Registration Dossier

Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Sperm abnormality assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data provided is from a peer-reviwed publication.

Data source

Reference
Reference Type:
publication
Title:
Gene mutation toxicity study of the test chemical
Author:
Bruce WR et al.
Year:
1978
Bibliographic source:
Canadian Journal of Genetics and Cytology

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
A significant increase in the proportion of abnormally shaped sperm cells has been observed in mice between four to eleven weeks after exposure to mutagenic agents (Proc. Natl. Acad. Sci. U.S.A. 1975; 72: 4425-4429). The abnormal sperm morphology is likely caused by small deletions, point mutations or both according to data from previous studies
GLP compliance:
not specified
Type of assay:
other: Sperm abnormality assay

Test material

Details on test material:
Name: Codeine phosphate
IUPAC name: 3-methoxy-17-methyl-7,8-didehydro-4,5-epoxymorphinan-6-ol phosphate (salt)
Molecular weight: 397.4 g/mol
Molecular formula: C18H24NO7P
Smiles: COc1ccc2C[C@@H]3[C@@H]4C=C[C@H](O)[C@@H]5Oc1c2[C@]45CCN3C.OP(=O)(O)O
Purity:No data
Impurity:No data

Test animals

Species:
mouse
Strain:
other: Hybrid mice (C57BL/6 X C3H/He)
Sex:
male
Details on test animals or test system and environmental conditions:
The rats were housed in suspension, wire bottom cages, in air conditioned rooms with automated light and dark cycles, and were allowed food (Purina Chow) and water ad lib. They were not treated with antibiotics or insecticides.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Water
Details on exposure:
The highest dose was selected by chosing a convenient value close to the LD50, which was initially established in the study.
Duration of treatment / exposure:
Five consecutive days
Frequency of treatment:
Once daily
Post exposure period:
30 days
Doses / concentrations
Remarks:
0 (vehicle), 125, 250 and 500 mg/kg bw/day
No. of animals per sex per dose:
3 to 4.
Control animals:
yes, concurrent vehicle
Positive control(s):
Benzo(a)pyrene

Examinations

Tissues and cell types examined:
Sperm cells
Details of tissue and slide preparation:
Sperm suspensions were prepared by mincing the cauda epididymis in 2 ml phosphate buffered physiological saline, pipetting the resulting suspension and filtering it through an 80 um stainless steel mesh to remove tissue fragments. A fraction of each suspension was mixed 10:l with 1% eosin-Y in water. Thirty min later smears were made, allowed to dry in air, and mounted under a coverslip with Permount. Three hundred and thirty-three sperm were examined at high magnification from each animal for a total of approx 1000 per treated group.
Evaluation criteria:
The results were plotted as percent abnormal sperm after subtraction of the frequency found in the control group. Threshold for biological significance was breached if the treated group exceeded the control group by 1% (10/1000). The test chemical was considered active when positive results were obtained consistently, i.e., when dose response curves could be constructed, or the results could be repeated.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Based on the presented data, the test chemical is non-mutagenic in mouse sperm cells following intraperitoneal injections at doses up to 500 mg/kg bw.
Executive summary:

The test chemical was tested for mutagenicity in this sperm abnormality assay by Bruce and Heddle. In brief, the assay was used as an in vivo method to study mutagenicity because it has been shown that mice exposed to mutagenic agents exhibit significantly increased numbers of abnormally shaped sperm cells. The abnormal sperm morphology is likely caused by small deletions, point mutations or both. Three male mice per dose level were exposed to the test chemical via intraperitoneal injection at 0 (vehicle), 125, 250 and 500 mg/kg bw/day for 5 consecutive days. The highest dose level was close to the pre-determined LD50 value. Mice treated with benzo(a)pyrene served as positive controls. All mice were sacrificed 30 days after the last injection. Sperm samples were then prepared accordingly to calculate the number of abnormally shaped sperm cells. A total of 333 sperm cells from each animal were examined under high magnification. The results were plotted as percent abnormal sperm after subtraction of the frequency found in the control group. Threshold for biological significance was breached if the treated group exceeded the control group by 1% (10/1000). The test chemical was considered active when positive results were obtained consistently, i.e., when dose response curves could be constructed, or the results could be repeated. No significant increase in the proportion of abnormal sperm cells could be observed following treatment with the test chemical. In contrast, the positive control group showed a distinct increase in the proportion of abnormal sperm cells at 125 mg/kg bw/day (lowest dose tested). Based on the presented data, the test chemical is non-mutagenic in mouse sperm cells following intraperitoneal injections at doses up to 500 mg/kg bw.