Theophylline

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Additional information

Theophylline was tested for its effects on reproduction and fertility in CD-1 mice (20 animals per group and sex; 40 animals in the control group per sex) using the Reproductive Assessment by Continuous Breeding (RACB, Task 2) protocol (Morrisey et al., 1988; NTIS 1985a).

Data on food and water consumptions, body weights, and clinical signs during a two week dose-range-finding study (Task 1) were used to set exposure concentrations for the Task 2 study (1 week premating exposure period (males and females), 14 weeks of cohabitation, 3 weeks post cohabitation) at 750, 1500, and 3000 ppm in feed (corresponding to126, 260, and 500 mg/kg bw/day). Food consumption was not altered by theophylline addition.

Alopecia occurred in both sexes of all groups of treated animals (20-25% in the 126 mg/kg bw/d group, and >50% in the 260 and 500 mg/kg bw/d groups). Seven mice died during Task 2: 3 controls, and 4 in the low dose group. A single control mouse showed alopecia; this was less severe than that seen in the treated mice.

Significant reproductive effects were observed: there was a 19 % reduction in the mean number of litters per pair in the high dose group, decreased number of live pups per litter in all dosed groups (reduction by 22 %, 29 %, and 42 % in the low, mid and high dose level, respectively), and, at the highest concentration level, a 6 % decrease in live pup weight adjusted for litter size. The number of days to delivery of each litter was consistently enhanced in the high dose group, being longer by 3 days for the first litter, and by 5 days for the last litter, and similarly increased for all others.

Given the significant adverse effects on reproductive performance during the cohabitation phase of the study, a one week crossover mating (Task 3) was conducted between mice of each sex in the high dose group and control mice of the opposite sex in an attempt to identify the affected sex. Thereby all mice were fed the control diet during the crossover mating and were then returned in the treatment groups. As results, there were no differences in the percent of pairs mating, or delivering a live litter.

However, in the group cohabiting control males and high dose exposed females, the proportion of pups born alive was reduced by 16%, and the adjusted live pup weight was reduced by 15%. The results suggest that the female mice may be more sensitive.

After the litters in Task 3 were delivered, evaluated, and discarded, the females were evaluated for vaginal cyclicity for 7 days, and then the parental (FO) mice in the control and high dose groups were killed and necropsied. In the high dose groups the terminal body weight was increased by 5 % in females and decreased by 7 % in males. In the high dose males and females, relative liver weights were increased (7 and 16 %, respectively). Additionally, in males, body weight adjusted seminal vesicle weight was decreased by 24% in the high dose group.

There were no changes in the length of the estrous cycle, or in the percent of time spent in the various estrous stages in females. In males, epididymal sperm density was reduced by 20% in the high dose group; the percent of motile and of abnormal morphologic forms were unchanged by 500 mg/kg bw/d theophylline exposure.

No second generation analysis was conducted (NTIS 1985a).

The reliability of the study was limited due to a high mortality rate in the treated and control groups, and since only the control and the high dose groups were the only groups studied at crossover mating and histopathology (OECD SIDS 2004). A NOAEL was not achieved in this study. Alopecia was not considered as adverse effect in the parental generation.

Based on the reproductive effects in absence of maternal toxicity, the LOAEL for reprotoxicity is 126 mg/kg bw/d.

 

Two experiments were conducted to investigate the effects of feeding theophylline to Osborn-Mendel and Holtzmann rats at a dietary level of 0.5% (ca. 230 mg/kg bw/d). Induction of testicular atrophy, oligospermatogenesis and aspermatogenesis was observed after75 weeks in Osborn-Mendel rats and after 19 weeks in Holtzmann rats (Friedman L. et al. 1979, J. Environ.Pathol. Toxicol, 2, 687 -706; Weinberger M.A. et al. 1979, J. Environ.Pathol. Toxicol. 1, 669 -688, both cited in OECD SIDS 2004), but not after 14 weeks. Because of the small group size of investigated animals (only 6-7 rats of 20 survived), the high mortality (up to 71 %) and the single dose tested, the studies are regarded to be inappropriate for assessing the toxicological potential of the substance.

In contrast, no histopathological changes were found in testes in well documented subchronic feeding/gavage studies in rats and mice (see Chapter 7.5).

  

Conclusion:

Reproductive toxic effects (reduced litter number and pup viability, decreased live pup weight) were seen in a continuous breeding study in mice at doses, which caused no toxicity. In 13 week-studies performed with rats and mice given theophylline by gavage or by feed, no significant differences in sperm morphology and vaginal cytology parameters or histopathological effects in the sex organs were found between control and exposed animals (see Chapter 7.5). Alltogether the data for toxicity to reproduction were inconclusive, but not sufficient for classification. 

Short description of key information:
In a reproductive assessment by continuous breeding study in mice, theophylline caused changes in parental body weight and significant reproductive effects to the offspring, whereas in the repeated dose studies no effects were observed in sperm morphology or in the estrous cycle of rats and mice. A potential of theophylline to impair fertility in high doses cannot be fully excluded.
In a continuous breeding reproductive assay study, theophylline was fed to CD-1 mice with the doses of 126; 260 and 500 mg/kg bw/day (corresponding to 0.075; 0.15 and 0.3 % in the diet) for 18 weeks (NTP 1985, NTIS PB85-204659, RACB84074; Morrissey R.E. et al., 1988, Fund. Appl. Toxicol. 10, 525-536). Subsequently, a cross over mating was performed to determine which sex was affected.
A NOAEL could not be achieved in this study. Based on adverse reproductive effects in absence of maternal toxicity, the LOAEL of 126 mg/kg bw/day were determined.

Effects on developmental toxicity

Description of key information

Theophylline was not shown to cause malformations in animal studies when given at doses that were not toxic to the parental animals. In the presence of maternal toxicity, fetotoxicity was observed in rats, mice and rabbits as well as teratogenicity in rabbits.  
oral, rat, feeding, gestation day 6 - 15, NTP Program (Lindstroem et al., 1990, Fund.Appl.Toxicol. 14, 167-178; NTIS 1985, PB86-108172, TER 8411 0): 
NOAEL maternal toxicity: 124 mg/kg bw/day (reduced body weight, reduced food and increased water consumption)
NOAEL fetotoxicity: 124 mg/kg bw/day (reduced average fetal body weight per litter)
NOAEL teratogenicity: ≥ 259 mg/kg bw/day (no malformations or variations observed)
oral, mouse, drinking water, gestation day 6 - 15, NTP Program, (Lindstroem et al., 1990, Fund.Appl.Toxicol. 14, 167-178; NTIS 1985, PB86-108172, TER 8411 0): 
NOAEL maternal toxicity: ca. 282 mg/kg bw/day (Clinical signs, reduced body weight and water consumption)
NOAEL embryotoxicity: ca. 282 mg/kg bw/day (Increased percentage of resorptions)
NOAEL fetotoxicity: ca. 282 mg/kg bw/day (Decreased average fetal body weight)
NOAEL teratogenicity: ≥ 396 mg/kg bw/day (slight, not statistical significant increase in malformations)
intravenous, rabbit, gestation day 6 - 15, basic data (Shibata et al., 2000): 
NOAEL maternal toxicity: 30 mg/kg bw/day (decreased body weight, clinical signs)
NOAEL fetotoxicity: 30 mg/kg bw/day (absorption, late death, decreased body weight)
NOAEL teratogenicity: 30 mg/kg bw/day (increased rate of fetal cleft palates)

Additional information

In a developmental toxicity study following the NTP standard method, groups of 20 – 21 timed-pregnant female Sprague -Dawley (CD) rats were treated with theophylline (NTIS 1985, Lindstroem 1990). Theophylline in the concentrations of 0, 1500, 3000, or 4000 ppm, corresponding to 0, 124, 218, and 259 mg/kg bw/d was administered continuously in the feed on gestational days (gd) 6 through 15.

During the treatment, dams exhibited clinical signs of toxicity consisting primarily of piloerection, transient weight loss, and rough coat, which were observed more frequently in the two higher treatment groups. No dose related maternal mortality occured.

Maternal body weight gain during gestation (gd 0 through 20), and treatment (gd 6 through 15), as well as maternal weight gain corrected for gravid uterine weight decreased about 19.5, 53.3, and 22.4 %, respectively, in a dose dependent manner and exhibited significant differences among treatment groups with the high dose group significantly (p < 0.05) being below controls.

Additionally, gravid uterine weight and absolute maternal liver weight showed a decreasing trend with an increasing theophilline concentration in the diet; there were, however, no differences between controls and any single treatment group. Relative maternal liver weight (i.e., percentage body weight) was unaffected by treatment.

During treatment and during the entire gestation period maternal food consumption (g/kg/day) decreased in a dose related manner and was significantly below controls in both the 3000 ppm and 4000 ppm dose groups during treatment, and in the 4000 ppm dose group for the entire gestation period. Maternal water consumption during treatment (g/day or g/kg/day) increased in a dose related manner up to 15% during gestation and up to 26% during treatment, with all theophylline treated groups significantly above controls.

 

There were no differences among treatment groups in the number of corpora lutea or implantation sites per dam, or in the percentage of preimplantation loss. Theophylline treatment had no effect on the percentage of dead fetuses per litter, or on the percentage of litters with one or more resorptions, dead fetuses, nonliving implants (i.e. resorptions and dead fetuses), or adversely affected implants (i.e. resorptions, dead fetuses and malformed live fetuses), or on the percentage of resorptions, dead fetuses, nonliving implants, or adversely affected implants per litter.

Theophylline treatment resulted in a significant decrease in live fetuses per litter in the high dose group (12) compared to the control (13.8). Average fetal body weight per litter (male, female, and combined) decreased in a dose- related manner, an effect significant in the mid (9%) and high dose (up to 15%) group.

Theophylline administered continuously from gd 6 to gd 15 had no effect on the percent of live fetuses malformed per litter or the percent of live male or female fetuses malformed per litter.

Malformed fetuses per litter occurred with an incidence of 1.38%, 0.92%, 0.33%, and 1.57% for the vehicle control, low, medium, and high dose groups, respectively.

The incidence of litters with one or more malformed live fetuses as well as the incidence of litters with one or more external, skeletal, or visceral malformations was unaffected by theophylline treatment.

 

Theophylline exposure resulted in significant dose-related fetotoxicity as evidenced by decreased average fetal body weight per litter at dose levels of 218, and 259 mg/kg bw/d and reduced number of live fetuses per litter at the high dose. These effects occurred in the presence of maternal toxicity (reduced corrected body weight gain 10% at 218 mg/kg bw/d, clinical signs like piloerection and rough coat), which was more pronounced at 259 mg/kg bw/d than at 218 mg/kg bw/d. (NTIS 1985, Lindstroem 1990)

 

According to the conditions of the test, the NOAEL for both, maternal toxicity and fetotoxicity is 1500 ppm (124 mg/kg bw/day) and the NOAEL for teratogenicity is ≥ 4000 ppm (≥ 259 mg/kg bw/day) in rats.

 

In a parallel study, theophylline was administered in the drinking water to pregnant CD 1 mice at the concentrations of 0, 750, 1500 or 2000 ppm corresponding to 282, 372, and 396 mg/kg bw/d) on gd 6 through 15. Clear direct signs of maternal toxicity occurred and were substantiated by distinct reduction of absolute and relative body weight gain (up to 31%) and reduction of water consumption in the mid and high dose group. Treatment at the 1500 ppm or 2000 ppm level resulted in an increase in the percent of resorptions (dead implants: 750 ppm: 14, 1500 ppm: 27, and 2000 ppm: 34%, respectively) and a decrease in the average fetal body weight (9 and 14%) per litter. The percentage of dead fetuses was not affected.

In the treated groups there was a slight, not statistically significant trend in the proportion of litters with malformed fetuses and for the incidence of external malformations in the mid and high-dose groups (cleft palates, exencephaly). Cleft palates also occurred in the control group, while exencephaly was only observed at the low and mid dose levels. However, it is well known from the literature (Schwetz B et al. 1977, Toxicol. Appl. Pharmacol. 40, 307 -315, cited in SIDS 2004; Beyer P.E . and N. Chemoff 1986, Teratogenesis, Carcinogenesis, and Mutagenesis 6: 419 -429, cited in SIDS 2004) that particularly in this species, stress and depreviation of water during gestation may induce these types of malformations in the offspring. Furthermore, this study was not designed to distinguish effects on the offspring caused by food and water deprivation from those caused by exposure to theophylline.

The authors, therefore, concluded that theophylline treatment was not associated with an increase in any particular malformation or group of malformations (Lindstroem, 1990)

According to the conditions of the test, the NOAEL for both, maternal toxicity and fetotoxicity is 750 ppm (282 mg/kg bw/day) and the NOAEL for teratogenicity is 2000 ppm (396 mg/kg bw/day) in mice.

 

A further study (Shibata et al., 2000) investigated the teratogenic and fetal toxicity of i.v. applications of theophylline and its relationship to maternal plasma levels in pregnant Kbl:JW rabbits. Theophylline was administered i.v. to pregnant rabbits (17 – 19 animals per group) at doses of 15, 30 and 60 mg/kg bw/d using an automatic infusion pump from days 6 to 18 of gestation.

In the highest dose group a significant decrease in body weight was observed from gestation day 11 onwards, and a decrease in food intake was noted during days 7-23.

Theophylline showed clear signs of maternal toxicity at 60 mg/kg bw/d like accelerated respiration, abortion, sluggish startle reactions, dilatation of the auricular vessels and polyuria. There were no signs of maternal toxicity in the dams given 15 and 30 mg/kg bw/d.

In the high dose group, 1 animal died on day 17 of gestation and 4 animals aborted between day 21 and 27.

Fetuses from the dam group treated with 60 mg/kg bw/d exhibited developmental toxicity, such as increased number of late deaths, increased fetal body weights (about 10% below concurrent controls) and effects on fetal morphology.

 

An increased rate of fetuses with cleft palates (8 out of 103 fetuses, in 2 of 14 litters) has to be considered as a malformation (teratogenicity), whereas a 13th rib (63 out of 103 fetuses, number of affected litters not exactly specified) is assessed as a variation because it appears quite frequently in control rabbit fetuses in the strain used for this study. No substance induced signs of developmental toxicology were observed in fetuses from the 15 and 30 mg/kg bw/d group.

In the 15, 30 and 60 mg/kg bw/d groups, maternal plasma concentrations (Cmax) during the treatment period were approximately 30, 56 and 106 μg/mL, respectively. These concentrations clearly exceed the effective therapeutic range of theophylline in clinical use (Shibata et al. 2000).

According to the conditions of the test, the NOAEL for maternal toxicity and fetotoxicity/teratogenicity is 30 mg/kg bw/day in rabbits.

Reproductive effects in humans

Theophylline is used as bronchodilator for asthma and chronic obstructive pulmonary disease in the pregnant patient (reviewed in Briggs et al. 1991, Williams and Wilkins, Baltimore). Theophylline crosses the placenta, and newborn infants may have therapeutic serum levels. Transient tachycardia, irritability, and vomiting have been reported in newborns delivered from mothers consuming theophylline. These effects are more likely to occur when maternal serum levels at term are in the high therapeutic range or above (therapeutic range 8-20 μg/ml). However, cord blood levels are approximately 100% of the maternal serum concentration.

In studies with female theophylline drug users, no association with congenital abnormalities was seen (Nelson M.M. and Forfar J.O., 1971: Br. Med. J. 1, 523-527; see Chapter 7.10.3) and women receiving theophylline during pregnancy did not deliver stillborn infants compared to controls (Neff R.K. and Leviton A. 1990, Chest 97, 1266-1267; see Chapter 7.10.3).

No effects on development of premature infants were seen (Nelson R.M. et al., 1980, Dev. Pharmacol. Ther. 1, 274-280; Ment L.R. et al. 1985, Am. J. Perinatol . 2, 223-227; see Chapter 7.10.3).

Theophylline therapy in surviving premature children of birth weight <1501 g showed at 14 years of age significantly higher rate of cerebral palsy compared to children not exposed. In contrast children who had received theophylline achieved higher psychological test scores. There was no association between theophylline therapy and growth (Davis P.G. et al. 2000, J. Pediatr. Child Health 36, 47-50; see Chapter 7.10.3).

Justification for classification or non-classification

Based on the available data for theophylline, theophylline has not to be classified for its potential to affect reproduction and development aversively according to requirements of the EU, Annex VI of Directive 67/548/EEC and of GHS.