Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 November 2015 - 21 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 439 without any deviation.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 17 June 2015 / signed on 24 September 2015)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
Brown red solid
Details on test material:
- Storage condition of test material: Room temperature, in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 15-EKIN-050
- Production date: not reported
- Shipping date: 15 December 2015
- Delivery date: 15 December 2015
- Expiry date: 21 Decemer 2015
- Date of initiation of testing: 16 Decembre 2015

Prior to dosing the test item was heated to 45 oC to aid handling.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not reported
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in DPBS
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without a reference filter)
- Filter: not applicable
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: no data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 0.765, 0.626 and 0.736 (historical control: mean OD = 0.808; range 0.626 to 1.245).
- Barrier function: IC50= 2.3 mg/mL ( ≥ 1.5 mg/mL)
- Morphology: well differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: : absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is less or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.
- Concentration (if solution): not applicable

VEHICLE
not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of DPBS
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL of SDS
- Concentration (if solution): 5% w/v aqueous solution



Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes at room temperature.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 h.
Duration of post-treatment incubation (if applicable):
- On Day 3, at the end of the 42 h post-treatment incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in air.
- On Day 6, at the end of the formazan extraction period: The optical density was measured at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.
Number of replicates:
Triplicate tissues for test item, negative and positive controls

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 h post-exposure incubation period.
Value:
101.8
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:
The relative mean viability of the test item treated tissues was 101.8% (calculated relative to the negative control group) after a 15 minute exposure period and 42 h post-exposure incubation period. It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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
- Range of historical values if different from the ones specified in the test guideline: For the previous 27 experiments conducted between 05 May 2015 and 21 October 2015 up to the start of this study (approximately 6 months data) using this test method, the mean OD of the positive control was 0.089; range 0.049 to 0.170 and the mean percentage viability was 11.3; range 6.2 to 21.5. In this same period the mean OD of the negative control was 0.808; range 0.629 to 1.245.
It was considered that comparison of the historical control data supports correct functioning of the test system. The results of this study are therefore considered acceptable.

Any other information on results incl. tables

Table 7.3.1/1: Mean OD562Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.765

0.709

0.073

107.9

100*

10.3

0.626

88.3

0.736

103.8

Positive Control Item

0.180

0.040

0.040

25.4

18.9

5.6

0.112

15.8

0.110

15.5

Test Item

0.700

0.722

0.019

98.7

101.8

2.7

0.732

103.2

0.733

103.4

SD=        Standard deviation

*=         The mean viability of the negative control tissues is set at 100%

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 18.9% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 5.6%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.709 and the standard deviation value of the percentage viability was 10.3%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues were 2.7%. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item is not classified according to Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.

This study was run twice due to a failed Quality Control result in the first run. The report is based on the results of the study generated in the acceptable second run. 

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 101.8% after the 15 minute exposure period and 42 h post-exposure incubation period.

 

The relative mean tissue viability for the positive control treated tissues was 18.9% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 5.6%. The mean OD562 for the negative control treated tissues was 0.709 and the standard deviation value of the percentage viability was 10.3%. The positive and negative controls acceptance criterion were therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues were 2.7%. The test item acceptance criterion was therefore satisfied.

 

Under the experimental conditions of this study, the test item is not classified according to Regulation (EC) No. 1272/2008 (CLP).