Ammonium 2-ethylhexyl sulphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-07 to 2019-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Version / remarks:

9th October 2017

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)

Version / remarks:

9th December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 2-ethylhexyl sulphate

EC Number:

274-625-4

EC Name:

Ammonium 2-ethylhexyl sulphate

Cas Number:

70495-37-3

Molecular formula:

C8H18O4S.H3N

IUPAC Name:

ammonium 2-ethylhexyl sulfate

Test material form:

cream / paste

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Tumkur slaughter house
- Characteristics of donor animals: 3.5 to 4.5 years old
- Storage, temperature and transport conditions of ocular tissue: To prevent exposure of the eyes to potentially irritant substances, the heads of the animals were not rinsed with detergent. Eyes were enucleated as soon as possible after death and immersed in the Hank's Balanced Salt Solution (HBSS) with 1% antibiotics (Penicillin and Streptomycin) in a suitable container and were transported to the test facility by placing in cool packs.

Test system

Vehicle:

water

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 10% w/v

VEHICLE
- Amount applied: 750 µL of distilled water

Duration of treatment / exposure:

The corneas were incubated for an exposure period of approximately 10 minutes. Similar procedure was followed for the vehicle and positive controls.

Duration of post- treatment incubation (in vitro):

1 hour and 55 minutes

Number of animals or in vitro replicates:

3

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS : Upon arrival to the test facility, eyes were examined for defects including opacity, scratches and neovascularization. Only corneas free of such defects were used in the experiment.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : No

SOLVENT CONTROL USED: Yes (distilled water)

POSITIVE CONTROL USED : Yes, Imidazole

APPLICATION DOSE AND EXPOSURE TIME : 750 µL, 10 min exposure

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE : After the exposure periode the test item and the vehicle and positive control substance were removed from the anterior chamber and the epithelium was washed with EMEM containing phenol red until no visual evidence of the test item was observed.

- POST-EXPOSURE INCUBATION: yes, 1 hour and 55 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
A test item was classified based on the obtained IVIS and categorized as mentioned below:
- Test item of IVIS score <3 would be considered as not requiring classification for eye irritation or serious eye damage according to UN GHS.
- Test item of IVIS score >3 and <55 would be considered as equivocal, subsequently testing with any other adequate method remains at the discretion of the sponsor.
- Test item of IVIS score >55 would be considered as severe irritant causing serious eye damage and classified as UN GHS category 1 without further testing.
A single testing run was considered as sufficient for assessment as the results were met according to the following criteria:
- Two of the three replicates did not give discordant predictions from the mean.
- None of the three replicates gave discordant prediction from the mean of all three rep-licates and the IVIS of the discordant replicate was not higher than 65.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

1-3

Value:

47.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Study Acceptance Criteria

The study was accepted:
- As the vehicle/negative control response resulted in opacity and permeability values less than the established upper limits of background opacity and permeability values for bovine corneas treated with the respective negative/vehicle control.
- As the positive control gave an IVIS that falls within two standard deviations of the historical mean.

Applicant's summary and conclusion

Interpretation of results:

other: Equivocal

Conclusions:

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item Ammonium 2-ethylhexyl sulfate induced an IVIS of 47.4 after 10 minutes of treatment. As the test item induced an IVIS >3 and <55, the resulting classification is equivocal and no prediction can be made regarding the corrosivity or severe irritancy to Bovine corneas.

Executive summary:

The test item, Ammonium 2-ethylhexyl sulfate was evaluated for ocular corrosivity or severe irritancy according to OECD guideline 437. Eyes of cattle were collected from a slaughter house by immersing them in the Hank's Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) in a suitable container and transported to the test facility by placing on cool packs. Eye balls free of defects were selected for the experiment. Empty cornea holder's opacity with pre-warmed Eagle's Minimum Essential Medium was measured and the mean opacity value obtained was determined as I0. Cornea holders with selected Corneas were equilibrated at 32±1°C for 1 hour 3 minutes with Eagle's Minimum Essential Medium with 1% Fetal Bovine Serum supplemented with 1% antibiotics and baseline opacity was recorded for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed for the treatment groups.

A volume of 750 µL of 10% w/v test item, vehicle (distilled water) and positive control (20% w/v imidazole) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32±1 °C for 10 minutes. Treated corneas were washed till no visual evidence of test item observed with EMEM containing phenol red and finally with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 4 mg/mL sodium fluorescein, post incubation of 85 minutes at 32±1 °C.

The test item resulted in the mean corrected opacity and mean corrected permeability values of 36.93 and 0.700, respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 47.4 and thus the result is considered as equivocal (no prediction can be made). Whereas the positive control resulted in mean corrected opacity and mean corrected permeability values of positive control are 83.56 and 1.601 respectively where the in vitro Irritancy Score (IVIS) of 107.6, indicating corrosivity or severe irritancy to Bovine cornea.

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item Ammonium 2-ethylhexyl sulfate induced an IVIS of 47.4 after 10 minutes of treatment. As the test item induced an IVIS >3 and <55, the resulting classification is equivocal and no prediction can be made regarding the corrosivity or severe irritancy to Bovine corneas.