Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-12-08 to 2009-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to a protocol which is comparable to guideline. It is also compliant with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD 439 (2010)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
human
Strain:
other: not applicable
Details on test animals and environmental conditions:
The test uses EPISKIN human epidermis skin constructs consisting of normal, human-derived epidermal keratinocytes, that have been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum.

Test system

Type of coverage:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
10 ul:
TS neat
Negative control = sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium.
Positive control was 5% Sodium Dodecyl Sulphate (SDS) in distilled water.
Duration of treatment / exposure:
15 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:

The principle of the test is that irritant substances are sufficiently cytotoxic to cause cell death in the cell layers. The cell viability is determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 minutes with test substance, negative or positive control at room temperature. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes application time. After 15 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

After 42 ± 1 hour each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. At the end of 3 hours ± 5 minutes the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube. When all tissues had been punched, the tissues were vortexed with 500 DL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted by storing at 2-8 oC, protected from light, for a minimum of 70 hours. After formazan extraction, duplicate 200 μL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm with acidified isopropanol solution as a blank.

The negative control was sterile Dulbecco’s Phosphate Buffered Saline with magnesium and calcium.

The positive control was 5% Sodium Dodecyl Sulphate in distilled water.

Results and discussion

In vivo

Irritant / corrosive response data:
MEAN TISSUE VIABILITY (compared to negative control) :
Test substance, ethylthioethanol 77.4% +/-6 (predicted non-irritant)
Positive control 13.3% +/-4 (predicted irritant)
Negative control 100% +/- 9
(Mean tissue viability greater than 50% is considered to indicate non-irritancy.)

Any other information on results incl. tables

No evidence of interaction of test substance with MTT.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
An in vitro study, conducted using EPISKIN (a human epidermis skin construct) in a manner similar to OECD 439 (2010) and with GLP reported mean tissue viability following exposure of 77.4%, indicating that the test material was unlikely to be irritant to the skin.