Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: In vitro skin irritation test using EpiDerm
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, available as unpublished research, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 439
Qualifier:
according to
Guideline:
other: EU Method B.46
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): monoethanolamine oleate
- Physical state: light brown, highly viscous/pasty liquid
- Analytical purity: 99.8%
- Lot/batch No.: SEALS 104/1
- Expiration date of the lot/batch: 1 September 2012
- Storage condition of test material: ambient temperature

Test animals

Species:
human
Strain:
other: in vitro EpiDerm reconstructed skin membrane

Test system

Vehicle:
physiological saline
Controls:
other: phosphate buffered saline (PBS) was used as a negative control and 5% sodium dodecylsulfate (SDS) was used as a positive control (3 skin replicates in each case)
Amount / concentration applied:
25 ± 1 mg test substance mixed with 25 μL of phosphate-buffered saline
Duration of treatment / exposure:
1 hour
Observation period:
42 hours
Number of animals:
3 skin replicates for the test substance, negative and positive controls'; 2 additional skin replicates for frozen test substance and frozen negative control
Details on study design:
Preliminary tests
The tissue staining potential of the test substance was investigated by incubating 30 μl of the test substance in 300 μL demineralised water for ca. 60
min (at ca. 37ºC and 5% CO2). At the end of the exposure period the presence and intensity of the staining (if any) was evaluated visually. No change in the colour of the solution was observed. Therefore, it was concluded that the test substance did not have the potential to stain the tissue.
Some chemicals are known to non-specifically reduce MTT, resulting in a blue precipitate. Therefore, prior to the start of the study, 30 μl of the test substance was incubated in 1 mL 1 mg/mL MTT solution for ca. 3 h at ca. 37ºC and 5% CO2 and the formation of a blue formazan product was assessed visually. During incubation the test substance turned blue/purple and therefore it was concluded that it was necessary to include freeze-killed tissues (frozen controls) treated with PBS and the test substance in the main study.
A nylon mesh was used to facilitate equal distribution of liquid test substances. To test if the test substance interacts with a nylon mesh, ca. 25 mg of the test substance was mixed with 25 μL PBS and applied to a nylon mesh. After ca. 60 min incubation at ambient temperature possible interaction with the mesh was checked under a microscope. The test substance did not show interaction with a nylon mesh. Therefore, the nylon mesh was applied in the main study.

Exposure to study substances
Prior to exposure, 25 ± 1 mg of the test substance was mixed with 25 μL PBS to obtain a suspension suitable for dosing. Following the second pre-incubation, the skin models were exposed to 30 μL of the negative control or positive control, or the total volume of the mixture of the test substance in PBS. Immediately after application a nylon mesh (provided with the EPI-200 kit) was placed on the skin model surface to facilitate an equal distribution of liquid substances. Exposure was initiated at ambient temperature in the flow cabinet. After dosing the last tissue, all plates were transferred to a humidified incubator (ca. 37ºC and 5% CO2). After 35 min, the plates were removed from the incubator and placed in the flow cabin until the period of 60 min was completed for the first dosed tissue. When the exposure period was completed, the inserts were removed from the well and the skin surface was carefully washed using excess of phosphate buffered saline (PBS). Subsequently, the insert was blotted dry and the skin membranes were carefully dried using a sterile cotton swab. The inserts were then transferred to a clean 6-well plate containing fresh medium (0.9 mL/well) and incubated in a humidified incubator (ca. 37ºC and ca. 5% CO2) for an additional 42 h. Medium was refreshed after 24 h. At the end of the additional incubation period, viability was determined using the MTT assay.

MTT assay
The MTT solution of 1 mg/mL was prepared by diluting MTT concentrate 5 times in MTT diluent (provided with the MTT-100 assay kit). The bottoms of the inserts were blotted dry, and the inserts were transferred to a 24-well plate containing 300 μL of MTT solution per well. After 180 min incubation in a humidified incubator at ca. 37ºC and 5% CO2, the skin membranes were rinsed three times with PBS. The formazan product was extracted from the tissue using 2 mL MTT extractant (provided with the MTT-100 assay kit). Extraction was performed in the dark for 2 days at room temperature.
Following 2 days of extraction, the skin membrane was pierced with a needle to allow the extract to run into the well from which the skin membrane was then taken. The optical density was measured in triplicate in 200 μL sub-fractions using a spectrophotometer set at 570 nm. Extractant solvent alone was used as blank and optical density of the skin membrane extract was corrected for the blank. The mean optical density was calculated and expressed as the percentage viability compared to the negative control (mean tissue viability).

Interpretation of the results
The test is considered valid if the optical density of the negative control is ≥ 1.0 and ≤ 2.5, the optical density of the extractant solvent alone is < 0.1, if skin models treated with the positive control demonstrate a mean tissue viability ≤ 20% compared to the negative control, and if the standard deviation (SD) calculated from individual tissue viability percentages of the three replicates is < 18%. Chemicals that provide tissue viabilities in a range of 30-70% may provide high SD. If the SD above the acceptance limit is typical for the chemical, and is consistent in independent experiments, the result is considered acceptable, although the third assay acceptance criterion is not met.
The test is considered invalid if the test does not meet one or more of these acceptance criteria.
The in vitro irritation potential of the test substance is determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:
Mean tissue viability ≤ 50 %: Irritant (I), UN GHS Category 2
Mean tissue viability > 50 %: Non-irritant (NI), UN GHS No Category

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: mean tissue viability
Basis:
mean
Time point:
other: 42 hours
Score:
93
Max. score:
100
Reversibility:
other: not applicable
Remarks on result:
other: test substance
Irritation parameter:
other: mean tissue viability
Basis:
mean
Time point:
other: 42 hours
Score:
100
Max. score:
100
Reversibility:
other: not applicable
Remarks on result:
other: negative control (PBS)
Irritation parameter:
other: mean tissue viability
Basis:
mean
Time point:
other: 42 hours
Score:
5
Max. score:
100
Reversibility:
other: not applicable
Remarks on result:
other: positive control (5% SDS)
Irritant / corrosive response data:
Optical density of the negative control (PBS) and positive control (5% SDS) were within the acceptable ranges and correctly indicated non irritancy and irritancy, respectively. In the range between 20% and 100% mean tissue viability the coefficient of variation (CV) was < 18%. This demonstrated validity of the study.
The optical density of the frozen controls (FC) treated with the test substance was comparable to the optical density of frozen controls treated with the negative control (PBS). Therefore, it was not necessary to correct data for MTT reduction of the test substance.

Applicant's summary and conclusion