1-(2-aminoethyl)imidazolidin-2-one

Skin irritation / corrosion

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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 material

Test material information

Constituent 1

Reference substance name:

1-(2-aminoethyl)imidazolidin-2-one

EC Number:

228-491-9

EC Name:

1-(2-aminoethyl)imidazolidin-2-one

Cas Number:

6281-42-1

Molecular formula:

C5H11N3O

IUPAC Name:

1-(2-aminoethyl)imidazolidin-2-one

Test material form:

other: solid

Details on test material:

Test substance name: AEEU

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: 3D-human epidermis model

Test animals

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.

Test system

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.

Results and discussion

In vivo

Results

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.

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.