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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-25 to 2012-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants), adopted: 7 September 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test system

Vehicle:
physiological saline
Amount / concentration applied:
The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.
Duration of treatment / exposure:
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed.
Details on study design:
Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals (from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were placed into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester foil no. 1 was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob. To test the linearity of the measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured. For these, the opacitometer was supposed to display 150 and 225, respectively (± 3%). If this had not been the case, the calibration procedure would have had to be repeated. The calibration procedure was performed before each test and was documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber and replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard deviations of the current historical mean.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
ca. 1.39
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
The eye irritancy potential of Fatty acids, C16-18-, reaction products with diethanolamine was investigated in the bovine corneal opacity
and permeability assay.
The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

The following mean in vitro irritation score was calculated:
1.39
Therefore the test item was non irritant to eyes.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean
and therefore this assay is considered to be valid.

Any other information on results incl. tables

Table:   Opacity

 

Cornea No.

 

Test Item

 

Initial Opacity

 

Final Opacity

 

Change of Opacity Value

 

Corrected Opacity Value

 

1

Negative control

 

4

3

-1

 

2

4

5

1

 

3

4

5

1

 

MV

4,00

4,33

0,33

 

4

Positive control

5

174

169

168,67

5

5

172

167

166,67

6

5

178

173

172,67

MV

5,00

174,67

169,67

169,33

7

Test item

3

5

2

1,67

8

3

4

1

0,67

9

3

4

1

0,67

MV

3,00

4,33

1,33

1,00

 

 

 Table:   Permeability

 

Cornea No.

 

Test Item

 

OD490

 

Corrected OD490 Value

 

1

Negative control

 

0,031

 

2

0,029

 

3

0,027

 

MV

0,029

 

4

Positive control

2,051

2,022

5

2,098

2,069

6

2,123

2,094

MV

2,091

2,062

7

Test item

0,081

0,052

8

0,053

0,024

9

0,031

0,002

MV

0,055

0,026

 

 

Table:   In Vitro Irritation Score

 

Cornea No.

 

Test Item

 

Corrected Opacity Value

 

Corrected OD490 Value

 

IVIS

 

1

Negative control

 

-1,00

0,031

 

2

1,00

0,029

 

3

1,00

0,027

 

MV

0,33

0,029

0,77

4

Positive control

168,67

2,022

 

5

166,67

2,069

 

6

172,67

2,094

 

MV

169,33

2,062

200,26

7

Test item

1,67

0,052

 

8

0,67

0,024

 

9

0,67

0,002

 

MV

1,00

0,026

1,39

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the evaluation criteria the test item Fatty acids, C16-18-, reaction products with diethanolamine is considered as non irritant to eyes.
Executive summary:

Summary Results

The eye irritancy potential of Fatty acids, C16-18-, reaction products with diethanolamine was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item:                     diluted with physiological saline 0.9% NaCl to gain a 20% concentration

Mean in vitro irritation score:                  1.39

Evaluation: non irritant

 

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

 

Conclusion

According to the evaluation criteria the test item Fatty acids, C16-18-, reaction products with diethanolamine is considered as non irritant to eyes.