Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 6th to April 16th, 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
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 (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra.
- Characteristics of donor animals (e.g. age, sex, weight): 1-5 years of age.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported under cold condition in Hank's Balanced Salt Solution containing antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Time interval prior to initiating testing: eyes were used within 24 h from the slaughtering.
- indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: yes.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 20% (w/v).

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 0.9% w/v sodium chloride (normal saline).
Duration of treatment / exposure:
4 h ± 5 min
Number of animals or in vitro replicates:
3 replicates.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: All eyes were examined prior to use, corneas free from defects were dissected to a 2-3 mm rim and transferred to a container with Hank's Balanced Salt Solution. Corneas from eyes free of visible defects and with an opacity value inferior than 7 were used. The selected corneas were mounted on the corneal holders with the endothelial side against the O-ring of the posterior half of the holder, the anterior half of the holder was then placed on top of the cornea and fixed with screws. Both chambers were filled with pre-warmed phenol red free Eagle's Minimum Essential Medium (EMEM) and equilibrated at 32 ± 1ºC for at least 1h.

QUALITY CHECK OF THE ISOLATED CORNEAS: Following the equilibration period, the medium was removed from both chambers and baseline opacity readings were taken for each cornea.

NUMBER OF REPLICATES: 3

SOLVENT CONTROL USED: yes. Normal saline (0.9% w/v sodium chloride), source: Axa Parenterals Ltd, lot: A161342.

POSITIVE CONTROL USED: yes. Imidazole (20% w/v solution), source: Sigma Aldrich, lot: SLBP2962V.

APPLICATION DOSE AND EXPOSURE TIME: 750 μL of 20% (w/v) test item solution in normal saline, 4h exposure.

TREATMENT METHOD: closed chamber.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the corneal epithelium was washed until no visual evidence of test item was observed using EMEM (containing phenol red) and, once the medium was free of test item, a final rinse with phenol red-free EMEM was performed.
- POST-EXPOSURE INCUBATION: no.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: corneal opacity was measured with an opacitometer BASF-OP3.0 (Duratec, Germany)
- 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: as indicated in the TG.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.36
Vehicle controls valid:
yes
Remarks:
1.99
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks:
202.34
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.66
Vehicle controls valid:
yes
Remarks:
1.38
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks:
179.56
Irritation parameter:
other: permeability
Run / experiment:
mean
Value:
-0.02
Vehicle controls valid:
yes
Remarks:
0.040
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks:
1.518
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean
- Acceptance criteria met for negative control: yes (historical value range: -1.68 - 2.52, SD: 1.19)
- Acceptance criteria met for positive control: yes (historical value range: 63.28 - 257.23, SD: 64.49; mean = 148.9, 2*SD = 128.99)

Any other information on results incl. tables

Table 1. In vitro Irritation Score.

 

Group : Normal Saline, 0.75 mL

Cornea Holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post Treatment)

(LUX)

Post Treatment Opacity

Value

Corr. Opacity

Value

OD490

Value

Corr. OD490

Value

IVIS

1

1142

1039

4.37

1012

5.54

1.17

0.076

0.031

1.64

2

1126

1038

3.80

1011

4.95

1.15

0.071

0.026

1.54

3

1117

1044

3.21

1001

5.04

1.83

0.108

0.063

2.78

Mean

1.38

-

0.040

1.99

SD

0.39

-

0.020

0.69

  

Group : Imidazole at 20% (w/v) in normal saline, 0.75 mL

Cornea holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity

Corr. Opacity

Final Opacity

OD490

Corr. OD490

Final OD490

IVIS

Score

4

1065

990

3.44

219

154.33

150.89

149.51

1.48

1.435

1.395

170.44

5

1146

1050

4.06

184

208.72

204.66

203.28

1.499

1.454

1.414

224.49

6

1089

1026

2.87

189

190.14

187.27

185.89

1.831

1.786

1.746

212.08

Mean

179.56

-

1.558

1.518

202.34

SD

27.44

-

0.197

0.197

28.31

Group : Neohesperidin (suspension) at 20% (w/v) in Normal Saline, 0.75 mL

 

Cornea holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity

Value

Corr. Opacity

Value

Final Opacity

Value

OD490

Value

Corr. OD490

Value

Final OD490

Value

IVIS

 

8

1098

1054

2.09

1001

4.28

2.19

0.81

0.064

0.019

-0.021

0.50

 

9

1093

1005

3.91

963

5.80

1.89

0.51

0.065

0.02

-0.020

0.21

 

11

1137

1068

3.00

1019

5.04

2.04

0.66

0.065

0.02

-0.020

0.36

 

Mean

0.66

-

0.020

-0.020

0.36

 

SD

0.15

-

0.001

0.001

0.15

 

Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX.Reading with Medium and Cornea, OD490= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD490value = 0.063.

- InitialOpacity Value = [((Io⁄I)-b)/a)], Where I = Initial LUX, Io = Baseline Reading, Note: a (0.0251) and b (0.9894) are constant.

- Post TreatmentOpacity Value = [((Io⁄I)-b)/a)], Where, I = Post Treatment LUX, Io = Baseline Reading.

- Corr. Opacity Value = Post Treatment Opacity Value - Initial Opacity Value

- Final Opacity Value = Corr. Opacity Value – Mean Corr. Opacity Value of Control (Group I)

- Corr. OD490Value = OD490Value – Blank OD490Value, Where Blank Value for this trial was 0.063

- Final OD490Value = Corr. OD490Value –Mean Corr.OD490Valueof Control (Group I)

- IVIS (Control) = Corr. Opacity Value + (15 x Corrected OD490)

- IVIS (Treatment) = Final Opacity Value + (15 x Final OD490)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean IVIS score for the test item was found to be 0.36. Therefore, the test item is not irritating to the eye (no category).
Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Three sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL normal saline; the second set was the positive control, and was treated with 750 μL of 20% (w/v) imidazole in normal saline; and the third set was treated with 750 μL of 20% (w/v) of test item suspension in normal saline for 4h at 32ºC. After exposure, opacity of the corneas was measured. Then, to determine permeability, 1 mL fluorescein solution (5 mg/mL) was applied on the anterior surface of the corneas, while fresh EMEM (phenol red-free) was added to the posterior chamber and, after 90 min incubation at 32ºC, the OD (490 nm) of the medium in the posterior chamber was measured. The mean corneal opacity for the test item treated corneas was 0.66, and the mean permeability was -0.020. The mean IVIS score for the control was 1.99, the mean score for the positive control was 202.34, and the mean IVIS score for the test item was found to be 0.36. Therefore, the test item is not irritating to the eye (no category).