Theophylline

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: very high doses, high mortality in controls.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Reproductive Effects of Theophylline in Mice and Rats

Author:

Morrissey, R.E. et al.,

Year:

1988

Bibliographic source:

Fund. Appl. Toxicol. 10, 525-536

Reference 2

Reference Type:

publication

Title:

Theophylline: Reproduction and Fertility Assessment in CD-1 Mice When Administered in Drinking Water/Feed.

Author:

NTP

Year:

1985

Bibliographic source:

Final Report, NTIS PB85-204659, RACB84074.

Reference 3

Reference Type:

secondary source

Title:

Reproductive Effects of Theophylline in Mice and Rats

Author:

Morrissey, R.E. et al.,

Year:

1988

Bibliographic source:

Fund. Appl. Toxicol. 10, 525-536; cited in: OECD SIDS for CAS-No. 58-55-9, 2004

Reference 4

Reference Type:

secondary source

Title:

Theophylline: Reproduction and Fertility Assessment in CD-1 Mice When Administered in Drinking Water/Feed.

Author:

NTP

Year:

1985

Bibliographic source:

Final Report, NTIS PB85-204659, RACB84074; cited in: OECD SIDS for CAS-No. 58-55-9, 2004

Materials and methods

Principles of method if other than guideline:

Reproductive Assessment by Continuous Breeding (RACB); study design according to Reel et al., 1985, J. Amer. Coll. Toxicol. 4,147-162 and Lamb et al., 1985, J. Amer. Coll. Toxicol. 4,172-184, Toxicol. Appl. Pharmacol. 81, 100-112.
RACB begins with a 14 day dose range-finding assay (Task 1), followed by the continuous breeding phase (Task 2) and in case of adverse effects by crossover mating (Task 3).

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Theophylline

EC Number:

200-385-7

EC Name:

Theophylline

Cas Number:

58-55-9

Molecular formula:

C7H8N4O2

IUPAC Name:

1,3-dimethyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione

Details on test material:

- Name of test material (as cited in study report): theophylline;
- Analytical purity: >99%.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 11 weeks;
- Housing: breeding pairs or individually per cage
- Diet (e.g. ad libitum): NIH-07 open formula mash feed for the dosed-feed studies; ad libitum;
- Water (e.g. ad libitum): tap water; ad libitum;
- Acclimation period: 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C;
- Humidity (%): 35 - 70 %;
- Air changes (per hr): 10;
- Photoperiod (hrs dark / hrs light): 10h/14h.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 98 days
- Other: After the 98 day cohabitation period, the pairs were separated for 21 days, during which time any final litters were delivered and kept for an additional 21 days. The reproductive capability of the offspring was not assessed.
Since an adverse effect of chemical treatment was detected during the cohabitation phase of the continuous breeding reproduction study, a 1-week crossover mating trial was carried out between mice of each sex in the 0.3% treatment group and control mice of the opposite sex in order to determine which sex was affected. This mating trial was conducted after continuous theophylline treatment for 19 weeks. All mice were fed the control diet during the mating trial and were then returned to the specified treatment groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosage analyses (ultraviolet spectrophotometric) both prior to and after dosing confirmed that the dosage formulation in feed were homogenous and were within ±10% of the target concentrations.

Duration of treatment / exposure:

Continuous breeding reproductive assay (Task 2): Treatment for a total of 18 weeks (1 week premating exposure period (males and females), 14 weeks of cohabitation, 3 weeks post cohabitation).
Cross over mating (Task 3): 19 weeks of continuous treatment before mating trial, control diet during mating trial, subsequently treatment according to the specified groups.

Frequency of treatment:

continuously in the diet

Doses / concentrations

Remarks:

Doses / Concentrations:
ca. 126, 260, 500 mg/kg bw/d (0.075, 0.15, 0.3 % in the diet)
Basis:
nominal in diet

No. of animals per sex per dose:

control: 40 mice of each sex; treatment groups: 20 animals of each sex.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale (Task 1): A portion of the animals was used for 14-day range-finding study, which utilized one control group and five groups of dosed animals (0.0375, 0.075, 0.15, 0.3, and 0.6% in drinking water; n = 8 males and 8 females per treatment level). Because a distinct precipitate was noted during 2 weeks of treatment, the route of administration was changed to feed for subsequent phases of the study. Mice of the 0.6% group were adversely affected with respect to body weight, therefore doses used in the continuous breeding phase were 0, 0.075, 0.15, and 0.3%.

Examinations

Parental animals: Observations and examinations:

At necropsy, the endpoints of target organ toxicity examined for the males included organ weights, percentage motile sperm, sperm concentration, and percentage abnormal sperm. Endpoints examined for the females included organ weights and estrous cycle length.

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 (F0) mice in the control and 500 mg/kg bw/d (0.3 % in the diet) theophylline groups were killed and necropsied.

Litter observations:

During the 98 days feeding study, data (body weight, proportion of males, numbers of litters per pair, number of live pups) were collected within 12 hr of birth on all newborns during this period, after which each litter was discarded.

After the crossover mating, litter size, sex, and pup weight was analyzed.

Statistics:

The Cochran-Armitage test (Armitage, 1971) was used to test for a dose-related trend. The χ2 test of homogeneity was used in the crossover mating trial since increasing dose levels were not being tested and only differences with respect to fertility and the presence of copulatory plugs were being evaluated. Pairwise comparisons involving mating and fertility indices were performed using Fisher's exact test. Differences were considered statistically significant if p < 0.05.
The number of litters and the number of live pups per litter were computed on a per fertile pair basis and then treatment group means were determined. The proportion of live pups was defined as the number of pups produced by each pair. The sex ratio was expressed as the proportion of male pups born to each fertile pair. Dose group means for these parameters were tested for overall differences by using the Kruskal-Wallis test and for ordered differences using Jonckheere's test, Pairwise comparisons of treatment group means were performed by applying the Wilcoxon-Mann-Whitney U test.
Since the number of pups in a Iitter may affect the average weight of the litter, an analysis of covariance was used to test for treatment differences in average pup weight, adjusting for average Iitter size (live and dead pups). Pairwise comparisons were done using a two-sided t test. A Kruskall-Wallis test was also performed.
For organ weights, least-squares treatment group means were generated from an analysis of covariance (with body weight as the covariate) and were tested for overall equality using the F test and for pairwise equality using a t test. All comparisons were two-sided. The Kruskall-Wallis and Wilcoxon-Mann-Whitney U tests were also used.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

- 3/40 control females died during week 15; 4/20 females of the low dose group died during cohabitation.

- Alopecia was observed in all treated animals (20-25% of the low dose mice and >50% in both mid and high dose mice) and in one control mouse (less severe than observed in the dosed groups). According to the authors, this was considered to be a sign of general toxicity.

Further parental effects were only investigated for the control and the high dose group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Feed consumption was not altered by theophylline addition.
- In the high dose females, terminal body weights were increased (5%).
- In high dose males, terminal body weights were decreased by 7% when compared with controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- The estrous cycle was unaffected.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- Epididymal sperm density was reduced by 20% in high dose males.
- The percent motile and the percent of abnormal morphologic forms was not affected in high dose males.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- In the high dose males and females, relative liver weights were increased (7 and 16 %, respectively).
- Relative seminal vesicle weights were decreased by 24 % in high dose males.

Effect levels (P0)

Dose descriptor:

LOAEL

Effect level:

126 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: Adverse reproductive effects (live pubs/litter, live pup weight) in absence of maternal toxicity.

Results: F1 generation

Details on results (F1)

With the breeding study, severe reproductive effects were found.
- The mean number of litter/pair was significantly reduced by 19% in the high dose group.
- The number of live pups/litter was significantly reduced in all dosed groups (reduction by 22%, 29%, and 42% at the low, mid and high dose level, respectively).
- Live pup weights adjusted for the litter size were significantly decreased in the high dose group (by 6%).
- The number of days to delivery was prolonged 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.

With the crossover mating, the following effects were observed.
- There were no differences in the percent of pairs mating, or delivering a live litter.
- In the group cohabiting control males and high dose 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.

No second generation analysis was performed.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

According to the authors, administration of the test substance resulted in significant adverse reproductive effects (affected offspring and changes of the male reproductive organs). The reproductive effects were not considered to be associated with the alopecia.

A NOEL was not achieved in this study.