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Toxicological information

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:
2015
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:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 50%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 50%) which does not affect the reliability of the study
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2013; signature: May 2013

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid.
- Storage condition of test material: At room temperature protected from light.
- Other: Colourless

Test animals

Species:
other: EpiDerm Skin Model (EPI-200, Lot no.: 21267 kit Q).
Strain:
not specified
Details on test animals and environmental conditions:
EpiDerm Skin Model). The 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.

Freeze-killed tissues. Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium. Further use of killed tissues was similar to living tissues.

All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 - 87%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.4°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 laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl
- Concentration (if solution): undiluted

Duration of treatment / exposure:
Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure.
In addition two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues were used for the cytotoxicity evaluation with MTT.
Number of animals:
The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure. In addition two freeze-killed tissues treated with test substance and two
freeze-killed non treated tissues were used for the cytotoxicity evaluation with MTT.
Details on study design:
TEST SITE
- Area of exposure: 50 μl of the undiluted test substance was added on top of the skin tissues.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Two tissues were washed after 3 minutes and two tissues were washed after 1 hour.

SCORING SYSTEM: Skin corrosion is expressed as the remaining cell viability after exposure to the test substance.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minutes exposure
Run / experiment:
mean
Value:
87
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Score in terms of percentage of control
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour
Run / experiment:
mean
Value:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Scored in terms as percentage of control
Other effects / acceptance of results:
Table 1 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with test substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 87% and 94% respectively. The values are corrected for non-specific MTT reaction.

The relative mean tissue viability of the positive control was after the 3-minute and 1-hour treatments compared to the negative control tissues was 8% and 10% respectively.

Any other information on results incl. tables

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

 

3-minute application (OD540)

1-hour application (OD540)

OD540measurement

A

B

Mean

SD

Mean tissue viability (% of control)

A

B

Mean

SD

Mean tissue viability (% of control)

Negative control 

1.651

1.640

1.645

±0.008

100

1.669

1.603

1.636

±0.047

100

Test Substance (1)

1.500

1.362

1.431

±0.098

87

1.549

1.517

1.533

±0.022

94

Positive control 

0.129

0.127

0.128

±0.001

8

0.184

0.153

0.169

±0.022

10

(1) The values are corrected for the non-specific MTT reaction

SD = Standard Deviation

Duplicate Exposures are indicated by A and B

Values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test substance is not considered to be corrosive in the in vitro skin corrosion test using EpiDerm Skin Model.
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 substance 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 substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure. The test substance did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Therefore in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test substance was 10% of the negative control tissues. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test substance treated viable tissues. Fifty μl of the undiluted test substance 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 8% after 3 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 10%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 87% and 94%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive. Under the conditions of this study the test material is not corrosive in the in vitro skin corrosion test.