Tall oil, sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The test was performed on 10 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, sodium salt

EC Number:

266-037-1

EC Name:

Tall oil, sodium salt

Cas Number:

65997-01-5

Molecular formula:

Not applicable. UVCB substance.

IUPAC Name:

Alkaline Aqueous Extraction of sodium salts of fatty acids and rosin acids from the Kraft Pulping Process

Test material form:

liquid

Test animals / tissue source

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance was tested undiluted.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3 cornea per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of the undiluted item for 10 minutes.
Following treatment of the corneas, the test material was rinsed off and the opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

Incubation Medium

The incubation medium consists of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% foetal calf serum (FCS).
The OECD guideline 437 recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

Opacity measurement

The opacitometer determines changes in the light transmission passing through the cornea, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the cornea was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the cornea to the test groups, after rinsing and further incubation of the cornea for two hours, the opacity value was determined again (t130).

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Cornea were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and thereafter was the optical density at 490 nm (OD490) determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control cornea is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control cornea is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.

Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine cornea treated with the respective negative control.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

main experiment

Value:

13.65

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: fulfilled, no category (IVIS 1.18)
- Acceptance criteria met for positive control: fulfilled, category 1 (IVIS 86.01)

Any other information on results incl. tables

Results after 10 Minutes Treatment Time

Test Group	Opacity value #		Permeability ##		IVIS	Mean IVIS	Proposed IVIS
		Mean		Mean
Neg. Control	1		0.049		1.74
	0	0.33	0.071	0.057	1.07	1.18	Not categorized
	0		0.050		0.75
Pos. Control	48.67*		1.544*		71.83
	61.67*		1.984*		91.43	86.01	Category 1
	70.67*		1.607*		94.78
Leuco Sulphur Blue 15	5.67*		0.302*		10.20
	7.67*		0.415*		13.90	13.65	No prediction can be made
	7.67*		0.612*		16.85

# Opacity value = Difference (t₁₃₀ - t₀) of Opacity

## Permeability at 490 nm (OD₄₉₀)

*corrected values

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (GHS) for Tall Oil Soap, TOS30.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Tall Oil Soap, TOS30 by means of the BCOP assay using fresh bovine cornea.

After a first opacity measurement of the fresh bovine cornea (t₀), the neat test item, the positive, and the negative controls were applied to cornea fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1°C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the cornea. Further, the corneas were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t₁₃₀).

After the opacity measurements permeability of the cornea was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horisontal position for 90 minutes at 32 ± 1°C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the cornea corresponding to a classification as serious eye damaging (EU/EPA/GHS(Cat 1)).

 

Relative to the negative control, the test item Tall Oil Soap, TOS30 caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 13.65. According to OECD 437 the test item’s hazard for eye damage cannot be predicted.