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Toxicological information

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:
Nov 2015
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2015)
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The test system epiCS is commercially available and the laboratory has been validated with this test system in a multicenter validation study.
Vehicle:
unchanged (no vehicle)
Details on test system:
DETAILS OF THE TEST PROCEDURE USED
The irritant potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item. All tests were performed in triplets for each time point. The test item was applied unchanged, i.e. 30 mg per insert for 20 min. (room temperature), plus 30 µL saline to moisten and ensure good contact to the tissue surface. After the exposure a post-treatment incubation period of 42 h of the rinsed tissue in the incubator followed. Then, cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density compared to negative control).

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): 37 ± 2° C (incubator temperature)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Incubation conditions: 37 ± 2° C, 5 % CO2, maximum humidity
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 for test substance, positive and negative control (for determination of cell viability the absorption of the isopropanol-extracts were measured in duplicates = 6 OD values for each)

DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- Classification according to UN GHS: The test substance is considered to be at least irritant to skin if the viability after 20 minutes exposure is less than or equal to 50% (Category 2 or 1; depending on outcome of corosivity test). The test substance is considered to be non-irritant to skin if the viability after 20 minutes exposure is greater than 50% .
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 mg per insert
Duration of treatment / exposure:
20 min. (room temperature)
Duration of post-treatment incubation (if applicable):
42 hours (37 °C)
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
91.75
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
The reliability of the test conduction was previously confirmed in an interlaboratory validation study.

Any other information on results incl. tables

Summary of results

Sample No. 

 Test item  OD mean*  StdDev  % Viability
 1 -3  Negative control (NaCl 0.9 %)  2.33  0.04  100.00
 4 -6  Positive control (SDS 5 %)  0.03  0.00  1.09
 10 -12  Test item, undiluted  2.14  0.11

91.75

*: 6 values

Applicant's summary and conclusion

Conclusions:
No irritant potential to the skin conlcuded when tested in an in vitro test according to OECD TG 439.
Executive summary:

An in vitro study was performed for the assessment of skin irritation on a reconstructed human epidermis (RhE; epiCS®). The experiment was carried out in accordance to OECD TG 439 with the neat substance. 30 mg of the test item was applied topically on the RhE, plus 30 µL saline to moisten and ensure good contact with the tissue surface. After an exposure period of 20 minutes (room temperature) and a post-exposure incubation of 42 hours (37 °C, 5 % CO2, maximum humidity), the cell viability was 91.8 %, as measured by a MTT conversion assay. The test item was thus not considered to have an irritant potential to the skin.