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Description of key information

Data for skin- and eye irritation/corrosion are available for the target substance.

Sodium azide was tested negative in vitro for acute dermal irritating properties in the Human Skin Model Test according to the OECD Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method (9 September 2009)

Further, Sodium azide is also tested negative for eye irritation/corrosion in the BCOP test according to OECD 437.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-20 to 2010-02-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, 9 September 2009, 3rd WNT circulation, Vers. 7.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EPISKIN™ reconstructed human epidermis model which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
water
Details on test system:
Prewarming of EpiSkin tissues: After about 22 hours 30 minutes incubation of the EpiSkin tissues, they were treated with the test item. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium.

Treatment: The negative and positive control, and the test item were added into the insert atop the
concerning EpiSkin triplicate tissues. Additionally, the test item tissues were wetted with
15 μL of deionised water. The 12-well plates were placed into the incubator for 15 ± 1 min
at 37 ± 1. 5 °C, 5 ± 0.5% C02.
After the end of the treatment interval the inserts were removed immediately from the 12-
well plate. Using a wash bottle the tissues were gently rinsed with PSS to remove any
residual test material. Excess PSS was removed by gently shaking the inserts and
blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL
maintenance medium. The tissues were incubated for 42 ± 1 hour at 37 ± 1. 5 °C, 5 ±
0.5% C02.

MTT Assay: The MTT concentrate was prepared freshly and diluted with the MTT diluent. A 12-well
plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues of each time point cell culture
inserts were transferred from the holding plates to the MTT-plates. After a 3 hour
incubation period (37 ± 1.5 °C, 5 ± 0.5% C02) MTT solution was aspirated from the wells
and the wells were rinsed three times with PBS. Tissue samples were cut out of the
inserts with a biopsy punch and transferred into plastic vials. The tissue samples were
immersed into extractant solution by gently pipetting 0.5 mL extractant solution
(isopropanol) into each vial. The tissue samples were completely covered by isopropanol.
The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted
for about 69 hours without shaking in the refrigerator.
Per each tissue sample 2 x 200 μL aliquots of the formazan blue solution were
transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader
(Versamax Molecular Devices, 85737 lsmaning, Germany) at 570 nm without reference
filter. Mean values were calculated from the 2 wells per tissue sample.
Some test chemicals may reduce MTT, which will result in a blue colour without any
involvement of cellular mitochondrial dehydrogenase. Although in the present assay the
test chemicals were rinsed off and the DMEM medium beneath the tissues was replaced
before contact with MTT medium, some amount of a test chemical may be released by
the tissues into the MTT medium and directly reduce the MTT, which would be interpreted
as "tissue viability". MTT reducing capability of the test item was tested as described in
section "8.5 Test for Direct MTT Reduction". No colour change could be observed in the
present study.

Evaluation of the results: The mean OD of the three negative control tissues was calculated. This value
corresponds to 100% tissue viability in the current test. For each individual tissue treated
with the test item or the positive control the individual relative tissue viability is calculated
according to the following formula:

Relative viability (%) [OD(test item)/OD(negative control)]*100

Acceptability of the Assay: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean
OD of the three tissues is ≥ 0.6.
An assay is meeting the acceptance criterion if mean relative tissue viability of the
Positive Control is ≤ 40%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 15 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 15 µL
Duration of treatment / exposure:
15 ± 1 min
Duration of post-treatment incubation (if applicable):
42 hours further incubation without test item
Number of replicates:
3 tissue samples were equally treated with either the test item, negative or positive control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
94.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
For detailed results see "Any other information on results" Table 2

Table 2: Results after treatment with Sodium azide 

Dose group

Treat-ment Inter-val

Absor-bance 570 nm
Tissue 1*

Absor-bance 570 nm
Tissue 2*

Absor-bance 570 nm
Tissue 3*

Mean Absor-bance of 3 Tissues

Stan-dard Devia-tion

Rel. Absor-bance

[% of Negative Control]**

Negative Control

15 min

1.2448

1.2532

1.0287

1.1755

0.1273

100.0

Positive Control

15 min

0.1718

0.2067

0.1372

0.1719

0.0348

14.6

Sodium azide

15 min

1.0338

1.1438

1.1437

1.1071

0.0635

94.2

*       Mean of three replicate wells after blank correction
**
      relative absorbance [rounded values]:

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

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 non irritant to skin.
Executive summary:

This in vitro study was performed to assess the irritation potential of Sodium azide by means of the Human Skin Model Test according to the OECD Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method (9 September 2009).

Three tissues of the human skin modelEpiSkin™ were treated with either the test item, the negative or the positive control for 15 minutes. About 15 mg of Sodium azide (99.3%) were applied to each tissue, wetted with 15 µL deionisedwater, and spread to match the tissue size. 15 µL of either the negative control (deionised water) or the positive control (5% sodium lauryl sulfate) were applied to each tissue.

After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.6 for the15 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system.

After treatment with the test item sodium azide the relative absorbance values did not decrease relevantly (94.2%) compared with the results of the negative control. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item sodium azide is non irritant to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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)
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.
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.
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.

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

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.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Sodium azide was tested negative in vitro for acute dermal irritating properties in the Human Skin Model Test according to the OECD Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method (9 September 2009) and for eye irritation according to OECD 437. In the Skin Model Test, a tissue viability of 94.2% was determined.

Further, a research study from Bassendowska et al. (1961) showed no signs of dermal irritation of Sodium azide after 10 hours observation period. In contrast to this, Potokar et al. (1985) reported a corrosive effect of Sodium azide. However, these two studies cannot be used for classification since there is a lack of information on methodology and individual results.

In conclusion, the in vitro study for acute dermal irritating properties in the Human Skin Model Test according to the OECD Draft Proposal for a New Guideline will be used as key study. Therefore, Sodium azide is not considered to possess an irritant potential.

Justification for selection of skin irritation / corrosion endpoint: 

GLP study according to OECD Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method (9 September 2009)

Justification for selection of eye irritation endpoint: 

GLP study according to OECD guideline 437

Justification for classification or non-classification

Sodium azide did not cause skin effects in a dermal irritation study according to OECD Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method (9 September 2009). The substance was tested negative according to OECD 437 (BCOP). Based on these results, no classification for Sodium azide is warranted.