Theophylline

Endpoint summary

Additional information

Valid experimental data were available to assess the genetic toxicity in vitro and in vivo.

In vitro studies:

Gene mutation in bacteria

In a standardized Ames test comparable to OECD 471, theophylline was tested in the preincubation assay with Salmonella typhimurium TA97, TA98, TA100, TA1535 in concentrations from 100 up to 10000 µg/plate, with and without metabolic activation using liver S-9 mix from Aroclor 1254 -induced male Sprague-Dawley rats or syrian hamster. No mutagenic and cytotoxic effects were observed (NTP 1998; Zeiger et al . 1988).

Mammalian chromosome aberration test

In an in vitro assay comparable to OECD 473, theophylline was tested in Chinese hamster ovary (CHO) cells for chromosomal aberrations in the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 mix at the concentrations of 510, 555 and 600 µg/mL. The high dose was limited by toxicity. Theophylline did not induce chromosomal aberrations in CHO cells with and without metabolic activation (NTP 1998).

Mammalian cell gene mutation assay

In a standardized mouse lymphoma assay comparable to OECD 476, theophylline was tested up to 5000 μg/ml with and without metabolic activation (S-9 mix from the liver of phenobarbital- and 5,6 -benzoflavone-pretreated Sprague-Dawley rats) for 3 hours and a negative result was described (Honma 1999a, Mutagenesis 14, 5-22). In a second mouse lymphoma assay with a longer treatment period of 24h the authors found weakly positive results in concentrations leading to reduced relative survival of less than 50 %. There was no information about colony size, therefore, no conclusions can be drawn as to whether the substance caused chromosomal aberrations or gene mutations (Honma 1999b, Mutagenesis 14, 23-29). Moreover, theophylline as a base analog yields mutations indirectly during chromosome segregation and DNA replication and does not directly damage DNA. Therefore the mutagenic potential may be cell cycle dependent and may require long-term treatment (Honma et al., 1999b). As well, long-term treatment indicates cytotoxicity, which was not observed after the short-term treatment.

Further in vitro tests have been performed with microorganisms, cell cultures and human lymphocytes. Negative and as well positive results were reported. Some positive results were found only at high, cytotoxic concentrations and without metabolic activation systems, whereas addition of metabolic activation systems resulted in negative or questionable results. Some inconclusive or positive responses were seen in non-validated test systems or in not well documented studies. Hence, these few questionable or positive results were judged not suitable to be taken into account in the overall evaluation (OECD SIDS 2004).

In vivo studies

Mammalian chromosome aberration assay

In a chromosome aberration assay comparable to OECD 475, theophylline, was administered to B6C3F1 mice by intra peritoneal injection with up to 250 mg/kg bw (62.5; 125 and 250 mg/kg bw/day). Theophylline did not induce chromosomal aberrations in bone marrow cells examined after 17 hours (standard procedure). Additionally, after delayed harvest (36 hours) which was performed to evaluate delayed clastogenic effects, theophylline was negative. Thereby, dose levels were limited by toxicity to 250 mg/kg in the standard harvest time study and 150 mg/kg in the extended harvest time study. In contrast, theophylline showed a modest, significant and dose-related increase in the number of sister chromatid exchanges at doses of 125 and 250 mg/kg bw (McFee, A.F., 1991 Mutat. Res. 264, 219 - 224, NTP 1998). However, the trial was not repeated and confirmed.

In a mammalian micronucleus test in peripheral blood erythrocytes, comparable to OECD 474, theophylline was administered by feed (males: 175 to 800 mg/kg bw/day; females: 225 to 850 mg/kg bw/day) and gavage (males: 75 to 300 mg/kg bw/day; females: 75 and 150 mg/kg bw/day) for 13 weeks. No significant increases in the frequencies of micronucleated normochromatic erythrocytes were seen in male or female mice (NTP 1998, Witt et al. 2000).

In a mammalian spermatogonial chromosome aberration test comparable to OECD 483, theophylline (ca. 230 mg/kg bw/day) was fed to male rats for 75 weeks. No chromosomal aberrations as well as no inhibition of mitotic ratio were observed in spermatogonial cells from testis (Friedman et al., 1979, J.Environ. Pathol. Toxicol. 2, 687 - 706; NTP 1998).

Negative results were described in a weakly documented dominant lethal assay after i.p. injection of 380 and 480 mg/kg bw in mice (Epstein S.S. et al., 1972, Toxicol. Applied Pharmacol. 23, 288 -325; Epstein S.S. and H. Safner, 1968, Nature 219, 385 -387) and in a host mediated assay after doses up to 300 mg/kg bw in mice (Gabridge M .G. and Legator M.S., 1969, Proc. Soc Exp. Biol. Med . 130, 831 -834).

A slight increase of sister chromatid exchanges in bone marrow cells of mice (factor 1 .3-1 .8 in comparison to the control, no inhibition of cell cycle) was observed after i .p. administration of theophylline up to 250 mg/kg bw (Giri A.K. et al. 1999, Mutat. Res. 444, 17 -23; Mc Fee A.F. 1991, Mutat. Res. 264, 219 -224) and after oral gavage of doses up to 600 mg/kg bw/day (Renner 1982, Experientia 38 (5), 600).

Conclusion :

Theophylline was not mutagenic or clastogenic in most of the standard in vitro tests. Positive results were found only at high, cytotoxic concentrations and without metabolic activations. Theophylline had no mutagenic or clastogenic effects in vivo.

Short description of key information:
Theophylline was not mutagenic or clastogenic in most of the standard in vitro tests and has no mutagenic or clastogenic effects in vivo.

In vitro:
Gene mutation in bacteria:
S. typhimurium TA97, TA98, TA100, TA1535, with and without metabolic activation (Ames test): negative (comparable to OECD 471; Zeiger et al., 1988 (Environ.Mol. Mutagen. 11, 1-158); NTP 1998).

Cytogenicity in mammalian cells:
Mammalian Chromosome Aberration Assay:
Chinese hamster Ovary (CHO) cells, with and without metabolic activation: negative (comparable to OECD 473; NTP 1998).

Gene mutation in mammalian cells:
Mouse lymphoma assay (L5178Y cells), with and without metabolic activation: negative/weak mutagenicity (comparable to OECD 476; Honma et al., 1999).

in vivo:
Mammalian chromosome aberration assay:
Micronucleus, mouse, 13 weeks, feed and gavage, peripheral blood: negative (comparable to OECD 474; NTP, 1998; Witt et al., 2000).

Chromosome aberration assay (cytogenetic), male rat, feeding (75 weeks), spermatogonial cells from testis: negative (comparable to OECD 483; Friedman et al., 1979, J.Environ. Pathol. Toxicol. 2, 687 - 706; NTP 1998).

Chromosome aberration assay (cytogenetic), mouse, i.p., bone marrow cells: negative (comparable to OECD 475; McFee et al., 1991, Mutat. Res. 264, 219-224; NTP 1998).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was predominantly not genotoxic in in vitro experiments using rodent and bacterial as well as human cells nor in in vivo experiments in rats and mice. Therefore, theophylline has not to be classified according to requirements of the EU Annex VI of directive 67/548/EEC and GHS.