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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 June - 09 August, 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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Phenol, 2,4-dinitro-, sulfurized, thiosulfonated
EC Number:
215-445-8
EC Name:
Phenol, 2,4-dinitro-, sulfurized, thiosulfonated
Cas Number:
1326-83-6
Molecular formula:
not applicable
IUPAC Name:
phenol, 2,4-dinitro-, sulfurized, thiosulfonated
Test material form:
solid: particulate/powder
Details on test material:
Test item: Solubilised Sulphur Black 1
Appearance: black powder
CAS No: 1326-83-6
Specific details on test material used for the study:
Expiration date: 30 November 2021

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 non-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), manufactured by EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model.
- Tissue batch number: 17-EKIN-025
- Date of initiation of testing: 2017-06-21

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the EpiSkinTMSM units were 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.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 15 min
- Spectrophotometer: 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
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.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: incubation in distilled water followed by freezing.
- N. of replicates: 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating/corrosive to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.
- The test substance is considered to be non-irritant to skin if to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 %
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1h)
Number of replicates:
In this assay 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates colour 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

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 1
Value:
110
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 2
Value:
132
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 3
Value:
122
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
122
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: relative viability %
Run / experiment:
mean
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The non-specific MTT reduction (NSMTT) was determined to be 28.378 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: As the test item has an intrinsic colour (dark black), 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 0.023. The Non Specific Colour % (NSC %) was calculated as 3.1 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded


DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

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 1: Summary of neg. and pos. control results

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

0.786

105

2

0.793

106

3

0.671

89

mean

0.750

100

standard deviation (SD)

9.13

Positive Control:
SDS (5 % aq.)

1

0.070

9

2

0.067

9

3

0.109

15

mean

0.082

11

standard deviation (SD)

3.10

Table 2: OD values and viability percentages of the test item (including corrected values)

Test Item

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

Solubilised Sulphur Black 1

1

0.827

0.614

110

82

2

0.994

0.781

132

104

3

0.918

0.705

122

94

mean

0.913

0.700

122

93

standard deviation (SD)

11.11

11.11

Table 3: OD values of additional controls for MTT-interacting test item

Additional controls

Optical Density (OD)

Negative control killed tissues:
1x PBS

1

0.033

2

0.052

3

0.009

mean

0.031

Test item treated killed tissues:
Solubilised Sulphur Black 1

1

0.242

2

0.231

3

0.260

mean

0.244

Table 4: OD values and NSC % of additional control

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSC %)

Solubilised Sulphur Black 1
(test item treated tissueswithout MTT incubation)

1

0.032

3.1

2

0.015

mean

0.023


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation asaay (RhE) according to OECD guideline 439, a relative cell viablility of 93.0 % was determined. The test item is considered to be not irritaitng to skin and is therefore not classified (UN GHS No Category).
Executive summary:

An in vitro skin irritation test (RhE) has been performed to predict the test item skin irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD guideline 439. Three replicates of EPISKIN tissue were treated with test item, positive or negative control and incubated for 15 minutes at room temperature. 10 µL SDS (5 % aq.) or 10 µL 1x PBS treated tissues (three replicatrees each) were used as positive and negative controls respectively. Exposure to the test item was terminated by rinsing with PBS 1x solution. The tissues were then incubated at 37 °C for 42 hours at 37 °C. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. For each treated tissue viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (dark black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. 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.

Since the test item is a possible MTT-reducer and has an intrinsic colour (dark black) a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double correction [TODTT (MTT and NSC)] for colour interference. Two killed treated tissues were used to avoid a possible double correction for colour interference. Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

In this in vitro skin irritation test using the EPISKIN model, cell viability was not significantly reduced (corrected value (mean relative viability: 93 %) after test item treatment compared to the negative control. Therefore the test item was considered to be not irritanting to the skin.

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