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Description of key information

Skin irritation/corrosion:

Under the conditions of the in vitro EPISKIN model test (OECD No. 431 guideline) with AEEU, the test item was found to be corrosive to the skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-25 to 2016-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed in accordance with OECD guideline 431.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: 3D-human epidermis model
Species:
human
Strain:
other: EPISKIN-SM: 3D-human epidermis model
Details on test animals or test system and environmental conditions:
EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:15-EKIN-039, Expiry Date: 05 October 2015 used in Experiment I., Batch No.:15-EKIN-044, Expiry Date: 09 November 2015 used in Experiment II.) is a three-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. 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 killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:15-EKIN-034, Expiry Date: 31 August 2015 used in Experiment I.,) Batch No.:15-EKIN-042, Expiry Date: 26 October 2015 used in Experiment II.) 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 on 28 August 2015 in Experiment I., 24 October 2015 in Experiment II. (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.

EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001).

The EPISKIN-SM kits were kept in their packaging at 37°C, the assay medium and maintenance medium supplied with the kits were stored at 2-8°C until the initiation of the test.
The EPISKIN-SM model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD Guideline 431); therefore, it was considered to be suitable for evaluation of this endpoint.

24 h before treatment, the Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere.
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: see 'Any other information on materials and methods incl. tables'
Amount / concentration applied:
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control substance at each time point in each experiment. Two epidermis units were used for positive control in 1 hour and 4 hour assays (the positive control incubated for 1 hour acted as control for both the 3 minutes and 1 hour treatment periods in Experiment II).

- 20 mg of the test item was applied evenly to the epidermal surface of each of two test units and two additional colour control skin units and then 100 µL physiological saline was added to the test item to ensure good contact with the epidermis. If necessary, the test item was spread gently on the skin surface with a curved flat spatula (or other appropriate tool) without damaging the epidermis.
- 50 µL of physiological saline was added to each of the two negative control skin units.
- 50 µL of glacial acetic acid was added to each of the two positive control skin units.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (23.2-23.9°C in Experiment I.), for 3 minutes and 1 hours (23.1-23.7°C in Experiment II.) covered with the plate lids.
Observation period:
Rinsing:
After the incubation time (3 minutes, 1 hours or 4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with approximately 25 mL PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly. The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis) and the culture medium was removed from the assay plate wells.

MMT test:
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5 % CO2 for 3 hours, protected from light.

Formazan extraction:
At the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurement:
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples were measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
Number of animals:
In each experiment, two replicates per test item per time point were used. Two negative controls were run in each assay. Two positive controls were run at 1 hour and 4 hour in assay. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation in each experiment. Furthermore, as the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used in the experiments.
Irritation parameter:
other: mean relative viability
Basis:
mean
Time point:
other: 4 hours
Score:
31.6
Max. score:
100
Reversibility:
other: not applicable
Remarks on result:
other: unit score: percentage
Irritant / corrosive response data:
Following exposure with AEEU, the mean relative viability after 3 minutes of treatment was 89.8%, the mean relative viability after 1 hour treatment was 81.3%, and the mean relative viability after 4 hours of treatment was 31.6% compared to the appropriate negative control values of the performed experiments (after adjustment for non-specific MTT reduction). The observed relative viability value was above the threshold of 35%, therefore the test item was considered as being non-corrosive in case of the 3 minutes and 1 hour exposure period. However, the observed relative viability value was below the threshold of 35%, therefore the test item was considered as being corrosive in case of the 4 hours exposure periods. Therefore, the test item is classified as Category 1B or 1C (or Corrosive class II or III) according to the GHS / CLP classification. Each experiment met the validity criteria, therefore the study was considered to be valid.

Validity of the test:

After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions in each case. The mean OD value of the two negative control tissues was in the recommended range (1.053 in Experiment I, and 0.874 and 0.908 in Experiment II in case of the 3 minutes and 1 hour treatments, respectively). The positive control treated tissues showed 1.4% relative viability (Experiment I and II, respectively) when compared to the relevant negative control values, thus demonstrated the proper performance of the assay in each case.

The variability of the calculated viability values of the test item treated replicates were 1.3% (3 minutes treatment), 4.4% (1 hour treatment), and 9.2% (4 hours treatment). All these parameters were within acceptable limits and therefore the study was considered to be valid.

Additional controls:

As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. In Experiment I. the mean optical density (measured at 570 nm) of these tissues was determined as 0.034, Non Specific Colour % was calculated as 3.2% in case of 4 hour treatment. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary. In Experiment II. the mean optical density (measured at 570 nm) of these tissues was determined as 0.014 and 0.012, Non Specific Colour % was calculated as 1.6% and 1.3% in case of 3 minutes treatment and 1 hour treatment. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

To determine the MTT interacting potential of the test item, additional controls and data calculations were used in each experiment to exclude the false estimation of viability. In Experiment I., the calculated NSMTT value was 21.4%, in case of 4 hour treatment (the observed mean OD value was 0.225). In Experiment II., the calculated NSMTT values were 2.5% and 4.5% in case of 3 minutes treatment and 1 hour treatment, respectively (the observed mean OD values were 0.022 and 0.041).

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
Under the conditions of the in vitro EPISKIN model test with AEEU, the test item was found to be corrosive to the skin.
Executive summary:

An in vitro skin corrosivity test of AEEU test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline. Disks of EPISKIN (two units per time point) were treated with test item and incubated for 3 minutes, 1 hour and 4 hours at room temperature. Exposure of test item was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative controls (two units/control) at each time point in each experiment. Glacial acetic acid treated epidermis were used as positive controls (two units/control) at 1 hour and 4 hour in treatments. Two additional disks were used to provide an estimate of colour contribution from the test item at each time point in each experiment. 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 at each time point in each experiment. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 3 minutes, 1 hour or 4 hours of exposure is below 35% of the negative control, the test substance is considered to be corrosive to skin. Following exposure with AEEU, the mean relative viability after 3 minutes of treatment was 89.8%, the mean relative viability after 1 hour treatment was 81.3%, and the mean relative viability after 4 hours of treatment was 31.6% compared to the appropriate negative control values of the performed experiments (after adjustment for non-specific MTT reduction). The observed relative viability value was above the threshold of 35%, therefore the test item was considered as being non-corrosive in case of the 3 minutes and 1 hour exposure period. However, the observed relative viability value was below the threshold of 35%, therefore the test item was considered as being corrosive in case of the 4 hours exposure periods. Therefore, the test item is classified as Category 1B or 1C (or Corrosive class II or III) according to the GHS / CLP classification. Each experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test with AEEU, the results indicate that the test item is corrosive to the skin. Based on the observed results the test item is classified as Category 1B or 1C.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation, other
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study need not be conducted because the available information indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to column 2 of REACH Annex VII, the study does not need to be conducted, because AEEU is corrosive.
In Vitro skin corrosivity test in the Episkin Model indicate corrosive properties for AEEU. Consequently, eye irritation potential does not need to be experimentally evaluated.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

One Klimisch score 1 study was available for dermal irritation and was used as a key study:

An in vitro skin corrosivity test of AEEU test item was performed in a reconstructed human epidermis model (2016). EPISKIN-SM is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline. Disks of EPISKIN (two units per time point) were treated with test item and incubated for 3 minutes, 1 hour and 4 hours at room temperature. Exposure of test item was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative controls (two units/control) at each time point in each experiment. Glacial acetic acid treated epidermis were used as positive controls (two units/control) at 1 hour and 4 hour in treatments. Two additional disks were used to provide an estimate of colour contribution from the test item at each time point in each experiment. 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 at each time point in each experiment. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 3 minutes, 1 hour or 4 hours of exposure is below 35% of the negative control, the test substance is considered to be corrosive to skin. Following exposure with AEEU, the mean relative viability after 3 minutes of treatment was 89.8%, the mean relative viability after 1 hour treatment was 81.3%, and the mean relative viability after 4 hours of treatment was 31.6% compared to the appropriate negative control values of the performed experiments (after adjustment for non-specific MTT reduction). The observed relative viability value was above the threshold of 35%, therefore the test item was considered as being non-corrosive in case of the 3 minutes and 1 hour exposure period. However, the observed relative viability value was below the threshold of 35%, therefore the test item was considered as being corrosive in case of the 4 hours exposure periods. Therefore, the test item is classified as Category 1B or 1C (or Corrosive class II or III) according to the GHS / CLP classification. Each experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test with AEEU, the results indicate that the test item is corrosive to the skin.

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

Based on the observed results in in-vitro EPISKIN model test, AEEU is classified as Skin Corr. 1B, H314: Causes severe skin burns and eye damage and Eye Damage 1,H318: Causes serious eye damage according to the Regulation (EC) 1272/2008 (CLP).

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