Theobromine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Test method similar to EPA OPPTS 870.5915 In vivo Sister Chromatid Exchange. No data on GLP

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.5915 In Vivo Sister Chromatid Exchange Assay

Deviations:

yes

Remarks:

animals are exposed to BdrU and then exposed to the chemical. 5 males are used.

Principles of method if other than guideline:

Paraffin-coated (approximately 80% of the surface) BrdU tables (50mg each) were implanted subcutaneously following the methodology of McFee et al.,and Sharief et al. for in vivo SCE study and cell replication kinetic analysis. Theobromine was injected as single intraperitoneal injection 1 hour after the tablet implantation. Colchicine (4mg/kg) was injected i.p. 22 hours after BdrU implantation. Two hours after, bone marrow cells were obtained,fixed and prepared with fluorescence-plus Giemsa to score SCE frequencies.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay

Test material

Test material information

Constituent 1

Reference substance name:

Theobromine

EC Number:

201-494-2

EC Name:

Theobromine

Cas Number:

83-67-0

Molecular formula:

C7H8N4O2

IUPAC Name:

theobromine

Details on test material:

- Name of test material (as cited in study report): Theobromine

Test animals

Species:

mouse

Strain:

other: Swiss albino

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-12 weeks old
- Weight at study initiation: 25-30 g- Housing: They were kept five per cage with husk bedding.
- Diet (e.g. ad libitum): standard rodent pellet diet Gold Mohar, Lipton India, Chandigarh, India.
- Water (e.g. ad libitum): yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28±2ºC
- Humidity (%): 60±5%
- Photoperiod (hrs dark / hrs light): Light cycle was 12 h light and 12 h dark.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO

Details on exposure:

TEST MATERIAL
- Concentration: 12.5 mg/kg, 25 and 50 mg/kg

Duration of treatment / exposure:

23 hours.

Frequency of treatment:

Once per animal.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

12.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose.

Control animals:

yes, concurrent vehicle

not specified

Positive control(s):

Mitomycin-C (1.5 mg/kg)- Route of administration: injection- Doses / concentrations: 1.5 mg/kg

Examinations

Tissues and cell types examined:

In vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:For SCE analysis, colchicine (4 mg/kg) was injected i.p.. 22 h after BrdU tablet implantation. Two hours later, bone marrow was expelled with 0.075 M KCl solution. After hypotonic treatment (0.075 M KCl at 37ºC) for 20 min, cells were fixed three times with methanol:acetic acid (3:1).

DETAILS OF SLIDE PREPARATION:The slides were prepared and chromosomes were differentially stained with fluorescence-plus-Giemsa technique. All the slides were coded and 30 division metaphase cells (40±2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored from five animals per dose tested.

Statistics:

Results of the in vivo sister chromatid exchanges assay were analysed using an analysis of variance test statistics with Dunnett’s multiple comparisontest with control. Trend test for the evidence of dose response effects was carried out following the method of Margolin et al..

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

A significant increase in sister chromatid exchange was observed in all the three doses tested. Trend test for the evidence of dose response effects was also significant. No significant changes in replicative indices were observed for any of the three doses tested for any of the drugs. Positive control compound mitomycin-C induced very high frequencies of sister chromatid exchange over those of solvent control and treated groups.

Table 1. Frequencies of sister chromatid exchanges induced by theobromine in vivo in bone marrow cells of mice

Chemicals (mg/kg)	Sister chromatid exchange/cell of five animals	Sister chromatid exchange/cell (mean ± S.D.)^a	Regression coefficient ± S.E.	Replicative indices (mean± S.D.)^a
Solvent control(75µl of DMSO)	3.4, 3.5, 4.9, 4.7, 4.2	4.1± 0.6		1.91± 0.07
Theobromine (mg/kg)
12.5	4.7, 5.9, 5.4, 6.1, 5.3	5.5± 0.5*		1.90± 0.06
25	5.8, 6.3, 6.4, 6.9, 6.7	6.4± 0.4*	0.057± 0.008*	1.93± 0.05
50	6.9, 7.3, 7.6, 6.5, 7.4	7.2± 0.4*		1.95± 0.08
Positive control
Mitomycin-C (1.5 mg/kg)	18.3, 19.8, 23.0, 25.6, 21.3	21.6± 2.7		1.89± 0.10

^aMean ± S.D. of five animals (30 cells per animal). Results of each concentration were compared with the solvent control by Dunnett’s multiple comparison with control.

*p<0.01.

Applicant's summary and conclusion

Conclusions:

Theobromine is able to increase sister chromatid exchanges in a dose related manner in bone marrow cells after an in vivo exposure from concentrations of 12.5 mg/kg b.w to 50 mg/kg b.w.

Executive summary:

An in vivo sister chromatid exchanges assay in bone marrow cells of mice was conducted with theobromine. 5 Swiss albino male mice were used for the test per dose group. Paraffin-coated BdrU tablets (approximately 50mg) were implanted subcutaneously in the flank of mice under anaesthesia. Theobromine was administered by intraperitoneal injection at three doses (12.5, 25, 50 mg/kg b.w.) with DMSO as vehicle to different groups of mice 1 hour after the tablet implantation. Negative control mice were injected with 75 µL of DMSO while mitomycin- C was used as a positive control at a dose of 1.5 mg/kg of body weight. Colchicine (4 mg/kg.) was injected i.p. 22 h after BrdU tablet implantation. Two hours later, slides of bone marrow cells were prepared and chromosomes were differentially stained with fluorescence-plus-Giemsa technique. All the slides were coded and 30 division metaphase cells (40±2 chromosomes) per animal were scored for sister chromatid exchange frequencies, i.e., a total of 150 cells were scored from five animals per dose tested. Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first, second and third division metaphases. Results indicate that theobromine can induce significant increases in sister chromatid exchanges in bone marrow cells of mice after an in vivo exposure from concentrations of 12.5 mg/kg b.w to 50 mg/kg b.w. No significant changes in replicative index were observed for any of the three tested doses.