Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 31 - October 10, 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:
Test item: Leuco Sulfur Red 14
Appearance: light red solid
CAS No: 68585-53-5
EC No: 271-559-8

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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiSkinTM Small Model (EpiSkinTMSM)
- Tissue batch number: 16-EKIN-037

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure:
room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
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.
- Observable damage in the tissue due to washing:
No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
0.3 mg/mL
- Incubation time:
3 h
- Spectrophotometer:
96-well plate spectrophotometer
- Wavelength:
570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
see "Any other information on Materials and Methods"

NUMBER OF REPLICATE TISSUES:
3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls,

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates :
2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.
- Method of calculation used:
Data calculation for normal test items
Blank
– The mean of the 6 blank OD values is calculated:
Negative control
– Individual negative control OD values are corrected with the mean blank OD: :
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 3 negative control values are calculated: this corresponds to 100% viability
Positive control
– Individual positive control OD values are corrected with the mean blank OD: :
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 3 positive control values are calculated
– The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item
– Individual test item OD values are corrected with the mean blank OD: :
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 3 test item values are calculated
– The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3

Data calculation for MTT-interacting items
Test items that interfere with MTT can produce non-specific reduction of the MTT. It is necessary to evaluate the OD due to non-specific reduction and to subtract it before calculations of viability %.
– Non-specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT - ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD (mean)
ODKT: test item treated killed tissues OD (mean)
ODNC: negative control OD (mean)
If NSMTT is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.
– True MTT metabolic conversion (TODTT) is undertaken if NSMTT is < 50%:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
– The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100
% RV 3 = [TODTT3 / ODNC] × 100
– The mean value of the 3 individual relative viability % for test item is calculated
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

Data calculation for dyes and chemicals able to colour the tissue
For test items detected as able to stain the tissues the non-specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability %.
– Non Specific Colour % (NSC %):
NSC % = (mean ODCT / mean ODNC) × 100
ODCT: test item treated tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
– True MTT metabolic conversion (TODTT) is undertaken if NSC % is > 5 % and ≤ 50 %.
TODTT = [ODTV – mean ODCT]
ODTV: test item-treated tissues (incubated with MTT)
ODCT: test item-treated tissues (not incubated with MTT)
– The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100
% RV 3 = [TODTT3 / ODNC] × 100
– The mean value of the 3 individual relative viability % for test substance is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3
– If NSC % is ≤ 5 % then the normal calculation mode is used.
– If NSC % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating or corrosive to skin if the viability after exposure is less than or equal to 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after exposure is greater than 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 mg of the test item was applied evenly to the epidermal surface.


NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 1x PBS

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aq. solution
Duration of treatment / exposure:
15 minutes (± 0.5 min) at room temperature.
Duration of post-treatment incubation (if applicable):
42 hours (± 1h) at 37 °C in an incubator with 5 % CO2, ≥95 % humidified atmosphere.
Number of replicates:
In this assay 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-3
Value:
>= 115 - <= 144
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: mean viability 128%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:
No
- Direct-MTT reduction:
As the test item has an intrinsic colour (light red), the check-method for possible direct MTT reduction with test item was impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with the MTT, an additional control was necessary.
The non-specific MTT reduction (NSMTT) was determined (0%)*, the correction of viability percentage was not necessary
- Colour interference with MTT:
Yes, as the test item has an intrinsic colour (light red), 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 0.012. The Non Specific Colour % (NSC %) was calculated as 1.4 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
Yes
- Acceptance criteria met for positive control:
Yes
- Acceptance criteria met for variability between replicate measurements:
Yes

Any other information on results incl. tables

Table 2: OD values and viability percentages of the controls

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

1.018

117

2

0.823

95

3

0.765

88

mean

0.869

100

standard deviation (SD)

15.22

Positive Control:
SDS (5 % aq.)

1

0.167

19

2

0.168

19

3

0.072

8

mean

0.136

16

standard deviation (SD)

6.34

Table 3: OD values and viability percentages of the test item

Test Item

Optical Density (OD)

Viability (%)

1

1.000

115

2

1.073

124

3

1.253

144

mean

1.109

128

standard deviation (SD)

14.93

 Table 4: OD values of additional controls for MTT-interacting test item

Additional controls

Optical Density (OD)

Negative control killed tissues:
1x PBS

1

0.065

2

0.078

3

0.079

mean

0.074

Test item treated killed tissues:

1

0.048

2

0.053

3

0.045

mean

0.048

 

Table 5: OD values and NSC % of additional control:

Additional colour control

Optical Density (OD)

Non Specific Colour % (NSC %)

(test item treated tissueswithout MTT incubation)

1

0.012

1.4

2

0.013

mean

0.012

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model according to OECD guideline 439, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Executive summary:

In an in vitro skin irritation test (EpiSkin SM) according to OECD guideline 492, the skin irritating potential of the test item was assessed.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. 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 (light red), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated for 42 hours in an incubator at 37°C with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (light red). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

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 this in vitro skin irritation test using the EPISKIN model, the test item did not significantly reduce cell viability in comparison to the negative control (mean viability: 128 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore 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 this in vitro skin irritation assay, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test is considered to be not irritating to skin and is therefore not classified (UN GHS No Category).