Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not stated but from multiple donors
Details on animal used as source of test system:
Not applicable
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
Test System Set Up
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22 hours at 37°C (Figure 1). Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

Killed tissues (EPISKIN-SMTM, 0.38 cm2, Lot no.: 17-EKIN-040).
Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.

MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

Environmental conditions
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 65 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.1°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 Item Preparation
No correction was made for the purity/composition of the test compound.
The solid test item was applied directly on top of the skin tissue. Mercaptamine was spread to match the size of the tissue.

Application/Treatment of the Test Item.
The test was performed on a total of 3 tissues per test item together with negative and positive controls. The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (48.78 to 82.44 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µl PBS (negative control) and 3 tissues with 25 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

Since the test item reacted with the MTT medium, in addition three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell Viability Measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS).
The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be b) The mean relative tissue viability of the positive control should be c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be d) The %NSCliving should be e) The non-specific MTT reduction should be All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
48.78 - 82.44 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
25µl

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
25µl
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours after rinsing
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
85
Vehicle controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
Mercaptamine was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that Mercaptamine did interact with the MTT endpoint.
In addition to the normal procedure, three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Mercaptamine was 0.8% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Mercaptamine compared to the negative control tissues was 85%. Since the mean relative tissue viability for Mercaptamine was above 50% Mercaptamine is considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 10%.

The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Appendix 3). The standard deviation value of the percentage viability of three tissues treated identically was ≤ 4.9%, indicating that the test system functioned properly

Any other information on results incl. tables

Table 1: Mean Absorption in the In Vitro Skin Irritation Test with Mercaptamine

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean

(OD570)

 

SD

Negative control

1.172

1.093

1.078

1.115

±

0.051

Mercaptamine

1.003

0.894

0.957

0.951

±

0.054

Positive control

0.109

0.125

0.110

0.115

±

0.009

OD = optical density

SD = Standard deviation

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

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

Table 2: Mean Tissue Viability in the In Vitro Skin Irritation Test with Mercaptamine

 

Mean tissue viability (percentage of control)

Standard deviation (percentage)

Negative control

100

4.5

Mercaptamine

85

4.9

Positive control

10

0.8


 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, Mercaptamine is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and CLP Regulation (EC 1272/2008, as amended).