Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
EPISKIN Model
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 June - 23 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: UGe-RS Kilo 1
Color: Yellow
Odor: Not specific
Storage conditions: room temperature
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiring date: 12.12.2018
Correction factor: 1.07
Specific details on test material used for the study:
Batch No.: UGe-RS Kilo 1
Appearance: Yellow powder
Expiration date: 12 December 2018
Storage: Room temperature

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:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and
no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
Units: EpiSkinTMSM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations.
(Batch No.: 16 MAIN3 041; Exp. Date: 06 July 2016)
A flask of sterile “Assay Medium” for use in MTT assays.
(Batch No.: 16 ESSC 026; Exp. Date: 06 July 2016)
The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8°C.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
The epidermal surface was first moistened with 5 µL deionised water* in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
Positive and negative control
A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.
Rinsing (day 0)
After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).
Duration of post-treatment incubation (if applicable):
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere.
MTT test after 42 hours incubation (day 2)
After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, ≥95% humidified atmosphere.
Number of replicates:
Three replicates were used for the test item and controls respectively.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
>= 88 - <= 121
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean OD value of the three negative control tissues was 0.761. The mean OD value obtained for the positive control was 0.139 and this result corresponds to 18 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the percentage viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
As the test item has an intrinsic colour (yellow), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.019. The Non Specific Colour % (NSC %) was calculated as 2.5 %. Therefore additional data calculation was not necessary. A false estimation of viability is precluded.

Any other information on results incl. tables

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated percentage viability of the cells is presented below:

 

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

0.672

88

2

0.798

105

3

0.814

107

mean

0.761

100

standard deviation (SD)

10.23

Positive Control:
SDS (5 % aq.)

1

0.077

10

2

0.151

20

3

0.190

25

mean

0.139

18

standard deviation (SD)

7.55

Test Item:
Yellow LF 6911

1

0.922

121

2

0.667

88

3

0.761

100

mean

0.783

103

standard deviation (SD)

16.91

OD values and NSC % of additional control:

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSC %)

Yellow LF 6911
(test item treated tissueswithout MTT incubation)

1

0.023

2.5

2

0.015

mean

0.019

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Yellow LF 6911 is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Executive summary:

EpiSkinTMSM test of Yellow LF 6911 has been performed to predict the irritation potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439,28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution atin 5% CO2protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item has an intrinsic colour (yellow), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

In thisin vitroskin irritation test using the EPISKIN model, the test itemYellow LF 6911did not show significantly reduced cell viability in comparison to the negative control(mean value: 103 %). Allobtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. The test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

The results obtained from thisin vitroskin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test itemYellow LF 6911is considered to be non-irritant to skin and is therefore not classified(UN GHS No Category).