3-(heptyloxy)propane-1,2-diol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Augustus 2019 - 24 September 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:

adopted October 09, 2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-octoxypropane-1,2-diol

Cas Number:

10438-94-5

Molecular formula:

C11H24O3

IUPAC Name:

3-octoxypropane-1,2-diol

Test material form:

liquid

Details on test material:

Storage Conditions: At room temperature protected from light container flushed with nitrogen.

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 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(s) applied: 750 µL, undiluted

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine
(Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen,
Germany) 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
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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). 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.

NEGATIVE CONTROL USED
Physiological saline

POSITIVE CONTROL USED
Ethanol

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.

REMOVAL OF TEST SUBSTANCE :
- Number of washing steps after exposure period: Once with cMEM and once with MEM. Possible pH effects of the test item on the corneas were recorded.

POST-EXPOSURE INCUBATION:
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.

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 (Sigma-Aldrich Chemie GmbH, Germany)/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).

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.

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

Run / experiment:

Experiment 1

Value:

68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.5

Positive controls validity:

valid

Remarks:

36

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

Experiment 1

Value:

5.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.5

Positive controls validity:

valid

Remarks:

16

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

other: Mean permeability

Run / experiment:

Experiment 1

Value:

4.161

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.000

Positive controls validity:

valid

Remarks:

1.319

Remarks on result:

positive 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 36 and within two standard deviations of the current historical positive control mean (mean 51.31, SD 13.72). The opacity and permeability values for the negative control varied between 2.0 - 3.3 and -0.004 - 0.011, respectively which is less than the upper limits of the laboratory historical ranges (3.0 for opacity and 0.1 for permeability). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

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	0.5	0.000	0.5
Positive control (Ethanol)	16	1.319	36
Saskine^TM80	5.9	4.161	68

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	2.7	2.1	-0.6			0.5
	2.9	3.8	0.9
	2.0	3.3	1.3
Positive control	2.0	17.6	15.6	15		16
	1.9	19.2	17.3	17
	1.8	18.1	16.3	16
Saskine^TM80	2.6	9.8	7.3	6.8		5.9
	2.6	7.3	4.8	4.2
	-0.8	6.3	7.1	6.6

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.

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	1	1.449	1.462	1.445	1.452	1.452	1.319
	1	0.808	0.796	0.787	0.797	0.797
	6	0.284	0.284	0.288	0.285	1.710
Saskine^TM80	6	0.767	0.772	0.777	0.772	4.630	4.161
	6	0.707	0.702	0.716	0.708	4.248
	6	0.598	0.607	0.598	0.601	3.604

Calculations are made without rounding off.

¹    OD₄₉₀values corrected for the mean final negative control permeability (0.000).

Table 4          
In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	-0.6	0.001	-0.6
	0.9	-0.003	0.8
	1.3	0.003	1.3
Positive control	15	1.452	37
	17	0.797	29
	16	1.710	41
Saskine^TM80	6.8	4.630	76
	4.2	4.248	68
	6.6	3.604	61

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	24.0 – 89.6
Mean	0.54	0.00	0.58	51.31
SD	1.23	0.01	1.26	13.72
n	120	120	120	108

SD = Standard deviation

n = Number of observations

Applicant's summary and conclusion

Interpretation of results:

other: Serious eye damage (category 1)

Remarks:

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

Conclusions:

In a Bovine Corneal Opacity and Permeability test performed according to OECD TG 437 the substance induced an IVIS ≥ 55 and is concluded to induce serious eye damage.

Executive summary:

A Bovine Corneal Opacity and Permeability (BCOP) test was performed according to OECD TG 437 and in accordance with 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. The mean in vitro irritancy score of the positive control (Ethanol) was 36 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.  The substance induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 68 after 10 minutes of treatment. In conclusion, the substance induced an IVIS ≥ 55 and is considered to induce serious eye damage in the BCOP and should be classified category 1 according to Regulation (EC) No. 1272/2008 and its amendments.