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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Sep - 14 Oct 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted Jul 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Reaction mass of tetra (potassium and sodium) (2R,3R)-2-hydroxy-3-(phosphonatooxy)succinate and potassium sodium (2S,3S)-2-hydroxy-3-(phosphonatooxy)succinate
- Physical state: white to yellowish powder of crystalline appearance
- Lot/batch No.: 010-2065
- Expiration date of the lot/batch: 16 Sep 2019
- Storage condition of test material: at temperatures < 40 °C, above fresh phosphorous pentoxide or in air tight sealed containers, reduce exposure to air to a minimum
- Other: very hygroscopic substance, pH = 9.45 (1% in solution)

Test animals

Species:
human
Strain:
other: EpiDermTM; reconstructed three-dimensional human epidermis
Details on test animals or test system and environmental conditions:
TEST SKIN MODEL
- Source: MatTek In Vitro Life Science Laboaratories, s.r.o, Bratislava, Slovak Republic

TEST METHOD
EPI-200-SCT: The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay. Histological examination to demonstrate appropriate morphology of the tissue was performed and the barrier properties of the tissue were confirmed by the manufacturer. The tissue was shown by the manufacturer to be free of contamination with virus, bacteria (including myoplasma) and fungi. The ET50 was shown to fall within the predefined range.

ADAPTATION TO CELL CULTURE CONDITIONS
EpiDerm tissue samples were conditioned by pre-incubation (1 hour) in Maintenance Medium for release of transport stress-related compounds and debris in the incubator (37 °C, 5% CO₂, 95% humidity). After pre-incubation the tissue was transferred to fresh Maintenance Medium and topically exposed with the test chemicals for 3 min and 60 min, respectively. Two tissue samples were used per treatment, negative and positive controls and exposure time (12 in total). After exposure, the tissue was rinsed, blotted and assay medium was replaced by MTT assay medium 2 (final concentration: 1 mg MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue)/mL medium). After a 3-h incubation, the tissue was washed with Dulbecco's phosphate buffered saline (D-PBS), blotted and the blue formazan salt was extracted with propanol-2. The optical density of the formazan extract was determined spectrophotometrically at 540 nm and cell viability was calculated for each tissue as % of the mean of the negative control tissues. Skin corrosivity potential of the test materials was classified according to the remaining cell viability obtained after 3 minutes or 1 hour exposure with the test chemical.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37
- CO₂ gas concentration (%): 5
- Humidity: 95%

Test system

Type of coverage:
other: in vitro cell system
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Controls:
other: concurrent control tissues treated with 50 μL sterile deionised water served as negative controls, positive controls were exposed to 50 μL 8N KOH
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
Duration of treatment / exposure:
3 and 60 min
Observation period:
Not applicable
Number of animals:
Not applicable. The test was performed in duplicates for each test or control group and treatment period (3 and 60 min).
Details on study design:
TEST SITE
- Area of exposure: 0.63 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: at the end of the exposure period the test substance was carefully washed from the skin surface with Dulbecco's phosphate buffered saline
- Time after start of exposure: 3 and 60 min

CELL VIABILITY MEASUREMENTS
MTT reduction assays were performed to determine the cell viability. Tissue samples were incubated in 1 mg/mL prewarmed MTT solution for 3 h at 37 °C, 95% humidity and 5% CO₂. The precipitated blue formazan product was extracted using the solvent propanol-2, and the concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer. Cell viability measurements were carried out at the end of the exposure period (1st period: 3 min; 2nd period: 60 min). The measurements were made for each of the two tissues in triplicate.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: cell viability
Basis:
other: mean value of negative controls (distilled water)
Time point:
other: 3 min
Score:
100
Reversibility:
other: not applicable
Irritation parameter:
other: cell viability (% of negative control)
Basis:
other: mean value of the positive controls (8 N KOH)
Time point:
other: 3 min
Score:
11.3
Reversibility:
other: not applicable
Irritation parameter:
other: cell viability (% of negative control)
Basis:
other: mean value of test item (undiluted)
Time point:
other: 3 min
Score:
102.4
Reversibility:
other: not applicable
Irritation parameter:
other: cell viability
Basis:
other: mean value of negative controls (distilled water)
Time point:
other: 60 min
Score:
100
Reversibility:
other: not applicable
Irritation parameter:
other: cell viability (% of negative control)
Basis:
other: mean value of test item (undiluted)
Time point:
other: 60 min
Score:
66.2
Reversibility:
other: not applicable
Irritation parameter:
other: cell viability (% of negative control)
Basis:
other: mean value of the positive controls (8 N KOH)
Time point:
other: 60 min
Score:
8.1
Reversibility:
other: not applicable
Irritant / corrosive response data:
The mean viability of cells exposed to the test item was 102.4% after a 3 min exposure period and 66.2% after a 60 min exposure period, compared with the negative controls (100%). The optical density for the 3-min negative control, 3-min treated tissue samples and 3-min positive control was 1.747 ± 0.091, 1.789 ± 0.046 and 0.198 ± 0.024, respectively. The optical density for the 60-min negative control, 60-min treated tissue samples and 60-min positive control was 2.072 ± 0.255, 1.372 ± 0.044 and 0.168 ± 0.007, respectively.
The 3-min and the 60-min exposure values were above the cut-off limit for cell viability values (> 50% and > 15%, respectively) that distinguish corrosive from non-corrosive test substances. The test substance was non-corrosive in this skin model and is therefore predicted to be non-corrosive to human skin. The negative and positive controls were shown to be valid and fell within the historical control data range.

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive
Remarks:
Criteria used for interpretation of results: EU