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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05.12.2016-19.01.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl salicylate
EC Number:
204-265-5
EC Name:
Ethyl salicylate
Cas Number:
118-61-6
Molecular formula:
C9H10O3
IUPAC Name:
ethyl 2-hydroxybenzoate
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- Source: MatTek Corporation
- Cell culture: The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm Ø).
- Pre-incubation period: pre-incubation phase of the EpiDerm™ tissues started on the day of receipt.
- Treatment: EpiDerm tissues were treated with the test substance
- Amount/test concentration: 50 µL
- Duration of treatment: 3 minutes, 60 minutes

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure : room tempeature
- Temperature of post-treatment incubation (if applicable): 37+-1.5°C

CONTROL
- Negative Control: 50 µL deionised water was used as negative control per tissue;
- Positive Control: 50 µL 8.0 N potassium hydoxide (Sigma) was used a positive control per tissue

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing (if done): Tissues washed with DPBS at least 20 times in order to remove any residual test material. Exess DPBS was removed by gently shaking the tissue inserts and blotting the lower surface with blotting paper.

SCORING SYSTEM:
Viability measured using MTT assay
mean tissue viability < 50% after 3 minutes exposure: corrosive
mean tissue viability ≥ 50% after 3 minutes exposure AND < 15% after 60 minutes exposure: corrosive
mean tissue viability ≥ 50% after 3 minutes exposure AND = 15% after 60 minutes exposure: non-corrosive
Test Item Identified as Corrosive:
< 25% after 3 minutes exposure: Optional Sub-category 1A
≥ 25% after 3 minutes exposure: A combination of optional Sub-categories 1B and 1C

Historical data positive control:
Mean Viability: 21.60% (3 min), 7.02% (1 h); CV: 9.52% and 14.72%, respectively;
Range of Viabilities: 4.60 – 39.83%.
Historical data negative control:
Mean Absorption: 1.68 (3 min), 1.65 (1 h); CV:
4.87 and 3.19, respectively;
Range of Absorbance: 1.34 – 1.93 (3 min) and 1.32 – 1.85 (1h)

Acceptability of the assay:
An assay met the acceptance criteria if
- the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
- the mean viability of the tissue replicates treated with the positive control for 1 hour is <15% compared to the negative control
- the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30%


Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Test item: 50 μL (79.4 μL/cm2 according to guideline) of undiluted test item
- Controls: each 50 μL
Duration of treatment / exposure:
3 minutes; 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min. incubation time
Value:
>= 111.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not considered to be corrosive.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour incubation time
Value:
>= 117.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not considered to be corrosive
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT:it did not change colour
- After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Any other information on results incl. tables

Results after treatment with the test item and the controls

Dose Group Exposure Interval (min) Mean Absorbance
(OD) of 2 tissues
Stand. Dev.
[%]
Mean Rel.
Absorbance [% of
Negative
Control]
Negative Control 3 1.577 3.1 100
Test Item  1.765 2.2 111.9
Positive Control 0.459 3.4 29.1
Negative Control 60 1.518 0.3 100
Test Item  1.782 1.9 117.4
Positive Control 0.153 12 10.1

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 reported experimental conditions, the test item is non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

This in vitro study was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models. The test was performed according to OECD 431 and EU-Method B.40 and in compliance to GLP.

The test item passed the colour and the MTT interference pre-tests. Independent duplicate tissues of EpiDerm were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for about 22.5 hours. The required acceptability criteria were met.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (29.1%) and for the 1 hour exposure period (10.1%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item the corrected relative absorbance value were not reduced both after 3 minutes exposure and after 1 hour exposure (111.9% and 117.4%, respectively). Therefore, the test item is not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item is non corrosive to skin according to EU CLP and UN GHS.