Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 January 2016 - 22 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according to OECD 439. GLP 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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Theobromine- Physical state: White powder.- Analytical purity: 99.8 %- Purity test date: 12/01/2015- Lot/batch No.: M150115C- Expiration date of the lot/batch: 01/2020- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: M150115C
- Expiration date of the lot/batch: 01/2020
- Purity test date: 12/01/2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

In vitro test system

Test system:
human skin model
Remarks:
three-dimensional human epidermis model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes
Cell source:
other: EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:16-EKIN-003, Expiry Date: 25 January 2016)
Source strain:
not specified
Justification for test system used:
The EPISKIN-SM model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (26.1-26.6°C)
- Temperature of post-treatment incubation (if applicable): room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS:
The EPISKIN-SM units were removed and rinsed thoroughly with PBS. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength:570 nm.

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:3PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin (Category 2) if the mean tissue viability % is ≤ 50 %
- The test substance is considered to be non-irritant to skin if the mean tissue viability % is > 50 %
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430: According to UN GHS/ CLP Classification
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
other: acidified isopropanol solution (200 μL/well) was used as blank during cell viability measurements
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg of the test item was applied evenly to the epidermal surface. The amount was sufficient to cover the epidermal surface. The test item was applied in its original form, no formulation was required (although the test item was ground to a fine powder).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of negative control Phosphate Buffered Saline

POSITIVE CONTROL
- Concentration (if solution):5% (w/v) Sodium Dodecyl Sulphate solution
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, three replicates were used for the test item. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: Cell viability (%)
Run / experiment:
1
Value:
82
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Compared to the Negative control, both being blank corrected
Irritation / corrosion parameter:
other: Cell viability (%)
Run / experiment:
2
Value:
79.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Compared to the Negative control, both being blank corrected
Irritation / corrosion parameter:
other: cell viability (%)
Run / experiment:
3
Value:
84.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Compared to the Negative control, both being blank corrected
Irritation / corrosion parameter:
other: cell viability (%)
Run / experiment:
mean
Value:
81.8
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: the test items did not interact with MTT according to the check-methods conducted.
- Colour interference with MTT: As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.017, Non Specific Colour % was calculated as 2.1% . This value was below 5%, therefore additional data calculation was not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
This proficiency verification with 10 reference chemicals, in the in vitro skin irritation test, in the EPISKIN model, used in OECD 439 (2010) demonstrated that the laboratory is fully proficient to perform this study. The 5 non-irritant chemicals all gave a clearly non-irritant response and the 5 irritant chemicals all gave a clearly irritant response.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.
- Acceptance criteria met for positive control:The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18.
- Acceptance criteria met for variability between replicate measurements:The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated Non Specific Colour % (NSC%)
of the Additional Control Tissues

 

Additional control

Optical density (OD)

Non Specific Colour (%)

Measured

Blank corrected

Treated with Theobromine

1

0.056

0.010

2.1

2

0.071

0.024

Mean

 

0.017

 Mean blank value was 0.047.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

Table 2: Optical Density (OD) and the calculated relative viability %

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative control
Phosphate buffered saline

1

0.868

0.821

103.5

2

0.842

0.796

100.3

3

0.811

0.764

96.3

Mean

 

0.794

100.0

Positive control
5 % (w/v) SDS Solution

1

0.112

0.065

8.2

2

0.086

0.039

4.9

3

0.090

0.044

5.5

Mean

 

0.049

6.2

Test item

Theobromine

1

0.698

0.651

82.0

2

0.675

0.628

79.1

3

0.715

0.669

84.2

Mean

 

0.649

81.8

 Mean blank value was 0.047.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Based on EU criteria.
Conclusions:
Following exposure to theobromine, the mean cell viability of reconstructed human epidermis was 81.8% compared to the negative control. This is above the threshold of 50% , therefore, theobromine was considered as being non-irritant to skin.
Executive summary:

An in vitro skin irritation test of Theobromine was performed in a reconstructed human epidermis model according to the OECD guideline 439 under GLP conditions. Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The 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 at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units/control). For each treated tissue, the viability was expressed as a % relative to the negative control. 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 item is considered to be irritant to skin. Following exposure with Theobromine, the mean cell viability was 81.8 %compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.