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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 431 and OECD 439
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: not applicable, in vitro system
Strain:
other: not applicable
Details on test animals 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 are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
It was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the EpidermTM human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov )

Test system

Type of coverage:
other: N/A
Preparation of test site:
other: N/A
Vehicle:
unchanged (no vehicle)
Controls:
other: PC and NC were concurrently applied
Amount / concentration applied:
Corrosion Test: Fifty microliter (50 μL) of the undiluted liquid test substance, negative control (NC, water) or positive control (PC, 8 n potassium hydroxide)
Irritation Test: Thirty microliter (30 μL) of the undiluted liquid test substance, NC (PBS) or PC (5% SDS)
Duration of treatment / exposure:
Corrosion Test: 3 min or 1 hr
Irritation Test: 1 hr (25 min at RT under the laminar flow hood and 35 min in the incubator)
Observation period:
Corrosion Test: Tissues were immediately washed after incubation time with PBS
Irritation Test: 42 hrs ± 2 hrs
Number of animals:
Corrosion Test: 2 tissues per exposure time (3 min at RT or 1 hr in the incubator) and test group (test material, NC, PC; 12 tissues per test)
Irritation Test: 3 tissues per test group
Details on study design:
Corrosion Test:
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 preincubation medium was replaced with fresh medium immediately before application of 50 µl of the test substance or controls. 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 by MTT solution and tissues were incubated for 3 hours. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with 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.
Irritation Test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 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. 30 µL of test material or controls were applied and tissues 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 post-incubation 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.
The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is corrosive or irritant. A test item is considered to be corrosive if the mean tissue viability (% of negative control) is < 50% after 3 min or >=50% after 3 min, but < 15% after 1 hr. Irritant potential of the test materials is predicted from the mean relative tissue viabilities
compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Value:
98
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 min. Max. score: 100.0. Reversibility: other: N/A. (migrated information)
Irritation / corrosion parameter:
other:
Value:
82
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hr. Max. score: 100.0. Reversibility: other: N/A. Remarks: Corrosion test: Mean tissue viability (% of negative control): 3 min: < 50 = corrosive; 3 min: ≥ 50 and 1 hour: < 15 = corrosive; 3 min: ≥ 50 and 1 hour: ≥ 15 = non-corrosive . (migrated information)
Irritation / corrosion parameter:
other:
Value:
5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hr. Max. score: 100.0. Reversibility: other: N/A. Remarks: Irritation test: Mean tissue viability (% of negative control) ≤ 50 = irritant; > 50 = non-irritant . (migrated information)

In vivo

Irritant / corrosive response data:
Based on the observed results and applying the evaluation criteria used in the descirbed test it was concluded, that 2-Butenoic acid, 4-oxo-4-(tridecylamino)-,(Z)-, branched shows a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: EU