Fatty acids, tall-oil, reaction products with diethylenetriamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Mar - Apr 2018

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

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:

09 Oct 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, reaction products with diethylenetriamine

EC Number:

263-160-2

EC Name:

Fatty acids, tall-oil, reaction products with diethylenetriamine

Cas Number:

61790-69-0

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

Fatty acids, tall-oil, reaction products with diethylenetriamine

Test material form:

other: solid, waxy, white to yellowish

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0017319511
- Expiration date of the batch: 18 Dec 2018
- Purity: > 99 %
- pH value: ca. 6
- Physical state / color: Solid, waxy / white to yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To improve application, the solid waxy test substance was heated at about 50 °C for about 15 minutes. Before application, the test substance was cooled down to room temperature.

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Straße 23, 55232 Alzey, Germany
- Characteristics of donor animals (e.g. age, sex, weight): min. 12 months and max. 60 months old
- indication of any existing defects or lesions in ocular tissue samples: no

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

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes are obtained as a by-product of freshly slaugthered cattle (age of the animals: minimum 12 months, maximum 60 months).

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle's MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 556 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups. Each corneal holder was uniquely identified with a number on the chambers.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
deionized water

POSITIVE CONTROL USED
Imidazole (CAS-No. 288-32-4) 20 % (w/v) solution in deionized water for non-surfactant solid test substances

APPLICATION DOSE AND EXPOSURE TIME
750 µL for 4 hours

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The NC and the PC were removed from the anterior chamber by using a syringe and the epithelium was washed at least 3 times with Eagle's MEM (containing phenol red) and once with Eagle's MEM (without phenol red). Because the test substance could not be removed by using a syringe the epithelium was rinsed with the open chamber method.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was visually observed and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria according to the OECD Guideline 437 (adopted 09 Oct 2017)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

9.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Opacity score of the test substance, the NC and the PC

Test substance identification	Cornea-No.	Initial opacity	Final opacity	Opacity change	Corrected opacity change*	Mean	SD
Test substance	7 8 9	3.2 4.0 4.9	8.9 8.2 8.5	5.7 4.2 3.5	0.0 0.0 0.0	0.0	0.0
NC	1 2 3	4.5 5.5 7.0	15.2 12.9 9.1	10.7 7.5 2.1	NA NA NA	6.7	4.3
PC	4 5 6	4.1 4.9 2.9	104.8 96.5 113.3	100.7 91.5 110.4	93.9 84.8 103.7	94.1	9.5

* negative values are set to zero for further calculation

Table 2: Permeability score of the test substance, the NC and the PC

Test substance identification	Cornea-No.	Mean OD490	Dilution factor	Mean corrected OD490	Mean	SD
Test substance	7 8 9	0.929 0.447 0.581	1 1 1	0.925 0.443 0.577	0.648	0.249
NC	1 2 3	0.005 0.004 0.003	1 1 1	NA NA NA	0.004	0.001
PC	4 5 6	0.266 0.491 0.345	5 5 5	1.328 2.449 1.721	1.833	0.569

Table 3: In Vitro Irritancy Score (IVIS) of the test substance, the NC and the PC

Test substance identification	Cornea-No.	Opacity per cornea*	Permeability per cornea	IVIS
				Per cornea	Per group
					Mean	SD
Test substance	7 8 9	0.0 0.0 0.0	0.925 0.443 0.577	13.9 6.6 8.7	9.7	3.7
NC	1 2 3	10.7 7.5 2.1	0.005 0.004 0.003	10.7 7.5 2.1	6.8	4.3
PC	4 5 6	93.9 84.8 10.37	1.328 2.449 1.721	113.9 121.5 129.5	121.6	7.8

* negative values are set to zero for further calculation

Applicant's summary and conclusion

Interpretation of results:

other: no severe eye irritant/no eye damage

Conclusions:

No prediction can be made for eye irritation according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL undiluted test substance to the epithelial surface of isolated bovine corneas.

The solid, waxy test substance could not be homogeneously prepared as a 20 % preparation in deionized water. Therefore, a piece of Parafilm (matched to the size of the application area) was covered with 750 µL of the undiluted (at ca. 50 °C heated) test substance and was applied directly to the epithelial surface of the cornea. Before application, the test substance was cooled down to room temperature. Three corneas were treated with the test substance for an exposure period of 4 hours. In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20 % imidazole in deionized water) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The following results were obtained in the BCOP Test:

Table 1: Mean values for opacity, permeability and IVIS of the test substance, the NC and the PC

Test substance identification	Mean Opacity Value	Mean Permeability Value	Mean In Vitro Irritancy Score
Test substance	0.0	0.648	9.7
NC	6.7	0.004	6.8
PC	94.1	1.833	121.6