Registration Dossier

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: The study was conducted according to the O.E.C.D. Draft Testing Guideline (2009) for "In Vitro Skin Irritation" with GLP compliance.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: O.E.C.D. Draft Testing Guideline (2009) for "In Vitro Skin Irritation", EPISKIN method.
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
As per IUCLI5 Sections 1.1. - 1.4.

In vitro test system

Test system:
human skin model
Source species:
human
Details on animal used as source of test system:
The study was conducted in vitro with human keratinocyte derived model tissue (EPISKIN) cultured in maintenance medium at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The test system in kit form was acquired from EPISKIN, SNC, Lyon, France.
Vehicle:
unchanged (no vehicle)
Details on test system:
The test involves the application of the test substance for 15 minutes to the EPISKIN threedimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38cm2. The EPISKIN kit included assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar. The principle of the assay 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 measured by Optical Density.

To initiate the assay the tissues were removed from the agar and placed into wells of 12 well plates containing 2 ml pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium and the positive control irritant was 5% Sodium Dodecyl Sulphate (SDS) in sterile water.

After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 ± 0.5 minutes with test substance, negative or positive control at room temperature. After 15 ± 0.5 minutes, each tissue was rinsed with 25 ml sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then 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.

The tissues were extracted by vortexing with 500 μl of acidic isopropanol (0.04 N HCl final concentration) and and storing at 2-8 ºC, protected from light, for 70 hours. Duplicate 200 μl aliquots of the extractant from each micro tube were assessed for absorbance at 540 nm with acidified
isopropanol solution as a blank.
Control samples:
yes, concurrent positive control
Amount/concentration applied:
10 uL at 100%.
Duration of treatment / exposure:
42 hours
Duration of post-treatment incubation (if applicable):
3 hours ± 5 minutes

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
8.4
Positive controls validity:
valid
Other effects / acceptance of results:
Following treatment with neat Crude 1,3-Dichloropropene, the individual relative percent viabilities of the epidermal cell cultures were, 9.8, 7.1, and 8.3 wwith a mean value of 8.4 ± 1.3%. The positive control 5% Sodium Dodecyl Sulphate (SDS) had a mean percent viability of 14.0 ± 3.0.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: O.E.C.D. Draft Testing Guideline (2009) "In Vitro Skin Irritation" with relative percent viability of < 50% is Irritant.
Conclusions:
With a relative percent viability of 8.4 ± 1.3, Crude 1,3-dichloropropene is judged to be a skin irritant. Therefore, Classification and Labeling as "Irritating to Skin" is required.
Executive summary:

Crude 1,3 -dichloropropene was assessed for skin irritating potential by O.E.C.D. Draft Testing Guideline "In Vitro Skin Irritation" with the human keratinocyte EPISKIN tissue model. With a relative percent viability of 8.4 ± 1.3, Crude 1,3-dichloropropene is judged to be a skin irritant. Therefore, Classification and Labeling as "Irritating to Skin" is required.