Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 16 July 2018 to 17 July 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Updated information. Study now available and provided ahead of extended submission date of 31/05/2019.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

EC Number:

271-672-2

EC Name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Cas Number:

68603-74-7

Molecular formula:

The substance is a UVCB so there is no molecular formula

IUPAC Name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Source of Bovine Eyes
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. Eyes were transported in physiological saline in a suitable container under cooled conditions.

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. The isolated corneas were mounted in a corneal holder (one cornea per holder) 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 °C. The corneas were incubated for the minimum of 1 hour at 32 °C.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied directly on the corneas in such a way that the cornea was completely covered. This required between 313.1 to 469.6 mg of test item. No correction was made for the purity/composition of the test item.

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

None

Number of animals or in vitro replicates:

Three replicates (corneas) per treatment (test substance, negative control, positive control)

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS: Yes

NUMBER OF REPLICATES: Three per treatment

NEGATIVE CONTROL USED: Yes, physiological saline

SOLVENT CONTROL USED (if applicable): Not Applicable

POSITIVE CONTROL USED: Yes, 20% v/v Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 240 minutes

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM (Earle’s Minimum Essential Medium).
- POST-EXPOSURE INCUBATION: None

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured using microtitre plate reader (TECAN Infinite® M200 Pro Plate Reader) at 490nm.
- Others (e.g, pertinent visual observations, histopathology): Each cornea was inspected visually for dissimilar opacity patterns and possible pH effects of the test item on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As specified in the test guideline.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Substance (mean of three replicates)

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- pH Effects: No pH effect of the test substance was observed on the rinsing medium at the post incubation washing stage.

- Visible damage on test system
The test substance treated corneas were translucent with clear spots after the 240 minutes of treatment.
The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the negative control item were clear after the 240 minutes of treatment.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

The Opacity, Permeability and In Vitro Irrtancy Scores are summarised in the table below:

Treatment	Mean Opacity	Mean Permeability	Mean In Vitro Irrtancy Score
Negative Control	1.5	0.006	1.6
Positive Control	123	2.387	159
Test Item	0.6	5.640	85

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test substance, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates, induced an IVIS = 55 and it was therefore concluded that it induced serious eye damage under the experimental conditions used in the study and that it should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

 Introduction

The objective of this study was to evaluate the eye hazard potential of the test substance, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates, using the Bovine Corneal Opacity and Permeability test (BCOP test). The method used was designed to meet the requirements of the following guideline:

• 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, (adopted October 09, 2017).

 

Method

The undiluted test item was applied via topical application for 240 minutes to isolated bovine corneas. The eye damage of the test item was tested through topical application for approximately 240 minutes. Negative and positive controls were tested concurrently. Two endpoints, corneal opacity and the passage of sodium fluorescein dye through the cornea (permeability) were assessed and used to generate an In Vitro Irritancy Score.

The test item was classified according to the prediction model below:

In Vitro Irritancy Score (IVIS)	UN GHS
= 3	No Category
> 3; = 55	No prediction can be made
>55	Category 1

 

Results

The test substance induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 85 after 4 hours of treatment.

 

Conclusion

The test substance, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates, induced an IVIS = 55 and it was therefore concluded that it induced serious eye damage under the experimental conditions used in the study and that it should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.