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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
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with OECD GLP regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The complete 60-min exposure to the test article was at ambient temperature, instead of 25 min at ambient temp and 35 min at 37 C as stated in OECD 439, to minimize evaporation of the test substance.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 948
- Substance type: Mono-constituent
- Physical state: Liquid
- Analytical purity: 94.4%
- Purity test date: 16 April, 2014
- Lot/batch No.: 20014, Unit 027
- Expiration date of the lot/batch: 11 August, 2016
- Storage condition of test material: Ambient temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Details on animal used as source of test system:
Commercially available EpiDerm reconstructuted human epidermis by MatTek was utilized for the test system.
Justification for test system used:
EpiDerm is an OECD-validated in vitro test system when tested according to OECD Test Guideline 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Ambient temperature to minimize test article evaporation.
- Temperature of post-treatment incubation (if applicable): 37 C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Three times with an excess of PBS
- Observable damage in the tissue due to washing: None reported
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 independent runs with tissues exposed in triplicate to the test article and controls.
PREDICTION MODEL / DECISION CRITERIA:
The in vitro irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:
- Mean tissue viability less than or equal to 50%: Irritant (GHS Category 2)
- Mean tissue viability greater than 50%: Non-irritant (Not Classified)

TEST ARTICLE EXPOSURE:
-The EpiDerm in vitro skin model was exposed to 30 uL of the test article, negative (PBS) or positive (5% aqueous SDS) control substance. Immediately after application, a nylon mesh was placed on the skin model surface to facilitate an equal distribution of these study substances. The complete 60-min exposure of all test groups was at ambient temperature, instead of 25 min at ambient temperature and 35 at 37 C as stated in OECD 439, to minimize evaporation of the test article. At the end of the exposure period, the skin models were removed from the well, carefully washed using an excess of PBS and blotted dry. The skin models were transferred to a clean 6-well plate containing fresh medium (900 uL/well) and incubated in a humidified incubator. Medium was refreshed after 24 hours. Following an additional 18 hour incubation period, viability was determined using the MTT assay.

Skin models were transferred to a 24-well plate containing 300 uL of 1 mg/mL MTT solution per well. After 180 min incubation in a humidified incubator at 37 C, the skin models were rinsed three times with an excess of PBS. The formazan product was extracted from the skin model using 2 mL MTT extractant. Extraction was performed at 2-10 C for 3 days. Following extraction, the optical density was measured in triplicate in 200 uL subfractions using a spectrophotometer set at 570 nm. MTT extractant was used as a blank. The mean optical density was calculated and expressed as a percentage viability compared to the negative control (mean tissue viability).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 uL

VEHICLE: None

NEGATIVE CONTROL: PBS
- Amount(s) applied (volume or weight): 30 uL
- Concentration (if solution): No data

POSITIVE CONTROL: 5% SDS Solution
- Amount(s) applied (volume or weight): 30 uL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 Minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Mean tissue viability
Value:
ca. 97.5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60. Max. score: 98.0. Remarks: Test article-treated tissues were had 97-98% viability of the negative control.. (migrated information)
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: None
- Colour interference with MTT: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

The mean viability of the test article-exposed EpiDerm tissues was 98 +/- 5% and 97 +/- 5% compared to the negative control group, in the first and second test, respectively.

In both tests, all acceptance criteria were met and therefore the study was considered valid.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of the study, the test article did not present evidence of dermal irritation potential and is classified as a non-irritant.
Executive summary:

The dermal irritation potential of the test article was determined using EpiDerm reconstructed skin membranes. The study was conducted in compliance with OECD GLP regulations. The test method was based on OECD Guideline 439 (2013). Two in vitro skin irritation tests were performed. In both tests, skin membranes were topically exposed to the undiluted test substance for 60 minutes. In the second test, the skin membranes were covered with a glass slide immediately after application to prevent evaporation of the test substance. In both tests, viability of the epidermal cells was assessed using the MTT test after 42 hours of culture. Negative and positive controls were run in parallel. In both tests, all acceptance criteria were met and therefore the study was considered valid. The mean viability of the test article-exposed EpiDerm tissues was 98 +/- 5% and 97 +/- 5% compared to the negative control group, in the first and second test, respectively. Based on the results of the study, the test article did not present evidence of dermal irritation potential and is classified as a non-irritant.