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Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 59%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS : In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 59%) which does not affect the reliability of the study
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light, container flushed with nitrogen. Use amber glassware or wrap container in
aluminum-foil
- Other: Colourless to pale yellow liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm Skin Model (EPI-200, Lot no.: 22226 kit N). The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EpiDerm Skin Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Preincubation:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM.

Application of test item and rinsing:
The plates were incubated for approximately 3 hours at 37 ± 1.0°C. The medium was replaced with fresh DMEM just before the test item was applied. Two tissues were used for a 3-minute exposure and two for a 1-hour exposure. Test item: 50 µl of the undiluted test item was added into the 6-well plates on top of the skin tissues. Negative Control: similarly treated with 50 µl Milli-Q water only. Positive control: 50 µl 8N KOH (potassium hydroxide 8 normal solution). After the exposure periods (3 minutes and 1 hour, respectively), the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed.

All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8N KOH(aq) (pre-diluted)
Duration of treatment / exposure:
Observations are made 3-minutes and 1-hour post-test item application
Duration of post-treatment incubation (if applicable):
The DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature.
Number of replicates:
Duplicate; used for a 3-minute exposure to the test substance and two for a 1-hour exposure.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minute exposure
Value:
112
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 3 minute exposure. Remarks: n=2 ; CV = 9.8 % ; Score in terms of percentage of negative control.
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour exposure
Value:
103
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: mean. Time point: 1 hour exposure. Remarks: n=2 ; CV = 11 % ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data: Negative Control OD570: 3-minutes (n=81): 1.324 – 2.615 ; 1-hour (n=83): 1.361 – 2.352. Positive Control HCD data are presented within the full study report and the concurrent PC was within the specified ranges.

Any other information on results incl. tables

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test item

 

3-minute application

1-hour application

A (OD570)

B (OD570)

Mean

(OD570)

SD

% viability

CV

A (OD570)

B (OD570)

Mean

(OD570)

SD

% viability

CV

Negative control

1.507

1.531

1.519

±0.017

100

1.5

1.848

1.812

1.830

±0.026

100

2.0

Test item

1.615

1.790

1.730

±0.124

112

9.8

1.767

1.988

1.877

±0.157

103

11.0

Positive control

0.165

0.163

0.164

±0.002

11

1.6

0.144

0.134

0.190

±0.008

7.6

7.5

Values are corrected for background absorption (0.0433). Isopropanol was used to measure the background absorption.

SD = Standard deviation

Duplicate exposures are indicated by A and B.

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria not met
Conclusions:
Under the conditions of this in vitro study, the test item is not considered to be corrosive to the skin.
Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μl of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 7.6% after 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of viability 20 - 100% the Coefficient of Variation between tissue replicates was less than 11%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 112% and 103%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item is not corrosive in the in vitro skin corrosion test.