Theobromine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test method according to OECD Guideline 471 with deviations. No data on GLP.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

four strains (TA97a, TA100, TA102 and TA104) of S. typhimurium are used.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation 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.

Method

Species / strain

Species / strain / cell type:

other: TA97a, TA100, TA102 and TA104

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1, 5*, 10, 50*, 100, 500*, 1000, 5000, 10000, 20000 µg/plate (*this concentrations were only tested with strains TA102, TA104).

Vehicle / solvent:

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: 4-nitro-o-phenylenediamine (NPD)

Remarks:

strain TA97a

Controls 3

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminofluoere

Remarks:

strain TA97a

Controls 4

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

Remarks:

strain TA100

Controls 5

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminofluorene

Remarks:

strain 100

Controls 6

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

cumene hydroperoxide

Remarks:

strain TA102

Controls 7

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: danthrone

Remarks:

strain TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two plates were used for each concentration tested and for both positive and negative controls. All the experiments were repeated once and a total of four plates were analysed for each concentration tested.

Statistics:

Results of the Ames mutagenicity assay were analysed using an analysis of variance test statistics with Dunnett’s multiple comparison test with control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

and TA104

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

Weak but significant increase in revertant colonies in some concentrations.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10,000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

and TA104

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10,000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

and TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10,000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Renner and Munzer and Brusick et al. reported theobromine to be non-mutagenic in Ames mutagenicity assay. Negative results in the present mutagenicity assay in the strains TA97a and TA100 of theobromine fully support the earlier observations of these researchers. Since strains TA102 and TA104 showed very weak mutagenic effects with S9 activation, later on, some more concentrations 5, 50 and 500 µg/plate were also carried out only for these two strains to see whether the mutagenicity was increased in these concentrations. For this reason, the solvent control value presented in the tables for both these two strains was the mean reading of eight plates.

Any other information on results incl. tables

Table 1: Number of revertants induced by theobromine in Salmonella plate incorporation test using TA97a, TA100, TA102 and TA104 both with or without S9.

-S9= Without metabolic activation.

+S9= With metabolic activation.

Theobromine (µg/plate)	Number of revertants/plate
	-S9^a	+S9^a
TA97a
Control (DMSO)	127±21	152±17
1	130±35	157±14
10	140±18	155±12
100	140±27	166±13
1000	119±9	134±11
5000	129±12	128±18
10000	Toxic	Toxic
Positive control
NPD (20 µg/plate)	1098±86
		1033±66
TA100
Control (DMSO)	170±13	172±11
1	162±18	156±27
10	168±14	167±23
100	169±35	159±20
1000	154±14	150±33
5000	154±18	140±26
10000	Toxic	Toxic
Positive control
SA (1.5 µg/plate)	1240±70
2-AF (10 µg/plate)		1096±68
TA102
Control (DMSO)^b	316±45	340±47
1	312±39	321±44
5	320±26	337±30
10	302±56	375±42
50	314±38	406±60
100	287±30	515±60**
500	319±33	396±50
1000	283±19	340±46
5000	263±34	312±28
10000	Toxic	282±36
20000		Toxic
Positive control
CH (100 µg/plate)	1114±65
DN (30 µg/plate)		1021±27
TA104
Control (DMSO)^b	358±47	356±49
1	349±34	366±54
5	429±48	432±44
10	415±50	521±29**
50	402±50	460±52*
100	387±86	535±42**
500	401±54	463±59*
1000	318±44	452±91*
5000	317±65	370±54
10000	277±59	314±35
20000	Toxic	Toxic

^aMean ±S.D. of four plates. Results of each concentration were compared with the solvent control by Dunnett’s multiple comparison with control.

^bMean of eight plates.

* p<0.05.

** p<0.01.

Applicant's summary and conclusion

Conclusions:

The results of Ames mutagenicity assay indicate that theobromine is very weakly mutagenic in bacterial strains TA102 and TA104 with S9 mix. Since none of the number of these revertant colonies in different concentrations of drug treatment was more than double when compared with the revertant colonies of the solvent control, the mutagenicity results may be considered as very weak or non-mutagenic in these strains.

Executive summary:

A reverse mutation assay was conducted according to OECD guideline 471 with theobromine. Salmonella typhimurium strains TA97a, T100, TA102 and TA104 were exposed to theobromine at concentrations ranging from 1 to 20000 µg/plate for 48 hours with and without metabolic activation, DMSO was used as the solvent. Positive and solvent controls were included however positive control for TA104 was not available. A total of four plates were used for each test concentration and control. Strains TA102 and TA104 showed very weak mutagenic effect in few concentrations with metabolic activation. Some more concentrations (5, 50 and 500 µg/plate) were included for these strains. Theobromine at different concentrations produced weak but significant increases in revertants colonies in TA 102 and TA104 with metabolic activation. Moreover theobromine was toxic to diverse bacteria strains at 10000 and 20000 µg/plate. The results of Ames mutagenicity assay indicate that theobromine is very weakly mutagenic in bacterial strains TA102 and TA104 with S9 mix. Since none of the number of these revertant colonies in different concentrations of drug treatment was more than double when compared with the revertant colonies of the solvent control, the mutagenicity results may be considered as very weak or non-mutagenic in these strains.