Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

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: Guideline study performed under GLP.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 72%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 72%) which does not affect the reliability of the study
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

Test animals

Species:
other: EPISKIN Small ModelTM
Strain:
not specified
Details on test animals and environmental conditions:
EPISKIN Small Model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 11%) occurred which were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Controls:
other: negative and positive controls were used
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 microlitres
- Concentration (if solution): undiluted

Duration of treatment / exposure:
Tissues were treated with the test material for 15 minutes and then washed with phosphate buffered saline to remove residual test material. Subsequently the skin tissues were incubated for 42 hours at 37°C.
Details on study design:
TEST SITE
- Area of exposure: Twenty five μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: Score in terms of percentage of negative control. (migrated information)

In vivo

Other effects:
The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT.

Any other information on results incl. tables

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test substance

  OD570 Mean  SD Mean tissue viability (% of control)
A B C
Negative control 0.966 1.118 1.135 1.073 ±0.093 100
Test Material 0.062 0.071 0.063 0.065 ±0.005 6
Positive control 0.084 0.108 0.135 0.109 ±0.025 10

OD = optical density SD = Standard deviation Triplicate exposures are indicated by A, B and C.

Values are corrected for background adsoption (0.041). Isopropanol was used to measure background adsorption.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test material is considered to be irritating to skin.
Executive summary:

The study was performed to OECD 439 and EU Method B.46 to assess the skin irritation potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 6%. Since the mean relative tissue viability for the test substance was below or equal to 50% after 15 minutes treatment it is considered to be irritant.