54 to 61. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

ACCEPTANCE OF RESULTS:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 55 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Summary of results

Treatment	Final opacity	Final OD490	In vitro Irritancy Score
Negative control	2.7	-0.005	2.6
	0.5	-0.003	0.5
	5.2*	0.029*	5.7*
Mean	1.6	-0.004	1.6
Positive control	21	2.218	54
	22	2.592	61
	30	1.370	50
Mean	24	2.060	55
Test material	0.5	0.013	0.7
	-0.2	0.006	-0.1
	0.2	0.018	0.5
Mean	0.2	0.012	0.4

Positive control and test material are corrected for the negative control.

*Excluded from analysis

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to EU criteria

Conclusions:

Under the conditions of the study, the test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.
In conclusion, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye irritation potential of the test material was evaluated, as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study was conducted in accordance with the standardised guideline OECD 437 and under GLP conditions.

During the study, the test material was applied as supplied (750 μL) directly on top of the corneas. The eye damage of the test material was tested through topical application for 10 minutes. After exposure the cornea was thoroughly rinsed to remove the test material and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 55 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Under the conditions of the study, the test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.

In conclusion, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.