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Key value for chemical safety assessment

Additional information

IN VITRO

1. Gene mutations

In a reverse gene mutation assay in bacteria (Mortelmans et al., 1986), strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium were exposed to Caffeine (>99 % pure) in DMSO at concentrations of 100 – 10000 µg/plate in the presence and absence of mammalian metabolic activation (preincubation method).

Caffeine was tested up to cytotoxic concentrations (cytotoxicity at concentrations >= 3333.3 µg/plate). The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

In another reverse gene mutation assay in bacteria (Dunkel et al., 1985), strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and strain WP2 uvrA of E. coli were exposed to Caffeine, (>99 % pure.) in DMSO or water at concentrations of 3.3 – 3333.3 µg/plate in the presence and absence of mammalian metabolic activation (plate incorporation method).

Caffeine was tested up to cytotoxic concentrations (cytotoxicity at concentrations >= 1000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background in the Ames test.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

In a mammalian cell gene mutation assay at the thymidine kinase locus (Amacher et al., 1980), L5178Y mouse lymphoma cells cultured in vitro were exposed to Caffeine (purity not given) in culture medium at concentrations of ca. 2950 - 10620 µg/mL in the absence of mammalian metabolic activation.

Caffeine was tested up to cytotoxic concentrations.

There was no evidence of induced mutant colonies over background in the mouse lymphoma assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

2. Chromosomal aberrations

2a. Chromosomal aberrations in animal cell cultures

In a mammalian cell cytogenetics assay in vitro (Chromosome aberration assay) (Ishidate, 1983), CHL cell cultures were exposed to Caffeine (purity not given) in physiol. saline at concentrations of 0, 250, 500, or 1000 µg/ml without metabolic activation.

Caffeine was not cytotoxic under the conditions of this test.

There was a concentration related positive response of Chromosome aberration induced over background.

Caffeine produced a dose-related increase in the frequency of chromosomal aberrations at the mid and high dose level. However, these concentrations are above the levels of 10 mM recommended by OECD/EU guidelines and could be the reason for the positive results. This study is classified as acceptable.

This study satisfies the requirement for Test Guideline In vitro mammalian cytogenetics OECD 473 for in vitro cytogenetic mutagenicity data.

2b. Chromosomal aberrations in human cells

In another cytogenetic assay in vitro (Chromosome aberration assay) (Aeschbacher et al., 1985), human primary lymphocyte cultures were exposed to pure Caffeine (purity not further stated) in water at concentrations of 0, 5, 10, 25, 50, 75, or 100 µg/ml with and/or without metabolic activation with Aroclor 1254-induced rat liver S-9.

Caffeine was tested up to limit concentration. Positive controls induced the appropriate response.

There was no evidence of Chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro mammalian cytogenetics OECD 473 for in vitro cytogenetic mutagenicity data.

Conclusion – in vitro:

There was no evidence of reverse gene mutation in bacteria.

There was no evidence of gene mutation in mammalian cells.

There was no definite evidence of clastogenicity (induction of chromosome aberrations in mammailian/human cells).

IN VIVO

1. Gene mutations

In a Host-mediated assay (King et al., 1979), NMRI mice were injected i.v.with E. coli K12 and treated i.p. with Caffeine (finest pure grade) at single doses of 0 or 194 mg/kg bw (vehicle not given).

There was no evidence for induction of gene mutations.

Data on toxicity are not given. The study is classified as acceptable; basic data are given.

2. Chromosomal aberrations

2a. Chromosomal aberrations in soma cells of animals

In a Sprague-Dawley rat blood cell chromosome aberration assay (Granberg-Oehman et al. 1980), (30 rats/dose; gender not stated) were treated orally with Caffeine (purity not given) at doses of 0 or ca. 46 mg/kg bw/d (0 or 0.102% in the diet) for 117 weeks. Whole blood cells were prepared post-sacrifice. No data are given on toxicity. Positive controls were not included.

There was not a significant increase in the frequency of chromosome aberrations in whole blood cells after chronic treatment.

This study is classified as acceptable. The study is used as a supporting study.

In a NMRI mouse bone marrow micronucleus assay (King et al., 1979), 4 mice/sex/dose were treated i.p. with Caffeine (finest pure grade) at doses of 0 or 97 mg/kg bw (two applications within 24 h). The vehicle was physiol. saline, olive oil, or 3% gum arabic (vehicle not definitely stated). Data on toxicity are not given. Caffeine was tested at an adequate dose. The positive control induced the appropriate response.

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

In another Swiss CD-1 and MS/Ae mouse bone marrow micronucleus assay (Aeschbacher et al., 1986), 4 mice/sex/dose of each strain were treated by gavage with pure Caffeine (purity not further stated) at doses of 0, 50, 75, or 100 mg/kg bw (1 or 2 doses). The vehicle was water. There were no signs of toxicity during the study. Caffeine was tested at an adequate dose. The positive control induced the appropriate response.

There was a weak, non-significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow at the high dose only. Overall, the result was judged to be ambiguous.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 474 for in vivo cytogenetic mutagenicity data.

In comparative cytogenetic studies (micronucleus test in mouse and hamster, chromosomal analysis in hamster, and sister chromatid exchange test in hamster) (Tsuchimoto and Matter, 1979), CD-1 mice and Chinese hamsters were treated i.p. with Caffeine (purity not given) at doses of 0, 100, 200, or 250 mg/kg bw (two doses within a 24-hour interval). The vehicle was physiol. saline.

There was not a significant increase in the frequency of micronuclei, chromosomal aberrations, or sister chromatid exchanges; however, a slight and non-significant increase in chromosome aberrations was considered an indirect effect of extremely high dosage.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

2b. Chromosomal aberrations in human cells

In a chromosome aberration assay (Weinstein et al., 1972), human volunteers ingested Caffeine (purity not given) at totally 800 mg/day (two does of 400 mg daily) for 4 weeks. Lymphocytes were collected weekly and prepared for chromosome analysis.

There was no evidence of a chromosome damaging effect.

This study is classified as acceptable.

3. Effects on germ cells in animals

In a CD-1 mouse dominant lethal test (Aeschbacher et al., 1978), 50 males/dose) were treated with Caffeine (purity not given) at doses of 0 or 90 mg/kg bw/d by gavage for 5 days. In a further test, 150 males/dose were treated with Caffeine (purity not given) at doses of 0 or ca. 112 mg/kg bw/ via the drinking water for 8 weeks. Treated males were mated with untreated, virgin females at 1, 3, and/or 6 weeks after treatment. No mutagenic induction of dominant lethals, pre-implantation egg loss or depression of the fertility of females, caused by caffeine at the dose levels administered was observed. The positive control induced the appropriate response.

There was neither a mutagenic induction whatsoever nor any significant depression of the fertility after any treatment time.

This study is classified as acceptable.

In an ICR mouse oocyte chromosome aberration assay (Mailhes et al., 1996), 10 or 20 females/dose were treated i.p. with a single dose of Caffeine (purity not given) at 0 or 150 mg/kg bw at different intervals relative to a single dose of HCG (- 4 to 5 h). Oocytes were harvested at 17 h post HCG-treatment. The vehicle was deuterized water (D2O).

There was no retardation of the rate of neither oocyte meiotic maturation nor increase in neither the rate of aneuploidy nor induction of structural chromosomal aberrations after any treatment time.

This study is classified as acceptable.

In a C3H mouse testicular tissue chromosomal aberration assay (Adler, 1966), (14 males/dose) were treated i.p. with Caffeine (purity not given) in physiol. saline at doses of 0 or 200 mg/kg bw, as single dose or once daily for 21 days. Toxicity was not examined during the study. Caffeine was tested at an adequate dose. Positive controls were not included.

There was not a significant increase in the frequency of chromosome aberrations in testicular tissues after any treatment time.

This study is classified as acceptable.

Conclusion – in vivo:

There was no evidence of clastogenicity (induction of micronuclei or chromosomal aberrations) in rodents and human soma cells.

There was no evidence of a mutagenic activity in germ cells of rodents.

There was no evidence of a dominant-lethal effect in mice.

Overall conclusion:

No induction of point mutations was observed in vitro and in vivo. There were some clastogenic effects observed in vitro at high concentrations. However, there was no clastogenic activity observed in numerous in vivo tests as well as no induction of chromosome aberrations in germ cells; and there was no induction of dominant-lethal mutations in mice.


Short description of key information:
IN VITRO
There was no evidence of reverse gene mutation in bacteria.
There was no evidence of gene mutation in mammalian cells.
There was no definite evidence of clastogenicity (induction of chromosome aberrations) in mammalian/human cells.

IN VIVO
There was no evidence of clastogenicity (induction of micronuclei or chromosomal aberrations) in rodents and human soma cells or in germ cells of rodents (spermatocytes, oocytes).
There was no evidence of a dominant-lethal effect in mice.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

There is no need to classify Caffeine for mutagenicity according to the Directive 67/548/EC or GHS criteria.