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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:
17 July 2017 - 21 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: white powder with lumps
- Test item storage: at room temperature protected from light
Specific details on test material used for the study:
Test substance was protected from light (amber glassware was used and container wrapped in aluminum-foil).
pH (1% in water, indicative range): 5.12 – 4.78.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Details on animal used as source of test system:
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.
- Source: MatTek Corporation, Ashland MA, U.S.A.
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
water
Remarks:
Milli-Q water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue lot number: 26708 kit L and M

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment/exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C (actual range 36,8 – 37,5°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration, one negative control, one positive contol.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze-killed tissues
- Number of replicates: 2
- Calculation: Nonspecific MTT reduction was calculated as the difference between the mean OD of the untreated freeze-killed tissues and test item treated freeze-killed tissues expressed as percentage of the mean of the negative control tissues. True tissue viability is calculated as the difference between the living test item treated tissues incubated with MTT medium and the difference between mean OD of the test item treated freeze-killed tissues and the mean OD of the untreated freeze-killed tissues.

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.

DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
other: concurrent control for MTT reduction by test item
Amount/concentration applied:
The solid test item was applied directly on top of the skin tissue. To protect the test item from light, glassware was wrapped in tin-foil.
- Amounts applied: 25.2 to 33.1 mg (skin was moistened with 25 µL Milli-Q water)

NEGATIVE CONTROL
- Amount applied: 50µL

POSITIVE CONTROL
- Amount applied: 50µL
- Concentration: 8N
Duration of treatment / exposure:
3-minute and 1-hour
Duration of post-treatment incubation (if applicable):
none
Number of replicates:
2/ exposure period

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
95
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.3%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
95
Vehicle controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6.2%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: Yes, but since the percentage nonspecific MTT reduction was ≤0.0, there was no need to correct for interference of the test item.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range;
- Acceptance criteria met for positive control: Yes, mean relative tissue viability following 1-hour exposure was 6.2%.
- Acceptance criteria met for variability between replicate measurements: Yes, in the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 11%, indicating that the test system functioned properly

Any other information on results incl. tables

Table 1 Historical data for positive and negative controls

 

Negative control

Positive control

Positive control

 

3-minute treatment

(OD570)

1-hour treatment

(OD570)

3-minute treatment

(OD570)

1-hour treatment

(OD570)

3-minute treatment

(% viability)

1-hour treatment

(% viability)

Range

1.324 – 2.615

1.361 – 2.352

0.0172 – 0.56

0.046 – 0.339

6 – 25

3 – 13

Mean

1.84

1.85

0.19

0.14

11.03

7.45

SD

0.26

0.22

0.09

0.06

4.39

2.51

n

81

83

80

77

38

38

SD = Standard deviation; n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008
Conclusions:
Based on an in vitro skin corrosion test conducted with ROC-118 according to OECD 431 guideline and GLP principles it is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test.
Executive summary:

In an in vitro skin corrosion test using a human skin model ( EpiDerm Skin Model), the influence of ROC-118 on the viability of human skin was tested. The test substance was applied directly to 0.6 cm^2 cultured skin (25.2 to 33.1mg, in presence of 25 μL Milli-Q water). After 3-minute and 1-hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. Viability of unexposed skin was set at 100%. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with TS compared to the negative control tissues was 95% and 95%, respectively. The positive control had a mean cell viability of 6.3% after 3 minutes exposure and 6.2% after 1 -hour exposure, respectively.

Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, it can be concluded that the test substance is not corrosive in the in vitro skin corrosion test.