Caffeine

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-03-17 to 1982-12-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (NTP study).

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1984

Reference 3

Reference Type:

publication

Title:

No information

Author:

Lamb IV J et al.

Year:

1997

Bibliographic source:

Environ Health Perspect 105 (Suppl. 1): 279-280. NTIS PB85101202 .

Reference 4

Reference Type:

publication

Title:

No information

Author:

Morrissey RE et al.

Year:

1989

Bibliographic source:

Fund Appl Toxicol 13: 747-777.

Reference 5

Reference Type:

secondary source

Title:

No information

Author:

Gulati DK et al.

Year:

1984

Bibliographic source:

NTP study (NTP-85-097). NIEHS, Research Triangle Park, NC. Cited in: IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, Vol. 51,Lyon, France (1991); pp. 291-390.

Reference 6

Reference Type:

secondary source

Title:

No information

Author:

Reel JR et al.

Year:

1984

Bibliographic source:

NTP study (NTP-84-158). NIEHS, Research Triangle Park, NC. Cited in: IARC Monographs on the Evaluation of Carcinogenic Risk to Humans, Vol. 51, Lyon, France (1991); pp. 291-390 .

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Caffeine

EC Number:

200-362-1

EC Name:

Caffeine

Cas Number:

58-08-2

Molecular formula:

C8H10N4O2

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): Caffeine
- Analytical purity: >98.5%
- Stability under test conditions: confirmed
No further data.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
male and female CD-1 mice [COBS, (ICR) BR, outbred albino]
- Source: Charles River Breeding Laboratory, Inc. (Kingston, NY)
- Age at study initiation: Task 1: 8 wks; Task 2: 11 weeks; Task 4: 21 days
- Water (ad libitum): tap water or dosing solutions
No further data.

ENVIRONMENTAL CONDITIONS: no data

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Exposure period: 21 weeks (Task 2);
Premating exposure period (males): 1 week;
Premating exposure period (females): 1 week;
Duration of test: 34 weeks (Tasks 1, 2, 4)

Frequency of treatment:

continuously in the drinking water

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
ca. 22, 44, 88 mg/kg be/d
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.012, 0.025, 0.05% in the drinking water
Basis:
nominal in water

No. of animals per sex per dose:

Task 1: 8 males and 8 females per group;
Task 2: 20 male and 20 female animals per group;
Task 4: no data

Control animals:

yes, concurrent no treatment

Examinations

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

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

22 mg/kg bw/day (actual dose received)

Sex:

male/female

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

22 mg/kg bw/day (actual dose received)

Sex:

male/female

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

88 mg/kg bw/day (actual dose received)

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

During the 14-day exposure period of Task 1, one male of the 0.012% group, 2 males of the 0.1% group, and 7 males and 8 females of the 1% group died. Both male and female CD-1 mice in the 1% group exhibited dehydration, piloerection, ptosis, and lethargy a few days prior to death. Daily water consumption per animal was slightly decreased in the 0.033% group and markedly reduced in the 0.1, 0.3 and 1% groups when compared to control. By the Chi-Square Test and Fisher's Exact Test, the percent lethality for the sexes combined was significantly higher (P<0.001) than for all other treatment groups. A Probit Analysis 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 percent body weight gain for the sexes was significantly decreased (P<0.01) in the 0.1 and 0.3% groups.

In Task 2, continuous exposure of 11 -week old mice to the test substance 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, the test substance had no influence on the number of litters produced per pair, proportion of pups born alive or sex of pups born alive, or live pup weight. In contrast, there was a significant (P<0.01) and 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% levels 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% level.

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. 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.

For Task 4, in most respects, the results obtained for the F1 parents were similar to those for the F0 parents. 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=0.065) and a reduced number of live pups per litter in the 0.05% caffeine group as compared to the 0% caffeine group, these differences were not statistically significant. It also was interesting that alopecia occurred to a lesser extent in the F1 parents (Task 4) as compared to the F0 parents (Task 2). Thus, hair loss was observed in 2/69 control F1 parents and 6/43 F1 parents given 0.05% caffeine in their drinking water.

Sperm assessment indicated no significant differences in the percent motile sperm, sperm concentration, or percent abnormal sperm in the cauda epididymis between male mice exposed to 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% 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. 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. Further, 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.

Conclusion:

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 cross-over mating trial (Task 3) was not conducted in this study.

Applicant's summary and conclusion

Executive summary:

Caffeine was tested simultaneously at two laboratories, each using a variation on the standard RACB study design. This study used Tasks 1, 2, and 4.

Data on body weights, clinical signs, and food and water consumptions were collected during the dose-range-finding phase (Task 1), and used to set exposure concentrations for Task 2 at 0.0, 0.012, 0.025, and 0.05% in drinking water. Water was chosen to mimic the route of human exposure.
Water consumption was not affected by addition of caffeine. These levels of caffeine, and measured water consumption and body weights, produced calculated consumption estimates nearly equal to 22, 44, and 88 mg/kg/d.
For the F0 animals, there were no effects on body weight. Alopecia occurred in 55% of the medium dose and 50% of the high dose animals. While there were no exposure-related changes in the number of litters/pair, viability, or adjusted pup weight, the number of live pups per litter, averaged over the 4-5 litters, dropped 15% at the medium dose and 20% for the high dose animals.
No crossover mating trial was conducted, and the offspring from the last litter of control and high dose mice were reared by their dams until weaning, when they were given the same treatment as their parents until mating at 74 ± 10 days of age.
At the second generation mating trial, there were no changes in any reproductive endpoint.
At necropsy, at 0.05% caffeine, male body weight was reduced by 8% while male adjusted liver weight increased by 8%. No change was found in female body or organ weights, or in any sperm endpoint.
In conclusion, 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.