Tall oil, maleated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 23 May 2017, experimental completion date: 23 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, maleated

EC Number:

268-859-6

EC Name:

Tall oil, maleated

Cas Number:

68152-93-2

Molecular formula:

UVCB

IUPAC Name:

3,7-dimethyl-14,16-dioxo-19-(propan-2-yl)-15-oxapentacyclo[10.5.2.0²,¹¹.0³,⁸.0¹³,¹⁷]nonadeca-13(17),18-diene-7-carboxylic acid; 8-(7-hexyl-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)octanoic acid

Test material form:

liquid

Details on test material:

Lot No.: HD0258QH13

Specific details on test material used for the study:

Viscous brown liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. As a consequence, a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

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.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was heated to 80 ºC to liquefy and was tested neat

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of either the negative control, positive control and test item

Duration of treatment / exposure:

10 ±1 min at 32 ±1 °C

Duration of post- treatment incubation (in vitro):

120 ±1 min 32 ±1 °C

Number of animals or in vitro replicates:

3

Details on study design:

Cornea Selection and Opacity Reading
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. Three corneas were selected at random for each treatment group.
Test Item Preparation
No correction was made for the purity/composition of the test item.
The test item was heated to 80 ºC to liquefy and was tested neat.
Treatment of Corneas and Opacity Measurements
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 (Earle’s Minimum Essential Medium, Life Technologies) 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.

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:
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 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.

Application of Sodium Fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) 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.

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 item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Test Item mean value

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: permeability

Run / experiment:

Test item mean value

Value:

0.015

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item mean value

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 1.2. The individual positive control in vitro irritancy scores ranged from 28 to 48 . The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from 1.9 to 2.7 and permeability values ranging from -0.005 to 0.047. The corneas were slightly translucent after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, thein vitro irritancy scores ranged from 1.8 to 3.4 after 10 minutes of treatment with the test item.

 
Opacity Score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	1.7	2.9	1.2			0.3
	1.5	1.1	-0.4
	4.4	4.4	0.0
Positive control	-0.5	12.8	13.3	13.0		13
	2.6	17.1	14.4	14.1
	0.7	12.8	12.1	11.8
The test item	1.4	3.7	2.3	2.0		2.2
	0.7	3.7	3.0	2.7
	2.5	4.7	2.2	1.9

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

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

³    Calculations are made without rounding off.

 

Table3          
Permeability ScoreIndividual Values (Uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2		OD490 3		Average OD		Final OD		Mean final negative control
Negative control	1	-0.004		-0.004		-0.002		-0.003		-0.003		0.007
	1	0.001		0.002		0.001		0.002		0.002
	1	0.024		0.024		0.022		0.023		0.023
Positive control	1	1.193		1.197		1.182		1.191		1.191
	6	0.382		0.379		0.374		0.378		2.270
	1	1.104		1.084		1.092		1.093		1.093
The test item	1	0.009		0.008		0.009		0.009		0.009
	1	0.054		0.055		0.052		0.054		0.054
	1	0.002		0.003		0.003		0.003		0.003

¹    Calculations are made without rounding off.

  Table4          
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	1.186	1.190	1.175	1.184	1.184	1.499
	6	0.375	0.372	0.367	0.371	2.227
	1	1.097	1.077	1.085	1.086	1.086
The test item	1	0.002	0.001	0.002	0.002	0.002	0.015
	1	0.047	0.048	0.045	0.047	0.047
	1	-0.005	-0.004	-0.004	-0.005	-0.005

¹    OD₄₉₀values corrected for the mean final negative control permeability (0.007).

²    Calculations are made without rounding off.

 

Table5          
In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	1.2	-0.003	1.2
	-0.4	0.002	-0.4
	0.0	0.023	0.3
Positive control	13	1.184	31
	14	2.227	48
	12	1.086	28
The test item	2.0	0.002	2.0
	2.7	0.047	3.4
	1.9	-0.005	1.8

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

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

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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 35 and within two standard deviations of the current historical positive control mean (Appendix 5, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment.
In conclusion, since EnvaMul 600 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
 

Executive summary:

The objective of this study was to evaluate the eye hazard potential of EnvaMul 600 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).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test item was tested through topical application for 10 minutes. 

The study procedures described in this report were based on the most recent OECD guideline.

Batch HD0379UD11 of the test item was a viscous brown liquid. 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 rangeindicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 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 test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment. 

In conclusion, since EnvaMul 600 induced an IVIS ≤3, no classification is required for eye irritation or serious eye damage.