Reaction mass of octan-2-ylbenzene, octan-3-ylbenzene and octan-4-ylbenzene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Apr 2019 - 25 Apr 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:

2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(octan-2-yl)benzene

EC Number:

950-576-0

Cas Number:

31047-57-1

Molecular formula:

C14H22

IUPAC Name:

(octan-2-yl)benzene

Test material form:

liquid

Details on test material:

Identification: SHR 1396
Physical Description: Clear colorless liquid
Storage Conditions: At room temperature

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Transport: 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: no
- 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: 750 µL, undiluted

CONTROLS
Positive control: Ethanol
Amount applied: 750 µL

Negative control: Physiological saline
Amount applied: 750 µL

Duration of treatment / exposure:

10 ± 1 minutes at 32 ± 1°C.

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes at 32 ± 1°C.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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. 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 (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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 the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS : Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0). 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 : 3 corneas per treatment group.

NEGATIVE CONTROL USED : Physiological saline

POSITIVE CONTROL USED : Ethanol

APPLICATION DOSE AND EXPOSURE TIME

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item 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 the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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 item 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 item 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.
- Corneal permeability: 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. 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).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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)

DECISION CRITERIA: The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in table 1 (see other information on materials and methods)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

Experiment 1

Value:

0.5

Negative controls validity:

valid

Remarks:

-0.1

Positive controls validity:

valid

Remarks:

48

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

Experiment 1

Value:

0.5

Negative controls validity:

valid

Remarks:

0.5

Positive controls validity:

valid

Remarks:

22

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

other: mean permeability

Run / experiment:

Experiment 1

Value:

0.002

Negative controls validity:

valid

Remarks:

-0.001

Positive controls validity:

valid

Remarks:

1.748

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The mean in vitro irritancy score of the positive control (Ethanol) was 48 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.

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: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity^1	Mean Permeability^1	MeanIn vitroIrritation Score^1, 2
Negative control	-0.1	-0.001	-0.1
Positive control (Ethanol)	22	1.748	48
SHR 1396	0.5	0.002	0.5

¹    Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.

²    In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

       
Table 2: Opacity Score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	2.1	2.4	0.3			-0.1
	3.1	2.5	-0.6
	2.9	3.1	0.2
Positive control	3.5	27.2	23.8	24		22
	3.5	24.4	21.0	21
	4.0	25.4	21.4	21
SHR 1396	3.2	2.9	-0.3	-0.3		0.5
	3.0	4.1	1.1	1.1
	3.1	3.8	0.7	0.7

Calculations are made without rounding off.

1    Final Opacity = Opacity after treatment – Opacity before treatment.

²    Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control. ³

³  In case the mean final opacity of the negative control is below zero, no correction will be made.

Table 3: Permeability Score Individual Values (Corrected)

Treatment	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	6	0.405	0.397	0.396	0.399	2.397	1.748
	6	0.347	0.350	0.352	0.350	2.099
	1	0.752	0.747	0.748	0.749	0.749
SHR 1396	1	0.002	-0.001	-0.002	0.000	0.000	0.002
	1	0.002	0.003	0.002	0.002	0.002
	1	0.003	0.003	0.006	0.004	0.004

Calculations are made without rounding off.

¹    OD₄₉₀values corrected for the mean final negative control permeability (-0.001).

Table 4: In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	0.3	-0.001	0.2
	-0.6	-0.002	-0.7
	0.2	0.000	0.2
Positive control	24	2.397	60
	21	2.099	52
	21	0.749	33
SHR 1396	-0.3	0.000	-0.3
	1.1	0.002	1.1
	0.7	0.004	0.8

¹  In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

²  Positive control and test item are corrected for the negative control.

Table 5: Historical Control Data for the BCOP Studies

	Negative control			Positive control
	Opacity	Permeability	In vitroIrritancy Score	In vitroIrritancy Score
Range	-2.0 – 3.0	-0.034 – 0.100	-2.2 – 3.0	26.0 – 89.6
Mean	0.49	0.00	0.54	52.96
SD	1.24	0.01	1.28	13.46
n	118	118	118	114

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Mar 2016 to Mar 2019.

 

 

Applicant's summary and conclusion

Interpretation of results:

other: Not eye irritating/corrosive

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

In conclusion, since SHR 1396 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

A Bovine Corneal Opacity and Permeability (BCOP) test was performed following OECD guidelines and GLP principles. The test item was applied as it is (750 µL) directly on top of the corneas. 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 meanin vitro irritancy score of the positive control (Ethanol) was 48 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. SHR 1396 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment. In conclusion, since SHR 1396 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.