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Description of key information

Skin irritation. Key study: OECD 439 under GLP conditions. The test item is predicted to be non-irritating to the skin.
Eye irritation. Key study: OECD 438 under GLP conditions. The test item is not expected to be corrosive or to cause severe eye damage, however no prediction can be made regarding the irritancy to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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%)
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.
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.

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).

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2016 - 25 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according to OECD 438. GLP study.
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material:M15015C
- 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: Room temperature
Species:
other: Eyeballs isolated from chikens killed for human consumption
Details on test animals or tissues and environmental conditions:
ANIMALS
The eyeballs used in the experiment were obtained from chickens killed for human consumption (Ex vivo).
- Source: Slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice.
- Age at study initiation: approximately 7 weeks.- Weight at study initiation: 1.5 - 2.5 kg.
- Other: After electric shock and incision of the neck for bleeding, the chicken heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container. The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes. All eyeballs used in the tests came from the same group of eyes collected on a specific day.Till the moment of their transfer, the eyeballs were kept in special containers at temperature of -18°C.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.03 g
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
240 minutes
Number of animals or in vitro replicates:
The test item and controls (positive and negative) were tested on three eyeballs each.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): With 20 mL physiological saline.
- Time after start of exposure: 10 seconds

SCORING SYSTEM:
Combination of fluoresceing retention, corneal opacity and corneal swelling.
Fluoresceing scores: Score Observations
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

Corneal opacity scores: Score Observations
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible
Corneal swelling was calculated as % as follows:corneal swelling = [corneal thickness at time t – corneal thickness at time t = 0/ corneal thickness at time t = 0] x100

TOOL USED TO ASSESS SCORE:
- Fluoresceing retention (30 minutes after end of exposure)
- Corneal opacity by assigning appropiate values to opaque areas, the mean corneal opacity value was calculated for all test eyeballs for all observation time points. Based on the highest mean score corneal opacity the final score was given.
- The degree of corneal swelling was determined by measuring corneal thickness using a slit-lamp microscope and an SP-100 pachymeter.The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, at any time point, the final category score was given.
Irritation parameter:
fluorescein retention score
Run / experiment:
Fist run
Value:
2.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
fluorescein retention score
Run / experiment:
Second run
Value:
2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
cornea opacity score
Run / experiment:
First run
Value:
1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
cornea opacity score
Run / experiment:
Second run
Value:
1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
percent corneal swelling
Run / experiment:
First run
Value:
-8.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
percent corneal swelling
Value:
-8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
In the first run histopathological examinations of the corneas treated with the test item revealed: karyorrhexis (eyeball No. 2), slight vacuolation of the anterior corneal epithelium (eyeballs No. 1, No. 2, No. 3), slight coagulation (eyeball No. 1), exfoliation (eyeball No. 2), necrosis (eyeballs No. 2, No. 3), slight erosions of the superficial layer of the anterior corneal epithelium (eyeballs No. 1, No. 2, No. 3), deposits of the test item on the corneal surface (eyeballs No. 2, No. 3), dissection of the corneal stroma (eyeballs No. 1, No. 2, No. 3). In the second run histopathological examinations of the corneas treated with the test item revealed: slight vacuolation and local coagulation of the anterior corneal epithelium (eyeballs No. 1, No. 2, No. 3), slight exfoliation (eyeballs No. 1, No. 2), necrosis and slight erosions of the superficial layer of the anterior corneal epithelium (eyeballs No. 1, No. 2, No. 3).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: No.

Evaluation of fluorescein retention– the first run.

observation after time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

2.3

III

3.0

IV

0.0

I

 

 Evaluation of fluorescein retention – the second run.

observation after time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

2.0

III

3.0

IV

0.0

I

 

Evaluation of corneal opacity– the first run.

observation after

time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

0.8

II

4.0

IV

0.0

I

75

1.0

II

4.0

IV

0.0

I

120

1.0

II

4.0

IV

0.0

I

180

1.0

II

4.0

IV

0.0

I

240

1.0

II

4.0

IV

0.0

I

 

Evaluation of corneal opacity – the second run.

observation after

time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

0.8

II

4.0

IV

0.0

I

75

1.0

II

4.0

IV

0.0

I

120

1.0

II

4.0

IV

0.0

I

180

1.0

II

4.0

IV

0.0

I

240

1.0

II

4.0

IV

0.0

I

 Evaluation of corneal swelling (%)– the first run.

observation after time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

-0.8

I

53.8

IV

-6.5

I

75

-0.5

I

63.2

IV

-5.0

I

120

-1.6

I

78.1

IV

-7.9

I

180

-4.5

I

94.7

IV

-7.9

I

240

-8.1

I

104.8

IV

-5.4

I

“-“          - percentage of corneal thickness decrease, no swelling

 

Evaluation of corneal swelling (%) – the second run.

observation after time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

-0.1

I

52.3

IV

-4.8

I

75

-2.4

I

64.1

IV

-4.8

I

120

-4.8

I

69.3

IV

-6.6

I

180

-8.0

I

79.3

IV

-9.9

I

240

-8.0

I

74.2

IV

-9.4

I

“-“          - percentage of corneal thickness decrease, no swelling

Interpretation of results:
irritating
Remarks:
Based on EU criteria
Conclusions:
The substance can not be classified as severe irritant or corrosive to the eyes and it is not possible to state that the test item is not irritant to the eyes according to the UN GHS classification which is in accordance with the CLP criteria. According to the histopathological findings the test can be regarded as moderately irritating to the eyes.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the substance theobromine according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to 0.03 g of the test item, the same amount of imidazole and 0.03 mL of physiological saline were applied to other eyeballs as positive and negative controls. Three eyeballs were used for the test item and for each control. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438 recomendations, histopathological evaluation of the corneal layers was conducted. Because the test item was a solid material and the first run lead to a no-classification outcome, a second run of three eyes for the test item was performed as recommended by the guideline, concurrent controls were included. According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints were 2xII and 1xIII in both runs, however taking into account the histopathological findings, the test can be considered as moderately irritating.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation: Key study: 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. Following exposure with Theobromine, the mean cell viability was 81.8% compared to the negative control. This is above the threshold of 50%, thereforethe test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Eye irritation: Key study: An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the substance theobromine according to the OECD guideline 438 under GLP conditions. According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints were 2xII and 1xIII in both runs. Taking into account the histopathological findings, the test can be considered as moderately irritating.

Justification for classification or non-classification

Skin irritation: Based on available data (not irritant based on OECD 439 test), the substance is not classified for skin irritation according to CLP Regulation (EC) No. 1272/2008.

Eye irritation: Based on the results on the OECD 438 test, the combination of the three ICE classes with the test item is not sufficient to classify the substance as corrosive/severe damaging to the eyes but it does not allow a no-classification, thus based on the histopathological evaluation, assuming a worst case, theobromine is classified as irritating to the eyes Category 2, H319 according to CLP Regulation (EC) No. 1272/2008.