Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 April 2018 - 30 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Extract obtained from the shell of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol
EC Number:
948-055-8
Molecular formula:
not applicable as it is a UVCB
IUPAC Name:
Extract obtained from the shell of Theobroma cacao (Malvaceae) by co-extraction with ethanol and propylene glycol
Test material form:
liquid: viscous
Details on test material:
- Physical appearance: dark brown to black viscous liquid
- Storage conditions: in refrigerator (2-8°C) protected from light

Method

Target gene:
Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9), prepared from male Sprague Dawley rats injected with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Dose range finding test (TA100 and WP2uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of the first experiment)
First experiment (TA1535, TA 1537 and TA98, with and without S9): 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (all strains, with and without S9): 52, 164, 512, 1600 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: The test item formed a clear brown solution in Milli-Q water. The vehicle was according to OECD guideline 471.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Remarks:
For more details see 'any other information on materials and methods'
Details on test system and experimental conditions:
Two individual experiments were performed: the first experiment was a direct plate assay and the second experiment was a pre-incubation assay. A dose-range finding study was performed and reported as part of the first experiment.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours (for both experiments)
- Pre-incubation period: 30 +/- 2 minutes (for the second experiment only)

NUMBER OF REPLICATIONS: 3

PLATE PREPARATION: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h.

NUMBER OF CELLS EVALUATED: 10^9 cells/mL

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this laboratory.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in both experiments, no precipitation was observed at the start or at the end of the incubation period in any of the strains and at any of the doses tested.

CYTOTOXICITY:
- Experiment 1: No cytotoxicity observed in any of the tester strains.
- Experiment 2: No cytotoxicity observed in any of the tester strains. Except in tester strain TA1537 in the absence of S9-mix where fluctuations in the number of revertant colonies below the laboratory historical control data range were observed at the dose level of 5000 µg/plate. However since the reduction in the mean number of revertant colonies was only minor when compared against relevant historical control data and no toxicity was observed in any of the other tester strains in both experiments, this reduction is caused by incidental fluctuations in the number of revertant colonies and is considered as not biologically relevant.

MUTAGENICITY: In both experiments, in all strains and at all doses tested, no biologically relevant increase in the number of revertants was observed upon treatment with the test item, with and without S9.

HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 2 Historical data for the solvent control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 – 29

3 – 27

3 – 20

3 – 23

8 - 41

8 – 55

61 – 176

60 - 160

10 – 59

9 - 67

Mean

10

11

6

6

16

22

110

106

26

33

SD

3

4

2

3

5

7

17

20

6

8

n

2458

2426

2402

2352

2416

2458

2473

2398

2237

2217

Table 3 Historical data for the positive control

 

TA1535

TA1537

TA98

S9-mix

-

+

-

+

-

+

Range

128 – 1530

73 – 1206

58 – 1407

54 – 1051

365 – 1978

250 – 1977

Mean

901

239

817

340

1355

903

SD

174

115

354

160

230

357

n

2400

2296

2051

2337

2357

2367

 

TA100

WP2uvrA

S9-mix

-

+

-

+

Range

439 – 1993

408 - 2379

93 – 1958

111 - 1359

Mean

905

1249

1059

444

SD

163

371

506

144

n

2402

2354

2153

2232

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Applicant's summary and conclusion

Conclusions:
The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that Cocoa Shell Extract (Pot) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.