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Diss Factsheets

Administrative data

Description of key information

NOAEL (rat, 91 days): 25% brewed and instant coffee dilutions, equivalent to 20 mg/kg bw/day of caffeine.

NOAEL (rat, 90 days): 1'500 ppm; males 151 mg/kg bw/day; females 174 mg/kg bw/day of caffeine

NOAEL (mouse, 90 days): 1'500 ppm; males 167 mg/kg bw/day; females 179 mg/kg bw/day of caffeine

NOAEL (rat, 2 years): 2'000 ppm, males 102 mg/kg bw/day; females 170 mg/kg bw/day of caffeine

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
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
Qualifier:
no guideline available
Principles of method if other than guideline:
90-day subchronic study (growth to sexual maturity)
GLP compliance:
no
Limit test:
no
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
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.
- Diet (e.g. ad libitum): Purina Laboratory Chow ad libitum
- Water (e.g. ad libitum): 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
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
The appropriate fluid was dispensed in clean calibrated bottles daily at about 10 AM and the consumption measured.
Analytical verification of doses or concentrations:
yes
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 solutions during growth to maturity phase (91 days)
Frequency of treatment:
Daily
Dose / conc.:
25 other: %
Remarks:
brewed and instant coffee
Dose / conc.:
50 other: %
Remarks:
brewed and instant coffee
Dose / conc.:
100 other: %
Remarks:
brewed and instant coffee
No. of animals per sex per dose:
20 males and 30 females per group
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
Observations and examinations performed and frequency:
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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 91 day
- Anaesthetic used for blood collection: animals were sacrificed
- Animals fasted: Not specified
- How many animals: 10 rats of each sex par testing group
- Parameters examined: WBC, RBC, hemoglobin, hematocrit, lymphocyte differencial count

CLINICAL CHEMISTRY: Not specified

URINALYSIS: Yes

IMMUNOLOGY: No

OTHER: none
Sacrifice and pathology:
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 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.
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.
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):
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.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
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.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological characteristics did not exhibit patterns indicative of a treatment-related effect.
Clinical biochemistry findings:
not specified
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The dicreased fluid intake of the rats given full-strength coffees resulted in a decreased urnary output with s 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 examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of 91-day growth period, among the males, the kidneys of the rats given both full-strength coffees were enlarged (based on both absolute and relative weights). The only other statistical difference among males was in the weight of the hearts of rats given 50% instant coffee, but this appeared to be without biological significance. Among the females, both the liver and the kidneys were heavier in all rats given brewed coffee, although the relative weights (ratios) were significantly higher only in the rats given the full-strength solution. Both the absolute and relative weights of these two organs were increased in the females given either 100 or 50% solutions of instant coffee. However, there were no clear patterns of dose-responses.

Significant differences also occurred in the heart weights of females given 50% brewed coffee and 100 or 50% instant coffee, as well as in the ovary/body weight ratio of rats given 25% instant coffee. These differences can be dismissed, since there is no dose-response, and they appear to be within normal limits.

Although there was liver and kidney enlargement in the females and kidney enlargement in males, no histopathology related to the ingestion of the coffees was discerned in any of the tissues examined in either males of females. The liver and kidney enlargement appeared to be adaptive hyperthophy.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
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:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
25 other: %
Based on:
test mat.
Remarks:
Brewed/instant coffee
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
other: caffeine
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Conclusions:
In a 91-day study in rats receiving high doses of brewed and instant coffee, the NOAEL for both male and female rats was 25% brewed and instant coffee dilutions, which was equivalent to 20 mg/kg body weight per day of caffeine.
Executive summary:

In a 91-day study, female and male rats 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.

At the end of 91-day period, male rats receiving both full-strength coffees had enlarged kidneys.

The only other statistical difference among males was in the weight of the hearts of rats given 50% instant coffee, but this appeared to be without biological significance. In females receiving either 100 or 50% solutions of instant coffee, the absolute and relative weights of liver and kidneys were increased; however, there were no clear patterns of dose-responses.

Although there was liver and kidney enlargement in the females and kidney enlargement in males receiving higher doses of coffees, no histopathology related to the ingestion of the coffees was discerned in any of the tissues examined in either males of females. The liver and kidney enlargement appeared to be adaptive hyperthophy.

The NOAEL for both male and female rats was 25% brewed and instant coffee dilutions, which was equivalent to 20 mg/kg body weight per day of caffeine.

Endpoint:
sub-chronic toxicity: oral
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
Qualifier:
according to guideline
Guideline:
other: NTP guideline
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine (batch no. 51589)
- Expiration date of the lot/batch: not available
- Purity: >99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: kept in shipping container and placed in the storage room at 18-25°C
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water, stable for 32 days
- 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: caffeine was dissolved in filtered deionized sterile (FDS) water
- 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
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Fischer 344 rats were obtained from the National Institute of Health and rederived (at NCTR) by cesarean delivery under gnotobiotic conditions. The production colony from which rats were allocated was maintained under specific pathogen-free (SPF) conditions so that no quarantine period was required.
- Age at study initiation: 31-40 days old
- Weight at study initiation: mean weight 114.0 g (males); 98.6 g (females)
- Fasting period before study:
- Housing: Rats were housed, three per cage, in polycarbonate cages capped with polyester filter bonnets. Hardwood chip bedding was used. Males and females were kept in separate columns on all five shelves of a stainless steel rack. The rack level position of dose groups was randomly determined except that the level location of a given dose group was the same for both sexes.
- Diet: autoclaved Purina 5010 meal ad libitum
- Water: control rats received water ad libitum; dosed animals received caffeine via the drinking water.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C
- Humidity: 50 %
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle with fluorescent lighting maintained at 100 foot candles

IN-LIFE DATES: From: To: not available
Route of administration:
oral: drinking water
Details on route of administration:
The animals were subjected to caffeine solutions in the drinking water for a period of 90 days. Water bottles were changed twice weekly.
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
188 ppm
Dose / conc.:
375 ppm
Dose / conc.:
750 ppm
Dose / conc.:
1 500 ppm
Dose / conc.:
3 000 ppm
No. of animals per sex per dose:
12
Control animals:
yes
yes, concurrent no treatment
Positive control:
none
Observations and examinations performed and frequency:
Body weight was recorded for each week the animals were on the test. Observations for signs of toxicity were recorded weekly, during the changing of the cages.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No marked changes in clinical signs of toxicity were observed up to 1500 ppm.
Mortality:
no mortality observed
Description (incidence):
No animals died during the course of the experiment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was little variation that occurred during the 90 day bioassay in female rats. Male rats show a significant difference in terminal body weight for both the 1500 and 3000 ppm dose groups, though only the 3000 ppm dose group had greater than a 10% differential. Mean weight gain determinations indicate that only the 3000 ppm caffeine dose group male and female animals had greater than a 10% depression in weight gain. In the 188, 375, 750 ppm dose groups (male and female) there was about a 7% increase in mean weight gain compared to control rats.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was decreased in rats given 3000 ppm, whereas it was increased in the 188, 375 and 750 ppm groups.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No dose-related changes in clinical chemistry were seen.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Female rats show a significant increase in liver/body weight ratios for the 375 ppm and above dose groups; this effect is not present for male rats. Kidney/body weight ratios increased for female rats from 375 ppm and up, and for males from 750 ppm and up dose groups. Brain/body weight ratios increased for male rats at the 3000 ppm level whereas thymus/body weigh ratios decreased for the 3000 ppm dose group female rats. Dose response trends appear for both the liver/body weigh and kidney body weigh ratios. Mean liver weights were significantly increased from control values (P<=0.04) for the 375, 750, 1500, and 3000 ppm female dose group. This effect was not observed for male rats. There was a significant decrease (P<=0.01) in mean thymus weights for female 3000 ppm dosed rats. Kidney mean weights were significantly increased (P<=0.01) from control for the 750, 1500, and 3000 ppm female dosed rats and for the 1500 ppm dose male rats. Covariance analysis of pathology receiving weights indicate a significant difference only for the 750 ppm female and the 3000 ppm male dose groups. Since there were no notable gross or microscopic lesions identified in this organ system, the biological significance of this data is speculative.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
With one exception (cellular enlargement in salivary gland), no pronounced significant changes in gross morphology or microscopic findings were observed. The effect observed in the salivary gland was described as dose dependent. Reversible effects in the salivary glands are a well known pharmacological effect of caffeine (sympathicomimetic). These morphological changes are not considered to be an adverse effect of the substance.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Detailed histopathology was performed initially on all surviving animals in the high dose (3000 ppm) and control rats, and subsequently for all dose levels. The only microscopic alterations which were treatment related appeared in the rat salivary gland. Cytoplasmic alterations were present in parenchymal cells of both the parotid and submandibular salivary glands. These changes were most marked in the high dose group and diminished with decreasing dose.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Sex:
male/female
Remarks on result:
other: male 151 mg/kg bw/day; female 174 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
In an adequately performed 90-day oral study in female and male Fisher 344 rats receiving 188, 375, 750, 1'500, and 3'000 ppm of caffeine in drinking water, the NOAEL was established at 1'500 ppm (male 151 mg/kg bw/day; female 174 mg/kg bw/day)
Executive summary:

In a 90-day oral toxicity study, caffeine was administered in drinking water to groups of Fischer 344 rats (groups of 12 animals/sex). Rats were given 188, 375, 750, 1'500, and 3'000 ppm (ca. 19.7, 42, 85.4, 151, 272 mg/kg bw/day for males and 23, 51, 104, 174, and 287 mg/kg bw/day for females). The control groups were given tap water. The body weight gains of all treated rat groups were decreased, however, the effect was significant in the highest dose only (reduction of 26%, males, 20%, females). Water consumption was decreased in rats given 3,000 ppm, whereas it was increased in the 375 and 750 ppm groups. No marked changes in clinical signs of toxicity were observed up to 1,500 ppm. No dose-related changes in clinical chemistry were seen with one exception (cellular enlargement in salivary gland), no pronounced significant changes in gross morphology or microscopic findings were observed. The effect observed in the salivary gland was described as dose dependent in rats; in mice it was observed only in the highest dose group. The effects in the salivary gland were considered adaptive. Reversible effects in the salivary glands are a well-known pharmacological effect of caffeine (sympathomimetic). These morphological changes were not considered to be an adverse effect of the substance. Finally, microscopic evaluation of sex organs revealed no significant differences between exposed and control rats. The NOAEL was established at 1,500 ppm (male 151 mg/kg bw/day; female 174 mg/kg bw/day).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: NTP guideline
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine (batch no. 51589)
- Expiration date of the lot/batch: not available
- Purity: >99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: kept in shipping container and placed in the storage room at 18-25°C
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water, stable for 32 days
- 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: caffeine was dissolved in filtered deionized sterile (FDS) water
- 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
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Original stocks of C57BL/JN females and C3H/Hen (MTV-) males were obtained di rectly from the Nati ona 1 Institutes of Health colonies. B6C3F1 mice were produced from these strains at NCTR under specificpathogen-free (SPF) barri er con,diti ons so that no quaranti ne peri od was required.
- Age at study initiation: 31 to 40 days old
- Weight at study initiation: average weight, 16 and 19 g, females and males respectively.
- Fasting period before study: not specified
- Housing: Mice were housed, four per cage, in polycarbonate cages capped with polyester filter bonnets. Hardwood chip bedding was used. Males and females were kept in separate columns on the bottom five shelves of a six-shelf stainless steel rack. The rack level position of dose groups was randomly determined except that the level location of a given dose group was the same for both sexes.
- Diet: autoclaved Purina 5010 meal ad libitum
- Water: control mice, ad libitum; dosed animals received caffeine via the drinking water.


DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C
- Humidity: 50%
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle with fluorescent lighting maintained at 100 foot candles

IN-LIFE DATES: From: To: not available
Route of administration:
oral: drinking water
Details on route of administration:
The animals were subjected to caffeine solutions in the drinking water for a period of 90 days. Water bottles were changed twice weekly.
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
94 ppm
Dose / conc.:
188 ppm
Dose / conc.:
375 ppm
Dose / conc.:
750 ppm
Dose / conc.:
1 500 ppm
No. of animals per sex per dose:
12
Control animals:
yes
yes, concurrent vehicle
Positive control:
none
Observations and examinations performed and frequency:
Body weight was recorded for each week the animals were on the test. Observations for clinical signs of toxicity were recorded weekly, during the changing of the cages. A detailed gross necropsy was performed on all treatment and control mice during the experiment.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No animals died during the dosing phase of the experiment. One animal died (male, 375 ppm) the day of removal and was cannibalized by its cagemates.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain was significantly, but not dose-dependently depressed for the 188, 375, and 750 ppm male dose group animals by 26%, 25%, 18% respectively, but not in the 1'500 ppm dose. Females showed little variation in weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was decreased by 10% and more in the 1500 and 750 ppm dose group animals, whereas the water consumption was increased by 10% and more in the 94, 188, 375 ppm dose group animals.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum amylase levels for male and female mice at the 1500 ppm caffeine level and for female mice at the 94 ppm caffeine level were significantly lower than control levels. Pancreatic infection or toxicity would, however, most likely result in increased serum amylase levels. The biological significance of this finding is unclear. No conclusive dose-related trends were evident.
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Male mice showed a significant increase (P<=0.05) for brain/body weight ratios from the 375 ppm dose group. This difference was not present in female mice. Liver/body weight and R. kidney/body weight ratios were significantly increased (P<=0.01), for both male and female mice, for the 750 and 1500 ppm dose groups. No other significant differences in organ/body weight rati os were found. Dose response trends appear for both liver/body weight and R. kidney/body weight ratios in male and female mice and for brain/body weight ratios in male mice. Mean R. kidney weight (P<=0.03) was significantly decreased from controls for the 94 and 375 ppm dose group (male) and significantly increased (P<=0.01) for the 750 ppm dose group (females). Liver mean weight was significantly increased (P<=0.01) from control values for the 750 ppm (male and female) and significantly decreased (P<=0.05) for the 94 ppm dose group. Only the mean brain weight in the 750 ppm female dose group showed a significant difference from controls (P<=0.01).
Covariance analysis of pathology receiving weights indicated a significant decrease for the 750 and 1500 ppm dose groups (females) and for the 375 ppm (males) dose group. Since there were no notable gross or microscopic lesions identified in the organ systems showing ratio differences, the biological significance of this data is speculative.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No statistically significant changes in gross morpology were observed.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Description (incidence and severity):
No statistically significant changes in microscopic findings (including the sex organs) were observed.
Histopathological findings: neoplastic:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Sex:
male/female
Basis for effect level:
other: the highest tested dose
Remarks on result:
other: male 167 mg/kg bw/day; female 179 mg/kg bw/day
Conclusions:
In an adequately performed 90-day oral study in B6C3F1 female and male mice receiving 94, 188, 375, 750 and 1'500 ppm of caffeine in drinking water, the NOAEL was established at 1'500 ppm (male 17 mg/kg bw/day; female 179 mg/kg bw/day).
Executive summary:

In a 90-day oral toxicity study, caffeine was administered in drinking water to groups of B6C3F1 mice (groups of 12 animals/sex). Mice were administered 94, 188, 375, 750, and 1'500 ppm in the drinking water (ca. 21, 44, 85, 130, 167 mg/kg bw/day for males; 25, 47, 88, 134, 180 mg/kg bw/day for females). The control groups were given tap water.

Mean body weight gain was significantly, but not dose-dependently depressed for the 188, 375, and 750 ppm male dose group animals by 26%, 25%, 18% respectively but not in the 1'500 ppm dose. Females showed little variation in weight gain. Food consumption was unaffected and water consumption was decreased by 10% and more in the 750 and 1'500 ppm dose group animals, however, the water consumption was increased by 10% and more in the 94, 188, 375 ppm dose group animals. Adaptive changes in the salivary glands were also observed in mice in the highest dose group only. Additionally, no tumors were seen in the salivary glands of mice, which were treated, with caffeine in the drinking water over 2 years (Mohr et al. 1984). Since no clinical signs of toxicity, or changes in clinical chemistry were seen, neither were statistically significant changes in gross morphology or microscopic findings (including the sex organs) observed, the highest dose of 1'500 ppm (male 167 mg/kg bw/day; female 179 mg/kg bw/day) was considered a No Observed Adverse Effect Level.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Test material
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: natural caffeine containing less than 0.01% theobromine
- 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: caffeine was dissolved in water
- 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
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wiga, Sulzfeld, FRG
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 28 days
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 3 animals per cage on sterile soft-wood chip bedding
- Diet: ad libitum (Hope Farms RMH-B, Woerden, Holland)
- Water: ad libitum
- Acclimation period: not specified

DETAILS OF FOOD AND WATER QUALITY:
Closed formula pelleted diet (Hope Farms RMH-B, Woerden, Holland)
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45-55%
- Air changes: 20 room-air changes/hour
- Photoperiod: 12-hour light/dark cycle

IN-LIFE DATES: From: To: not available
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 years
Frequency of treatment:
daily
Dose / conc.:
200 mg/L drinking water
Dose / conc.:
430 mg/L drinking water
Dose / conc.:
930 mg/L drinking water
Dose / conc.:
2 000 mg/L drinking water
No. of animals per sex per dose:
80
Control animals:
yes
yes, concurrent vehicle
Details on study design:
Animals were allocated into six groups randomly, each group containing 80 males and females. Two groups served as controls and received plain water only. In each group, 30 males and 30 females were pre-designated for interim kills after 3, 6 and 12 months of treatment (10 per sex at each stage).
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, 7 days/week

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week throughout the entire study

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weekly
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: Not specified

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weekly
- Animals fasted: Not specified
- How many animals: Not specified

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified
Sacrifice and pathology:
Complete autopsy was done on all rats found dead or moribund, and on those killed during or at the end of the study period. Tissues and organs were fixed in 10% neutral buffered formalin and processed for routine histology; special stains were used when necessary. Blood samples were collected from all animals killed (except when moribund) and standard haematological and biochemical parameters were measured.
Statistics:
Body weight, food consumption, fluid intake, haematological and clinical chemical data were subjected to multivariate analysis of variance (MANOVA). Cumulative mortality data were tested for significance by the Kaplan-Meier method. The chi-square statistic was generally applied to frequency distributions of the incidence of pathological findings, as was the Mann-Whitney U-test. The Scheff multiple range test was additionally used in some instances. All these statistical analyses were performed according to SPSS computer programs.
Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No significant difference in mortality rate between test and control groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body-weight gain was considerably depressed by high caffeine intake. Terminal body weights of treated animals were lower than terminal control weights by 25% at the highest dose and by approximately 11% at the next lower level. There was no difference between the sexes in this respect. Although food consumption was somewhat lower at the highest caffeine dose, there was no effect on the efficiency of food utilization (calculated as g weight gain/100 g food eaten).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Fluid-consumption measurements showed that the females drank about 1.6 times more fluid in proportion to body weight than did the males. This resulted in a higher caffeine intake in females than in males. The overall means for the four treatment levels were 12, 26, 49 and 102 mg/kg/day for males, and 15, 37, 80 and 170 mg/kg/day for females.While there was a slight reduction in fluid consumption at the highest dose (2000 mg/L), the maximum intake occurred at 200 mg/L).
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related trends in hematological findings.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment-related trends in chemical biochemistry findings.
Urinalysis findings:
no effects observed
Immunological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related trends in non-neoplastic histopathology findings.
Description (incidence and severity):
Total tumor incidence, survival time, tumor latency and tumor multiplicity are provided in Table 1 (see attached document).
There was an inverse dose-response with respect to both frequency and multiplicity of tumors.

The distribution of benign and malignant tumors in the treatment groups and controls is shown in Table 2 (see attached document).
Compared with controls, the number of benign tumors in the highest-dose group was significantly decreased in both sexes. At the same time, the highest incidence of malignant tumors occurred in the two lower-dose groups (200 and 430 mg/L). High numbers of neoplasms (more than 10%) occurred only in the pituitary and thyroid glands of both sexes and in the mammary glands of female rats.
Details on results:
There were no statistically significant differences between control and treated animals for tumors of any type except for mammary fibroadenomas, the incidence of which was 50% in the controls compared with 26% in the highest-dose group. The incidence of mammary fibroadenomas showed a significant inverse dose-response relationship (chi-square test: P < 0.001).
The first tumor to be found among control rats was a malignant lymphoma in a female control rat in week 20. In the caffeine-treated rats, the first tumor observed was an epidermoid carcinoma of the skin found at week 16 in a female of the group given 430 mg/L. A number of organs in control and experimental animals showed a low incidence (< 10%) of neoplasms.
The frequency of C-cell (medullary) carcinomas of the thyroid was about 20% in both sexes and was evenly distributed across all treatment groups. Other types of thyroid tumors, such as cystic papillary adenomas and carcinomas of follicular-cell origin, occurred at a low incidence rate (up to 4%).
There were five ovarian granulosa-cell tumors, of which two were in control rats. Of the 20 cases of uterine polyps found, 14 were in treated rats and six were in controls. There were two adenocarcinomas of endometrial origin together with four neoplasms of the uterine wall (leiomyofibromas and/or sarcomas), which occurred in different groups.
Of a total of seven pancreatic islet-cell adenomas and four islet-cell carcinomas, all were in male rats, with five adenomas and two carcinomas occurring in controls. Pancreatic ductal adenomas were found in three cases, one of which was in a control rat. One malignant mesothelioma of pericardial origin was observed. Of the five tumors that arose in mediastinum and mesentery, three were liposarcomas and two were fibrosarcomas.
In the urogenital tract, a renal-cell carcinoma (clear and granular cell type) was observed in one male control rat. A transitional-cell papilloma and a transitional-cell carcinoma occurred in the renal pelvis of each of two male caffeine-treated rats. One male control had papillomatosis of the urinary bladder and one female control had a leiomyofibrosarcoma originating in the bladder wall.
Key result
Dose descriptor:
other: maximum tolerated dose
Effect level:
430 mg/L drinking water
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Remarks on result:
other: it corresponds to 30-40 mg/kg bw/day

See attached additional information on results

Conclusions:
The exposure to caffeine at 200, 430, 930 and 2000 mg/L for 2 years did not enhance or induce neoplasia in the Sprague-Dawley rats.
A maximum tolerated dose was established at 30 - 40 mg/kg body weight (430 mg/L drinking water) based on the reduced terminal body weights and reduced food and fluid consumption.
Executive summary:

In a 2-year chronic study, Sprague-Dawley rats received food-grade natural caffeine in drinking water. Groups of 50 animals per sex received levels of 200, 430, 930 and 2000 mg caffeine/L, while two control groups (50 animals per sex) received plain water.

There was no statistically significant increase in tumor incidence in treated animals as compared to controls even at doses exceeding the maximum tolerated dose and given to animals over a major portion of their lifespan.

A maximum tolerated dose was established at 30 - 40 mg/kg body weight (430 mg/L drinking water) based on the reduced terminal body weights and reduced food and fluid consumption.

Additional information

In a 91-day study, female and male rats 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. At the end of 91-day period, male rats receiving both full-strength coffees had enlarged kidneys. The only other statistical difference among males was in the weight of the hearts of rats given 50% instant coffee, but this appeared to be without biological significance. In females receiving either 100 or 50% solutions of instant coffee, the absolute and relative weights of liver and kidneys were increased; however, there were no clear patterns of dose-responses. Although there was liver and kidney enlargement in the females and kidney enlargement in males receiving higher doses of coffees, no histopathology related to the ingestion of the coffees was discerned in any of the tissues examined in either males of females. The liver and kidney enlargement appeared to be adaptive hyperthophy. The No Observed Adverse Effect Level (NOAEL) for both male and female rats was 25% brewed and instant coffee dilutions, which was equivalent to 20 mg/kg body weight per day of caffeine.

In a 90-day oral toxicity study, caffeine was administered in drinking water to groups of Fischer 344 rats and B6C3F1 mice (groups of 12 animals/sex). Rats were given 188, 375, 750, 1,500, and 3,000 ppm (ca. 19.7, 42, 85.4, 151, 272 mg/kg bw/day for males and 23, 51, 104, 174, and 287 mg/kg bw/day for females). Mice were administered 94, 188, 375, 750, and 1,500 ppm in the drinking water (ca. 21, 44, 85, 130, 167 mg/kg bw/day for males; 25, 47, 88, 134, 180 mg/kg bw/day for females). The control groups were given tap water. The body weight gains of all treated rat groups were decreased, however, the effect was significant in the highest dose only (reduction of 26%, males, 20%, females). Water consumption was decreased in rats given 3,000 ppm, whereas it was increased in the 375 and 750 ppm groups. No marked changes in clinical signs of toxicity were observed up to 1,500 ppm. No dose-related changes in clinical chemistry were seen with one exception (cellular enlargement in salivary gland), no pronounced significant changes in gross morphology or microscopic findings were observed. The effect observed in the salivary gland was described as dose dependent in rats; in mice it was observed only in the highest dose group. The effects in the salivary gland were considered adaptive. Reversible effects in the salivary glands are a well-known pharmacological effect of caffeine (sympathomimetic). These morphological changes were not considered to be an adverse effect of the substance. Finally, microscopic evaluation of sex organs revealed no significant differences between exposed and control rats. The NOAEL for rats was established at 1,500 ppm (male 151 mg/kg bw/day; female 174 mg/kg bw/day). In the mouse study mean body weight gain was significantly, but not dose-dependently depressed for the 188, 375, and 750 ppm male dose group animals by 26%, 25%, 18% respectively but not in the 1,500 ppm dose. Females showed little variation in weight gain. Food consumption was unaffected and water consumption was decreased by 10% and more in the 750 and 1,500 ppm dose group animals, however, the water consumption was increased by 10% and more in the 94, 188, 375 ppm dose group animals. Adaptive changes in the salivary glands were also observed in mice in the highest dose group only. Additionally, no tumors were seen in the salivary glands of mice, which were treated, with caffeine in the drinking water over 2 years (Mohr et al. 1984). Since no clinical signs of toxicity, or changes in clinical chemistry were seen, neither were statistically significant changes in gross morphology or microscopic findings (including the sex organs) observed, the highest dose of 1,500 ppm (male 167 mg/kg bw/day; female 179 mg/kg bw/day) was considered a NOAEL.

Caffeine in drinking water was administered to Sprague-Dawley rats (groups of 50 animals/sex) at concentrations of 200, 430, 930 and 2,000 ppm (12, 26, 49, 102 mg/kg bw/day males and 15, 37, 80, 170 mg/kg bw/day, females) for 104 weeks. A slightly increased mortality at 2,000 ppm was observed in rat males and decreased body weights were found in both sexes at 930 ppm (11%) and 2,000 ppm (25%), associated with reduced food and water consumption. There were no statistically significant differences between control and treated animals for tumor incidences of any type except for mammary fibroadenomas. The incidence of mammary fibroadenomas showed a significant inverse dose-response relationship (26% high dose vs. 50% control). There was no statistically significant increase in the tumor incidence of caffeine treated rats as compared to controls, even at doses exceeding the maximum tolerated dose and given to animals over a major portion of their lifespan

Justification for classification or non-classification

Appropriate repeated dose toxicity studies were performed on the suitable analogue substances of Coffee, bean, roasted, ext.

No clinical signs of toxicity and significant gross lesion or microscopic findings were seen in either rats or mice receiving coffee or caffeine. Liver, kidney and salivary gland enlargement appeared to be an adaptive hypotrophy, therefore, it can be concluded that the classification for specific target organ toxicity resulting from the repeated exposure is not warranted for Coffee, bean, roasted, ext.