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Skin irritation / corrosion

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Administrative data

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

Data source

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

Materials and methods

Test guideline
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 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

In vitro test system

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.

Results and discussion

In vitro

Results
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

Any other information on results incl. tables

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.

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 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.