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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:
2010-02-23 to 2010-03-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted September 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium azide
EC Number:
247-852-1
EC Name:
Sodium azide
Cas Number:
26628-22-8
Molecular formula:
N3Na
IUPAC Name:
sodium azide
Test material form:
solid: crystalline

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES

- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Number of animals: 9
Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed
from the excised eyes. The isolated eyes were transported to the laboratory in Hank's BSS
supplemented with streptomycin penicillin at ambient temperature. The corneae were
isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation
medium composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and
Taurine, and stored in the refrigerator at 2-8 °C until the following day. Shortly before use,
Dextran was added to the medium.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of a 20% (w/v) solution of the test item were applied to the isolated corneas
Duration of treatment / exposure:
The corneas were exposed to the test item for 4 hours.
Observation period (in vivo):
After incubation with the test item, the anterior side of the corneas were incubated with sodium fluorescein solution for additional 90 minutes before the fluorescence of the posterior compartment was determined.
Number of animals or in vitro replicates:
3 cornea for each tretment: test item, positive and negative control
Details on study design:
Corneas were mounted in a cornea holder and laced in a chamber with an anterior and a posterior side which were both filled with medium and equilibrated at 32°C in a water bath. The anterior department was then exposed to the test item, dissolved in saline, for 4 h.
10% benzalconium chloride was used as positive control and saline as negative control.
After 4 hour treatment the test item was rinsed off the application side with fresh medium and the opacity of the corneas was determined.
Medium was changed and the anterio side was incubated for additional 90 minutes with sodium fluorescein solution. Subsequently the fluorescein concentration in the mposterior side medium was measured at 490 nm to serve as measure for the permeability of the cornea.

EVALUATION OF RESULTS
The change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea. The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
The corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

The following formula was used to determine the in vitro score of the negative control:
In vitro Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro score of the positive control and the test item:
In vitro Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro score was calculated for each individual treatment and positive control cornea. The mean in vitro score value of each treated group was calculated from the individual in vitro score values. Depending on the score obtained, the test item was classified into one of the categories listed in table 1.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
-0.67
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The acceptance criteria for the negative control were met. With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The mean in vitro score was calculated as 1.37.
- Acceptance criteria met for positive control: The acceptance criteria for the positive control were met. The positive control (10% (w/v) Benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The mean in vitro score was calculated as 148.56.

Any other information on results incl. tables

Table 2: Results after 240 Minutes Incubation Time


Test Group

Opacity value = Difference (t240-t0) of Opacity*

Permeability at 490 nm (OD490)*

In vitro Score

Mean in vitro Score

Proposed in vitro Irritation Scale

 

 

Mean

 

Mean

 

 

 

Negative Control

1

0.67

0.046

0.047

1.69

1.37

Non eye irritant

Negative Control

1

0.045

1.68

Negative Control

0

0.049

0.74

Positive Control

153.33*

0.020*

153.64

148.56

Very severe eye irritant

Positive Control

136.33*

0.050*

137.09

Positive Control

154.33*

0.040*

154.94

Sodium azide

- 1.67*

0.080*

- 0.46

- 0.67

Non eye irritant

Sodium azide

- 1.67*

0.056*

- 0.82

Sodium azide

- 1.67*

0.062*

- 0.73

*corrected values

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sodium azide is not considered to be an eye irritant.
Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of sodium azide by means of the BCOP assay using fresh bovine corneae according to the OECD Guideline for Testing of Chemicals 437 (September 2009).

After a first opacity measurement of the fresh bovine corneae (t0), the 20% solution in saline (w/v) of the test item sodium azide, the positive and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 2 °C. The posterior chamber contained MEM medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum (FCS) (complete medium = cMEM). After the incubation phase the test item, the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 2 °C in a horizontal position. The liquid coming out was measured spectrophotometrically.

With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (10% (w/v) benzalconium chloride) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The test item sodium azide did not cause any opacity or permeability of the corneaecompared with the results of the negative control.

The calculated mean in vitro score was -0.67 and therefore, the test item was classified as non eye irritant.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item sodium azide is not considered to be an eye irritant.