Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

opacity & permeability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 January 2018 to 07 March 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:

other: No. 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”, adopted on 9th October 2017

Version / remarks:

9th October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

EC Number:

947-922-8

Molecular formula:

n.a. (UVCB)

IUPAC Name:

Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

Test material form:

liquid

Details on test material:

clear yellowish liquid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Slaughter House
- Age at study initiation: 3.5 to 4.5 years

Test system

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 Hours

Number of animals or in vitro replicates:

Triplicates

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS: Done

NUMBER OF REPLICATES: Triplicates

NEGATIVE CONTROL USED: Normal saline

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME : 750 µL & 10 mins
TREATMENT METHOD: [closed chamber]

POST-INCUBATION PERIOD: yes, duration: 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times
- POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Done
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry] (OD490) : Done
- Others: histopathology

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1
SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES

NEGATIVE CONTROL USED

SOLVENT CONTROL USED (if applicable)

POSITIVE CONTROL USED

APPLICATION DOSE AND EXPOSURE TIME

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [ spectrophotometry] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean-Test item

Value:

95

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean-Positive control

Value:

116.4

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: No

In vivo

Other effects:

- Histopathological findings: Epithelium: Mild focal squamous cell coagulation
Stroma: Apparently normal
Endothelium: Apparently normal

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on the results obtained in the Bovine Corneal Opacity Test, the test item induced an IVIS of 95.0 after 10 minutes of treatment. The results indicated an appreciable increase in both the endpoints “permeability” as well as “opacity”. As the test item resulted in IVIS >55, considered as severe irritant causing serious eye damage and classified as UN GHS category 1, and no further testing will be carried out.

Executive summary:

Eyes of cattle were collected from 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±1ºC for 1 hour 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.Quantity of 750 µL of test item, normal saline (negative control) and ethanol (positive control) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32±1ºC for 10 minutes. Treated corneas were washed with EMEM with 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 90 min at 32±1ºC. Baseline opacity and permeability values obtained for negative control (normal saline) treated corneas were used for correction.The mean corrected opacity and mean corrected permeability values of test item is 71.31 and 1.581 respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 95.0 indicating corrosivity or severe irritancy to Bovine corneas.

Whereas the positive control resulted in the mean corrected opacity and mean corrected permeability values is 91.91 and 1.633 respectively. Thein vitroIrritancy Score (IVIS) of positive control resulted in 116.4, indicatingcorrosivity or severe irritancy to Bovine corneas.

In histopathological examination, the corneas treated with negative control did not show any abnormalities, whereas in thehistopathological examination ofthe corneas treated with test item resulted in mild focal squamous cell coagulation in epithilium. Stroma and endothelium of all corneas were apparently normal. 

In positive control, histopathology data of epitheliumshowed mild diffuse and moderate multifocal cytoplasmic and nuclear vacuolization in the wing and basal layers.In stroma minimal multifocal expansion of the superficial collagen was observed. Endothelium was apparently normal in all three corneas.