1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June, 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

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:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

EC Number:

268-761-3

EC Name:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

Cas Number:

68139-30-0

Molecular formula:

C9H19N2O5S(CnH(2n+1)), (n=7,9,11,13,15,17)

IUPAC Name:

1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-coco acyl derivs., hydroxides, inner salts

Test material form:

solid

Specific details on test material used for the study:

Batch: DSP-001.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Local abbatoir.
- Characteristics of donor animals: 12-60 months old.
- Storage, temperature and transport conditions of ocular tissue: Placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL).
- Time interval prior to initiating testing: Transported to the test facility over ice packs on the same day of slaughter.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL.
- Concentration: The test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

10 minutes.

Duration of post- treatment incubation (in vitro):

4 hours.

Number of animals or in vitro replicates:

Triplicate (3).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES
3 for test item, 3 for negative control, 3 for positive control.

NEGATIVE CONTROL USED
Sodium chloride 0.9% w/v.

POSITIVE CONTROL USED
Imidazole 20% w/v.

APPLICATION DOSE AND EXPOSURE TIME
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

TREATMENT METHOD: Closed chamber.

POST-INCUBATION PERIOD: Yes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed at least 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

- POST-EXPOSURE INCUBATION: 32 ± 1 ºC for 240 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of Labtech LT-4500 microplate reader (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) .

DECISION CRITERIA:
IVIS = mean opacity value + (15 x mean permeability OD492 value).
Additionally, the opacity and permeability values are evaluated independently to determine whether the test item induced a response through only one of the two endpoints.
The condition of the cornea is visually assessed post treatment and post incubation.
The test item is classified according to the following prediction model:
IVIS Classification:
≤ 3: No category. Not requiring classification to UN GHS or EU CLP;
> 3: ≤55 No prediction of eye irritation can be made;
> 55: Category 1. UN GHS or EU CLP Causes serious eye damage.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

15.1

Negative controls validity:

valid

Remarks:

The negative control gave opacity of ≤2.4 and permeability ≤0.072.

Positive controls validity:

valid

Remarks:

The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3.

Remarks on result:

not determinable

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Test item / 9

Value:

8

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Test item / 10

Value:

15

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

Test item / 11

Value:

7

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

Negative control / 2

Value:

0.008

Results 6

Irritation parameter:

cornea opacity score

Run / experiment:

Negative control / 2

Value:

0

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

Negative control / 4

Value:

1

Results 8

Irritation parameter:

cornea opacity score

Run / experiment:

Positive control / 5

Value:

53

Results 9

Irritation parameter:

cornea opacity score

Run / experiment:

Positive control / 6

Value:

70

Results 10

Irritation parameter:

cornea opacity score

Run / experiment:

Positive control / 7

Value:

64

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were partly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean (n=3) corrected test item corneal opacity was measured at 10.0.

Mean corrected permeability (OD492) of test item was 0.341.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Baed on the results of the study, no prediction of eye effects could be made.

Executive summary:

A GLP-compliant in vitro eye damage study was performed on the substance in accordance with the OECD Testing Guideline 437. The IVIS score obtained was >3 and ≤ 55, therefore, no prediction could be made and a subsequent test was comissioned following OECD Testing Guideline 492.