Coffee, bean, roasted, ext.

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Investigation of coffee in sister chromatid exchange and micronucleus test in vivo

Author:

Aeschbacher HU et al.,

Year:

1984

Bibliographic source:

Fd Chem. Toxic. Vol. 22, No. 10, pp 803-807

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: original method

Version / remarks:

The micronucleus test. Schmid W. Mutation Research 31 (1975) 9-15

Principles of method if other than guideline:

- Principle of test: micronucleus test
- Short description of test conditions: test substance was applied sub-acutely to mice, and the effect was read in direct smears from bone marrow
- Parameters analysed / observed: bone-marrow metaphase chromosomes

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

Molecular formula:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

IUPAC Name:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

Constituent 2

Reference substance name:

not applicable - Inorganic constituents (including K, Mg and Ca) of Coffee, bean, roasted, ext.

Molecular formula:

not applicable - Inorganic constituents (including K, Mg and Ca) of Coffee, bean, roasted, ext.

IUPAC Name:

not applicable - Inorganic constituents (including K, Mg and Ca) of Coffee, bean, roasted, ext.

Constituent 3

Reference substance name:

Caffeine

EC Number:

200-362-1

EC Name:

Caffeine

Cas Number:

58-08-2

Molecular formula:

C8H10N4O2

IUPAC Name:

1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione

Constituent 4

Reference substance name:

1-methylpyridinio-3-carboxylate

EC Number:

208-620-5

EC Name:

1-methylpyridinio-3-carboxylate

Cas Number:

535-83-1

Molecular formula:

C7H7NO2

IUPAC Name:

1-methylpyridinium-3-carboxylate

Constituent 5

Reference substance name:

5-O-(3,4-dihydroxycinnamoyl)-L-quinic acid

EC Number:

206-325-6

EC Name:

5-O-(3,4-dihydroxycinnamoyl)-L-quinic acid

Cas Number:

327-97-9

Molecular formula:

C16H18O9

IUPAC Name:

3-{[3-(3,4-dihydroxyphenyl)acryloyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Instant coffee was obtained in sealed 100 g jars
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: coffee was always freshly prepared just before use, with boiling tap water and cooled to room temperature.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

Test animals

Species:

mouse

Strain:

Swiss

Remarks:

OF-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa Credo, France
- Age at study initiation: 10 weeks
- Weight at study initiation: not available
- Assigned to test groups randomly: yes
- Fasting period before study: not available
- Housing: individually in cages after random distribution
- Diet: ad libitum, Nafag diet 875
- Water: ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 55±5
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): not available

IN-LIFE DATES: From: To: not available

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Tap water was used as a test substance vehicle and negative control, administered at 1 mL/40 g bw/day

Details on exposure:

Instant coffee was administered to the animals by gavage in volumes up to 1 mL. Volumes were adjusted for body weight. Coffee doses were applied up to the highest tolerated dose level, which were determined in the preliminary assay. Instant coffee was administered for five consecutive days at 24-hour intervals, the last dose was given 6 hours before killing. The negative-group animals received water at the same time as the test animals were treated. The positive control animals were always treated 30 minutes and 6 hours before killing by intraperitoneal injection.

Duration of treatment / exposure:

5 days

Frequency of treatment:

24-hour intervals

Post exposure period:

6 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Remarks:

instant coffee

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

instant coffee

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

instant coffee

Doses / concentrations 4

Dose / conc.:

3 000 mg/kg bw/day

Remarks:

instant coffee

No. of animals per sex per dose:

9-10

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenethiophosphoramide
- Justification for choice of positive control(s): not available
- Route of administration: intraperitoneal
- Doses / concentrations: 1.5 mg/kg bw, injected at 30 and 6 hours prior to killing

Examinations

Tissues and cell types examined:

marrow cells from femurs

Details of tissue and slide preparation:

After killing, the femurs were removed and the marrow expelled with 2 ml of fetal bovine serum. After one or two flushings, the marrow cells obtained were dispersed in the serum as a fine suspension, which was centrifuged at 200 g for 10 minutes. The suparnatant was removed and the cells were resuspended in a small drop of serum and the slides were prepared. After drying for 24 hours, the slides were fixed for 5 minutes in absolute methanol and stained in 10% Giemsa for 45 minutes. They were then thoroughly rinsed with distilled water, air dried, cleared in xylitol and covered with Merckoglas spray.

Evaluation criteria:

1000 polychromatic were scored for micronuclei

Statistics:

Counts were checked for their mathematical distribution. Analysis of variance (ANOVA) based on the Poisson distribution was used to compare the difference in micronuclei frequency between animals receiving water only and those receiving instant coffee.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Instant coffee induced no significant effect in the micronucleus test when mice received up to 3 g coffee/kg body weight/day during five consecutive days. There was no difference in micronuclei frequency between the mice receiving water only and those receiving instant coffee.

Any other information on results incl. tables

See attached additional information on results.

Applicant's summary and conclusion

Conclusions:

Administration of oral doses of instant coffee given to Swiss OF-1 mice up to 3000 mg/kg/ body weight per day for five executive days did not induce increases in micronuclei above spontaneous levels.

Executive summary:

Instant coffee was administered to Swiss OF-1 mice by gavage up to 3000 mg/kg/ body weight per day for 5 consecutive days. The negative-group animals received water at the same time as the test animals were treated and positive control animals received triethylenethiophosphoramide at 1.5 mg/kg bw 30 minutes and 6 hours before killing by intraperitoneal injection.

After killing, the femurs were removed, bone marrow expelled, washed, stained and 1000 polychromatic were scored for the presence of micronuclei.

Instant coffee did not have any significant effect in the micronucleus test when mice received up to 3000 mg coffee/kg body weight/day during five consecutive days. There was no difference in micronuclei frequency between the mice receiving water only and those receiving instant coffee.

Under conditions of this study, instant coffee was considered non-genotoxic in mice.