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Toxicological information

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:
February 2018
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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human SkinModel Test”,
Version / remarks:
Official Journal of the European Union No. L142 (31 May 2008)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch 44034
Description: Cream to light grey powder
Expiry date: 30 November 2019
Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%).

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult humanderived epidermal keratinocytes
Justification for test system used:
The EPISKINTM(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 No. 431); therefore, it was considered to be suitable for this study.
Details on test system:
Kit Contents
Units: EPISKINTM(SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM(SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”
(Batch No.: 18 MAIN3 004; Exp. Date: 07 February 2018)
A flask of sterile “Assay Medium”
(Batch No.: 18 ESSC 004; Exp. Date: 07 February 2018)

Number of Replicate Wells
In this assay, two replicates for test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD
evaluation.

Kit Reception
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM(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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test item was applied as supplied, no formulation was required (although it was grounded to fine powder).

Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was 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.

Application (Day 0)
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was 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 materials.
- 20 mg of powdered test item was applied evenly to the epidermal surface of each of two test item treated skin units and each additional control skin units and then 100 μL physiological saline was added to the test item to ensure good contact with 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.
Note: The negative and positive controls were also part of a concurrent study (Citoxlab study code: 17/361-039B) performed in the same experimental period using the same batch of chemicals and same batch of skin units.
The plates with the treated epidermis units were incubated for 4 hours at room temperature (23.1-25.2°C) covered with the plate lids.
Duration of treatment / exposure:
1 day

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
optical density (OD) measured at 570 nm
Value:
104.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

VALIDITY OF THE TEST

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the two negative control tissues was in the recommended range (0.928).

The two positive control treated tissues showed -0.2% viability* demonstrating the proper performance of the assay.

*Note: The positive control material completely destroyed the skin cells. Thus the minor negative viability values were considered acceptable, and the result were considered to meet the validity criterion.

The difference of viability between the two test item-treated tissue samples in the MTT assay was 1.0%.

The difference of viability between the two negative control tissue samples in the MTT assay was 5.1 %.

The mean OD value of the blank samples (acidified isopropanol) was 0.046. All these parameters were within acceptable limits and therefore the study was considered to be valid.

INTERPRETATION OF TEST RESULTS

The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016).

The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).

For 2 disks:

If both disks have mean viability of ≥35% = Non Corrosive

If both disks have mean viability of <35% = Corrosive (at the corresponding incubationperiod)

For more than 2 disks:

If the mean value is ≥35% and the variability is less than 50% = Non Corrosive

If the mean value is <35% and the variability is less than 50% = Corrosive

Otherwise:

If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

VIABILITY RESULTS

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2 (see attached). The mean OD value for the test item treated skin samples showed a 104.6% relative viability compared to the negative control.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure with sodium [(6-hydroxy-4-methyl-2-oxo-2H- 1-benzopyran-7-yl)oxy]acetate, the mean cell viability was 104.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN™(SM) model test with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (Batch number: 44034), the results indicate that the test item is non-corrosive to the skin.
Executive summary:

An in vitro skin corrosivity test of sodium [(6-hydroxy-4-methyl-2-oxo-2H-1 -benzopyran-7-yl)oxy]acetate test item was performed in a reconstructed human epidermis model. EPISKINTM(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 EPISKINTM(SM) (two units) were treated with powdered sodium [(6-hydroxy- 4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate test item and incubated for 4 hours at room temperature. Exposure of test material 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) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (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 % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7 - yl)oxy]acetate, the mean cell viability was 104.6% compared to the negative control.

This is above the threshold of 35%, therefore the test item was considered as being non corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (Batch number: 44034), the results indicate that the test item is non-corrosive to the skin.