Theophylline

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1998

Reference 2

Reference Type:

publication

Title:

Micronucleated Erythrocyte Frequency in Peripheral Blood of B6C3F1 Mice from Short-Term, Prechronic, and Chronic Studies of the NTP Carcinogenesis Bioassay Program.

Author:

Witt K.L.

Year:

2000

Bibliographic source:

Environ. Mol. Mutagen. 36: 163-194

Reference 3

Reference Type:

secondary source

Title:

Unnamed

Year:

1998

Reference 4

Reference Type:

secondary source

Title:

Micronucleated Erythrocyte Frequency in Peripheral Blood of B6C3F1 Mice from Short-Term, Prechronic, and Chronic Studies of the NTP Carcinogenesis Bioassay Program.

Author:

Witt K.L.

Year:

2000

Bibliographic source:

Environ. Mol. Mutagen. 36: 163-194, cited in: OECD SIDS for CAS-No. 58-55-9, 2004

Materials and methods

Principles of method if other than guideline:

According to MacGregor, J.T. et al. (1990): Fundam. Appl. Toxicol. 14, 513-522

GLP compliance:

not specified

Type of assay:

micronucleus assay

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: according to NTP, 1998, the purity was >99%:

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 4 weeks;
- Housing: 1 per cage
- Diet (e.g. ad libitum): NIH-07 open formula rodent feed; mash for the dosed-feed studies; ad libitum;
- Water (e.g. ad libitum): tap water; ad libitum;
- Acclimation period: 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 25 °C;
- Humidity (%): 35 - 70 %;
- Air changes (per hr): 10;
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: feed

Vehicle:

- Vehicle(s)/solvent(s) used: none;

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dose formulations were prepared weekly during the 13-week studies by mixing theophylline with feed to the
the required concentrations. Homogeneity and stability studies of the dose formulations were performed using HPLC and ultraviolet/visible spectroscopy. Homogeneity was confirmed for the feed formulations.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously in the diet

Post exposure period:

none

Doses / concentrations

Remarks:

Doses / Concentrations:
ca. 175, 400, 800 mg/kg bw/d (males); ca. 225, 425, 850 mg/kg bw/d (females) (1000, 2000, 4000 ppm in the diet)
Basis:
nominal in diet
Peripheral blood samples were collected from groups of 10 mice/sex at the end of a 13-week feeding study (see chapter 7.5.1).

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Positive control(s):

none;

Examinations

Tissues and cell types examined:

peripheral blood; normochromatic erythrocyte (NCE) and polychromatic erythrocyte (PCE) populations.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose levels for the studies were selected after evaluation of the results of previous 14-day repeated-dose studies (Lindamood et al.,
1988, Fundam.Appl.Toxicol.10,477-489).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The National Toxicology Program has conducted 13-week subchronic toxicity studies in B6C3F mice (10 animals/group) following administration of theophylline via the diet (feed): 0, 1000, 2000 and 4000 ppm.
Blood was obtained immediately before or at the time of sacrifice by retro-orbital bleeding; less often other methods were employed, including heart puncture, puncture of the ventral tail vessels, or tail clip.

DETAILS OF SLIDE PREPARATION:
Peripheral blood smears were prepared from B6C3F1 mice. Drops of blood were spread on precleaned standard glass microscope air dried, and fixed in absolute methanol for 5 min. Staining was performed with either Hoechst 33258/pyronin Y or acridine orange, except in cases where all study slides had previously been stained with a Giemsa-based stain. All slides were coded prior to scoring.

METHOD OF ANALYSIS:
Slides stained with acridine orange or Hoechst 33258/pyronin Y were scored at 630x or 1000x magnification by epifluorescence microscopy, and slides stained with Giemsa were scored by Koehler microscopy at 1000x.
Polychromatic erythrocytes (PCE) were scored by direct manual counting. Normochromatic erythrocytes (NCE) were scored using a semiautomated
method described by Jauhar et al. (1988), in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Routine micronucleus frequency scores were based on approximately 10,000 NCE or 1000 PCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 10,000 or 5,000 erythrocytes.

Evaluation criteria:

The decision to classify a test as negative, equivocal, or positive for induction of micronuclei in this in vivo assay was based on a broader evaluation of a number of factors that determined the biological relevance of the results, including the appropriateness of the concurrent control data, the magnitude of the observed response and the presence of a dose-dependent increase in the frequency of micronucleated cells.
Test chemicals were evaluated separately in each gender; results from tests in male and female mice were not combined to reach an overall conclusion.
Positive controls were not included, because the toxicity assays from which the test animals were obtained did not include positive control groups.

Statistics:

The frequency of micronucleated cells among NCE or PCE was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group.
Pairwise comparisons between each treatment group and the concurrent solvent control group were performed using an unadjusted one-tailed Pearson χ2-Test (Chi-Square-Test).
A test was considered positive if (1) the trend test P value was 0.025 or less or (2) the P value for any single exposure group was 0.025/N or less where N is the number of test chemical treatment groups. Trend test P values between 0.025 and 0.05 were considered to be equivocal if accompanied by a monotonic increase in the frequency of micronuclei over the dose range investigated. All other responses were considered to be negative.
The percentage of polychromatic erythrocytes (%PCE) data were analyzed by a standard ANOVA to determine if significant PCE suppression or stimulation occurred.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

A slight cytotoxic effect was observed based on the decrease in percent PCE.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Theophylline induced increases (weak to marginal) in micronucleated cells only in male mice. However, the effect noted with theophylline in the dosed-feed study, despite producing a significant trend test (P = 0.009), was judged not to be of sufficient magnitude to be considered positive and therefore the dosed-feed study with theophylline in male mice was concluded to be equivocal.

Any other information on results incl. tables

Dose (ppm)	Number of animals	Gender	micronuclei / 1000 NCE	P value (Witt)	% PCE
Control	10	Males	1.90 ± 0.17		1.95 ± 0.29
1000	10	Males	1.72 ± 0.12	0.8641	1.87 ± 0.18
2000	10	Males	2.02 ± 0.09	0.2348	1.91 ± 0.22
4000	10	Males	2.25 ± 0.19	0.0445	1.65 ± 0.17
Trend test (Witt)			P = 0.009
Trend test (NTP)			P = 0.096
ANOVA					P = 0.613
Control	10	Females	1.31 ± 0.13		1.81 ± 0.11
1000	10	Females	1.70 ± 0.20	0.0320	1.53 ± 0.11
2000	10	Females	1.56 ± 0.10	0.1292	1.55 ± 0.17
4000	10	Females	1.85 ± 0.24	0.0128	1.63 ± 0.21
Trend test (Witt)			P = 0.032
Trend test (NTP)			P = 0.034
ANOVA					P = 0.384

% PCE = percentage of polychromatic erythrocytes 5000 or 10000 total erythrocytes.
NCE = normochromatic erythrocytes
The NCE/PCE ratio is based on the scoring of 1000 erythrocytes
P value: Comparison of treated group to control χ2-Test (Chi-Square-Test).
Trend test: One-tailed trend test, significant at P ≤ 0.025.
ANOVA Test: Analysis of variance, significant at P ≤ 0.025.