Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Oct 2018 - 08 Oct 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
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
aqueous solution
Details on test material:
Substituted amino acid (2) solution, FC-C 13587
Appearance: clear colorless solution

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
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).
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™
- Tissue batch number: 18-EKIN-040
- Surface: 0.38 cm^2
- Expiration date: 8 October 2018

TEST FOR THE INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- The test substance was checked for possible color interference and reduction of MTT before the study. No interference was found.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure to the test item: room temperature
- Temperature of incubations(°C): 37.0 ± 1.0°C (actual range 36.8 – 37.3°C)
- Humidity(%): 80 - 100% (actual range 71 - 86%)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: tissues were washed with phosphate buffered saline (1 washing step)
- Observable damage in the tissue due to washing: no

APPLICATION/TREATMENT OF TEST ITEM
- Number of replicate tissues: 3 for the test substance. Additionally, 3 tissues for the negative and the positive control each.
- Volumen test item: 25 μL
- The positive control was re-spread after 7 minutes contact time.
- Exposure period: 15 minutes.
- Incubation period after exposure: 42 hours at 37°C.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 single run

MEASUREMENT OF CELL VIABILITY
- After incubation, tissues were dried and incubated with 2 mL MTT-solution (0.3 mg/mL in PBS).
- Incubation time: 3 hours at 37°C in air containing 5% CO2.
- Formazan was extracted with 500 μL isopropanol (MatTek corporation)
- The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

INTERPRETATION (see Table 1)
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.

ACCEPTABILITY 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 ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The liquid test item was applied undiluted (25 μL) directly on top of the tissue.
Duration of treatment / exposure:
15 ± 0.5 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 hours at 37°C +3 hours with MTT
Number of replicates:
The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Test system

Duration of treatment / exposure:
15 minutes

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
82
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean cell viability: 100%
Positive controls validity:
valid
Remarks:
Mean cell viability: 17%

Any other information on results incl. tables

Table 2:  Individual OD Measurements at 570 nm

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570 measurement 1

OD570 measurement 2

 

1.2691

1.4125

 

1.1130

1.2929

 

1.3486

1.6808

Test item

OD570 measurement 1

OD570 measurement 2

 

0.9845

1.1103

 

1.1447

1.1547

 

1.1466

1.1748

Positive control

OD570 measurement 1

OD570 measurement 2

 

0.4137

0.4675

 

0.2014

0.2271

 

0.1325

0.1521

OD = Optical density

Triplicate exposures are indicated by A, B and C.

OTHER EFFECTS:

- Visible damage on test system: no

- Direct-MTT reduction: no

- Colour interference with MTT: no

 

ACCEPTANCE OF RESULTS:

- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 17 %

- Acceptance criteria met for variability between replicate measurements: yes, the variability between tissues treated with the test item was ≤ 12%

- Since the mean relative tissue viability for Substituted amino acid (2) solution FC-C 13587 was above 50% is considered to be non-irritant.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Small model (EPISKIN-SM ™)), the influence of Substituted amino acid (2) solution FC-C 13587 on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin. After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 7% whereas the test substance showed cell viability of 82%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test. Based on these results, Substituted amino acid (2) solution FC-C 13587 needs not to be classified for skin irritating properties.

Categories Display