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Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 March 2020 to 24 April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

1
Chemical structure
Reference substance name:
Reaction mass of 1-hydroxydecan-3-one and 3-(hydroxymethyl)nonan-2-one and nonan-2-one
EC Number:
909-017-6
Molecular formula:
C10H20O2 C9H28O
IUPAC Name:
Reaction mass of 1-hydroxydecan-3-one and 3-(hydroxymethyl)nonan-2-one and nonan-2-one
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
PRE-TESTS
To check the MTT-reducing capability of the test item, 50 μL of the test item were mixed per 1 mL MTT medium and incubated for 1 h at 37 ± 1 °C, 5.0% CO2 / 95% air. As the mixture turned blue/purple, the test item was 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 per exposure period were treated with 50 μL of the test item (KT) and two killed tissues were left untreated per exposure period as a control (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NC) per treatment period according to the following formula: NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100.
If non-specific MTT reduction was ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was calculated for each treatment period according to the following formula: TODTT = ODTM – (ODKT – ODKU). If the test item was classified as non-corrosive and if non-specific MTT reduction was ≤ 30% relative to the negative control of living epidermis, the correction procedure was not necessary. If non-specific MTT reduction was > 30% relative to the negative control of living epidermis, the test item was considered as incompatible with the test method.
To check the colouring potential of the test item, 50 μL of the test item were mixed per 300 μL Aqua dest. and per 300 μL isopropanol each in a transparent recipient and incubated for 1 h at 37 ± 1 °C, 5.0% CO2 / 95% air. The mixture showed no colouring detected by unaided eye-assessment. Thus, the additional test with viable tissues and the quantitative corrections were not necessary. The test item showed non-specific reduction of MTT, but no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

MAIN TEST
Upon receipt of the EpiDerm, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.
- 60 min experiment: The tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 20 sec. was kept between dosing. Then the 6-well plate was incubated at 37 ± 1 °C, 5.0% CO2 / 95% air. After 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37 ± 1 °C, 5.0% CO2 / 95% air.
- 3 min experiment: The tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing. After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL pre-warmed assay medium per well. All inserts were treated in the same manner. Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37 ± 1 °C, 5.0% CO2 / 95% air.
- 3 min and 60 min experiment: After the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert: thus, the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature. After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min. Per each tissue 3 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

NUMBER OF REPLICATE TISSUES: 2

DATA EVALUATION
- Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%) according to the following Prediction Model:
- < 50% after 3 min exposure: Corrosive
- ≥ 50% after 3 min exposure AND < 15% after 60 min exposure: Corrosive (A combination of optional Sub-categories 1B and 1C)
- ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure: Non-corrosive
- < 25% after 3 min exposure: Optional Sub-category 1A* (Prediction as optional subcategory 1A bears 29% risk of over-prediction)
- ≥ 25% after 3 min exposure: A combination of optional Sub-categories 1B and 1C

QUALITY CRITERIA
The test meets acceptance criteria if:
- Mean absolute OD570 nm of the two negative control tissues of the 3 min and 60 min treatment period is between 0.8 and 2.8
- Mean relative tissue viability of the two positive control tissues of the 60 min treatment period is ≤15%
- Coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤30%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μL
Duration of treatment / exposure:
3 min and 60 min
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
103.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
5.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
PRE-EXPERIMENTS
The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The test item turned blue/purple.
For quantitative correction of results, the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. Therefore, two tissues per treatment period were treated with the test item (KT) or left untreated (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NC) per treatment period according to the following formula:
- NSMTT (3min) = [(ODKT - ODKU)/ODNC] * 100 = [(0.125 - 0.088)/ 1.490] *100 = 2.5%
- NSMTT (60 min) = [(ODKT - ODKU)/ODNC] * 100 = [(0.146 - 0.128)/ 1.892] *100 = 1.0%
NSMTT was ≤ 30% relative to the negative control of living epidermis. In the 3 min experiment NSMTT was 2.5%, in the 60 min experiment 1.0%. This means that the test item was washed away almost completely before the addition of the MTT solution. The true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected for each treatment period according to the follwing formula:
- TODTT (3 min) = ODTM - (ODKT - ODKU) = 1.810 – (0.125 - 0.088) = 1.773
- TODTT (60 min) = ODTM - (ODKT - ODKU) = 0.102 – (0.146 - 0.128) = 0.084
The mixture of 50 μL test item per 300 μL Aqua dest. and per 300 μL isopropanol showed no colouring as compared to the solvent. Therefore, NSC equalled 0%.
The test item showed non-specific reduction of MTT, but no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570nm of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period (1.798, 2.021).
- Acceptance criteria met for positive control: The mean relative tissue viability (% negative control) of the positive control was ≤ 15% (5.9%) after 60 min treatment.
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (0.7% - 3.7%).

Applicant's summary and conclusion

Interpretation of results:
other: Skin corrosive
Remarks:
according to EU CLP (EC 1272/2008 and its amendments)
Conclusions:
Under the conditions of this study the test item was considered to corrosive to the skin, because the mean relative tissue viability (% negative control) was reduced below 15% after 60 min treatment.
Executive summary:

The skin corrosive potential of the test substance was tested in vitro using the EpiDerm™ model after a treatment period of 3 and 60 minutes. The study procedures were according to OECD TG 431 and GLP principles. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The test item turned blue/purple. For quantitative correction of results, the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. Therefore, two tissues per treatment period were treated with the test item or left untreated, respectively. NSMTT was calculated relative to the negative control of living tissues (NC) per treatment period. In the 3 min experiment NSMTT was 2.5%, in the 60 min experiment 1.0%. NSMTT was ≤ 30% relative to the negative control of living epidermis. This means that the test item was washed away almost completely before the addition of the MTT solution. The true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected for each treatment period. The mixture of 50 μL test item per 300 μL Aqua dest. and per 90 μL isopropanol showed no colouring as compared to the solvent. Therefore, non-specific color (NSC) equalled 0%. The test item showed non-specific reduction of MTT, but no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary. The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% (4.2%, NSMTT-corrected) after 60 min treatment but not below 50% (101.4%, NSMTT-corrected) after 3 min treatment. The test item is therefore corrosive when exposure last longer than 3 minutes.

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