Theobromine

Endpoint summary

Description of key information

Key study: OECD 429. GLP study. The substance was determined to be not sensitizing to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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:

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.

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)

Positive control results:

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

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Key study: A local lymph node assay was conducted according to the OECD guideline 429 under GLP conditions with the test substance theobromine. The stimulation indexes 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 and historical range. It was concluded that theobromine had no skin sensitisation potential in the LLNA study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation: Based on the available experimental results, the test substance is not classified for skin sensitisation in accordance with CLP Regulation (EC) No. 1272/2008.