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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
March, 2002 to July, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
other: Draft guidelines of what became OECD 487 in vitro micronucleus assay
Qualifier:
equivalent or similar to guideline
Guideline:
other: Although no definitive guidelines existed for this assay at the time, the methodology was in accordance with current literature and accepted scientific/regulatory principles in current guidelines for clastogenicity testing in vitro. Please Refer to CSR.
Deviations:
yes
Remarks:
Minor deviations from study plan which did not affect study are quoted in Appendix 7 of Final Study Report.
Principles of method if other than guideline:
Name of test material (as cited in study report): Fat Reduced cocoa powder
- Molecular formula (if other than submission substance):complex mixture
- Molecular weight (if other than submission substance): N/a complex mixture
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:
- Physical state:Powder
- Analytical purity: Suitable for human consumption
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:10-12% Fat, shell content 1.75% max
- Isomers composition: Not applicable
- Purity test date: 16/07/2001
- Lot/batch No.:XXXX
- Expiration date of the lot/batch: 24 January 2003
- Stability under test conditions: 1 year
- Storage condition of test material: Below 25oC in the dark
- Other:

GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Reference substance name:
Fat reduced cocoa powder
IUPAC Name:
Fat reduced cocoa powder
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Fat Reduced cocoa powder
- Molecular formula (if other than submission substance):complex mixture
- Molecular weight (if other than submission substance): N/A complex mixture
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance):
- Substance type:UVCB
- Physical state:Powder
- Analytical purity: Suitable for human consumption
- Composition of test material, percentage of components:10-12% Fat, shell content 1.75% max
- Isomers composition: Not applicable
- Purity test date: 16/07/2001
- Lot/batch No.:XXXX
- Expiration date of the lot/batch: 24 January 2003
- Stability under test conditions: 1 year
- Storage condition of test material: Below 25oC in the dark

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Cell cultures
V79 cells, supplied by Dr Eian Massey, BAT, Southampton, England, were maintainedat Covance Laboratories Limited in tissue culture flasks containing DMEM medium including 10% (v/v) foetal calf serum (FCS), 1% glutamine and 0.52% Penicillin/Streptomycin. They are subcultured regularly at low density, and before overgrowth occurs, to maintain low aberration frequencies. Stocks of cells preserved in liquid nitrogen are reconstituted for each experiment so as to maintain karyotypic stability. The cells are routinely screened for mycoplasma contamination.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA. The batches of MolTox S-9 were stored frozen
Test concentrations with justification for top dose:
Treatment
(hours + recovery) Doses analysed(micro g/mL) Cytotoxicity (%) +

3+17, -S-9 1582, 2813, 3750 55%
3+17, +S-9 2813, 3750, 5000 31%
20+0, -S-9 500.6, 889.9, 1582 57%


+ Reduction in cytokinesis-block proliferation index (CBPI) at highest concentration analysed
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; water

-Justification for choice of solvent/vehicle:
Water is known to be compatible with the test system. Cocoa was not soluble in any common solvents it was prepared Preliminary solubility data indicated that Fat Reduced Cocoa Powder formed a suspension in water for injection (purified water) with the aid of warming at 37°C and
vortexing at a concentration of at least 300 mg/mL. A top concentration of 5000 µg/mL was selected as the maximum for this study and was achieved by formulating the test article as a suspension at 50.00 mg/mL prior to a 10-fold dilution into culture medium.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Remarks:
Sterile purified water was added to cultures designated as negative controls as described in the methods section of this report. The positive control chemicals (for 3+17 hour treatments only) were dissolved in sterile anhydrous analytical grade DMSO.
Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; test article was formulated in water for injection (purified water)

DURATION
- Preincubation period: - Approximately 20 hours prior to treatment cells were removed from stock cultures using Trypsin/EDTA solution, and subcultured at a density of approximately 6 x 105 cells per flask into 25 cm2 tissue culture flasks. The final volume of culture medium in each flask was 10 mL. Flasks were gassed with 5% (v/v) CO2 in air for approximately 10-20 seconds, sealed and incubated at 37ºC for 20 hours. Cultures at a suitably low level of confluence were selected for treatment.

Exposure duration: 3 hours with and without S9 followed by 17 hours recovery (short) , and 20 hours with out S9 (long)
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours all treatments



SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays):Acridine orange in phosphate buffered saline.

NUMBER OF REPLICATIONS: 1.6-2 (replication time is 10-12 hours)

NUMBER OF CELLS EVALUATED: 2000 per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytokinesis block proliferation index treated cells compared to control.

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Other:

Evaluation criteria:
Evaluation criteria
The test article was to be considered as positive in this assay if:
1. A statistically significant increase in the proportion of cells with micronuclei occurred at one or more concentrations
2. The proportion of micronucleated binucleate cells (MNBN) at these concentrations exceeded the current historical negative control (normal) range in both replicate cultures
Statistics:
[The proportions of micronucleated cells in each culture were used to establish acceptable homogeneity between the replicates by means of a binomial dispersion test (11).
The proportion of cells with micronuclei for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test (11). Probability values of p <= 0.05 were accepted as significant.

Results and discussion

Additional information on results:
Controls
Sterile purified water was added to cultures designated as negative controls as described in the methods section of this report. The positive control chemicals (for 3+17 hour treatments only) were dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO) immediately prior to use, as follows:
Chemical
Supplier Concentration of treatment solution (mg/mL) Final concentration (µg/mL) S-9

Cyclophosphamide
(CPA) Sigma Chemical Co,
Poole, UK 0.6
0.8 6.0
8.0 +
+
4 Nitroquinoline 1-oxide
(NQO) Aldrich Chemical Co, Gillingham, UK 0.0125
0.025 0.125
0.25 -
-


Cells treated with 0.25µg NQO/mL and 8.0µg CPA/mL gave satisfactory responses in terms of quality and quantity of micronucleated binucleate cells. These were selected for analysis.
Metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA. The batches of MolToxTM S 9 were stored frozen in aliquots at -80°C and thawed just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities). The quality control statement relating to the batch of S-9 preparation used, is included in Appendix 4 of this report.
Preparation of S-9 mix
Treatment was carried out in both the absence and presence of S-9, prepared in the following way.
Glucose 6 phosphate, 0.2 M (61 mg/mL), NADP, 0.04 M (32 mg/mL), KCl, 1 M (74.55 mg/mL), MgCl26H20, 0.25 M (18.64 mg/mL), NaHPO4, 0.2 M (24 mg/mL), water and rat liver were mixed as indicated below to achieve a 10% S-9/Co-factor mix:
Chemical
Molarity
(M) Concentration
(mg/mL) Volume
(mL)
Glucose 6 phosphate 0.2 61.00 1.75
NADP 0.04 32.00 7
KCl 1.0 74.55 2.31
MgCl26H20 0.25 18.64 2.24
NaHPO4 0.2 24.00 35
Water - - 14.7
S-9 - - 7

An aliquot of the resulting S 9 mix was added to each cell culture designated for treatment in the presence of S-9 to achieve the required final concentration in a total of 10 mL. The final concentration of the liver homogenate in the test system was 1%.
Treatment
Immediately prior to treatment, the culture medium from all culture flasks was removed and the flasks washed with 5 mL Hanks Balanced Salt solution (HBSS) at room temperature.
All cultures had pre-warmed DMEM medium including 10% (v/v) heat activated foetal calf serum (FCS), and 0.52% Penicillin/Streptomycin added. This medium included test article or vehicle control treatments. Positive control treatments were not included with the 20+0 hour –S-9 treatment. Final treatment volume was 10 mL.
Quadruplicate cultures (A, B, C and D) were treated with the solvent, and duplicate cultures treated with the test article. Duplicate cultures were treated with the positive control chemicals, where appropriate. This scheme is illustrated below:
Treatment S-9 Number of cultures
3+17* 20+0*

Negative control -
+ 4
4 4
Test article
(doses as appropriate) -
+ 2
2 2
Positive controls
(all doses) -
+ 2
2

* Hours treatment + hours recovery

Immediately following the addition of treatment medium to the culture flasks, cultures were gassed with 5% (v/v) CO2 in air for approximately 10 seconds prior to returning to an incubator at 37C.
Treatment media remained on cultures receiving the continuous treatment until sampling, that is, 20 hours after the beginning of treatment. Cultures received pulse treatments (both in the absence and presence of S-9) for 3 hours only. Treatment medium was then removed; cell monolayers washed twice with HBSS (at room temperature). Cultures were then re-fed with fresh DMEM medium containing heat inactivated FCS (10%), 0.52% Penicillin/Streptomycin and Cytochalasin B (at a final concentration of 3 g/mL). Additionally, Cytochalasin B, formulated in DMSO was added to all continuous 20+0 hour treatment flasks (0.1 mL) to give a final concentration of 3 g/mL. All culture flasks were then gassed with 5% (v/v) CO2 in air for approximately 10 seconds prior to returning to an incubator at 37C. Cultures were then incubated for a further 17 hours prior to harvesting.
Summary of Treatment Conditions
Treatment S-9 Duration of treatment (hours) Harvest time (hours after start of treatment)

Continuous 20+0 - 20 20

Pulse 3+17 + 3 20
3+17 - 3 20

Harvesting
At the defined sampling time, the monolayers of these cultures were then removed using trypsin/EDTA.

The suspension from each flask was transferred to a plastic centrifuge tube containing fresh medium and an aliquot was taken for determination of cell number by using a Coulter Counter. The remaining suspension was stored at room temperature prior to slide preparation.
Preparation of microscope slides
A further aliquot of the cell suspension was removed for slide preparation and the cell concentration adjusted to approximately 6 x 104 cells/mL. Slides labelled with appropriate study details (e.g. study number, experiment number, dose, treatment, replicate and date of preparation) were loaded into a Cytospin® cytocentrifuge and 250 µL of cell suspension from the corresponding culture used per funnel (double funnel slides). Two slides were prepared per culture. The samples were then spun at 1000 rpm (approximately 110 x 'g') for 5 minutes at high acceleration. When the cycle was complete the slides are removed and allowed to air dry. Slides were then fixed by immersion into a bath of ice-cold 90% methanol for 9 minutes. Slides were then removed and allowed to air dry and stored at room temperature.
Slides were then stained by immersion in 125 g/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds. Slides were then washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approximately 10 minutes. The quality of the stain was then checked. Slides were then air-dried and stored in the dark at room temperature prior to analysis. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.
Selection of doses for micronucleus analysis
Slides were initially examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 500 cells per culture (where possible). From these data the cytokinesis-block proliferation index (CBPI) was determined using the following formula:

CBPI = number of binucleate cells + 2 (number multinucleate cells)
Total number of cells


Selections of random fields were observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

Rationale for dose selection
Doses for analysis were selected based on the CBPI determinations. The highest dose for micronucleus analysis from cultures sampled at 20 hours was one at which at least 50% (approximately) cytotoxicity had occurred or was to be the highest dose tested as illustrated in the Test article section. Analysis of slides from highly cytotoxic concentrations was avoided, if possible. Slides from the highest selected dose and two lower doses, such that a range of toxicity from maximum to little or none was covered, were taken for microscope analysis.
For each treatment regime, two solvent control cultures were initially analysed for micronuclei. Slides from the remaining solvent control cultures (C and D) were only to be analysed for micronuclei if necessary to help resolve an equivocal result or increased frequencies of micronuclei were observed in solvent controls, in which case the Sponsor was to be contacted.
A single positive control dose level, which gave satisfactory responses in terms of quality and quantity of binucleate cells and extent of micronucleated binucleate cells, was analysed (see Controls section).
CBPI data and the results of dose selection are presented in the results section of this report.
Slide analysis
Acceptance criteria
Binucleate cells were only included in the analysis if all the following criteria were met:
1.the cytoplasm had remained essentially intact, and
2.the daughter nuclei were of approximately equal size, and
3.any micronuclei present were separate from the main nucleus (micronuclei may be touching the main nucleus but must have clear boundaries).
Procedure
Slides from the positive control treated cultures were initially examined for micronuclei to ensure that the system had responded satisfactorily. Slides from the selected treatments and from solvent and positive controls were then coded, using randomly generated letters, by a person not connected with the scoring of the slides. Labels bearing only the study reference number, experiment number and the code were used to cover treatment details on the slide.
Analysis was performed using fluorescence microscopy. Nuclei and micronuclei appeared as a bright orange/yellow colour and the cytoplasm as a dull red colour.
Where possible, one thousand binucleate cells from each replicate (2000 per dose level) were analysed for micronuclei. Observations were recorded on raw data sheets and the microscope stage co-ordinates of the first six cells containing one or more micronuclei recorded (excluding positive control slides).
Slide analysis was performed off-site by an analyst trained in accordance with Covance Laboratories Limited Standard Operating Procedures. Details of the analyst are included in the responsible personnel section. All slides and raw data were returned to Covance Laboratories Limited for archiving in accordance with the Archive statement.
Analysis of results
Treatment of data
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei in each culture were obtained and tabulated (Appendix 1).
The proportions of micronucleated cells in each culture were used to establish acceptable homogeneity between the replicates by means of a binomial dispersion test (11).
The proportion of cells with micronuclei for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test (11). Probability values of p  0.05 were accepted as significant.
Acceptance criteria
The assay was to be considered valid if the following criteria were met:
1.the binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures, particularly where no positive responses were seen, and
2.the positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei, and
3.negative control cultures exhibited a proportion of binucleate cells which was at least 50% of the total cell population.
Evaluation criteria
The test article was to be considered as positive in this assay if:
1. A statistically significant increase in the proportion of cells with micronuclei occurred at one or more concentrations
2. The proportion of micronucleated binucleate cells (MNBN) at these concentrations exceeded the current historical negative control (normal) range in both replicate cultures.

Any other information on results incl. tables

Preparation of S-9 mix

Treatment was carried out in both the absence and presence of S-9, prepared in the following way.

Glucose‑6‑phosphate, 0.2 M (61 mg/mL), NADP, 0.04 M (32 mg/mL), KCl, 1 M  (74.55 mg/mL), MgCl26H20, 0.25 M (18.64 mg/mL), NaHPO4, 0.2 M (24 mg/mL), water and rat liver were mixed as indicated below to achieve a 10% S-9/Co-factor mix:


Chemical

Molarity
(M)

Concentration
(mg/mL)

Volume
(mL)

Glucose‑6‑phosphate

0.2

61.00

1.75

NADP

0.04

32.00

7

KCl

1.0 

74.55

2.31

MgCl26H20

0.25

18.64

2.24

NaHPO4

0.2

24.00

35

Water

-

-

14.7

S-9

-

-

7

 

An aliquot of the resulting S‑9 mix was added to each cell culture designated for treatment in the presence of S-9 to achieve the required final concentration in a total of 10 mL. The final concentration of the liver homogenate in the test system was 1%.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation

Treatment of cells with Fat Reduced Cocoa Powder in the absence and presence of metabolic activation (S-9) (all treatment regimes), resulted in frequencies of micronucleated binucleate (MNBN) cells that were similar to, and not statistically different from, those observed in concurrent vehicle controls for all concentrations analysed (Appendices 1 and 3). The MNBN cell frequency of all Fat Reduced Cocoa Powder treated cultures fell within historical negative control (normal) ranges.
Executive summary:

It is concluded thatFat Reduced Cocoa Powderdid not induce micronuclei in cultured Chinese hamster V79 cells when tested either at concentrations up to 5000mg/mL (3+17 hour, +S-9) or its limit of cytotoxicity (3+17 hour and 20+0 hour, -S-9) when treated in the absence and presence of metabolic activation (S‑9).

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