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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:
July 2018 - Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
18 June 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Sulfuric acid, mono-C8-18-alkyl esters, magnesium salts, compds. with triethanolamine
EC Number:
287-840-3
EC Name:
Sulfuric acid, mono-C8-18-alkyl esters, magnesium salts, compds. with triethanolamine
Cas Number:
85586-38-5
IUPAC Name:
85586-38-5
Test material form:
solid
Remarks:
dried from of a reaction mixture, water and cyclohexanol where evaporated
Details on test material:
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: February 2020
Storage conditions: Room temperature
pH-value: Approx. 7 (moistened with water, determined by Bioassay Laboratories)
Specific details on test material used for the study:
Solid / white

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Vehicle:
unchanged (no vehicle)
Details on test system:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour, but not more than 1.5 hours before test substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The pre-incubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. In addition, two killed control tissues per exposure time were treated with the test substance and the NC, respectively, to detect direct MTT reduction.

Due to the texture of the solid test substance the application with a sharp spoon was not
possible. Therefore, a metal pin (matched to the size of the tissue) was covered with
ca. 25 mg ground undiluted test substance and was applied with direct contact to the skin.
Before application the tissues were wetted with 25 μL deionized water

Control tissues were treated concurrently with 50 μL deionized water (NC, NC KC) or with 50 μL 8 N potassium hydroxide solution (PC) or test substance (KC).A nylon mesh was placed carefully onto the tissue surface of the NC afterwards (2nd test run).

The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.

Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced with MTT solution, and the tissues were incubated for 3 hours.

After incubation, the tissues were washed with PBS to stop the MTT incubation. The formazan that was produced metabolically by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
Control samples:
yes, concurrent negative control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
(Skin irritation test, SIT)
- Amount(s) applied (volume or weight with unit):
25mg solid ground material per tissue

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


POSITIVE CONTROL
(KOH)
- Amount(s) applied (volume or weight):
50 µL



Duration of treatment / exposure:
3 minutes and 1 h
Duration of post-treatment incubation (if applicable):
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed.
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2 (SCT 1h 2nd run)
Value:
25.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1 (SCT 1h 2nd run)
Value:
25.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2 (SCT 3 min 2nd run)
Value:
42.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1 (SCT 3 min 2nd run)
Value:
31.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1 (SCT, 3min)
Value:
27.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 1st test run
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2 (SCT, 3min)
Value:
26.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 1st test run
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1 (SCT, 1h)
Value:
38.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 1st test run
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2 (SCT, 1h)
Value:
34.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 1st test run
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:
no
- Direct-MTT reduction:
no
- Colour interference with MTT:
no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes


Acceptance criteria for the positive control (PC) in Skin corrosion test:
8 N potassium hydroxide solution is used as positive reference.
A tissue viability of ≤ 30% is acceptable for the 3-minute exposure. Mean viability of the tissues exposed for 1 hour should be <15%.

Acceptance criteria for the variability of the tissues in Skin corrosion test:
For every treatment, two tissues are treated in parallel. In the range of 20% and 100% viability, variability between the tissues is considered to be acceptable if the coefficient of variation (CV) of % viability is ≤ 30%.

Acceptance criteria for the killed controls (KC)
The OD570 of the tissues for the KC of the NC should be ≤ 0.35. The OD570 value for direct MTT reduction of a test substance should be ≤ 30% of the OD570 of the NC.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
Based on the results observed and by applying the evaluation criteria described, it was concluded the test item has an intermediate or a weak corrosive potential and may be assigned to UN GHS skin corrosivity subcategories 1B or 1C as cited in current OECD TG 431
Executive summary:

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of ca. 25 mg ground undiluted test substance to a reconstructed threedimensional human epidermis model (EpiDerm™) according to OECD 431 (BASF 2019).


Due to the texture of the solid test substance the application with a sharp spoon was not possible. Therefore, a metal pin (matched to the size of the tissue) was covered with ca. 25 mg ground undiluted test substance and was applied with direct contact to the skin. Before application the tissues were wetted with 25 μL deionized water or PBS. For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each.


Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.


In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and of a positive control (8 N KOH) were applied to two tissues each per exposure period.


The test substance is not able to reduce MTT directly.


Two test runs of the Skin Corrosion Test were performed.
Results of the 1st test run:
The relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 27.2%, and it was 36.8% after an exposure period of 1 hour. The results indicated a corrosive potential of the test substance at the 3-minutes exposure period. However, the result of the 1-hour exposure lies above the cutoff for skin corrosion. To clarify the inconclusive result the study was repeated.


Results of the 2nd test run:
The relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 36.8%, and it was 25.4% after an exposure period of 1 hour. Thus, the 2nd test run verified the results of the 1st test run.


The acceptance criteria for the variability of the tissues are met.


Overall, the results of the skin corrosion test indicate a corrosive potential of the test substance. The results after the 3-minutes exposure period indicates that the test substance has an intermediate or a weak corrosive potential and may be assigned to UN GHS skin corrosivity subcategories 1B or 1C as cited in current OECD TG 431


Based on the results observed it was concluded that the test item shows a skin corrosion potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.