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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes

Test material

Constituent 1
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
Name of the test substance used in the study report: Sovermol Pol 1100
batch: L82

Test animals

Species:
other: Three dimensional human epidermis model
Details on test animals or test system and environmental conditions:
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
Negative and positive controls and raw material were testes in triplicate.
- Negative control: 10µl of PBS+
- Positive control: 10µl of 5% aqueous solution of Sodium Dodecyl Sulfate


Duration of treatment / exposure:
Three EpiDerm™ tissue samples were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.
Details on study design:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the
tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced 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 6 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Viability %
Value:
96
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42h. (migrated information)

In vivo

Irritant / corrosive response data:
The mean viability of the test-substance treated tissues determined after an exposure period of 15 minutes with about 42 hours post-incubation was 96%. This assay suggests that the undiluted test item is not considered as potentially irritant to skin.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information
Conclusions:
Based on the observed results it was concluded, that the test item does not show a skin irritation potential in the EpiDerm test under the test conditions chosen.
Executive summary:

The potential of the test subsatnce to cause dermal irritation was assessed by a single topical application of 30 μL of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissue samples were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/ post-incubation 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 testsubstance treated epidermal tissues is compared to that of negative control tissues. The

quotient of both values indicates the relative tissue viability. The EpiDerm skin irritation test showed the following results:

The test substance is able to reduce MTT directly. Subsequent testing of MTT reduction control was not performed, because no visible residues of the test substance remained on the tissues after washing. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 96%.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.