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Toxicological information

Genetic toxicity: in vitro

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
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
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-o-phenylenediamine (NPD)
Remarks:
strain TA97a
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminofluoere
Remarks:
strain TA97a
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
strain TA100
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminofluorene
Remarks:
strain 100
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
cumene hydroperoxide
Remarks:
strain TA102
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 resultsopen allclose all
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
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
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

-S9a

+S9a

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.