Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January 2016 - 2 Februaray 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according to OECD 429. 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 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

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: 12/02/2015- Lot/batch No.: M15015C- 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: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

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formerly: Harlan Laboratories S.r.l.), San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy.
- Age at study initiation: 8-weeks old.
- Weight at study initiation: 18.9 - 20.8 g.
- Housing: Group caged, mice were provided with glass tunnel-tubes, bedding consisted of certified wood chips, nest building material was also provided.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7 - 24.3 ºC
- Humidity (%): 27 - 77 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
50, 25 and 10 % (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
The preliminary toxicity test was conducted under identical conditions to the main test (See any other information on material and methods incl. tables) except that was finalized on day 6 and no assessment of lymph node proliferation was performed. 2 animals per dose were used and 2 doses were tested (50 and 25%). All mice were observed daily for clinical signs of systemic toxicity and local irritation, ear thickness measures were taken at day 1, 3 and 6 and body weights were recorded at the start and at the end of the test.
- Compound solubility: The solubility of the test item was examined in a Preliminary Compatibility Test. The following OECD vehicles were assessed: Acetone:Olive oil 4:1 (v:v), DMF, EMK, Propylene glycol, DMSO and 1% Pluronic. Due to the physical characteristics of the test item (powder), the 100 % (w/v) concentration was not achievable. The formulation at 50 % (w/v) using DMSO as vehicle was suitable for the test. As DMSO is one of the vehicles recommended by the relevant OECD guideline, it was selected for vehicle of the study.- Irritation: No indications of irritancy at the site of application.

MAIN STUDY (See any other information of material and methods inc. tables)

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response: DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group/ DPN value of the negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION (See any other information of material and methods inc. tables)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Stimulation index Positive control (25 % (w/v) HCA in DMSO)= 7DPM Positive control (25 % (w/v) HCA in DMSO)= 26214.0

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
Thebromine 10%
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
Theobromine 25%
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
Theobromine 50%
Cellular proliferation data / Observations:
See "Any other information on results" below.

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured DPM / group

DPM

Number
lymph nodes

DPN

Stimulation Index

Background

(5 % (w/v) TCA)

30 / 26

-

-

-

-

Negative control

(DMSO)

3747

3719.0

8

464.9

1.0

Theobromine
50 % (w/v)

in DMSO

5672

5644.0

8

705.5

1.5

Theobromine

25 % (w/v)

inDMSO

4605

4577.0

8

572.1

1.2

Theobromine

10 % (w/v)

inDMSO

6262

6234.0

8

779.3

1.7

Positive control

(25 % (w/v) HCA
inDMSO)

26242

26214.0

8

3276.8

7.0

The stimulation index values were 1.5, 1.2 and 1.7 at concentrations of 50%, 25 % and 10% (w/v), respectively.

No mortality or signs of systemic toxicity were observed during the study. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the experimental animals in the 50% and 25 % (w/v) dose groups on Days 1‑4 and in the 10%.(w/v) dose group on Days 1-3. There were no indications of any irritancy at the site of application.

No treatment related effects were observed on the mean body weight changes of experimental animals. Individual and mean bodyweights are given in the following table:

Individual Body Weights for all Animals with Group Means

Animal

Number

Identity

Number

Test Group

Name

Initial Body

Weight (g)

Terminal Body

Weight* (g)

Change#

(%)

915

1

Negative (vehicle) control

DMSO

 

 

20.7

20.4

-1.4

928

2

19.3

20.4

5.7

910

3

20.1

20.5

2.0

936

4

19.2

19.3

0.5

 

 

Mean

19.8

20.2

1.7

941

5

Theobromine

50 (w/v) %

in DMSO

20.8

21.2

1.9

912

6

18.9

20.3

7.4

933

7

19.3

20.1

4.1

943

8

19.4

20.6

6.2

 

 

Mean

19.6

20.6

4.9

934

9

Theobromine

25 (w/v) %

in DMSO

20.1

21.7

8.0

921

10

20.2

20.5

1.5

938

11

19.1

19.7

3.1

944

12

19.6

19.8

1.0

 

 

Mean

19.8

20.4

3.4

939

13

Theobromine

10 (w/v) %

in DMSO

20.5

20.8

1.5

924

14

19.3

19.3

0.0

942

15

19.9

20.0

0.5

923

16

20.0

19.5

-2.5

 

 

Mean

19.9

19.9

-0.1

950

17

Positive control

25 (w/v) % HCA

in DMSO

 

20.7

20.6

-0.5

961

18

19.0

19.1

0.5

946

19

19.5

20.3

4.1

948

20

19.5

19.2

-1.5

 

 

Mean

19.7

19.8

0.7

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Based on EU criteria
Conclusions:
Theobromine did not show skin sensitisation potential under the tested contitions in the LLNA assay. The stimulation indeces were 1.5, 1.2 and 1.7 at concentrations of 50, 25 and 10% in DMSO.
Executive summary:

A local lymph node assay was conducted with theobromine according to the OECD guideline 429 under GLP conditions. In the preliminary studies 50 % (w/v) of the test item in DMSO was selected as the top dose for the main experiments. Two animals were used for each concentration in the preliminary studies. On day 1, 2 and 3, 25 µL of each of the theobromine solutions (50, 25 and 10%) and positive and negative controls were applied to the dorsal surface of each ear of the mice. There was no treatment on day 4, 5 or 6. Clinical signs and signs of irritancy were monitored during the 6 days, there were no signs of irritancy or signs of systemic toxicity. Additionally ear thickness measures were performed at day 1, 3 and 6 in the preliminary test; there were no significant changes which could indicate excessive local irritation. Four mice were used per concentration and for each of the controls in the main experiment. On day 6 the weight was determined, the preliminary test finished at this point. In the main tests 250 µL of sterile phosphate buffered saline (PBS) containing approximately 20 µCi of 3HTdR was intravenously injected via tail vein. Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide. The draining auricular lymph nodes were prepared and pooled. Single cell suspensions (SCS) of pooled lymph node cells (LNCs) were prepared and collected in disposable tubes for each group of pooled lymph nodes. The determination of incorporated3HTdR was determined as DPM for each pooled group. The measured DPM values were corrected with a background blank DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes). The stimulation index was calculated (SI = DPN value of a treated group/ DPN value of the negative control group) for each treatment group. A stimulation index of 3 or greater was considered a positive result. The stimulation indices of theobromine at concentrations of 50, 25 and 10 % were 1.5, 1.2 and 1.7 respectively, no dose-related response was observed, the SI of the positive and negative controls were within the acceptable range. It was concluded that theobromine was not as a skin sensitizer under the conditions of this study.