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Description of key information

Non-corrosive to skin


Irritating to the skin


Information on eye irritation/corrosion lacking

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Start: 24 September 2020, End: 25 September 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiSkinTM (Manufacturer: SkinEthic, France, Batch No.: 20-EKIN-036) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a collagen type I matrix coated with type IV collagen.
Justification for test system used:
EpiDerm™, a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum was selected for this in vitro test, as it is recommended by international guidelines.
Vehicle:
unchanged (no vehicle)
Details on test system:
Disks of EpiDerm™ (two units) were treated with 50 µL CA5697A and incubated for 1 hour at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere and 3 minutes at room temperature. Exposure of the test material to the EpiDerm™ surface was terminated by rinsing the units with Phosphate buffered Saline (PBS) solution. The cell viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically at 570 nm. The negative control epidermis units were treated with distilled water, whilst the positive control epidermis units were treated with 8 M KOH (two units/ control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue, viability was expressed as a percentage relative to the negative control. If the mean relative cell viability after 3 minutes is below 50% or after 3 minutes is above 50% as well as being below 15% after 1 hour, when compared to the negative control, the test item is considered to be corrosive to skin.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
50 µL of neat test substance
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours with MTT solution
Number of replicates:
2 for test substance and controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure time
Value:
>= 34.4 - <= 43.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure time
Value:
>= 10.4 - <= 13.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
The results of the in vitro study indicate that the substance is corrosive to skin.
Executive summary:

The skin corrosion potential of the substance was studied under GLP in an in vitro assay performed to OECD TG 431. Disks (two units) of the EpiDerm model, based on human keratocytes, were treated with neat test substance and incubated for 1 hour at 37 °C in an incubator with 5% CO2, in a >95% humidified atmosphere and 3 minutes at room temperature. Exposure of the EpiDerm model surface to the substance was terminated by rinsing the units with phosphate buffered saline (PBS) solution. The cell viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically at 570 nm. The negative control epidermis units were treated with distilled water, whilst the positive control epidermis units were treated with 8 M KOH (two units per control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue, viability was expressed as a percentage relative to the negative control. If the mean relative cell viability after 3 minutes is below 50% or after 3 minutes is above 50% as well as being below 15% after 1 hour, when compared to the negative control, the test item is considered to be corrosive to skin. Following exposure to the test item, the mean cell viability after 3 minutes of treatment was 38.8% and the mean cell viability after 1 hour of treatment was 12.1% compared to the negative control. These values are below the thresholds in both cases, therefore the test item was considered to be corrosive to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiSkin (Manufacturer: SkinEthic, France, Batch No.: 20-EKIN-036) is a 3-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Justification for test system used:
The EpiSkin has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. For the preparation of the killed epidermis units, living epidermis units were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 48 hours (± 1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen approximately 48 hours later; the frozen units can be used up to 6 months. Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.
The model is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number: 298-93-1) assay.
Disks of the human skin model (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the skin model's surface to the substance was terminated by rinsing the units with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2 in a >95% relative humidified (RH) atmosphere. After the 42-hour incubation, MTT solution was added to the units and incubated for a further 3 hours to determine cell viability. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm.
The negative control epidermis units were treated with PBS, whilst the positive control epidermis units were treated with 5% (w/v) sodium dodecyl sulphate (SDS) (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls (two additional disks) for non-specific colour in killed tissues (NSCkilled), were used. For each treated tissue, the viability was expressed as a percentage relative to the negative control. If the mean relative cell viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be an irritant to skin.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
10 μL of test item was applied evenly to each of three test units and each additional control skin unit. The 10 μL test item was sufficient to cover the epidermal surface. 10 μL of negative control (PBS) or positive control (5% (w/v) sodium dodecyl sulphate solution) were added to each skin unit by using a suitable pipette.
Duration of treatment / exposure:
Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis). The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 minute) at room temperature (26.2-27.8 °C). After the 15 minutes incubation time, the units were removed and rinsed thoroughly with PBS (25 mL) to remove as much of the remaining test material as possible from the epidermal surface. The remaining PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
Duration of post-treatment incubation (if applicable):
Main test: After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) and then incubated for 42 hours (± 1 hour) at 37 °C in an incubator with 5% CO2, in a >95 RH % humidified atmosphere.
MTT test: After the 42 hours incubation, all units (except the two living and two killed colour control units which were incubated with Assay Medium) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred units were incubated for 3 hours at 37 °C in an incubator with 5% CO2 protected from light, in a >95 RH % humidified atmosphere.
Number of replicates:
Number of replicates: three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. As the test item was coloured, two additional tests item-treated living tissue was used for the non-specific optical density (OD) evaluation. Furthermore, as the test item might have an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks were used.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean viability of three replicates, 15 minutes of exposure
Value:
4.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All the validity and acceptability parameters were met and the study was considered to be acceptable and valid.
Direct MTT reduction: After the test item had been incubated with MTT for three hours, yellow colour with purple precipitate of the mixture was detected in the test tube. Thus, the test item was considered to react with MTT and therefore, additional controls were used in the experiment.
Additional controls: As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues was 0.027, the non-specific colour percent was calculated as 3.9%. This value was below 5%, therefore an additional data calculation to account for non-specific colouring was not necessary.
As colour change was observed (yellow colour with purple precipitate) after a three hour incubation period of the test item in MTT working solution, the potential for the test material to interact with MTT was identified. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on the non-specific MTT reduction (NSMTT) value (-0.069), the calculated NSMTT% was -10.0%. Negative values were not taken into account in the calculation, therefore no correction with NSMTT was used in this study.
As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.017 and the Non-Specific Colour % (NSCkilled%) was calculated as 2.4%. The NSCliving% was not used for calculation, therefore the correction with NSCkilled% was not necessary.

Optical density (OD) and calculated relative cell viability (%) compared to negative control







































































































SubstanceReplicateOptical density Viability 
  MeasuredBlank corrected(% RV)
Negative control10.7260.68199.7
Phosphate buffered saline20.7060.66196.7
 30.7530.708103.6
Positive controlMean--0.683100.0
5% (w/v)10.1950.15021.9
Sodium dodecyl sulphate20.1680.12318.0
 30.1710.12618.4
 Mean--0.13319.4
Test substance10.0790.0345.0
 20.0700.0253.7
 30.0780.0334.8
 Mean--0.0314.5
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Based on the mean cell viability of 4.5% compared to the negative control, the substance was considered to be irritating to skin.
Executive summary:

The in vitro skin irritation potential of the substance was investigated under GLP in a reconstructed human epidermis model to OECD TG 439. The used model is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number: 298-93-1) assay.
Disks of the human skin model (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test model's surface to the substance was terminated by rinsing the units with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2 in a >95% relative humidified (RH) atmosphere. After the 42-hour incubation MTT solution was added to the units and incubated for a further 3 hours to determine cell viability. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm.
The negative control epidermis units were treated with PBS, whilst the positive control epidermis units were treated with 5% (w/v) sodium dodecyl sulphate (SDS) (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls (two additional disks) for non-specific colour in killed tissues (NSCkilled), were used. For each treated tissue, the viability was expressed as a percentage relative to the negative control. If the mean relative cell viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be an irritant to skin. Following exposure to the substance, the mean cell viability was 4.5% compared to the negative control. Therefore the test item was considered as being irritant to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))
Deviations:
no
GLP compliance:
no
Test system:
isolated skin discs
Source species:
rat
Cell type:
non-transformed keratinocytes
Cell source:
other: pelt of humanely killed young Wistar strain rat
Source strain:
Wistar
Details on animal used as source of test system:
Not available
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Not reported
Duration of treatment / exposure:
24 hours
Number of replicates:
Three
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
Mean
Value:
22.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
13.4
Positive controls validity:
valid
Remarks:
0.97
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the in vitro TER study, the substance is unlikely to be corrosive to skin in vivo.
Executive summary:

An in vitro skin corrosion study following the principles of OECD TG 430 (transcutaneous electrical resistance assay) was conducted with the substance under non-GLP conditions. Due to the poor level of documentation, the reliability of the test cannot be assessed. Skin discs were prepared from the pelt of a humanely killed young Wistar strain rat. Three discs were used to test the corrosion potential of the test substance, a negative and a positive control. All substances were applied as such to the surfaces of the skin discs for a period of 24 hours, and then removed with a jet of warm water. The electrical resistance of the skin was then measured. The mean electrical resistance measured was 22.7 kOhms for the test substance, 13.4 kOhms for the negative control and 0.97 kOhms for the positive control. Based on these results, the substance is considered unlikely to be corrosive to the skin in vivo.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A Transcutaneous Electrical Resistance (TER) assay was performed in 2013. The test item was applied to 3 skin discs for 24 hours and the TER measured using a low voltage alternating current electronic data bridge. The mean electrical resistance of skin discs treated with the substance was 22.7 kΩ, which was clearly above the threshold value indicative of potential skin corrosion (5 kΩ). The substance was considered unlikely to have the potential to cause corrosion.
In a bottom-up approach, a skin corrosion study was performed under GLP in 2020 (EpiDerm Model, OECD 431). The substance was applied to 2 EpiDerm disks that were incubated at room temperature for 3 minutes or at 37 °C for 1 hour before being rinsed with PBS. MTT was added, disks were incubated, and formazan extracted and quantified. The mean cell viability after 3 minutes of treatment was 38.8% and after 1 hour of treatment was 12.1% compared to the negative control. Based on these results, the test item was considered to be corrosive to skin. The EpiDerm corrosion result was unexpected since the TER study showed the test item was non-corrosive and the pH value of the substance was 6.13. Furthermore, a screening test on the acute dermal toxicity, in which a dose of 1000 mg/kg bw of substance was applied to the skin of three rats, produced no clinical signs and only minor dermal reactions, including very slight erythema, small superficial scattered scabs and glossy skin. No oedema was observed in this screening study. Taking all the data into consideration, it suggested the EpiDerm study was giving a false positive result. Factors that may contribute to inaccurate viability assessment in the EpiDerm model include increased metabolism in response to cell damage (hormesis) and test substance induced direct reduction of MTT.
In 2020, an in vitro skin irritation assay (EpiSkin Model, OECD 439) was therefore performed under GLP. Substance was applied to 3 EpiSkin disks, which were incubated for 15 minutes at room temperature, rinsed with PBS and incubated for 42 hours at 37 °C. MTT was then added and the disks were incubated for a further 3 hours. Formazan precipitate, resulting from the reduction of MTT, was extracted and quantified to determine cell viability. After exposure to the substance, the mean cell viability was 4.5%, which is clearly less than the threshold of 50%. The test item was considered to be a skin irritant.

Justification for classification or non-classification

Based on the available data and applying a weight-of-evidence approach, the substance is considered to be not corrosive to the skin. In consideration of the positive result obtained in the in vitro study using the EpiSkin model performed to OECD TG 439, the substance should be classified for skin irritation in accordance with Regulation (EC) No 1272/2008.