Registration Dossier
Registration Dossier
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EC number: 272-823-5 | CAS number: 68916-18-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
NOAEL (rat): 25% brewed and instant coffee dilution, equivalent to 20 mg/kg bw/day of caffeine
NOAEL (mouse) (NTP protocol):
ca. 22 mg/kg bw/day (F0 parental)
ca. 22 mg/kg bw/day (F1 offspring)
NOAEL (mouse) (NTP protocol):
ca. 22 mg/kg bw/day (F0 parental) (Task 2)
ca. 22 mg/kg bw/day (F1 offspring) (Task 2)
ca. 88 mg/kg bw/day (F1 parental) (Task 4)
ca. 88 mg/kg bw/day (F2 offspring) (Task 4)
NOAEL (rat) (NTP protocol): >12.5 mg/kg bw/day
Link to relevant study records
- 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
- 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
- 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. 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 - 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.
- 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 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
- 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 - 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: 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. - 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
- 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
- Critical effects observed:
- no
- 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.
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- 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
- 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
- Key result
- Reproductive effects observed:
- no
- 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.
- Endpoint:
- fertility, other
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From March 17 to December 21 1982
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- NTP guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: NTP protocol
- Principles of method if other than guideline:
- NTP Reproductive Assessment by Continuous Breeding (RACB), according to Reel JR et al. (1985). J Am Coll Toxicol 4: 147-162.
- GLP compliance:
- not specified
- 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 from Taylor Chemical Companyvia Midwest Research Institute (MRI) , Kansas City, Mo
- Expiration date of the lot/batch: not available
- Purity test date: >98.5%
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 deionized/sterile-filtered tap 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:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston, NY)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: not specified
- Fasting period before study: none
- Housing: solid bottom polypropylene or polycarbonate cages with stainless steel wire lids
- Diet: Purina Certified Pelleted Rodent Chow ad libitum
- Water: deionized/filtered water ad libitum
- Acclimation period: not specified
ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 1°C
- Humidity: not specified
- Air changes (per hr): 12 – 14
- Photoperiod (hrs dark / hrs light): 14-hour light and 10-hour dark cycle
IN-LIFE DATES: From: To: not available - Route of administration:
- oral: drinking water
- Vehicle:
- water
- Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- Dose-range finding (Task1) : 14 days
Continuous breeding phase (Task 2): 18 weeks, 1 week premating, 14 weeks cohabitation, 3 weeks segregation
Offspring assessment (Task 4): 14 weeks - Frequency of treatment:
- Continuously in the drinking water
- Dose / conc.:
- 0 other: %
- Remarks:
- water
- Dose / conc.:
- 0.012 other: %
- Remarks:
- in drinking water
- Dose / conc.:
- 0.025 other: %
- Remarks:
- in drinking water
- Dose / conc.:
- 0.05 other: %
- Remarks:
- in drinking water
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 21.9 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 43.8 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 87.9 mg/kg bw/day
- Remarks:
- actual ingested
- No. of animals per sex per dose:
- Dose range-Finding (Task 1): 8
Continuous breeding phase (Task 2): 20 (caffeine) and 40 (vehicle control)
Offspring assessment (Task 4): not specified - Control animals:
- yes, concurrent vehicle
- Details on study design:
- During the Preliminary Range-Finding Study (Task 1), eight week old animals were housed four per cage by sex, eight males and eight females per treatment group. There were six treatment groups: vehicle control and five dose groups. Treatment was administered in the drinking water for 14 days. Dosed drinking water (deionized and filtered) was available ad libitum.
For the Reproduction and Fertility Study (Task 2), the mice (11 weeks of age) were matched by weight and randomly assigned into four treatment groups: a vehicle control group (40 males and 40 females) and three dose groups (20 males and 20 females per group); housed as previously described. For the first week, animals were housed five per cage by sex. Subsequently, the females and males from the same dose group were randomly paired and cohabited for 100 days, one breeding pair per cage (i.e., 20 breeding pairs per dose group). Thereafter each male and female was housed individually for a period of 20 days. The litters delivered during this time was counted, sexed and weighed by sex.
Offspring assessment (Task 4) study was a continuation of the 120-day mating trial. The offspring obtained between days 100 and 120 from high dose and control groups were mated beginning at 70 ± 10 days of age and their reproductive capacity evaluated. Caffeine exposure via the drinking water continued throughout Task 4 (via lactation until weaning) and then directly until the breeding pairs (offspring of Task 2 animals) were sacrificed. Twenty male offspring (at least one male from each litter if possible) were randomly assigned to 20 female offspring from the same dose levels (in Task 2) (at least one female from each litter if possible). Sibling matings were avoided. - Positive control:
- none
- Statistics:
- Chi-square test, Fisher's Exact Test, Probit analysis, ANOVA, Duncan's Multiple Range Test, Jonckheere's Test for ordered differences, analysis of covariance
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- 11/80, 7/40, 22/40, and 20/40 mice in the 0%, 0.012%, 0.025% and 0.05% group, respectively, exhibited alopecia during Continuous Breeding. Chi-Square analysis revealed an overall significant difference (P<0.01) in alopecia for the dosed groups. Pairwise comparison by Fisher's Exact Test indicated that the number of mice with hair loss was significantly increased (P<0.01) in the 0.025 and 0.05% groups.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- The random distribution of the deaths across treatment groups indicated that they were not treatment-related.
- Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- Reduced numer of live pups per litter.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 21.9 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- reproductive performance
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 21.9 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- other: Reduced number of live pups per litter
- Remarks on result:
- other: F1 offspring
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Hair loss was observed in 2 of 69 control F1 parents and 6 of 43 F1 parents given 0.05% caffeine in their drinking water.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Male body weight was significantly depressed (p<0.01) in the 0.05% caffeine group relative to the 0.0% caffeine group, whereas there was no significant difference in the female body weight between these two groups.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Following adjustment of organ weights for body weight by analysis of covariance, the liver weight of male mice exposed to 0.05% caffeine in the drinking water proved to be significantly greater (p<0.05) than that for control male mice. Conversely, liver weight was unaffected by caffeine exposure in female mice. Neither brain weight, pituitary weight, nor reproductive organ weights were affected in F1 male and female mice given 0.05% caffeine in their drinking water.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related gross or histopathologic lesions were noted for the pituitary, testis, epididymis, prostate or seminal vesicles in male mice, or for the pituitary, ovary, oviduct, uterus or vagina in female mice.
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 87.9 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Remarks on result:
- other: none
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- 87.9 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- other: none
- Remarks on result:
- other: F2 offspring
- Key result
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 21.9 mg/kg bw/day (actual dose received)
- Treatment related:
- yes
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
- Conclusions:
- Under the conditions of this reproductive toxicity study caffeine (0.025 and 0.05% in the drinking water) was a reproductive toxicant in breeding pairs of CD-1 mice as evidenced by a significantly reduced number of live pups per litter in the F1 generation (Task 2). Further, the F1 generation exposed to 0.05% caffeine tended to have a reduced fertility rate and there was a tendency toward a decreased number of live pups per litter in the F2 generation (Task 4). It should be noted, however, that these reproductive effects were accompanied by changes in water consumption and by decreased male and female body weights in the 0.05% caffeine group relative to the control group. It was not possible to determine whether this effect was due to an effect on the male, female or both since a crossover mating trial was not conducted in this study.
- Executive summary:
Caffeine was simultaneously tested by two independent laboratories using the RACB protocol.
During the 14-day exposure period of dose-range finding (Task 1) 8 male and 8 female CD-1 mice (8 weeks of age) per dose group were given 0.0, 0.012, 0.033, 0.1, 0.3, or 1.0% caffeine in their drinking water. During the exposure period, one male in the 0.012% caffeine group, two males in the 0.1% caffeine group, and 7 males and 8 females in the 1% caffeine group died.
Both the male and female CD-1 mice in the 1% caffeine group exhibited dehydration, piloerection, ptosis and lethargy a few days prior to death. Daily water consumption per mouse was slightly increased in the 0.033% caffeine group and markedly reduced in the 0.1, 0.3, and 1% caffeine groups relative to the control group.
Based on the acute toxicity results (Task 1), drinking water levels of 0.0, 0.012, 0.025, and 0.05% caffeine were selected for the continuous breeding phase of the study (Task 2). Assuming that a male or female CD-1 mouse weighing 40 g consumed 7 g of water per day, then these drinking water levels represented daily intakes of 0.0, 21.9, 43.8, and 87.5 mg/kg body weight/day, respectively.
Continuous exposure of CD-1 mice (11 weeks of age at outset) to these dose levels of caffeine in the drinking water during the 7-day premating, 98-day cohabitation, and 21-day segregation periods had no effect on the number of pairs able to produce at least one litter In addition, caffeine, at the dose levels given, had no influence on the number of litters produced per pair, proportion of pups born alive or sex of pups born alive (males/total), or live pup weight. In contrast, there was a significant (p<0.01) dose-related decrease in the number of live pups per litter as indicated by Jonckheere's Test for ordered differences. In particular, 0.025 and 0.05% caffeine levels in the drinking water resulted in a significantly decreased number of live pups per litter for female pups and for the sexes of pups combined. The number of male pups per litter was significantly reduced only at the 0.05% caffeine level relative to the control group.
Due to the lack of an effect of caffeine on fertility and its minimal effects on reproductive performance in the Task 2 parental mice (F0 generation), it was decided to assess fertility and reproductive performance in the F1 offspring (Task 4). That is, continuous exposure to 0.05% caffeine via the drinking water had no significant effects on mating behavior, proportion of pups born alive or sex of pups born alive (males/total), or live pup weight. Although there was a tendency toward a decreased fertility rate (p=O.065) and a reduced number of live pups per litter in the 0.05% caffeine group as compared to the 0.0% caffeine group, these differences were not statistically significant.
Sperm assessment indicated no significant differences in the percentage motile sperm, sperm concentration, or percentage abnormal sperm in the cauda epididymis between male mice exposed to 0.0 or 0.05% caffeine in the drinking water. In contrast, male body weight was significantly depressed (p<0.01) in the 0.05% caffeine group relative to the 0.0% caffeine group, whereas there was no significant difference in the female body weight between these two groups. Following adjustment of organ weights for body weight by analysis of covariance, the liver weight of male mice exposed to 0.05% caffeine in the drinking water proved to be significantly greater (p<0.05) than that for control male mice. Conversely, liver weight was unaffected by caffeine exposure in female mice.
The NOAEL of ca. 22 mg/kg body weight/day was derived for F0 parental and F1 offspring generation from Task 2. The NOAEL of ca. 88 mg/kg bw/day (the highest tested dose) was derived for F1 parental and F2 offspring generation from Task 4.
- Endpoint:
- fertility, other
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 31 March 1982 to 01 December 1983
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- NTP guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: NTP protocol
- Principles of method if other than guideline:
- NTP Reproductive Assessment by Continuous Breeding (RACB)
- GLP compliance:
- yes
- 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 from Taylor Chemical Company via Midwest Research Institute (MRI) , Kansas City, Mo
- Expiration date of the lot/batch: not available
- Purity test date: >98.5%
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 deionized/sterile-filtered tap 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:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston, NY)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: not specified
- Fasting period before study: none
- Housing: polycarbonate cages with stainless steel wire lids
- Diet: Purina Certified Rodent Chow ad libitum
- Water: deionized/filtered water ad libitum
- Acclimation period: not specified
ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 20-70%
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 14-hour light and 10-hour dark cycle. Lights on between 5 am to 7 pm.
IN-LIFE DATES: Animals received March 16, 1982 to between October 26- November 19, 1982 - Route of administration:
- oral: drinking water
- Vehicle:
- water
- Details on exposure:
- Dose-range finding (Task 1): 14 days
Continuous breeding phase (Task 2): 18 weeks, 1 week premating, 14 weeks cohabitation, 3 weeks segregation
Crossover mating (Task 3): 14 weeks - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- Dose-range finding (Task 1): 14 days
Continuous breeding phase (Task 2): 18 weeks, 1 week premating, 14 weeks cohabitation, 3 weeks segregation
Crossover mating (Task 3): 14 weeks - Frequency of treatment:
- Continuously in the drinking water
- Dose / conc.:
- 0 other: %
- Remarks:
- water
- Dose / conc.:
- 0.012 other: %
- Remarks:
- in the drinking water
- Dose / conc.:
- 0.025 other: %
- Remarks:
- in the drinking water
- Dose / conc.:
- 0.05 other: %
- Remarks:
- in the drinking water
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 21.9 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 43.8 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 87.3 mg/kg bw/day
- Remarks:
- actual ingested
- No. of animals per sex per dose:
- Task 1: 16 animals per group;
Task 2: 20 males and 20 females per dose group, 40 males and 40 females in the control group
Task 3: not specified - Control animals:
- yes, concurrent vehicle
- Details on study design:
- During the Preliminary Range-Finding Study (Task 1), eight week old animals were housed four per cage by sex, eight males and eight females per treatment group. There were six treatment groups: vehicle control and five dose groups. Treatment was administered in the drinking water for 14 days. Dosed drinking water (deionized and filtered) was available ad libitum.
For the Reproduction and Fertility Study (Task 2), the mice (11 weeks of age) were matched by weight and randomly assigned into four treatment groups: a vehicle control group (40 males and 40 females) and three dose groups (20 males and 20 females per group); housed as previously described. For the first week, animals were housed five per cage by sex. Subsequently, the females and males from the same dose group were randomly paired and cohabited for 100 days, one breeding pair per cage (i.e., 20 breeding pairs per dose group). Thereafter each male and female was housed individually for a period of 20 days. The litters delivered during this time was counted, sexed and weighed by sex. Parameters evaluated: body weight, litter weight, fixed brain and pituitary weight, weight of complete reproductive tract (ovaries, oviducts, uterus and vagina).
Determination of affected sex (Task 3) designed to determine whether fertility in the male, female or both sexes has been affected during Task 2. Male mice, previously exposed to the maximum tolerated dose were mated with control female mice (Group 1). Similarly, female mice previously treated with the maximum tolerated dose of test article were matched with control males (Group 2). The remaining twenty control mice were randomly matched with the remaining 20 control females (Group 3). Cohabitation of matched mice was continued for 7 days and they did not receive caffeinated water during this time. After separation males and females were held in separated cages for 21 days. During this period the fertility data was obtained as previously described in Task 2. To determine the affected sex, the data from Group 1 and 2 was compared against data from Group 3. - Positive control:
- none
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Loss of body hair, especially the facial hair.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Five of 200 animals died during Task 2; three in the control group, 1 in the 0.012% dose group and 1 in the 0.025% dose group. Three of five animals died during cohabitation phase, and two during the holding period.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Caffeine administered in drinking water at 0.012 to 0.05% dose levels had no apparent effect on male or female body weights.
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- The average daily water consumption in the control and treatment groups was essentially the same.
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- 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
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Caffeine treatment did not produce any histopathologic changes in male or female CD-1 mice.
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- effects observed, treatment-related
- Description (incidence and severity):
- Caffeine treatment had no effect on the incidence of abnormal sperm (P>0.05). The average sperm motility for the control animals was significantly higher (P<0.05) than the treatment group (47 +/- 1 vs. 37 +/- 3). Sperm density in the treatment group was significantly higher (p<0.05) than the control group.
- Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- Task 2: The fertility index in the control and three treatment groups varied between 95-100%, all breeding pairs except 1 in the 0.012% dose group delivered at least one litter. The mean number of litters per fertile breeding pair was not affected by the treatment at up to 0.05% dose level (p>0.05). The average number of pups per litter, sex ratio, and mean live pup weight values were also not significantly affected by caffeine (p>0.05) except for the number of live male pups in the 0.05% dose group (p<0.05). When the live pup weight was adjusted for total number of live and dead pups per litter, the average male and the combined pup weight values in the 0.05% dose group were significantly decreased (p<0.05).
Even though the average number of litters per pair after 100 days of cohabitation in the 0.05% dose group was not significantly different than the control group, the group mean number of live pups from the first to fifth litter decreased with continued treatment.
During Task 3, animals from the 0.05% dose group were tested in a crossover mating trial to determine whether the males or females or both sexes had compromised reproductive performance when matched with control animals. There was no statistically significant difference (p>0.05) with respect to the percent fertile matings for both the treated males (0.05% caffeine) cohabited with control females (68%) and the treated females (0.05% caffeine) cohabited with control males (55%) as compared to the control pairs (61%). No significant effects (p>0.05) were noted for other reproductive parameters such as the litter size, the proportion of pups born alive, and the live pup weight. Sex ratio was, however, altered; the proportion of males in the pups born alive by the control fertile pairs was 0.68. The corresponding values for treated males cohabited with control females and treated females cohabited with control males were 0.55 and 0.51, respectively. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 21.9 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- reproductive performance
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 21.9 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- other: pup weight
- Remarks on result:
- other: F1 offspring
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 21.9 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- organ weights and organ / body weight ratios
- Key result
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 21.9 mg/kg bw/day (actual dose received)
- Treatment related:
- yes
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Caffeine administered at 0.05% level in drinking water caused a weak reduction in fertility in CD-1 mice.
- Executive summary:
Caffeine was tested simultaneously at two laboratories, each using a variation on the standard RACB study design.
During the 14-day exposure period of dose-range finding (Task 1) 8 male and 8 female CD-1 mice (8 weeks of age) per dose group were given 0.0, 0.01, 0.03, 0.1, 0.3, or 1.0% caffeine in their drinking water. All 16 animals exposed to 1.0% caffeine died within 2 weeks of treatment. The cause of death was diagnosed as cardiac myodegeneration and tubular nephrosis. Animals in the 0.30% dose group lost nearly 25% of their body weight after 14 days of treatment.
During Reproduction and Fertility Study (Task 2), the mice (11 weeks of age) were matched by weight and randomly assigned into four treatment groups: a vehicle control group (40 males and 40 females) and three dose groups (20 males and 20 females per group). Animals were administered caffeine at 0.012, 0.025 and 0.05% in drinking water. Five of 200 animals died during Task 2; three in the control group, 1 in the 0.012% dose group and 1 in the 0.025% dose group. Caffeine administered in drinking water at 0.012 to 0.05% dose levels had no apparent effect on male or female body weights.
The average number of pups per litter, sex ratio, and mean live pup weight values were also not significantly affected by caffeine except for the number of live male pups in the 0.05% dose group. When the live pup weight was adjusted for total number of live and dead pups per litter, the average male and the combined pup weight values in the 0.05% dose group were significantly decreased. Additionally, the group mean number of live pups from the first to fifth litter decreased with continued treatment.
During Task 3, no significant effects were noted for parameters such as the litter size, the proportion of pups born alive, and the live pup weight, however, sex ratio was altered.Female body weight at necropsy was reduced by 5%, while body-weight-adjusted organ weights were unchanged.Caffeine treatment had no effect on the incidence of abnormal sperm. The average sperm motility for the control animals was significantly higher than the treatment group. Caffeine treatment did not produce any histopathologic changes in male or female CD-1 mice.
Even though the overall effect of caffeine on fertility appeared negative, the following significant findings have been noted: a significant number of animals drinking caffeinated water lost body hair, especially facial hair. During Task 2 the number of live pups per litter in the 0.05% dose group decreased from 11.1 ± 0.5 in the first litter to 8.6 ± 1.0 in the fifth litter.
The NOAEL of ca. 22 mg/kg body weight/day (the lowest tested dose) was determined for F0 parental and F1 offspring generation (Task 2).
- Endpoint:
- fertility, other
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From April 1994 to May 1995
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP guideline study (NTP)
- Qualifier:
- according to guideline
- Guideline:
- other: NTP protocol
- Principles of method if other than guideline:
- NTP Reproductive Assessment by Continuous Breeding (RACB)
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: was purchased from Pfaltz & Bauer (Waterbury, Connecticut)
- Expiration date of the lot/batch: Not available
- Purity: 98.6 % (HPLC)
- Purity test date: 1993-05-27
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated at 1-7°C
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble
- 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: disolved in deionized water
- Preliminary purification step (if any): none
- Final preparation of a solid: not applicable - Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- Crl:CD BR
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories (Portage, Michigan)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 11 weeks; (F1) 81 +/- 12 days
- Weight at study initiation: Task 1: males: 359.3 - 424.1 g; females: 208.4 - 262.8 g; Task 2: (P) Males: 361.2 - 423.1 g; Females: 198 .1 - 268 .6 g; Task 3: (P) naive Males: 345.8 - 412.1 g; naive Females 219.0 - 269.0 g; Task 4: no data
- Fasting period before study: not specified
- Housing: polycarbonate cages suspended on stainless-steel racks
- Diet: Pelleted Zeigler™ NIH 07 Small Animal Feed at libitum
- Water: filtered tap water ad libitum
- Acclimation period: 13-15 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
IN-LIFE DATES: From: 1994-05-05 To: 1995-02-30; depending on the test group - Route of administration:
- oral: gavage
- Vehicle:
- water
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The identity and purity of the compound was confirmed by high performance liquid chromatography.
- Duration of treatment / exposure:
- Task 1 (dose range finding phase): 4 weeks
Task 2 (continuous breeding phase): 1 week pre-cohabitation exposure, 16 weeks cohabitation, 6 weeks lacation phase
Task 3 (male/female crossover mating): 1 week
Task 4 (fertility assessment): 1 week cohabitation, until post natal day 1 - Frequency of treatment:
- once a day
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 12.5 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 25 mg/kg bw/day
- Remarks:
- actual ingested
- Dose / conc.:
- 50 mg/kg bw/day
- Remarks:
- actual ingested
- No. of animals per sex per dose:
- Task 1: 8
Task 2: 20
Task 3: 50 naive males and 50 naive females in addition to Task 2 animals
Task 4: 2 males and 2 females from each litter - Control animals:
- yes, concurrent vehicle
- Details on study design:
- Task 1, the limited dose range-finding phase, was conducted to determine the appropriate dose levels for the continuous breeding phase of the study. Caffeine at dose levels of 0, 50, 100, 150, and 200 mg/kg (8 animals/sex/group) was administered in deionized water via oral gavage for 28 consecutive days. Clinical observations, body weights, feed and water consumption data were collected.
Task 2 dose levels were set so that the highest dose was expected to depress weight gain by approximately 10% and permit greater than 90% survival. The middle dose was selected to produce little or no systemic toxicity, whereas the low dose was designed to be a no-effect level. Task 2, the continuous breeding phase, consisted of a control group and three treated groups (20 pairs/group). F0 animals were dosed daily via oral gavage from Study Day 1 until the day before necropsy. Following seven days of premating exposure to caffeine, the animals were housed as breeding pairs for 112 days (16 weeks). Litters produced during the cohabitation period were counted and weighed by sex on PND 1 and then euthanized. At the end of the 112 days, the pairs were separated. Any litters born (F1) after the continuous breeding phase were reared by the dam until weaning on PND 21. Selected weanlings were reared in the same sex groups until 81 ±12 days of age. F1 animals were dosed via oral gavage, after weaning, with the same level of caffeine as their parents received during Task 2. These F1 animals were used for assessment of second-generation reproductive toxicity.
Because decreased pup weights were observed during Task 2, a one-week crossover mating trial (Task 3) was performed on the parental animals from the control and high-dose groups to determine the affected sex. Low-and mid-dose animals were maintained at the same dosing regimen until the control and high-dose animals were euthanized/necropsied. After Task 3 crossover mating, F0 (Task 2/3) animals were necropsied and terminal body weights and organ weights were obtained, sperm analysis was performed, and tissues were saved.
Task 4, the assessment of the F1 generation, was conducted using offspring from all four dose groups. At sexual maturity (81 ±12 days of age), twenty control animals of each sex and 20 treated animals of each sex in each dose group were randomly assigned to breeding pairs, avoiding sibling matings, and cohabitated for up to seven days. Offspring were counted and weighed by sex on PND 1. At necropsy, F1 terminal body weights and organ weights were obtained, sperm analysis performed, and tissues were saved. - Positive control:
- none
- Parental animals: Observations and examinations:
- Task 1
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
FOOD and WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly
BODY WEIGHT: Yes
- Time schedule for examinations: at initiation of treatment and weekly thereafter. and at termination
OTHER: Animals were euthanized via carbon dioxide asphyxiation on Study Day 29 and discarded without necropsy.
------------------------------------------
Task 2:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
BODY WEIGHT: Yes
- Time schedule for examinations: The total pup weight of each sex were obtained on PND 1, 4, 7, 14, and 21. The dam and sire were weighed following delivery. The dam was also weighed on PND 4, 7, 14, and 21.
FOOD CONSUMPTION: Feed consumption measurements for lactating dams were obtained on PND 1, 4, 7, 11, 14, 18, and 21. Feed consumption of the males was measured during Study Weeks 1, 6, 12 and 18.
----------------------------------------
Task 3
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
BODY WEIGHT: Yes
- Time schedule for examinations: Naive males and females were not treated and were weighed at randomization, cohabitation, littering, and termination. The naive females were weighed one additional time, however, it was not required by the protocol.
FOOD CONSUMPTION: No data
WATER CONSUMPTION: No data
------------------------------------
Task 4
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION: Yes
- Time schedule for examinations: during Weeks 2 and 4 of the F1 fertility assessment - Postmortem examinations (parental animals):
- Task 2/3 - Terminal Procedures
Following the completion of the Task 3 crossover mating, terminal body weights were obtained from all surviving Task 2/3 animals. All animals were euthanized via carbon dioxide asphyxiation and animals not scheduled
to be necropsied were discarded. A gross necropsy was performed on all of the control animals and the first surviving 10 per sex from each of the treated groups.
The folIowing organs were weighed: liver, kidneys, right cauda epididymis, right epididymis, prostate, seminal vesicles with coagulating glands, right testis, and ovaries.
Sperm motion parameters were measured using the right vas deferens (in phosphate buffered saline and bovine serum albumin) using the CellSoft™ computer-assisted sperm motion analysis system . Sperm density and morphology were determined using the right cauda epididymis.
Task 4 - Offspring Assessment
Animals were weighed and then euthanized via CO-asphyxiation. Animals not scheduled to be necropsied were discarded. A gross necropsy was performed on all control animals and the first 10 surviving animals from the treated groups. The following organs were weighed: liver, kidneys, right cauda epididymis, right epididymis, prostate, seminal vesicles with coagulating glands, right testis, and ovaries.
Sperm motion parameters were measured using the right vas deferens (in phosphate buffered saline and bovine serum albumin) with the CellSof computer-assisted sperm motion analysis system. Epididymal sperm density and morphology were determined using suspensions prepared with the right cauda epididymis - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical signs noted during the study included abrasions, swellings, discharge, thinness, lacrimation, tissue masses, vaginal discharge, paleness, and urine stains. The incidence of these observations was low to moderate (less than 26% per group) and no dose-related differences were observed. Alopecia was observed more frequently and the incidence was higher in all treated groups compared to controls, beginning Study Week 6.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Mortality was observed in only three animals (one control and two low-dose females during Tasks 2 and 3). Two 12.5 mg/kg females died or were removed from the study as a result of parturition difficulties. One control female died as a result of gavage error.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Sire weights, obtained on delivery day for corresponding dam, decreased with increasing dose. The sire weights of the 25 and 50 mg/kg males were 9-14 and 9-18% less than controls, respectively, for Litters 1-5. Mean dam weights at delivery, also decreased with increasing dose. Body. weights of the F0 males and females, obtained on Study Weeks 6, 12, and 18, decreased with increasing dose and the magnitude of the effect increased over time
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Mean relative feed consumption expressed as g/kg body weight/day was decreased by 3, 15, and 20% in the 12.5, 25, and 50 mg/kg males and by 6, 14, and 24% in the 12.5, 25, and 50 mg/kg females, respectively, during the first week of the study (Week 1). The mean relative feed consumption was increased by -8, 9, and 14% in the 12.5, 25, and 50 mg/kg males and females, respectively, during and after continuous breeding (Study Weeks 6, 12, and 18 (males only). Mean feed consumption when expressed as g/animal/day was decreased by 16 and 21 % in the 25 and 50 mg/kg males and by 14 and 23% in the 25 and 50 mg/kg females, respectively, during the first week of the study (Week 1). No differences were observed in mean feed consumption values during Study Weeks 6 and 12 for the cohabitated males and females. Mean feed consumption values were decreased by 6 and 7% for the 25 and 50 mg/kg males, respectively, during Study Week 18.
During lactation of the final litter, the mean dam weights decreased with increasing dose by 8. 10. and 15% for females in low-to-high dose groups, respectively. - Food efficiency:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean feed consumption data (g/animal/day) for lactating darns at the final litter on PND 4, 7, 14, and 21 were comparable among dose groups. Mean feed consumption (g/kg body weight/day) was increased in the 50 mg/kg females on PND 4; this was probably due to the decreased body weights.
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- 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
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- effects observed, treatment-related
- Description (incidence and severity):
- Evaluation of F0 computer-assisted sperm analysis (CASA) data revealed a treatment-related decrease (3 %) in percent motile sperm in the 50 mg/kg F0 males compared to controls. The sperm velocity (micrometer/sec) was decreased (7-12 %) in all treated males. The average radius (micrometer) was also decreased (22-36 %) in all treated males. The percent normal sperm was decreased slightly in the 50 mglkg males when compared to controls. The remaining sperm endpoints were comparable among all groups.
- Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- The pregnancy index (number delivering/number cohabitated) was comparable among dose groups. The pregnancy index declined with increasing litter number for both the control and treated dose groups. No differences were observed in the mean average litters per pair, number of live pups per litter, proportion of pups born alive, or sex ratio (number of males/total number of pups). Absolute live pup weight and adjusted live pup weight were decreased by 6-8% in all dose groups compared to controls. The mean cumulative days to litter was not affected by caffeine
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- < 12.5 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- body weight and weight gain
- Remarks on result:
- not determinable
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical observations noted during Task 4 included abrasions, swelling, and head tilt. The incidence of these observations was low to moderate (5-25% per group) and no dose-related differences were observed. Alopecia was observed at a higher incidence in all treated groups beginning Task Week 5 (males) and 4 (females).
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- No mortality was observed during the maturation and Task 4 phases of the study.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Adult body weights of the F1 males and females were decreased (11-23 %) in all treated groups when compared to controls.
- Food efficiency:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean feed consumption relative to body weight was increased by 7-13, 8-17, and 10-24% for the 12.5. 25, and 50 mglkg males, respectively, compared to control values. Mean relative feed consumption values were increased by 9% for the 25 mg/kg females during Task Week 2 and by 16% for the 50 mg/kg females during Task Week 4. Mean feed consumption values expressed as g/animal/day were decreased by 7, 14, and 16% for the 12.5,25, and 50 mglkg males during Task Week 2 and by II % for the 25 and 50 mglkg males, respectively, during Task Week 4 compared to control values. Mean daily feed consumption values for the F1 females were decreased by 10, 11, and 19% for the 12.5, 25, and 50 mg/kg females during Task Week 2 and by 10% in the 50 mg/kg females during Task Week 4, respectively.
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- Urinalysis findings:
- not specified
- Sexual maturation:
- not specified
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean relative liver weights (mg/g body weight) decreased by 10 and 6% in the 25 and 50 mg/kg males compared to controls. No differences were observed in female relative liver weights. Mean absolute kidney weights were decreased by 10% in the 25 and 50 mg/kg males and by 9,8, and 17% in the 12.5, 25, and 50 mg/kg females, respectively, compared to the controls. Mean relative kidney weights (mg/g body weight) were increased by 16 and 17% (males) and by 20 and 13% (females) in the 25 and 50 mg/kg dose groups.
The absolute weight of the right testis was decreased by 8, 9, and 6% in the 12.5, 25, and 50 mg/kg F1 males. The relative right testis weight was increased for the 25 (20%) and 50 (23%) mg/kg males. The absolute weights of other organs were comparable among dose groups, with the exception of a 14% decrease in the right epididymis weight of the 25 mg/kg males. The relative weights of the right cauda epididymis, right epididymis, prostate. seminal vesicles with coagulating glands, and ovaries were increased in all treated groups when compared to controls. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No gross lesions were observed in the F1 males at necropsy. Gross lesions noted in the F1 females included dilated renal pelvis, pale ovaries, and ovarian cyst, and masses on the cervix. These lesions were not considered to be treatment related because no dose relationship was observed.
- Histopathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No microscopic lesions were observed in the kidneys, liver, left testis, or left epididymis in the males examined or in the liver of females examined. Microscopic lesions noted in the kidneys for the F1 females examined included mineralization, renal pelvis and renal tubule dilatation, and renal tubule regeneration. These were not considered treatment-related due to a lack of dose response.
- Other effects:
- not specified
- Behaviour (functional findings):
- not specified
- Developmental immunotoxicity:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- < 12.5 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Remarks on result:
- not determinable
- Key result
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 12.5 mg/kg bw/day (actual dose received)
- Treatment related:
- yes
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- no
- Conclusions:
- Results of this study show that caffeine is not a selective reproductive toxicant, because the minor effects on sperm motion parameters and pup weight/viability occurred concomitant with, or at doses greater than the doses that reduced body weight gain. It was demonstrated that exposure to caffeine reduced pup weights in the F0 females at ~ 12.5 mg/kg and litter size and viability in the F1 generation at 50 mg/kg. The delay to conception observed in humans consuming caffeine was not observed in this study. The NOAEL was not established as the animals receiving 12.5 mg/kg displayed reduced body weight gain.
- Executive summary:
The potential reproductive toxicity of caffeine in Sprague-Dawley rats was evaluated using the Reproductive Assessment by Continuous Breeding (RACB) protocol. Based on decreased body weights and feed consumption, increased water consumption, and mortality noted during Task 1, dose levels for the continuous breeding phase for this study were set at 12.5, 25, and 50 mg/kg.
Male and female Sprague-Dawley rats were exposed to caffeine in deionized water by oral gavage at a dose volume of 5 mL/kg. Individual dose volumes were adjusted weekly. During 16 weeks of cohabitation, live pup weight adjusted for litter size was decreased by 7, 7, and 8% in the 12.5, 25, and 50 mg/kg dose groups, respectively. No differences were observed in other reproductive endpoints.
A crossover mating trial (Task 3) revealed no changes on male or female fertility or in pup weight. Reproductive parameters were comparable between dose groups when naive males were mated with control or 50 mg/kg females and when naive females were mated with control or 50 mg/kg males.
The decreased pup weight was observed concomitant with reduced dam weight gain. Throughout the study, the body weights were less than controls in the F0 12.5 (4-7 %), 25 (9-15 %), and 50 (9-18 %) mg/kg males and 12.5 (5-18%), 25 (5-19%) and 50 (8-19%) mg/kg females. Mean feed consumption (g/animal/day) was decreased by approximately 17 and 20% in the 25 and 50 mg/kg animals of both sexes, respectively, during the first week of the study. At necropsy, no differences were noted in F0 male or female absolute organ weight data; however, many relative organ weights (mg/g body weight) were increased in all dosed groups when compared to controls. These differences were attributed to the decrease in terminal body weights. No treatment-related gross or microscopic lesions were observed in the F0 animals.
Evaluation of F0 computer-assisted sperm analysis (CASA) data revealed a treatment-related decrease (3 %) in percent motile sperm in the 50 mg/kg F0 males compared to controls. The sperm velocity (µm/sec) was decreased (7-12 %) in all treated males. The average radius µm) was also decreased (22-36 %) in all treated males.
The percent normal sperm was decreased slightly in the 50 mg/kg males when compared to controls. The remaining sperm endpoints were comparable among all groups.
In Task 4 (second generation evaluation), no treatment-related differences were observed in pup weights during lactation (PND 1-21). From the initiation of dosing (PND 22) through the maturation phase up to the termination of Task 4, mean body weights of the F1 low-to-high dose males and females were decreased by approximately 12%, 18%, and 23% when compared to controls. Mean feed consumption values (g/animal/day) were decreased (5-19%) in all treated groups during Task 4. No mortality was observed in any of the F1 animals.
Measures of reproductive performance of second generation breeding pairs revealed a decrease in the number of live F2 pups per litter (21 %) and proportion of pups born alive (4%) in the 50 mg/kg dose group. No differences were observed between dose groups in other endpoints.
- Endpoint:
- screening for reproductive / developmental toxicity
- 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:
- no guideline available
- Principles of method if other than guideline:
- The study was designed to evaluate the effects of caffeine on the prenatal and post-natal development of bones in rats.
The specific purpose was to determine whether the ossification delay seen in the offspring of caffeine treated rats is reversible up to 6 days post-natally. - GLP compliance:
- no
- Limit test:
- no
- Justification for study design:
- Postnatal day 0, was selected to observe bone development in the offspring at a time past that of rapid ossification. 6 days after birth, was considered an appropriate time at which to measure bone development, on the assumption that if the delayed ossification seen after caffeine treatment was truly reversible, the bone development of pups from treated groups would be similar to that of those from control groups.
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine from Eastman Kodak Co. Rochester, NY
- Expiration date of the lot/batch: not available
- Purity test date: 98.7%
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 distilled 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:
- Osborne-Mendel
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: FDA rat-breading colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 3-5 months old (females), 3-6 months (males)
- Weight at study initiation: 220-270 g (females)
- Fasting period before study: no
- Housing: single hanging cages, on gestation day 20, group B and C were placed in plastic tubes
- Diet: Purina Rat Chow ad libitum
- Water: ad libitum
- Acclimation period: not specified
ENVIRONMENTAL CONDITIONS
- Temperature: 21-26°C
- Humidity: 17-76 %
- 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
- Details on exposure:
- The caffeine solution or distilled water was poured into glass bottles fitted with stainless-steel ball-point non-drip tubes and the fluid was provided ad libitum from gestation day 0 to gestation day 20.
- Details on mating procedure:
- On mating days, females of the proper age and weight were placed in cages with males at approximately 16h30. The following morning, a vaginal smear was done on each female to determine if copulation had occurred. Mating was continued until a sufficient number of females had been assigned to each group. The day of finding sperm was designated day 0 of gestation.
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- Gestation day 0 to 20
- Frequency of treatment:
- Daily
- Details on study schedule:
- Group A litters were killed on gestation day 20; group B neonates were killed on post-natal day 0; and group C pups were killed on post-natal day 6.
- Dose / conc.:
- 0.018 other: %
- Remarks:
- in drinking water
- Dose / conc.:
- 0.036 other: %
- Remarks:
- in drinking water
- Dose / conc.:
- 0.07 other: %
- Remarks:
- in drinking water
- No. of animals per sex per dose:
- Dose 1: 60
Dose 2: 30
Dose 3: 30 - Control animals:
- yes
- yes, concurrent vehicle
- Details on study design:
- Because the delayed ossification was seen in small numbers in the low-dose level in the preceding study (Collins et al. 1983), the number of animals in the 0.018% group was set at 60. The two higher dose levels had produced greater numbers of ossification defects in the preceding study, and the number of animals was set at approximately 30.
- Positive control:
- none
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: The animals were observed twice daily for behavioral variations or gross changes
BODY WEIGHT: Yes
- Time schedule for examinations: at 3-day intervals
FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: at 3-day intervals - Oestrous cyclicity (parental animals):
- Not specified
- Sperm parameters (parental animals):
- Not specified
- Litter observations:
- Animals from groups B and C were allowed to litter. On Postnatal (PN) day 0 (group B) and PN day 6 (group C), neonates were weighed, sexed and observed for external abnormalities.
- Postmortem examinations (parental animals):
- On day 20 of gestation, the females of group A were examined for gross abnormalities and then killed by CO2 asphyxiation. The abdomen was opened and the uterus examined in situ for the presence and position of resorption sites and fetuses (dead or alive) and the number of implantation sites.
Females from group B and C were allowed to litter and then were killed by CO2 asphyxiation. The uterus of each dam was visually inspected and the sites of implantation were counted and recorded. - Postmortem examinations (offspring):
- On day 20 of gestation, the females of group A were examined for gross abnormalities and then killed by CO 2 asphyxiation. The abdomen was opened and the uterus examined in situ for the presence and position of resorption sites and fetuses (dead or alive) and the number of implantation sites. Each live fetus was promptly weighed, sexed and examined for gross external malformations under magnification. The length between crown and rump was also measured. All fetuses were fixed and stained in Alizarin Red S and examined for skeletal development.
Neonates from group B and C were fixed and stained in Alizarin Red S and examined for skeletal development. - Statistics:
- Data (except incidence data) were checked for homogeneity of variance using Levene’s test. A one-way analysis of variance was performed, either a straightforward analysis of variance (ANOVA), a nested ANOVA or an analysis of covariance (ANOCOVA), depending on the parameter. Incidence data, in general (i.e. number of litters having foetuses with one or more parameters analysed), were analysed by the asymptotic approximation of the Cox trend test to test for a linear trend as defined above. A Fisher exact test (one-tail) was used for pairwise comparisons between the treated groups and the control group. Data on average numbers of foetuses per litter with skeletal, sternebral or missing plus incomplete plus bipartite variations were transformed using the Freeman-Tukey arc-sine transformation, with a one-tailed LSD. Data on fertility (number pregnant divided by number mated), number of foetuses with one or more, two or more, etc., early deaths, late deaths, skeletal variations, sternebral variations and missing, incomplete or bipartite sternebrae were analysed by the Cox and Fisher exact test.
- Offspring viability indices:
- Group B:
Control group: 97.37 ± 0.78
Caffeine 0.018%: 98.77 ±0.48
Caffeine 0.036%: 97.45 ± 1.11
Caffeine 0.07%: 97.43 ± 0.94
Group C:
Control group: 98.45 ± 0.54
Caffeine 0.018%: 98.24 ±0.64
Caffeine 0.036%: 97.02 ± 1.15
Caffeine 0.07%: 94.81 ± 3.14 - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Alopecia was present in groups A, B and C. The findings did not appear to be compound related.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Initial body weights on day 0 were similar in groups A, B and C. In group A animals, ingestion of 0.018% caffeine had no effect on maternal body weight. Ingestion of 0.036% caffeine produced significantly less overall gain on days 0-20 and significantly decreased gain compared with the controls specifically on days 12-15. The animals that ingested the highest dose of caffeine gained significantly less weight during the whole of gestation (days 0-20), and also gained significantly less weight during days 0-3 than did the controls.
In group B, animals receiving 0.018% gained significantly more weight than the controls during the first 3 days of gestation and during the entire gestation period. The group B animals from the mid-dose group also gained significantly more weight than the controls on days 0-3, but no other effects on weight were seen. A significant amount of weight was lost during days 0-3 by animals in the 0.07% group who also gained significantly less weight than controls during days 3-6 and 9-12 and significantly more weight during days 6-9 and 12-15. During the whole of gestation, the group B mid-dose animals gained significantly less weight than did the controls.
In group C animals, there was no significant effect on body weight considering the whole gestation period in low- and mid-dose animals, but low-dose animals gained significantly more weight on days 0-3. Animals in the 0.07% group gained significantly less weight than controls 0-3, 9-12 and 0-20.
Maternal body weight on Post natal (PN) day 0 was different in the low-dose animals in groups B and C. The low-dose animals in group B weighed significantly more than the controls; the weight of the group C animals in the same dose level was greater than that of the controls, but not significantly. PN day 0 body weight of the 0.036% animals in both groups B and C was similar to that of the controls.
In both groups B and C, the PN day 0 weight of the animals given 0.07% caffeine was significantly less than that of the controls. By PN day 6, the weight of all the group C treated animals was similar to the weight of the controls. - 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
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- No difference was seen between control and treated groups in the number of implantations, early deaths or late deaths (singly or combined). The number of viable fetuses was not affected in the 0.018% group but was significantly decreased in the 0.036 and 0.07% groups. When the fetuses were analyzed by sex, the number of viable females was not affected but the number of viable males was significantly less than the control value in all treated groups. The number of females with at least one resorption was not affected in any treated group, but there were significantly more females with at least two resorptions in the 0.036% group than in the control group. No litters were totally resorbed.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 0.018 other: %
- Based on:
- test mat.
- Remarks:
- corresponding to 24.7-29.0 mg/kg bw/day
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- reproductive performance
- Clinical signs:
- not examined
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- The number of viable fetuses was not affected in the 29.0 mg/kg bw/day group but was significantly decreased in the 48.8 and 70.6 mg/kg bw/day groups. When the fetuses were analyzed by sex, the number of viable females was not affected but the number of viable males was significantly less than the control value in all treated groups
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The body weights of fetuses in the 0.036 and 0.07% groups were similar to the weights of the controls, but the weights of both male and female fetuses of the 0.018% group were significantly greater than those of the controls. Fetal length of males from crown to rump was significantly greater in the 0.018% fetuses than in the controls but no difference was seen in the 0.036 and 0.07% fetuses. Fetal length of females was not affected in the 0.018 and 0.036% groups, but the length of the 0.07% females was significantly greater than the controls. The incidence of runts was not affected by the compound. Sex distribution of the fetuses was not affected.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Group B:
Within the control group, three anomalies were observed: one encephalocele, one dome-shaped head, and one micromelia. In the 0.018% group, one animal from each of two litters had a kinked tail. Within the 0.036% dose group, one animal had a small tail, one had edema, one had partially opened eyes and one remained in the dam. Within the 0.07% group, one animal had edema.
Group C:
One control neonate had an indented skull; one neonate from 0.018% group had edema and one had ectrodactyly; and three neonates from a single litter in 0.07% group had dome-shaped heads. - Histopathological findings:
- not examined
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Skeletal variations:
Group A:
Neither the incidence of incomplete sternebrae nor of malaligned sternebrae was affected by the compound. The average number of fetuses per litter with at least one sternebral ossification delay was increased significantly in all treated groups and the average number of fetuses per litter with at least two sternebral variations was significantly increased in the mid- and high-dose groups, The percentages of litters containing fetuses with at least two and at least three sternebral variations and the average number of fetuses per litter with at least three sternebral variations were significantly increased only in the high-dose group. Fetuses from the mid- and high-dose groups also had significant increases in certain skeletal defects, namely missing centra and reduced ossification of the dorsal arch. Fetuses from the high-dose group also had significant increases in bipartite supraoccipital, and reduced ossification of the hyoid, metacarpals and metatarsals.
Group B:
Neonates in the 0.018 and 0.07% groups showed significant increases in the numbers of fetuses with incomplete sternebrae, but they do not appear to be related to caffeine dose, as neonates in the 0.036% group were not affected. The 0.036% group produced no effect on sternebral variations.
Dosing with 0.07% caffeine produced a significant increase in the number of neonates with missing sternebrae. The day 0 neonates from group B showed fewer sternebral variations than did the group A fetuses and if the increase in 0.018% neonates with incomplete sternebrae is discounted as random variation, the high-dose neonates are the only ones affected.
Only the 0.07% caffeine dose produced any effect on sternebral development--a significant increase in the average number of neonates per litter with at least one sternebral variation and in the percentage of litters containing neonates with at least one variation. The numbers of neonates with at least two sternebral variations were not affected in any treatment group. This result contrasts with that for group A in which animals from all dose levels were affected.
The percentage of litters containing neonates with at least one affected sternebra was increased in all treated groups compared with the control value, but the increase was not related to dosage. The number of litters containing neonates with at least two affected sternebrae was increased significantly in the 0.07% neonates.
A dosage of 0.018% of caffeine produced a significant increase in supernumerary rib bud (first lumbar) and a dosage of 0.036% produced a significant increase in reduced ossification of the metatarsals. Dosing with the highest caffeine dose produced a significant increase, compared with the controls, in supernumerary rib bud (first lumbar), and significant increases in reduced ossification of the metacarpals, metatarsals and calcaneus.
Dosing with either 0.018 or 0.036% caffeine did not affect the incidence of skeletal variations. Dosing with the highest caffeine dose produced a significant increase in the number of neonates per litter with at least one, two, three, four or five skeletal variations and in the average number of litters containing neonates with at least two, three, four or five skeletal variations. The average numbers of vertebrae ossified per neonate in the 0.018 and 0.036% dose groups (14.15 and 13.77, respectively) were similar to the control value (13.98) but the number for the 0.07% caffeine group (12.99 vertebrae) was significantly less than that for the controls.
Group C:
Dose levels of 0.018 and 0.036% caffeine produced no effect on sternebral ossification. Dosing with 0.07 % produced a significant increase in the number of neonates with incompletely ossified sternebrae. When compared with day 0 neonates from group B, the incidence of missing sternebrae was less, but the incidence of incomplete sternebrae was still increased significantly compared with the control group in the high-dose group.
Neither the average number of neonates per litter with sternebral variations nor the incidence of litters containing such neonates was affected in any group. This contrasts with the results for group B in which there were significant increases in the high-dose group in the neonates with at least one sternebral variation and in the litters containing neonates with at least one sternebral variation
No specific bone in day 6 neonates showed any effect of caffeine dosage. This lack of effect contrasts with the significantly decreased ossification of bones in group B offspring of the 0.036 and 0.07% dosage groups.
The incidence of skeletal variations in day 6 neonates was unaffected by caffeine dosing, which also contrasts with the significant increases in the numbers of neonates with one to five skeletal variations and of litters containing neonates with two to five skeletal variations seen in the day 0 animals in the 0.07% dosage group in group B.
No effect of caffeine dosage on vertebral ossification was seen in any day 6 neonates (28.33, 28.51, 28.45 and 28.46 vertebrae ossified in the control, 0.018, 0.036 and 0.07% groups, respectively), unlike day 0 neonates from group B in which the highest dosage level showed a significant decrease in vertebral ossification compared with the controls. - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- > 0.018 other: %
- Based on:
- test mat.
- Remarks:
- corresponding to 24.7-29.0 mg/kg bw/day
- Sex:
- not specified
- Basis for effect level:
- other: delayed sternebral ossification
- Remarks on result:
- other: Group B (neonates)
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 0.036 other: %
- Based on:
- test mat.
- Remarks:
- corresponding to 24.7-29.0 mg/kg bw/day
- Sex:
- not specified
- Basis for effect level:
- other: delayed sternebral ossification
- Remarks on result:
- other: Group C (6-day-old pups)
- Conclusions:
- Based on the results from the in utero exposure to caffeine during 20 days of gestation, it has been concluded that caffeine delayed the ossification of several bones such as dorsal arch, centra, metacarpals, metatarsals, supraoccipital and hyoid in fetuses and in day 0 neonates, and that the ossification delay observed in bones other than sternebrae has reversed by postnatal (PN) day 6. Even though the sternebral ossification did not totally reverse by PN day 6, it has been concluded that the decrement in the degree of delayed sternebral ossification between day 0 neonates and PN day 6 suggested that the reversibility would have continued and would have been complete if a longer post-natal period had been studied.
- Executive summary:
Groups of Osborne-Mendel rats, killed at three time intervals following mating, were studied to determine whether prenatal skeletal ossification delays observed following low-level caffeine administration represent transient or persistent ossification problems.
Group A litters were killed on gestation day 20; group B neonates were killed on post-natal day 0; and group C pups were killed on post-natal (PN) day 6.
Within each group, dose levels of 0, 0.018, 0.036 or 0.07% caffeine in distilled water were available ad libitum to groups of 30-60 dams from gestation day 0 to day 20.
Average daily caffeine consumption was 24.7-29.0 mg/kg body weight for the 0.018% group, 42.7-48.8 mg/kg body weight for the 0.036% group and 70.6-75.1 mg/kg body weight for the 0.07% group.
No maternal toxicity was observed during the course of the study.
In group A litters, the mean number of viable fetuses was significantly less in the mid-dose and high-dose animals than in the controls. In the same group, the average number of fetuses per litter with at least one sternebral ossification delay was increased significantly in all treated groups and the average number of foetuses per litter with at least two sternebral variations was significantly increased in the mid- and high-dose groups, The percentages of litters containing fetuses with at least two and at least three sternebral variations and the average number of fetuses per litter with at least three sternebral variations were significantly increased only in the high-dose group. Fetuses from the mid- and high-dose groups also had significant increases in certain skeletal defects, namely missing centra and reduced ossification of the dorsal arch. Fetuses from the high-dose group also had significant increases in bipartite supraoccipital, and reduced ossification of the hyoid, metacarpals and metatarsals.
In group B, day 0 neonates from all treated groups showed a significantly increased incidence of delayed sternebral ossification (average number of fetuses per litter with one or more missing, incomplete or bipartite sternebrae). The percentages of litters containing neonates with delayed sternebral ossification were also increased significantly in all treated groups. Neonates from the 0.07% level in group B also exhibited a significant increase in the incidence of supernumerary rib bud, and in reduced ossification of the metacarpals, metatarsals and calcaneus bones. Significant increases were also seen, in group B, in the low- and mid-dose animals, respectively, in supernumery rib bud and in reduced ossification of the metatarsals.
In group C, the 6-day-old pups of the 0.07% level showed a significant increase in incompletely ossified sternebrae, but no other skeletal effects were seen. Thus the previously observed delay of skeletal ossification seen in day 20 fetuses and in day 0 neonates after caffeine ingestion was confirmed. By post-natal day 6, the ossification delay had been nearly reversed.
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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.
Both the male and female CD-1 mice in the 1% caffeine group exhibited dehydration, piloerection, ptosis and lethargy a few days prior to death. Daily water consumption per mouse was slightly increased in the 0.033% caffeine group and markedly reduced in the 0.1, 0.3, and 1% caffeine groups relative to the control group. By the Chi Square Test and Fisher's Exact Test, the percent lethality for the sexes combined in the 1% caffeine group was significantly higher (p<0.001) than for all other treatment groups. A probit analysis of the lethality data for the sexes combined projected an acute LD50 of 0.25% caffeine and an LD50 of 0.61% caffeine when administered ad libitum in the drinking water. By ANOVA and Duncan's Multiple Range Test, the % weight gain for the sexes combined (excluding the 1% caffeine group) was significantly decreased (p<0. 01) in the 0.1 and 0.3% caffeine groups versus the 0.0,0.012, and 0.033% caffeine groups.
Based on the acute toxicity results (Task 1), drinking water levels of 0.0, 0.012, 0.025, and 0.05% caffeine were selected for the continuous breeding phase of the study (Task 2). Assuming that a male or female CD-1 mouse weighing 40 g consumed 7 g of water per day, then these drinking water levels represented daily intakes of 0.0, 21.9, 43.8, and 87.5 mg/kg body weight/day, respectively. Continuous exposure of CD-1 mice (11 weeks of age at outset) to these dose levels of caffeine in the drinking water during the 7-day premating, 98-day cohabitation, and 21-day segregation periods had no effect on the number of pairs able to produce at least one litter. In addition, caffeine, at the dose levels given, had no influence on the number of litters produced per pair, proportion of pups born alive or sex of pups born alive (males/total), or live pup weight. In contrast, there was a significant (p<0.01) dose-related decrease in the number of live pups per litter as indicated by Jonckheere's Test for ordered differences. In particular, 0.025 and 0.05% caffeine levels in the drinking water resulted in a significantly decreased number of live pups per litter for female pups and for the sexes of pups combined. The number of male pups per litter was significantly reduced only at the 0.05% caffeine level relative to the control group.
11/80, 7/40, 22/40, and 20/40 mice in the 0%, 0.012%, 0.025% and 0.05% group, respectively, exhibited alopecia during continuous breeding phase. Chi-Square analysis revealed an overall significant difference (P<0.01) in alopecia for the dosed groups. Pairwise comparison by Fisher's Exact Test indicated that the number of mice with hair loss was significantly increased (P<0.01) in the 0.025 and 0.05% groups. Further, 1, 2, 2, and 1 parental female(s) of the 0, 0.012, 0.025, and 0.05% groups, respectively, died during Task 2. The random distribution of the deaths across treatment groups indicated that they were not treatment-related.
Evaluation of the vaginal smears revealed a difference between the control and 25 mglkg group in the amount of time spent in the different estrous stages and there was a decrease in number of females with a regular cycle in the 25 mglkg group but not at 50 mglkg. No differences were observed in cycle length, number of cycles, or number of cycling females.
Measures of reproductive performance of second generation breeding pairs revealed a 21% decrease in number of live pups per litter and a 4% decrease in the proportion of pups born alive in the 50 mglkg group when compared to the controls. Other endpoints, including pup weight, were unaffected by caffeine exposure.
Additional information
In a reproduction study, rats were exposed to instant or brewed coffee as 100, 50 or 25% solutions in drinking water (representing approximately 80, 40 or 20 mg/kg bw/day of caffeine) for 28 weeks. 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. Animals 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 50 and 100% of brewed and instant coffee.
Caffeine was tested for its effects on reproduction and fertility in Swiss CD-1 mice at two laboratories, each using a variation on the standard RACB study design. One laboratory performed Tasks 1, 2, and 3, while the other laboratory performed Tasks 1, 2, and 4. In the first study, the slight but significant reductions in (male) pup number, pup viability, and adjusted pup weight suggested that caffeine produced some slight reproductive toxicity. This occured in the presence of slight indications of general toxicity (body or organ weight changes). The NOAEL of ca. 22 mg/kg bw/day (120 ppm) was derived for F0 parental, F1 offspring generation (Task 2). In the second study, a reduction in the number of live pups/litter for the F0 generation was the only reproductive effect observed in this study. This occurred in the absence of a change in body weights in the F0 parental mice, but in the presence of clinical findings in form of alopecia. The NOAEL was ca. 22 mg/kg bw/day (120 ppm) F0 parental and F1 offspring generation (Task 2). In addition, the NOAEL of ca. 88 mg/kg bw/day (500 ppm) was derived for F1 parental and F2 offspring generation (Task 4).
In another NTP study in Sprague-Dawley rats caffeine was not a selective reproductive toxicant. It was demonstrated that exposure to caffeine reduced pup weights in the F0 females at ~ 12.5 mg/kg and litter size and viability in the F1 generation at 50 mg/kg. The NOAEL was not established as the 12.5 mg/kg (the lowest tested dose) animals displayed reduced body weight gain.
In an in utero exposure study, groups of Osborne-Mendel rats, killed at three time intervals following mating, were studied to determine whether prenatal skeletal ossification delays observed following low-level caffeine administration represent transient or persistent ossification problems. Group A litters were killed on gestation day 20; group B neonates were killed on post-natal day 0; and group C pups were killed on post-natal (PN) day 6. Within each group, dose levels of 0, 0.018, 0.036 or 0.07% caffeine in distilled water were available ad libitum to groups of 30-60 dams from gestation day 0 to day 20. Average daily caffeine consumption was 24.7-29.0 mg/kg body weight for the 0.018% group, 42.7-48.8 mg/kg body weight for the 0.036% group and 70.6-75.1 mg/kg body weight for the 0.07% group. No maternal toxicity was observed during the course of the study. In group A litters, the mean number of viable fetuses was significantly less in the mid-dose and high-dose animals than in the controls. In the same group, the average number of fetuses per litter with at least one sternebral ossification delay was increased significantly in all treated groups and the average number of foetuses per litter with at least two sternebral variations was significantly increased in the mid- and high-dose groups, The percentages of litters containing fetuses with at least two and at least three sternebral variations and the average number of fetuses per litter with at least three sternebral variations were significantly increased only in the high-dose group. Fetuses from the mid- and high-dose groups also had significant increases in certain skeletal defects, namely missing centra and reduced ossification of the dorsal arch. Fetuses from the high-dose group also had significant increases in bipartite supraoccipital, and reduced ossification of the hyoid, metacarpals and metatarsals. In group B, day 0 neonates from all treated groups showed a significantly increased incidence of delayed sternebral ossification (average number of fetuses per litter with one or more missing, incomplete or bipartite sternebrae). The percentages of litters containing neonates with delayed sternebral ossification were also increased significantly in all treated groups. Neonates from the 0.07% level in group B also exhibited a significant increase in the incidence of supernumerary rib bud, and in reduced ossification of the metacarpals, metatarsals and calcaneus bones. Significant increases were also seen, in group B, in the low- and mid-dose animals, respectively, in supernumery rib bud and in reduced ossification of the metatarsals. In group C, the 6-day-old pups of the 0.07% level showed a significant increase in incompletely ossified sternebrae, but no other skeletal effects were seen. Thus the previously observed delay of skeletal ossification seen in day 20 fetuses and in day 0 neonates after caffeine ingestion was confirmed. By post-natal day 6, the ossification delay had been nearly reversed.
Mode of Action Analysis / Human Relevance Framework
Recently, EFSA panel concluded that the caffeine intakes from all sources including coffee, up to 200 mg per day consumed throughout the day by pregnant women in the general population do not give rise to safety concerns for the fetus. This conclusion was based on prospective cohort studies showing a dose-dependent positive association between caffeine intakes during pregnancy and the risk of adverse birth weight-related outcomes in the offspring (EFSA, 2015).
Justification for classification or non-classification
Adequate reproductive toxicity studies were performed on the suitable analogues of Coffee, bean, roasted, ext.
Coffee or caffeine intake produced minimal effects in the parent animals, but none of treatments had an adverse effect on reproduction or teratogenicity. The only effect seen was the retardation of sternebral calcification; however, it has been shown to be reversible.
Based on these data, it is concluded that the classification for reproductive or developmental toxicity endpoint is not warranted Coffee, bean, roasted, ext.
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