Coffee, bean, roasted, ext.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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:

The effect of brewed and instant coffee on reproduction and teratogenesis in the rat.

Author:

Nolen GA

Year:

1981

Bibliographic source:

Toxicology and Applied Pharmacology, 58, 171-183

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The purpose of this study was to determine the effects of high intakes of coffee on reproduction and teratogenesis in the rat. The administration of the coffee solutions began shortly after weaning and continued for about 30 weeks, through two pregnancies.

GLP compliance:

no

Limit test:

no

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: Folgers instant coffee and ground vacuum coffee
- 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: stable
- 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: the brewed coffee was prepared freshly each day of the study by brewing 648 g of ground coffee in 16’244 ml of water for 47 min. The instant coffee solutions were made by dissolving 14 g of instant coffee crystals in 1000 ml of water heated up to 150ºF (65.5ºC).
- 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:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ARS/Sprague-Dawley, Inc. Madison, Wisconsin.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 21-23 days
- Weight at study initiation: not available
- Fasting period before study: no
- Housing: individually in stainless-steel suspended wire cages with raised floors. Pregnant rats were transferred into nesting cages equipped with plywood liners and shredded paper to make a nest
- Diet: Purina Laboratory Chow ad libitum
- Water: ad libitum during acclimation period
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2°C
- Humidity: 50 ±10 %
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): 12-hour light and 12-hour dark cycle

IN-LIFE DATES: From: To: not available

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

The appropriate fluid was dispensed in clean calibrated bottles daily at about 10 AM and the consumption measured.

Details on mating procedure:

- M/F ratio per cage:
- Length of cohabitation:
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility
- After successful mating each pregnant female was caged (how): nesting cages

Analytical verification of doses or concentrations:

yes

Remarks:

other

Details on analytical verification of doses or concentrations:

The brew solids were determined daily for each brew with Fuchs hydrometer and biweekly on a composite sample by reftactive index measurements.
These composite samples were also analyzed for pH, titratable acidity, and caffeine. Typical analyses showed brew solids of 0.90%, a caffeine level of 0.057%, pH 5.23 and titratable acidity of 13.65 ex-
pressed as milliliters of 0.1 N NaOH required to neu-tralize 1.5% solids in 100 ml of the coffee brew.

Duration of treatment / exposure:

Rats were exposed to coffee during growth to maturity phase (91 days), during mating and reproduction phase and during second phase of reproduction until pregnancy day 13 or 21.

Frequency of treatment:

Daily

Details on study schedule:

At the end of 91-day growth phase, some animals were sacrificed and the remaining rats were mated (one male to two females). Ten days after weaning their first litters, the females were mated the second time and were sacrificed at Day 13 or 21 of pregnancy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 other: %

Remarks:

brewed and instant coffee

Doses / concentrations 2

Dose / conc.:

50 other: %

Remarks:

brewed and instant coffee

Doses / concentrations 3

Dose / conc.:

100 other: %

Remarks:

brewed and instant coffee

No. of animals per sex per dose:

20 males and 30 females per dose per coffee type

Control animals:

yes

yes, concurrent vehicle

Details on study design:

Groups of animals were randomly assigned to the following treatments:
1) Distilled water
2) 100% brewed coffee
3) 50% brewed coffee
4) 25% brewed coffee
5) 100% instant coffee
6) 50% instant coffee
7) 25% instant coffee

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: no data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes

- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

- Time schedule for examinations: weekly food consumption and daily fluid consumption records

OTHER: During first 12 weeks of the study, 10 randomly selected rats of each sex per group were transferred to stainless-steel metabolism cages, where they stayed for 48 hours without feed. Their urine was collected for two consecutive 12-hour period. The total volume for 24 hours was measured and the second 12-hour collection was measured with Ames “Clinitest” strips for pH, sugar, ketones and total protein. Afterward the animals were returned to their regular cages.

After mating, vaginal smears were done daily until sperm were found in vaginal lavage. At about Day 15 or 15 of gestation, the pregnant females were transferred into nesting cages and inspected twice daily for the birth of the young

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

No data

Litter observations:

At birth, the number of live and dead pups were recorded but otherwise, they were not disturbed. Four days after birth the pups were counted, the litter weighted as a unit, and the sex of the pus was determined. If litter contained more than 8 pups, the litter was reduced to that number, discarding the extremely light and heavy pups but divided as evenly as possible between the sexes. The litter was weighted again. On Day 21 of lactation, the young rats were weighted individually and discarded.

Postmortem examinations (parental animals):

After the end of the 91-day growth phase, 10 rats of each sex per group were randomly selected and sacrificed by excessive ether.The rats were examined grossly and blood was removed from the vena cava for WBC, RBC, hemoglobin, hematocrit and lymphocyte differential counts. The heart, liver, kidneys and gonads were removed and weighted; these and the following organs were removed, fixed in 10% neutral, buffered formalin, mounted in paraffin, sectioned, and stained with haematoxylin-eosin: lung, stomach, pancreas, jejunum, colon, mesenteric lymph node, urinary bladder, spleen, and brain. The slides were examined for histopathology.

After second mating, female rats were subdivided into two groups, one subgroup was sacrificed at Day 13 and the other at Day 21 of pregnancy. The numbers of corpora lutea, implantation and resorption were counted.

Postmortem examinations (offspring):

The fetuses from the second mating were removed by C-section from the dams sacrified on Day 21 were weighed, their sex was determined and they were inspected for gross abnormalities.
One third of the fetuses were randomly seleted, cleared in alcoholic KOH, stained with Alizarin, and examined for skeletal abnormalities. The remaining fetuses were fixed whole in Bouin’s fixative for 2-4 weeks, razorblade sectioned, and examined for soft-tissue abnormalities.

Statistics:

The continuous data were analyzed by the analysis of variance, least significant difference test, Bartlett’s test for homogeneity, Wilcoxon’s rank sum test and t test for unequal variance. The discrete data were analyzed by X2.

Offspring viability indices:

Overall average: 5.6
Lacation indice overall average: 83

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four rats died and four moribund were sacrificed during the study (thee males and five females). Five had severe pulmonary disease; one female given 25% brewed coffee died during parturition; one female given 25% instant coffee was sacrificed because of a mammary carcinoma and one male given 100% brewed coffee because of a nephroblastoma. These deaths and/or conditions were scattered through the test and control groups and were not related to the coffee treatments.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Sub-chronic study:
During 91-day period, rats given either brewed or instant coffee solutions gained as much as or more weight than control rats given water. In fact, the three groups of female rats given brewed coffee solutions and the two groups of females given either the 100 or 50% instant coffee solutions, gained significantly more body weight than controls. All of the female rats given coffee more feed than the controls, while only the males given 50% brewed coffee or 50 and 25% instant coffee ate significantly more. The male rats fed 100% and 25% brewed coffee and 100% instant coffee solutions also ate more than controls although the difference was not statistically significant. Thus, in general, the rats that ate more, gained more weight, resulting in no significant differences in the feed efficiencies among groups of either sex.

Chronic study:
The pattern of growth and feed consumption of males seen at 13 weeks continued throughout the study. There were no significant differences for the gain in the body weight, but the animals given the coffee solutions continued to eat more. The increased feed consumption was statistically significant for the groups fed 100 and 50% brewed coffee and 50 and 25% instant coffee This increased consumption of feed by the rats drinking coffee solutions resulted in lower feed efficiencies, although only the group given 50% instant coffee was significantly different.

There were no significant differences in the gain in body weight of females at 26-28 weeks as there were at 13 weeks, although the tendency of rats drinking coffee solutions to eat more feed than the controls continued. They, too, had somewhat lower feed efficiencies, although none were statistically different.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Sub-chronic study:
The rats given either brewed or instant coffees at full strength (100%) drank significantly less than controls from the very beginning of the study. However, the rats given 50 or 25% solutions drank more than controls. Among the males, the difference was statistically significant only for the group fed the 25% dilution of brewed coffee, but among the females the differences were significant for the groups fed the two dilutions (50 and 25%) of both coffees.

The male rats ingested less caffeine per unit body weight than the females because of their larger size and of course, the rats given the instant coffee ingested less caffeine than those given brewed coffee.

Chronic study:
The pattern in fluid consumption seen at 13 weeks for both male and female rats continued through 28 weeks, although it was not quite as pronounced and showed somewhat different statistical significances. The male rats given the 100% coffee solutions drank less than the water controls, while the rats of both sexes given the two coffee dilutions drank more. The exception at 28 weeks was the group of females on 100% instant coffee; these drank more than the controls, although not statistically significantly more. The females given 100% brewed coffee drank amounts similar to the controls.

The daily intake of caffeine plateaued at approximately the values shown for week 13. Thus, the average daily caffeine consumption for the groups of males drinking brewed coffee was about 50, 30, and 17 mg/kg and those drinking instant coffee was 40, 28, and 13 mg/kg. The corresponding values for the female groups were 80, 45, and 30 mg/kg/day of caffeine from brewed coffee and 70,40, and 25 mg/kg/day of caffeine from instant coffee.

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

Sub-chronic study:
Hematological characteristics did not exhibit patterns indicative of a treatment-related effect.

Chronic study:
Hematological characteristics measured during the final necropsy were within the normal range of the rat and demonstrated no pattern considered to be related to the coffee treatments.

Clinical biochemistry findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The decreased fluid intake of the rats given full-strength coffees resulted in a decreased urnary output with concomitant increase in urinary protein. Otherwise the urinary characteristics measured were within the normal range of the rat and did not exhibit a dose-resonse relationship.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histopathology associated with the coffee treatments was observed in either sex.

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

The percentage of conceptions was unaffected by the coffee treatments and there were no significant differences in the numbers of pups born, nor in the numbers alive at 4 days after birth. At 4 days after birth, the pups of dams given either 25% brewed coffee or 50% instant coffee were significantly heavier than the controls. The fluid intake increased in all females during lactation, but the caffeine exposure remained similar to what it was during pregnancy. A portion of this was undoubtedly consumed by the pups themselves as they began to take solid feed and to drink during the final stages of lactation just prior to weaning.

The numbers of pups weaned (overall average of 5.6) and the lactation indices (overall average of 83) were somewhat lower than normal in this study (usually litter sizes of just under 8 and lactation indices averaging 90). However, all of the groups given coffee solutions weaned more pups and had similar or higher lactation indices than the control group. Thus, none of the coffee solutions had an adverse effect on fetal or neonatal viability. The mean weights of the pups from the coffee-treated groups were lower than the control pups at weaning. These differences were statistically significant only for the pups from the groups receiving 100% solutions of both coffees.

The conception rate during the second mating was about 90% and did not vary significantly among the groups. There were no significant differences in the numbers of corpora lutea or implantations in the dams sacrificed on either Day 13 or Day 21 of pregnancy.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

25 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day

Based on:

other: Caffeine consumption

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Litter from 1st pregnancy:
The mean weights of the pups from the coffee-treated groups were lower than the control pups at weaning. These differences were statistically significant only for the pups from the groups receiving 100% solutions of both coffees.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Description (incidence and severity):

On Day 21 of lactation pups were diccarded.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pups from the second litter sacrificed on Day 21 of pregnancy were examined for soft tissues and skeletal abnormalities. No significant differences were seen in the numbers of fetuses with soft tissue defects. In addition, no frank abnormalities were seen in the skeletons, although the incidence of pups with developmental variations in the stemebrae (missing or hypoplastic 2nd and 5th) was significantly increased in the two groups fed full strength coffees and in the group fed 50% brewed coffee. Thus, there seemed to be a retardation in the ossification and calcification of bone in the fetuses exposed to very high levels of coffee. This phenomenon appeared to be related to the caffeine intake, since the incidence was less, and not statisticallysignificant, in the group given 50% instant
coffee.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was a single significant difference in the number of resorptions in dams given 25% instant coffee and sacrificed on Day 13, but since no significant differences were seen in the groups given higher levels of coffee and none were seen in the dams sacrificed on Day 21, this increase in resorptions is not related to the coffee treatment. Thus, these high levels of coffee had no embryotoxic effect.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

There were no significant differences in the number of live or dead fetuses, indicating that the coffee treatments had no lethal effects on the late development of the fetus. There were no significant differences in the weights of the fetuses, another indication of the lack of effects of coffee ingestion during pregnancy.
These data, together with those from the reproduction phase (first litters), indicate that very high intakes of coffee (and therefore, caffeine) have no deleterious effects on the implantation, growth, or viability of the embryo or fetus.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

25 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: retardation in ossification and bone calcification

2

Key result

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

20 mg/kg bw/day

Based on:

other: caffeine cosumption

Sex:

male/female

Basis for effect level:

other: retardation in ossification and bone calcification

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a reproductive and teratogenicity study in rats receiving high doses of brewed and instant coffee, no deleterious effects on reproduction or embryogenesis was observed; only slight retardation of fetal calcification confined to the sternebrae was seen in pups from 50 and 100% of brewed and instant coffee groups.

Executive summary:

The purpose of this study was to determine the effects of high intakes of coffee (and caffeine) on reproduction and teratogenesis in the rat.

Animals received brewed or instant coffee as 100, 50, or 25% solutions instead of their drinking water. For the brewed coffee, this represented approximately 80, 40 or 20 mg/kg body weight per day of caffeine with slightly lower doses for the 3 solutions of instant coffee. Control animals received water.

The replacement of the rats’ drinking water with the coffee solutions exposed animals to very high doses of coffee and caffeine, albeit distributed throughout most of the day in small increments. This consumption was equivalent to up to 50-60 cups per day in the human.

The rats started receiving coffee solutions from shortly after weaning until they completed the second pregnancy, an interval of about 28 weeks. Thus, the reproductive organs were continually exposed to coffee and caffeine during maturation.

The higher doses (50 and 100%) produced minimal effects (enlarged kidneys and livers) in the dams, but none of the coffee treatments had an adverse effect on reproduction or teratogenicity. The only retardation of sternebral calcification at was seen in the pups from 50 and 100% of brewed and instant coffee groups.