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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Expiry Date: 20 December 2020
Storage Conditions: at room temperature, protection from light
Description: Colourless to yellow liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EpiDermTM reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
- Pre-Experiments: To check the non-specific MTT-reducing capability of the test item 30 μL of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator. If the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. For quantitative correction of results, two killed tissues were treated with 30 μL of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. All steps were performed exactly as described in the chapter below. NSMTT was then calculated relative to the negative control of living tissues (NK) according to the following formula: NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100. If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was corrected according to the following formula: TODTT = ODTM – (ODKT – ODKU). If non-specific MTT reduction is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method. To check the colouring potential of the test item 30 μL of the test item were mixed per 300 μL aqua dest. and per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min. If the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues treated with 30 μL of the test item (TVT). All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which were incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCliving) was then calculated according to the following formula: NSCliving [%] = [ODTVT/ODNK]*100. If NSCliving is ≤ 5% relative to the negative control of living epidermis, no correction of the results is necessary. If NSCliving is > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formula: TODTT = ODTM – ODTVT. If NSCliving is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method. For test items which are classified as non-irritant and which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 30 μL of the test item (TKT). All steps were then performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which was incubated in medium without MTT. The non-specific colour of additional killed tissues (NSCkilled) was then calculated according to the following formula: NSCkilled [%] = [ODTKT/ODNK]*100. The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled. If the coloured test material or MTT reducing substance is classified as “irritant” i.e. mean tissue viability is < 50%, the correction procedures are not necessary.
- Experimental Procedure: Upon receipt of the EpiDermTM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for 60 ± 5 min. Subsequently the tissues were transferred into new wells containing 0.9 mL pre-warmed assay medium per well and were incubated for 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2. After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over. Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The plates were post-incubated at 37 ± 1 C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h. After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 μL pre-warmed MTT medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%. After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 h with gentle shaking on a plate shaker. Before using the extracts, the plate had been shaken for at least 15 min on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 μL aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Data Analysis: Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance if the tissue viability after exposure and post-treatment incubation is more than 50%.
- Test Acceptance Criteria: The test meets acceptance criteria if: mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8; mean relative tissue viability of the three positive control tissues is ≤ 20%; standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
77.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixture of 30 μL of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (77.3%) after 60 min treatment and 42 h post-incubation. The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.385). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.5%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (1.4% - 15.4%).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met